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Chapter 1 The cerebellum: chemoarchitecture and anatomy
CHAPTER I
The cerebellum: chemoarchitecture and anatomy J. VOOGD, D. JAARSMA AND E. MARANI
......... but the Spirits inhabiting the Cerebel perform unperceivedly and silently their Work of Nature without our Knowledge or Care. Thomas Willis. Of the Anatomy of the Brain. Englished by Samual Pordage, Esquire, London. Printed for Dring, Harper, Leigh and Martyn, 1681. Facsimile Edition, McGill University Press, Montreal, 1965. p. 111.
1. INTRODUCTION During the last 150 years the morphology of the cerebellum attracted numerous histologists. Its relatively simple structure, with its three-layered cortex and clearly defined afferent and efferent connections made it one of the favourite sites in the brain to test out new hypotheses on the connectivity, the development and chemical interaction in nervous tissue. We have attempted to review present knowledge about the external and internal morphology of the cerebellum and to relate the 'classical' topography of the cerebellum to the more recently discovered chemical specificity of its neurons and afferent and efferent pathways. Not all what is new in the histochemistry of the cerebellum is relevant to a better understanding of its chemoarchitecture. This review, therefore, does not pretend to be complete. It is focussed on afferent and intrinsic connections of the cerebellum. The efferent connections of the cerebellum to the brain stem and the spinal cord have not been systematically covered.
2. CYTOLOGY OF THE CEREBELLAR CORTEX A complete description of the histology of the cerebellar cortex was given by Ramon y Cajal (1911) (Figs 1 and 4). More recently the anatomy of the cortex including its ultrastructure was reviewed by Braitenberg and Atwood (1958), Eccles et al. (1967), Fox et al. (1967), Mugnaini (1972), and Palay and Chan-Palay (1974). Three layers are distinguished in the cortex (Fig. 3). The granular layer borders on the central white matter of the cerebellum. The Purkinje cell layer contains the cell bodies of the Purkinje cells, that are arranged in a single row. The perikarya of the Bergmann glia (the Golgi epithelial cells) are intercallated between the larger Purkinje cells (Fig. 9A). The molecular layer has a low cell content. It contains the dendritic arbors of the Purkinje cells and the Bergmann glial fibers, which run up to the pial surface where they constitute the external glial limiting membrane. The morphology of the cerebellar cortex can be characterized as a lattice: '... it can only be represented in two planes perpendicular to each other and having definite relations to the longitudinal and transversal axes of the
Handbook of Chemical Neuroanatomy, Vo112. Integrated Systems of the CNS, Part IH L.W. Swanson, A. Bj6rklund and T. H6kfelt, editors 9 1996 Elsevier Science B.V. All rights reserved.
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Fig. 1. Cerebellar cortical circuits. Top. Diagram showing the main mossy fiber-granule cell-Purkinje cell circuit and the innervation of the granule cells by the axonal plexus of the Golgi cell. A: mossy fiber; a: granule cell; B: Purkinje cell axon; b: parallel fiber; c: Golgi cell; d: Purkinje cell. Bottom. Similar diagram showing the main cortical circuit and the connection of the basket cell with the Purkinje cell somata. A: mossy fiber; a: granule cell; B: Purkinje cell axon; b: basket cell; C: climbing fiber; c: Purkinje cell soma. Redrawn from Ramon y Cajal (1911).
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A Fig. 2. D i a g r a m s of the cerebellar circuit. Inhibitory neurons are indicated in black. A. M a i n circuit. B. Cortical interneurons and recurrent pathways. Abbreviations: B = basket cell; cf = climbing fiber; G = Golgi cell; G R = granule cell; IO = inferior olive; m f = mossy fiber; nc = nucleocortical axons; no = nucleo-olivary axons; pcc = recurrent Purkinje cell axon collaterals; P cell = Purkinje cell; P C N = precerebellar nuclei; p f - parallel fiber; pi = pinceau of basket cell axons; S = stellate cell; U B C = unipolar brush cell; 1 = extracerebellar mossy fiber; 2 - nucleo-cortical mossy fiber; 3 - mossy fiber collateral of uni-polar brush cell.
animal. The whole three dimensional structure, therefore, cannot be obtained by rotation but by translation in two directions, thus producing a lattice' (Braitenberg and Atwood, 1958, p. 1). The elements of the main cerebellar circuit were discovered by Ramon y Cajal (1888, 1911). The electrophysiological properties of the circuit were established by Eccles et al. (1967). The main circuit (Figs 1 and 2) consists of the mossy fiber afferent system, that terminates on the granule cells; the granule cell axons that ascend to the molecular layer and bifurcate into parallel fibers, that run in the long axis of the folium and terminate on the Purkinje cells and the projection of the Purkinje cells to the cerebellar or vestibular nuclei. Each Purkinje cell is innervated by a single climbing fiber (Ramon y Cajal, 1911; Eccles et al., 1966a) that takes its origin from the contralateral inferior olive. The synaptic connections of mossy fibers, parallel fibers and climbing fibers are excitatory. The Purkinje cells are inhibitory and use gamma aminobutyric acid (GABA) as a transmitter (Ito and Yoshida, 1964). Small interneurons of the cerebellar cortex (stellate, basket and Golgi cells) receive a parallel fiber input and constitute inhibitory feed back and feed forward loops terminating on the granule cells and the Purkinje cells (Figs 1, 2 and 4). The main determinant of the firing rate of Purkinje cells is the mossy fiberparallel fiber system. Excitatory coupling between climbing fibers and Purkinje cells is very strong, but the frequency of the complex spikes evoked in Purkinje cells by the climbing fiber is too low to contribute significantly to its firing rate. The function of the climbing fibers, therefore, is one of the main problems in cerebellar neurobiology. Purkinje cells project to the cerebellar or the vestibular nuclei, where their axons terminate with inhibitory synapses. The cerebellar nuclei receive their excitatory drive from collaterals of the mossy and the climbing fibers.
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Fig. 3. A. Nissl-stained section of the cerebellar cortex of the cat. G = Golgi cell; Gr = granule cells; P = Purkinje cell, asterisks: protoplasmatic islands of Held. Bar = 20 r Purkinje (1837).
B. diagram of the cerebeUar cortex of
Granule cells are small neurons located in cell nests in the granular layer. Cell-free spaces in the granular layer, that are known as the protoplasmatic islands of Held, contain the terminals of the mossy fibers (Fig. 3A, asterisks). Mossy fibers originate from many different sites in the spinal cord and the brain stem and constitute the main afferent system of the cerebellar cortex. Mossy fibers are myelinated fibers that branch extensively within the cerebellar white matter and the granular layer. They terminate with large irregular swellings (the mossy fiber rosettes, Figs 1, 5 and 6) that are located along or at the end of the axon. Each rosette forms the center of a complex synapse (cerebellar glomerulus) between the mossy fiber rosette, the dendrites of several granule cells and the terminals of one type of short axon (Golgi) cell of the cerebellar cortex. More than one mossy fiber rosette may be present within a protoplasmatic island. Granule cells possess 3-4 short dendrites, terminating in claw-like excrescenses (Fig.7). The thin, unmyelinated axon ascends towards the molecular layer, where it bifurcates in the form of a T. The two branches, that are known as the parallel fiber, pursue a straight course in the long axis of the folia, parallel to the thousands of other parallel fibers that constitute the bulk of the molecular layer. Parallel fibers synapse with dendrites of Purkinje cells and short axon cells in the molecular layer. Both the ascending portion of the granule cell axon and the parallel fiber are beaded. These varicosities probably correspond to the synaptic sites (Fig. 7D-E). Parallel fibers are very long. In monkeys their length varied between 0.8 and 5 mm. (Fox and Barnard, 1957). Maximal lengths of parallel fibers of 4.6-5.0 mm were reported for the rat (Brand et al., 1976; Schild, 1980; Mugnaini, 1983). The mean length
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Fig. 4. Semidiagrammatic parasagittal section through a folium of the mammalian cerebellum, based on data
from Golgi-stained material9 A: molecular layer; B: granular layer; C: white matter; a: Purkinje cell; b: basket cells of the lower molecular layer; d: terminal basket formation of the basket cell axon; e: superficial stellate cells; f: Golgi cell; g: granule cells with their ascending axons; h: mossy fibers; i: the bifurcation of the granule cell axons; j: epithelial glial cell; m: astrocyte of the granular layer; n: climbing fiber; o: branching point of Purkinje cell recurrent axon collaterals. Redrawn from Ramon y Cajal (1911).
of parallel fibers of 4.4 mm, measured after microinjections of biocytin in the granular layer in the rat (Pichitpornchai et al., 1994) is close to the mean length of these fibers of 5 mm, estimated with stereological techniques by Harvey and Napper (1988). The two branches of the parallel fiber are of equal length (Pichitpornchai et al., 1994). Shorter parallel fibers are located at the base of the molecular layer (mean branch length 2.08 mm), they become progressively longer as they approach the pial surface (mean branch length 2.35 mm: Pichitpornchai et al., 1994). Parallel fibers in the superficial molecular layer are of a smaller calibre than deep parallel fibers (Fox and Barnard, 1957, monkey). A similar increase in size of the parallel fibers from superficial to deep laminae of the molecular layer was noticed by Pichitpornchai et al. (1994) in the rat. They also observed proximo-distal tapering of parallel fibers. Van der Want et al. (1985a,b) observed corresponding differences in synaptic size in superficial and deep layers of the molecular layer in the cat. The size and the spacing of the varicosities along the parallal fibers was found to be correlated with their caliber. The mean interval between two varicosities was 5.2 ~tm for the parallel fibers, 4.02 ~tm for the ascending axon of the granule cell (Pichitpornchai et al., 1994). The lamination in the molecular layer may be the expression of a deep to superficial gradient in the development of the parallel fibers
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Fig. 5. Mossy fiber rosettes in the granular layer. Left. Mossy fiber rosettes from neurons of the lateral reticular nucleus, labelled with antegrade transport of Phasaeolus vulgaris lectin. Bar = 25/lm. Right: Mossy fibers, Golgi impregnation. Cajal (1911). Abbreviations: a = large, terminal rosettes; b = rosettes 'en passage'; c = small rosette 'en passage'; G = granular layer; M - molecular layer; W = white matter. Courtesy of Dr. T.J.H. Ruigrok.
(Pellegrino and Altman, 1979). A population of thick, short parallel fibers was noticed by Pitchitpornchai et al. (1994) in the deep parts of the molecular layer. Deep lying parallel fibers may be myelinated and are one of the constituents of the supraganglionic plexus located above the Purkinje cells (Mugnaini, 1972). The mossy fiber-parallel fiber-Purkinje cell pathway is characterized by a large divergence. Each mossy fiber terminates on a great number of granule cells and each granule cell contacts hundreds of Purkinje cells along its parallel fiber. An average parallel fiber with a length of 6 mm forms approximately 1100 boutons (Brand et al., 1976). A portion of the molecular layer 6 mm wide contains approximately 750 Purkinje cell dendritic sheets (Brand and Mugnaini, 1976). This number is somewhat lower than the number of available boutons, when a parallel fiber would synapse once with each Purkinje cell it meets on its way (Brand et al., 1976). It is higher than the estimate of Napper and Harvey (1988b) in the rat that 15% of the boutons on parallel fibers synapse with non-Purkinje cells and that the rest synapses once with half of the Purkinje cell dendritic sheets it meets on its way. The granule cell/Purkinje cell ratio was estimated at 274/1 by Harvey and Napper (1988) and at 350-500/1 for different lobules of rat vermis by Drfige et al. (1986). Napper and Harvey (1988) concluded that there are some 175.000 parallel fiber synapses on a single Purkinje cell of the rat. Fox et al. (1967) arrived at a number of 120.000 in monkeys. The actual strength of the convergence of individual mossy fibers to Purkinje cells depends on the distribution of their mossy fiber rosettes. Electrophysiological studies of Bower and Woolston (1983) in the rat demonstrated that Purkinje cells are most responsive to mossy fiber input that reaches the granule cells located immediately below them. Llinas (1982) explained this strong radial connectivity by the greater number of
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Fig. 6. Drawing of horizontal section through rat cerebellum showing orientation of mossy fibers. A. Elliptical segment or stripe of mossy fiber terminals in the medial portion of the anterior lobe showing the strong caudal-rostral organization of the terminal neuropil. Note the small cluster of granule cell bodies at the open arrow. B. Single mossy fiber from the next adjacent section showing the almost linear caudal-rostral pattern of the related terminals and small groups of parallel fibers (pf). a: View of rat cerebellum from the above showing approximate position of the field illustrated (note square and arrow), b: Medial sagittal section through cerebellum showing approximate location and plane of section. Abbreviations: fp = fissura prima; Isim = lobulus simplex; crI = crus I; fsp = fissura superior posterior; fpl = fissura posterolateralis; pf - parallel fiber. Golgi modification; 21-day-old rat. Scheibel (1977).
synapses with Purkinje cells on the ascending portion of the parallel fiber. However, according to Napper and Harvey (1988) the synapses on ascending portions of parallel fibers would account for only 3% of the total number of synapses of these fibers. Pichitpornchai et al. (1994), who observed a closer spacing of varicosities on the ascending axon and the proximal branches of the parallel fibers than on their distal branches, concluded that parallel fibers will exert a graded synaptic influence on their target Purkinje cells, with the most powerful influence occurring on cells located around the proximal regions of the fibers where they bifurcate. Mossy fiber terminal branches in the granular layer are oriented longitudinally, in the same plane as the Purkinje cells (Scheibel, 1977), (Fig. 6) (see also Section 6.4.2.). Mossy fibers, therefore, preferentially activate longitudinally oriented patches of Purkinje cells. Different types of mossy fiber rosettes were described by Brodal and Drablos (1963) with the Glees and Rheumont-Lhermitte silver impregnations and the Golgi method in rat and cat. Highly branching mossy fibers terminating in small, relatively simple rosettes, located along or at the end of the fiber, occur in all parts of the cerebellum. Large rosettes, consisting of aggregations of larger and smaller argyrophilic particles, interconnected by fiber fragments occur exclusively in nodulus and adjoining uvula, lingula and flocculus. The dendritic tree of the Purkinje cell is flattened in a plane perpendicular to the long
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Fig. 7. Granule cells and parallel fibers after an injection of biocytin in lobule X of the cerebellum of the rat. A. Biocytin labelled granule cell. B. Golgi-impregnated granule cells and parallel fibers in transverse section. Cajal (1911). C. biocytin injection site in granular layer and labelled parallel fibers in molecular layer. D. bifurcation site of labelled parallel fibers. E. labelled varicose parallel fibers. Abbreviations: A: molecular layer; B: granular layer; C: white matter; a: granule cell axon; b: bifurcation of granule cell axon; d: Purkinje cell; f: Purkinje cell axon; g: granular layer; I: injection site; m: molecular layer. Bars in A = 12/~m, in C = 500 /~m, in D and E - 50/~m. Courtesy of Dr. T.J.H. Ruigrok. (
axis of the folia (Figs 8 and 9). The soma and the proximal dendrites of the Purkinje cell are relatively smooth, the distal dendrites (spiny branchlets) bear long-necked spines (Fox and Barnard, 1957). When the parallel fibers traverse the Purkinje cells they terminate with boutons en passage on the spines of their spiny branchlets. Climbing fibers terminate on short, stubby spines on the proximal dendrites of the Purkinje cells (Larramendi and Victor, 1967; Palay and Chan-Palay, 1974) (Figs 10, 11 and 14). The axon of the Purkinje cell is myelinated (Fig. 9) and gives rise to recurrent collaterals (Bishop, 1982, 1988; Bishop and O'Donoghue, 1986; Bishop et al., 1987; O'Donoghue and Bishop, 1990). The collaterals form a plexus of beaded axons, mainly at the level of the Purkinje cell layer (Fig. 8a and b). They terminate on neighbouring 9
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Fig. 8. Purkinje cell of rat cerebellum. Intracellular injection with lucifer yellow and staining with anti-lucifer yellow antibody. PAP method, cresyl violet counterstained. Note plexus of beaded axon collaterals in A and B and spiny branchlets in C. Courtesy of Dr. T.J.H. Ruigrok. Bars in B = 50 ~m, in C = 5/~m. Abbreviations: a = Purkinje cell axon; cp = collateral plexus.
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Fig. 9. Purkinje cell in sagittal section. H~iggqvist stain. A. The small, densely stained nuclei in the Purkinje cell layer belong to the Bergmann glial cells. B. Initial segment of Purkinje cell myelinated axon (A) surrounded by pinceau of terminal basket cell axons. Abbreviations: A = Purkinje cell axon; B = Bergmann glial fiber; D = Purkinje cell dendrite. Bar - 25 pm.
10
The cerebellum: chemoarchitecture and anatomy
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Fig. 10. Synapses on mouse Purkinje cell. Climbing fibers terminate on short, stubby spines of proximal dendrites (Ds), parallel fibers terminate on spiny branchlets (Bs). Stellate cell and basket cell axons terminate on proximal dendrites and soma. Larramendi and Victor (1967).
Purkinje cells. The recurrent collaterals extend into the molecular layer where they contact basket cells (O'Donoghue et al., 1989). The whole collateral arborization is oriented perpendicular to the long axis of the folia, i.e. in the same plane as the dendritic tree of the Purkinje cell. In the cat it measures 300-700 #m in the sagittal and 100-400 #m in the transverse direction (Bishop, 1988). The width of the arborization and its penetration in the molecular and granular layers varies for different parts of the cerebellum. Recurrent collaterals of Purkinje cell axons are constituents of the infra- and supraganglionic plexus. The main Purkinje cell axon enters and traverses the white matter to terminate on cells of the cerebellar or the vestibular nuclei. Climbing fibers (Fig. 14) innervate the Purkinje cells, each Purkinje cell receiving only one climbing fiber (Ramon y Cajal, 1911). The olivocerebellar parent fibers of the climbing fibers branch extensively in the cerebellar white matter. For the adult rat the ratio of climbing fiber innervated Purkinje cells to neurons of the inferior olive is approximately 10:1 (Schild, 1970; Delhaye-Bouchaud et al., 1985). During their development the Purkinje cells receive more than one climbing fiber, it is not known how these supernumary climbing fibers are eliminated. Branching of olivocerebellar fibers occurs 11
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Fig. 11. Diagram of the interaction of Purkinje cell dendrite with a climbing fiber and several parallel fibers. A proximal Purkinje cell dendrite (pd) shows stubby thorns contacted by a climbing fiber (cf), whereas parallel fibers (pf) synapse on spines protruding from a spiny branchlet (sb). Rossi et al. (1991).
in the parasagittal plane (Armstrong et al., 1973; Brodal et al., 1980; Rosina and Provini, 1983). This is one of the reasons for the longitudinal, strip-like organization of the olivocerebellar projection (see Section 6.3.3.). Transverse branching is limited to climbing fibers terminating in certain longitudinal strips (Ekerot and Larson, 1982). Climbing fibers take their origin from the contralateral inferior olive. For a long time the origin of the climbing fibers remained obscure. Their ultrastructure and their mode of termination were first recognized by Larramendi and Victor (1967) (Figs 10 and 11) in the mouse as beaded fibers, with boutons en-passage, filled with rounded vesicles terminating on short spines on Purkinje cell proximal dendrites. The clear intervesicular axoplasm distinguishes climbing fibers from the neurofilamentous basket cell axons. Earlier Scheibel and Scheibel (1954) had reviewed Ramon y Cajal's (1888) original description of the morphology of the climbing fiber. They concluded that climbing fibers emit collaterals in the granular and molecular layer, that terminate in glomeruli, on somata of Golgi, basket and stellate cells and on neighbouring Purkinje cells. Szentagothai and Rajkovits (1959) subsequently identified climbing fibers in axonal degeneration studies from their 'Scheibel-collaterals' and concluded that the climbing fibers originate from the inferior olive. Hamori and Szentagothai (1966b) described the climbing fibers as packed with neurofilaments and making synaptic contacts with few vesicles on the smooth parts of the dendrites. They probably mistook ascending collaterals of basket cell axons for the climbing fibers. The origin of the climbing fibers from the inferior olive was finally settled by Desclin (1974), who observed their degeneration with axonal silver impregnation methods after lesioning the inferior olive of the rat with 3-acetylpyridin (3-AP) administrated intra-peritoneally. In an exhaustive analysis of the normal light- and ultrastructural morphology of the climbing fiber, Palay and Chan-Palay (1974) observed the existence of climbing fiber glomeruli and synapses with Golgi cells in the granular layer and synaptic contacts of climbing fiber tendrils with basket and stellate cells. Desclin and Colin (1980) were unable to confirm these types of collateral contacts, outside the Purkinje cells, in an 12
The cerebellum." chemoarchitecture and anatomy
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Fig. 12. Purkinje cells from the cerebellum of, from left to right, birds (Gallus domesticus, Feirabend, 1983); mammals (cat, Cajal, 1911) and fish (Gnathonemus petersii, Nieuwenhuys, 1969). Note different length, orientation and position in the molecular layer of the spiny dendritic branchlets.
ultrastructural study of the cerebellar cortex of 3-AP-treated rats. O'Donoghue et al. (1989) found intracellularly stained basket cells of the cat to lack climbing or mossy fiber terminals on their somata. During postnatal maturation of the cerebellum of the mouse, Mason and Gregory (1984) found many axons that combine the morphology and synaptic connections of both climbing and mossy fibers. These combination fibers are rare in the adult. Purkinje cell dendritic trees in the molecular layer remain oriented perpendicular to the parallel fibers irrespective of the changes in direction of the folial chain. Their dendrites share this orientation with the climbing fibers terminating on them. This type of spatial organization is found in all vertebrates and is the main condition which determines the morphology of the cerebellum. Purkinje cells in fish and amphibians are not arranged in a monolayer, but can be clustered in specific parts of the cortex, reminiscent of the clustering of the Purkinje cells during early stages of cerebellar development in all vertebrates (Nieuwenhuys, 1967). Purkinje cells in lower vertebrates differ from the mammalian type by the disposition of their smooth, proximal branches and their spine-loaden terminal branches in the molecular layer (Fig. 12). In fish the proximal smooth branches are found at the same level as the somata of the Purkinje cells, and the distal spiny branchlets extend as straight spikes into the molecular layer. This condition was extensively studied by Nieuwenhuys and Nicholson in the cerebellum of mormyrid fish (Nieuwenhuys and Nicholson, 1969a,b). As a consequence the climbing fibers, that synapse with the smooth proximal part of the dendrites, do not 'climb' 13
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into dendrites within the molecular layer, but terminate at the same level as the perikarya of the Purkinje cells (Kaiserman-Abramof and Palay, 1969). In reptiles and birds the smooth, proximal dendrites with their climbing fiber terminals do not extend beyond the lower third of the molecular layer (Mugnaini, 1972; Freedman et al., 1977; Kfinzle 1985). Only in mammals the smooth branches and the climbing fiber arborizations reach the pial surface (Fig. 12). Cerebellar nuclei that contain the target cells of the Purkinje cell axons have been described in species of all vertebrate classes. In some species of fish target cells of the Purkinje cell axons are also located within the cortex among the Purkinje cells (the 'eurodendroid' cells of Nieuwenhuys et al., 1974). The cells of the 'fourth cortical layer' in some aquatic mammals, that are located below the granule cells in the white matter, can be considered as displaced cerebellar nuclear cells (Ogawa, 1934). Interneurons in the cerebellar cortex are inhibitory and constitute various feed-back and feed-forward circuits between parallel fibers, granule cells and Purkinje cells (Figs 1, 2 and 4). Their dendrites are located in the molecular layer, where they are contacted by parallel fibers. Golgi cells are most numerous in the upper part of the granular layer. Some of their dendrites ramify in the granular layer, where they are contacted by mossy fiber terminals in the glomeruli. The dendritic tree of Golgi cells is not oriented in a specific plane. Recently it was shown by De Zeeuw et al. (1994c) that axons of Golgi cells course for some distance in the supra- or infraganglionic plexus in the direction of the long axis of the folia, before they branch into a dense telodendrion in the granular layer. Their terminals are located at the periphery of the glomeruli, where they synapse with granule cell dendrites (Fox et al., 1967). Their ratio was estimated in the rat as 4-6 Golgi cells for each Purkinje cell. The number of Golgi cells is about three times higher in lobule X than in other lobules (Drfige et al., 1986). However, unipolar brush cells (see below and Section 3.6.3.) may have been mistaken for Golgi cells by these authors.
Fig. 13. Orthogonal arrangement of basket cell axons (thick horizonal fibers oriented in the plane of the Purkinje cells in A and B) and parallel fibers (thin, vertical fibers in A and B). A. Drawing from Golgiimpregnated section, Cajal (1911). B. Bodian-stained section of rat cerebellar cortex. Abbreviations: A and B = stellate cells; C = basket cell axon; E = pericellular basket; F = Purkinje cell dendritic tree; G = climbing fiber; Pb = pericellular baskets. Bar = 100/~m.
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The cerebellum." chemoarchitecture and anatomy
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Fig. 14. Phaseolus vulgaris lectin-labelled climbing fibers of rat cerebellum. A. Sagittal section. B. Coronal section. Abbreviations: G = granular layer; M = molecular layer; P = Zebrin- labelled Purkinje cells. Bar = 100/lm. Courtesy of Dr. T.J.H. Ruigrok.
Apart from the parallel fiber boutons on the dendrites, the soma of Golgi cells is contacted by Purkinje cell recurrent collaterals (Hamori and Szentagothai, 1966a, 1968, Palay and Chan Palay, 1974). Mossy- and climbing fiber terminals on Golgi cells, that were mentioned by several authors (Hamori and Szentagothai, 1966a, 1968, Palay and Chan Palay, 1974) have not yet been confirmed in experimental axonal tracing studies. Myelinated fibers, indicated as mossy and climbing fibers, and recurrent collaterals of Purkinje cell axons, terminate on Golgi cell somata with large, crenelated synapses ('synapse en marron': Palay and Chan-Palay, 1974). The synapse en marron recently was identified by Mugnaini and Floris (1994) as a synapse of the mossy fibers with the unipolar brush cells of the cerebellar cortex. Stellate cells are located in the entire molecular layer, basket cells constitute a special population located in its lower one third. Dendrites of stellate and basket cells are oriented in a direction perpendicular to the long axis of the folium. Axons of stellate cells terminate on Purkinje cell dendrites. The basket cell axon increases in thickness after it emerges from its soma (Figs 1 and 13). It runs, perpendicular to the long axis of the folium, above the perikarya of the Purkinje cells and gives off descending and ascending collaterals. The descending collaterals branch and surround and synapse with the somata of Purkinje cells. The axons of these pericellular baskets of the Purkinje cell terminate in a periaxonal plexus (the pinceau) surrounding the initial segment of the Purkinje cell axon. Ascending collaterals of the basket cell axon terminate on the smooth surface of the proximal dendrites of Purkinje cells. O'Donoghue et al. (1989) who studied the connections of intracellularly stained basket cells and Purkinje cells in the cat concluded that each basket cell soma received input from recurrent collaterals from a single Purkinje cell. Other afferents of the basket cell include parallel fibers, climbing fibers and stellate and basket cell axons (Palay and Chan Palay, 1974). The infra- and supraganglionic plexus, are located on either side of the layer of Purkinje cell somata. They contain myelinated Purkinje cell collaterals. Most myelinated fibers in the supraganglionic layer are oriented in the long axis of the folia and, therefore, represent myelinated granule cell axons or, possibly, axons of candelabrum cells (Lain6 and Axelrad, 1994) or Golgi cells (De Zeeuw et al. 1994c). In silver impregnations these axons are distinctly smaller than the basket cell axons, that cross them at right angles 15
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(Fig. 13). The so-called multi-layer fibers also contribute to the plexus surrounding the Purkinje cells. These fibers were traced from several sources, including the noradrenergic, serotoninergic and cholinergic cell groups of the brain stem. They ramify in all layers of the cortex, but constitute the densest plexus at the level of the Purkinje cells. They are discussed more fully in Sections 3.8., 3.9. and 3.10.1. Several other neuronal cell types have been identified in the cerebellar cortex. The Lugaro cell is a relatively rare fusiform neuron, located just below the Purkinje cell layer (Lugaro, 1894; Fox, 1959; Palay and Chan-Palay, 1974). Its dendrites stretch out along the boundary of the granular and the Purkinje cell layer, the destination of its axon is not known. Lugaro cells can be discriminated from Golgi cells immunocytochemically with specific antibodies (Section 3.6.2., Fig. 67). The candelabrum cell has been recognized in Golgi-impregnated sections from rat cerebellum by Lain6 and Axelrad (1994). The neuron is rather frequently encountered in all lobules of the cerebellum. Its medium sized perikaryon is sqeezed in between the somata of Purkinje cells. Its dendritic tree is somewhat flattened and mainly extends in the parasagittal plane. One or two dendrites course through the molecular layer, dividing into few, slightly oblique branches, that are covered with irregularly distributed spines. A few slender dendrites branch in the upper granular layer. The axon courses in the direction of the long axis of the folium in the Purkinje ceil - or the supraganglionic layer, and gives off terminal, beaded branches that ascend in the molecular layer at regular parasagittal intervals. The chemical anatomy of the candelabrum cell has not yet been studied (Section 3.6.2). The unipolar brush cells were first identified in the rat by Altman and Bayer (1977) as the 'pale cells' of the granular layer. These cells are intermediate in size between the granule and the Golgi cells, and possess a typical, pale nucleus. They are concentrated in the nodulus, the ventral uvula, the flocculus and parts of the paraflocculus. They are born after the Purkinje cells, but before the stellate, basket and granule cells. The cells were sporadically recognized as monodendritic neurons in a number of immunocytochemical studies (see Section 3.6.3), but have been characterized with Golgi impregnation and electron microscopic methods only recently (Floris et al., 1994; Mugnaini and Floris, 1994; Mugnaini et al., 1994). The name 'unipolar brush cell' was given by Mugnaini and colleagues (Mugnaini and Floris, 1994) after the tip of the stubby dendrite, that forms a tightly packed group of branchlets resembling a paint brush (Fig. 68). The soma of unipolar brush cells is spherical to oval and carries thin appendages. The axon only can be impregnated for a short distance, suggesting that its distal, unimpregnated part is myelinated. Side branches of the axon terminate in rosette-like formations in the granular layer (Fig. 2) (Berthi6 and Axelrad, 1994; Floris et al., 1994; Rossi et al., 1995), the main stem of the axon may enter the white matter. Unipolar brush cells are innervated by one or two mossy fiber rosettes, in the form of particularly extensive contacts. Mossy fibers end on the perikaryon as well as on the dendritic brush (Mugnaini et al., 1994). These large synapses correspond to the 'synapse en marron' of Palay and Chan-Palay (1974), originally identified as a mossy fiber-Golgi cell synapse (see also Monteiro et al., 1986). Unipolar brush cells also receive symmetrical synapses from boutons containing pleomorphic vesicles, presumably originating from Golgi cells or Purkinje cell recurrent axons. Some of the dendritic branchlets may be presynaptic to dendrites of other cells in the granular layer (Floris et al., 1994). Pale cells, monodendritic and unipolar brush cells are all more frequent in the vestibulocerebellum. The chemical identity of the unipolar brush cell will be discussed in Section 3.6.3.
16
The cerebellum." chemoarchitecture and anatomy
Ch. I
3. CHEMICAL ANATOMY OF THE CEREBELLAR CORTEX By virtue of its laminated and relatively simple structure the cerebellar cortex has served as the playground for every student who wanted to test a histochemical reaction or a new antibody on the brain. From this large body of data we have selected those which are important for the understanding of the morphology and the connections of the cerebellum. The localization in the cerebellar cortex of neurotransmitters, peptides, second-messenger systems, calcium-binding proteins and other biochemical markers is reviewed separately for each cell type of the cortex and for the mossy and climbing fibers. Glutamate and GABA receptors, nitric oxide, adenosine, the monoamine afferent systems and receptors, the hypothalamo cerebellar and histaminergic afferents and the cholinergic systems and acetylcholinesterase are discussed in separate sections. The chemoarchitecture of the cerebellar cortex has been reviewed by Schulman (1983), Nieuwenhuys (1985) and Oertel (1993). 3.1. PURKINJE CELLS
3.1.1. Gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD) and the GABA-transporters in Purkinje cells Purkinje cells use gamma-aminobutyric acid (GABA) as their main neurotransmitter and exert a postsynaptic inhibitory effect on cells of the cerebellar and vestibular nuclei (Ito and Yoshida, 1964; Obata et al., 1967; Obata, 1969, 1976; Obata and Takeda, 1969). GABA in rabbit Purkinje cells was first demonstrated using a histochemical method, demonstrating the conversion of GABA into succinic acid (Van Gelder, 1965). In selective uptake studies of [3H]GABA in cerebellar slices, only a low activity was present over the Purkinje cells (H6kfelt and Ljungdahl, 1970, 1971; Schon and Iversen, 1972). Minimal uptake of [3H]GABA was also observed for Purkinje cell axon terminals (Storm-Mathisen, 1975). All Purkinje cell somata of the cerebellum of the rat and their primary and secondary dendrites were immunoreactive for antisera against glutamic acid decarboxylase (GAD), the synthesizing enzyme of GABA (Fig. 62D,E). Varicose fibers and terminals in the cerebellar nuclei were densely stained (Saito et al., 1974; McLaughlin et al., 1974; Oertel et al., 1981b; Perez de la Mora et al., 1981; Somogyi et al., 1985). Immunoreactivity in Purkinje cell somata was generally found to be weak, or to be dependent on blocking of axonal transport by colchicine (Ribak et al., 1978). Strong immunoreactivity in Purkinje cell somata was, however, reported by Mugnaini and Oertel (1985) with an anti-GAD antiserum produced by Oertel et al. (1981 a). The presence of GAD mRNA in Purkinje cells has been demonstrated with in situ hybridization histochemistry in rodents and primates resulting in dense labelling over somata of Purkinje cells (Wuenschell et al., 1986; Julien et al., 1987; Ferraguti et al., 1990; Herrero et al., 1993). Two forms of GAD with apparent molecular weights in the range of 59-67 kDa, that differ by 2-4 kDa, were identified by Chang and Gottlieb (1988) and Martin et al. (1991). In situ hybridization histochemistry with probes for the high molecular weight form, GAD67, and the low molecular weight form, GAD65, showed a prevalent localization of GAD67 over GAD65 in Purkinje cell bodies of rat cerebellum. The reverse localization was reported for Golgi cells (Esclapez et al., 1993; Feldblum et al., 1993). A differential distribution of GAD67 and GAD65 in Purkinje cells was found in immunocytochemical studies with specific antibodies for GAD67 and GAD65. Antibody K2, which is specifc 17
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for GAD67 , strongly immunoreacted with Purkinje cell perikarya, their proximal dendrites and their axon terminals in rat cerebellum (Kaufman et al., 1991; Moffett et al., 1994). The monoclonal antibody GAD-6, which is specific for GAD65 (Chang and 18
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 15. Localization of GABA-like immunoreactivity in semithin sagittal sections of rat (A,B) and chick (C,D) cerebellum. A. Low magnification of cell bodies and neuronal processes reacting with monoclonal anti-GABA antibodies. B. Higher magnification of an area partially included in the frame in A. Note strong immunoreactivity in stellate cell bodies (open arrow), in Golgi neurons (thick arrow), in the basket terminals surrounding Purkinje cell bodies, in puncta at glomeruli (dotted line) and in axons in the white matter. Immunoreactivity in Purkinje cells is weak. C and D. Two typical patterns of GABA-like immunoreactivity observed in Vibratome slices of the chick cerebellum. C: Intensely (thick arrow) and weakly (open arrow) immunoreactive Purkinje cells together with the staining in their dendritic arborization (thick arrowhead). D. Basket terminals around two weakly stained Purkinje cells (open arrowheads). Molecular layer (MO); Purkinje cell layer (P); granular cell layer (GL); white matter (WM). Bar in A = 100 r bar in B, C and D = 25 r (Matute and Streit, (1986). (
Gottlieb, 1988), i m m u n o r e a c t e d with axon terminals of Purkinje cells, but p o o r l y imm u n o s t a i n e d the p e r i k a r y a of Purkinje cells ( K a u f m a n et al., 1991). Antibodies against conjugates of G A B A were first applied to d e m o n s t r a t e specific G A B A - l i k e i m m u n o r e a c t i v i t y in Purkinje cells by S t o r m - M a t h i s e n et al. (1983). Imm u n o r e a c t i v i t y of the cell b o d y and the dendrites with antibodies against conjugates of G A B A was generally f o u n d to be weak or absent, but strong in the a x o n and the myelinated axon collaterals in the infraganglionic, but especially in the supra-ganglionic plexus, and in their terminals in rat (Ottersen and S t o r m - M a t h i s e n , 1984a,b, 1987; Ottersen et al., 1987; M a d s e n et al., 1985; Sdgudla et al., 1985; G a b b o t t et al., 1986; M a t u t e and Streit, 1986; S o m o g y i et al., 1986; A o k i et al., 1986) cat (Somogyi et al., 1985) and m o u s e ( T a k a y a m a , 1994). Staining in Purkinje cell s o m a t a in the chicken was stronger t h a n in m a m m a l s ( M a t u t e and Streit, 1986) (Figs 15, 62, 63). Several G A B A t r a n s p o r t e r proteins, that are active in the high-affinity u p t a k e of GABA
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Fig. 16. Schematic diagram to illustrate the concept of dynamic interrelationships between taurine, motilin, and gamma-aminobutyric acid (GABA) in a single neuron. A neuron with both substances in coexistence may have fluctuating levels of one or both substances depending upon parameters of rhythm, time, and physiologcal demands for one or another mediator during specific types or phases of activity. Chan-Palay (1984). 19
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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The cerebellum." chemoarchitecture and anatomy
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Fig. 17. Immunoreactive staining with taurine (Tau2) antibody in rat cerebellum fixed with 4% paraformaldehyde. A. Tau2-immunoreactive staining on a coronal section though lobule 6 of the vermis. Purkinje cells and dendrites exhibiting Tau-LI were separated by bands of unstained Purkinje cells. M, molecular layer; P, Purkinje cell layer; G, granule cell layer; W, white matter; P", Purkinje cell layer out of plane focus. Location, bregma = 11,5 mm. B. High-power photomicrograph of area indicated in A demonstrating taurine-like immunoreactivity in Purkinje cells (short solid arrows) and dendrites (long solid arrows) separated by unstained Purkinje cells (open arrows). C. Tau2-immunoreactive staining in a horizontal section through lobule 3 of the vermis. D. Adjacent section to (C) demonstrating that taurine-like immunoreactivity was completely absorbed by incubation of Tau2 (1 : 40) with original antigen, taurine conjugated to KLH using glutaraldehydeborohydride. Bars in A, C, and I) = 100/.tm, in B = 50 r Magnusson et al. (1988).
GABA, have been cloned (GAT1-4: Guastella et al., 1990; Lopez-Corcuera et al., 1992; Borden et al., 1992; Liu et al., 1993; and GAT-B: Clark et al., 1992). All transporters occur in brain tissue. The regional distribution of GAT1 was studied by Rattray and Priestly (1993) with in situ hybridization in rat cerebellum. GAT1 mRNA is not expressed by Purkinje cells, but strongly by Bergmann glial cells. GAT-2 may be confined to glia (Liu et al., 1993), but detailed studies of their localization have not been published thus far. 3.1.2. Motilin and taurine in Purkinje cells
Certain inconsistencies in the results on the localization of GABA in Purkinje cells were discussed by Chan-Palay (1984). She concluded that GABA is present in varying amounts in different Purkinje cells and that it may co-exist with other neuroactive substances, notably with motilin and taurine, that also produce an inhibitory action on postsynaptic cells (Fig. 16). The presence of motilin in Purkinje cells was demonstrated with an antibody directed against conjugates of motilin (Chan-Palay et al., 1981; Nilaver et al., 1982). More than half of the Purkinje cells of the rat are immunoreactive for this antibody and in human cerebellum their proportion was even higher (Nilaver et al., 1982). Chan-Palay et al. (1981) found coexistence of GAD and motilin in 10-20% of the Purkinje cells of the rat. The presence of motilin in Purkinje cells has, however, been disputed by Lange (1986), who was unable to demonstrate the presence of motilin using radioimmuno-assay and reversed phase HPLC in extracts of rat cerebellum. Only one of Lange's anti-motilin antibodies, all of which had been demonstrated to be effective in demonstrating motilin-like immunoreactivity in rat duodenum, was found to immunoreact with Purkinje cells in immunocytochemical studies with rat cerebellum. Taurine has been proposed as a neurotransmitter in certain fiber systems. In the guinea pig cerebellum it was found to exert a hyperpolarising effect on Purkinje cell dendrites and was proposed as a neurotransmitter in stellate cell-Purkinje cell synapses (Okamoto et al., 1983). [3H]Taurine was found to accumulate in Purkinje cells. Immunocytochemical studies with antibodies specific for cysteine-sulfonic acid decarboxylase (CSADCase), the enzyme involved in taurine synthesis, by Chan-Palay et al. (1982a,b), showed that CSADCase immunoreactivity was present in most, but not all the Purkinje cells of rat cerebellum, and was more prominent in the main dendritic arbor than in the perikarya and the axon. CSADCase, motilin and GAD-like immunoreactivities were found to co-exist in Purkinje cells located near the midline. In contrast to the observations of Chan-Palay et al. (1982a,b), Almarghini et al. (1991) found CSADCase immunoreactivity to be localized in Bergmann glia and interfascicular oligodendrocytes and to be absent from Purkinje and stellate cells. Most authors who used antisera against conjugates of taurine to localize taurine-like 21
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immunoreactivity found staining in all Purkinje cells of the cerebellum of the rat (Madsen et al., 1985; Campistron et al., 1986b; Tomida and Kimura, 1987; Ida et al., 1987; Ottersen et al., 1988b; Ottersen, 1988, 1989). Magnusson et al. (1988) recognized a zonal distribution of taurine-immunoreactivity similar to the zonal labelling of CSADCase immunoreactivity observed by Chan-Palay et al. (1982a,b) (Fig. 130) in paraformaldehyde-fixed brain tissue (Fig. 17), but not in glutaraldehyde-fixed tissue. Analysis of semithin sections and immunogold electron microscopy indicated that taurine-immunoreactivity is selectively enriched in the somata, proximal and distal dendrites and axon terminals of the Purkinje cells (Fig. 18). Stellate and basket cell somata and their axon terminals are only weakly immunolabelled (Ottersen et al., 1988b; Ottersen 1988, 22
The cerebellum." chemoarchitecture and anatomy
Ch.I
Fig. 18. Photomicrographs showing the distribution of taurine-like immunoreactivity in the rat cerebellum, and the results of different control experiments. A,B. Semithin (0.5 r sagittal sections through vermis posterior treated with taurine (Tau) antiserum 20 diluted 1:3000 and subsequently processed according to the peroxidase-antiperoxidase procedure. A. Intense labelling of the somata, dendrites (small arrowheads), and axons (crossed arrow) of the Purkinje cells. The neurons (large arrowheads) and glial processes (small arrows) of the molecular layer appear immunonegative. Small asterisks indicate pial surface, large asterisk indicates Purkinje cell enlarged in B. B. Note staining of Purkinje cell dendritic spines (large arrow heads). Inset: Semithin test section mounted on the same slide as the tissue section shown in A and B and incubated in the same drops of sera. The test section contains brain protein-glutaraldehyde conjugates of different amino acids, separated by brain tissue that appears as darkly stained zones. Code: 1, GABA; 2, glutamate; 3, taurine; 4, glycine; 5, none (i.e., no amino acid in the reaction mixture); 6, aspartate; 7, glutamine (only small part of section represented in this particular view). The taurine conjugate is selectively stained. C. Transverse semithin section through nucleus interpositus anterior showing intense staining of axons (arrows) and nerve terminal-like dots, some of which (large arrowheads) appear to contact unstained cell bodies (asterisks). Same procedure as in A. D. Thin layer chromatograms (5 mm width) of soluble brain extracts fixed with glutaraldehyde and subsequently stained with an antiserum against glutamate (left strip) or taurine (right strip). The taurine antiserum reveals a single spot which has comigrated with free taurine and which is separate from the spot labelled by the glutamate antiserum. E, T and G indicate the application sites of the brain extract, taurine and glutamate, respectively. E. Adjacent section to that shown in A and accompanying test section (inset) incubated with the taurine antiserum after preabsorption with glutaraldehyde-taurine complexes (200/tM with respect to taurine). There is virtually no staining. Abbreviations: MO, molecular layer; GC, granule cell layer. Bars 25/~m. Ottersen (1988). (
1989). Following hypo-osmotic stress there is a transient shift of taurine from the Purkinje cells to the B e r g m a n n glial c o m p a r t m e n t , where taurine-immunoreactivity now becomes a p p a r e n t (Nagelhus et al., 1993).
3.1.3. Calcitonin gene-related peptide (CGRP), acetylcholinesterase (ACHE), somatostatin and tyrosine hydroxylase in Purkinje cells Some neuroactive substances have been reported to be present in Purkinje cells only for a certain period during development. Calcitonin gene-related peptide ( C G R P ) is almost undetectable in adult rat cerebellum, but a transient immunoreactivity in i m m a t u r e Purkinje cells in rat cerebellum has been detected with antisera against C G R P ( K u b o t o et al., 1987, 1988; Chedotal and Sotelo, 1992). In adult rats, however, C G R P - l i k e immunoreactivity, co-localized with G A D immunoreactivity, can be detected in m a n y Purkinje cells near injections of colchicine (Kawai et al., 1985, 1987). C G R P receptors measured autoradiographically with [125I]CGRP as the ligand, are a b u n d a n t in adult rat and h u m a n cerebellum. [125I]CGRP-binding is dense over the molecular and Purkinje cell layers and low over the granular layer and the cerebellar nuclei (Inagaki et al., 1986). Binding to the molecular layer occurs in a pattern of longitudinal stripes (Kruger et al., 1988) and increases after intraperitoneal administration of harmalin (Rosina et al., 1990, 1992). Choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) have been found to be transiently expressed in Purkinje cells during development: Purkinje cells in certain parts of the i m m a t u r e rat and guinea pig cerebellum, including the lobules IX and X of the caudal vermis, display a transient reactivity for A C h E (Csillik et al., 1963, 1964; A l t m a n and Das, 1970; Odutola, 1970; Brown et al., 1986). The authors suggested that this transient A C h E activity in Purkinje cells is due to a transient cholinoceptive stage, when they are contacted by cholinergic mossy fiber afferents. A similar, transient expression of C h A T was observed in Purkinje cells of the rat vestibulocerebellum (Gould and Butcher, 1987). Pseudo-cholinesterase was localized in adult Purkinje cells of the 23
Ch. I
J. Voogd, D. Jaarsma and E. Marani
lobules IX and X (Robertson et al., 1991). These cells are arranged in multiple, sagittal bands (Gorenstein et al., 1987). Robertson et al. (1991) were unable to confirm the transient staining with AChE in rat Purkinje cells. Somatostatin was located in rat Purkinje cells using polyclonal and monoclonal antibodies against conjugates of somatostatin (Johansson et al., 1984; Vincent et al., 1985; Villar et al., 1989). Reactive Purkinje cells were especially numerous in parts of the vermis and paraflocculus and flocculus during early postnatal stages, but mostly disappeared later on (Figs 19 and 20). In part of the vermis they were located in bands. In the adult rat Purkinje cells can be stained on the ventral aspect of the paraflocculus (Gonzalez et al., 1988) and in the vermis, after interventricular administration of colchicine (Villar et al., 1989). Somatostatin-like immunoreactivity was also observed in climbing fibers, that were correlated with the patches of immunoreactive Purkinje cells and, more diffusely distributed, in Golgi cells (Villar et al., 1989). The presence of somatostatin in adult rat Purkinje cells of the paraflocculus was confirmed with nonradioactive in situ hybridization for somatostatin mRNA (Kiyama and Emson, 1990). Specific binding of iodinated agonists of somatostatin was studied in rat, using ligands for short, 14 amino-acid ([125I]SS-14) and long forms ([125I]SS-28). Binding in the cerebellar cortex was found to be low, but strong binding of both ligands was observed over the cerebellar nuclei (Uhl et al., 1985). Binding to somatostatin receptors in the human cerebellar cortex was higher. Different distribution patterns were noted among the patients studied, with higher densities over the granular layer (Laquerriere et al., 1994). Leroux et al. (1985) and Gonzalez et al. (1988) failed to demonstrate specific binding over the cerebellar nuclei of the rat of a different SS-14 ligand, but confirmed binding of SS-28 (Leroux et :al., 1985). Binding of an octopeptide somatostatin analogue was reported to be almost absent in rat cerebellum (Reubi and Maurer, 1985) and low over the cerebellar cortex of the human cerebellum, with intermediate values in the molecular layer (Reubi et al., 1986). Tyrosine hydroxylase, the synthesizing enzyme of dopamine, is expressed by Purkinje cells of the ventral vermis (lobules I and X) and the hemisphere (ansiform lobule, paraflocculus) of rat cerebellum (Takada et al., 1993). Expression of tyrosine hydroxylase by Purkinje cells is increased in the mutant tottering and leaner mice (Austin et al., 1992). 3.1.4. The localization of the IP3 receptor and the intracellular calcium stores of Purkinje cells
The phosphoinositide system is a second messenger system coupled to metabotropic, G protein-linked receptors (see Ross et al. (1990), Mayer and Miller (1990), Ferris and Snyder (1992) and Berridge (1993), for reviews). Receptor-mediated hydrolysis of phosphatidylinositol (PIP2) is catalyzed by phospholipase C and leads to the formation of inositol-l,4,5-triphosphate (IP3) and diacylglycerol (DAG), two second messengers that function in a bifurcating signal pathway. Other inositol phosphates (inositol 1,3,4,5tetrakiphosphate, IP4; inositol 1,3,4,5,5-pentakiphosphate, IPs; and inositol hexakiphosphate, IP6) have been localized in rat cerebellum (Vallejo et al., 1987; Theibert et al., 1987, 1991). Phosphorylation of IP 3 by the enzyme IP 3 3-kinase leads to the formation of IP4. IP3, through activation of IP3 receptors, causes Ca 2+ mobilization from intracellular sources, whereas DAG, together with Ca 2+, activates the enzyme protein kinase C that phosphorylates regulatory proteins. The localization of phospholipase C, IP 3 receptors and protein kinase C has been extensively studied in Purkinje cells. 24
The cerebellum: chemoarchitecture and anatomy
Ch. I
B
C
f
Fig. 19. Schematic illustration of the zonal distribution of somatostatin immunoreactive Purkinje cells at
different levels of the cerebellum of a 21 day old rat. Drawings have been made from frontal, cresyl-violet stained sections. Each dot represents 2-5 cells. Abbreviations: 5-9, cerebellar lobules V-IX; 4V, 4th ventricle; COP, copula pyramis; CR2, crus 2, ansiform lobule; FL, flocculus; PFL, paraflocculus; PM, primary fissure; SF, secondary fissure. Villar et al. (1989).
IP 3 3-kinase, the enzyme that produces IP 4 from IP3, was exclusively localized in Purkinje cells of the rat using immunohistochemistry (Mailleux et al., 1991 a, Mizuguchi et al., 1992) and in situ hybridization in rat and human cerebellum (Mailleux et al., 1991 b, 1992). Immunoreactivity was present in Purkinje cell dendrites more than in the perikarya. Intense immunolabelling of the dendritic spines was observed in the rat (Yamada et al., 1992; Go et al., 1993) (Fig. 21) but a specific role of IP 4 in Purkinje cell dendritic spines has not been disclosed. A similar localization in Purkinje cell dendritic spines was described for the mGluR1 subunit of the metabotropic glutamate receptor (Section 3.3.2., Fig. 52). Different isoenzymes of the phospholipase C (PLC) family, belonging to three major groups (fl, ~ and d), have been identified (Rhee et al., 1989; Rhee and Choi, 1992). PLC-fll, PLC-y and PLC-~ have been localized with in situ hybridization in the brain of the rat. Moderate activity was found for PLC-fll in the granular layer and strong activity in Purkinje cells and granule cells for PLCT'. The activity of PLC-d is low and may be localized in glial cells (Choi et al., 1989). PLC-A m R N A that was localized in rat Purkinje cells by Ross et al. (1989b), probably codes for a thiol-protein disulphide oxido-reductase and not for a PLC (Berridge, 1993). The IP 3 receptor has been found to be identical to the Purkinje cell-specific P400 25
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The cerebellum." chemoarchitecture and anatomy
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protein (Mignery et al., 1989) (Fig. 22). The P400 protein was originally isolated by Mallet et al. (1976) as a Purkinje cell-specific protein, that was reduced in homozygous Purkinje cell-deficient (pcd, Mullen et al., 1976) and 'staggerer' (Sidman et al. 1962) mice, but relatively enriched in the cerebella of 'reeler' and 'weaver' mutant mice, with a loss of granule cells (Mikoshiba et al., 1979). Immunocytochemical studies with a monoclonal antibody specific for P400 protein, indicated that the protein was localized in somata, dendrites and axons of Purkinje cells in rodents (Maeda et al., 1988; Nakanishi et al., 1991; Rodrigo et al., 1993). The development of Purkinje cells could be traced with P400-immunostaining of staged cerebella of mouse embryos (Maeda et al., 1989) (Fig. 24). At the ultrastructural level it was identified on the plasma-membranes and the endoplasmatic reticulum, including the subsurface cisterns (Maeda et al., 1989). Notably Purkinje cells of'staggerer' mice, that are defective in synaptic contacts of parallel fibers and lack dendritic spines, do not express P400-immunoreactivity, whereas P400-immunoreactivity was found at 'normal' levels in ectopic Purkinje cells of 'reeler' cerebellum (Mariani et al., 1977; Mikoshiba et al., 1980; Maeda et al., 1989) (Fig. 23), and in the few remaining Purkinje cells of 'pcd' mutant mice. Cloning of the P400 protein cDNA revealed that it was identical to the IP 3 receptor protein, as well as the Purkinje cell-specific PCPP-260 protein isolated by Walaas et al.
Fig. 21. IP3-3-kinase immunoreactivity in the rat cerebellum. Electron micrograph showing intense immunoreactivity in the dendritic spines of Purkinje cells. Bar 2 r Yamada et al. (1992).
27
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 22. Localization of inositol 1,4,5-triphosphate receptor with PCD6 antibody in frozen sections of rat cerebellum by immunofluorescence. Sagittal section of the cerebellar cortex. Small arrows in the granule cell layer (GL) point to segments of immunoreactive axons which represent recurrent collaterals of Purkinje cell axons. Mignery et al. (1989).
(1986) and the PDC6 protein of Nordquist et al. (1988). The localization of P400 (= IP3 receptor) mRNA in Purkinje cells was confirmed by in situ hybridization (Furuichi et al., 1989) (Fig. 25). The IP3 receptor was purified from rat cerebellum as a protein with a molecular weight of 260 kDa (Supattapone et al., 1988). The primary structure of the mouse IP3 receptor protein, and its partial homology to the skeletal muscle ryanodine receptor were elucidated by Mignery et al. (1990). The IP3 receptor is composed of four identical subunits of a molecular weight of 320 kDa, and forms a calcium-permeable channel (Maeda et al., 1991). Three additional cDNAs encoding for the IP3 receptor, 28
The cerebellum." chemoarchitecture and anatomy
Ch. I
named IP~R-II, III and IV, were identified by Sfidhof et al. (1991) and Ross et al. (1992), but were not found to be expressed at significant levels by Purkinje cells. The presence of the IP~ receptor in Purkinje cells was confirmed immunocytochemically. In immunocytochemical studies with gold-conjugates, that allow precise ultrastructural localization of the immunoreactivity, it was shown that gold particles were located on membranes of the endoplasmatic reticulum in somata, dendrites, dendritic spines and axons of the Purkinje cells (Mignery et al., 1989; Ross et al., 1989a; Sharp et al., 1993a,b) (Fig. 26). Immunolabelling predominated in the smooth-surfaced endoplasmatic reticulum, including the subsurface cisterns, but was also found on portions of the perinuclear and rough endoplasmatic reticulum, and on the cis-cisternae, but not the intermediate and trans-cisternae, of the Golgi apparatus. IP~-receptor immunoreactivity was also observed in a subpopulation of spherical or elongated, membrane-bound structures, named calciosomes (Volpe et al., 1989), that are present throughout the cytoplasm of the Purkinje cells (Volpe and Villa, 1991; Nori et al., 1993). Strong immunoreactivity for the IP~ receptor was found on stacks of flattened cisternae of the endoplasmatic reticulum (Otsu et al., 1990; Satoh et al., 1990; Takei et al., 1992, 1994). The labelling on the cisternal stacks was mostly located in the spaces between the cisternae and between the cisternae and the plasmalemma or mitochondria (Satoh et al., 1990; Takei et al., 1992, 1994). It should be noted that the amount of cisternal stacks in Purkinje cells may depend on the conditions of perfusion fixation. The presence of cisternal stacks in healthy Purkinje cells, therefore, has been disputed (Takei et al., 1994).
9
.:.
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.
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9
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.
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Fig. 23. Section of reeler m u t a n t m o u s e cerebellum stained with m o n o c l o n a l a n t i b o d y 4C11 against the P400 protein. N o t e stained Purkinje cells in the cortex (CX) and in the central mass o f dislocated cells (DP). Bar = 200/~m. M a e d a et al. (1989).
29
Ch. I
J. Voogd, D. Jaarsma and E. Marani
9 ~~
-~A~
~
'
~'~-
E
~,:.. ~....
., t.
..r. .... .
~!~
.
.
.
i~ ~i!,,~~............,~ ~i~ii~ 'D
.
9
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~.
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~~ H
"
Fig. 24. Sagittal sections of mouse cerebella of various ages stained with a monoclonal antibody (4C 11) against the P400 protein.The samples were from (A) postnatal day O (PO), (B) P3, (C) P5, (D) P7, (E) P10, (F) P15, and (G) P20 cerebellum. A section from a P20 old mouse cerebral cortex did not react with same antibody (H). Magnification 100• Maeda et al. (1989).
30
The cerebellum." chemoarchitecture and anatomy
Ch. I
Nevertheless the formation of stacks of cisternae of the endoplasmatic reticulum could be induced by overexpression of IP3 receptors in fibroblasts, which indicates that cisternal stacks may exist as special organelles related to the IP3 receptor (Takei et al., 1994). The localization of the IP3 receptor has been compared to the localization of other luminal or membrane components of the endoplasmatic reticulum related to Ca 2+ homeostasis. The membrane pump CaZ+-ATPase, immunolabelled with antibodies against cardiac CaZ+-ATPase, was found to be located in regular cisternae of the endoplasmatic reticulum, the lateral tips of cisternae of the Golgi complex and in calciosomes of Purkinje cells (Kaprielian, 1989; Michelangeli et al., 1991; Villa et al., 1991; Takei et al., 1992) (Fig. 27). Distal axons of Purkinje cells, however, lacked CaZ+-ATPase immunoreactivity (Takei et al., 1992). Appreciable levels of calsequestrin, the main intraluminal calcium-binding protein of muscle, were present in Purkinje cells of the chicken. Calsequestrin-immunoreactivity was present over the lumen (Villa et al., 1991) and membranes (Takei et al., 1992) of stacked and isolated cisternae of the endoplasmatic reticulum and in a subpopulation of calciosomes (Volpe et al., 1988; Volpe and Villa, 1991). Mammalian Purkinje cells do not have calsequestrin but, instead, express calretic-
d
b
9
~..
ML PL
]cL " ;7-'.-11
Fig. 25. Localization of the P400-specific mRNA by in situ hybridization, a. Autoradiograph of a sagittal section of mouse cerebellum, b. Higher magnification of a. ML, molecular layer; PL, Purkinje cell layer; GL, granular layer. Furuichi et al. (1989).
31
Ch. I
~.
J. Voogd, D. Jaarsma and E. Marani
:.5.. ,~ -.
Fig. 26. Electron-microscope immunocytochemical localization of InsP3 receptor in Purkinje cells of rat cerebellum using pre-embedding avidin-biotin labelling and InsP3 receptor antiserum. A, B. Nuclear membrane and some, but not all, portions of endoplasmic reticulum (ER) are labelled. C. Higher magnification of the dendritic pole of a labelled Purkinje cell. Note the unlabelled ER very close to labelled ER. D. Labelled portions of endoplasmic reticulum (L-ER) immediately subjacent to an unlabelled presynaptic terminal (U-T). Cell membrane indicated with triangles in (D) and (E). E. InsP3 receptor antiserum. Labelled portions of ER near plasma membrane, but not directly subjacent to presynaptic terminal. F. Preimmune serum. No specific label is present, even though the section is very close to the surface of the vibratome section. Abbreviations: L-ER, labelled endoplasmic reticulum; L-G, labelled Golgi apparatus; L-NM, labelled nuclear membrane; U-ER, unlabelled endoplasmic reticulum; U-G, unlabeled Golgi apparatus; U-M, unlabelled mitochondrion; U-NM, unlabelled nuclear membrane. Scale bars for all panels 1 r (Ross et al., 1989a).
ulin (Treves et al., 1990). Calreticulin-immunoreactvity was located in stacks of rough and smooth endoplasmatic reticulum in rat Purkinje cells (Nori et al., 1993). Calsequestrin and calreticulin are not exclusively present in Purkinje cells, but also in other cell types of the cerebellar cortex. 3.1.5. Protein kinase C in Purkinje cells
Protein kinase C (PKC) plays an important role in the control of several cellular processes, such as the short-term modulation of membrane excitability and transmitter release, positive or negative interaction with the conductance through various ion channels and the regulation of gene expression and cell proliferation (Shearman et al., 1989, 1991; Farago and Nishizuka, 1990; Nishizuka et al., 1991). PKC, that through phosphorylates multiple target protein including neurotransmitter receptors, and has been implicated in long-term depression (LTD) of glutamate sensitivity of Purkinje cells (Cr~pel and Krupa, 1988). Breakdown of PIP2 by phospholipase C (see Section 3.1.4) in Purkinje 32
The cerebellum." chemoarchitecture and anatomy
Ch. I
a
b
C
Fig. 27. Immunofluorescence localization of the cerebellar Ca2+-ATPase in a transverse cryosection of adult chicken cerebellum. CaS/CI-IgG localizes the Ca2+-ATPase to the Purkinje cell bodies in the Purkinje layer (b), and the dendritic trees in the molecular layer (a). Very faint immunofluorescence was detected in the granule cell layer (c). Bar 50/~m. Kaprielian et al. (1989).
cells can activate PKC through the production of DAG and the mobilization of C a 2+ from the endoplasmatic reticulum. Alternative routes for the production of DAG and the mobilization of C a 2+ from extracellular sources are available (Nishizuka et al., 1991). Three isoenzymes of PKC have been distinguished on the basis of the analysis of the sequence homology of complementary DNA clones from different sources. The PKCtypes I, II and III of Huang et al. (1987a,b) are the products of the 7', fl and ~ genes respectively (Ono et al., 1987; Nishizuka, 1988). The PKC fl isoenzyme occurs in two forms, flI and flII, generated through alternative splicing (Ono et al., 1987; Nishizuka, 1988; Saito et al., 1989; Shimohama et al., 1990; Farago and Nishizuka, 1990). PKC ~, fl and 7' are calcium-dependent forms. In addition, calcium-independent isoenzymes of PKC have been identified: ~, e, e' and ( (Ono et al., 1988). Non-specific antibodies against PKC were found to strongly immunostain Purkinje cell perikarya, dendrites and axons (Mochly-Rosen et al., 1987; Kitano et al., 1987; Saito et al., 1988). Immunocytochemical studies with subtype specific antibodies and in situ hybridisation histochemistry have shown that several PKC subtypes are located in Purkinje cells (Figs 28 and 29, Table 1) (Brandt et al., 1987; Huang et al., 1987a,b, 1988, 1991; Ase et al., 1988; Hashimoto et al., 1988; Hidaka et al., 1988; Kose et al., 1988; Shimohama et al., 1990; Wetsel et al., 1992; Chen and Hillman, 1993a; Garcia et al., 1993; Merchenthaler et al., 1993). PKC~' immunoreactivity occurs at high levels in both the somatodendritic and axonal domains of Purkinje cells, and is absent from other cell types of the cerebellar cortex. Immunoreactivity for PKC ~ is also present in Purkinje 33
Ch. I
J. Voogd, D. Jaarsma and E. Marani
cells. PKC d-immunoreactive Purkinje cells are distributed in immunopositive and immunonegative columns (Fig. 133) (Chen and Hillman, 1993a). According to Wetsel et al. (1992) Purkinje cells were stained with antisera against PKC e, but Chen and Hillman's (1993a) found Purkinje cells to be unlabelled for PKC e. PKC/6 and e' were not located in Purkinje cells (Table 1). 3.1.6. cGMP; cGMP-dependent protein kinase and nitric oxide synthase in Purkinje cells Purkinje cells are the only cerebellar cell type containing cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGK) (Walter et al., 1981; Walter, 1984; Lohmann et al., 1981; De Camilli et al., 1984; Wassef and Sotelo, 1984). cGK-immunoreactivity is present throughout the entire Purkinje cell, including its dendrites and its axon (Fig. 30). During development Purkinje cells display a heterogeneity in their expression of immunoreactivity for cGK (Wassef and Sotelo, 1984, rat; Levitt et al., 1984, monkey) (see Section 6.2.). A 23 kD protein, which is likely to be a substrate of cGK was found to be concentrated in Purkinje cells (Walter, 1984; Nairn and Greengard, 1983). Immunoreactivity for guanylate cyclase, the synthesizing enzyme of cGMP, was
Fig. 28. Developmental expression of protein kinase C (PKC) isoenzymes in rat cerebellum. Immunofluorescent staining of cerebellar cortex by antibodies specific for PKC 1, corresponding to PKC~" (panels A, B and C), PKCfl (panels D, E and F) and PKC~ (panels G, H and I). Sagittal sections of cerebellum of 1-week-old (A, D and G), 2-week-old (B, E and H) and 3-week-old (C, F and I) rats were used. PKCz- antibody stained mainly the Purkinje cell bodies and dendrites throughout the development. PKCfl antibody stained the cerebellar granule cells in the external germinal layer (EGL) of the 1- and 2-week-old rats and mainly the granular layer of the 3-week-old rats. PKC~ antibody stained both granule cells and Purkinje cells throughout the development. Huang et al. (1991).
34
The cerebellum." chemoarchitecture and anatomy
Ch. I
....
.,
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9
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Fig. 29. Immunostaining for different isozymes of PKC in the rat cerebellum. PKC0~ (A) is present in Purkinje cells (P). The dendrites of these cells can be followed as far as the top of the molecular layer (m). The granular layer (g) and the white matter (w) are not stained. To avoid crowding, the abbreviations for layers of the cerebellum are indicated only in (A); however, the layer of Purkinje cells (P) is indicated in each figure for orientation. PKCfl (B) and PKCflI I (C) are present only in cells of the granular layer. PKCg is present in Purkinje cells and Bergmann glial cells in the molecular layer (D). Not only the perikarya but also the dendrites of Purkinje cells in the molecular layer and their axons in the granular layer are immunopositive. The antiserum for PKC~ stained Purkinje cells (E) and presently unidentified cells below the unstained Purkinje cells (F). The dorsally located folia contain mainly unstained Purkinje cells. Their axonal origin is surrounded by immunopositive cells. In the basal folia, the Purkinje cells are immunostained. PKCe is present in Purkinje cell (G), whereas PKCe' is present in cells in the molecular and granular layers and in the nerve fibers surrounding the unstained Purkinje cells (H). Antiserum against PKC~" stained only Purkinje cells in the cerebellum (I). Bar 100 •m. Wetsel et al. (1992).
35
Ch. I
J. Voogd, D. Jaarsma and E. Marani
localized in Purkinje cells, but does also occur in other cell types of the cerebellar cortex (Zwiller et al., 1981; Ariano et al., 1982; Nakane et al., 1983; Poegge and Luppa, 1988). cGMP was, however, found to be absent from rat Purkinje cells, using antibodies against conjugates of cGMP in combination with sodium nitroprusside-stimulation of cGMP synthesis. Prominent cGMP-immunoreactivity within the molecular layer was detected in Bergmann glial cells (Fig. 56) (Berkelmans et al., 1989; De Vente et al., 1989, 1990). Soluble guanylate cyclase is activated by nitric oxide (NO) (see Section 3.4). NO has been implicated in the generation of long term depression (LTD) of parallel fibermediated EPSP's in Purkinje cells. LTD can be prevented by the application of haemoglobin that absorbs NO, or by the inhibition of NO synthesis (CrGpel and Jaillard, 1990; Shibuki and Okada, 1991; Ito, 1991). However, nitric oxide synthase, the synthesizing enzyme of NO, appears to be absent from the Purkinje cell (Section 3.4.).
3.1.7. Calcium-binding proteins in Purkinje cells Calbindin-D28K, parvalbumin and calmodulin are cytosolic, calcium-binding proteins of the EF-hand family (see Baimbridge et al., 1992 and Andressen et al., 1993 for reviews), that are present in high amounts in Purkinje cells. Calretinin is a calciumbinding protein closely related to calbindin-D28K, that is absent from the Purkinje cells, but present in other neurons and in afferent mossy and climbing fibers of the cerebellar cortex (Rogers, 1989; Arai et al., 1991; RGsibois and Rogers, 1992; Floris et al., 1994). One of the calcium-binding proteins, the 28 kDa vitamin-D-dependent calcium-binding protein (calbindin-D28K), occurs in most, if not all, Purkinje cells in rat and chicken cerebellum (Lawson, 1981; Roth et al., 1981; Jande et al., 1981a,b; Baimbridge and Miller, 1982; Legrand et al., 1983; Schneeberger et al., 1985; Kosaka et al., 1993; Amenta et al., 1994). Its presence in soma, dendrites and axon was demonstrated with polyclonal and monoclonal antibodies raised against calbindin-D28K (Fig. 31A). Its exclusive presence in the cerebellum in Purkinje cells was confirmed with in situ hybridization with cDNA probes in rat and mouse (Nordquist et al., 1988; Iacopino et al., 1990; Abe et al., 1992a; Kadowaki et al., 1993). According to Garcia-Seguera et al. (1984) only 74% of the rat Purkinje cells was immunoreactive for a polyclonal antibody raised against chick duodenal calbindin-28K. This antibody also stained Golgi cells in the granular layer in rat and human cerebellum (Fournet et al., 1986). Developmental gradients in the expression of immunoreactivity for calbindin-28K by Purkinje cells were studied by Legrand et al. (1983) and Wassef et al. (1985) (see Section 6.2.). TABLE
1. Immunoreactivities o f P K C in cerebellar neurons
Isoenzymes
P u r k i n j e cells
Basket &
G r a n u l e cells
Cerebellar nuclei
+
++
+
s t e l l a t e cells Alpha
++
Beta
-
+
++
+
Gamma
+++
-
-
-
Delta
+++
+++
-
-
Epsilon
-
+
++
++
Zeta
++
+
++
++
Chen and Hillman (1993a)
36
The cerebellum." chemoarchitecture and anatomy
Ch. I
P 21
Fig. 30. A. Frontal section through the cerebellum and attached brainstem of an adult rat. All the Purkinje cells are stained by cyclic 3',5'-guanosine monophosphate-dependent protein kinase (cGK) antiserum, including their dendrites in the molecular layer and their axon terminals in the deep nuclei and in the brainstem (arrow). Bar = 1 mm. B. Higher magnification of the neurons indicated by an arrow head in A. Like a few other isolated labelled cells found in variable locations, these cells are considered as ectopic Purkinje cells. Bar = 50/lm. C. cGK immunoreactive neuron in the cerebellum of 1 day-old rat. This ectopic Purkinje cell is located in the white matter and its appearance mimics that of 1-day-old Purkinje cells as visualized in Golgi impregnated material. Bar = 25 ~m. Wassef and Sotelo (1984).
Calmodulin-immunoreactivity was observed both in Purkinje cells and in cells of the cerebellar nuclei of the rat (Lin et al., 1980; Means and Dedman, 1980; Seto-Oshima et al., 1983, 1984). During postnatal development calmodulin-immunoreactivity was transiently present in the inner part of the external germinative layer and in fibers in the white matter of P3-P11 rat pups (Seto-Oshima et al., 1984). Polyclonal antibodies against parvalbumin stain all Purkinje cells and stellate and basket cells in the molecular layer of rat and avian cerebellum (Figs 31B and 32) (Celio and Heizmann, 1981; Heizmann, 1984; Braun et al., 1986; Endo et al., 1985; Schneeberger et al., 1985; Seto-Oshima et al., 1983; Rogers, 1989; Kosaka et al., 1993). The localization of parvalbumin in Purkinje, stellate and basket cells was confirmed in the rat with non-radioactive in situ hybridization (Kadowaki et al., 1993). Parvalbumin 37
Ch. I
J. Voogd, D. Jaarsma and E. Marani
. .
Fig. 31. A. Calbindin-D28k immunoreactivity. B. Parvalbumin-immunoreactivity in rat cerebellar cortex. Purkinje cells react with both antibodies; arrows in B indicate parvalbumin- immunoreactive stellate and basket cells. Bar - 50/zm. Courtesy of Dr. M.P.A. Schalekamp.
supposedly occurs in GABAergic neurons (Celio and Heizmann, 1981) and/or neurons with characteristically high firing rates (Karmy et al., 1991). Karmy et al. (1991) studied the co-localization of parvalbumin and cytochrome oxidase, as an indicator of metabolic activity, in many regions of the brain. They found only weak immunoreactivity with antibodies against cytochrome oxidase in parvalbumin immunoreactive Purkinje cells of the rat. A developmentally regulated polypeptide (PEP-19), that is a presumptive neuronspecific calcium binding protein, was identified in adult and neonatal rat cerebellum and its amino acid sequence was determined (Ziai et al., 1986). PEP-19-like immunoreactivity is expressed by Purkinje cells and by the cartwheel cells of the dorsal cochlear nucleus of the mouse (Mugnaini et al., 1987). Berrebi et al. (1991) drew attention to the expression of PEP-19, CaBP and other Purkinje cell markers (cerebellin, L7: see below) by bipolar cells and other neurons of the retina.
3.1.8. Other specific biochemical markers for Purkinje cells Several polypeptides, that are present in all Purkinje cells, but not in other cells of the cerebellum, have been mentioned in the previous sections of this chapter. They include the IP3 receptor (identical to the P400 protein and to the PCPP-260 protein of Walaas et al., 1986) (see Section 3.1.4), IP3-3-kinase (Section 3.1.4), cGMP-dependent protein kinase (Section 3.1.5), PEP-19 and calbindin-D28K (Section 3.1.7). Two other 38
The cerebellum." chemoarchitecture and anatomy
Ch.I
proteins, cerebellin and L-7 that occur in all Purkinje cells, are dealt with in this section. Other proteins only occur in certain subpopulations of Purkinje cells. Zebrin I and II (Hawkes et al., 1985) are the prototypes of this group. The restriction of the Zebrins to a subpopulation of Purkinje cells is the more remarkable because they are originally present in all Purkinje cells of rat neonates (Leclerc et al., 1988). The developmental histories of cGMP-dependent protein kinase, calbindin-D28K and L-7 are quite different, in that these proteins make their first appearance in subpopulations of fetal Purkinje cells and only in later stages become expressed by all Purkinje cells of the cerebellum (Wassef and Sotelo, 1984; Smeyne et al., 1991) (Section 6.2.). Purkinje cell-specific markers include several glyco- and phosphoproteins, peptides, antigenic determinants that have not been identified or determinants that Purkinje cells share with other, non-cerebellar cell types. One of the first sera specific for rat Purkinje cells was obtained, using immunohistochemical screening, by Woodhams et al. (1979), but the antigen corresponding to this antibody has not been identified. Reeber et al. (1981) isolated a Purkinje cell specific 24 kDa glycoprotein from rat, that was present (Reeber et al., 1981) throughout the whole somatodendritic extent of the Purkinje cells, associated with the plasma membrane, as well as with the rough endoplasmatic reticulum and polysomes, the cytoplasmic side of the nuclear envelope and subsurface cisterns (Langley et al., 1982). Visinine, a soluble, 24 kDa protein, isolated from chicken retina, was found to be an exclusive marker for Purkinje cells in rat cerebellum (Yoshida et al., 1985). Specific staining of Purkinje cells was also found with monoclonal antibodies directed against human T cells (Garson et al., 1982), against certain cytoplasmic antigens in Purkinje cells (Weber and Schachner, 1982) and against antigenic determinants on trypanosomes (Wood et al., 1982). One of the antibodies (UCHT 1), isolated by Garson et al. (1982) is remarkable because its antigen is not present in Purkinje cells from 'staggerer' mutant mice (Caddy et al., 1982), a property the UCHT 1 antigen shares with the IP3 receptor protein (Section 3.1.4). One group of Purkinje cell-specific markers, the cerebellins, has been studied in more detail. A Purkinje cell-specific hexadecapeptide called 'cerebellin' and its metabolite, des-Serl-cerebellin were isolated and sequenced by Slemmon et al. (1984). Cerebellin immunoreactivity as studied with polyclonal antibodies in rat was found in soma and dendrites of nearly all Purkinje cells, but was absent beyond the initial axon segment (Slemmon et al., 1984). Cerebellin-immunoreactivity could also be demonstrated in cerebella of different species, including human and chick (Morgan et al., 1988), and in cartwheel cells and basal dendrites of pyramidal neurons of the dorsal cochlear nucleus (Fig. 33) (Mugnaini and Morgan, 1987). Cerebellin differs from most other markerproteins of Purkinje cells in being absent from other sites in the CNS, including the retina (Berrebi et al., 1991). Slemmon et al. (1988) and Morgan et al. (1988) concluded from an analysis of cerebellin immunoreactivity in Purkinje cells of different mutant mice with a varying loss of the granule cells, that the amount of cerebellin is correlated with the formation and the number of parallel fiber-Purkinje cell synapses. L-7 is a protein specific for Purkinje cells. Labelling with polyclonal antibodies against predicted L-7 sequences was present in somata, including the nucleus, in dendrites and dendritic spines, and in axon and axon terminals of Purkinje cells. All Purkinje cells, but no other types of cerebellar neurons appeared to be labelled (Berrebi and Mugnaini, 1992). The expression of the L-7 gene by all adult Purkinje cells of the rat cerebellum was reported by Nordquist et al. (1988, their PCD5 clone), Oberdick et al. (1990) Vandaele et al. (1991, their Purkinje cell protein-2) and Smeyne et al. (1991). According to Oberdick et al. (1990) and Berrebi et al. (1991) the L-7 gene is also expressed by retinal 39
Ch.I
J. Voogd, D. Jaarsma and E. Marani
Fig. 32. Parvalbumin immunoreactivity in the developing cerebellar cortex of the zebra finch. A. Incubation day D 16: Clusters of labelled Purkinje cells of varying staining intensity. Stained Purkinje cells axons are seen in the internal granular layer (IGL). Note the areas containing unstained or only slightly stained cells and the dot-like staining pattern in the external granular layer (EGL). B. Adult: The dendrites of the Purkinje cells have reached the cerebellar surface and are now fully branched. Between them many immuno-stained basket and stellate cells are visible. Parvalbumin immunoreactivity in Purkinje cell axons is no longer visible except for a few fragments lying in the internal granular layer (IGL). The layer of Purkinje cells is still interrupted by parvalbumin immunonegative areas. Calibration bar in A - 50/lm, in B = 100/~m. Braun et al. (1986).
bipolar cells. The initial expression of the L-7 gene by zonally distributed Purkinje cells during prenatal and early postnatal development was studied by Vandaele et al. (1991), Smeyne et al. (1991) and Oberdick et al. (1993) (see also Section 6.2.). 40
The cerebellum." chemoarchitecture and anatomy
Ch. I
Several other markers are only present in zonally distributed subpopulations of Purkinje cells (see also Section 6.1.3.). The monoclonal antibody B1 of Ingram et al. (1985) was raised against rat embryonic forebrain membranes. Purkinje cells in broad parasagittal bands, alternating with B 1-negative zones, were immunoreactive in the cerebellum of Macaca fascicularis. Other neurons in the molecular layer and cells of the cerebellar nuclei were also stained by this antibody. A similar pattern of B l-immunoreactivity was present in the cerebellum of the rat. The monoclonal antibody mabQ 113 was developed, specified and used in anatomical studies by Hawkes et al. (1985), Hawkes and Leclerc (1986, 1987), Hawkes and Gravel (1991), Hawkes (1992) and Leclerc et al. (1992). It is directed against a 120 Kda protein (Zebrin I); the function of this protein is still unknown. A specific subpopulation of Purkinje cells displays immunoreactivity for Zebrin I in their dendrites, soma, axon and axon terminals. Zebrin I-positive and negative Purkinje cells are distributed in parasagittal bands (Fig. 34) (see also Section 6.1.3.). Ultrastructurally Zebrin I-immunoreactivity in rat Purkinje cells is localized in the cytosol. It is absent from membrane-bound organelles such as the mitochondria and the synaptic vesicles. In large dendrites reaction product is associated with microtubuli, in spines it is located at the postsynaptic densities. An antibody raised against the cerebellum of the weakly electric fish Apteronotus (anti-Zebrin II: Brochu et al., 1990) recognizes the same Purkinje cells as anti-Zebrin I in the cerebellum of the rat, and is effective in staining these neurons in a large number of other species such as the opossum (Fig. 137). The epitope of the Zebrin II antibody is associated with a 36 kDa polypeptide identified as the glycolytic enzyme aldolase C. In situ hybridization of Zebrin II mRNA showed a strong signal in mouse Purkinje cells with normal regional heterogeneity (Hawkes, 1992; Ahn et al., 1994). Rat Purkinje cells containing low affinity nerve growth factor receptor protein (Sotelo and Wassef, 1991; Dusart et al., 1994) (see Section 3.1.10 and Fig. 38C,D), PKC delta (see Section 3.1.10 and Fig. 133), or the monoclonal antibody B30 of Stainier and Gilbert (1989), that recognizes two minor gangliosides, show the same distribution as Zebrin-stained Purkinje cells. Although the distribution of the enzyme 5'-nucleotidase in the molecular layer of rat and mouse cerebellum (Scott, 1963; Marani, 1982a,b) is identical to that of the Zebrins (Eisenman and Hawkes, 1993) (Fig. 135), it may be located in Bergmann glial and not in Purkinje cells (see Section 3.5.). Several proteins are distributed in more or less complementary patterns, either in Zebrin-negative Purkinje cells (Ppath, HNK, cytochrome oxidase) or in Bergmann glia (3a-fucosyl-N-acetyl lactosamine [FAL], glycogen phosphorylase). The antibody P-path is directed against acetylated gangliosides (Edwards et al., 1989, 1994; Leclerc et al., 1992) and reacts with Zebrin-negative Purkinje cells in mouse cerebellum (Fig. 134). The localization of cytochrome oxidase was described by Hess and Voogd (1986), Leclerc et al. (1990) and Harley and Biejalew (1992) in the cerebellum of macaques, the squirrel monkey and the rat. The localization of HNK was studied by Eisenman and Hawkes (1993) in the mouse. The FAL-epitope (Fig. 94; Bartsch and Mai, 1991) and the enzyme glycan phosphorylase (Marani and Boekee, 1973; Harley and Bielajew, 1992) have been located in subsets of mouse Bergmann glial cells, that are distributed in a complementary manner with respect to the Zebrin pattern. Gangliosides are glycolipids, concentrated in the outer layer of neural plasma membranes. Biochemical analysis showed a correlation between the selective degeneration of Purkinje cells in pcd and nervous mutant mice with the loss of the ganglioside GT~A. GT~A was more concentrated and the ganglioside GD~A was diminished in weaver mutant mice with a selective loss of the granule cells (Seyfried et al., 1983, 1987; Marani 41
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Fig. 33. Light photomicrographs showing cerebellin immunoreactivity in rat cerebellum (A-C) and the dorsal cochlear nucleus (D-F) in parasagittal sections. A. Cerebellar hemisphere with part of the underlying dorsal cochlear nucleus (DCoN, arrowhead). CN, cerebellar nuclei. B. Immunostaining in DCoN. The cell bodies of cartwheel neurons in the superficial layers (layers 1 and 2) of the DCoN and the plexus in the deeper region (layer 3) predominate. The plexus is most dense in the upper portion of the deep region, which may correspond to layer 3 of the feline nuclei, a zone that contains the basal dendritic arbors of the bipolar pyramid neurons, one of which is indicated by an arrow. C. Immunoreaction product is present in Purkinje cell body and main dendrites. D. In the axon, immunostaining is restricted to the initial axon segment (arrowhead). E. Three subependymal displaced Purkinje cells in DCoN. Smaller cell bodies of several cartwheel neurons (arrowheads) are also shown. F. Portion of the ventral cochlear nucleus in which immunostaining is restricted to rare cartwheel cell bodies (arrowheads) displaced in the superficial granular layer. Bars in A and B = 0.5/~m, in C-F = 50 r Mugnaini et al. (1987).
a n d M a i , 1992). A n o t h e r g a n g l i o s i d e , GD3, was localized in i m m a t u r e P u r k i n j e cells o f the rat, u s i n g a m o n o c l o n a l a n t i b o d y (Fig. 35). I m m u n o r e a c t i v i t y d i s a p p e a r e d f r o m the cell b o d y in the adult, b u t r e m a i n e d p r e s e n t in the m o l e c u l a r layer ( R e y n o l d s a n d Wilkin,
42
The cerebellum." chemoarchitecture and anatomy
Ch. I
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Fig. 34. 50/lm horizontal sections through the cerebellar cortex of the rat at postnatal day 25 to show the distribution of mabQ113 (Zebrin I) immunoreactivity. A. The peroxidase reaction product is confined exclusively to a subset of Purkinje cells that are distributed symmetrically into parasagittal compartments in both the vermis and hemispheres. Labelling of the bands of Zebrin I-immunoreactive Purkinje cells P l+ to P7+ according to Hawkes and Leclerc (1987). Scale bar = 500/~m. B. A higher-power view of P5 + and P6 + of the posterior lobe hemisphere, in the lobules bordering the intercrural fissure. Immunoreactivity is seen to extend throughout the Purkinje cell, and no other cell types in the cerebellum are stained. Scale bar = 200/zm. C. In addition to the regular band display, additional narrow 'satellite' bands are also common. The arrowheads indicate two such satellites in the posterior lobe vermis. Scale bar = 100/lm. Leclerc et al. (1988).
1988). Levine et al. (1986), who used another monoclonal antibody against GD3, found immunoreactivity of reactive astrocytes in mouse mutants, but failed to observe a reaction within the Purkinje cells. These different results probably are due to differences in fixation (Reynolds and Wilkin, 1988). 3.1.9. Cytoskeleton and metabolism of Purkinje cells The DNA content of mature Purkinje cells is high. Feulgen-DNA or propidiumiodideDNA reveal hyperdiploid values (Bernocchi, 1986; Bernocchi et al., 1986). Purkinje cells stand out by their high content of enzymes, mostly dehydrogenases (Adams, 1965). Their content of the glycolytic enzyme enolase is low (Pelc et al., 1986; Vinores et al., 43
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1984). However, Purkinje cells of the human cerebellum stand out from other nerve cells by their high content of aldolase-C (Royds et al., 1987). Purkinje cells do not react with antibodies against the phosphorylated forms of the 70, 150 and 200 kDa neurofilament proteins (Pelc et al., 1986; Matus et al., 1979; Marc et al., 1986; Langley et al., 1988). The phosphorylated form of the 200 kDa protein is present in axons in the granular layer, that were identified as Purkinje cell axons by Marc et al. (1986) and as mossy fibers by Langley et al. (1988), both in the rat (Fig. 36). The non-phosphorylated form of the neurofilament proteins was found to be present in the entire Purkinje cell with the exception of distal dendrites. According to Marc et al. (1986) the protein is present as filamentous aggregates. Langley et al. (1988) stated that a monoclonal antibody against the non-phosphorylated form of the 200 kDa protein is present in soma and dendrites as patches of diffuse immunoreactivity without a filamentous substructure. In Friedreich's ataxia neurofilament, mainly the phosphorylated form, is expressed by human Purkinje cells within their soma and dendrites (Marani, unpublished results) (Fig. 37). The process of endocytosis in Purkinje cell has been studied in relation to synaptogenesis of the Purkinje cell dendrites. Glycoproteins located on the parallel fiber are also pinocytosed into the Purkinje cell. Lysosomal action degradates these glycoproteins. In this process alpha-D-massosidase plays an important role, which is selectively present in the Purkinje cell dendrites (Dontenwill et al., 1983). Other glycoproteins, like K+Na+ATP-ase are not taken up, indicating a receptor-mediated recognition of some glycans of the glycoproteins. The specificity of the pinocytosis for certain molecules suggests that this recognition is the preliminary event in the establishment of Purkinje cell synapses.
3.1.10. Nerve growth factor and nerve growth factor-receptor protein in Purkinje cells Nerve growth factor-like immunoreactivity was present in Purkinje cell somata and dendrites, with dense labelling in the paraflocculus, and in neurons of the cerebellar nuclei and the lateral vestibular nucleus of rat cerebellum (Nishio et al., 1994). All but a few of the Purkinje cells of the adult rat cerebellum stain with an antiserum against basic fibroblast growth factor. Staining was observed in all cellular compartments (Matsuda et al., 1992). P75 nerve growth factor-receptor protein (NGF-R) is present in developing and adult Purkinje cells. Yan and Johnson (1988) and Cohen-Cory et al. (1989) described and reviewed the development of NGF-R in rat cerebellum. Low affinity NGF-R immunoreactivity has been demonstrated with species-specific monoclonal antibodies in Purkinje cells of adult rats (Pioro and Cuello, 1988, 1990; Pioro et al., 1991; Fusco et al., 1991; Dusart et al., 1994), monkey and human brain (Mufson et al., 1991). Immunoreactivity was present in the somata, dendrites and the proximal axon of the Purkinje cells. Additional immunoreactivity in granule cells was reported by Vega et al. (1994), using Bouin's fixative. NGF-R mRNA is expressed during early development in neurons of the rat external granular layer and in Purkinje cells. It peaks at postnatal day 10 and declines afterwards (Cohen-Cory et al., 1989; Lu et al., 1989) but also can be demonstrated in Purkinje cell somata in adult rodents (Fig. 38) (Koh et al., 1989) and primates (Mufson et al., 1991). NGF-R immunoreactivity was found to be highest in the flocculonodular lobe (Pioro and Cuello, 1988, 1990; Fusco et al., 1991). A distribution with strong expression in the flocculonodular lobe, the ventral parts of the anterior lobe and the lobules VII, VIII and 44
The cerebellum." chemoarchitecture and anatomy
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Fig. 35. Double-immunofluorescent staining of 20-day rat cerebellar sections with antibodies to GD 3 ganglioside and glial acidic fibrillary protein (GFAP). Purkinje cell dendrites are intensely GD3-immunoreactive (A) but do not extend to the pial surface, unlike the Bergmann glial fibers (B), which project brightly GFAPimmunoreactive end-feet onto the pial membrane. Scale bar is 35 ~tm. Reynolds and Wilkin (1988).
45
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Immunocytochemical staining patterns of two monoclonal anti-bodies directed against nonphosphorylated and phosphorylated neurofilaments were studied in the cerebellum of developing normal rats. A. Non-phosphorylated neurofilaments on postnatal day 11. B. Day 21. Basket cell axons form a characteristic brush-like plexus around the initial segment of the Purkinje cell axon. C. Phosphorylated neurofilaments on postnatal day 13. D. Postnatal day 21. Stained filaments are restricted to Purkinje cell and basket cell axons and are absent from the Purkinje cell cytoplasm. Calibration bars in A and C 30/lm, in B and D 10 ~tm. Marc et al. (1986).
Fig. 36.
46
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IX of the caudal vermis and low activity in the hemisphere, was described by Mufson et al. (1991) for primates and man. The administration of colchicine results in the expression of N G F - R in most cerebellar Purkinje cells (Pioro and Cuello, 1988, 1990; Pioro et al., 1991). Koh et al. (1989) and Fusco et al. (1991) found N G F - R mRNA expression and NGF-R immunoreactivity in adult rat~ to be present in alternating Purkinje cell zones of strong and weak activity (Fig. 38C,D). This zonal pattern was also observed by Pioro and Cuello (1990). Its correspondence to the pattern of mabQ113 (Zebrin) immunoreactive zones (Hawkes and Leclerc, 1987) was noticed by Sotelo and Wassef (1991) and verified by Dusart et al. (1994) in adult rats. Lesions of the white matter, or knife cuts isolating the dorsal portion of the vermis of the rat cerebellum induces NGF-R immunoreactivity in previously unstained Purkinje cells (MartinezMurillo et al., 1993; Dusart et al., 1994).
3.1.11. Immunoreactivity of Purkinje cells in paraneoplastic diseases Specific forms of immunoreactivity of Purkinje cells have been discovered in human paraneoplastic conditions. Subacute cortical cerebellar degeneration in man may be associated with several types of carcinoma (see Vecht et al., 1991 for review). It has been most frequently observed in association with ovarian or endometrial carcinoma, but it also occurs as a rare sequal of small-celled bronchial carcinoma. It is generally characterized by a diffuse or patchy loss of Purkinje cells; granule cells also can be affected (Brain et al., 1951; McDonald, 1961; Brain and Wilkinson, 1965; Schmid and Riede, 1974; Steven et al., 1982). Strong labelling of Purkinje cells and weak staining of the granular layer was observed in sections of human cerebellum with a serum of patient with cerebellar degeneration with Hodgkin's disease using the indirect fluorescent staining procedure (Trotter et al., 1976). Sera of patients with carcinoma of the ovary were found to react with human Purkinje cells and neurons of the cerebellar nuclei using the same method (Greenlee and Sun, 1985). Jaeckle et al. (1985) distinguished a granular cytoplasmic and a diffuse form
9
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Fig. 37. Expression of phosphorylated neurofilament localization in normal human cerebellar cortex (A) and in Friedreich's disease (B). Note the strong positivity of the white matter and the molecular layers in a case of Friedreich's ataxia. No expression was found in the normal folium that was Nissl counterstained to demonstrate the granular and Purkinje cell layer. M = molecular layer, P = Purkinje cell layer, G = granular layer, F = fiber layer. Marani, unpublished.
47
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Fig. 38. A. Nerve growth factor-R (NGF-R) transcripts are localized within Purkinje cells in the paraflocculus of rat cerebellum. B. NGF-R immunocytochemistry shows the perikarya of the Purkinje cells as well as the dense staining of the molecular layer, where the dendritic trees of the Purkinje cells arborize. Arrows in C and D point to parasagittal zones of intense labelling interdigitated with weaker labelling. Bar = 90 r Koh et al. (1989).
of immunoreactivity of human Purkinje cells with sera from patients with cerebellar degeneration suffering from ovarian or breast cancer. The diffuse form of Purkinje cell staining also was observed at higher concentrations with some sera of normal controls. Moreover the diffuse staining is not restricted to Purkinje cells, but also involves stellate, basket and some granule cells (Andersson et al., 1988). Cunningham et al. (1986) further analysed the sera causing granular deposits in the Purkinje cell cytoplasm with immunoblotting of extracts of human Purkinje cells. This so-called anti-Yo serum recognizes a 62 kDa and a 34 kDa protein. Antibodies raised against both proteins react with Purkinje cells in tissue sections (Fig. 39). The strongest reaction was observed for the antibody against the 62 kDa protein. The specificity of this reaction and the presence of anti-Yo immunoreactivity in tumor tissue was demonstrated by Furneaux et al. (1990). The 34 kDa antigen was found to correspond to the c D N A sequence of a clone recognized from a cerebellar expression library by a serum from a patient with paraneoplastic cerebellar degeneration (Dropcho et al., 1987; Furneaux et al. 1989). Other forms of immunoreactivity, with different cerebellar epitopes and a different localization of the immunoreactivity have been described (Tanaka et al., 1986; Smith et al., 1988; Rodriguez et al., 1988; Tsukamoto et al., 1989; Szabo et al., 1991). Differences in the localization of the immunoreactivity and in the characterization of the epitopes may be due to the use of rat cerebellum instead of human cerebellum in testing the sera by Tanaka et al. (1986), Smith et al., (1988) and Tsukamoto et al. (1989). Szabo 48
The cerebellum." chemoarchitecture and anatomy
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et al. (1991) isolated the (NuD) neuronal antigen recognized by sera from patients with paraneoplastic encephalomyelitis associated with small-celled bronchus carcinoma. This serum, also designated as anti-Hu, reacts with nuclei of neurons in the CNS, including the cerebellum (Andersson et al., 1988). 3.2. EXCITATORY PATHWAYS The cerebellar cortex is innervated by two types of excitatory afferents, the mossy and climbing fibers, and an intrinsic excitatory fiber system, the parallel fibers. An additional excitatory intrinsic pathway may be formed by unipolar brush cells, that give rise to mossy fiber-like fibers. The excitatory amino acid glutamate is the most likely neurotransmitter candidate for these pathways. An inherent problem in the localization of glutamate as a neurotransmitter is that there is no unequivocal marker for glutamatergic neurons and fibers since glutamate also participates in several metabolic pathways of nerve cells (Van den Berg and Garfinkel, 1971; Fonnum, 1984; Erecinska and Silver, 1990). The identification of glutamatergic pathways, therefore, is based upon a combination of anatomical, biochemical and physiological techniques (Fonnum, 1984). Immunocytochemistry with antibodies against glutamate (Storm-Mathisen et al., 1983) and physiological studies have proven to be particularly fruitful in the identification of glutamate as the neurotransmitter of the cerebellar excitatory pathways. These methods, however, do not totally exclude the possibility that other excitatory amino acids, such as aspartate or homocysteate, also participate as excitatory neurotransmitters. This holds in particular for the climbing fibers that have been frequently proposed to use aspartate as their primary neurotransmitter (see below). A major problem with 'nonglutamate' excitatory neurotransmitter candidates is that, as yet, no vesicular uptake
Fig. 39. Immunofluorescence of rat Purkinje cells with anti-Yo serum of a patient suffering from a cerebellar syndrome with ovarian carcinoma. Courtesy Dr. Ch. J. Vecht and Dr. J.W.B. Moll, Department of Neurology, Erasmus University Medical Center, Rotterdam.
49
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system has been isolated for these compounds. Instead, glutamate has been shown to accumulate in synaptic vesicles by a proton-driven vesicle transporter. This vesicle transporter is highly specific for glutamate, and in contrast to the cytoplasma membrane 50
The cerebellum." chemoarchitecture and anatomy
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Fig. 40. Immunostaining in rat cerebellar cortex produced by anti-glutamate(Glu) mAb 2D7 (A,C,C',E and F) or by 'anti-GABA' mAb 3A12 (B and D). A, B (overview) and C, D (details) are from a pair of consecutive semithin sections. C and C' are enlargements of areas indicated in A and C, respectively. A and B. Note drastic difference in labelling patterns obtained with the two antibodies, gr, granule cell layer; P, Purkinje cell layer; mol, molecular layer. C. Frame indicating part of area shown enlarged in C'. C and D. Complementary labelling in stellate cells (stars), Golgi cell (arrows), pinceau formed by basket cell terminals (double arrow heads). Mossy fiber terminal-like structures in C (arrow head) fit into glomerular arrangements outlined by dots in D (arrow head). C'. Densely packed puncta probably represent parallel fiber terminals in molecular layer. E. Numerous strongly stained patches (arrow heads) and some fibers (arrow) are reminiscent of mossy fiber terminals. Granule cells with unlabelled nuclei appear less immunoreactive than those in C. F. Large mossy fiber terminal with several synaptic contacts (arrows) shows higher surface density of gold granules (EM immunogold procedure) than another terminal nearby (stars). Bars 100 ~tm in B, D and E, 1 r in F. Liu et al. (1989). (
transporter, does not transport aspartate (reviewed by Nicholls and Atwell, 1990; Jahr and Lester, 1992). 3.2.1. Mossy fibers Glutamate-like immunoreactivity in mossy fibers
Although subpopulations of mossy fibers may be peptidergic or cholinergic (see Sections 3.10. and 6.4.5.), it is now generally accepted that most if not all of the mossy fibers use L-glutamate as their principal neurotransmitter. The glutamatergic nature of mossy fibers has been evidenced with immunocytochemistry with antibodies against glutamateglutaraldehyde (Storm-Mathisen et al., 1983) or carbodiimide-glutamate conjugates (Madl et al., 1986). The rationale of this method is that glutamate, although ubiquiteously present throughout the neuronal cytoplasm at relatively high concentrations (~ 10 mM; Van den Berg and Garfinkel, 1971; Nichols and Attwell, 1990), is particularly enriched in glutamatergic nerve terminals, because of the presence of synaptic vesicles that concentrate glutamate to at least 60 mM. When electron microscopic post-embedding immunogold protocols are employed, quantitative and statistical analysis of the distribution of immunolabelling can be performed (e.g. see Ottersen, 1989). Glutamate immunoreactivity is widely distributed throughout the granular layer, but is enriched over mossy fiber rosettes in rat (Figs 40 and 41) (Ottersen and Storm-Mathisen, 1984a,b, 1987; Ottersen et al., 1987, 1990; Liu et al., 1989; Ji et al., 1991), cat (Somogyi et al., 1986) and monkey (Zhang et al., 1990). Mossy fiber rosettes contained significant higher levels of immunoreactivity than Golgi cell terminals and granule cell dendrites. Enriched glutamate-like immunoreactivity was also demonstrated in anterogradely horseradish peroxidase-wheat germ agglutinin (WGA-HRP) labelled spinocerebellar mossy fiber terminals. Notably, the density of glutamate-like immunoreactivity showed a strong positive correlation with the density of synaptic vesicles in these mossy fiber terminals (Ji et al., 1991). The anterogradely labelled mossy fiber terminals had a similar density of glutamate-like immunoreactivity as other mossy fiber rosettes. Mossy fiber terminals were not enriched in aspartate- or GABA-like immunoreactivities (Ji et al., 1991; Zhang et al., 1990). Data from physiological studies including recent patch-clamp studies are in line with the assumption that glutamate is the neurotransmitter of mossy fibers (Garthwaite and Brodbelt, 1989, 1990; Silver et al., 1992; D'Angelo et al., 1993; Rossi et al., 1995). 51
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Glutamine and glutaminase
Ottersen et al. (1992) quantified the compartmentalization of glutamate and glutamine in the cerebellar cortex of the rat, using post-embedding immunogold immunocytochemistry. They found the highest ratios of glutamate/glutamine in parallel fibers, high ratios in mossy and climbing fibers, low ratios in Purkinje and granule cells and in basket cell and Golgi cell terminals and the lowest ratios in Bergmann glia and astrocytes. This distribution is in accordance with uptake of glutamate from the extracellular space by glial cells, and its conversion into glutamine by the enzyme glutamine synthase, that is exclusively present in glia (Van den Berg and Garfinkel, 1971; Norenberg and MartinezHernandez, 1979; Fonnum, 1984; Erecinska and Silver, 1990). The glutaminase-glutamine loop is closed by diffusion of glutamine into neurons, that contain glutaminase, the enzyme that catalyzes the hydrolytic cleavage of glutamine to form glutamate. Wenthold et al. (1986) and Kaneko et al. (1987, 1989) used antibodies against glutaminase as an alternative approach to determine the cellular localization of glutamate. In the granular layer glutaminase-like immunoreactivity was present in granule cell somata (Wenthold et al., 1986) and in in small clusters, that probably represent mossy fiber rosettes (Fig. 42e) (Wenthold et al., 1986; Kaneko, 1987, 1989). Intense glutaminase-like immunoreactivity was also detected in several precerebellar nuclei, that give rise to mossy fibers, such as the pontine nuclei, the reticular nucleus of the pons, the lateral reticular nucleus, the vestibular nuclei and the external cuneate nucleus (Fig. 42a-d). Neurons in some of these nuclei have also been shown to react with antibodies against conjugates of glutamate (Beitz et al., 1986; Clements et al., 1986; Raymond et al., 1984). Glutamate transporters
The major mechanism by which synaptically released glutamate is inactivated is by highaffinity, sodium-dependent transport (Fonnum, 1984; Nicholls and Attwell, 1990). The sodium-dependent glutamate transporters are present in both neurons and astroglial cells, and have been assumed to be enriched on nerve terminals of glutamatergic axons. [3H]D-aspartate, a metabolically inert substrate of the glutamate transporter with very low affinity for glutamate receptors, has been widely used to locate glutamate or aspartate using fiber systems in the brain (Fonnum, 1984). Autoradiographic studies on cryostate sections indicate that [3H]D-aspartate binding sites are particularly enriched in the molecular layer, but are also present in the granular layer (Greenamyre et al., 1990; Anderson et al., 1990). Studies in cerebellar slices, however, show that [3H]Daspartate is not taken up by mossy fiber terminals (Garthwaite and Garthwaite, 1988). Accordingly, [3H]D-aspartate is not retrogradely transported by mossy fibers, allthough it is efficiently transported by climbing fibers (Wiklund et al., 1984). Three high-affinity sodium-dependent glutamate transporters have been cloned in rat: GLT-1 (Pines et al., 1992; Tanaka, 1993), EAAC1 (Kanai and Hediger, 1992), and GLAST (Storck et al., 1992). Recently, also four subtypes of human glutamate transporters, EAAT1-EAAT4, have been cloned with similar properties as their rat counterparts (Arriza et al., 1994; Fairman et al., 1994). In situ hybridisation and immunocytochemistry showed a differential distribution of the three transporters throughout the cerebellum. GLT1 is concentrated in the Bergmann glial fibers, but also occurs in the glial processes of the granular layer and in the cerebellar nuclei (Danbolt et al., 1992; Rothstein et al., 1994). High levels of GLAST are present in Bergmann glial fibers, but it is essentially absent from the granule cell layer. In the cerebellar nuclei it is mostly 52
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Fig. 41. Electron micrographs of serial sections through a glomerulus in the granular layer of the cat cerebellar cortex. The section shown in (A) was reacted with antiserum to glutamate (GLU), the section in (B) with antiserum to GABA. The electron-dense gold particles show immunoreactive sites. For GLU the highest density of gold appears to be over the mossy fiber terminal (mt) and the lowest over glial processes and Golgi cell terminals (1-3). This was confirmed by statistical comparison of the populations. The same Golgi cell terminals are strongly reacting for GABA, while other processes have only a low surface density of gold. Scale (A and B) 0.5/Ira. Somogyi et al. (1986).
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I
Fig. 42. Phosphate-activated glutaminase-like immunoreactivity (PAG-LI) in the precerebellar nuclei and the cerebellar cortex of the rat. Intensely labelled neuronal somata are seen in the pontine tegmental reticular nucleus of Bechterew (a), pontine nuclei (b), external cuneate nucleus (c), and lateral reticular nucleus of the medulla oblongata (d). Small clusters of grains, possible axon terminals, with PAG-LI are seen in the granular layer of the cerebellar cortex (e). Fine grains with PAG-LI are densely distributed, but no cell bodies are seen in the inferior olivary nucleus (f). CM, cerebellar medulla; G, granular layer; M, molecular layer; ML, medial lemniscus; P, pontine longitudinal fibers; Py, pyramidal tract; R, raphe. Scale bar 200 pm in a-d, f, 50 pm in e. Kaneko et al. (1987).
54
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associated with neurons (Rothstein et al., 1994). EAAC1 has been exclusively localized in neurons, with high densities in Purkinje cells and granule cells. Interestingly, immunocytochemical data show that EAAC1 is enriched in axon terminals of Purkinje cells, indicating that EAAC1 is not selective for glutamatergic nerve terminals. In accordance with the biochemical data, there was no immunocytochemical evidence for the presence of EAAC 1 or one of the other glutamate transporter proteins in mossy fiber terminals. Taken together the above data indicate that mossy fiber terminals are not provided with high-affinity glutamate transporters. Consequently, glutamate released by mossy fibers is likely to be predominantly cleared through glial cells (Wilkin et al., 1982; Garthwaite and Garthwaite, 1988). However, since glial processes do not enter the glomeruli (e.g. see Palay and Chan-Palay, 1974), an exclusive glial uptake implies that 'mossy fiber glutamate' molecules have to travel throughout extracellular space of the glomeruli before being inactivated. The clearance of 'mossy fiber glutamate' may be particularly slow at the giant mossy fiber-unipolar brush cell synapses, that may extend over 12-40 ,um2 with multiple clusters of presynaptic vesicles apposed to continuous regions of postsynaptic densities (Mugnaini and Floris, 1994). In fact, unusually long excitatory postsynaptic responses have been observed in unipolar brush cells following mossy fiber stimulation, consistent with a slow clearance of synaptically released glutamate (Rossi et al., 1995). 3.2.2. Climbing fibers Aspartate and glutamate
Several observations have led to the assumption that L-aspartate is the principal neurotransmitter of climbing fibers. (1) Destruction of the inferior olive in the rat with 3-acetylpyridine resulted in a small decrease in cerebellar aspartate concentration in total tissue homogenate (Nadi et al., 1977) and synaptosomal fractions (Rea et al., 1980). However, these observations were not confirmed by Perry et al. (1976). (2) It was demonstrated that after 3-acetylpyridine treatment Ca2+-dependent and K+-induced release of aspartate was significantly decreased (Toggenburger et al., 1983). Glutamate release was more dramatically decreased (e.g. see Cu6nod et al., 1989). (3) It was observed that climbing fibers but not mossy fibers in rat (Wiklund et al., 1984) and monkey (Matute et al., 1987) retrogradely transported [3H]D-aspartate. These experiments, however, only showed that climbing fibers are provided with high-affinity sodium-dependent glutamate transporter protein, and did not give information about the kind of transmitter used by the climbing fibers (see 3.2.1.). It should be noted that high affinity glutamate transporters have not yet been located at synapses of climbing fibers in immunocytochemical studies with antibodies against high-affinity glutamate transporters, although this possibility is still open since a detailed electron microscopical analysis of the cerebellar molecular layer has not yet been done (Rothstein et al., 1994). (4) Physiological studies suggested that the distal region of the Purkinje cell dendrites was relatively less sensitive towards aspartate as compared to glutamate than the proximal dendrites (Cr6pel et al., 1982). Since climbing fibers chiefly innervate the proximal two-thirds of the Purkinje cell dendritic tree (Palay and Chan-Palay, 1974), these data would be consistent with the proposal that aspartate is a climbing fiber transmitter, whereas glutamate is the transmitter of the parallel fibers (see Cu6nod et al., 1989). Voltage-clamp studies of Purkinje cells in slices, however, suggest that climbing fibers 55
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Fig. 43. Photomicrographs of consecutive semithin sections from rat inferior olive stained with antisera to aspartate, glutamate and GABA, respectively. All neurons (arrows) in this field are labelled for aspartate and glutamate, but unlabelled for GABA. Glial cells (arrowheads; identity established on the basis of electron microscopic analysis of adjacent sections) contain little or no GABA and glutamate immunoreactivities, but are moderately stained with the aspartate antiserum. Asterisks indicate fiber bundles. Scale bar = 50 r Insets show test sections incubated together with the respective tissue sections. The test antigens are GABA (1), glutamate (2), taurine (3), glycine (4), 'none' (5), aspartate (6), and glutamate (7). Note selective staining of the respective amino acid conjugates. Zhang et al. (1990). (
and parallel fibers activate the same type of glutamate receptors (Llano et al., 1991). Summarizing, one may conclude that the case for aspartate as the principal neurotransmitter of climbing fibers is far from being conclusive. Zhang et al. (1990), who compared glutamate- and aspartate-like immunoreactivities in the neurons of the inferior olive and climbing fibers in rat and baboon (Papaio anubis), showed that glutamate and aspartate-like immunoreactivities were co-localized in all neurons of the inferior olive, with a slightly heavier staining in the principal olive (Fig. 43). Significant glutamate-like, but little aspartate-like labelling, however, was recognized over climbing fiber profiles and, therefore, it was concluded that glutamate and not aspartate is the most likely transmitter of the climbing fibers (see also Zhang and Ottersen, 1993). It was also concluded that the presence of aspartate-like immunoreactivity in cell bodies is an unreliable indicator of transmitter identity.
Homocysteate Cu6nod et al. (1989) reported on the results of a series of experiments on K+-induced release of different transmitters by the cerebellum of the rat, after previous destruction of the inferior olive by 3-acetylpyridine. Release of aspartate was found to be decreased compared to the controls, with the main decrease occurring in the hemisphere. Values for the vermis were only slightly lower than in normal rats. This difference might be explained by a relative sparing of neurons in the caudal inferior olive, that project to the vermis. Decreased values after 3-acetylpyridine treatment were also found for adenosine (see Section 3.5) and for homocysteic acid. For the release of these substances no differences were noticed between vermis and hemisphere. Homocysteic acid was originally considered as a transmitter of the climbing fibers (Grandes et al., 1989), but proved to be located in Bergmann glia (Figs 44 and 45) (Cu6nod et al., 1990; Grandes et al., 1991). Climbing fibers, therefore, interact with Bergmann glia, both in the release of homocysteic acid and in 5'-nucleotidase-regulated adenosine release (see Section 3.5). Immunocytochemical studies have shown that subpopulations of climbing fibers may use peptides as a neurotransmitter, including somatostatine, corticotrophin-releasing factor and enkephalin. Their distribution and characteristics will be discussed in Section 6.3.4.
3.2.3. Granule cells and parallel fibers In early studies it was found that in 'staggerer', 'weaver' and 'reeler' mutant mice which have almost complete or partial loss of their granule cells (McBride et al., 1976a; Hudson et al., 1976) and in rats or mice that lost their granule cells by a viral infection or 57
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postnatal X-irradiation (McBride et al., 1976b; Rohde et al., 1979), glutamate was depleted. However, the interpretation of this finding is not immediately clear, because mossy fiber terminals and inhibitory interneurons of the cerebellar cortex also may have been affected. Furthermore, it proved difficult to exclude aspartate as a transmitter of granule cells (Rohde et al., 1979; Roffler-Tarlov and Turey, 1982). Also the demonstration of Garthwaite and Garthwaite (1985) that granule cells in slices accumulate [3H]Daspartate did not provide conclusive evidence about the nature of the neurotransmitter used by parallel fibers. Immunocytochemical studies strongly support glutamate as the neurotransmitter of the parallel fibers. Thus, glutamate-like immunoreactivity but no other amino acids were enriched over parallel fiber terminals in rat (Ottersen and Storm-Mathisen, 1984a,b, 1987; Ottersen et al., 1987, 1990; Liu et al., 1989) (Fig. 40), cat (Somogyi et al., 1986) and monkey (Zhang et al., 1990). Also electrophysiological experiments are in favour of glutamate as the neurotransmitter at the parallel fiber-Purkinje cell synapse (Barbour, 1993 and references therein).
~
[!2s .
Fig. 44. Immunocytochemical localization of homocysteate (HCA) in Bergmann glia with polyclonal antiHCA antibodies. A. Test system mimicking immunocytochemical procedure. Conjugates are assembled in 'sandwich' construction with tissue as spacer and contain the following compounds (from top to bottom): HCA, Glu (glutamate), Asp (aspartate), Tau (taurine), Gly (glycine), GABA (~,-aminobutyric acid), L-Ala (L-alanine), fl-Ala (fl-alanine), Htau (homotaurine), Hypotau (hypotaurine), Gline (glutamine), Ca (cysteate), CSA (cysteine sulphinate), HCSA (homocysteine sulphinate), Cys (cysteine), Cyt (cystine), Met (methionine), carnosine, Hcys (homocysteine), cystathionine, gluta-thione, homocarnosine, y-Glu-Glu (y-glutamyl glutamate), fl-L-Asp-Gly (fl-L-aspartyl glycine), no AA (no amino acid conjugated to glutaraldehyde-treated rat brain protein). B. Pattern of HCA-like immunoreactivity in low-power view of rat cerebellar cortex in semithin section. Double arrow: fibrous, radially oriented immunoreactive element. Arrowheads, stained varicosities in association with Purkinje cell dendrites. C. Pattern of HCA-like immunoreactivity in rat cerebellar section pretreated with 3-acetylpyridine 10 days previously and degeneration of the inferior olive. No changes in the distribution of HCA are apparent. Bars 50/lm. Grandes et al. (1991).
58
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 45. Immunocytochemical localization of homocysteate (HCA) with polyclonal anti-HCA antibodies. A. Staining pattern in section close to that in Fig. 44B at higher magnification. Cell (asterisk) and capillary (circle) used as landmarks in A and B. B and C. Electron micrographs from ultrathin section immediately preceding semithin section in A. The HCA-immunoreactive varicosities indicated with arrows in (A) were identified as parts of the glial sheath surrounding Purkinje cell dendrites (d) in B and C. Bars: 10/lm in A, 5/lm in B, 1 j~m in C. Grandes et al. (1991).
Specific markers for granule cells are few. Seyfried et al. (1983), concluded from biochemical analysis in 'weaver' mutant mice that the ganglioside GDIA was more concentrated in granule cells. Webb and Woodhams (1984) developed three monoclonal 59
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J. Voogd, D. Jaarsma and E. Marani
antibodies (G-l-3; 7-8D2 and 8-20-1), that recognize cell surface antigens expressed by rat granule cells and their axons (see also Reynolds and Wilkin, 1988). Calcium-binding proteins, with the exception of calretinin (Rogers, 1989; Arai et al., 1991; Kadowaki et al., 1993; Floris et al., 1994) have not been localized in granule cells. Proteine kinase C (PKC) e, flI and II, e and ~"are expressed by rat granule cells (Ase et al., 1988; Wetsel et al., 1992; Chen and Hillman, 1993) (Table 1). 3.3. LOCALIZATION OF GLUTAMATE RECEPTORS
3.3.1. lonotropic glutamate receptors Glutamate activates two main classes of glutamate receptors, the ionotropic and metabotropic glutamate receptors. The ionotropic receptors are receptor/channel complexes that can be categorized into three groups according to their differential sensitivity to agonist ligands, as ~-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors, formerly known as quisqualate receptors, kainate receptors, and N-methylD-aspartate (NMDA) receptors (Monaghan et al., 1989; Mayer and Miller, 1990; Westbrook, 1994). The non-NMDA (AMPA and kainate) receptors display rapid kinetics. They are typically inhibited by 7-cyano-7-nitroquinoxaline-2,3-dione (CNQX), are permeable to monovalent cations (Na+, K+), but mostly impermeant to Ca 2+, and have been implicated in fast excitatory synaptic transmission (Mayer and Westbrook, 1987; Jahr and Lester, 1992). NMDA receptor channels, instead, have relatively slow kinetics, are also permeable to Ca 2+ ions, and are typically inhibited by D-2-amino-5-phosphonovalerate (APV). NMDA receptors are characterized by a voltage-dependent channel block by MgZ+-ions. They are dependent on, and are equipped with a coagonist site for glycine. Apart from their role in excitatory synaptic transmission, NMDA receptors have been implicated in synaptic plasticity and in developmental processes like cell migration and synaps formation (Collingridge and Singer, 1990). AMPA receptors
AMPA receptors have been autoradiographically labelled with [3H]AMPA and the antagonist [3H]CNQX: [3H]AMPA binding is moderately high over the rodent (Rainbow et al., 1984b; Monaghan et al., 1984; Nielsen et al., 1990; Garcia-Ladona et al., 1991; Makowiec et al., 1991) and human (Jansen et al., 1990) cerebellum, and is higher over the molecular than over the granular layer. [3H]CNQX binding sites are preferentially localized over the molecular layer, but cerebellar [3H]CNQX binding is relatively higher than [3H]AMPA binding, when the two are compared to binding levels of both ligands in other brain areas (e.g. see Fig. 6 in Nielsen et al., 1990). This difference is not due to [3H]CNQX binding to kainate receptors since these receptors are preferentially localized in the granular layer. Both [3H]AMPA and [3H]CNQX binding in the molecular layer was decreased in Purkinje cell deficient (pcd) mutant mice, but strongly upregulated in granuloprival mice (Makowiec et al., 1991). These observations favour a primary localization of AMPA receptors on Purkinje cells and an upregulation of the number of AMPA receptors on Purkinje cells as a consequence of deafferentation (Makowiec et al., 1991). Originally, AMPA receptors were assessed as quisqualate-sensitive [3H]glutamate binding sites (Cha et al., 1988, and references therein). Quisqualate-sensitive [3H]glutamate binding is strongly increased by the presence of CaC12, and is relatively high in the cerebellar molecular layer. CaC12-dependent quisqualate-sensitive [3H]glutamate bind60
The cerebellum." chemoarchitecture and anatomy
Ch. I
ing over the molecular layer, however, is largely insensitive to AMPA (Cha et al., 1988). These sites most likely correspond to the quisqualate-sensitive metabotropic glutamate receptors (Young et al., 1991), that have been recently demonstrated to be expressed at high levels by Purkinje cells (see Section 3.3.2.). Kainate receptors
High-affinity [3H]kainate binding sites predominate in the granular layer in rat (Monaghan and Cotman, 1982; Olson et al., 1987; Cambray-Deakin et al., 1990; Bahn et al., 1994) and man (Jansen et al., 1990). Low to moderate levels of [3H]kainate binding occur in the rat cerebellar nuclei. [3H]Kainate binding is not affected in Purkinje cell deficient (pcd) or 'nervous' mutant mice, but is decreased in granuloprival mice (Griesser et al., 1982). This decrease concerns the granular but not the molecular layer (Olson, 1987; Makowiec et al., 1991). Henke et al. (1981) noted a high level of low-affinity [3H]kainate binding sites in the molecular layer of pigeon cerebellum. Similar [3H]kainate binding sites were also labelled in the chicken cerebellum (Henley and Barnard, 1990), in fish (Maler and Monaghan, 1991) and in amphibian cerebellum, although in the amphibian kainate-binding sites seems to have somewhat different pharmacological and functional properties (reviewed in Henley, 1994). The chicken kainate binding sites could also be labelled by [3H]CNQX (Henley and Barnard, 1990). Several non-mammalian vertebrate kainate-binding proteins have been purified and cloned. These proteins display some homology towards mammalian ionotropic AMPA and kainate receptor subunits (see below), but are smaller (40-50 kDa instead of 100 kDa), and do not form functional receptors channels (reviewed by Hollman and Heinemann, 1994; Henley, 1994). In situ hybridisation and immunocytochemistry has shown that avian kainate-binding protein is localized in Bergmann glia (Fig. 95) (Somogyi et al., 1990; Gregor et al., 1992 and others). Somogyi et al. (1990) showed that immunostaining with a monoclonal antibody (IX-50) against chicken kainate-binding protein, was also localized in Bergmann glia in the cerebellum of fish. Frog kainatebinding protein, however, is widely distributed throughout the frog brain. High receptor densities were found in cerebellum, but their cellular distribution has not yet been reported (Dechesne et al., 1990; Wenthold et al., 1990). N M D A receptors
The distribution of NMDA receptors has been autoradiographically determined as NMDA-replaceable [3H]glutamate binding sites. In rat (Greenamyre et al., 1985; Monaghan and Cotman, 1985) and human cerebellum (Jansen et al., 1990), moderate densities of binding sites are found over the granular layer and in the cerebellar nuclei, whereas binding over the molecular layer is low. Olson et al. (1987) and Makowiec et al. (1991) reported that NMDA-sensitive [3H]glutamate binding is unchanged in Purkinje cell deficient (pcd) mutant mice, but that the density of binding sites is considerable reduced over the granular layer in granuloprival mice. These data suggest that NMDAbinding sites are absent on Purkinje cell dendrites and, instead, are present on granule cells and perhaps on stellate, basket and Golgi cells. Using different ligands including the competitive antagonist [3H]-2-carboxypiperazine-4-yl-propyl-l-phosphonic acid ([3H]CPP), [3H]glycine that specifically binds to the glycine coagonist site of NMDA receptors, and the non-competetive channel blockers [3H]MKS01 and [3H]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP), it was found that the pharmacological properties of NMDA receptors in the cerebellar 61
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J. Voogd, D. Jaarsma and E. Marani
cortex were different from those in other brain areas (reviewed in Monaghan and Anderson, 1991). Notably, cerebellar NMDA receptors label poorly with the noncompetetive channel blockers [3H]MK801 and [3H]TCP (Maragos et al., 1988; Monaghan and Anderson, 1991). Monaghan and coworkers recognized at least 4 pharmacologically distinct NMDA receptor types throughout the brain and recently demonstrated that their pharmacological heterogeneity reflects differences in subunit composition (see below; Buller et al., 1994). They identified two populations of NMDA receptors in the cerebellar cortex. One population of 'antagonist-prefering' sites, that can be labelled by [3H]CPP, is present throughout the brain, and represents NMDA receptors containing NR2A subunits. The second population consists of the 'cerebellar-like' sites, where competitive antagonists and the agonist homoquinolinate are relatively ineffective in displacing the NMDA-sensitive [3H]glutamate binding. They reflect the presence of NR2C subunit, that is uniquely expressed by cerebellar granule cells (Buller et al., 1994). The low level of [3H]MK801 and [3H]TCP binding in the cerebellum remains to be explained. Distribution of subunits
Like other classes of ionotropic receptors functional glutamate receptor channel complexes are multimeric proteins. Recent molecular cloning studies have revealed families of AMPA (GluR1-GluR4, also named GluRA-GluRD), kainate (GluR5-GluR7, and KA1 and KA2), NMDA (NR1, named ~'1 in mice, and NR2A-NR2D, named el-e4 in mice) and orphan (~1 and ~2) glutamate receptor subunits (reviewed in Nakanishi, 1992; Sommer and Seeburg, 1992; Hollman and Heinemann, 1994). The diversity of glutamate receptor subunits is further increased through alternative splicing that primarily involves the AMPA receptor subunits GluR1-GluR4, each of which exists in two versions, i.e. flip or flop, and the NR 1 subunit, that has eight splice variants. Combinatorial expression studies have demonstrated that the subunits aggregate into functional receptor channels in the homomeric as well as the heteromeric configuration. Thus multiple functionally distinct forms of each receptor type can be formed through different combinations of subunits (see below). In situ hybridisation (KeinS.nen et al., 1990; Monyer et al., 1991, 1994; Araki et al., 1993; Sato et al., 1993; Wisden and Seeburg, 1993; Akazawa et al., 1994; Laurie and Seeburg, 1994; Watanabe et al., 1994; and others) and immunocytochemical studies with antibodies for specific subunits (Martin et al., 1992, 1993; Petralia and Wenthold, 1992; Brose et al., 1993; Baude et al., 1994; Nusser et al., 1994; Petralia et al., 1994a,b,c; Jaarsma et al., 1995b) have shown that subunits are heterogeneously distributed throughout the cerebellum, each cell type expressing a characteristic set of subunits (see Table 2). AMPA subunits
AMPA receptor subunits are not only expressed by cerebellar neurons, but also by Bergmann glia, that express high levels of GluR 1 (GluRA) and GluR4 (GluRD) mRNA (Table 2, Fig. 46). GluR1 subunit mRNA is also expressed by Purkinje cells but not by other cerebellar cells (Kein~inen et al., 1990; Monyer et al., 1991; Sato et al., 1993). GluR4 mRNA in addition to Bergmann glial cells, is produced by granule cells and neurons of the deep nuclei. Granule cells express a GluR4 splice variant exclusively found in the cerebellum, GluR4c, consisting of GluR4 with the flop module and a truncated C-terminus (Gallo et al., 1992). GluR2 (GluRB) mRNA is found over the granular and molecular layers, in Purkinje cells, and in cells of the deep nuclei (Fig. 46). 62
The cerebellum." chemoarchitecture and anatomy
Ch. I
GluR3 (GluRC) mRNA is not expressed in granule cells, but occurs in Golgi cells, stellate and basket cells, Purkinje cells and cells in the deep nuclei (Fig. 46) (KeinS.nen et al., 1990; Monyer et al., 1991; Sato et al., 1993). The distribution of AMPA subunits has been studied immunocytochemically with antibodies specific for GluR1, GluR2/3, and GluR4 (Martin et al., 1992; Petralia and Wenthold, 1992; Baude et al., 1994; Nusser et al., 1994; reviewed in Jaarsma et al., 1995b). The processes of the Bergmann glia are densely immunostained for GluR1 and GluR4 (Fig. 47) confirming in situ hybridisation. Electron microscopy showed that GluR1 immunoreactivity was localized throughout the cytoplasma membrane (Baude et al., 1994). Dense immunostaining was associated with the processes of Bergmann fibers ensheathing PC spines and the attached synaptic varicosities of parallel fibers and climbing fibers (Fig. 48). This indicates that AMPA receptors on Bergmann glia may be activated by glutamate released by parallel fibers or climbing fibers, allthough there is no clue as yet of the functional role of glial cell activation (see discussion Baude et al., 1994, but also Mfiller et al., 1992). Purkinje cells are weakly-to-moderately immunopositive for GluR1. Dense GluR1 immunolabelling was found at the post-synaptic membrane specialisations of the dendritic spines of Purkinje cells, facing parallel and climbing fiber boutons (Fig. 48). The post-synaptic membranes of the parallel fiber-Purkinje cell and the climbing fiberPurkinje cell synapses are also stongly immunoreactive for GluR2/3 (Nusser et al., 1994; Jaarsma et al., 1995b). The GluR2/3 antibodies immunoreact with all cerebellar neurons. The perikarya and dendritic arbors of Purkinje cells densely immunostain, whereas
TABLE Type
AMPA
Kainate
NMDA
orphan
2.
Distribution of glutamate receptor subunit mRNAs in rat cerebellum Subunit
Cell t y p e PC
GrC
GoC
GluR1
+ flip
-
-
-
+ + flip
-
GluR2
+ + flip/flop
+ flip
+
+
-
++
GluR3
+ flip
-
++
++
-
+
GluR4
-
+ 4c-flop
-
-
+ + flip
+
GluR5 GluR6
+ -
. ++
GluR7
-
-
-
+
KA1
+
.
.
BC/Stc
. .
Bg
.
.
.
.
-
.
+
.
.
DCN
.
.
KA2
-
++
-
-
-
+
NR1
+(NRI-b)
++(NRI-a)
+
+
-
++
NR2A
-
+
-
-
-
+
NR2B
.
NR2C
-
++
.
NR2D
-
-
+
-
+
delta 1
+
.
delta2
.
.
.
.
.
.
.
.
.
+
. .
.
. .
. .
. .
S y m b o l s : - , n o t d e t e c t e d ; +, p o s i t i v e ; + + , s t r o n g l y p o s i t i v e ; P C , P u r k i n j e cells; G r C , g r a n u l e cells; G o C , G o l g i cells; B C , b a s k e t cells; St, s t e l l a t e cells; Bg, B e r g m a n n
glia; D C N ,
deep cerebellar nuclei.
B a s e d o n d a t a f r o m K e i n ~ n e n et al., 1990; M o n y e r et al., 1991, 1994; L a m b o l e z et al., 1992; A r a k i et al., 1993; L o m e l i et al., 1992, 1993; S a t o et al., 1993; W i s d e n a n d S e e b u r g , 1993; A k a z a w a S e e b u r g , 1994; W a t a n a b e
et al., 1994; L a u r i e a n d
et al., 1994.
63
.,
9
0
Ch. I
64
.
._0_
..
~:~.',,.
0
".~.
..
\;
9
9
..
...
i
t~
"~ ~
~~
.,.,~
c~
.~~
,,-q
"~' "E'
0
,,.~
c~
~.~.~
"~ o .~ ~c~
~.~
o m
0
o
J. Voogd, D. Jaarsma and E. Marani
.........
9
~4
p
~/ 9 .
The cerebellum." chemoarchitecture and anatomy
Ch. I
5',.2 uletl
Fig. 47. Sagittal sections of the rat cerebellar cortex immuno-labelled with antibodies to GluR1 (a), GluR2/3 (b,e), and GluR4 (c,d). As, astrocyte-like cells; BG, Bergmann glial processes; Go, Golgi cell; Gr, granular layer; L, Lugaro cell; Mo, molecular layer; Pj, Purkinje cell body; WM, white matter; small arrow, Purkinje cell dendrite; asterisks, Bergmann glial cell body; arrow head, basket/stellate cell. Petralia and Wenthold (1992).
light-to-moderate staining neurons occur in basket/stellate cells, Golgi cells and granule cells (Fig. 47) (Martin et al., 1992, 1993; Petralia and Wenthold, 1992; Jaarsma et al., 1995b). Unipolar brush cells are also strongly GluR2/3-immunopositive (Jaarsma et al., 1995b). Dense and moderate GluR2/3-staining was found in the perikarya and neuropil of the deep nuclei, respectively. Using electronmicroscopic immunogold protocols, that allow precise ultrastructural localization of the immunoreaction product, Nusser et al. (1994) obtained stong proof that GluR2/3 immunoreactivity is associated with postsynaptic membrane specialisations of excitatory synapses in the cerebellar cortex (Fig. 49B, C, F). Their data indicate that GluR2/3 immunoreactivity is considerably stronger at parallel fiber-Purkinje cell, climbing fiber-Purkinje cell and parallel fiber-stellate cell synapses than at mossy fiber-granule cell synapses (compare Figs 49B and C with F). Conventional peroxidase-DAB (3,3'-diaminobenzidine tetrahydrochloride)-immuno65
Ch. I
J. Voogd, D. Jaarsma and E. Marani
electron microscopy also indicates that GluR2/3 immunoreactivity is relatively weak at the mossy fiber-granule synapses (Jaarsma et al., 1995b). The post-synaptic membranes of the giant mossy fiber-unipolar brush cell synapses are, however, strongly GluR2/3 immunopositive (Jaarsma et al., 1995b). The GluR4 antibodies, in addition to the Bergmann glia, moderately immunostain the granular layer, and the neuropil and perikarya in the the deep nuclei (Fig. 47). It was originally assumed that GluR4-immunostaining in the granular layer was associated with granule cells (Martin et al., 1992, 1993; Petralia and Wenthold, 1992), but electron microscopy showed that GluR4 immunoreactivity is localized in the astroglia (Jaarsma et al., 1995b). Thus granular layer astroglia like Bergmann glia express AMPA receptor subunits, but unlike the Bergmann glia, do not have GluR1. The absence of GluR4-immunoreactivity in granule cells can be explained by the fact that granule cells primarily express an atypical form of GluR4, GluR4c (see above), that is not recognized by the GluR4 antibodies currently available. AMPA receptors are believed to mediate most of the fast excitatory neurotransmission in the brain, including the cerebellum. Concordantly the types of AMPA receptor subunits expressed by a cell largely determine the characteristics of the fast excitatory responses (see Jonas and Spruston, 1994). The GluR2 subunit dominate the AMPA receptor channel behavior, in that homomeric GluR2 channels as well as heteromeric
Fig. 48. Electron micrograph of the synaptic distribution of immunoreactivity for the GluR 1 subunit of the AMPA receptor in rat cerebellum as detected by an antibody against the carboxy-terminal (intracellular) region of GluR1. A. A spine (s) emerging from a Purkinje cell dendrite (Pd) establishes an immunopositive type 1 synapse (solid arrows) with a parallel fiber terminal (pft). Intra-cellular immunoreactivity is present inside Bergmann glial cell processes along dendritic elements (e.g., open arrow). B. The peroxidase reaction end-product labels the postsynaptic density (psd) at the intracellular face of the postsynaptic membrane (pom) and not the synaptic cleft between the presyaptic (pem) and postsynaptic (pom) membranes. Scale bars in A = 0.5 r in B = 0.1 r Baude et al. (1994).
66
The cerebellum." chemoarchitecture and anatomy
Ch. I
channels formed with the participation of GluR2 show the properties of 'typical' AMPA receptors, i.e. linear current-voltage relations and a relative impermeability to Ca 2+. Homo- or heteromeric channels without GluR2, instead, display inward rectification and are permeable to Ca 2+ and other divalent cations (for references see Sommer and Seeburg, 1992; and Hollman and Heinemann, 1994). These channels usually are not found in neuronal cells, concordant with the notion that most neuronal cells express GluR2, but are present in Bergmann glial cells (Mfiller et al., 1992; but see also Burnashev et al., 1992). In accordance with the presence of high levels of GluR 1 and GluR4 but absence of GluR2 in these cells. By combining two powerful methods, i.e. the patch-clamp technique to characterize the properties of native receptor channels in single cells in brain slices, followed by single cell PCR-amplification methods to analyse the mRNA contents of the respective cells semiquantitatively, Jonas et al. (1994) recently showed that the CaZ+-permeability of native AMPA receptor channels in cerebral cortical cells is related to the relative abundance of GluR2 subunit mRNA in the respective cells. Thus inhibitory interneurons of the cerebral cortex have low GluR2/non-GluR2 ratios (-- 0.3) and highly Ca 2+permeable AMPA receptors (which, however, display linear current-voltage relations unlike 'Bergmann-glial' AMPA receptors), whereas pyramidal cells, which have a relatively high GluR2/non-GluR2 mRNA ratio (-- 3), contain CaZ+-impermeable AMPA receptors. PCR-amplification analysis of AMPA subunit m R N A of Purkinje cells, indicates that GluR2 mRNAs are more abundant than GluR1 and GluR3 mRNAs (Lambolez et al., 1992), implying that Purkinje cells express weakly CaZ+-permeable AMPA receptors. Also in (pooled) granule cells GluR2 mRNA is more abundant than non-GluR2 (GluR4) mRNA. PCR-amplification analysis has not yet been done for other cerebellar cells. One might speculate that the basket, stellate and Golgi cells express CaZ+-permeable AMPA receptor like the inhibitory interneurons of the cerebral cortex, since according to in situ hybridisation data they seem to express relative high levels of GIuR3 compared to GluR2 (see Table 2). AMPA receptors made from different subunits may have different desensitization kinetics. Desensitization is particularly fast for AMPA receptors formed with GluR3flop or GluR4-flop (Mosbacher et al., 1994). Granule cells produce GluR4-flop and GluR2 (Table 2), and therefore are likely to have fast (submillisecond) desensitizing AMPA receptors. This could explain the very fast decay kinetics of non-NMDA component of the excitatory post-synaptic currents (EPSCs) at the mossy fiber-granule cell synapses (Silver et al., 1992; Rossi et al., 1995). If this holds true this would imply that the length of the excitatory responses at the mossy fiber-granule cell synapses is largely controlled by the desensitization properties of the AMPA receptors and does not depend upon the time course of transmitter removal, that may be relatively slow at these synapses (Jonas and Spruston, 1994) (see Section 3.2.1.). Purkinje cells express GluRl-flip, GluR2-flip and -flop, and GluR3-flip mRNA (Lambolez et al., 1992) that form AMPA receptors with desensitization time constants 3-5 times slower than 'GluR4-flop-GluR2' channels (Mosbacher et al., 1994). Concordantly, AMPA receptors in Purkinje cells appear to have relatively slow desensitization kinetics (Barbour et al., 1994). Also the decay phases of AMPA receptor-mediated EPSCs in Purkinje cells after parallel fiber or climbing fiber activation, have slow time constants (Perkel et al., 1990; Llano et al., 1991; Barbour et al., 1994). Interestingly stellate/basket cells, that express AMPA receptors with the same desensitization kinetics as Purkinje cell AMPA receptors, showed much faster decaying parallel fibers EPSCs (Barbour et al., 1994). Barbour et al. (1994) concluded that glutamate is rapidly cleared 67
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J. Voogd, D. Jaarsma and E. Marani
at the parallel fiber-stellate/basket cell synapses, resulting in rapid deactivation of postsynaptic AMPA receptors, whereas synaptically released glutamate seems to be present during a prolonged time at Purkinje cell synapses. According to H/~usser (1994) EPSCs of climbing fiber-Purkinje cell synapses have decay time constants that are slower than parallel fiber-evoked EPSCs in Purkinje cells, which may be explained by the fact that clearance of glutamate at climbing fiber synapses is slower due to their larger size.
68
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 49. Electron micrographs showing the subsynaptic segregation of GluR2/3 AMPA receptor subunits (GluR B/C/4c) and the metabotropic mGluRl~ glutamate receptor (mGluR1; see Section 3.3.2) as revealed by post-embedding immunogold labelling. A and B. Consecutive sections of the same synaptic junctions showing that immunoparticles for mGluRl~ (double arrows in A) are concentrated at the edge, whereas immunoparticles for GluR2/3 (arrows in B) are concentrated in the main body of synaptic junctions established by parallel (pft) and climbing (cft) fiber terminals with spines (s) of Purkinje cell dendrites (Pd). Note that mGluRlcz is often localized extrasynaptically (double arrow heads in A). C. Immunoreactivity for GluR2/3 (arrows) was always very strong on basket and stellate (Stc) cells. D and E. Double immunolabelling of mGluRl~ (large particles, double arrows) and GluR2/3 (small particles, arrows) immunoreactivity in the synapses on spines (s) of Purkinje cells, confirming synaptic segregated subsynaptic localization of mGluR1 and GluR2/3. The synapse in E is from Triton treated material. F. Generally a lower density of immunoparticles for GluR2/3 (arrows) has been found in synapses between mossy fiber terminals (mt) and granule cell dendrites (d) than in the parallel fiber synapses (compare to B and C). Scale bars = 0.1 r in A,B,D,E, 0.2 r in C,F. Nusser et al. (1994). (
Kainate subunits
Kainate receptor subunits are most prominent in granule cells, which express high amounts of both GluR6 and KA2 mRNA (Fig. 50) (Wisden and Seeburg, 1993). Purkinje cells express moderate levels of GluR5 and low levels of KA1 mRNAs; basket and stellate cells express GluR7 mRNA, and neurons of the deep cerebellar nuclei produce GluR7 and KA2 mRNAs (Table 2, Fig. 50) (Wisden and Seeburg, 1993). The high level of kainate receptor mRNA expression by granule cells is in accordance with the preferential binding of [3H]kainate over the granular layer (see above, but also Bahn et al., 1994). Immunocytochemical studies with antibodies specific for GluR6 and GluR7 show that dense GluR6/7-immunostaining occurred over the granule cell layer, where it was associated with granule cell perikarya and dendrites (Petralia et al., 1994c; Jaarsma et al., 1995b). The post-synaptic membranes of the mossy fiber-granule cell synapses were strongly immunoreactive for GluR6/7 (Jaarsma et al., 1995b). Stellate and basket cells and cells in the deep cerebellar nuclei were also immunoreactive for GluR6/7. KA2 immunoreactivity was relatively low in the cerebellar cortex and was concentrated over the glomeruli and neurons in the deep nuclei (Petralia et al., 1994c; Jaarsma et al., 1995b). Recombinant expression studies have shown that GluR5 and GluR6 form glutamategated channels in the homomeric configuration as well as in the heteromeric configuration with KA1 and KA2, whereas KA1 and KA2 do not assemble into functional receptor channel complexes (reviewed in Wisden and Seeburg, 1993). Thus functional kainate receptors can be formed in several cerebellar cells, in particular in granule cells expressing significant levels of GluR6 and KA2. Unequivocal physiological evidence for the presence of kainate receptors in the cerebellum (as well as in other brain areas) is, however, still lacking (see discussion Wisden and Seeburg, 1993). It should be noted that kainate in spite of its low affinity for AMPA receptors, potently activates AMPA receptors, and that excitatory responses evoked by kainate in brain tissue are generally mediated through AMPA receptors. Recombinant kainate receptors have been demonstated to desensitize very rapidly, which in part may explain why kainate receptor responses have not been detected (Wisden and Seeburg, 1993). Another possibility is that kainate receptor responses are masked by AMPA receptors, which are assumed to be present in much higher concentrations in neurons (Wenthold et al., 1994 and references therein). Autoradiographic (see above) and immunocytochemical studies (Jaarsma et al., 1995b), however, suggest that kainate receptors predominate over AMPA recep69
Ch. I
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Fig. 50. Distribution of GluR5 (A), GluR6 (B), GluR7 (C), KA-1 (D), KA-2 (E) of subunits in RNAs of high-affinity kainate receptor mRNAs in coronal sections at level of the cerebellum of the rat. Gr, granular layer; LC, locus coeruleus; Mol, molecular layer; P, Purkinje cell layer; Po, pontine nuclei. Scale bars: 2.3 mm. Wisden and Seeburg (1993).
tors in granule cells, and may significantly contribute to excitatory neurotransmission at the mossy fiber-granule cell synapses. N M D A subunits Functional NMDA receptors are believed to be generated as heteromeric assemblies of NR1 subunits with members of the NR2 subunit family. The pharmacological and kinetic heterogeneity of NMDA receptors seems to be primarily dependent upon the type of NR2 subunit (Monyer et al., 1992; Meguro et al., 1992; Nakanishi, 1992; Buller et al., 1994), although NR1 diversity generated through alternative splicing may also contribute to NMDA receptor heterogeneity (Buller et al., 1994; Hollman and Heineman, 1994). Essentially all cerebellar neurons seem to express significant levels of NR1 mRNA (Table 2, Fig. 51E) (Moriyoshi et al., 1991). The main splice variants produced in the cerebellum are NR 1-2 (with 3'-end deletion 1) and to a lesser extent NR1-4 (with 3'-end deletions 1 and 2; Laurie and Seeburg, 1994). There is a remarkable difference between the Purkinje cells and the other cells of the cerebellar cortex, in that Purkinje cells express high levels of the NRI-a forms (without 5'-insertion), whereas in the other cells the N 1-b splice variants (with 5'-insertion) predominate (Laurie and Seeburg, 1994). NR2 subunit mRNAs are heterogeneously expressed throughout cerebellar neurons (Table 2, Fig. 51) and show pronounced changes during development (Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1994). NR2 subunit mRNAs are most prominent in granule cells, that express high levels of NR2C mRNA and moderate levels of NR2A mRNAs in adult rodent cerebellum (Fig. 51). Interestingly, whereas NR2A mRNA expression in rodent granule cells begins early postnatally, NR2C first appears in later stages (postnatal day 10-11 in rat) in post-migratory cells of the internal granular layer. It apparently replaces NR2B, which is transiently expressed by cerebellar granule cells (Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1994). The expression of NR2C starts in granule cells of the caudal vermis (lobules VIII-X) and subsequently extends throughout the whole cerebellar cortex by postnatal day 13 (see Fig. 3N and O in Watanabe et al., 1994 and Fig. 7 in Akazawa et al., 1994). This pattern is compat-
70
The cerebellum." chemoarchitecture and anatomy
Ch. I
ible with the sequence of maturation of the granule cells (Altman, 1972). According to Akazawa et al. (1994) and Watanabe et al. (1994), but not Monyer et al. (1994), NR2C mRNA is also expressed in the external granular layer during the first postnatal days. NMDA receptors have been demonstrated to be critically involved in granule cell migration (Komuro and Rakic, 1993; Rossi et al., 1993). Since NR2B is transiently expressed by granule cells during the period of migration, one may speculate that receptors made with NR2B and NR1 (and possibly NR2A) may act as 'migration receptors'. The presence of multiple NMDA receptors in granule cells is consistent with the presence of multiple NR2 subunits and has recently been demonstrated with patchclamp methods (Farrant et al., 1994): Pre-migratory and migratory granule cells were shown to express NMDA receptor channels with conductancy properties of recombinant NMDA receptors formed by co-expression of NR 1 and NR2A or NR2B. Mature post-migratory cells, in addition, express 'low-conductance' NMDA receptor channels, which have the properties of NMDA receptors with NR2C (Monyer et al., 1994). Basket, stellate cells, Golgi cells and neurons in the cerebellar nuclei express NR2D mRNAs (Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1994). Neurons of the cerebellar nuclei also produce NR2A mRNA, but it is not clear whether NR2A and NR2D producing cells reflect distinct neuronal populations. It should be noted that NMDA receptors composed of NR1 and NR2D have very slow deactivation kinetics (roll = 4.8 s) (Monyer et al., 1994) and, therefore, may modulate the cell activity during many seconds even when the receptor channel has been briefly activated by glutamate (see discussion Monyer et al., 1994). Quinlan and Davies (1985) have provided indirect physiological evidence for the presence of NMDA receptors in stellate and basket cells, by showing that NMDA may induce inhibition of Purkinje cells. Also neurons of the deep cerebellar nuclei have been shown to display prominent NMDA responses in cerebellar slice cultures (Audinat et al., 1990).
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Fig. 51. Bright-field micrographs showing cellular distributions of the NMDA receptor channel subunit mRNAs in the cerebellar cortex of the adult mouse: (A) el (mouse homologue of NR2A) mRNA; (B) e2 (NR2B); (C) e3 (NR2C); (D) e4 (NR2D); and (E) ~'1 (NR1). Each photograph in the figure was taken from lobule V of the cerebellar vermis, and the expression patterns of the respective subunit mRNAs are identical to those in remaining regions of the cerebellum. Sections were counter-stained with toluidine blue. Arrows indicate cell bodies of the Purkinje cells. Gr, granular layer; Mol, molecular layer. Scale bar = 50 r Watanabe et al. (1994).
71
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J. Voogd, D. Jaarsma and E. Marani
The presence of NMDA receptors on Purkinje cells has been disputed. Some studies have supported the presence of NMDA-receptors on Purkinje cells (Sekiguchi et al., 1987), but in most studies no evidence of NMDA-receptors on Purkinje cells has been found (e.g. Audinat et al., 1990; Perkel et al., 1990; Llano et al., 1991; Farrant and Cull-Candy, 1991). Studies of Krupa and Cr6pel (1990) and Rosenmund et al. (1992) have indicated that NMDA receptors are present on most Purkinje cells during early post-natal life, but disappear with age. Both in situ hybridisation in rat and mouse and immunocytochemical studies in rat have shown that the NR1 subunit is expressed at high levels by Purkinje cells (Brose et al., 1993; Akazawa et al., 1994; Monyer et al., 1994; Petralia et al., 1994a; Watanabe et al., 1994). Petralia et al. (1994a) further demonstrated that NRl-immunoreactivity occur at the post-synaptic membrane specialisations in Purkinje cell spines. With respect to the NR2 subunits, Akazawa et al. (1994) found that rat Purkinje cells may express NR2D mRNA until post-natal day 8 and thereafter express low levels of NR2A mRNA. Accordingly Petralia et al. (1994b) observed that Purkinje cells display a low level of NR2A/B immunoreactivity, also in the post-synaptic densities of Purkinje cell dendritic spines, indicating that low levels of 'NR1-NR2A' receptors may be present at parallel fiber or climbing fiber synapses. Watanabe et al. (1994), however, found that mouse Purkinje cells only express low levels of NR2B (indicated as e2, which is the mouse homolog of NR2B) until one day postnatally, but not at any later stage, whereas according to Monyer et al. (1994) Purkinje cells do not produce any NR2 mRNA at any age. One may conclude from the in situ hybridisation and immunocytochemical data, that in spite of the presence of high levels of NR1 subunit, Purkinje cells both during development and in adulthood are likely to express none or only low amounts of functional NMDA receptors, which is in line with the aut0radiographic data. NR1 subunits can also form receptor-channel complexes in the homomeric configuration, but these channels produce very small currents and are, therefore, unlikely to contribute significantly to the excitatory actions of glutamate in Purkinje cells (Moriyoshi et al., 1991). Orphan receptors
51 and 52 are two related subunits isolated by homology screening. 51 is not produced in the rodent cerebellum, but 52 is selectively expressed by Purkinje cells (Araki et al., 1993; Lomeli et al., 1993). The subunit protein is distributed throughout the somatodendritic domain of the Purkinje Cells, similar to other glutamate receptor subunits (Araki et al., 1993). The function of S1 and 52 is not yet understood. The subunit protein does not bind glutamate receptor agonists and does not aggregate into functional receptors (Lomeli et al., 1993).
3.3.2. Metabotropic glutamate receptors Metabotropic glutamate receptors are coupled to G-proteins and modulate intracellular second messenger systems. The metabotropic glutamate receptors consist of at least seven subtypes that can be subdivided into three subgroups on the basis of sequence homology, agonist selectivity, and second messenger system (Nakanishi, 1992; Tanabe et al., 1992): (1) mGluR1 and mGluR5, that are coupled primarily to activation of phosphoinositide hydrolysis and are activated by quisqualate (QA) and 1S,3R-aminocyclopentane dicarboxylate (1S,3R-ACPD); (2) mGluR2 and mGluR3, that are coupled to inhibition of the cAMP cascade, are sensitive to pertussis toxin, and are activated by 72
The cerebellum." chemoarchitecture and anatomy
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1S,3R-ACPD, but are insensitive to QA; and (3) mGluR4, mGluR6 and mGluR7, which are also coupled to inhibition of the cAMP cascade, and are potently activated by L-2-amino-4-phosphonobutyrate (L-AP4), but are insensitive to QA and 1S,3R-ACPD. Metabotropic glutamate receptors have been implicated in multiple neuronal processes including modulation of transmitter release, plasticity phenomena such as long term potentiation and long term depression, and other long term changes of neuronal functions (see Schoepp, 1994 for a review). With the exception of mGluR6 that is expressed only in retina, all metabotropic receptors are expressed in the cerebellum. mGluR1 mRNA is expressed to some extent by most cerebellar neurons (Shigemoto et al., 1992), but is found at very high levels in Purkinje cells. Immunocytochemistry shows that the mGluR1 protein is localized in the spines of Purkinje cell dendrites (Fig. 52) (Martin et al., 1992; Baude et al., 1993; Shigemoto et al., 1994). Dense mGluR1 immunostaining is also associated with the brushes of unipolar brush cells (Jaarsma, Mugnaini, Shigemoto et al., in preparation). Interestingly, mGluR1 immunostaining is not associated with the post-synaptic region of the giant mossy fiber-unipolar brush cell synapses, but instead, occurs at very high levels in spiny appendages and small branchlets that emanate from the dendritic stem that do not have synaptic specialisations (see Mugnaini et al., 1994). Recently workers from Somogyi's group (Baude et al., 1993; Nusser et al., 1994) demonstrated, with immunogold techniques, that mGluR1 immunoreactivity in Purkinje cell spines (as well as in other neurons) was never localized to the postsynaptic membrane specialisations of the synapses, but was associated with perisynaptic and extrasynaptic regions. This is in marked contrast with ionotropic glutamate receptor subunits that are primarily located at the postsynaptic membrane (Fig. 49) (Nusser et al., 1994). It has been proposed that, as a consequence of its peri-and extrasynaptic localization, mGluR1 is only activated during high frequency stimuli, because low frequency stimuli may not release enough glutamate to reach the perisynaptic receptors at significant concentrations (Baude et al., 1993; Nusser et al., 1994). It was originally reported by Kano and Kato (1987) that a QA/transAPCD-sensitive glutamate receptor is critically involved in the induction of long term depression (LTD) of parallel-fiber-Purkinje cell synapses, a cerebellar paradigm of synaptic plasticity that is induced following repetitive stimulation of parallel fibers in conjunction with climbing fiber input (Ito, 1989; Linden and Connor, 1993). Recently strong evidence has been obtained that mGluR1 plays a major role in cerebellar LTD: (1) the induction of LTD could be inhibited with antiserum that inactivated mGluR1 in an in vitro model of LTD (Shigemoto et al., 1994); and (2) LTD could not be induced in a mutant mouse lacking mGluR1 (Aiba et al., 1994). In these animals the anatomy of the cerebellum was not overtly disturbed. The Purkinje cells showed some minor morphological alterations, but had normal excitatory responses upon parallel fiber and climbing activation. Interestingly, the animals showed characteristic cerebellar symptoms such as ataxic gait and intention tremor, which suggest that mGluR1, possibly through its role in LTD, is important in cerebellar function. The mGluR5 receptor is selectively localized to a subpopulation of Golgi cells with the receptor protein localized throughout the somato-dendritic domain of the cells (Abe et al., 1992b; Shigemoto et al., 1993). Also mGluR2 and mGluR3 mRNA's are selectively expressed by Golgi cells (Ohishi et al., 1993, 1994), although mGluR3 may also occur in glial cells (Ohishi et al., 1994; Tanabe et al., 1993). Using an antibody selective for mGluR2 and mGluR3, Ohishi et al. (1994) found that mGluR2/3 immunoreactivity was strongest in Golgi axon terminals in the glomeruli (Figs 53 and 54). The Golgi axon terminals are not in close contact with mossy fibers, but the distance between mossy fiber 73
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 52. A. Photomicrograph of semithin 3/lm thick plastic section of the nodulus of rat cerebellar cortex immunostained with an antibody against the carboxyterminus of the metabotropic glutamate receptor, mGluRl~z (antibody A52) (Shigemoto et al. 1994). Immunoreaction product in the molecular layer (ml) has a punctate distribution. Very little staining occur in the perikarya and primary dendrites of Purkinje cells (PC). In the granular layer moderate and dense immunoreactivity is localized to the perikarya and 'brushes' (open arrows) of unipolar brush cells (asterisks in cell nucleus), respectively. Bar = 20/lm. B. Electron micrograph of the molecular layer showing that puncta within the cerebellar molecular layer correspond to mGluRlctimmunoreactive spines (arrows) of Purkinje cells. Curved arrow point to an immunoreactive spine branching from an unlabelled Purkinje cell dendrite (PCd). pf, parallel fiber terminal. Bar = 0.5/lm. Courtesy of Jaarsma, Dino, Mugnaini, Ohishi and Shigemoto.
terminals and Golgi axon terminals is usually less than 1 ~tm, and it is possible that glutamate released from mossy fibers may diffuse into the intercellular space to activate mGluR2/3 on the Golgi cell axons (see Section 3.2.1.). mGluR2/3 in Golgi axon terminals may be involved in the regulation of inhibitory neurotransmitter release, which would imply that mossy fibers may directly influence inhibitory neurotransmission on granule cell dendrites (e.g. see discussion Ohishi et al., 1994). Both mGluR5 and mGluR2/3 antibodies immunostain subpopulations of Golgi cells (Shigemoto et al., 1993; Ohishi et al., 1994). mGluR2/3 immunoreactive Golgi cells constitute three-quarters of the total population of Golgi cells (defined as GABApositive, parvalbumin-negative cells of the granular layer), whereas only a small population of Golgi cells appears mGluR5 positive. Large mGluR2/3-positive Golgi cells were frequently encountered in the Purkinje cell layer and the superficial part of the granular layer (Fig. 53), and at least in part may represent the candelabrum cells as described by Lain6 and Axelrad (1994, see section 2). In contrast, large mGluR5-positive immunoreactive Golgi cells were mostly found deeper in the granular layer. This indicates that mGluR2/3 and mGluR5 positive Golgi cells represent different subpopulations of Golgi cells. It remains to be determined whether mGluR2/3 and mGluR5 positive cells are entirely exclusive or overlapping populations, and whether yet another 74
The cerebellum." chemoarchitecture and anatomy
Ch. I
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Fig. 53. a. Immunocytochemicallocalization of mGluR2/3 in a parasagittal section through the vermis of rat cerebellum. The most intense immunoreactivity is seen in the granular layer. Staining in the molecular layer is associated with Golgi cell dendrites. B. Drawing of the section shown in (a) showing presumed Golgi cell bodies with mGluR2/3 immunoreactivity (closed circles) and those without mGlu2/3 immunoreactivity (open circles). G, granular layer; M, molecular layer; P, Purkinje cell layer; W, white matter. Bar = 500 #m. Ohishi et al. (1994).
subpopulation exists that is both mGluR5 and mGluR2/3 negative. It is important to realize that mGluR2/3 as well as mGluR5 immunoreactivity is detected in both small and large Golgi cells, and that, therefore, the segregation of Golgi cells into mGluR2/3 respectively mGluR5-positive and negative cells does not correspond to previous classifications which were based on size (e.g see Palay and Chan-Palay, 1974; but also section 3.6.2.). The L-AP4-sensitive mGluR, m G l u R 4 is expressed at high levels by granule cells (Kristensen et al., 1993; Tanabe et al., 1993), whereas mGluR7 m R N A is produced by Purkinje cells (Okamoto et al., 1994; Saugstadt et al., 1994). Physiological data indicate that the L-AP4 sensitive mGluRs are predominantly located presynaptically, where they may act as autoreceptors to regulate glutamate release. Studies in turtle suggest the presence of a L-AP4-sensitive presynaptic glutamate receptor at the parallel fiber-Purkinje cell synapse (Larson-Prior et al., 1989). Thus, possibly, mGluR4 is located presynaptically on parallel fiber boutons. The ultrastructural localisation of m G l u R 4 and m G l u R 7 remained to be determined at the time of writing this manuscript. 75
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J. Voogd, D. Jaarsma and E. Marani
3.4. NITRIC OXIDE: THE CEREBELLAR LOCALIZATION OF NITRIC OXIDE SYNTHASE, GUANYLATE CYCLASE AND CYCLIC GMP Nitric oxide (NO) (see Dawson et al., 1992 and Vincent and Hope, 1992 for reviews) has gained importance as an intracellular and diffusible intercellular messenger in the cerebellum, since the demonstration by Garthwaite et al. (1988, 1989) that N-methyl-Daspartate (NMDA) receptor activation caused an increase in cyclic guanosine 3',5'monophosphate (cyclic GMP) in the cerebellum by stimulating the release of a diffusible messenger with properties similar to a endothelium-derived relaxing factor which was identified as NO. They considered granule cells as the main source of NO and glial cells as the main target for the activation of soluble guanylate cyclase by NO and the production of cyclic GMP. The enzyme nitric oxide synthase (NOS), that produces NO and citrullin from arginine, occurs as several isoenzymes (Knowles et al., 1989). Type I NOS is a constitutive, calcium and calmodulin-dependent enzyme, present in neurons and, possibly, in glia. Type II NOS is calcium-independent and can be induced in macrophages and glial cells by exposure to bacterial lipopolysaccharide (Galea et al., 1992; Murphy et al., 1993). Type III NOS is the endothelial iso-enzyme. NOS-I, II and III are produced by different genes (Bredt et al., 1991; Lamas et al., 1992; Xie et al., 1992; Lowenstein et al., 1992; Lyons et al. 1992; Ogura et al., 1993). NOS displays NADPH-dependent diaphorase
Fig. 54. Ultrastructural localization of mGluR2/3 immunoreactivity in the granular layer of rat cerebellar cortex. Dense immunoreaction products accumulate in axon terminals of Golgi cells, which often make synaptic contacts (curved arrows) with possible granule cell dendrites around a mossy fiber terminal (MT) in the cerebellar glomerulus. Bar = 0.5 r Ohishi et al. (1994).
76
The cerebellum." chemoarchitecture and anatomy
Ch. I
activity and can be demonstrated in aldehyde-fixed tissue by NADPH-dependent reduction of tetrazolium salts to visible formazans (Hope et al., 1991). NOS-I has been localized with antisera to the purified enzyme (Bredt et al., 1990) and by in situ hybridization to NOS-I mRNA (Bredt et al., 1991) in basket cells and in granule cells and their axons, where NOS-I is co-localized with NADPH diaphorase (Bredt et al., 1991; Vincent and Kimura, 1992; Schmidt et al., 1992; Schilling et al., 1994). NADPH-diaphorase-positive granule cells are distributed in a symmetrical pattern of heavily stained clusters, separated by granule cells that were stained weakly, or not at all (Fig. 55) (Schilling et al., 1994). The NADPH-diaphorase-positive granule cell clusters were correlated with the Zebrin pattern in the overlying molecular layer by Hawkes and Turner (1994). A sparse axonal network and a few cells were stained in the cerebellar nuclei (Vincent and Kimura, 1992). Schmidt et al. (1992) also found weak NOS-I immunoreactivity in Bergmann glia and astrocytes where it co-localized with NADPH-diaphorase. NOS-II was expressed by astrocytes in lipopolysaccharide-stimulated cultures. These cells also double-label for NADPH-diaphorase (Galea et al., 1992). Guanylate cyclase, the enzyme responsible for the synthesis of cyclic GMP from guanosine triphosphate, was localized with immunofluorescence in Purkinje, granule stellate and Golgi cells and in oligodendrocytes, astroglia and Bergmann glial fibers of the cerebellar cortex of the rat (Zwiller et al., 1981). The localization in Purkinje and granule cells and in astrocytes was confirmed by Ariano et al. (1982), Nakane et al. (1983) and Schmidt et al. (1992). Bergmann glia and small cells in the molecular and granular layers were weakly stained. Expression of the soluble guanylyl cyclase mRNA in rat cerebellum was moderate in Purkinje, basket, stellate and Golgi cells, weak in granule cells, but could not be demonstrated in glial cells (Matsuoka et al., 1992, see also Burgunder and Cheung, 1994). Cyclic GMP was located with immunohistochemical methods in Bergmann glia (Cumming et al., 1977, 1979; Chan-Palay and Palay, 1979; Ariano et al., 1982) and in a subpopulation of stellate and basket cells (Chan-Palay and Palay, 1979). Its preferential localization in Bergmann glia and cerebellar astrocytes was stressed by Berkelmans et al. (1989) and De Vente et al. (1989, 1990), using antibodies against conjugates of cyclic GMP and activation of cyclic GMP by sodium nitroprusside in slices of rat cerebellum. They observed a patchy distribution of the reactive Bergmann glia in the molecular layer (Fig. 56). Purkinje and granular cells remained unstained. Immunoreactive varicose (mossy?) fibers and astrocytes and/or Golgi cells were observed in the granular layer. Owing to the differential localizations of NOS, guanylate cyclase and cyclic GMP, the cellular basis for the actions of cerebellar NO remains difficult to establish. Basket and stellate cells appear to be the only cell types that can be stimulated by NMDA receptors (Quinlan and Davis, 1985; Hussain et al., 1991) that contain both NOS-I, guanylate cyclase and cyclic GMP. It has been suggested that carbon monoxide (CO) is an activator of soluble guanylyl cyclase in Purkinje cells. Heme oxygenase-2, which degrades heme to biliverdin and releases carbon monoxide in the process, was shown to be co-localized with guanyl cyclase in rat Purkinje and granule cells with in situ hybridization histochemistry (Verma et al., 1993). 3.5. ADENOSINE, 5'-NUCLEOTIDASE AND ADENOSINE DESAMINASE Adenosine-like immunoreactivity was found in rat Purkinje cells, using polyclonal anti77
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 55. Coronal section through the copula pyramidis (lobule VIII). In the adult rat granule cells in the lateral tip of the copula pyramidis show strongly reduced staining intensity for NADPH-diaphorase, in contrast to the medial copula, where a cluster of heavily stained granule cells can be seen. g, granular layer; m, molecular layer. Scale bar = 200/lm. Schilling et al. (1994).
sera against a conjugate of the adenosine derivative laevulinic acid (Braas et al., 1986). Staining was present in the cell soma outside the nucleus, extending in the dendrites. Weaker staining was observed in the granular layer. Adenosine is co-released with adenosine triphosphate (ATP) and certain neurotransmitters (Richardson and Brown, 1987). High affinity uptake sites for adenosine are present in all layers of the cerebellar cortex (Marangos et al., 1982; Nagy et al., 1985; Biss6rbe et al., 1985). Steady state concentrations of adenosine are maintained through the activities of only three enzymes, 5'-nucleotidase (5'-N), adenosine kinase and adenosine deaminase. Adenosine kinase and adenosine deaminase were located mainly in the soluble fractions of rat cerebellar homogenates, whereas 5'-N was present in subcellular fractions (Philips and Newsholme, 1979), mainly in the synaptosomal fraction (Marani, 1977). Adenosine deaminase-immunoreactivity in rat cerebellum was present with one out of five polyclonal sera prepared by Nagy et al. (1988). Staining was present in most Purkinje cells with a variation in intensity. Staining was observed in the Purkinje cell axons and terminals in the cerebellar and vestibular nuclei. The localization of 5'-N will be discussed below. Adenosine blocks the parallel fiber-induced simple spike discharge in Purkinje cells (Kostopoulos et al., 1975) but not the climbing fiber-mediated synaptic transmission (Kocsis et al., 1984). The effect of adenosine is presynaptic and is mediated by A1adenosine receptors that are located on parallel fibers. A 1-adenosine receptors are coupled to pertussis toxin-sensitive G proteins and inhibit adenyl cyclase. Activation of A 1-adenosine receptors decreases transmitter release from the terminals (Dolphin and Prestwich, 1985, see Fredholm and Dunwiddie, 1988, for a review). The presence of Al-adenosine receptors on parallel fibers was demonstrated autoradiographically by 78
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 56. cGMP-immunostaining of adult rat cerebellum. A. Section of a cerebellar slice that was incubated with cyclic GMP antiserum, in the presence of 1 mM isobutyl-methylxanthineto inhibit phosphodiesterase activity and 10 r nitroprusside and post-fixed in paraformaldehyde. B. Same areas of the same section as shown in (A) after removal of cGMP-immunostaining using the methods of Tramu et al. (1978) and reincubation of the section with glial fibrillary acidic protein-antiserum. Note presence of cGMP-immunoreactivity in Bergmann glial fibers and in thin varicose fibers (arrows in A) and in astrocytes or Golgi cells (arrow head in A). Bars = 100 r De Vente et al. (1989).
Goodman and Snyder (1982) and Goodman et al. (1983) using specific binding of [3H]cyclohexyladenosine ([3H]CHA) and Weber et al. (1990), using the antagonist [3H]DPCPX (Fig. 57). Binding was highest over the molecular layer, with lower concentrations in the granular layer. Binding was absent in the granuloprival cerebellum of 'weaver' mice (Goodman et al., 1983; Wojcik and Neff, 1982, 1983). Al-adenosine receptors were present over the entire molecular layer; no bands of high activity, corresponding to the 5'-N pattern, were observed (Fastbom et al., 1987). Adenosine is released in a Ca2+-dependent manner by K + stimulation from rat cerebellar slices (Cu6nod et al., 1989; Do et al., 1990). The stimulated release of adenosine was decreased by 60-70% in vermis and hemisphere, in slices from 3-acetylpyridine-treated rats, which may indicate that the released adenosine, at least in part, is released by climbing fibers. The 'climbing fiber-dependent' adenosine release, however, occurs with some time delay after the K + stimulus. Adenosine, therefore, has been proposed to be derived from extracellular degradation of released nucleotides by ectonucleotidases. Inhibition of 5'-nucleotidase (5'-N) by ~,fl-methylene-ADP and GMP, indeed, decreased stimulated adenosine release by 50-60%. 5'-Nucleotidase (5'-N) is an integral glycoprotein of the cellular plasma membrane in a wide range of animal cells. Its functional role is still unclear. 'Possibilities .... include recovery of purines and pyrimidines from the extracellular space, the extracellular formation of neuromodular adenosine from released nucleotidases and non-enzymatic functions related to the interaction of 5'-nucleotidase with compartments of the cytoskeleton and extracellular matrix' (Schoen et al., 1987). 5'-N catalyses the production of adenosine by the hydrolytic cleavage of 5'-nucleotide monophosphates (i.e. adenosine5'-monophosphate). The development of 5'-N in the cerebellum was studied by Schoen et al. (1987, 1988, 1990). 5'-N in the molecular layer of mouse cerebellum is distributed in positive and negative parasagittal bands (Scott, 1963). The distribution of cerebellar 5'-N has been reviewed by Marani (1986). Its zonal distribution in mice is very similar to the distribution of the m a b Q l l 3 (Zebrin)-positive dendrites of Purkinje cells in the molecular layer (Marani, 1986; Eisenman and Hawkes, 1989) (Figs 58A, 130, 131, 135) (Section 6.1.4.). The 79
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Fig. 57. Light microscopic autoradiographic localization of A l adenosine receptors using [3H]DPCPX binding
to a section of rat cerebellum. Left. Photomicrograph of a pyrorine Y-stained section of rat cerebellum showing the molecular layer (ML), the Purkinje cell perikarya (PC), the granule cell layer (GL), and some white matter ( W M ) . R i g h t . Darkfield photomicrograph of the tissue section incubated with 0.8 nM [3H]DPCPX and apposed to nuclear emulsion-coated coverslips. The silver grains are from the same area shown on the left. Note the high density of A~ adenosine receptors in the molecular layer and moderate labelling in the granule cell layer. The white matter and the Purkinje cell bodies showed background levels of labelling. Bar = 5 0 / l m . Weber et al. ( 1 9 9 0 ) .
reaction product in the histochemical procedure of Scott (1964, 1965, 1967) is uniformly distributed within the bands of high 5'-N activity in the molecular layer. In the hemisphere the staining in some of the 5'-N bands is less uniform and assumes the aspect of radially disposed striations. Hess and Hess (1986) tentatively identified these striations as the processes of the Bergmann glia. These authors found 5'-N in the molecular layer of Purkinje cell-deficient mice (pcd and nr strains) to be reduced and the residual enzyme activity to be localized in approximately the same position as the surviving Purkinje cells. This would imply that the expression of 5'-N in Bergmann glia is regulated by the adjoining Purkinje cells. Marani's electron microscopic enzyme-histochemical studies (Marani, 1981, 1982a,b, 1986), favoured a localization of the enzyme in the subsurface cisterns and the spine apparatus of the spines of the Purkinje cell dendrites and within boutons of parallel fibers (Fig. 58B-D). A non-zonal distribution of 5'-N in the somata of Purkinje cells and other large cells of the cerebellar cortex was observed when different substrates for the enzyme histochemical reaction for 5'-N were used (Scott, 1967; Marani, 1982a and b, 1986; Hess and Hess, 1986). According to Marani this represents a rest-activity of non-specific phosphatases, that disappears when the appropriate inhibitors are used. The presence of 5'-N in parallel fibers was disputed by Hess et al. (1983), who showed that 5'-N remains at significant levels in the molecular layer in agranular 'weaver' 80
The cerebellum." chemoarchitecture and anatomy
Ch. I
cerebellum. According to Kreutzberg et al. (1978) 5'-N is predominantly associated with glial membranes. Schoen et al. (1987, 1988), used monoclonal and polyclonal sera directed against rat liver 5'-N in the localization of cerebellar 5'-N in addition to the enzyme-histochemical techniques. They found the enzyme to be situated at the outer border of the plasma membranes of Bergmann glial fibers in the molecular layer, astroglial endfeet around blood vessels and glial processes surrounding Purkinje and granule cells (Fig. 59). They were unable to confirm Marani's observations of an intracellular localization of the enzyme. The study of Schoen et al. (1987) was done in rats, which do not have the longitudinal band pattern of 5'-N with their antibody directed against this enzyme. Balaban et al. (1984) observed an increase of cerebellar 5'-N in the P2 (synaptosome) fraction after climbing fiber activation with harmaline in rats (Fig. 60). Harmaline synchronizes the discharge in climbing fibers from certain parts of the inferior olive and induces a rhythmic tremor (Sj61und et al., 1977, 1980). Two different climbing fiber induced effects, therefore, may be involved in adenosine-mediated blockade of transmission in parallel fiber-Purkinje cell synapses: an increased release of nucleotides and an increase of cerebellar 5'-N. Loss of climbing fiber-induced 5'-N and/or adenosinemediated blockade of transmission in the parallel fiber-Purkinje cell synapses (see Marani, 1986) would explain the long-term increase of simple spike activity that occurs when complex spikes are suppressed by destruction or inactivation of the inferior olive in rats (Colin et al., 1980; Montarolo, 1982). Bloedel and Lou (1987), however, observed a short-term facilitation of transmission in the mossy fiber-parallel fiber-Purkinje cell pathway on stimulation of climbing fibers in the cat. This difference may be due to species-dependent differences in 5'-N mediated formation of adenosine or to a facilitation at the level of mossy fiber-granule cell synapse. If the formation of adenosine is largely dependent on the degradation of nucleotides by 5'-N, the zonal distribution of this enzyme in different species and of the climbing fibers which promote their release would be of crucial importance (see Marani (1986) and Section 6.1.4.). 3.6. INTERNEURONS OF THE CEREBELLAR CORTEX Stellate, basket and Golgi cells are inhibitory (Eccles et al., 1964a, 1966a,b,c,d, 1967). It was against this background that Uchizono (1965) (see also Uchizono, 1969 for a review) formulated and tested his hypothesis that excitatory and inhibitory axon terminals in aldehyde fixed tissue can be distinguished by the shape of their synaptic vesicles (Fig. 61). Inhibitory boutons contain flattened vesicles (F-type boutons) and excitatory boutons contain spherical vesicles (S-type boutons). Earlier Gray (1959) distinguished two types of synaptic junction, which were also supposed to represent the excitatory and inhibitory synapse (Landis and Reese, 1974). Gray type 1 junctions are characterized by a widening of the synaptic cleft that contains dense material and a distinct asymmetry caused by the presence of a dense undercoating of the postsynaptic membrane. It was considered to be excitatory. The thickening of the pre- and postsynaptic membranes in the Gray type 2 junction is symmetrical and the cleft is narrow; this type was supposed to be inhibitory. According to Uchizono (1969) there is an excellent correlation in the cerebellar cortex of the cat of S-type boutons with Gray's type 1 synaptic junctions and of F-types with a synapse of Gray's type 2. For the excitatory connections of the mossy and climbing fibers and for the parallel fiber-Purkinje cell synapse the correlation with S-type terminals and Gray 1 synaptic junctions still is valid. For the terminals of the inhibitory interneurons of the cerebellar cortex (Golgi cells: pleomorphic vesicles, synap81
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 58. Light and electron micrographs of incubations for 5-nucleotidase according to Scott (1967). A. Detail of the light microscopic location of 5'-nucleotidase in uvula (IX) and pyramis (VIII). B. Electron microscopic location of 5'-nucleotidase reaction products in the subsurface cisternae of a Purkinje cell dendrite. C. Electron microscopic localization of 5'-nucleotidase in the spine apparatus of Purkinje cell dendritic spines (asterisks). D. Localization of reaction product in a parallel fiber bouton, synapsing on a Purkinje cell dendritic spine. Bars in A = 1 mm, in B,C = 0.5 ~tm, in D = 0.25 ~tm. Marani (1977).
82
The cerebellum. chemoarchitecture and anatomy
Ch.I
Fig. 59. 5'-Nucleotidase immunohistochemical staining of rat cerebellum. A. Immunofluorescence. B. PAPmethod. Enzyme activity is predominantly found within the molecular layer on Bergmann glial fibers (long arrows). Purkinje cells are surrounded by fine rims of reaction product (small arrows). Within the granular layer 5'-nucleotidase activity is diffusely scattered between granule cells (arrow heads). Vibratome sections. C. Longitudinally sectioned Bergmann glia cell processes (B) of the molecular layer of rat cerebellum. Fine DAB reaction product is located on adjacent membranes of these processes (arrows). Bars in A,B = 50 ~tm, in C = 0.5/lm. Schoen et al. (1987).
tic junction resembles Gray type 1; basket cells: ellipsoid, irregular and spherical vesicles, Gray type 2; stellate cells: flattened vesicles, Gray type 2) there is a greater variation in morphology (Palay and Chan-Palay, 1974). Cell bodies and terminals of the Golgi, basket and stellate cells can be labelled with selective uptake of [3H]GABA (H6kfelt and Ljungdahl, 1970, 1971; Schon and Iversen, 1972), immunostaining with antibodies against GAD (Saito et al., 1974; McLaughlin et al., 1974; Oertel et al., 1981b; Mugnaini and Oertel, 1985) and in situ hybridization for G A D 6 5 and G A D 6 7 (Wuenschell et al., 1986; Julien et al., 1987; Esclapez et al., 1993; Feldblum et al., 1993). They are also immunostained with antibodies against conjugates 83
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Fig. 60. Harmaline-induced changes in 5'-nucleotidase (5'-N) activity of cerebellar fractions in rats with an intact inferior olive (vehicle injection on day 1) or a 3-acetyl-pyridine (3-AP) olivectomy. Data are represented as means + S.E. for 8 animals in each group. The changes in 5'-N levels in each fraction (CH-crude homogenate, P 1, P2, P3 and $3) are shown for intact and 3-AP olivoectomized animals that served as either controls (C) or received harmaline injections (H) 45 min prior to decapitation. Harmaline evoked an increase in 5'-N activity in the CH and P2 fraction of rats with an intact olive; it evoked a decrease in the activity after 3-AP olivectomy. No significant effects appear in the P1, P3 or $3 fractions in either intact or 3-AP olivectomized animals. Balaban et al. (1984).
of GABA (Figs. 15, 40, 62 and 63) (Ottersen and Storm-Mathisen, 1984a,b; Somogyi et al., 1985; Aoki et al., 1986; Gabbott et al., 1986; Matute and Streit, 1986; Ottersen et al., 1987). 3.6.1. Stellate and basket cells Stellate cells are located in the entire, and basket cells in the deep part of the molecular layer. The dendritic arborizations of both cell types are flattened in a plane perpendicular to the long axis of the folium. Both receive synapses from parallel fibers on their dendrites. The axon of the stellate cell terminates on shafts of dendrites from Purkinje, basket, Golgi and stellate cells in the molecular layer. The immunoreactivity of stellate cells for antibodies against conjugates of taurine (Madsen et al., 1985; Magnusson et al., 1988; Ottersen etal. 1988b) is low. This is in contrast with the selective uptake by stellate and basket cells of [3H]taurine and the immunoreactivity of these cells with antibodies against CSADS, the synthesizing enzyme of taurine (Chan-Palay et al., 1982a,b) (see Section 3.1.2.). The localization of CSADS in basket and stellate cells has, however, been disputed, since Almerghini et al. (1991) found CSADS-immunoreactivity to be exclusively localized in glial cells and not in neurons of the cerebellar cortex. Basket axons have been studied extensively (Palay and Chan-Palay, 1974). They extend in a direction across the axis of the folium (Fig. 13) and terminate with ascending 84
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Fig. 61. Size and shape analysis of synaptic vesicles in S-type and F-type synapse. Diameters of both major and minor axis in each synaptic vesicle in both types of synapses were measured. Ordinate shows the length of the major axis, while the abscissa that of the minor axis of vesicles in each type of synapse. Diameters of vesicles in S-type synapse (white) are distributed around the 45-degree line between ordinate and abscissa, while those in F-type synapse (black) of both white and black circles indicates the relative frequency of occurrence. Elongation index ratio of average length of major versus minor axis of vesicles in S-type synapse was about 1.2, while that of F-type synapse was about 1.7. Uchizono (1965).
branches on the primary dendrites of Purkinje cells and constitute the baskets surrounding the cell body that end in the pinceau around the initial part of the axon of the Purkinje cells. GABA-like immunoreactivity was present in boutons of stellate and basket cell axons on Purkinje cell dendritic shafts, in basket cell terminals on dendrites of stellate cells and on Purkinje cell somata (but not in all of them) and in some of the axons of the pinceau (Gabbott et al., 1986). No specific neurochemical properties seem to distinguish the basket cells from the stellate cells. According to Somogyi et al., (1986) GABA-like immunoreactivity is weaker in stellate cells than in basket and Golgi cells. Basket and stellate cells are immunoreactive for antibodies against parvalbumin, like the Purkinje cells (Fig. 31B). No reactivity for these antibodies or m R N A probes for parvalbumin was mentioned for the Golgi cells (Celio and Heizmann, 1981; Heizmann, 1984; Schneeberger et al., 1985; Endo et al., 1985; Braun et al., 1986; Kadowaki et al., 1993; Kosaka et al., 1993). Calretinin immunoreactivity is present in a subpopulation of stellate and basket cells in the cerebellum of the chicken, where it is co-localized with parvalbumin in some of the cells (Rogers, 1989). Immunoreactivity with antibodies against PKC ~, fl, g, e and possibly ~"is present in stellate and basket cells (Fig. 29, Table 1). The localization in basket and stellate cells of nitric oxide synthase, guanylyl cyclase and cyclic GMP has been reviewed in Section 3.4.
3.6.2. Golgi cells and Lugaro cells Golgi cells are located in the granular layer, and have been roughly subdivided into large Golgi cells, which are located in the superficial part of the granular layer and small Golgi cells located more deeply. The dendrites of both types extend into the molecular layer, where they are not confined to a single plane. The axons branch repeatedly to form a 85
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Fig. 62. Cerebellar cortex of cat reacted by the unlabelled antibody enzyme method. A and B. Serial semithin (1/lm) sections reacted under postembedding conditions with an anti-GABA serum (A) or with the same serum after solid phase absorption (B). Basket cells (BC) and Golgi cells (GC) are strongly immunoreactive, Purkinje cells (PC) and stellate cells (SC) reacted less strongly. GABA-immunoreactive terminals are present in all layers, but the terminals of basket cells around the Purkinje perikarya and in the pinceau (p) and the Golgi cell terminals in the glomeruli (gl) are especially strongly reacting. C-E. Preembedding demonstration of GABA and GAD in vibratome sections. The distribution of amino acid and its synthesizing enzyme are very similar, but perikarya of basket, stellate, and Golgi cells, and basket cell axons (ba) stain stronger for the amino acid than for G A D in animals not treated with colchicine. F and G. Electron micrographs of glomeruli demonstrating GABA (F) and GAD (G) in the terminals (asterisks) of Golgi cells. The dendritic digits of granule cells (diamonds) receive synapses (arrows) from the immunoreactive terminals as well as from the mossy fiber terminals (mft). Bar in A-E - 50/~m, in F and G = 0.5 r Somogyi et al. (1985).
86
The cerebellum." chemoarchitecture and anatomy
Ch. I
dense plexus in the granular layer. The terminals participate in the formation of the glomeruli where they make synaptic contact with the granule cell dendrites. Golgi cells in the upper molecular layer in rat and cat are selectively recognized by a monoclonal antibody (rat-303, Hockfield, 1987) (Figs. 66 and 69B). Uptake studies have shown that [3H]GABA and [3H]glycine uptake result in similar patterns of axonal labelling in the granular layer, in circular deposits resembling the periphery of the glomeruli, whereas no [3H]glycine labelling was found over the pericellular baskets of the Purkinje cells (Wilkin et al., 1981a). Concordantly it was shown that a large proportion of the Golgi cells, in addition to GABA, was also immunoreactive for antibodies against conjugates of glycine (Ottersen et al., 1987, 1988a; Campistron et al., 1986a; Takayama, 1994). In a high percentage of these glycine containing Golgi cells (40%) glycine-like and GABA-like immunoreactivity co-exist (Ottersen and StormMathisen, 1987). GABA and glycine-like immunoreactivity co-exist in most Golgi cell
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Fig. 63. Photomicrographs of semithin (0.5 r tissue and test sections incubated with GABA antiserum 26 diluted 1:100 (A) or glycine antiserum 31 diluted 1:60 (B). Three of the four Golgi neurons that are glycine immunoreactive (thick arrows) are also stained with the GABA antiserum in the adjacent section; the fourth glycine-positive neuron (crossed arrow) is virtually immunonegative for GABA. Most if not all glomeruli (arrowheads) show GABA-like-immunoreactive as well as glycine-like-immunoreactive positive Golgi cell terminals. The molecular layer contains no glycine-like immunoreactive positive structures except for a few fibrous processes (small arrows in B). The terminals of the basket and stellate cells and their respective cell bodies (double arrowhead) are glycine immunonegative, but GABA immunopositive. Asterisks, Purkinje cell bodies. Other abbreviations: MO and GC, molecular and granule cell layers, WM, white matter. Bar = 50 r Ottersen et al. (1988a).
87
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 64. Electron micrograph showing GABA-LI in a glomerulus in the granular layer of the cerebellar cortex (rat). After incubation in rabbit primary antiserum, the section was treated with sheep anti-rabbit immunoglobulins bound to colloidal gold particles (16 nm). Axon terminals of the inhibitory Golgi cell (GO) show a high density of gold particles, whereas the densities of such particles over the mossy fibre boutons (MF) and granule cell dendritic digits (GC) are close to back-ground level. Scale bar = 0.5 ~tm. Ottersen and StormMathisen (1987).
terminals located at the periphery of the glomeruli (Somogyi et al., 1986; Ottersen et al., 1987) (Fig. 63) (Ottersen et al., 1988a) (compare Figs 41 and 65). A similar localization of GABA-like and glycine-like immunoreactivity was found in the rat and the baboon (Ottersen et al., 1987) (Fig. 65). Some displaced Golgi cells were present in the molecular layer of the baboon and many fibers running in the supraganglionic plexus in the direction of the long axis of the folium displayed glycine-like immunoreactivity. In summary the majority of the Golgi cells may use glycine in addition to GABA. Golgi cells in the rat stain strongly for acetylcholinesterase (Brown and Palay, 1972; 88
The cerebellum." chemoarchitecture and anatomy
Ch. I
Altman and Das, 1970). They share this property with a group of displaced Golgi cells located in the lower molecular layer in the rabbit (Ramon y Cajal, 1911; Spa~;ek, 1973). A subpopulation of Golgi cells in the cat and man, but not in rat or rabbit, is immunoreactive for choline acetyltransferase (see Section 3.10.1., Fig. 86). Certain Golgi cells in rats were found to be immunostained with antibodies against conjugates of somatostatin (Johansson et al., 1984; Vincent et al., 1985; Villar et al., 1989) (Fig. 20) or enkephalin (Schulman et al., 1981; Ibuki et al., 1988). Calcium binding proteins have not been found in Golgi cells, with the exception of a single antibody against calbindin-D28k, that stains Purkinje cells and Golgi cells in rat and human cerebellum (Garcia-Segura et al., 1984; Fournet et al., 1986), and the presence of calretinin in some Golgi cells (Arai et al., 1991; Floris et al., 1994). Of the PKC subtypes only PKC e' has been localized in Golgi cells (Wetsel et al., 1992). None of the immunoreactive subpopulations of Golgi cells seem to correspond exclusively to one of the anatomical subtypes distinguished by Palay and Chan-Palay (1974). Similarly, Golgi cell heterogeneity due to differential expression of metabotropic glutamate receptors, does not correspond to any anatomical subdivision (see Section 3.3.2.). It remains to be elucidated to what extent the different immunocytochemical markers overlap. Lugaro cells are fusiform cells located below the Purkinje cell layer, with dendrites arising from opposite poles of the cell and somata extending for long distances beneath the Purkinje cell layer. Lugaro cells are chemically distinct from Golgi cells in that they are selectively recognized by two monoclonal antibodies (cat 301 and 304, Sahin and Hockfield, 1990) (Fig. 67). Like Golgi cells they are immunoreactive for antibodies against GABA (Aoki et al., 1986). Ottersen et al. (1988a) included the Lugaro cells with the Golgi cells and found GABA-like and glycine-like immunoreactivity to be colocalized in Lugaro cells. Lugaro cells are assumed to primarily innervate the granule cells (Palay and Chan-Palay, 1974), although according to Fox (1959) their axon may enter the molecular layer. 3.6.3. Unipolar brush cells
Unipolar brush cells often have been interpreted as small Golgi cells. They are, however, non-GABAergic and non-glycinergic (Aoki et al., 1986; Mugnaini et al., 1994). Since they give rise to mossy fiber rosette-like terminals in the granular layer they are likely to be glutamatergic (Berthie and Axelrad, 1994; Rossi et al., 1995) (Section 3.2.1.). Unipolar brush cells can be distinguished from other granular layer neurons by a number of immunocytochemical markers (reviewed by Mugnaini and Floris, 1994). Following their original characterization as pale cells by Altman and Bayer (1977), they were first recognized by Hockfield (1987) using a monoclonal antibody against spinal cord gray matter, Rat-302 (Fig. 69). A study of Harris et al. (1993) showed that Rat-302 is directed against high molecular weight neurofilament protein, and that unipolar brush cells are strongly immunostained by several different antibodies against high molecular weight neurofilament protein. Unipolar brush cells are essentially unstained and moderately stained with antibodies against middle and low molecular neurofilament protein, respectively. Cozzi et al. (1989) and Munoz (1990) identified unipolar brush cells in the rat and human cerebellum, respectively, on the basis of their immunoreactivity to antisera against proteins of the secretogranin (or chromogranin) family. Unipolar brush have a relatively high density of large dense core vesicles, which in conjunction with the 89
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Fig. 65. Details from sagittal vibratome section of baboon cerebellum (vermis) treated with the glycine antiserum diluted 1:250. A. One of the immunopositive Golgi cells shows a dendrite (small arrowheads) extending into the molecular layer, and a thinner process (axon?) which seems to engage in a glomerulus-like structure (large arrowhead). Note the presence of thick and thin immunoreactive fibers (thin arrows) in the deep part of the molecular layer, which also contains scattered immunoreactive neurons (thick arrow). B. Radial fibrous processes (small arrows) interpreted as Bergmann glia, show weak glycine-like immunoreactivity. Large arrow points at an immunostained Golgi cell. Asterisks indicates immunonegative Purkinje cell. C. Immunopositive Golgi-like neurons (arrows) occur in or just below the layer of the unstained Purkinje cells (asterisks). Large arrowhead indicates a stained Golgi cell slightly outside the plane of focus. Inset: Arrowhead, fusiform cell with horizontal dendrites situated directly beneath the Purkinje cells (asterisk). G, granular layer; M, molecular layer. Bar = 100 ~tm. Ottersen et al. (1987).
presence of chromogranins might indicate that they are neurosecretory of some kind of peptide (see discussion Mugnaini et al., 1994). Particularly dense immunostaining in unipolar brush cells was obtained with antibodies against calretinin (R6sibois and 90
The cerebellum." chemoarchitecture and anatomy
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Fig. 66. A. Monoclonal antibody Rat-303 recognizes neurons in the granule cell layer (g), but not in the Purkinje cell (p) or molecular (m) layers of the rat cerebellar cortex. Scale bar = 100/~m. B. The morphology of Rat-303-positive neurons matches that described for Golgi II cells: a large ceil body emitting relatively stout dendrites from many points over the cell circumference (I3). Scale bar = 10 ~m. Hockfield (1987). R o g e r s , 1992; B r a a k a n d B r a a k , 1993; Floris et al., 1994). C a l r e t i n i n - i m m u n o r e a c t i v i t y in u n i p o l a r b r u s h cells was u s e d for a d e t a i l e d c h a r a c t e r i z a t i o n o f this cell type, a n d to
Fig. 67. Lugaro cells are molecularly distinct from Purkinje cells. In the pairs illustrated in A and B, the same fields were photographed under fluorescent optics separately for FITC (anti-calbinding, A) and Texas Red (Cat-301 in B). The blood vessel (triangle) passing through the field can be used to align the photographs. The cell type-specific antibody Cat-301 recognizes Lugaro, but not Purkinje cells in cat cerebellum, while anticalbindin recognizes Purkinje, but not Lugaro cells. Scale bar = 50/lrn. Sahin and Hockfield (1990). 91
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 68. Immuno-electron micrograph of an unipolar brush cell stained with antiserum to calretinin. The micrograph was obtained near the surface of the immunoreacted slice, as indicated by open areas in the tissue and over the cell nucleus (stars). The unipolar cell body has an irregular contour. The nucleus (N) shows a deep indentation (T) and the cytoplasm contains an array of ringlet subunits (R). The short dendrite forms an extensive synapse (arrows) with a calretinin-negative mossy fiber ending (mf). At the aspect opposite the synapse the dendrite contains numerous mitochondria. Granule cells (CG) are immunonegative. Bar = 1/~m. Floris et al. (1994).
92
The cerebellum." chemoarchitecture and anatomy
Ch. I
study its distribution in the cerebella of different species (Fig. 68) (Floris et al., 1994; Dino and Mugnaini, in preparation). 3.7. LOCALIZATION OF GABA RECEPTORS A N D GLYCINE RECEPTORS 3.7.1.
GABA A
receptors
GABA receptors can be divided in two main classes, GABAA and GABA~ (Bowery et al., 1980; Barnard et al., 1987; Sieghart, 1989; Sivilotti and Nistri, 1993; Mody et al., 1994). G A B A A receptors are ionotropic receptors that gate chloride channels. They are inhibited by bicuculline and picrotoxin and activated by muscimol. G A B A A receptors are the target of for a variety of drugs including benzodiazepine tranquilizers, and barbiturates. Benzodiazepines bind to a modulatory site that facilitates G A B A A receptor function. Different types of G A B A A receptors can be pharmacologically distinguished based on their differential sensitivity to benzodiazepine ligands and barbiturates (Doble and Martin, 1992). Originally the distribution of G A B A A receptors was studied autoradiographically with [ 3 H ] G A B A in the presence of the baclofen and the absence CaC12 or with [ 3 H ] m u s cimol. G A B A A receptors in rat and mouse cerebellum were found to be highly concentrated in the granular layer and relatively low in the Purkinje cell and molecular layers
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~
i
Fig. 69. A. Monoclonal antibody Rat-302 recognizes a subset of neurons, later identified as unipolar brush cells, restricted to the granular layer of the flocculus and the vermis of rat cerebellum (arrows), whereas in other areas of the cerebellum no positive cells are found. B. In contrast, antibody Rat-303 recognizes Golgi-II cells in the granular layer (g) in the entire cerebellum. C. Rat-302 also recognizes Purkinje cells outside the caudal vermis and the flocculus. D. Rat-302 positive cells in the vermis. E and F. Unipolar brush cells recognized by Rat-302 have a round cell body and short dendrites ending in a spray of appendages (arrows). g, granular layer; m, molecular layer; p, Purkinje cell layer. Scale bars: 500 p m in A and B, 50 p m in C and D, 10 p m in E and F. Hockfield (1987).
93
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(Fig. 70) (Palacios et al., 1980, 1981a; Kingsbury et al., 1980; Bowery et al., 1987; Rotter et al., 1988; Wilkin et al., 1981b). Binding to benzodiazepine receptors of [3H]flunitrazepam (Young and Kuhar, 1979, 1980; Vaccarino et al., 1985) or [3H]Ro15-1788 (Schoch et al., 1985), that would be expected to follow the pattern of [3H]muscimol binding to GABAA receptors, with the highest values in the granular layer, actually is reversed, with higher binding over the molecular layer. The inverse partial benzodiazepine agonist [3H]Ro 15-4513, however, exhibit higher binding to the granular layer than to the molecular layer (Sieghart et al., 1987). [3H]Ro15-4513 binding sites in the cerebellar granular layer differ from [3H]Ro15-4513 binding sites in the molecular layer and other brain regions in that they are insensitive to diazepam and other benzodiazepine agonists (see below). Yet another type of GABAA receptor is labelled with the antagonists [3H]bicuculline methochloride and [3H]SR95531, which preferentially bind to the low-affinity GABAA sites, and which bind at low density to the cerebellum (Bristow and Martin, 1988). Autoradiographic studies with mutant mice have revealed different changes in [3H]flunitrazepam and [3H]muscimol binding: [3H]flunitrazepam binding in the molecular layer in 'Purkinje cell degeneration' (pcd) mutant mice was decreased at 45 days, after the degeneration of the Purkinje cells. A further decrease was observed at 300 days, concomitant with the loss of the granule cells. Benzodiazepine receptors, therefore, may be located on Purkinje cells, and on parallel fibers in the molecular layer (Vaccarino et al., 1985; Kahle et al., 1990). An increase of [3H]flunitrazepam binding was found in 'weaver' cerebellum, with a loss of the granule cells (Chang et al., 1980; Kahle et al., 1990). Fry et al. (1985) found [3H]flunitrazepam binding to homogenates and tissue sections of the cerebellum of 'lurcher' mutant mice, with loss of their Purkinje cells and most granule cells, to be unchanged, whereas [3H]muscimol binding was reduced. Rotter et al. (1988)concluded that the decrease in [3H]muscimol binding in 'weaver', 'staggerer' and 45 days 'pcd' mutant mice was associated with a loss of granule cells and granule cell-Purkinje cell contacts. No [3H]muscimol labelling was observed over the deep Purkinje cell clusters in 'reeler' mutants, suggesting that these sites are absent on Purkinje cells. The electron microscopical studies of Chan-Palay (1978) and Chan-Palay and Palay (1978) in the rat indicate that [3H]muscimol binding to GABAA receptors is found on the plasma membrane of Purkinje cell somata, primary dendrites and initial axonal segment, and is also present on basket cells, their axons and the pinceau formation, on stellate cells and on dendrites of Golgi and granule cells. GABA A receptor subunits
To date five subunit classes of GABAA receptors, and several isoforms of each class have been cloned: ~1-~6, fll-fl3, 7'1-7'3, ~, and el and e2 (reviewed in Wisden and Seeburg, 1992; DeLorey and Olsen, 1992; Doble and Martin, 1992). GABAA receptors are constructed as hetero-oligomeric (presumably pentameric) assemblies of subunits. The ~-subunits in particular determine the different affinities of benzodiazepine ligands (Doble and Martin, 1992; Wisden and Seeburg, 1992). The y subunits are required for benzodiazepine-sensitivity, which indicates that the benzodiazepine binding site probably resides at the interface between the ~- and y-subunits. The fl-subunits are an essential structural component of the GABAA receptors, since without fl subunits, recombinant GABAA receptors are poorly expressed. The distribution of the distinct subunit mRNAs throughout the rodent cerebellum has been investigated in many studies and has been reviewed by Laurie et al., (1992) and Persohn et al. (1992) (Table 3). Most cerebellar neurons, including basket/stellate cells, Purkinje cells, and neurons of the deep nuclei 94
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 70. Bright-field (A) and dark-field (B) photomicrographs of a cerebellar folium of rat cerebellum labelled with [3H]muscimol to demonstrate the GABAA receptors. Note the clustering of grains over the area corresponding to the glomeruli of the granule cell layer and the low level of autoradiographic grains over the molecular layer (m) and the Purkinje cell layer (arrows). W, white matter. Bar = 100 r Palacios et al. (1981a).
express ~1, f12 and the 72 subunit mRNAs (Fig. 71, Table 3). Many types of subunits are produced by granule cells, which express c~l, ~z6,f12, f13, ?'2, fi mRNA and possibly also ill, cz4 and 7"3 mRNA (Fig. 71). No data are available on the subunit mRNAs expressed by Golgi cells. Bergmann glial cells express distinct levels of ~z2, 7'1, and possibly fll m R N A (Fig. 72, Table 3). The distribution of G A B A A receptor subunits has also been studied immunocytochemically with subunit specific antibodies. The monoclonal antibodies bd-24, which is selective for the ~zl subunit (but does not immunoreact with the rat antigen) and bd-17, which reacts with f12 and f13, preferentially stain the granular layer in rat, cat and monkey (Fig. 73) (Richards et al., 1987; Somogyi et al., 1989; Baude et al., 1992), and conform to the distribution of [3H]muscimol binding. A similar pattern was obtained with the monoclonal antibody 62-3G1 of De Bias et al. (1988), which also react with f12 and f13, and with several antibodies specific for the cz1 subunit (Meinecke et al., 1989; Gutidrrez et al., 1994 and references therein). Immunostaining in the granular layer prevails in the glomeruli, but granule cell membranes are also lightly stained (Fig. 73). Golgi cells were found to be unstained. Immunostaining in the molecular layer is compatible with a localization on Purkinje cell dendrites and stellate and basket cell somata. Purkinje cell somata and the basket cell-pinceau formations are lightly stained or unstained. In the deep nuclei, the majority of the cell bodies and also the dendrites were outlined by immunoreactivity. Electron microscopy by Somogyi et al. (1989) 95
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J. Voogd, D. Jaarsma and E. Marani
TABLE
3.
Distribution of GABAA receptor subunit mRNAs in rat cerebellum
Subunit
Cell type PC
GrC
al
++
a2
.
a3
-
o~4
.
.
~5
.
.
a6
-
fll
.
.
++ -
yl ?'2 y3
Based
-
not detected; stellate
on data
+
-
.
.
.
or +
+
-
-
. . -
.
DCN
or +
.
-
.
or +
++
+
-
+
+
-
-
+
-
-
-
+
-
++
+
+
-
++
-
-
-
-
-
-
++
-
_
_
or +
or +
+, positive;
++, strongly
cells; Bg, Bergmann
glia; DCN,
from
Bg
.
++
f13
Symbols:
. -
f12
cells; StC,
++ .
BC/StC
Laurie
et al., 1992; Persohn
positive; deep
PC, Purkinje
cerebellar
cells; GrC,
granule
cells; BC, basket
nuclei.
et al., 1992.
showed that both bd-24 (al) and bd-17 (,82/3) immunoreactivity occurs at the synapses established by Golgi cells with granule cell dendrites. Immunoreactivity, however, is also present at non-synaptic sites throughout the surface of the granule cell including the somatic membrane which does not receive synapses, but is absent from the postsynaptic membrane specialisations of mossy fiber-granule cell synapses. No immunoreactivity is present in the Bergmann glia and granular layer astroglia. In Purkinje cells, the synaptic junctions formed by basket cells are often immunopositive. Immunoreactivity was also found along most of the dendritic surface including the dendritic spines. No bd-24 and bd-17 immunoreactivity could be observed at the parallel fibers synapses (Somogyi et al., 1989). A different subcellular localization than that of the a l and fl2/3 subunits was observed for the a6 subunit: Baude et al. (1992) showed that a6 immunoreactivity, which was confined to the glomeruli in the granular layer, was detectable only at the postsynaptic membranes facing Golgi cell boutons, and did not occur on extrasynaptic membranes. g-subunit immunoreactivity, which was also specifically localized in the granular layer, was found in both the glomeruli and on granule cell bodies (Benke et al., 1991a). Immunocytochemical studies with antibodies specific for the y2 subunit (Benke et al., 1991 b) or for each of the two splice variants, ~ ' 2 L ( o n g ) and ) " 2 S ( h o r t ) (Guti6rrez et al., 1994), showed that 7'2 immunostaining is most abundant over the molecular layer, moderately dense over the deep nuclei, and low over the granular layer. This distribution is similar to the distribution of [3H]flunitrazepam binding sites, which is consistent with the idea that the 7' subunit is required for the binding of [3H]flunitrazepam and other benzodiazepine agonists, y2L and y2s immunoreactivities seem to be similarly distributed, but their exact cellular localisation remains to be described. The subunit compositions of functional GABAA receptors in the different cerebellar cells have been discussed by Laurie et al. (1992) and Pershon et al. (1992). Purkinje cells are likely to contain alf127'2 GABAA receptor/channel complexes, and possibly also 96
The cerebellum. chemoarchitecture and anatomy
Ch. I
~6
,13 ilili-l~1ill :lllll!llll ::l ll l li:lii:~l:l"
i........
~li
Fig. 71. Bright-field photomicrographs showing cellular distribution of mRNAs of ~1, ~6, f12, f13, y2 and GABAA receptor subunits in the cerebellum of the rat. Arrows indicate examples of labelled stellate/basket cells; arrowheads delineate Purkinje cells. Gr, granule cells; Mol, molecular layer; P, Purkinje cells. Scale bar = 50 r Laurie et al. (1992).
~lf13y2 or otlfl2fl3y2 receptors. These receptors would have a B E 1 subtype of benzodiazepine site, which is consistent with the observations that BE1, but not B E 2 benzodiazepine receptors, are found at high levels in the cerebellum (Doble and Martin, 1992). ~ l f 1 2 y 2 G A B A A Receptors are also likely to be present in basket/stellate cells and neurons of the deep nuclei. Granule cells producing at least six different subunits may have multiple subtypes of G A B A A receptors. The high level of [ 3 H ] m u s c i m o l and [ 3 H ] R o 15-4513 binding in combination with a low level of [3H]flunitrazepam binding to the granular layer can be explained by the presence of o~6(fl2and/orfl3)y2 receptors, which exhibit benzodiazepine agonist-insensitive Ro 15-4513 binding as well as high-affinity [3H]muscimol binding. It has been recently demonstrated that an alcohol-non-tolerant rat line, which is highly susceptible to impairment of postural reflexes by benzodiazepine agonists, has a point 97
Ch. I
J. Voogd, D. Jaarsma and E. Marani
i~}ii!::i~!i'iiiiili~ii]i!ii!~ili!iiiiiii::ili~ii::i~tliii:t ) 'ill
i
~t:, ....................... i~ii:~'~!:::.i";i::., " ,:~ ......
,
.........
~9
9
..............
!
J......................................................... :.~ :'':
~:':::::::::..t........:.. ~
..... ~:::
i,~,, .....
9.................
....
,., ,:::,,~:, ....... ~:,,,,~
~
" ......
~:,s
':;:::
::::,.::~:~
" " " ~Ji:"!~ . ~
~
~
'~
9
Fig. 72. ~2 (A) and 7'1 (B and C) GABAA subunit mRNAs are localized in Bergmann glial cells in rat cerebellum. A and B are low-power dark-field, C is high-power bright field of the image in (B)./3, putative Bergmann glia; Gr, granule cells; Mol, molecular layer; P, Purkinje cells. Arrowheads in A and B indicate 'halo' of silver grains along the boundary of the granule cell/molecular layers. In a high-power bright-field view of the 7'1 probe autoradiographic signal (C), Purkinje cells (arrow heads) and granule cells appear to be unlabelled, whereas other small cells (arrows) in the Purkinje layer have clusters of silver grains over them. There is also a density of grains higher than background over the molecular layer in areas having no cell bodies. Scale bars in A and B = 100 ~m, in C = 35/lm. Laurie et al. (1992).
98
The cerebellum." chemoarchitecture and anatomy
....~
.
. ...~,~.,-~c"q ~ .:.~..-
. ,~,-"
Ch. I
..,~.~
.+.<
......
.~.
Fig. 73. Low-power dark-field (A) and microscopic brigh-field (B, Normarski optics) images of the distribution of immunoreactivity in the rat cerebellum using mAb bd-17 against the fl2/3 GABA A receptor subunits. Immunostaining is very intense over the granular layer (PMgr), moderately dense over the molecular layer (PMmo) and the deep nuclei and absent in the white matter (my). In B, note the intense staining of the glomeruli in the granular layer, and virtually absence of staining in Purkinje cells (Pc). Bars 1 mm in A, 50/~m in B. Richards et al. (1987).
99
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J. Voogd, D. Jaarsma and E. Marani
mutation in the ~6 subunit, which dramatically increases the sensitivity of ~6fl27'2 receptors for benzodiazepine agonists (Korpi et al., 1993). Since ~6 subunits are exclusively expressed by cerebellar granule cells these data indicate that the increased susceptibility to benzodiazepine impairment of postural relexes is mediated at the level of the granule cells, and imply that ~6 subunit containing G A B A a receptors are important in the cerebellar circuits controlling movements (Korpi et al., 1993). Immunoprecipitation indicate that ~6 mostly colocalize with ~1, f12 or/33, and 7'2 (Khan et al., 1994). It has also been proposed that ~6 assemble into GABAA receptor complexes without ~1, and into receptors without 7'2, but with the g subunit (see Laurie et al., 1992). Granule cells may also make G A B A A receptors without the e6 subunit. Mfiller et al. (1994) characterized the properties of GABAA receptors in Bergmann glial cells in developing rat cerebellum. Significant benzodiazepine-insensitive GABAA receptor conductancies were detected in Bergmann glial cells between post-natal days 7 and 10, but were small or undetectable in the adult. It was also observed that Bergmann glial cells in addition to c~l-immunoreactivity, transiently express g-subunit immunoreactivity, indicating that the transient GABAA responses are mediated by a GABAA receptor composed of ~1 and ~ subunits. In view of the in situ hybridisation data also fll and yl may contribute to the Bergmann glial GABAA receptor (Laurie et al., 1992; Pershon et al., 1992).
3.7.2. GABAB receptors GABAB receptors are insensitive to the GABAA-antagonists bicuculline or picrotoxin, and are selectively activated by baclofen. GABAB receptors are believed to be G-protein coupled receptors similar to the metabotropic glutamate receptors. Their molecular structure remains, however, to be elucidated (Bowery, 1993). GABAB receptors are mostly coupled to adenylate cyclase, and exert their inhibitory action through the activation of potassium channels or inhibition of CaZ+-channels (Bowery, 1993). Ca 2+sensitive binding of [3H]GABA, with suppression of GABA A binding by isoguvacine, or binding of [3H]baclofen were used to study the distribution of GABAB receptors. GABAB sites are present at high density in the molecular layer (Wilkin et al., 1981b; Gehlert et al., 1985; Bowery et al., 1985, 1987), where they are distributed in a pattern of alternating, parasagittal zones of high and low [3H]GABA binding (Albin and Gilman, 1988; Turgeon and Albin, 1993). GABAB receptor binding in the granular layer is very low, but above background (Turgeon and Albin, 1993). A low amount of GABAB receptor binding was found over the cerebellar nuclei in adult rats. The cerebellar nuclei, however, transiently express a very high density of GABAB receptors during development (Turgeon and Albin, 1993). The cellular localization of GABAB receptor binding in the molecular layer is controversial (reviewed in Turgeon and Albin, 1993). Wojcik and Neff (1984) reported that GABAB induced inhibition of adenylate cyclase was reduced in 'weaver' mutant mice with a loss of their granule cells. It was not affected by Purkinje cell loss in the appropriate mutants or by climbing fiber deafferentation by 3-acetylpyridine in rats. Kato and Fukuda (1985) found [3H]baclofen binding to GABAB receptors to be decreased in homogenates of rat cerebellum, after destruction of the inferior olive with 3-acetylpyridine. They concluded that GABAB receptors were located on climbing fibers and that the residual binding could be associated with granule cells. They explained the apparent discrepancy with the results of Wojcik and Neff (1984) by assuming that the high affinity binding sites, which were mainly affected in their experiments, were not 100
The cerebellum." chemoarchitecture and anatomy
Ch. I
coupled to adenylate cyclase and, therefore, would have escaped to be noticed in the experiments of these authors. Bowery et al. (1987), who analyzed GABAB binding in several strains of mutant mice, however, advocated that the majority of GABAB receptors had a postsynaptic localization on Purkinje cell dendrites. Turgeon and Albin (1993), who analyzed GABAB receptor binding in rats with methyl azoxymethanol lesions of granule cells, or with 3-acetylpiridine lesions, and in stumbler mutant mice lacking Purkinje cell dendrites, also concluded that GABAB sites are primarily located on Purkinje cell dendrites. Finally, a recent immunoelectron microscopic study with monoclonal antibodies to L-baclofen indicate that GABAB receptors are found on both Purkinje cell dendrites and parallel fibers (Martinelli et al., 1992).
3.7.3. Glycine receptors Glycine-mediated inhibition of neuronal activity results from activation of the glycine receptor, a ligand-gated chloride channel (Betz, 1991). The distribution of this receptor has been autoradiographically studied with the antagonist [3H]strychnine. These studies indicate that the glycine receptor is almost absent from the cerebellum (Zarbin et al., 1981). The cerebellum, however, displays a significant number of [3H]glycine binding sites mainly distributed over the granular layer (Bristow et al., 1986), but these sites are assumed to represent the glycine coagonist sites of NMDA receptors (see Section 3.3.1 .). The glycine receptor has been demonstrated to be a pentameric protein composed of ligand binding ~ and structural fl subunits (Langosch et al., 1990). Variants of the ligand-binding ~ subunit (~1, ~2, ~3) have been cloned, that modify the pharmacological and the physiological properties of the glycine receptors. In situ hybridisation has revealed a hybridisation signal of the probe for the/3 subunit in all layers of the cerebellar cortex and the cerebellar nuclei, with a particular strong signal in the Purkinje cell and granular layers (Malosio et al., 1991). The ~l-subunit mRNA is expressed at low level in the cerebellar nuclei, and by 'rare single cells' in the granular layer, whereas ~3 mRNA appears to be selectively expressed by cerebellar granule cells. The ~2-subunit mRNA is not produced in the adult cerebellar cortex (Malosio et al., 1991). A monoclonal antibody selective for the N-terminal sequence of the ~ subunits (mAb2b), immunostained sparse puncta on cell somata in both the granular and molecular layer (Kirsch and Betz, 1993). Another monoclonal antibody, mAb4a, which bind to ~ and fl subunits, in addition to sparse puncta in the granular and molecular layer also produced diffuse labelling over Purkinje cell perikarya and occasional cell bodies in the granular layer (Kirsch and Betz, 1993). The punctate labelling is believed to reflect subunit staining at post-synaptic membrane specialisations (Kirsch and Betz, 1993). The diffuse labelling, instead, probably represents the fl subunit. Gephyrin is a 93 kDa peripheral membrane protein that co-purifies with glycine receptors. It has been proposed to anchor the glycine receptor to sub-synaptic tubulin (Betz, 1991). In situ hybridisation (Kirsch et al., 1993) and immunocytochemistry (Araki et al., 1988; Kirsch and Betz, 1993) show that gephyrin is widely distributed throughout the brain, including the cerebellum. Gephyrin mRNA is expressed by Purkinje cells and granule cells. Punctae of gephyrin immunostaining were concentrated over the molecular layer, and on cell bodies in the granular layer, but were virtually absent on Purkinje cell somata. The functional implications of the presence of glycine receptor subunits and gephyrin in the cerebellum are poorly understood. Granule cells express 0~3, the fl subunit and gephyrin and, therefore, have all the ingredients to produce functional glycine receptors. 101
Ch. I
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These receptors could be involved in the inhibitory actions of Golgi cells, in view of the fact that a subpopulation of Golgi cells may be glycinergic (see above). However, the presence of glycine receptors in granule cells is difficult to reconcile with the absence of any strychnine labelling in the cerebellum (Zarbin et al., 1981; see discussion Malosio et al., 1991). Gephyrin and/or the fl subunit are also produced by cells that do not express ~ subunits, such as the Purkinje cells. It has been postulated that gephyrin and/or the fl-subunit participate in other transmitter-gated ion channels, like the GABAAreceptors (see Malosio et al., 1991; Kirsch et al., 1993). For instance, Triller al. (1987) showed that gephyrin-immunoreactive postsynaptic specialisation face GABAergic synapses in the cerebellar cortex (Triller et al., 1987). Chen and Hillman (1993b) demonstrated that gephyrin-immunoreactive postsynaptic membrane specialisations (labelled with antibody R7A) faced a subpopulation of glutamate decarboxylase (GAD) immunoreactive boutons in the cerebellar nuclei. Since glycine often colocalizes with GABA it is possible that GABAergic boutons facing gephyrin-immunoreactive postsynaptic membrane specialisations, are also glycinergic. 3.8. M O N O A M I N E R G I C A F F E R E N T SYSTEMS AND RECEPTORS Several monoaminergic pathways terminate in the cerebellum (And6n and Ungerstedt, 1967). Their terminations in the cerebellar nuclei and the inferior olive will be considered elsewhere (Sections 5.2. and 6.3.2.2.). The monoaminergic fibers are primarily serotoninergic and noradrenergic (NA). The serotoninergic and noradrenergic fibers are present in all three layers of the cerebellar cortex. Their morphology differs from the classical mossy and climbing fiber afferents. Serotonin
Serotoninergic fibers in the cerebellar cortex were first demonstrated with the histofluorescence method, and were mainly recognized as transverse fibers in the molecular layer (H6kfelt and Fuxe, 1969). In studies using specific uptake, [3H]serotonin accumulated in small calibre, varicose axons. The varicosities were filled with minute, 15-25 nm round or flattened agranular vesicles and contained some large granular vesicles with a diameter of 50-90 nm (Beaudet and Sotelo, 1981). Synaptic contacts of the varicosities were few, most of them being present in the molecular layer. These, and other studies
A
B
Fig. 74. Camera lucida drawing of serotonin immunostainingin Lobule X of the cerebellum of the rat (A) and
the paramedian lobule (PML) (B). Sagittal sections. Grl = granular layer; ML = molecularlayer; WM = white matter. Calibration bar = 200/lm. Bishop and Ho (1985). 102
The cerebellum: chemoarchitecture and anatomy
Ch. I 9
t.-,~
......
Fig. 75. Photomicrographs of serotonin immunoreactive fibers in selected regions of rat cerebellar cortex. A. Dark-field photomicrograph. B. High-power bright-field photomicrographs illustrating the beaded nature of the immunoreactive fibers (arrows). Vermal lobule X. Abbreviations: see Fig. 74. Calibration bar = 200 tim. Bishop and Ho (1985).
using specific uptake of [3H]serotonin also reported labelling over mossy fiber rosettes (Chan-Palay, 1975; Bloom et al., 1971). The presence of serotoninergic mossy fibers could, however, not be confirmed immunocytochemically with antibodies directed against conjugates of serotonin in rat, cat and opossum (Takeuchi et al., 1982; Bishop et al., 1985; Kerr and Bishop, 1991). Serotonin-like immunoreactivity was present in randomly oriented fibers in the molecular layer in the rat. Long transverse fibers were present in superficial strata of the molecular layer in the rat (Takeuchi et al., 1982) or in deep parts of the molecular layer in the opossum (Bishop et al., 1985), radially oriented fibers were most numerous in the vermis of rat (Figs 74 and 75) (Bishop and Ho, 1985). The granular layer contains a loose serotoninergic plexus. A layer of fine, beaded serotonin-containing fibers is present below the Purkinje cells in rat and opossum (Bishop and Ho, 1985; Bishop et al., 1985). The Purkinje cell layer only contains passing fibers. In the caudal vermis of the oppossum the serotoninergic innervation of the cortex is concentrated in midsagittal and parasagittal bands. In the cat serotonin-immunoreactive fibers are dense in the granular and Purkinje cell layers with only a few fibers in the molecular layer (Kerr and Bishop, 1991). These serotoninergic axons and varicosities have a uniform distribution throughout all lobules of the cerebellum with the exception of lobule X were the fiber density is low (Kerr and Bishop, 1991). A dense plexus of serotoninergic fibers is also found in all of the deep nuclei of the cat. In addition, there appear to be serotonin positive cell bodies in the deep nuclei (Kerr and Bishop, 1991) (Section 5.7.). Double-labelling experiments in rat (Bishop and Ho, 1985) (Fig. 76), opossum (Walker et al., 1988) and cat (Kerr and Bishop, 1991) revealed that the serotoninergic innervation of the cerebellum originates from the reticular formation. In the rat these neurons are distributed over the medullary and the pontine reticular formation (nucleus reticularis gigantocellularis, nucleus reticularis paragigantocellularis and nucleus pontis oralis). The origin of these fibers from the gigantocellular reticular formation and from a few neurons located in the medullary pyramids is more restricted in the opossum. Kerr and Bishop (1991) found that the serotoninergic projection to the cerebellar cortex of the cat shows some degree of topographical organization: Serotoninergic fibers in the anterior vermis arise from neurons located within the paramedian reticular nucleus, the 103
Ch. I
J. Voogd, D. Jaarsma and E. Marani
lateral reticular nucleus and the lateral tegmental field, whereas fibers to the caudal vermis and the paramedian lobules exclusively derive from the lateral reticular nucleus. The hemispheres receive serotoninergic input from cells in the lateral tegmental field, the periolivary reticular formation and the paramedian reticular formation. Allthough retrogradely labelled cells were found in the raphe, in no case these cells were serotoninergic (Kerr and Bishop, 1991). Serotonin receptors
Physiological studies indicate that serotonin may play a modulatory role in the cerebellar circuitry (see Gardette and Cr6pel, 1993; Kerr and Bishop, 1992; and references therein). Serotonin interacts with a particularly large number of functionally and pharmacologically diverse receptor types. The serotonin receptors have been placed into five subgroups, 5HT1, 5HT 2, 5HT3, 5HT4 and 5HT5 (Tecott and Julius, 1993). The 5HT3 receptors are ionotropic receptors that mediate rapid excitatory responses in neurons, whereas the other serotonin receptor types are coupled to G-proteins, through which they activate second messenger cascades. The 5HT1 receptors comprise a heterogeneous group with at least six subtypes, named 5HT1A-5HT1F receptors. Ligand binding autoradiography with [3H]serotonin, which preferentially labels 5HT1 receptors, shows that a low level of [3H]serotonin binding occurs over the cerebellar cortex with the highest density over the molecular layer (Pazos and Palacios, 1985). The deep nuclei presented a higher density of [3H]serotonin binding with a lateral-to-medial decrease in receptor density. The cerebellar [3H]serotonin binding sites have the pharmacological properties of 5HT1B receptors (Pazos and Palacios, 1985). More recent autoradiographic studies with ligands specific for the 5HT1B and the 5HT1D receptors, which are closely related to 5HT1B receptors, indicate that the cerebellar cortex has 5HT1B, but no 5HT1D receptors (Bruinvels et al., 1993). The presence of 5HT1B receptors in rat cerebellum was confirmed by in situ hybridisation studies showing that 5HT1B receptor mRNA is expressed at high levels by Purkinje cells, but also by cells in the molecular layer, and in the deep cerebellar nuclei (Appel et al., 1990; Voigt et al., 1991; Maroteaux et al., 1992). In view of the findings that 5HT1B mRNA levels are relatively high in Purkinje cells, whereas receptor binding is low in the molecular and Purkinje cell layers, but relatively high in the deep nuclei, one is tempted to speculate that the 5HT1B receptor is localized presynaptically on Purkinje cell axon terminals. The ultrastructural localization of the 5HT1B receptors remains, however, to be determined. No 5HT1A receptors were found in the rat cerebellum with receptor autoradiography or in situ hybridisation (Pompeiano et al., 1992). Using immunocytochemistry with an antibody specific for the 5HT1A receptor, Matthiesen et al. (1993), however, found that the 5HT1A receptor occurs in Purkinje cells of immature, but not in adult cerebellum. The 5HTlc and 5HT 2 receptors were found at low concentrations in the deep nuclei in autoradiographic studies (Pazos et al., 1985; Molineaux et al., 1989). Since 5HT 2 and 5HTlc are structurally related they have been recently renamed as 5HTzA and 5HTzc receptors, respectively. In situ hybridisation studies have shown that mRNAs of both receptor types are expressed in the deep cerebellar nuclei (Pompeiano et al., 1994). The other known serotonin receptors (e.g. 5HT3 and 5HT4) seem to be essentially absent from rodent cerebellum (Tecott and Julius, 1993; Domenech et al., 1994). Summarizing, the known serotonin receptors are expressed at low levels or are not expressed at all in the cerebellum, with the exception of the deep nuclei which express significant levels of different types of serotonin receptors.
104
The cerebellum." chemoarchitecture and anatomy
Ch. I
/
I.U
ji.
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Fig. 76. Schematic drawings showing the distribution of labelled neurons in the medullary and pontine reticular formation in the rat, after injections of H R P in the vermis and adjacent parts of the hemisphere. Filled triangles represent neurons retrogradely labelled with H R P alone; closed circles represent neurons immunolabelled for serotonin alone; and asterisks represent double-labelled neurons. Each symbol represents one neuron in each population. Abbreviations: BC = brachium conjunctivum; BP = brachium pontis; CP = cerebral peduncle; D R = dorsal raphe nucleus; EC = external cuneate nucleus; ICP = inferior cerebellar peduncle; IN = interpeduncular nucleus; IOC = inferior olive; M R = nucleus raphe magnus; MV = medial vestibular nucleus; PG = nucleus paragigantocellularis; PH = nucleus pre-positus hypoglossi; PO = nucleus pontis oralis; R G = gigantocellular reticular nucleus; R M = nucleus raphe magnus; RO = nucleus raphe oralis; RP = nucleus raphe pallidus; SV = superior vestibular nucleus; V = nucleus of spinal tract of the trigeminal nerve; VII = nucleus of facial nerve. Redrawn from Bishop and Ho (1985).
Noradrenalin
The noradrenergic (NA) innervation of the cerebellum is mostly directed at the cortex. In the original studies of H6kfelt and Fuxe (1969) with a histochemical fluorescence method for the demonstration of catecholamines (Falck et al., 1962), NA containing fibers were found to be present as a randomly oriented plexus in all three layers of the cortex of rat cerebellum. The density of the NA innervation was lower in the flocculonodular lobe and the uvula than in the corpus cerebelli. A similar plexus was illustrated in rat cerebellum by Grzanna et al. (1989), using an antibody against a conjugate of NA (Geffard et al., 1986), and by Fritschy and Grzanna (1989) with an antibody against dopamine-fl-hydroxylase (Fig. 77A), the synthetic enzyme that converts dopamine to 105
Ch. I
J. Voogd, D. Jaarsma and E. Marani
NA. A heavy plexiform innervation of the granular layer by thin varicose axons and parallel fiber-like fluorescence in the molecular layer was also found in chicken cerebellum. The parent fibers in the cerebellar white matter were concentrated in two parasagittal bundles (Mugnaini and Dahl, 1975). Axons containing dark core vesicles, which were labelled by specific uptake of [3H]NA terminated on Purkinje cell proximal dendrites and spines in the molecular layer (Bloom et al., 1971; Yamamoto et al., 1977). The inhibitory action of NA on Purkinje cells and the transduction of this response by cyclic AMP were reported in the subsequent papers of Hoffer et al. (1971) and Siggins et al. (1971). Purkinje cells therefore seem to be the main target of the NA innervation (Felten et al., 1986; see also Llano and Gerschenfeld, 1993). The ultrastructure of NA fibers of the cerebellar cortex and other parts of the rat CNS was analyzed with pre-embedding dopamine-fl-hydroxylase immunohistochemistry by Olschowka et al. (1981). Immunoreaction product was present in the axoplasm, associated with smooth endoplasmatic reticulum, Golgi apparatus, synaptic and large dense core vesicles and the outer membranes of mitochondria. Large varicosities were interconnected by narrow intervaricose axon segments. Varicosities, filled with clear, round synaptic vesicles and large dark-core vesicles, made asymmetric contacts with dendrites, but never with somata or axons. More than 50% of the labelled varicosities in the cerebellum made synaptic contacts: most of them with dendritic shafts, fewer on spines. Kimoto et al. (1981) used the histofluorescence method for light microscopy and potassium permanganate fixation in their electron microscopic studies of NA in rat cerebellum. They found NA-containing nerve terminals making contact with granule cell dendrites and secondary and spiny branchlets of the Purkinje cell dendritic tree. The terminals contained a large number of small dark core vesicles and a couple of larger ones. No synaptic contacts with the Purkinje cell somata were observed. Triarhoe and Ghetti (1986) are of the opinion that these results should be reconsidered because a distinction between NA and serotonin is not possible with this fixation method. The cerebellar NA fibers in the rat take their origin from the dorsal part of the locus coeruleus (group A6 of Dahlstr6m and Fuxe, 1964) and the A4 groups in the roof of the fourth ventricle. Single NA neurons innervate both the cerebellum and the forebrain (see also Steindler, 1981). The fibers enter the cerebellum close to the fourth ventricle. According to Pickel et al. (1973) the main entrance route is through the superior cerebellar peduncle. Pasquier et al. (1980) localized the cells of origin of the NAprojections to the cerebellum in the rat with specific retrograde transport of dopaminefl-hydroxylase. They traced the main projection from the entire caudal pole of the locus coeruleus and smaller contributions from the nucleus subcoeruleus and the cell groups A7 and A5; all projections are bilateral. The cerebellar projections in the cat take their origin from NA-containing cells located around the superior cerebellar peduncle, and the dorsolateral part of the locus coeruleus (Chu and Bloom, 1974; Somana and Walberg, 1978; see also Dietrichs, 1985, 1988). Grzanna et al. (1989), Fritschy and Grzanna (1989) and Grzanna and Fritschy (1991) reported a selective loss of the terminal portions of NA axons originating from the locus coeruleus in rats treated with the neurotoxin DSP (N-(2-chloroethyl-N-ethyl-2 bromobenzylamine)). DSP affects the NA innervation of the cerebellum, the cerebral cortex, the pontine nuclei, the inferior olive, the vestibular and cochlear nuclei, the sensory nuclei of the trigeminal nerve and the dorsal horn but spares the NA axonal plexus in the basal forebrain, the hypothalamus and most of the brain stem and the cord. After 24 hours loss of NA-staining is most-pronounced in the molecular layer, but with longer survival times most NA fibers have disappeared (Fig. 77B). 106
The cerebellum." chemoarchitecture and anatomy
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Fig. 77. Effects of DSP-4 (50 mg/kg) on the noradrenaline innervation of the cerebellar cortex of the rat. A. Control section incubated with an antibody against dopamine-fl-hydroxylase. B. Section from a DSP-4-treated rat. Parasagittal plane; dark-field photomicrographs. Magnification 90x. Fritschy and Grzanna (1989).
Adrenergic receptors Adrenergic receptors can be subdivided in ~l,0t2 and fll,fl2 receptors on the basis of p h a r m a c o l o g i c a l criteria (Bylund and Pritchard, 1983; M i n n e m a n et al., 1981). M o s t 107
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receptors in the cerebellum are of the fl type, low levels of specific binding of the ~2 agonist [3H]para-aminoclonidine are associated with the granular layer in rat cerebellum (Unnerstall et al., 1984). Binding assays with the fl-adrenergic antagonist [3H]dihydroalprenolol in homogenates of cat cerebellum showed that the receptors are mainly of the f12 type and that they are evenly distributed over the vermis, the hemisphere and the cerebellar nuclei (Pompeiano et al., 1989). Elevated binding to the molecular layer was found in autoradiographs of tissue sections of rat cerebellum with ligands which bind non-selectively to fl receptors (Palacios and Kuhar, 1980). The same pattern emerged when either fll or f12 receptors were eliminated by specific inhibitors, with high levels of f12 binding in the molecular layer and much lower levels over the Purkinje and the granule cells. The overall contribution of fll receptor binding was low (Rainbow et al., 1984a). Beta adrenergic receptor binding was evenly distributed over the molecular layer, but at the level of the Purkinje cell somata the ligand is bound in irregular patches (Fig. 78) (Sutin and Minneman, 1985). These patches are most prominent in vermis and paravermal zones of the lobules I-IX and are less frequent in the medial hemisphere. Incubations with specific fll and f12 antagonists showed that the receptors in these patches are of both subtypes. 109
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Polyclonal antibodies which recognize domains of the f12 adrenergic receptor other than the NA-binding site, immunoreact with distal segments and dendritic spines of rat Purkinje cells. Immunoreactivity is present throughout the dendritic cytoplasm and accumulates at the dendritic membrane, opposite synaptic clefts (Strader et al., 1983). Dopamine
Dopaminergic projections to the cerebellum are still controversial. Low levels of dopamine (DA) supposedly represent a precursor of noradrenaline. DA-like immunoreactivity, as demonstrated with an antibody against a conjugate of DA, was present in fibers with irregularly spaced varicosities in all layers of the cerebellar cortex of the rat (Panagopoulos et al., 1991). Radially oriented DA fibers were abundant in the molecular layer. These fibers may take their origin from the A10 dopaminergic cell group of Dahlstr6m and Fuxe (1964) in the medial tegmental area. Cerebellar DA levels in rat cerebellum decreased by 50% after lesions of the A10 area (Kizer et al., 1976) and connections of the A10 area with the cerebellar nuclei and the granular and Purkinje cell layers were demonstrated with antegrade and retrograde axonal transport methods by Simon et al. (1979). Recently Ikai et al. (1992) reported a dopaminergic projection of the A10 group in the rat to restricted portions of the cerebellar hemisphere (Figs 79A,B and 80). Projections of the A10 group to the lateral cerebellar nucleus, however, appeared to be non-dopaminergic, as demonstrated immunocytochemically with an antibody against tyrosine hydroxylase, the enzyme involved in the conversion of tyrosine to DA. Fibers and terminals in the cortex were most numerous in the granule and Purkinje cell layer and scarce in the molecular layer. Their distribution differs, therefore, from the DA fibers described by Panagopoulos et al. (1991), which predominate in the molecular layer. Dopamine receptors
Five different receptors for dopamine have been distinguished, D~-Ds, which are often grouped as Dl-like (D1 and Ds) and Dz-like (D2, D3, and D4) receptors with high affinities for [3H]SCH23390 and spiperone, respectively (Grandy and Civelli, 1992). The D~ receptor is considered to be stimulatory, whereas the D 2 receptor is either inhibitory or unlinked to adenylate cyclase. Autoradiographic studies using [3H]SCH23390 as the ligand show that D~ receptor binding in the cerebellar cortex is low (Camps et al., 1990; Mansour et al., 1992). According to Camps et al. (1990), who compared the distribution of D~ and D 2 receptors in the cerebellum of rat, mouse, guinea pig, cat, monkey and man, Dl-binding was only detected in the Purkinje cell and molecular layer in rat and cat. Dz-receptors, labelled with [3H]CV 205-502, were only found at significant levels in rat cerebellum, specifically localized in the molecular layer of lobule X and IXc. In lobule IX high activity is distributed in sagittal and parasagittal columns. Although similar labelling has been repeatedly found with other 'D2-1igands' it was recently shown by both autoradiography and in situ hybridisation that the dopamine receptors in the rat nodulus and ventral uvula are of the D3-type. Thus Bouthenet et al. (1991) noticed a very low D 2 signal over the granular layer of all cerebellar lobules, and a high expression of D3 mRNA by Purkinje cells in lobule X and IXc. Autoradiographic studies with D3-preferring ligands, [3H]quinpirole (Gehlert, 1993; Levant et al., 1993) and 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin (L6vesque et al., 1992) showed a similar type of labelling in rat cerebellum (Fig. 81). The functional role of the cerebellar D3 receptor is puzzling in view of the lack of significant dopaminergic innervation. It is 110
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Fig. 79. Photomicrograph of camera lucida drawing (inset) of anterogradely labelled axons and terminal boutons in the crus I ansiform lobule of rat cerebellum after cholera toxin (b fragment) injection into the contralateral ventral tegmental area. Arrows point to 'rosettes' characteristic of mossy fiber endings in the granular layer (G). R Purkinje cell layer; WM, white matter. Scale bar = 40/xm. Ikai et al. (1992).
notable that substance P receptors have a very similar distribution in rat cerebellum (Nakaya et al., 1994). 3.9. H Y P O T H A L A M O CEREBELLAR CONNECTIONS AND HISTAMINERGIC PROJECTIONS Hypothalamo cerebellar fibers were demonstrated with different anterograde axonal transport techniques in squirrel monkey, cat and rat (Dietrichs, 1984; Dietrichs and Haines, 1985; Haines et al., 1984, 1985, 1986). Most fibers enter the cerebellum bilaterally with an ipsilateral dominance from the central grey, passing medial to the superior cerebellar peduncle. They enter the granular layer, where they branch. Fibers passing around the Purkinje cell somata enter the molecular layer, where most of them assume a longitudinal course in the long axis of the folia (Fig. 82). Few labelled mossy fibers and no labelled climbing fibers were seen. These 'multilayer' hypothalamo cerebellar fibers are more frequent in the cortex of the flocculus and the vermis, but are also present in parts of the hemisphere. Some fibers project to the cerebellar nuclei (Dietrichs and Haines, 1985; Haines et al., 1990). The projection takes its origin from cells in the posterior, lateral and dorsal hypothalamic areas, the lateral mammillary nucleus and the periventricular nucleus (Dietrichs and Zheng, 1984; Dietrichs, 1984; Haines and Dietrichs, 1984, 1987; Dietrichs et al., 1985b). Immunocytochemical studies have demonstrated the existence of histaminergic neurons, which are concentrated in the tuberomammillary nucleus of the posterior hypothalamus, and which give rise to fibers to almost all parts of the brain (reviewed by Wada 111
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et al., 1991). A low and low to moderate density of histamine-immunopositive fibers has been observed in rat (Inagaki et al., 1988) and guinea pig cerebellum (Airaksinen and Panula, 1988), respectively. Like the hypothalamo cerebellar fibers, the histaminergic fibers were sparsely distributed to all cortical layers, and were more frequent in the vermis and the flocculus than in other lobules. A moderate density of histaminergic fibers occurred in the cerebellar nuclei and the inferior olive. Three subtypes of histamine receptors have been pharmacologically identified in the brain: H1, H2 and H3 receptors. H1 receptors are coupled to phospholipase C, through which the inositol 1,4,5-trisphosphate-Ca 2+ and diacylglycerol-protein kinase C cascades are activated. Receptor autoradiographic studies with [3H]mepyramine and [125I]iodobolpyramine show that essentially no H~ receptors occur in the rat and human cerebellar cortex (Palacios et al., 1981b), whereas high densities are found over the molecular layer of guinea pig and mouse cerebellum (Palacios et al., 1981b; Rotter and Frostholm, 1986; Bouthenet et al., 1988). Moderate densities were found in mouse and guinea pig deep cerebellar nuclei (Rotter and Frostholm, 1986; Bouthenet et al., 1988). H1 receptor density over the molecular layer was greatly decreased in Purkinje cell deficient mice, whereas in reeler mice, which contain malpositioned Purkinje cells, high H~ receptor density was found over regions with heterotopically located Purkinje cells. No change in H1 receptor density was found in weaver mouse cerebellum, which is almost devoid of cerebellar granule cells. These data indicate that H~ receptors are predominantly localized on Purkinje cell dendrites (Rotter and Frostholm, 1986). 112
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Fig. 81. Bright-field autoradiographs showing [3H]quinpirole binding to dopamine D 3 receptors to coronal sections of the caudal cerebellum of rat. Note high-density of binding sites over the molecular layer (m) of the nodulus (X). Also note stripes of increased [3H]quinpirole binding over molecular layer of the uvula (IX). g, granular layer; IO, inferior olive; MVe-medial vestibular nucleus; 12, hypoglossal nucleus. Gehlert (1993).
H 2 receptors, which are coupled to adenylate cyclase were also found in the molecular layer of guinea pig cerebellum (Ruat et al., 1990). H3 receptors are autoreceptors involved in the inhibition of the release of histamine. A low level of H3 receptors were found to be homogeneously distributed throughout the rat cerebellar cortex (Arrang et al., 1987; Pollard et al., 1993). 3.10. CHOLINERGIC SYSTEMS AND ACETYLCHOLINESTERASE (ACHE) IN THE CEREBELLUM 3.10.1. Distribution of choline acetyltransferase Biochemical measurement of distinct levels of acetylcholine (McIntosh, 1941; Kfisa et al., 1982) and its biosynthetic enzyme, choline acetyltransferase (CHAT) in cerebellar tissue (K/tsa and Silver, 1969; Salvaterra and Foders, 1979; Hayashi, 1987; and others) indicated the presence of a cholinergic innervation in the cerebellum. ChAT activity varies among different lobules with the highest levels in the nodulus and ventral uvula. Following deafferentation of the cerebellar cortex, ChAT activity is considerably de113
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Fig. 82. A. Semidiagrammatic representation showing the correlation between the patterns of individually wheat germ agglutinin (WGA)-HRP labelled hypothalamo-cerebellar fibers and the diffuse patterns seen just above and below the Purkinje cell layer. B. Darkfield microphotograph of the termination of WGA-HRPlabelled hypothalamo-cerebellar fibers. Note the clusters of label just above (solid arrows) and below (open arrows) the Purkinje cell layer. GL, granular layer; ML, molecular layer. Squirrel monkey. Scale bar = 75 ~m for B. Haines et al. (1986)
114
The cerebellum." chemoarchitecture and anatomy
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creased, indicating that the cholinergic innervation is mostly of extracerebellar origin (K/tsa and Silver, 1969; Asin et al., 1984). Acetylcholine and ChAT were found in the glomerular fraction consistent with the view that acetylcholine may be the transmitter in a subpopulation of mossy fibers (Isr~iel and Whittaker, 1965; Bal/tzs et al., 1975). Early anatomical studies have employed acetylcholinesterase (ACHE) histochemistry. However, AChE activity is disproportionally high in the cerebellum (reviewed in Silver, 1974), and its widespread distribution in non-cholinergic cells and fiber systems precludes its use as a marker for cholinergic neurons. More reliable methods to unravel the anatomy of the cerebellar cholinergic system became available with the development of antibodies specific for CHAT. Early immunocytochemical studies revealed a subpopulation of ChAT-positive mossy fibers in rabbit and man (Kan et al., 1978, 1980). However, subsequent studies with monoclonal antibodies, failed to demonstrate ChAT positive staining in the cerebellum (Armstrong et al., 1983; Houser et al., 1983). Yet more recent studies by Ojima et al. (1989) in rat, by Illing (1990) in cat, by Barmack et al. (1992a) in rat, rabbit, cat, and monkey, and by DeLacalle et al. (1993) in man, demonstrated the presence of at least four types of cholinergic innervation in the cerebellum: (1) a subpopulation of ChAT-positive mossy fibers which primarily innervate the nodulus and the ventral uvula of the vermis; (2) a sparse plexus of thin beaded fibers which is present in all layers; (3) thin ChAT-positive fibers that innervate the cerebellar nuclei; and (4) a still controversial subpopulation of cholinergic Golgi cells. In addition, Ikeda et al. (1991) reported some typical granule cells with their bifurcating parallel fibers to be ChAT-immunoreactive in the cat, but this finding was not confirmed by others. Ojima et al. (1989) showed that a significant number of ChAT-positive mossy fiber rosettes was present throughout most vermal lobules, but that the density in the nodulus and ventral uvula was approximately ten times that of other vermal lobules (Fig. 85A). ChAT-positive mossy fiber rosettes were also enriched in the flocculus and the ventral paraflocculus. The distribution of ChAT-positive rosettes in rat observed by Barmack et al. (1992a) was essentially similar, although they suggested that the ChAT-positive rosettes were also enriched in lobules I, II and III (Fig. 83). They also demonstrated that ChAT-positive rosettes were concentrated in the nodulus and the ventral uvula of rabbit (Fig. 84), cat and monkey, and that the distribution of ChAT-positive profiles correlated with the distribution of ChAT activity. Large and small mossy fiber rosettes and transitional forms with the plexus of beaded fibers were distinguished in different species (Barmack et al., 1992a). Illing (1990) noted a 'wealth of ChAT-immunoreactive mossy fibers' that were slightly enriched in the ventral folia I to III, IX and X. ChAT-positive mossy fibers were also observed in human cerebellum, but their spatial distribution remains to be described (Kan et al., 1980; DeLacalle et al., 1993). By combining retrograde tracing and ChAT-immunocytochemistry Barmack et al. (1992b) demonstrated that the ChAT-positive mossy fibers in the nodulus, ventral uvula and the flocculus are likely to be secondary vestibular afferents. After injections of horseradish peroxidase in the lobules X and IX in rat and rabbit, neurons in the caudal medial vestibular nucleus and the nucleus prepositus hypoglossi were double labelled (Fig. 198). The flocculus and the paraflocculus of the rabbit receive a small, cholinergic projection, mainly from ChAT-positive neurons of the nucleus prepositus hypoglossi (Barmack et al., 1992b). Csillik et al. (1964) already drew the conclusion from the preferential distribution of AChE-positive glomeruli in the 'archi-cerebellum', that they represent terminals of a cholinergic system of secondary vestibulo cerebellar mossy fibers. Ikeda et al. (1991) noticed that in cat the number ChAT-positive mossy fibers 115
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Fig. 83. Choline-acetyltransferase (ChAT)-like immunoreactivity in the rat uvula-nodulus. A,B. Photomicrographs taken of lobule Xb. These photomicrographs demonstrate varieties of ChAT-positive mossy fiber rosettes in the granule cell layer and in the molecular layer of the rat nodulus. C. Illustration of ChAT-positive staining of mossy fiber rosettes and fine fibers innervating both the granule cell layer and molecular layer of the nodulus (lobule Xb). D. Illustration of three kinds of ChAT-positive mossy fiber rosettes found in the rat cerebellum: (1) Large grape-like terminals found with highest density in the uvula-nodulus (lobules IXb, Xa,b), (2) smaller mossy fiber rosettes that are characteristic of both the uvula (lobule IXa) and the anterior vermis (lobule I), (3) finely beaded ChAT-positive fibers that are found in both the granule cell layer and molecular layer throughout the cerebellum. Primary antibody: rat-~pig-ChAT. Barmack et al. (1992a). (
were reduced following excitotoxic and electrolytic lesions in the cerebellar nuclei (see below). The distribution of ChAT-positive mossy fibers roughly corresponds to that of unipolar brush cells (see Section 3.6.3.), which raises the question whether these mossy fibers innervate these cells. Electron microscopic analysis of ChAT-immunoreactivity in the nodulus showed that a minority (10-20%) of ChAT-immunoreactive mossy fiber terminals synapse on brush cell profiles, and that a minority (10-30%) of the mossy fiber terminals contact unipolar brush cells that are immunoreactive for ChAT (Jaarsma, 1995c). Barmack et al. (1992a) noted a dense population of small ChAT-positive mossy fiber-like terminals in the tip of lobules IXa,b of rat cerebellum that are significantly smaller than the other ChAT positive rosettes (Fig. 83). This type of labelling was not described by Ojima et al. (1989) but could be observed in their Fig. 4. The source and characteristics of this peculiar population of terminals is still unknown. Ojima et al. (1989), Illing (1990), Ikeda et al. (1991), Barmack et al. (1992a), and DeLacalle et al. (1993) observed thin ChAT-positive beaded fibers which were distinct from mossy fiber profiles. According to Ojima et al. (1989) these fibers in rat were most frequent immediately beneath or within the Purkinje cell layer. A substantial number of varicose fibers could also be identified in the molecular layer (Fig. 85B). Illing (1990) in cat noted that thin beaded fibers in the molecular layer mostly course at right angles from the Purkinje cell layer to the surface of the cerebellar cortex, whereas in the granular layer these fibers have an irregular course. The source of the sparse plexus of 'non-mossy' ChAT-immunoreactive fibers is presently unknown. It has been proposed that they originate in the ponto-mesencephalotegmental cholinergic complex (Illing, 1990), but at least for rat this seems to be untrue, since not a single retrogradely labelled, cholinergic neuron was found in the pontomesencephalic tegmentum after tracer injections in the cerebellar cortex (Woolf and Butcher, 1989). The cerebellar nuclei, instead, receive afferents from tegmental cholinergic neurons (see next paragraph). Ojima et al. (1989) showed that all cerebellar nuclei in rat are innervated by CHATimmunoreactive fibers. The density of these fibers varies between the different nuclei. Moderately dense innervation was found in most of the medial nucleus and in the magnocellular part of the lateral nucleus, whereas only a few ChAT-immunoreactive fibers invade the ventromedial parvicellular portion of the lateral nucleus and most of the interposed. Also in the cerebellar nuclei of man a moderate density of ChAT-positive fibers has been observed (DeLacalle et al., 1993). Both in rat and man these fibers did not form pericellular networks. DeLacalle et al. (1993) and Ikeda et al. (1991) also found 117
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sporadic ChAT-positive neurons in the cerebellar nuclei. As demonstrated by Woolf and Butcher (1989) with retrograde tracing, at least a portion of the cholinergic fibers in the cerebellar nuclei are likely to originate from the pedunculopontine tegmental nucleus. The projections are bilateral, but most prominently ipsilateral (Woolf and Butcher, 1989). Cerebellar Golgi cells display strong AChE activity (Shute and Lewis, 1965; Brown and Palay, 1972 and others), but there has been no evidence of ChAT-immunoreactive Golgi cells in the rat (Ojima et al., 1989; Barmack et al., 1992a; Jaarsma et al., 1995c). 118
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Fig. 85. A. Drawing displaying the distribution of mossy rosettes (dots) immunoreactive to monoclonal choline-acetyltransferase (CHAT) antibody. The section (40/Jm thick) was cut sagittally through the middle vermis of rat cerebellum. A considerable number of immunoreactive mossy terminals are observed in lobules I through IXab, although they are much fewer than in lobules IXc and X. Calibration bar = 1 mm. B. Drawing of part of lobule IXab shows the overall distribution of immunoreactive fibers. Arrows indicate mossy fibers with glomerular rosettes. Small and large arrowheads point to some varicose fibers distributing in or near the Purkinje cell layer (PCL) and in the molecular layer (ML), respectively. The M L fibers are most frequently observed in this lobule and tend to be restricted to the inner half of the layer. Calibration bar - 200/Jm. Ojima et al. (1989).
ChAT-positive Golgi cells have been found in cat (Illing, 1990; Ikeda et al., 1991) and in man (DeLacalle et al., 1993), although Barmack et al. (1992a) did not see them in their cat and monkey material. Illing (1990) found that the CHAT119
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Distribution and density of choline-acetyltransferase (ChAT)-immunoreactive Golgi cells (dots) in two subsequent 100 pm thick parasagittal sections through the vermis of the cerebellar of the cat. Roman numbers indicate lobules according to Larsell; pcs, pedunculus cerebellaris superior. Scale bar - 2 mm. B and C. Drawings of immunoreactive Golgi cells from 100 pm thick cerebellar sections. The uppermost cell is from the hemisphere, the lower one from the dorsal vermis. The arrows point to processes thought to be axons. ML, molecular layer; GL, granular layer. The border of gray to white matter is marked by a dashed line. Illing (1990). 120
The cerebellum." chemoarchitecture and anatomy
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positive Golgi cells mostly are positioned close to or in the Purkinje cell layer of both the vermal and hemispheral lobules (Fig 86). Also in man, ChAT-positive Golgi cells seem to be preferentially located beneath the Purkinje cell layer, but they were found to have a heterogeneous lobular distribution, with higher densities in the flocculonodular lobules (DeLacalle et al., 1993). In cat the ChAT positive Golgi cells have been estimated to represent only 5% of the total Golgi cell population (Illing, 1990). Ikeda et al. (1991) and DeLacalle et al. (1993) found ChAT immunoreactive neurons in the cerebellar nuclei in cat and man, respectively. According to Ikeda et al. (1991) cholinergic neurons in the cerebellar nuclei project to the cerebellar cortex in the form of mossy fibers, and to the thalamus and the red nucleus (see Section 5.2.).
3.10.2. Cholinergic receptors Nicotine receptors Both nicotine and muscarine receptors have been detected in the cerebellum. Nicotine receptors are cation-gating ion channel complexes and consist of five subunits, like the nicotine receptors in skeletal muscles. The subunit composition of neuronal nicotine receptor is found to be different from the muscle receptor, which is made up of four types of subunits (o~fle~) with two copies of the alpha subunit per receptor. A number of putative nicotine subunits are expressed in the mammalian brain, including at least five alpha subunits, named cz2, cz3, cz4, cz5, and cz7 and three subunits homologous to the muscle beta subunit, /32, /33 and/34 (Deneris et al., 1989; Duvoisin et al., 1989; Wada et al., 1989, 1990; Seguela et al., 1993). The subunits ~3, ~4, and/32 are expressed in rat cerebellum, the granular layer displaying low levels of cz3, cz4 and/32 mRNA, and Purkinje cells expressing high levels of/32 (Wada et al., 1989). Most Purkinje cells do not express any of the alpha subunits, but a few Purkinje cells display strong hybridisation signals with the cz2, ~3 or ~4 probes. Cells in the cerebellar nuclei express moderate levels of cz4 and/32 mRNA. A recent study with antibodies against the/32 subunit showed that the intensity of fl2-immunostaining was high in the perikarya and main dendritic arbors of Purkinje cells, low in granule cells and moderate in neurons of the cerebellar nuclei, thus confirming the results from in situ hybridisation (Hill et al., 1993). Swanson et al. (1987) using a iodinated monoclonal antibody to chicken neuronal acetylcholine receptors ([125I],mAB270) that is assumed to specifically immunoreact with the fl2-subunit of rat, obtained a somewhat different immunodistribution offl2-subunits, with labelling concentrated in the granular layer. Functional channels gated by nicotinic agonists are formed by certain combinations of alpha and beta subunits (Deneris et al., 1991). Receptors made of cz4 and f12 subunits are most common in the brain, and are believed to represent high-affinity [3H]acetylcholine binding sites, (that can be labelled in the presence of atropine to block muscarinic sites), and the [3H]nicotine binding sites of the brain (Clarke, 1993). Accordingly in rat, high-affinity [3H]nicotine binding sites are present at low density in the granular layer and the cerebellar nuclei, which are the structures expressing both cz4 and //2 subunits, but are essentially absent in the Purkinje cell and molecular layer, where ~4 subunits are lacking (Clarke et al., 1985). It should be noted that Purkinje cells, in spite of the fact that they express high levels of fl2 subunit, may lack functional nicotine receptors, because of the absence (as far as known) of alpha subunits. The cz7 subunit is different from the other neuronal nicotine receptor subunits in forming functional channels in the homomeric configuration (S6guala et al., 1993) that, unlike other nicotine receptors of the brain, are sensitive to ~-bungarotoxin (czBTX).
121
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The distribution of ~7-mRNA throughout the brain roughly correspond to that of [125I]~BTX binding sites. It is therefore assumed that nicotine receptors made up of ~7 subunits represent the major portion of brain ~BTX binding sites (Clarke et al., 1985; S6gu61a et al., 1993). The level of [125I]~BTX binding in most of the rat cerebellum is low. Patches of intense labelling were, however, found over glomeruli in the lobules I, IXd, X and the flocculus (Fig. 87; Hunt and Schmidt, 1978; Frostholm and Rotter, 1986). Frostholm and Rotter (1986) postulated that these [125I]0~BTX binding sites were located presynaptically on mossy fibers, based on the observation that developmental 122
The cerebellum." chemoarchitecture and anatomy
Ch. I
appearance of the binding sites corresponds to the appearance of the mossy fibers. Notably, the distribution of [125I]~BTX binding sites is similar to that of ChAT-positive mossy fibers rosettes, which raises the possibility that cerebellar '~BTX-receptors' are localized presynaptically on the subpopulation of cholinergic mossy fibers. The distribution of ~7 mRNA has yet to be studied systematically in the vestibulo-cerebellum, but data from in situ hybridisation presently available suggest that no ~7-mRNA is expressed in the cerebellum, which is consistent with the idea of a presynaptic localisation of '~7-receptors' (S6gu61a et al., 1993). Recently an immunocytochemical study on the distribution of the ~7 protein has been reported, in which three different monoclonal antibodies raised against chicken ~7 were employed (Dominguez del Toro et al., 1994). The results of this study, however, are puzzling since immunolabelling in the cerebellar cortex was mainly localized in Purkinje cells, which disagrees both with the distribution of ~7-mRNA and [125I]~BTX binding sites. Muscarine receptors
Muscarine receptors are coupled to G-proteins and may activate various second messenger cascades. At least four subtypes (M1, M2, M3 and M4) could be distinguished according to pharmacological criteria (e.g. see Waelbroeck et al., 1990), whereas molecular cloning has revealed five distinct subtypes (ml-m5; Hulme et al., 1990). Membrane binding and autoradiographic studies have shown that cerebellar muscarine receptors have the pharmacological properties of the cardiac (M2)-type (Mash and Potter, 1986; Spencer et al., 1986; Waelbroeck et al., 1987, 1990; Araujo et al., 1991; Aubert et al., 1992). Concordantly, the cerebellum has been demonstrated to primarily express m2-mRNA (Vilar6 et al., 1992, 1993) and m2 receptor protein (Levey et al., 1991; Li et al., 1991). It has been proposed that also m3 (respectively M3) receptors may be expressed in the cerebellum, because granule cells in culture express m3 mRNA (Fukamauchi et al., 1991). Immunochemical and pharmacological studies, however, indicate that m3 receptors represent less than 10% of the cerebellar muscarine receptor population (Waelbroeck et al., 1990; Wall et al., 1991). Moreover, no high-affinity binding sites for [3H]4-DAMP, a ligand with preference for M3 receptors, were found in the cerebellum (Araujo et al., 1991). Early autoradiographic studies on the topographic distribution of muscarine receptors in rat cerebellum with [3H] propylbenzilylcholine mustard as the ligand showed that muscarine receptors were confined to the molecular layer of the nodulus and the ventral uvula (Rotter et al., 1979a,b). Subsequent autoradiographic studies in rat with the more potent antagonist [3H]quinuclinidyl benzylate ([3H]QNB, Neustadt et al., 1988) and with M2-specific ligands [3H]oxotremorine (Spencer et al., 1986; Vilar6 et al., 1992), and [3H]AF-DX384 (Aubert et al., 1992), all gave identical results. Muscarine receptors were found to be localized throughout the whole cerebellar cortex, with the highest densities in the molecular layer of the nodulus and the ventral uvula (Fig. 88). The amount of muscarine receptors in the other lobules was relatively low, with higher densities in the granular and Purkinje cell layers than in the molecular layer (Fig. 88). Muscarine receptors also occur in the cerebellar nuclei (Spencer et al., 1986; Neustadt et al., 1988; Vilar6 et al., 1992) and in some parts of the white matter. In the caudal folia of the uvula (lobule IXb) muscarine receptors appear to be distributed in five parasagittal columns of high receptor density that traverse the granular layer (Fig. 88D, Neustadt et al., 1988). In situ hybridisation shows that m2 mRNA in rat cerebellum is localized in the granular layer as well as in the deep nuclei (Vilar6 et al., 1992). In accordance with the autoradiographic data the highest signal is present in the nodulus and ventral uvula. 123
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J. Voogd, D. Jaarsma and E. Marani
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Fig. 88. Bright-field photograph showing cresyl-violet staining (A and C) and corresponding dark-field photomicrographs showing the distribution of muscarine receptors using [3H]QNB binding (B and D, respectively) in the caudal cerebellum of the rat. A and B. Coronal section through the nodulus (X) and the rostral uvula (IX) showing high density of [3H]QNB binding over the molecular layer (m) of lobule X and ventral region of lobule IX. Also note higher binding densities at the level of the Purkinje cell layer (P) in most lobules. C and D. Coronal section at the level of the dorso-caudal uvula (IXa,b). Note five parasagittal columns (large arrows in D) of high receptor density that traverse the granular layer (g); w, white matter. Scale bars = 1 mm. Neustadt et al. (1988).
Since in this region the mRNA signal is in the granule cell layer, whereas receptor binding is high over the molecular layer (Fig. 89), one is tempted to speculate that the muscarine receptors are associated with parallel fibers. In fact, analysis of sections immunostained by Levey et al. (1991) with a polyclonal antibody specific for the m2 receptor, showed that parallel fibers are immunostained (Jaarsma et al., 1995a). Parallel fiber staining was seen in all lobules. It remains to be determined whether the amount of parallel fiber staining is most prominent in the nodulus and ventral uvula, concordant with the density distribution of receptor binding. In addition to parallel fibers the m2-antibody also immunostained Golgi cells and some mossy fiber rosettes (Jaarsma et al., 1995a). Neustadt et al. (1988) using receptor autoradiography noted that the topographic distribution of muscarine receptors in rabbit and guinea pig cerebellum is characterized by the presence of parasagittal columns of very high receptor density over the molecular layer (Fig. 90). In rabbit the bands of high muscarine receptor density were most prominent in the anterior lobe (lobules I-V), in Crus I and II, and in the flocculus and the ventral paraflocculus. Their distribution only partially corresponded to the Zebrinpositive zones (Jaarsma et al., 1995a). The receptors in the parasagittal bands were 124
The cerebellum." chemoarchitecture and anatomy
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Fig. 89. Comparison between the distribution of m2 muscarine receptor mRNA (A) and [3H]oxotremorine-M binding to muscarine M2 receptor sites (B) in approximately equivalent coronal sections of rat brain at the level of the caudal cerebellum. Note that m2 mRNAs in the ventral uvula (IX) and the nodulus are localized in the granular layer, whereas M2 receptor binding sites are in the molecular layer (m). 12, hypoglossalnucleus. Scale bar - 3 ram. Vilar6 et al. (1992).
located in Purkinje cell dendrites as demonstrated immunocytochemically with a monoclonal antibody specific for the m2 receptor protein. A second characteristic feature of the distribution of rnuscarine receptor in rabbit cerebellum, is the presence of a stripe of very high receptor density immediately above the Purkinje cell layer in the nodulus and the ventral uvula (Fig. 90B) (Neustadt et al., 1988). Immunocytochemistry showed that the receptors in this transverse stripe were localized on a population of densely packed parallel fibers (Jaarsma et al., 1995a). In addition to these parallel fibers and Purkinje cell dendrites, also Golgi cells, that in rabbit in part are located in the molecular layer (Spa~ek et al., 1973), and a subpopulation of mossy fiber rosettes are immunostained with m2-receptor antibody (Jaarsma et al., 1995a). Autoradiographic data from human (Cortes et al., 1987) and cat cerebellum (D. Jaarsma, unpublished observations), and immunocytochemical data from monkey (Jaarsma and Levey, unpublished) indicate that the distribution of muscarine receptors in the cerebellum of these species is different from that in rodents and rabbit. The level of ligand binding was very low with slightly higher receptor densities over the granular layer. There is no increased receptor density in the nodulus and the ventral uvula. The overall distribution in monkey was similar. Only Golgi cells and a subpopulation of mossy fibers rosettes, (mainly located in the vermal lobules III-VI), were immunostained (Jaarsma and Levey, unpublished). Summarizing it appears that the cellular and regional distribution of muscarine receptors in the cerebellum is different between different species. Golgi cells and subpopulations of mossy fibers seem to express muscarine receptors most constantly. An interesting aspect about the presence of muscarine receptors in parallel fibers in rat and rabbit, is that the lobular distribution of m2-containing parallel fibers, is the same as that of ChAT-positive mossy fiber rosettes (see above). This raises the possibility that muscarine m2 receptor are specifically expressed by those granule cells that are innervated by cholinergic mossy fibers. If this proves to be true, this would imply that there 125
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is some regulatory interaction between the ChAT-positive mossy fibers and granule cells through which the expression of muscarine m2-receptor is regulated. Although lightmicroscopic analysis suggest that the muscarinic receptors are absent in granule cell dendrites at the synapses with ChAT-positive mossy fibers, this possibility can not be excluded without electron microscopic examination. The function of muscarine receptors in the parallel fibers is puzzling since there seems to be little cholinergic innervation in the molecular layer (see above). 126
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Physiological responses to acetylcholine Only a few physiological studies on the actions of acetylcholine in the cerebellar cortex have been reported and the results of these studies were often contradictory: Iontophoretic application of acetylcholine has been observed to mildly excite Purkinje cells via a muscarinic (Crawford et al., 1966) or a nicotic (McCance and Phillis, 1968) mechanism, or to inhibit them via a mixed muscarinic and nicotinic mechanism. For interneurons no effect of acetylcholine was found by Crawford et al. (1966), whereas De la Garza et al. (1987) found a strong nicotinic excitatory effect. Cr6pel and Dhanjal (1982) reported slow depolarisations of Purkinje cells, accompanied by an increase in membrane resistance, with application of high doses of acetylcholine in slices of the nodulus and the ventral uvula of the rat's cerebellum, that was mediated through muscarine receptors.
3.10.3. Acetylcholinesterase Based on their substrate specificity cholinesterases can be subdivided into the acetylcholinesterases (AChE's) and the butyryl or pseudocholinesterases. Several isoenzymes of AChE have been detected in brain tissue. Three to four isoenzymes of AChE can be distinguished in developing brain (Henderson, 1977). In the chicken the faster migrating forms predominate in embryos. Their composition changes progressively into the slowly migrating forms of the adult tissues. Pseudocholinesterase can be distinguished from the acetylcholinesterases by specific inhibitors (iso-OMPA or ethopropazine) and by using butyrylcholiniodide as a substrate, which is split predominantly, though not exclusively by pseudocholinesterase. Most other esterases can be inhibited using eserine (Marani et al., 1977). Butyrylcholinesterase has also been localized with immunocytochemical methods (Barth and Ghandour, 1983). AChE-staining in the molecular layer of the mammalian cerebellum is generally lower than in the granular layer, but great variations occur and the pattern is reversed in the human and the avian cerebellum (Friede and Fleming, 1964). AChE-staining generally is higher in the vestibulocerebellum than in other parts of the cerebellum. The reactivity in the glomeruli in rat (Csillik et al., 1964; Brown and Palay, 1972 and many others) and rhesus monkey (Robertson and Roman, 1989) follows this general pattern, but the distribution of AChE-positive Golgi cells is more uniform (Brown and Palay, 1972). In the cerebellar white matter of monkey cerebellum AChE-rich fibers are distributed in parasagittal compartments, that are aligned with concentrations of strongly ACHEreactive regions in the granule cell layer and narrower, AChE-rich 'spikes' in the molecular layer (Hess and Voogd, 1986; Marani, 1986). Purkinje cells in certain parts of rat and guinea pig cerebellum, including the lobules IX and X of the caudal vermis, display a transient reactivity for ACHE, which disappears later. AChE was localized in adult Purkinje cells of the lobules IX and X (Robertson et al., 1991); these cells are arranged in multiple, sagittal bands (Gorenstein et al., 1987). Robertson et al. (1991), however, were unable to confirm the transient staining with AChE in rat Purkinje cells. AChE-positive displaced Golgi cells (Ramon y Cajal, 1911) are present in the lower one third of the entire molecular layer of the rabbit cerebellum (Spa~ek et al., 1973) and strong AChE-staining is present in this stratum in the vermis and certain lobules of the hemisphere in the same species (Tan et al., 1995a). An AChE-band pattern was detected in the molecular layer of the vermis of the anterior and posterior lobes in 2-4 month old cats (Marani and Voogd, 1977) (Fig. 120). 127
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Reaction product in transverse sections was present in narrow striations reaching from the AChE-negative Purkinje cell somata to the pial surface; in sagittal sections the distribution of AChE was more uniform. The same banding pattern could be visualized in adult cats, with AChE-histochemistry on aldehyde-fixed tissue (Brown and Graybiel, 1983). No such banding was present in the cerebellum of rats or primates (Marani, 1986). Spikes of strong AChE-activity sometimes extend from heavily stained portions of the granular layer into the molecular layer, at certain preferred localizations (Hess and Voogd, 1986). The ultrastructural localisation of AChE has methodological problems, because of uncontrolled diffusion of reaction product (e.g. see discussion Brown and Palay, 1972). In the molecular layer AChE-reaction product was located in subsurface cisterns of Purkinje cell dendrites and in the spine apparatus of their dendritic spines and, extracellularly, associated with the dendritic plasma membrane and around parallel fibers (Marani, 1982a, 1986). AChE was never found in the synaptic cleft of parallel fiberPurkinje cell synapses, lntra-axonal deposits in parallel fibers were considered as an artefact. Neurons in deep parts of the molecular layer that may represent basket or stellate cells, contain AChE-reaction product in their rough endoplasmatic reticulum. AChE in cat molecular layer, therefore, is associated with neurons and not with glial cells. AChE in the molecular layer of the rat is preferentially located in Bergmann glia (Friede and Fleming, 1964; Barth and Ghandour, 1983; Gorenstein et al., 1987; Robertson et al., 1991). Brown (1985a) demonstrated an increase in AChE-staining in the cat molecular layer, 2-6 weeks after lesions of the inferior olive. Upregulation of AChE was found in the projection zones of the lesioned olivary neurons. These plastic changes are reminiscent of the re-appearance of neonatal AChE in Purkinje cells after the transection of their axons (Phillis, 1968). AChE-staining in the granular layer is distributed in 'An alternating fine mosaic of stained and unstained spots, with regional differences in staining intensity' that represent AChE-positive Golgi cells and moderately to strongly stained glomeruli. Granule cell somata remain unstained in the rat cerebellar cortex (Altman and Das, 1970). The presence of AChE in glomeruli has been generally acknowledged (Gerebtzoff, 1959; Csillik et al., 1964; Shute and Lewis, 1965). AChE-reaction product in rat cerebellum is located outside the mossy fiber terminal, between the axonal plasma membrane and the dendrites of the granule cells and the Golgi cell terminals. AChE in Golgi cells is associated with the cisterns of the rough endoplasmatic reticulum and the perinuclear cisterns. Granule cells, Purkinje cells and glia are AChE-negative (Brown and Palay, 1972). The preferential staining of the borders of white matter compartments (the 'raphes', Voogd, 1964) for AChE (Hess and Voogd, 1986; Marani, 1986; Voogd, 1995; Fig. 126) and its use in topographical analysis of the corticonuclear and olivocerebellar projections will be considered in Sections 6.1.1., 6.1.2., 6.1.5. and 6.3.3. Several authors have attempted to devise other functions for AChE than the hydrolytic cleavage of acetylcholine; their proposals were reviewed by Greenfield (1984) and Appleyard and Jahnsen (1992). 3.11. NEUROGLIA The morphology of the neuroglia in the cerebellar cortex was reviewed by Palay and Chan-Palay (1974) and Ghandour et al. (1980). Three forms were distinguished, the Bergmann glial fibers in the molecular layer that take their origin from Golgi epithelial cells, located in the Purkinje cell layer, the astroglia and the oligodendroglia. The 128
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Fig. 91. Bergmann glial fibers in the rat cerebella of various ages. PAP immunocytochemistry with anti-GFAP antiserum. The ages of the rats are 3, 5, 8 days and 30 months in A, B, C and D, respectively. EG, external granule cell layer; M, molecular layer; G, granule cell layer. Scale bars = 20/lm. De Blas and Cherwinski (1985).
enzyme histochemistry of cerebellar glia was studied by Sotelo (1967). Neuroglia has its own metabolism, that is qualitatively different from that of neurons. The glial anaerobic glycolysis is high in neuronal neuroglia and interfascicular neuroglia. The aerobic glycolysis is secondary and much weaker than in nerve cells. The hexose129
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Fig. 92. Micrograph of adult cerebellar cortex, incubation with vimentin antibody V9 counterstaining with cresylviolet. Sagittal unfixed cryostat section. Bergmann glial fibers (arrowheads) and large astrocytes (arrows) are seen in the molecular and granular layer, respectively, pc, Purkinje cell; ml, molecular layer; gl, granular layer. Bar = 100 pm. Roeling and Feirabend (1988)
monophosphate shunt is also important in glia. In oligodendroglia and astroglia different enzyme patterns were found. The main difference concerns glycogen, which is metabolised almost exclusively in astroglia (Sotelo 1967). Bergmann glia and astroglia are immunoreactive for anti-glial fibrillary acidic protein (GFAP) (Fig. 91). The development and the adult configuration of the Bergmann glia has been studied, using antibodies against GFAP (Bignami and Dahl, 1974; De Blas, 1984; De Blas and Cherwinsky, 1985; Levitt and Rakic, 1980; Gr~iber and Kreutzberg, 1985; Pelc et al., 1986). Bergmann glia can also be demonstrated with anti-vimentin (Dupouey et al., 1985; Roeling and Feirabend, 1988) (Fig. 92) and with cell-specific monoclonal antibodies (De Blas, 1984; De Blas and Cherwinsky, 1985; Edwards et al., 1986). Butyrylcholinesterase has been localized in Bergmann glia and in glial cells in the granular layer using an immunocytochemical method in the rat (Barth and Ghandour, 1983). Carbonic anhydrase is a specific marker for oligodendrocytes (Cammer, 1984). The distribution of oligodendrocytes in the cerebellar cortex of the mouse was studied with immunostaining for carbonic anhydrase II by Ghandour et al. (1980, 1981). Occasionally oligodendrocytes are present in the molecular layer, they are more common in the granular layer and abundant in the white matter, sn-Glycerol-3-phosphate-dehydrogenase (GPDH) in mice is present in adult oligodendrocytes (De Vellis et al., 1977), but also in Bergmann glia (Fisher et al., 1981) (Fig. 93). The expression of GPDH in Bergmann fibers is dependent on the adjoining Purkinje cells (Fisher, 1983). A similar interdependency may exist for 5'-N in Bergmann glia (Hess and Hess, 1986) (see Section 130
The cerebellum." chemoarchitecture and anatomy
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Fig. 93. Sagittal section through the cerebellum of a 25-day-old BALB/cBy mouse, with anti-sn-glycerol-3 phosphate dehydrogenase (GPDH) serum (1:10,000) viewed with a dark-field condenser. The molecular layer is densely stained although clear areas representing non-staining cell bodies and processes are numerous. Intensely stained cell bodies of the Bergmann glia are located between large unstained Purkinje cell somata, at the boundary between the molecular and granular layers. Magnification x l00. Fisher et al. (1981). 3.5.), for t a u r i n e t h a t shifts f r o m P u r k i n j e cells to B e r g m a n n glia u n d e r c o n d i t i o n s of h y p o - o s m o t i c stress ( N a g e l h u s et al., 1993) (see Section 3.1.2.) a n d Z e b r i n I, t h a t a p p e a r s in B e r g m a n n glia after lesions of the cerebellum ( D u s a r t et al., 1994).
Fig. 94. A and B. Distribution of 3-fucosyl-N-acetyl-lactosamine (FAL)-immunoreactive Bergmann glial cells in adult mouse cerebellum. Note zonal distribution of immunoreactive neuroglia in molecular layer (MOL) in A. GCL, granular layer. Bars in A = 800/~m, in B = 100 ~tm. Courtesy of Dr. J.K. Mai, Department of Neuro-anatomy, Heinrich Heine University, Dfisseldorf. 131
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Fig. 95. Chicken cerebellum, 1 day old, 1-/lm thick sections. Post-embedding immunocytochemistry with antibody IX-50 against a putative kainate receptor shows immunoreactivity outlining Purkinje cells (Pc), their main ascending dendrites (arrows) as well as their spiny branchlets. Immunoreactivity is associated with Bergmann glial cells (BG) which lie beneath and amongst the Purkinje cells. Scale bar = 20/~m. Somogyi et al. (1990).
Several substances are distributed in zonally distributed Bergmann glial cells. This may be the case for 5'-N, and has been observed for 3-fucosyl-acetyl-lactosamine (FAL) immunoreactivity in mouse cerebellum (Fig. 94) (Bartsch and Mai, 1991; Marani and Mai, 1992). The protein kinase types ~, flII, y and ( have been localized in Bergmann glia (Shimohama et al., 1990; Hidaka et al., 1988; Wetsel et al., 1992). Bergmann glial cells 132
The cerebellum." chemoarchitecture and anatomy
Ch. I
contain both nitric oxide synthase and guanylcyclase and are the main cerebellar store for cyclic GMP (see Section 3.4.). As mentioned before also homocysteic acid (Cu6nod et al., 1990; Grandes et al., 1991; Tschopp et al., 1992) (Figs 44 and 45) and 5'nucleotidase (Kreutzberg et al., 1978 and Fig. 59; see, however, Marani 1986 and Fig. 58) are associated with Bergmann glia. Climbing fiber-induced release of homocysteic acid and adenosine, therefore, involve this glial compartment (see Sections 3.2.2. and 3.5.). Bergmann glial cells and granular layer astroglia are provided with glutamate receptors and, therefore, may be actively involved in the cerebellar neural transmission (Fig. 95) (see Section 3.3.1.).
4. GROSS ANATOMY OF THE MAMMALIAN CEREBELLUM The gross anatomy of the cerebellum is the morphology of its cortical sheet. The cerebellum in mammals, birds and some reptiles is subdivided by transverse fissures of varying depth in lobes, lobules and folia. Two paramedian sulci demarcate the vermis from the hemispheres in the mammalian cerebellum. The present nomenclature for the mammalian cerebellum evolved from older purely descriptive studies of the human cerebellum (reviewed by Glickstein, 1987), elaborate comparative anatomical studies of the adult cerebellum (Elliot Smith, 1903; Bolk, 1906; Riley, 1928, reviewed by Larsell 1970; Larsell and Jansen, 1972) and comparative embryological investigations of the development of the folial pattern (Stroud, 1895; Bradley 1903, 1904; Larsell, 1947, 1952, 1953, 1954, 1970). The cerebellum of the rat served as the prototype for Larsell's subdivision of the mammalian cerebellum in 10 lobules (Larsell, 1952, 1970) (Fig. 96).
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Larsell extensively studied its adult morphology and the development of its folial pattern. More recent accounts of the cerebellum of the rat (Voogd, 1995) (Fig. 97) and the mouse (Marani and Voogd, 1979) are available. The cerebellum of the cat was described by Larsell (1953, 1970) and Voogd (1964) (Fig. 98). The gross morphology of the cerebellum in different species of macaques was described by Larsell (1953, 1970), and by Madigan and Carpenter (1971) for Macaca rhesus. The anatomy of the cerebellum of macaques is very similar to other primates (Macaca fascicularis, Fig. 99) (Larsell, 1970: Cercocebus sp. Cebus sp., Ateles, Saimiri sciurus) and subprimates (Haines, 1969). It is possible to recognize the main subdivisions in nearly all mammalian cerebella by inspection of the branching pattern of the arbor vitae in a midsagittal section of the vermis (Fig. 98) and the characteristic convolutions of the folia of the hemisphere. For the lobules of the vermis either the classical nomenclature of the human cerebellum or Larsell's (1952, 1970) numbering system can be applied. The anterior lobe consists of lingula (lobule I), the central lobule (II, III), and the culmen (IV, V). The base of the lingula is continuous with or embedded in the anterior medullary velum. The subdivision of the anterior lobe by the preculminate fissure into the lobules I-III and the lobules IV-V is of prime relevance to its connectivity. It is usually deep and its walls are subfoliated. The anterior lobe is separated from the posterior lobe by the deep primary fissure. The posterior lobe vermis can be subdivided in most mammals in the declive (VI), folium and tuber (VII A and VII B), the pyramis (VIII), the uvula (IX) and the nodule (X). The secondary and posterolateral fissures in between the lobules VIII, IX and X usually are quite distinct. The prepyramidal fissure separating lobule VII and VIII is less obvious in some species and the borders between the lobules VI, VII A and VII B often are difficult to recognize. For the lobules of the hemisphere of the mammalian cerebellum both Bolk's (1906) descriptive terms and Larsell's numerals can be used. In Larsell's nomenclature the hemispheral lobules bear de prefix H to the number of the vermal lobule with which they are continuous. Bolk's (1906) and Larsell's (1952, 1970) nomenclature for the hemisphere are not readily interchangeable because they are based on conflicting views on the morphology of the mammalian cerebellum (Fig. 96). Bolk (1906; see Glickstein and Voogd, 1995, for a recent discussion of Bolk's views) stressed the difference between vermis and hemisphere that should be considered as independent folial chains, which have a tendency to local longitudinal expansion. For the cat the folial chains are illustrated in Fig. 98. In the anterior lobe the folial chains of vermis and hemisphere develop in parallel. The inter- and most of the intralobular fissures continue uninterruptedly from the vermis into the hemispheres and a paramedian sulcus is either absent or shallow. The same situation prevails in the region of the posterior lobe immediately behind the primary fissure. Bolk coined the name 'lobulus simplex' to express the continuity of vermis and hemisphere in this part of the cerebellum. The vermal portion of the simple lobule corresponds to the declive (Larsell's lobule VI). Caudal to the lobulus simplex the folial chain of the hemisphere deviates from the vermis and forms a series of loops. The first loop is the ansiform lobule, that can be subdivided in the Crus I and the Crus II. The cortex in the center of the ansiform lobule, in between folium vermis (lobule VIIA of Larsell) and the hemisphere, usually is interrupted (Figs 97 and 99). This interruption may involve all layers of the cortex, with white matter coming to the surface, or only affect the parallel fibers of the molecular layer. At the level of the paramedian lobule the folial chains of vermis and hemisphere are aligned and the cortex between them usually is continuous. The cortex of the pyramis (Larsell's lobule VIII) 135
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continues into the caudal part of the paramedian lobule as the copula puramidis. The paraflocculus and the flocculus are the caudal segments of the folial chain of the hemisphere. The cortex between the paraflocculus and flocculus and the caudal vermis 136
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(uvula and nodulus: lobules IX and X of Larsell) usually is completely interrupted. White matter invades the centre of the successive loops of the folial chain of the hemisphere and separates the successive segments of the flocculus and the paraflocculus. The dorsal and ventral segments of the paraflocculus are designated as the dorsal and ventral paraflocculus and the portion of the paraflocculus located in the subarcuate fossa of the petrosal bone is known as the petrosal lobule. This nomenclature is purely descriptive and these terms do not necessarily refer to identical segments of the paraflocculus in different species (see Fig. 97e for the topology in the rat and Fig. 149 for a comparison of rabbit, monkey and cat). The border between the paraflocculus and the flocculus is the posterolateral fissure. This fissure is one of the first to develop in the hemisphere. Its localization was established by Larsell (1970) for many mammalian species. Larsell tried to verify the relations between vermis and hemisphere by tracing the development of the transverse fissures. He attached great importance to the confluence of the vermal and hemispheral parts of fissures which, in the adult, would indicate the transverse continuity of the lobules of vermis and hemisphere. Larsell's nomenclature for the cerebellar hemisphere is rather unpractical and the significance of some of his many subdivisions (significance in the sense that a portion of the hemisphere has something in common with the vermal lobule to which it belongs) is questionable.
5. THE CEREBELLAR NUCLEI The (central) cerebellar nuclei and the lateral vestibular nucleus of Deiters receive the axons of the Purkinje cells of the cerebellar cortex and serve as the main output stations of the cerebellum. The vermis and the flocculus also project to other vestibular nuclei, but here the Purkinje cell axons compete with vestibular root fibers, intrinsic and commissural vestibular connections and projections from the medial cerebellar nucleus and, therefore, are not the dominant afferent system. Large numbers of Purkinje cells axons converge upon the cerebellar nuclei. The Purkinje cell/dentate nuclear cell convergence ratio is about 14:1 in the rhesus monkey and about 30:1 in the rat (Chan-Palay, 1977). It was estimated from Golgi impregnated Purkinje cell axons of the rat that between 20-50 perikarya of central nuclear neurons are included within the conical terminal field of a single axon (Chan-Palay, 1977), but the actual number of central nuclear neurons contacted by a single axon must be far greater. A considerable overlap in the corticonuclear projection of adjacent cortical areas was observed in axonal tracing studies (Courville and Diakew, 1976; Armstrong and Schild, 1978a,b; Haines and Koletar, 1979; Haines et al., 1982). Convergence and overlap are greatest in the rostrocaudal dimension where the entire length of the cortical sheet is compressed in the small volume of the cerebellar nuclei. Convergence is much less in the transverse direction and overlap may be even absent at the borders of neighbouring longitudinal zones where the Purkinje cells project to different nuclei (Voogd, 1964; Voogd and Bigar6, 1980). The convergence of the Purkinje cells from apex and base of a lobule onto the same cerebellar nuclear neurons is usually taken for granted. This type of convergence is of potential interest, because it combines the output of Purkinje cells that are under the influence of quite different mossy fiber-parallel fiber systems: corticopontine and exteroceptive systems at the apex and vestibular and proprioceptive systems at the base of the lobules (see also Section 6.4.2.). The subdivision of the cerebellar nuclei is closely related to the longitudinal, zonal 138
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organization of the corticonuclear and olivocerebellar projections. In this view the cerebellar nuclei are the output stations of corticonuclear modules and their independent olivocerebellar control systems. Certain cytochemical correlates of this longitudinal organization exist, but there are no signs of a corresponding cytochemical differentiation in the output of the cerebellar nuclei. The main input to the cerebellar nuclei from the Purkinje cells is GABAergic and inhibitory (Sections 3.1.1. and 5.5.). The nuclear cells receive their excitatory drive from collaterals from afferent mossy and climbing fiber systems (Section 5.6.). Collaterals of olivocerebellar fibers are organized according to the same longitudinal principle as their climbing fiber terminals, i.e. collaterals terminate in the cerebellar nucleus that receives axons from Purkinje cells that receive climbing fibers from the same parent axons (Groenewegen and Voogd, 1977). Mossy fiber input to the cerebellar nuclei is more diffuse, i.e. not limited to a single nucleus, and more selective, i.e. not present in all mossy fiber systems. Mossy fibers, therefore, are diverse in origin and dissimilar with respect to their collateral projections to the cerebellar nuclei, but as yet neurochemical correlates of this diversity are lacking. The monoaminergic input to the central nuclei 139
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J. Voogd, D. Jaarsma and E. Marani
is diffuse and independent of the mossy and climbing fiber collaterals. It is mainly represented by serotonin in a varicose, all pervading plexus (Section 5.7.). Neurons of the cerebellar nuclei are a mixed population of cells of all shapes and sizes. Several authors noticed a binominal distribution for cell size in the cerebellar nuclei. According to Courville and Cooper (1970) all four central nuclei of the monkey contain cells of all sizes. They noticed a peak in the size distribution for the lateral and interposed nuclei at 22 ~tm cell diameter. Palkovits et al. (1977) distinguished two populations of neurons, one with small and one with large nuclei, in the fastigial and posterior interposed nuclei and less clearly so in the lateral nucleus of the cat. Chan-Palay (1977) classified the cells of the lateral cerebellar nucleus in approximately equal groups of small and large neurons, with a separation at 180 t i m 2 surface area for the rat and 270 t i m 2 for the monkey. These binominal distributions can be explained by the presence of a population of small, GABAergic neurons and a non-GABAergic population of putative glutamatergic cells of different sizes, as recently reported by Batini et al. (1992) for the rat cerebellar nuclei (Section 5.1.3.) (Fig. 110). The targets of the projections of individual cerebellar nuclei in the brain stem and the cord differ, but some of their projections i.e. to the thalamus, show a remarkable degree of overlap. As yet there exists no corresponding differentiation in the chemoarchitecture of the cerebellar nuclei. The only system for which the neurotransmitter is known is the nucleo olivary projection that takes its origin from the population of small, GABAergic neurons (Mugnaini and Oertel, 1985). GABA also may be present in the intrinsic connections of the cerebellar nuclei and in their nucleocortical projection (Section 5.3.). 5.1. SUBDIVISION OF THE CEREBELLAR NUCLEI The cerebellar nuclei with their efferent tracts border on the ventricular surface of the cerebellum. Ventrolaterally they are continuous with the vestibular nuclei. They are surrounded by a sheet of afferent fibers from the restiform body and the middle cerebellar peduncle. This sheet is perforated by the bundles of Purkinje cell axons that terminate in the cerebellar nuclei or continue through or along these nuclei to the vestibular nuclei. The cerebellar nuclei of mammals can be subdivided according to Brunner (1919) into three, medio-laterally arranged nuclei or according to Weidenreich (1899) and Ogawa (1935) into a rostrolateral and a caudomedial nuclear group. Cytoarchitectonic criteria can be used to subdivide the cerebellar nuclei, but the presence of fiber bundles, and the disposition of their efferents in the nuclear hilus and in their efferent tracts are especially important in this respect. Brunner's (1919) mediolateral subdivision into the medial (fastigial), lateral (dentate) and interposed nuclei is based on the contours of the cerebellar nuclear mass. The three nuclei are part of a continuum and the nuclear borders therefore remain arbitrary. Although Brunner's concept of the central nuclei as a single mass has been disproved, his names for the nuclei have been retained. Weidenreich's (1899) comparative studies in mammals had already shown that fiber bundles subdivide the nuclei into a caudomedial group, that includes the medial nucleus and the caudal part of the Brunner's interposed nucleus (the nucleus interpositus posterior of Ogawa, 1935) and a rostrolateral group that is composed of the lateral cerebellar nucleus and the rostral part of the interposed nucleus (the nucleus interpositus anterior). The nuclei within each group are interconnected by cell bridges. The subdivision of Weidenreich-Ogawa can be readily appreciated in lagomorpha (Ono and Kato, 1938; Snider 1940) in carnivores (Flood and 140
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Jansen, 1961; Voogd 1964) and primates (Courville and Cooper, 1970; Chan-Palay, 1977). In rodents and insectivores the separation between the two nuclear groups is less distinct and the connections between the nuclei of each group are more extensive (Korneliussen, 1968a; Ohkawa 1957). The subdivision of Weidenreich-Ogawa received strong support from the localization of the fibers in the superior cerebellar peduncle. A small medial and a large lateral portion can be distinguished in this pathway in most mammals at its exit from the central nuclei (see* in Fig. 102). Experiments in cat (Verhaart, 1956; Voogd, 1964) and rat (Haroian et al., 1981) have shown that the medial part of the superior cerebellar peduncle takes its origin from the nuclei of the caudomedial group, mainly from the ipsilateral posterior interposed nucleus, and the lateral portion from the ipsilateral anterior interposed and lateral cerebellar nucleus. The efferent connections of the cerebellar nuclei were reviewed by Voogd et al. (1990), Ruigrok and Cella (1995), Voogd (1995) and Glickstein and Voogd (1995). The efferent connections of the lateral and interposed nuclei are summarized in Fig. 101. They give rise to nucleo olivary fibers which, at their exit from the nuclei, occupy a more ventral and medial position than the main bundle of the superior cerebellar peduncle (Legendre and Courville, 1987). After its decussation at the border of met- and mesencephalon, the superior cerebellar peduncle splits in ascending and descending branches. An uncrossed descending branch that detaches from the peduncle prior to its decussation, is only present in rodents (Ramon y Cajal, 1911). The main target of the crossed descending branch is the nucleus reticularis tegmenti pontis. This nucleus gives rise to a recurrent 141
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Fig. 102. A. Course of the direct fastigiobulbar tracts (left) and the uncinate tract (u) (right) in the stereotaxic planes P6-P8 in the cat. bc, brachium conjunctivum; bp, brachium pontis; CO, cochlear nuclei; cr, restiform body; dfb, direct fastigiobulbar tract; drV, spinal tract of the trigeminal nerve; DV, spinal vestibular nucleus; F, fastigial nucleus; FLO, flocculus; gVII, genu of the facial nerve; IA, anterior interposed nucleus; L, lateral cerebellar nucleus; LV, lateral vestibular nucleus; mlf, medial longitudinal fasciculus; MV, medial vestibular nucleus; sad, dorsal acoustic striae; SV, superior vestibular nucleus; vma, anterior medullaryvelum; vsc, ventral spinocerebellar tract. Modified from Voogd (1964). B. Hfiggqvist-stainedsection at the level of P7. Note the uncinate tract (u) arching over the brachium conjunctivum and the medial course of the direct fastigiobulbar tract (dfb). bc, brachium conjunctivum; cr, restiform body; dfb, direct fastigiobulbar tract; IA, anterior interposed nucleus; oc, olivocerebellar fibers; SV, superior vestibular nucleus; u, uncinate tract; vsc, ventral spinocerebellar tract; asterisk, medial one-third of the brachium conjunctivum, containing fibers from the posterior interposed nucleus. Voogd et al. (1990) (
mossy fiber pathway to the cerebellar cortex, with a strong collateral projection to the cerebellar nuclei. The ascending branch terminates in the red nucleus, in nuclei at the mesodiencephalic junction, including Darkschewitsch nucleus, that give rise to the medial and central tegmental tracts, which terminate in the inferior olive. The ascending branch terminates in the thalamus. Not all the nuclei contribute equally to each connection. The lateral and the anterior interposed nuclei project heavily to the nucleus reticularis tegmenti pontis and the red nucleus, whereas the posterior interposed nucleus is preferentially connected with the nuclei of the mesodiencephalic junction. The fastigial nucleus gives rise to fibers of the uncinate tract, that decussate within the cerebellum, and to the ipsilateral fastigiobulbar tract, that passes medial to the superior cerebellar peduncle in the lateral wall of the fourth ventricle (Fig. 102) (Voogd, 1964; Batton et al., 1977). The uncinate tract gives rise to a small, ascending bundle, that terminates in the central grey, the mesencephalic tegmentum and the thalamus. The majority of the uncinate- and direct fastigiobulbar tract fibers terminate in the vestibular nuclei and the reticular formation of pons and medulla oblongata. Regions containing predominantly small cells have been distinguished in the ventral parts of the cerebellar nuclei of cat (Flood and Jansen, 1961; Voogd, 1964), rat (Korneliussen, 1968a; Beitz and Chan-Palay, 1979; Voogd, 1995), several subprimates (Haines, 1977b) and primates (Courville and Cooper, 1970). These parvicellular regions are not well-defined and difficult to compare in different species. Even where they were designated as subnuclei (Flood and Jansen, 1961) these subdivisions had no clear functional basis. Data on the connections of the small cells are scanty and conflicting. Haines (1977b) proposed direct and indirect projections of the parvicellular medial and lateral cerebellar nuclei to the oculomotor nuclei. Itoh and Mizuno (1979) found a projection of the parvicellular dentate in the cat to the pulvinar and Mugnaini and Oertel (1985) noticed a concentration of small, GABAergic, nucleoolivary neurons in the ventral parvicellular part of the lateral nucleus of the rat. The group y of Brodal and Pompeiano (1957) and the basal interstitial nucleus of Langer (1985) are two nuclei which are located within the cerebellum, outside the 'classical' cerebellar nuclei. The group y was defined as a small-celled subgroup of the vestibular nuclei of the cat, that caps the restiform body dorsally. Ventrolaterally it is in contact with the dorsal cochlear nucleus. Dorsally scattered cells form strands extending to the dentate nucleus. Medially it is continuous with the small cells of the caudal pole of the superior vestibular nucleus, located dorsolateral to Deiters' nucleus also known as the group 1 of Brodal and Pompeiano (1957). In transverse sections the cells of the y group are fusiform because they are located between the fibers of the floccular peduncle (Voogd, 1964). 143
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144
The cerebellum." chemoarchitecture and anatomy
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Fig. 104. Two transverse, AChE-incubated sections through the cerebellar nuclei of the cat. A. Rostral section. B. Caudal section. Note medium-sized cells of dorsal group y in floccular peduncle and strongly AChE positive ventral group y along dorsal border of restiform body in (A); U-shaped nucleus between IP and F in (B). cr = restiform body; F = fastigial nucleus; flo+y = floccular peduncle with group y; IA = anterior interposed nucleus; IP - posterior interposed nucleus; IP/F = U-shaped nucleus between F and IP; L = lateral cerebellar nucleus; sad = stria acoustica dorsalis.
Graybiel and Hartweg (1974) found retrograde labelling in the cells of the y group of the cat after injection of retrograde tracers in the oculomotor nucleus. Matters have been further complicated by the introduction of the term 'infracerebellar nucleus' for the y group by Gacek (1977, 1979)l, and the distinction of ventral and dorsal divisions in the group y (Kevetter and Perachio, 1986; Highstein and Reisine, 1979; Highstein and 145
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J. Voogd, D. Jaarsma and E. Marani
Fig. 105. The cerebellar nuclei of Macaca fascicularis. Upper diagram is a graphical reconstruction of the cerebellar nuclei in a dorsal view. Levels of the transverse sections are indicated. The U-shaped, transitional nucleus located between the fastigial (F) and posterior inter-posed nucleus (IP) is indicated with double hatching, bc = brachium conjunctivum; BIN = basal interstitial nucleus of Langer; cr = restiform body; DV = descending vestibular nucleus; F = fastigial nucleus; IA - anterior interposed nucleus; IP = posterior interposed nucleus; L = lateral cerebellar nucleus; LV = lateral vestibular nucleus (Deiters'); MV = medial vestibular nuleus; SV = superior vestibular nucleus; Y = group y; asterisk = medial one-third of the brachium conjunctivum. )
McCrea, 1988). A more realistic approach would be to consider the cells of the y group as the bed-nucleus of the floccular peduncle, and to realize that a distinction between the group y and the superior vestibular nucleus, that receives a major part of the fibers of the floccular peduncle, remains arbitrary (Tan et al., 1995a). The basal interstitial nucleus was described by Langer (1985) in the monkey as a broadly distributed interstitial population of neurons in the white matter ventral to the cerebellar nuclei and extending from the white matter of the nodulus, into the peduncle of the flocculus. The nucleus was distinguished from the group y in the monkey. It is reciprocally connected with the flocculus. It seems likely from their descriptions that the group y of Brodal and Pompeiano corresponds to the portion of Langer's basal interstitial nucleus that lies embedded in the floccular peduncle. The 'group y' of the monkey can be considered as the enlarged, caudal pole of the superior vestibular nucleus (corresponding to the group 1 of Brodal and Pompeiano, 1957, in the cat). Some features of the cerebellar nuclei of the cat, the monkey, and the rat shall be discussed in the next sections. The cerebellar nuclei of birds were described by Feirabend (1983) and Arends and Zeigler (1991 a,b). 5.1.1. The cerebellar nuclei of the cat (Figs 103 and 104) The subdivision of Weidenreich-Ogawa can be applied to the central nuclei of the cat. This is not amazing, because Ogawa's (1935) description of the central nuclei of pinnepedia is based on earlier, unpublished, material from the cerebellum of cat and dog. Moreover the parasagittal organization of the corticonuclear and olivocerebellar projections, that provided important clues for the subdivision of the central nuclei, was first and most extensively studied in the cat (Hohman, 1929; Voogd, 1964, 1969; Courville et al., 1974; Groenewegen and Voogd, 1977). Large cells are prominent in the rostral part of the medial nucleus, small cells predominate in its ventromedial and caudal parts (Flood and Jansen, 1961). The lateral border of the medial nucleus is flush with the AChE-positive raphe which forms the lateral border of the medial A-compartment of the anterior vermis. AChE is concentrated in the lateral and ventral parts of the medial nucleus and in the neuropil of cell groups scattered in between the medial and the anterior interposed nucleus. Caudally these AChE-positive clusters coalesce in a U-shaped nucleus located at the transition of the medial and the posterior interposed nucleus (Fig. 104B). The medial limb of the U forms the lateral border zone of the fastigial nucleus, the lateral limb usually is included with
1 Gacek (1977, 1979) used the term infracerebellar nucleus for the cells embedded in the fasciculus angularis (i.e. the floccular peduncle) as it arches over the restiform body and applied the term 'y group' to the cells of the group 1 of Brodal and Pompeiano (1957).
146
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the posterior interposed nucleus. The large cells in this area are the target of the Purkinje cells in the X-zone of the anterior vermis (Trott and Armstrong, 1987a,b). The cells in the lateral limb of the U, that extend rostrally as the AChE-positive cell groups medial to the anterior interposed nucleus, may provide the target for the 'C ~' zone, the lateral portion of the Cl-zone, that shares its afferent, climbing fiber input with the X-zone (Ekerot and Larson, 1982; Campbell and Armstrong, 1985; Trott and Armstrong, 1987a) (Section 6.3.3.1., Fig. 174). The cells of this area project to the spinal cord (Matsushita and Hosoya, 1979) and can be double-labelled from the cord and the thalamus (Bharos et al., 1981). Apart from the medial limb of the U-shaped subnucleus, the medial nucleus at this level consists of a dorsally directed tail that extends into the white matter of the posterior lobe vermis. It receives Purkinje cell axons from the vermal visual area of lobule VII (Voogd, 1964; Courville and Diakew, 1976). It constitutes one of the targets of the collateral projection from the nucleus reticularis tegmenti pontis (Gerrits and Voogd, 1987). Other parts of the medial nucleus receive their mossy fiber collaterals from the lateral reticular nucleus (Ktinzle, 1975; Russchen et al., 1976) and the spinal cord (Matsushita and Ikeda, 1970). The ventral, parvicellular part of the medial nucleus is located at the base of the lobules IX and X. The posterior and anterior interposed and the lateral cerebellar nucleus are clearly delimited. The border between the anterior and posterior interposed nucleus is not located in a frontal plane, but passes obliquely forward, from caudolaterally to rostromedially (Fig. 103). The posterior interposed nucleus, therefore, extends far rostrally, medial to the anterior interposed nucleus. The caudal pole of the anterior interposed nucleus is located far laterally, adjacent to and merging with the lateral nucleus. AChE is concentrated along the borders of the nuclei and in the ventrolateral portions of the lateral and posterior interposed nuclei. Elongated, AChE-positive cells in the floccular peduncle, ventral to the lateral nucleus, belong to cell group y of the vestibular nuclei of Brodal and Pompeiano (1957) (Fig. 104A). The medium-sized cells of group 1 which are located dorsolateral to Deiters' nucleus, should be distinguished from the group y. They constitute a caudal extension of the superior vestibular nucleus and display strong AChE activity.
5.1.2. The eerebellar nuclei of primates The cerebellar nuclei in primates were described by Courville and Cooper (1970) and Chan-Palay (1977, Macaca mulatta), Haines (1971, Galago), and Haines and Dietrichs (1991, Saimiri sciurus). The cerebellar nuclei of the human cerebellum were reviewed by Larsell and Jansen (1972) and Voogd et al. (1990). The four nuclei of the subdivision of Weidenreich-Ogawa can be recognized in macaque fascicularis and their topograph-
Fig. 106. Horizontal (A) and transverse (B,C) AChE-stained sections through the cerebellar nuclei of Macaca fascicularis. Note connection of IA and dorsal pole of dentate nucleus in (B) and of medial lamella of the dentate nucleus and border region of IA and IP in (A), U-shaped nucleus located between F and IP in (B) and (C) and strong AChE-reactivity in this nucleus in (A); localization of medial limb of this U-shaped nucleus in X compartment (B and C); extension of AChE-positive C2 compartment in border region of 1A and IP in B; interstitial nucleus of Langer in (B) and large group y in (C). C2 = C2 compartment; F = fastigial nucleus; IA = anterior interposed nucleus; IP = posterior interposed nucleus; IP/F = U-shaped nucleus between F and IP; L = lateral cerebellar nucleus; Lp = parvocellular part of lateral cerebellar nucleus; y = group y; X = X compartment. 148
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Fig. 107. The cerebellar and vestibular nuclei of the rat. A graphical reconstruction of a dorsal view of the nuclei is placed at the top. The levels of the transverse sections are indicated with numbers. The different nuclei are indicated with different shadings. The transitional region between the fastigial (MM) and posterior interposed (IP) nuclei (the interstitial cell groups of Buisseret-Delmas et al., 1993) indicated by double hatching. Asterisk indicates efferents from posterior interposed nucleus. 8vn = interstitial nucleus of the vestibular nerve; bp = brachium pontis; CM = caudomedial part of the medial cerebellar nucleus; CO = cochlear nuclei; cr = restiform body; das = dorsal acoustic striae; DLB = dorsolateral protuberance of themedial cerebellar nucleus; DLH = dorsolateral hump; DLP = dorsolateral protuberance; DMC = dorsomedial crest; DV = descending vestibular nucleus; IA = anterior interposed nucleus; IP = posterior interposed nucleus; L = lateral cerebellar nucleus; LV = lateral vestibular nucleus of Deiters; M = medial cerebellar nucleus; M/IP = transitional region of M and IP; MM = medial part of the medial cerebellar nucleus; MV = medial vestibular nucleus; PH = nucleus prepositus hypoglossi; scp = superior cerebellar peduncle; SV = superior vestibular nucleus; unc.= uncinate tract; Y = group y. Redrawn from Ruigrok and Voogd (1990). 150
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of a core and a shell; the neuropil of the shell is connected with the medial limb of the dentate nucleus. The anterior interposed nucleus consists of medial and lateral portions, separated by a notch in the dorsal surface. The caudal pole of the anterior interposed nucleus merges with the medial limb of the dentate nucleus. The dentate nucleus displays ventrolaterally and caudally directed bulges. The hilus of the dentate nucleus is directed rostromedially, caudally it is closed by the medial limb of the dentate nucleus. In its caudal portion the structure of the medial limb is compact, with AChE-positive cell bodies and neuropil concentrated along its borders, similar to the rest of the dentate nucleus. Rostrally where the medial limb surrounds and merges with the anterior interposed nucleus, its structure is loose and AChE-staining less prominent, A subdivision of the dentate nucleus of the monkey in the two parts that were distinguished in the human dentate nucleus, i.e. in the rostromedial neodentatum with large neurons and the caudolateral palaeodentatum, with densely packed, smaller neurons (Gans, 1924; Demol6, 1927a,b) is not feasible and is not warranted by Chan-Palay's (1977) detailed cytological analysis of this nucleus. The so-called group y is large and compact and occupies the rostral part of the floccular peduncle. Medially it is continuous with the caudal pole of the superior vestibular nucleus. The small AChE-positive cells of Langer's (1985) basal interstitial nucleus appear more caudally and extend from the flocculus into the roof of the fourth ventricle, ventral to and in between the dentate and posterior interposed nuclei. 5.1.3. The cerebellar nuclei of the rat
The cerebellar nuclei of the rat were described by Goodman et al. (1963), Korneliussen (1968a) and Voogd (1995). They are very similar to the nuclei of the cerebellum of the mouse, illustrated by Marani (1982a). The morphology and the cytology of the lateral and the medial nucleus of the rat were analysed in Chan-Palay's (1977) monograph on the dentate nucleus and in the paper of Beitz and Chan-Palay (1979). The cerebellar nuclei of the rat are difficult to subdivide according to the scheme of WeidenreichOgawa. They constitute, more or less, a single mass, with a number of protrusions, that were first named by Goodman et al. (1963) as the dorsolateral protuberance of the medial nucleus, and the dorsomedial crest and the dorsolateral hump, two excrescences of the interposito-dentate complex (Figs 107 and 108). Similar subnuclei were distinguished in the cerebellum of the mouse (Marani, 1986). The connections of the cerebellar nuclei in the rat differ in some respects from carnivores and primates. The medial nucleus was subdivided by Korneliussen (1968a) into a caudoventral parvicellular part, the magnocellular dorsolateral protuberance and a 'middle' part that contains cells of intermediate size. The dorsolateral protuberance is situated more laterally. It projects into the white matter of the posterior lobe and receives Purkinje cell fibers from a medial zone of the lobules VI and VII of the posterior lobe hemisphere (Goodman et al., 1963; Armstrong and Schild, 1978a,b; Buisseret-Delmas, 1988a,b) (see also Section 6.1.4.). The posterior interposed nucleus is best distinguished in horizontal sections. A cellfree zone separates it from the anterior interposed and lateral nuclei. Medially the posterior interposed nucleus is continuous with the fastigial nucleus. Cells at the junction of these two nuclei project to the spinal cord (Matsushita and Hosoya, 1978 and Bentivoglio and Kuypers, 1982). This region was distinguished as the interstitial cell group, the target nucleus of the X zone, by Buisseret-Delmas et al. (1993). At the junction of the anterior interposed and lateral nuclei the dorsolateral hump 151
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forms a distinct ridge on the rostral aspect of these nuclei. A more caudally located cluster of large neurons usually is indicated by the same name. Some authors included the dorsolateral hump with the anterior interposed nucleus (Goodman et al. 1963; Voogd, 1995), others delegated it to the lateral nucleus (Chan-Palay, 1977; Angaut and Cicirata, 1982), sometimes it was considered as a separate subdivision, peculiar to rodents. It gives rise to the uncrossed descending limb of the superior cerebellar peduncle (Woodson and Angaut, 1984; Bentivoglio and Molinari, 1986; Rubertone et al., 1990), that detaches from the superior cerebellar peduncle before its decussation. The uncrossed descending limb terminates in the lateral reticular formation (Ramon y Cajal, 1911; Mehler, 1967, 1969; Achenbach and Goodman, 1968; Faull, 1978; Chan-Palay, 1977). The ventromedial part of the lateral nucleus consists of smaller cells. Those in the region of the hilus correspond to the small intrinsic neurons described by Chan-Palay (1977), the densely packed small cells located ventral to the hilus may be identical to the concentration of GABAergic nucleo-olivary cells that was illustrated in this region by Mugnaini and Oertel (1985). AChE staining does not distinguish the parvicellular part of the lateral nucleus, but the medium-sized cells and the neuropil of the group y, located ventral to it display a high AChE activity. In a rostral direction the group y (Brodal and Pompeiano, 1957) becomes continuous with the superior vestibular nucleus). Deiters' nucleus with its large AChE-positive perikarya in an unstained neuropil is wedged in between the AChE-rich areas of the group y and the medial vestibular nucleus and reaches far dorsally into the hilus region of the central nuclei. Purkinje cell fibers enter Deiters' nucleus as perforating fibers, passing in between the dorsolateral protuberance and the anterior interposed nucleus, and through the middle part of the medial nucleus. More rostrally, where Deiters' nucleus has disappeared, the AChE-rich neuropil of the superior and medial vestibular nuclei meet at the oblique border between the two nuclei. 5.2. THE GABAERGIC NUCLEO-OLIVARY PROJECTION NEURONS OF THE CEREBELLAR NUCLEI Cell size has generally been considered of less importance for the subdivision of the central nuclei, than the presence of local differences in cell density resulting from sheets or bundles of fibers between and around the nuclei. Interest in the question whether different cell-types can be distinguished in the cerebellar nuclei on the basis of size, dendritic and axonal morphology and neurotransmitter content was renewed since the observation that the projection of the cerebellar nuclei to the inferior olive arises from a population of small GABAergic neurons. The nucleo-olivary projection was discovered with anterograde tracing with tritiated amino acids by Graybiel et al. (1973) and with retrograde tracing by Gould and Graybiel (1976) and Tolbert et al. (1976b), all in the cat. The nucleo-olivary and olivo nuclear projections are reciprocally organized (see Section 6.3.3.). Tolbert's (Tolbert et al., 1978a) and Courville and Cooper's (1970) quantitative analysis clearly showed that all sizes of neurons were present in all central nuclei in monkey and cat. Histograms of the soma diameter of the nuclear neurons projecting to the thalamus and the cerebellar cortex are very similar to the overall size distribution of these neurons. The cells in the cerebellar nuclei of the cat that project to the inferior olive, however, constitute a population of small, spindle shaped neurons (Fig. 109). This 154
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population of small cells was recognized in retrograde tracer experiments in opossum (Martin et al., 1976, 1980) cat (Tolbert et al., 1976b; Tolbert et al., 1978a; Bharos et al., 1981; Legendre and Courville, 1987), rat (Angaut and Cicirata, 1982; Hess, 1982b; Brown et al., 1977; Bentivoglio and Kuypers, 1982; Buisseret et al., 1989; Swenson and Castro, 1983b) and monkey (Chan-Palay, 1977). The small cells were depleted in the cerebellar nuclei of lurcher mutant mice, probably due to the degeneration of the inferior olive (Heckroth, 1994). According to Tolbert et al. (1978a) the nucleo-olivary neurons in the cat are concentrated in the ventral parts of the dentate and posterior interposed nuclei, they are scarce in the fastigial nucleus. Concentrations of these small neurons were also reported in the rostral and caudal poles and the hilar portion of the dentate nucleus and the lateral parts of the interposed nucleus in the rat (Brown et al., 1977; Chan-Palay, 1977). A more diffuse distribution of these cells was noticed in experiments with retrograde tracing of Martin et al. (1976) in the dentate and interposed nuclei of the opossum and in double labelling studies with fluorescent dyes in rat (Bentivoglio and Kuypers, 1982) and cat (Bharos et al., 1981). Neurons of the cerebellar nuclei and the lateral cerebellar nucleus of rat and cat that react with antibodies against GAD or conjugates of GABA, generally are small (Mugnaini and OerteI, 1981, 1985; Houser et al., 1984; Gabbott et al., 1986; Kumoi et al., 155
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Fig. 110. Diameter distributions of GABA-like immunoreactive (GABA-IR) and glutamate-like immunoreactive (Glu-IR) neurons in the nucleus medialis (NM), the nucleus interpositus (NI) and the nucleus lateralis (NL) of rat cerebellum. The populations of GABA-IR (A) and Glu-IR (B) neurons were clearly peaked and similar in size in all three nuclei. In C the spectra from the three nuclei are averaged for both populations and plotted together. The size range of the GABA and Glu overlap is the same as for cells positively identified as colocalizing GABA and Glu. Abscissae: diameter of the neurons in p m (class interval 2.5/lm). Ordinates: percentage of neurons in each diameter class. Batini et al. (1992).
1987, 1988; Buisseret-Delmas et al., 1989; Walberg et al., 1990; Takayama, 1994; Moffett et al., 1994). Batini et al. (1992), who measured cell diameters of GABA and single labelled glutamate-immunoreactive neurons, showed that they represent two populations with little overlap in all cerebellar nuclei of the rat (Figs 110 and 111). Consistent with glutamate being a metabolic precursor for GABA, most of the GABAergic neurons co-localized glutamate. GAD or GABA-like immunoreactive cells are scattered through all parts of the cerebellar nuclei, but have been reported to be more sparse in the fastigial nucleus, where they are concentrated in its ventral portion and to be preferentially located in the hilus and in the ventral parvocellular subnucleus of the lateral cerebellar nucleus of the rat (Mugnaini and Oertel 1985; Buisseret-Delmas et al. 1989). These small 156
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 111. Typical distribution of glutamate-like and GABA-like immunoreactive neurons in the nucleus lateralis of rat cerebellum. Consecutive sections immunostained for glutamate (A) and GABA (B) are shown as mirror images. Note the difference in size of the stained neurons. Batini et al. (1992).
neurons proved to be the origin of the GABAergic, nucleo-olivary pathway in the rat (Nelson et al. 1984) and the rabbit (Nelson and Mugnaini, 1989). De Zeeuw et al. (1988, 1989b) demonstrated that all the terminals in the inferior olive that could be traced from the cerebellar nuclei by anterograde transport of WGA-HRP could be labelled with a polyclonal antibody against GABA. The nucleo-olivary projection, therefore, seems to be entirely GABAergic. Some of the small GABAergic perikarya of the cerebellar nuclei and the lateral vestibular nucleus also contain glycine (Ottersen et al., 1987; Walberg et al., 1990; Chen and Hillman, 1993b; Takayama, 1994), but glycine could not be detected in terminals of the nucleo-olivary pathway in the rat (De Zeeuw, unpublished observations). Projections to certain subdivisions of the inferior olive also take their origin from the vestibular nuclei (Saint Cyr and Courville, 1979; Gerrits et al. 1985a) that contains a population of small GABAergic neurons in all its subnuclei (Kumoi et al., 1987, guinea pig). Some of these connections are also GABAergic (Nelson et al., 1986), but it is not known whether all vestibulo-olivary connections use this neurotransmitter. One of the main sources for the vestibulo-olivary projection is the nucleus prepositus hypoglossi (Saint Cyr and Courville, 1979; McCrea and Baker, 1985; Gerrits et al., 1985a). Both GABA (de Zeeuw et al., 1993) and acetylcholine (Barmack et al., 1991) have been identified as neurotransmitters in this pathway. At present it seems unlikely that the small nucleo-olivary neurons possess collaterals that terminate in other targets. Tolbert et al. (1978a), however, found many of these neurons in the cat to be antidromically activated from the thalamus and the cerebellar cortex in addition to the inferior olive. Ban and Ohno (1977) also produced electrophysiological evidence for collateralization of nucleo-olivary cells to the red nucleus or more rostral structures. Bharos et al. (1981) and Bentivoglio and Kuypers (1982), were unable 157
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to double label these small neurons, after combined injections of one fluorescent tracer in the caudal medulla, in a region including the inferior olive, and a second injection with another fluorescent tracer in either thalamus with tectum or the spinal cord. These results were corroborated for the combination of thalamus and inferior olive by Legendre and Courville (1987) for the cat and the red nucleus and the inferior olive by Teune et al. (1995) for the rat. The existence of GABAergic projections of the cerebellar nuclei to other precerebellar relay nuclei is not excluded by these experiments. Projections from GABAergic neurons of the lateral cerebellar nucleus of the rat to the basal pontine nuclei were demonstrated by Border et al. (1986). 5.3. NUCLEOCORTICAL AND INTRINSIC NEURONS OF THE CEREBELLAR NUCLEI Nucleocortical neurons occasionally have been described in Golgi material (Ramon y Cajal, 1911) and were considered as the origin of the climbing fibers by Carrea et al. (1947). Their existence was confirmed by retrograde transport of HRP injected in various parts of the cerebellar cortex in the cat (Gould and Graybiel, 1976; Tolbert et al., 1976a, 1978b; Dietrichs, 1981a,b, 1983a; Dietrichs and Walberg, 1979a, 1980; Trott and Armstrong, 1990), in primates (Tolbert et al., 1977, 1978b; Tolbert and Bantli, 1979; Haines, 1978b, 1988; Haines and Pearson, 1979), and in the rat (Chan-Palay, 1977; Hess, 1982a; Buisseret-Delmas and Angaut, 1988, 1989a). The nucleocortical projection was reviewed by Tolbert (1982). The size of the nucleocortical cells in the cat follows the same frequency distribution as the central nuclear cells as a whole and as the neurons that could be retrogradely labelled from the thalamus (Fig. 109). Single neurons, intracellularly stained with HRP, with axons leaving the central nuclei in the superior cerebellar peduncle, were shown to emit collaterals, that could be traced to the cerebellar cortex (McCrea et al., 1978). Antidromic invasion and collision experiments (Tolbert et al., 1977, 1978a) also favoured a collateral origin of the nucleocortical fibers from projection neurons. There is some evidence for GABA or acetylcholine as the neurotransmitter of the nucleocortical fibers, but the majority, probably, uses glutamate or aspartate, in accordance with their collateral origin from large relay cells of the cerebellar nuclei. 20% of the nucleocortical neurons in the medial cerebellar nucleus that were WGA-HRP labelled from the cerebellar cortex of the rat, reacted with an antibody raised against GABA (Angaut et al., 1988). Most if not all nucleocortical neurons in the rat were found to be immunoreactive for an antibody to a conjugate of GABA in the experiments of Batini et al. (1989). She labelled GABAergic cells of all sizes from the cortex, although fewer retrogradely labelled cells were present among the smaller GABAergic neurons, most of which presumably project to the inferior olive. The proportion and the size distribution of nucleocortical neurons of the rat labelled with the same antibody to a conjugate of GABA were reported to be different in a later publication of the same authors (Batini et al., 1992). Again, the retrogradely labelled nucleocortical neurons were of all sizes with a peak around a diameter of 20-25 r With almost 50%, the population of single labelled glutamate-immunoreactive nucleocortical neurons far exceeded the GABAergic population. The glutamate-immunoreactive neurons were also larger and peaked at a diameter of 20-25 r The GABAergic-nucleocortical cells may be identical to the small nucleocortical neurons that were identified in the posterior interposed nucleus and along the boundaries of the lateral nucleus of the rat by retrograde transport of an antibody to GAD 158
The cerebellum." chemoarchitecture and anatomy
Ch. I
(Chan-Palay et al., 1979). Antegrade transport of an antibody to GAD in nucleocortical fibers resulted in labelling of a small number of mossy fiber rosettes in the cortex (Chan Palay et al., 1979). Antegrade transport in nucleocortical fibers of other, non-specific tracers also resulted in the labelling of mossy fiber rosettes (Kultas-Illinsky et al., 1979; Tolbert et al., 1980). The morphology of these nucleocortical mossy fiber rosettes was not distinctive and they never displayed any of the features of GABAergic terminals, such as pleomorphic vesicles and symmetrical synapses. H~mori and Tak~cs (1989) and H~.mori et al. (1990) distinguished 4 types of mossy fiber rosettes in the cerebellar cortex of rat and cat with immunohistochemistry of glutamate and GABA and deafferentation of the cerebellum. Two of their types of mossy fiber rosettes, i.e. large, GABA-immunoreactive rosettes, with round or pleomorphic synaptic vesicles that accounted for 3% of all mossy fiber rosettes in the cat and small rosettes with small, pleomorphic synaptic vesicles, that reacted with an antibody to a conjugate of glutamate and accounted for less than 10% of the mossy fiber rosettes in the rat, presumably represented the endings of nucleocortical fibers. Mossy fibers of extracerebellar origin terminated as large, glutamate-immunoreactive rosettes; a fourth type of small, GABAergic rosettes with small synaptic vesicles was of intracortical origin. According to Ikeda et al. (1991) the cholinergic, ChAT-immunoreactive afferent fibers in the cortex of the cerebellum of the cat, that include mossy fibers, disappear after electrolytic or kainate lesions of the cerebellar nuclei. Different types of nucleocortical neurons have been distinguished on the basis of their localization with respect to the corticonuclear projection. Most nucleocortical cells were considered to be of a reciprocal type since they were located within an area receiving Purkinje cell projections from the same region of the cortex from which they can be retrogradely labelled (Dietrichs, 1981a,b, 1983a; Dietrichs and Walberg, 1979a, 1980; Tolbert, 1982; Buisseret-Delmas and Angaut, 1988, 1989a; Haines, 1988). Other nucleocortical neurons were found outside the corticonuclear projection area, either ipsilaterally (the non-reciprocal neurons of Buisseret-Delmas and Angaut, 1988, 1989a, see also Hess, 1982a, Dietrichs and Walberg, 1979a, 1980) or even contralaterally (symmetrical neurons: Buisseret-Delmas and Angaut, 1988, 1989a rat; Tolbert et al., 1978b cat and monkey; Haines, 1978a,b; Haines and Pearson, 1979, treeshrew; Haines, 1988, Galago; Dietrichs, 1983a; Dietrichs and Walberg, 1980, cat). Non-reciprocal and contralateral neurons in cat and rat were most numerous in the fastigial and posterior interposed nuclei. Contralateral neurons in the posterior interposed nucleus of Galago were small and fusiform and resembled the nucleo-olivary neurons (Haines, 1988). Non-reciprocal connections were the rule in primates where they originated mainly from the ventral part of the dentate nucleus. Reciprocity in general was more noticeable in cat and rat. A more restricted origin of the sparse nucleocortical projection to the electrophysiologically identified C~ and C2 zones and the strong bilateral projection to the C2 zone of the paravermal cortex of the cat from the posterior interposed and fastigial nuclei was advocated by Trott and Armstrong (1990). The existence of a rest group of neurons that remains unaffected by large lesions of the efferent cerebellar pathways in the kitten has been claimed as evidence in favour of the presence of intrinsic or nucleocortical neurons in the central nuclei (Jansen and Jansen 1955). Many of these neurons were found to be large and to be located in the posterior interposed nucleus. Intrinsic neurons of the cerebellar nuclei have been observed in Golgi preparations of the rat by Chan-Palay (1973a, 1977) as small multipolar neurons in the dentate nucleus. The terminals of these intrinsic, inhibitory neurons on the soma and dendrites of cerebellar nuclear cells were tentatively identified as small 159
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boutons with few elliptical vesicles in a light matrix (E-type, Chan-Palay, 1973b, 1977). Wassef et al. (1986), however, were unable to distinguish between these E-types and Purkinje cell terminals (Chan-Palay's A-type) in the cerebellar nuclei of mutant Pcd mice. Some 15% of the small and large GAD-positive terminals in the cerebellar nuclei of these mutants were spared, although these mice lost 99% of their Purkinje cells. Intrinsic GABAergic connections of the nuclei should have been spared in these animals, but the possibility that these 15% represent sprouting from another GABAergic cell population cannot be excluded. Non-GABAergic intrinsic connections through boutons with spherical vesicles carrying Gray-type I synapses were postulated by Chan-Palay (D-type, 1973b); no other evidence is available on this system. Strong evidence for the presence of a population of small glycinergic interneurons in the cerebellar nuclei of the rat was supplied by Chen and Hillman (1993b). Glycine immunoreactivity was restricted to a population of small neurons throughout the cerebellar nuclei and was present in boutons outlining somata of large relay cells. Intense glycine receptor immunoreactivity was observed in these large cells, opposite the glycinergic terminals. Many, but not all of these small, glycinergic neurons colocalize GABA. Since glycine-immunoreactivity was not observed in nucleo-olivary terminals (De Zeeuw, unpublished observations) it seems likely that these glycine and glycine/GABA containing neurons are interneurons. Rather similar observations were made for calretinin in a subpopulation of small nuclear cells, that give rise to a dense plexus in the cerebellar nuclei of the rat (Floris et al., 1994). 5.4. NON-GABAergic PROJECTION N E U R O N S OF THE CEREBELLAR NUCLEI
Glutamate and aspartate have been suggested as the neurotransmitter of the nonGABAergic projection neurons. Immunoreactivity with an antibody to a conjugate of glutamate was found in neurons of all sizes in all cerebellar nuclei of the rat (Batini et al., 1992) (Fig. 110) and with antibodies to conjugates of aspartate in large neurons of the cerebellar nuclei of the rat (Kumoi et al., 1988; Chen and Hillman, 1993b). A reaction product of glutamate with carbodiimide (gamma-Glu-Glu) has been localized with immunocytochemistry in neurons of all sizes in all the cerebellar nuclei of carbodiimide-perfused rats (Monaghan et al., 1986, see also Madl et al., 1986, 1987). Moderate, glutaminase (GLNase)-like immunoreactivity was present in scattered small cells of the cerebellar nuclei of the rat (Kaneko et al., 1989). Cells reacting with an antibody against aspartate aminotransferase (AATase), that catalyses the conversion of glutamate to aspartate, are more numerous than the GLNase and gamma-Glu-Glucontaining cells, but not all cerebellar nuclear neurons were found to contain AATase (Monaghan et al., 1986). Since AATase-like immunoreactivity has been found in Purkinje, basket and stellate cells of the cerebellar cortex, this enzyme apparently is also present in GABAergic neurons. Monaghan et al. (1986) tentatively concluded that three types of neurons can be distinguished in the cerebellar nuclei of the rat. The first group includes putative glutamatergic neurons containing GLNase- and gamma-Glu-Glu-like immunoreactivity, the second group corresponds to the GABAergic neurons that contain AATase-like activity and the third group are the neurons that do not react with any of the three antibodies and whose neurotransmitters remain to be determined. They could not exclude the presence of aspartate-containing neurons in the cerebellar nuclei because elevated levels of AATase and GLNase can be present both in aspartatergic as well as in glutamatergic neurons. The large cells of Deiters' nucleus in guinea pig (Kumoi 160
The cerebellum." chemoarchitecture and anatomy
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Fig. 112. Retrograde transport of [3H]choline (A,B) to perikarya (arrow-heads) in medial part of nucleus interpositus (Int) or in lateral cerebellar nucleus (LatC) and of wheat germ agglutinin-coupled HRP (E,F), but absence of retrogradely labelled elements in corresponding regions after application of D-[3H]aspartate to the red nucleus (C,D). Rat, bright-field (A,E) as well as dark-field (B,F) illumination. Cresyl violet counterstaining. Bars = 0.5 mm. Bernays et al. (1988).
et al., 1987) and cat (Walberg et al., 1990), reacted with antibodies raised against conjugates of aspartate. The same giant cells in the cat also stained with antibodies to conjugates of glutamate (Walberg et al., 1990). The presence of excitatory amino acid transmitters in the projection neurons of the cerebellar nuclei was also investigated by retrograde transport of D-[3H]aspartate in the cerebellorubral pathway, but no such transport was observed in the rat (Bernays et al., 1988 (Fig. 112C,D)). Interruption of this pathway, however, resulted in a decrease of high affinity glutamate uptake in the caudal, magnocellular portion of the red nucleus (Nieoullon et al., 1984), which could be explained by a loss of cerebellorubral, glutamate containing terminals. High affinity uptake of glutamate in other cerebellar target nuclei 161
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162
J. Voogd, D. Jaarsma and E. Marani
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 113. A. Darkfield micrograph of a frontal section through the cerebellar white matter and nuclei of an experiment with an injection of Phaseolus vulgaris leucaglutinin into part of the principal nucleus of the inferior olive and the rostral dorsal accessory olive in the rat. Labelled axons can be seen in the restiform body (cr). Bundles of labelled axons directed toward the cerebellar hemispheres seem continuous with innervated areas of the lateral cerebellar nucleus (L), the dorsolateral hump (DLH) and the anterior interposed nucleus (NIA). At this low magnification, the plexuses of nerve terminals in the nuclei appear as 'clouds' of fine dots representing individual varicosities. In the lateral cerebellar nucleus, the sector receiving labelled terminals is sharply demarcated from neighbouring tissue that does not receive labelled innervation, suggesting a detailed topographical organization of the PO projection to the lateral cerebellar nucleus. Bar = 250/tm. B. Photomontage of olivary axon in the border of the white matter and interposed nuclei of rat cerebellum which gives off a thin collateral toward an area of innervation. The branching point is indicated by an arrow. The thin collateral follows a tortous course (for a short distance it is obscured by an underlying thicker axon) and demonstrates several varicosities that may be the sites of synaptic interaction. Bar = 20/Lm. C. Darkfield micrograph of a sector of the lateral cerebellar nucleus in a sagittally sectioned specimen. Thicker olivocerebellar axons (arrow) run in the overlying white matter and some traverse the neuropil in the right part of the micrograph. These arriving olivary axons seem to give rise to the dense plexus of thin varicose terminal fibers, which extend over most of the illustrated neuropil. Bar = 20/tm. Van der Want et al. (1989a). (
such as the rostral parvocellular portion of the red nucleus and the ventrolateral nucleus of the thalamus, however, shows an increase rather than a decrease after lesions of the cerebellorubral and thalamic pathway. Several explanations were offered for this phenomenon, one of which involves the loss of a cholinergic, cerebellar efferent pathway that would facilitate glutamate release from corticothalamic or corticorubral terminals in these nuclei (Nieoullon and Dusticier, 1981; Nieoullon et al., 1984). The existence of cholinergic efferents among the projections of the cerebellar nuclei received support from observations by the same authors of a temporary decrease of ChAT activity in the red nucleus, especially in its rostral part, after lesions of the cerebellorubral fibers (Nieoullon and Dusticier, 1981). Moreover specific retrograde transport of [3H]choline in the cerebellorubral pathway to large perikarya in the anterior half of the lateral cerebellar nucleus of the rat was reported by Bernays et al. (1988) (Fig. 112) and to cerebellar nuclei in the cat by Stanton and Orr (1985). Evidence for the existence of cholinergic neurons in the cerebellar nuclei is controversial; ChAT was found to be absent in these cells (Kimura et al., 1981, cat; Armstrong et al., 1983, rat), but to be present in the large cells of Deiters' nucleus (Kimura et al., 1981). Ikeda et al. (1991) reported the presence of ChAT-immunoreactive neurons in the cerebellar nuclei of the cat; these cells give rise to both thalamic projections and nucleocortical collaterals. One group of small neurons in the rhesus monkey, which was described by Langer (1985) under the name of the basal interstitial nucleus (see Section 5.1 .), displays uniform and strong AChE activity. These small cells lie dispersed in the white matter of the flocculus and the nodulus and ventral to the dentate and the posterior interposed nucleus, in the roof of the fourth ventricle. The presence of strongly AChE-positive but ChAT-negative small cells in the white matter of the flocculus of the rat, that may correspond to Langer's interstitial nucleus, was noticed by Komei et al. (1983). The interstitial nucleus should be distinguished from the group y (Brodal and Pompeiano, 1957), which is a lateral extension of the superior vestibular nucleus, located ventral to the dentate nucleus. The cells of group y are slightly larger AChE-positive neurons. In accordance with other parts of the superior vestibular nucleus, group y contains a small number of GAD-positive cells, but most of its cells display aspartatelike immunoreactivity (Kumoi et al., 1987). 163
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Reports on the presence of other transmitters or transmitter-related substances have been published. Some neurons in the medial and posterior interposed nucleus of the opossum were immunoreactive with antibodies to CCK (King and Bishop, 1990, Fig. 116). Some somatostatin-like immunoreactive neurons were present in the medial part of the fastigial nucleus of the rat (Vincent et al., 1985). Occasional cells labelled with antibodies against conjugates of taurine were observed by Ottersen et al. (1988b) in rat cerebellum. Glycine-immunoreactivity was observed in large neurons of the ventral fastigial nucleus of the rat (Chen and Hillman, 1993b). PKC subtypes in neurons of the cerebellar nuclei have been specified by Kose et al. (1988), Shimohama et al. (1990), Huang et al. (1991), Merchenthaler et al. (1993), Garcia et al. (1993) and Chen and Hillman (1993a) (Table 1). 5.5. A F F E R E N T CONNECTIONS OF THE CEREBELLAR NUCLEI: P U R K I N J E CELL AXONS The Purkinje cell axons constitute the main afferent system of the cerebellar nuclei. Other afferents include collaterals of mossy and climbing fibers, a projection from the contralateral red nucleus (see Section 5.6. and 6.4.4.) and certain monoaminergic systems (see Section 3.8.). The terminals of the Purkinje cell axons in the cerebellar nuclei and the lateral vestibular nucleus contain pleomorphic synaptic vesicles, most of which are elliptical and are provided with Gray type II or an intermediate type of synapse. These terminals cover most of the soma and the large dendrites, extend on the spines and the small dendrites and are found as axo-axonal synapses on the initial segment. These terminals first were identified by antegrade axonal transport of [3H]-GABA in the rat (McGeer et al., 1975) and tritiated leucine in the cat (Walberg et al., 1976). Most of the boutons lining the surface of cells of the cerebellar nuclei contain GAD or GABA-like immunoreactivity (Saito et al., 1974; Wassef et al., 1986; Fonnum et al., 1970; Fonnum and Walberg, 1973; Houser et al., 1984; Roffler-Tarlov et al., 1979; Kumoi et al., 1987; Hawkes and Leclerc, 1986). Most if not all Purkinje cells are GABAergic (Chan-Palay, 1984; Mugnaini and Oertel, 1985), but with respect to other substances they constitute a heterogeneous population (see Sections 3.1.2. and 3.1.3.). The possibility of a differential distribution in the cerebellar nuclei of the terminals of chemically different populations of Purkinje cells has been studied for taurine and for the Purkinje cell specific antibody mabQ 113 (anti-Zebrin). For taurine the results were conflicting. The distribution of taurine in parasagittal bands of Purkinje cells in the cerebellar cortex was based upon the presence in these cells of the synthesizing enzyme of taurine CSADase (Chan-Palay et al., 1982a,b). No such a differential distribution was noticed, however, for taurine-like immunoreactivity in Purkinje cells (Madsen et al., 1985; Ottersen and Storm-Mathisen, 1987) or for taurine-containing terminals in the cerebellar nuclei (Ottersen et al., 1988b). Zebrin-immunoreactive and non-immunoreactive Purkinje cells are distributed in parallel longitudinal bands in the cortex of rat cerebellum (Hawkes et al., 1985) and distribute their axons to different parts of the cerebellar nuclei (Hawkes and Leclerc, 1986). Light microscopical observations showed that all Zebrin positive boutons on the soma and dendrites of large central nuclear cells contained GAD, and that most GADpositive boutons on individual cells either were Zebrin-positive or -negative. The two populations of Purkinje cells, therefore, terminate on different central nuclear cells. Zebrin-positive Purkinje cells of the vermis projected to the caudal part and a majority of Zebrin-negative Purkinje cells to the rostral part of the fastigial nucleus of the rat. 164
The cerebellum." chemoarchitecture and anatomy
Ch. I
Hawkes and Leclerc (1986) estimated that Zebrin-positive terminals made up the great majority of the GAD-positive boutons on the class of Zebrin receptive neurons. This suggests that intrinsic GABAergic terminals are few and that extracerebellar and intrinsic non-GABAergic connections account for at most 20% of the terminals on these cells. GABA receptors in the cerebellar nuclei were not systematically studied (see Section 3.7.). 5.6. EXTRACEREBELLAR AFFERENTS OF THE CEREBELLAR NUCLEI: COLLATERALS OF MOSSY AND CLIMBING FIBERS Extracerebellar afferents of the cerebellar nuclei consists of the collaterals of mossy and climbing fiber systems, that provide their excitatory drive (Eccles et al., 1967), the cholinergic (Section 3.10.), and the monoaminergic afferent systems. The existence of extracerebellar afferents to the central nuclei is known from antegrade axonal tracing experiments and was verified in ultrastructural studies. Injection of [3H]-leucine or other antegrade tracers in the inferior olive in the cat and the rat resulted in labelling over climbing fiber strips in the molecular layer and over parts of the cerebellar nuclei and the lateral vestibular nucleus (Courville, 1975; Groenewegen and Voogd, 1977; Groenewegen et al., 1979; Kawamura and Hashikawa, 1979; Balaban, 1984, 1988; Van der Want and Voogd, 1987; Van der Want et al., 1989a and b). The presence of projections of the inferior olive to the vestibular nuclei outside the lateral vestibular nucleus was denied by Groenewegen and Voogd (1977), but advocated by Balaban (1984, 1985, 1988) in experiments on rabbits. When the projection of the inferior olive to the fastigial nucleus was analysed with ultrastructural autoradiography of [3H]-leucine in the cat, the labelled boutons were predominantly found on small, distal dendrites, but never on somata. These terminals contain spherical vesicles and occasional dense core vesicles in an electron-lucent matrix and were provided with asymmetrical synapses (Van der Want and Voogd, 1987) (Fig. 115). The origin of these terminals as collaterals from olivocerebellar fibers terminating as climbing fibers in the cortex was first suggested by electrophysiological studies (Eccles et al., 1967) and by the observation of a strict topographical relation between the olivocerebellar climbing fiber zones and the termination of olivocerebellar fibers in the cerebellar nuclei (Groenewegen and Voogd, 1977). Direct proof of a collateral origin of olivonuclear fibers was provided by the retrograde transport to the central nuclei of [3H]-D-aspartate, injected in the cerebellar cortex of rats (Wiklund et al., 1984); the double-labelling studies in the cat with fluorogold implants in the cerebellar nuclei combined with injections of rhodamine-spheres in the cortex (Qvist, 1989b) and the observations of collaterals in antegrade tracing with the lectin from Phaseolus vulgaris in the rat (Van der Want et al., 1989a,b) (Fig. 113). Collaterals from olivocerebellar fibers generally terminate in the particular cerebellar nucleus that receives the axons of the Purkinje cells innervated by the same set of olivocerebellar fibers. It is not known whether all subdivisions of the inferior olive project to the cerebellar nuclei. The topographical distribution of the olivonuclear projections is reviewed in section 6.3.3. Some of the nuclei in the brainstem and the spinal cord, that give rise to mossy fibers terminating in the granular layer, also project to the cerebellar nuclei. Antegrade and retrograde axonal tracing experiments demonstrated projections in rat and cat from the spinal cord (Szentagothai in Eccles et al., 1967; Voogd, 1969; Matsushita and Ikeda, 1970; Robertson et al., 1983; Ikeda and Matsushita, 1973; Matsushita and Yaginuma, 1990, 1995), the lateral reticular nucleus and from the nucleus reticularis tegmenti pontis 165
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J. Voogd, D. Jaarsma and E. Marani
Fig. 114. EM autoradiogram of a spiny dendrite in the fastigial nucleus of the cat. A labelled mossy fiber terminal, originating from an injection of tritiated leucine into the nucleus reticularis tegmenti pontis, is densely filled with uniform spherical vesicles. Boutons with flattened vesicles form synaptic contacts on the same dendrite. Cat. Van der Want et al. (1987).
and adjacent regions of the pontine nuclei (Ktinzle, 1975; Russchen et al., 1976; ChanPalay et al., 1977; Matsushita and Ikeda, 1976; Eller and Chan-Palay, 1976; Martin et al., 1977; Ruggiero et al., 1977; Hoddevik, 1978; Dietrichs and Walberg, 1979a, 1987; Dietrichs, 1983b; Brodal et al., 1986; Gerrits and Voogd, 1987; Qvist, 1989a,b; Shinoda et al., 1992; Mihailoff, 1993). Other mossy fiber systems, such as the cuneocerebellar tract and the basal pontine nuclei provide only few or no collaterals to the cerebellar nuclei. Their distribution is reviewed in Section 6.4.4. Terminals in the fastigial nucleus of the cat originating from the reticular and the vestibular nuclei ranged widely in size and formed asymmetric synapses with small and large dendrites but not with somata. These boutons contained clear, spherical vesicles, and they occurred in two types, that differed in the aggregation of their vesicles (ChanPalay, 1977) which are present in equal numbers among the terminals from all sources (Van der Want et al., 1987) (Fig. 114). Morphologically these boutons are indistinguishable from the olivonuclear boutons that were described in Van der Want's companion study (Van der Want and Voogd, 1987) (Fig. 115). The differences in distribution between the climbing fiber and the different types of mossy fiber terminals in the rat and monkey lateral cerebellar nucleus reported by Chan-Palay (1973a, 1977), that were based on the similarity in morphology of these terminals in the cerebellar cortex and the nuclei, were not confirmed. According to the ultrastructural degeneration studies of Ikeda and Matsushita (1973) spinocerebellar fibers terminate both on dendrites and on somata. 166
The cerebellum." chemoarchitecture and anatomy
Ch. I
5.7. EXTRACEREBELLAR A F F E R E N T S OF THE CEREBELLAR NUCLEI: SEROTONINERGIC, N O R A D R E N E R G I C , D O P A M I N E R G I C AND PEPTIDERGIC PROJECTIONS Serotonin-like immunoreactivity resides in a fine network of varicose fibers in the neuropil of all cerebellar nuclei. This plexus is most dense in the hilar region of the lateral cerebellar nucleus of the rat (Takeuchi et al., 1982), among the small, intrinsic neurons of this region (Chan-Palay, 1977) and in the caudal and dorsal regions of the central nuclei of the opossum (Fig. 117) (Bishop et al., 1985). The noradrenergic innervation of the cerebellar nuclei was studied with histochemical fluorescence methods by H6kfelt and Fuxe (1969) in the rat and Landis et al. (1975) in mice and by Mugnaini and Dahl (1975) in the chicken and with selective uptake of [3H]noradrenalin (Chan-Palay, 1977) and dopamine-fl-hydroxylase immocytochemistry (Pasquier et al., 1980) in rats. The network of varicose noradrenergic fibers in the cerebellar nuclei is much less dense than the serotoninergic plexus and in the chicken a noradrenergic innervation of the central nuclei even is completely lacking (Mugnaini and Dahl, 1975). The possibility should be considered that this plexus mainly consists of passing fibers on their way to the cortex (Sachs et al., 1973). A heterogeneous population of terminals, containing large dense-core vesicles with a diameter of 900 A, were found to be labelled in the cerebellar nuclei after intraventricular infusions of 3H-serotonin in the rat. Few of these boutons show synaptic specializations (Chan-Palay, 1975, 1977). The ultrastructural morphology of nor-
Fig. 115. EM autoradiogram showing two climbing fiber boutons with spherical and pleomorphic vesicles, labelled from an injection with tritated leucine into the inferior olive make synaptic contact with a dendrite of a neuron of the fastigial nucleus of the cat. Van der Want and Voogd (1987).
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adrenergic terminals in the cerebellar nuclei has not been studied. Ultrastructural studies of the central nuclei using antibodies against conjugates of serotonin or noradrenalin have not yet been reported. Neurons of the ventral tegmental area of the rat project to the cerebellar nuclei. These neurons do not contain tyrosine-hydroxylase and this projection, therefore, is nondopaminergic, contrary to the projection of the dopaminergic A10 group located in the same area, to the cerebellar cortex of the rat (Ikai et al., 1992). Peptides, such as enkephalin, corticotropin-releasing factor (CRF) and cholecystokinin (CCK) that occur in both mossy and climbing fibers(see Sections 6.3.2.2. and 6.4.3.) have been demonstrated in the cerebellar nuclei of several mammals. The distribution of these peptides in a plexus of beaded fibers in these nuclei is very similar the plexiform distribution of serotonin and noradrenalin, substances that do not occur in climbing or mossy fibers. Enkephalin-like activity was concentrated in fibers along the lateral and dorsal borders of the dentate nucleus and in the rostral part of the fastigial nucleus and in varicosities in all parts of the cerebellar nuclei of the opossum (King et al., 1987) (Fig. 188). Enkephalin-like activity in cat and rat was found in some mossy fiber rosettes, but not in climbing fibers (King et al., 1987); the cerebellar nuclei have not yet been studied in these species. CRF-like immunoreactivity was present in a diffuse plexus of beaded fibers in all cerebellar nuclei of the opossum (Cummings et al., 1989) and in a similar plexus with concentrations in the anterior interposed nucleus and the ventral dentate in the cat (Cummings, 1989). CCK-like immunoreactivity was present in a similar plexus 168
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J. Voogd, D. Jaarsma and E. Marani
of varicose fibers in the cerebellar nuclei of the opossum (King et al., 1986b; King and Bishop, 1990).
6. EFFERENT AND AFFERENT CONNECTIONS OF THE CEREBELLAR CORTEX: CORTICONUCLEAR, OLIVOCEREBELLAR AND M O S S Y FIBER C O N N E C T I O N S AND CYTOCHEMICAL MAPS
Morphological, embryological, physiological and cytochemical studies have revealed longitudinal, zonal patterns in the cerebellar cortex. The cytoarchitecture of the cerebellar cortex is uniform, without clear signs of a regional differentiation. Differences in the size of granule and Purkinje cells between vermis and hemisphere, with larger and less densely packed cells in the vermis, and smaller more densely packed cells in the hemisphere, were noticed by Lange (1982) and Drfige et al. (1986) for human and rat cerebellum. Local differences also exist for the Golgi and/or the unipolar brush cells, which are more closely packed in the flocculus and the vermis of the posterior lobe, than in the anterior vermis and the hemisphere. Maps showing local differences in density of the Purkinje cells have been published for the cerebellum of the turtle Pseudomys scripta elegans and the lizard Varanus exanthematicus (Gerrits and Voogd, 1973; Bangma et al., 1983). The cerebellum of these species is rather simple and consists of a single leaf. Three zones with a different density of the Purkinje cells, that also differ in their projection to the cerebellar and vestibular nuclei were distinguished. Similar density maps are not available for the mammalian cerebellum, probably because the curvature of the cortex makes this technically difficult. There are indications in the mammalian cerebellum for medio-lateral, zonally distributed differences in the size of the Purkinje cells (Chan-Palay et al. 1981; Voogd, 1989) and an even better case can be made for the existence of systematic differences in the size of the fibers taking their origin from different Purkinje cell zones (Fig. 118) (Voogd, 1964, 1967, 1969; Voogd and Bigar6, 1980). The mediolateral compartmentalization of the Purkinje cells and their axons is related to the zonal organization of the corticonuclear projection and was first recognized by Klimoff (1899). He also concluded from his Marchi experiments in the rabbit that the corticonuclear projections are uncrossed, each hemivermis projecting to the medial cerebellar nucleus and to the vestibular nuclei, and the hemisphere to the dentate/interposed complex. The differential projection of the left and right hemivermis to the medial nuclei of either side implies that a sudden transition in the corticonuclear projection, in this case across the midline, is perfectly compatible with the uniform structure of the adult cortex. During early, fetal stages of cerebellar development the cortex is not uniform and discontinuities in the anlage of the Purkinje cell layer are present that subdivide it into a number of bilateral symmetrical parasagittal zones or clusters (see Section 6.2.). The first to demonstrate the paired origin and the zonal pattern in the development of the cortex were Hayashi (1924) and Jakob (1928) who distinguished an intermediate zone (the 'pars intermedia', Zwischenstiick or B convolution) located between the anterior vermis and the hemisphere in the 3-4 month human fetus (Fig. 154). The anlage of the anterior vermis was paired and consisted of the two A convolutions. Its histogenesis was more advanced than in the pars intermedia. Development of the cortex in the hemisphere lagged far behind. Caudally the triangular intermediate zone extended into the posterior lobe in the region of the paramedian sulcus where it degenerated and disappeared. The dentate nucleus developed in the hemisphere, the emboliform nucleus 170
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 118. Myeloarchitectonic compartments of the ferret cerebellum. In A the thin fibers at the border of the A and B compartments (R) are shown. In B the small Purkinje cell fibers of the C2 compartment are flanked by larger Purkinje cell fibers of C, and C3. H~iggqvist stain. Bar = 100/lm. Voogd (1969). (anterior interposed nucleus, Hayashi 1924) and the globose nucleus (posterior interposed nucleus, Jakob, 1928) belonged to the pars intermedia and the medial nuclei occupied the white matter of the vermis. Jansen and Brodal (1940, 1942) studied the corticonuclear projection of the corpus cerebelli with the Marchi method in cat, rabbit and macaque. They confirmed the uncrossed projection of each hemivermis to the medial nucleus and the vestibular nuclei. In the hemisphere they distinguished an intermediate zone, that projects to Brunner's (1919) interpositus nucleus and a lateral zone, that is connected with the lateral nucleus. They noticed the correspondence between their three-zonal arrangement in the corti171
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conuclear projection of the anterior lobe and the corticogenetic zones of Hayashi and Jakob, but at the level of the cortex the exact borders of the three zones of Jansen and Brodal remained arbitrary. The paramedian sulcus, which is the border between vermis and pars intermedia, is often absent or indistinct and the border between the intermediate and lateral zones in Jansen and Brodal's scheme depends upon the arbitrary border between the interpositus and lateral nuclei. Moreover the longitudinal topology of their zones was defective. In the anterior lobe the orientation of the three zones was approximately perpendicular to the interlobular fissures, but when they were extrapolated to include the posterior lobe, the intermediate and lateral zones did not follow the curved axis of the folial chain of the hemisphere, and their common border cut through the centers of the folial loops of the ansiform lobule and the paraflocculus. Brodal's (1940; see also Jansen and Brodal, 1958) earlier studies of the olivocerebellar projection supported the distinction of the three zones in the anterior lobe. In the cat the dorsal accessory olive was found to project to the vermis and to the entire hemisphere of the anterior lobe. An additional projection of the rostral pole of the principal olive to the extreme lateral part of the anterior lobe could be observed in rabbit, monkey and man, but not in the cat. In the anterior lobe of rabbit and man, therefore, three zones can be distinguished on each side of the midline: the (hemi)vermis and the intermediate zone, that receive fibers from the dorsal accessory olive, and the lateral zone, that receives a projection of the principal olive and is absent in the cat. On the basis of these observations Jansen and Brodal (1940) called 'attention to the striking conformity in the arrangement of the corticonuclear and olivocerebellar projections, both systems apparently being arranged according to principles entirely different from those prevailing within the spinocerebellar and vestibulocerebellar projections'. The similarity in the organization of the olivocerebellar and corticonuclear projections remained one of the central concepts in the anatomy of the cerebellum that received ample support from later studies on longitudinal zonation. At the level of the cortex the borders between zones containing Purkinje cells that project to different target nuclei usually cannot be distinguished, but in the white matter the Purkinje axons from these zones collect in parasagittally oriented sheets that appear as white matter compartments in transverse sections. The sheets or compartments containing the Purkinje cell axons remain separated by narrow spaces. Voogd (1964, 1969) in cat and ferret, Marani (1982a, 1986) in the mouse and Feirabend and Voogd (1986) in the chicken traced these fiber compartments in myelin-stained (Hfiggqvist's method, 1936; Voogd and Feirabend, 1981) sections throughout the cerebellum. Large caliber fibers, that were identified as the axons of Purkinje cells, with an admixture of smaller fibers were present within the compartments and small myelinated fibers accumulate at their borders (Fig. 118). These borders usually are continuous with the borders between the subdivisions of the cerebellar nuclei and this configuration, therefore, made it possible to predict the longitudinal zonal organization in the corticonuclear projection (Figs 119 and 120). Corticonuclear projection zones are continuous from lobule to lobule and they are oriented perpendicular to the long axis of the folia: they follow the curved axis of the folial chains of vermis and hemisphere. The large caliber fibers within the compartments of the white matter could be identified as Purkinje cell axons with axonal tracing methods and immunohistochemistry with Purkinje cell-specific antibodies. They appear as discrete bundles, separated by narrow gaps with antibodies against cyclic GMP-dependent protein kinase (De Camilli et al., 1984) and calbindin-D28K (Paxinos, unpublished observations) in the rat and other species, with anti-Zebrin in cats and macaques (where Zebrin reacts with all Purkinje 172
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173
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Fig. 120. Compartments in the white matter of the cerebellum of the cat. Drawings and reconstructions from H~iggqvist and AChE-stained sections. Compartments are indicated with different symbols. A-D. Graphical reconstructions of the rostral aspect of the anterior lobe (A) and the posterior lobe (B), the dorsal aspect (C) and the caudal aspect (D) of the cerebellum. Compare Fig. 98. E-G. Transverse sections. A = A compartment; ANS = ansiform lobule; A N T = anterior lobe; B = B compartment; C1-3 = C1-3 compartments; cr = restiform body; D(1,2) = D(1,2) compartments; F = fastigial lateral cerebellar nucleus; P F L = paraflocculus; P M D = paramedian lobule; SI = simple lobule; vest - vestibular nuclei; X - X compartment; III-IX = lobules III-IX. (.
cells) and with parvalbumin in the immature avian cerebellum (Braun et al., 1986) (Fig. 32). Monoamino oxidase in the white matter of the cerebellum of the chicken, is also restricted to the large caliber fiber areas (Feirabend, 1983; Marani, 1981). Systematic differences in the caliber of the Purkinje cell fibers were noticed in myeloarchitectonic studies in cat and ferret (Voogd, 1964, 1969; Voogd and Bigar6, 1980) and retrograde tracing of Purkinje cell axons (Voogd et al., 1991a). The small fibers located in the gaps between the bundles of Purkinje cell axons were called 'raphes' when they were positively stained with the H~iggqvist method (Fig.118; Voogd, 1964) and with AChE-histochemistry (Figs 122 and 127) (Hess and Voogd, 1986). Within the medullary core of the monkey cerebellum Hess and Voogd (1986) described fibers that stain densely for AChE and which are distributed in longitudinally oriented sheets, that appear as stripes in cross-sections (Fig. 127). These stripes in the white matter were aligned with AChE-rich bands, containing concentrations of reactive glomeruli in the granular layer, and narrow AChE-rich stripes in the molecular layer. A prominent midline band of AChE-rich axons flanked on each side by 5 or 6 ACHErich bands delineated a number of compartments in the cerebellar white matter. Within the white matter and the granular layer, cytochrome oxidase-rich axons and glomeruli were distributed in a longitudinally banded pattern, topographically identical to, but of lower contrast than, the banded distribution of ACHE. Both in the monkey and the cat the number and the disposition of the AChE-rich zones in the cerebellar white matter corresponded exactly with the raphe-like concentrations of small fibers at the borders of large fiber compartments in myelin-stained sections (Fig. 122). The highest amount of AChE was found in dense strips at the borders of the compartments; within the compartments the reactivity of the fibers for AChE differed. Compartments that contained large axons usually displayed very little AChE activity whereas the activity of AChE was high in compartments with smaller fibers. An explanation for the entire banded distribution of AChE in the molecular and granular layers and in the white matter as yet is not available, but it is clear that this distribution faithfully reflects the basic plan of the longitudinal zonation of the mammalian cerebellum. The possibility that zonally distributed differences in size and connections of the Purkinje cells are correlated with specific chemical properties of these cells was first raised by Marani (Marani and Voogd, 1977; Marani, 1981, 1982a; Marani, 1986) on the basis of the distribution of 5'-nucleotidase and acetylcholinesterase in the molecular layer and by Chan-Palay (1984) who reported a restricted distribution of certain peptides in subsets of Purkinje cells. More recently a complete pattern of alternating zones of immunoreactive and non-immunoreactive Purkinje cells was described by Hawkes and Leclerc (1986, 1987) with a Purkinje cell-specific antibody (anti Zebrin-I) in the rat and by Brochu et al. (1990) with anti-Zebrin II in the rat and other species (see Section 6.1.3.). The distribution of the Zebrin-positive Purkinje cells was very similar to the distribution of the enzyme 5'-nucleotidase in the molecular layer of certain rodents (Eisenman and Hawkes, 1989). 175
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The concept of the similarity in the organization of the corticonuclear and olivocerebellar projections, that dates from the work of Jansen and Brodal, received strong support from the observation that the distribution of the olivocerebellar fibers from certain subdivisions of the inferior olive was congruent with the parasagittal compartmentalization of the white matter (Voogd, 1969). The zonal termination of the climbing fibers could be visualized in autoradiograms of the olivocerebellar fibers using anterograde transport of [3H]leucine (Courville et al., 1974; Groenewegen and Voogd, 1977; Groenewegen et al., 1979). The olivocerebellar fibers located within a compartment were found to terminate both as climbing fibers on the Purkinje cells of the corresponding zone and on the cells of their cerebellar or vestibular target nucleus. The corticonuclear projection zones were experimentally verified in the antegrade degeneration and transport studies of Haines in primates and subprimates (see Haines et al., 1982 for a review) and Dietrichs in the cat (Dietrichs 1981a,b, 1983a; Dietrichs and Walberg, 1979a, 1980; Dietrichs et al., 1983b), and by retrograde labelling of the Purkinje cells and their axons from their target nuclei (Courville and Faraco-Cantin, 1976; Voogd and Bigar6, 1980; Balaban, 1984). A similar longitudinal pattern emerged from the electrophysiological studies of Oscarsson (1969, 1973, 1979, 1980; Oscarsson and Sj61und, 1974, 1977a,b,c; Andersson and Oscarsson, 1978a; Ekerot and Larson, 1979a,b; Ekerot et al., 1987, 1991a,b; Andersson and Eriksson 1981; Andersson and Nyqvist, 1983; Garwicz, 1992) and Armstrong et al. (1974) of the distribution of the climbing fibers in the cerebellar cortex. The electrophysiological identification of these climbing fiber zones rests on their laterality, their topography and on differences in latency between the different spino-olivocerebellar climbing fiber paths. These studies greatly extended our knowledge on the branching of the climbing fibers (Armstrong et al., 1973; Ekerot and Larson, 1982) and on the internal topography of the zones and lead to the distinction of the 'micro zone' as the smallest somatotopically defined unit engaged in the processing of information (Andersson and Oscarsson, 1978b; Garwicz, 1992). Direct proof of the congruence of the corticonuclear and olivocerebellar projections was obtained in the antegrade tracing studies of the Purkinje cells in electrophysiologically identified climbing fiber zones by Trott and Armstrong (1987a,b). The olivocerebellar and nucleo-olivary projections are reciprocally organized in the sense that a cerebellar target nucleus of one or more particular Purkinje cell zones is connected with the subdivision of the inferior olive that provides climbing fibers to the Purkinje cells of the same zone and a collateral projection to the target nucleus (see Ruigrok and Voogd, 1990, for a review of the literature). The zonal organization of the cerebellar cortex and the compartmental subdivision of the cerebellar white matter, therefore, are the manifestations of the modular organization of the output systems of the cerebellum. A module consists of a Purkinje cell zone, or a set of zones, its cerebellar or vestibular target nucleus with its GABAergic nucleo-olivary projection and its nonGABAergic output system and a sustaining olivocerebellar projection. Is the number of modules constant among the mammalian species and are they correlated with the different forms of chemoarchitectonic zonation in the cortex and the white matter? These questions only can be answered after a more thorough review of the corticonuclear projection (Sections 6.1.1., 6.1.2., 6.1.4. and 6.1.5.), the chemoarchitectonic evidence on zonation (Section 6.1.3.) and the olivocerebellar and nucleocerebellar projections (Section 6.3.) in different mammalian species. The existence of such a basic plan has been questioned (Boegman et al., 1988), not so much because the different maps for parasagittal zonation are mutually exclusive, but mainly because most 176
The cerebellum." chemoarchitecture and anatomy
Ch. I
of the studies on the topography of afferent and efferent connections were conducted in carnivores and primates, whereas the immunocytochemical studies, that revealed a finer grain in the parasagittal organization were mostly restricted to the rat. Evidence on the corticonuclear projection in cat and primates and its correlation with white matter compartments, mostly dating from the 1960s to the early '80s, will be discussed in Sections 6.1.1. and 6.1.2. The compartmentalization of the cerebellar cortex for Zebrin (Hawkes et al., 1985) and other markers, such as 5'-Nucleotidase (Scott, 1963), that was mostly studied in rodents, will be considered next (Section 6.1.3.). Correlations of the corticonuclear projection in the rat with these cytochemical maps are reviewed in Section 6.1.4. and efferent connections of the vestibulocerebellum in Section 6.1.5. Some regional differences in the development of the cerebellum, mainly concerning the transient, chemical heterogeneity of the Purkinje cells, are considered in Section 6.2. The inferior olive and the large body of anatomical and electrophysiological evidence on the olivocerebellar projection, that has contributed so much to the ideas on the zonal organization of the cerebellar cortex, are discussed in Section 6.3. 6.1. COMPARTMENTS AND CORTICONUCLEAR PROJECTION ZONES: CORRELATIONS WITH CYTOCHEMICAL MAPS 6.1.1. Corticonuclear projection zones in the cat: Correlation with white matter compartments and cytochemical zones
In the cerebellum of the cat at least 8 zones have been defined on the basis of the corticonuclear and olivocerebellar projections (Fig. 119). The corresponding white matter compartments have been delineated in myelin-stained or AChE-stained sections in ferrets (Voogd, 1967, 1969), cats (Voogd, 1964, 1989) and monkeys (Hess and Voogd, 1986; Voogd et al., 1987a,b; Voogd and Hess, 1989). AChE in the anterior vermis of the cerebellum of young cats is present in bands in the molecular layer (Marani and Voogd, 1977; Voogd and Bigar6, 1980) (Fig. 121). An AChE-positive band at the midline is separated by an AChE-negative area from a strongly reactive, paramedian band. At the lateral, sharp border of the paramedian band a narrow AChE-negative band is found, that merges into the hemisphere, which is uniformly AChE-positive. The bands diverge in the dorsal part of the anterior lobe and in the simple lobule. AChE in the molecular layer is present in parallel striations. The general direction of these striations corresponds to the orientation of the Purkinje cells and to the direction of the cortical zones. A similar, but less distinct pattern of AChE positive and negative zones is present in the lobules VIII-X of the caudal vermis. The AChE-stained material illustrated in this chapter differs from Marani and Voogd's (1977) original illustrations because it is derived from aldehyde-fixed tissue from the adult animal (Brown and Graybiel, 1983). Sections prepared in this way display the borders between the compartments in the white matter in addition to the banded pattern in the molecular layer. Three compartments can be distinguished on both sides of the midline in the region of the vermis of the anterior lobe: a medial A compartment, a lateral B compartment and a wedge-shaped X compartment in between A and B (Figs 120 and 122). Compartments A and B are present in all lobules of the anterior lobe, but an X compartment is only present in its dorsal part (lobules IV and V). Ventrally the fused AChE-positive borders of the X compartment continue in the lateral border of the fastigial nucleus. The fibers of the B compartment pass lateral to the fastigial nucleus and medial to the interposed nuclei to enter Deiters' lateral vestibular nucleus from dorsally. It is clear from a comparison of 177
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J. Voogd, D. Jaarsma and E. Marani
the AChE-positive borders between the compartments and the zonal distribution of AChE in the molecular layer, that the medial border of the B compartment is exactly in register with the lateral border of the first paramedian band of high AChE activity in the molecular layer. Consequently, the cortical X zone corresponds to the lateral part of this first paramedian band of AChE-activity, but its border towards the A zone cannot be recognized in the distribution of AChE in the molecular layer. The B zone corresponds to the second, AChE-negative strip of the molecular layer. Previously, the correspondence between the localization of AChE in the molecular layer and the corticonuclear projection has been studied by Brown and Graybiel (1983), but they failed to recognize the X zone. The molecular layer of the hemisphere of the anterior lobe is uniformly ACHEpositive. In the white matter of the hemisphere of the anterior lobe C1,C2,C3 and one or more D compartments can be delineated in AChE or HS.ggqvist-stained material (Figs 120 and 122). The C2 compartment is present in the dorsal part of the anterior lobe (lobules III, IV and V). More ventrally the C1 and C3 compartments are contiguous. The anterior interposed nucleus is located within the fused C1 and C3 compartments, the C2 compartment can be traced into the more caudally located posterior interposed nucleus. In H/iggqvist-stained sections of the cerebellum the fibers of the A,B,C1 and C3 compartments were larger than those of the X and C2 compartments. Staining for AChE generally is more intense in C2 and X than in the large fiber compartments. The AChE-banding pattern in the molecular layer of the anterior vermis and the white matter compartments of the anterior lobe continue, across the primary fissure, into the posterior lobe. A wide A compartment, flanked by diverging X and B compartments is present in the vermis of the simple lobule (Figs 119 and 120). The B compartment ends at the area without cortex in the center of the ansiform lobule, where it abuts on the pontocerebellar fibers of the cerebellar commissure that reach the surface at this point. It is not clear whether the X compartment continues from the simple lobule into lobule
Fig. 121. Zonal distribution of A C h E in the cerebellum of the cat. A. Whole mount preparation of the anterior lobe. B. Transverse section through the anterior lobe. H E M = hemisphere; m = midline AChE-positive band; L = parasagittal AChE-positive band; arrows = lateral border of L. Marani (1986).
178
The cerebellum." chemoarchitecture and anatomy
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Fig. 122. Photographs of white matter of the anterior lobe of the cerebellum of the cat. A. Borders of compartments and midline (m) are delineated by AChE-reactive raphes. The lateral border of the X compartment is in line with the lateral border of the parasagittal AChE-positive strip in the molecular layer; the molecular layer over the B compartment is AChE-negative. B. H~iggqvist-stain. Compartments A-D are delineated by dark stripes containing small calibre fibers. Same level as Fig. 120E.
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CORTICO~/#cSTT'## LAR ~ CORpTdCjONcUC~EAR Ve~#Y" CAUDAL ANTERIOR Fig. 123. D i a g r a m of the corticonuclear projection in the cat. Based on retrograde labelling of Purkinje cells and their axons after injections of H R P in their target nuclei. A = A zone; A = Anterior interposed nucleus; A N S = ansiform lobule; B = B zone; C1-C3 = C1-C3 zones; C R I I = crus II of the ansiform lobule; D1-D2 = D1-D2 zones; DV = descendng vestibular nucleus; F - fastigial nucleus; F L O C = flocculus; IP = posterior interposed nucleus; L = lateral cerebellar nucleus; LOB POST = posterior lobe; LOB A N T = anterior lobe; LV = Deiters' nucleus; MVmc = magnocellular medial vestibular nucleus; MVpc = parvicellular medial vestibular nucleus; P F L = paraflocculus; P F L D = dorsal paraflocculus; PFLV = ventral paraflocculus; P M E D = paramedian lobule; SV = superior vestibular nucleus; I-X = lobules I-X. Bigar6 (1980).
VII. Usually the entire white matter of lobule VII is considered to belong to the A compartment. 180
The cerebellum: chemoarchitecture and anatomy
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C1,C2,C3 and D compartments can be distinguished in the hemisphere of the simple lobule. C1 and C3 compartments are narrow or interrupted in the white matter of the ansiform lobule. A medial C2 and more laterally located D1 and D 2 compartments can be recognized in the white matter of this lobule. The complete set of compartments is present again in the white matter of the caudalmost folial rosette of the Crus II of the ansiform lobule (the ansula). From here these compartments pass into the paramedian lobule. C3 is present in the dorsal part of paramedian lobule only, and ends at the border with the pars copularis. C~ continues into the ventral most folia of the paramedian lobule as the most medial compartment of the hemisphere. C2 and the narrow, laterally located D1 and D2 compartments continue from the paramedian lobule into the dorsal paraflocculus (Figs 119 and 120). The dorsal and ventral paraflocculus contain C2, D1 and D 2 compartments. Concentrically arranged AChE-positive raphes, that stain strongly for ACHE, separate the compartments of the dorsal and ventral paraflocculus in the rostral part of the parafloccular loop (Figs 125 and 126). The rostral (dorsomedial) dentate nucleus is located within the D 2 compartment, the caudal (ventrolateral) dentate within the D~ compartment and the C2 compartment enters the lateral pole of the posterior interposed nucleus. Distinct compartments cannot be delineated in the white matter of the flocculus of the cat. The zonation in the corticonuclear projection that could be predicted from the disposition of the white matter compartments, was confirmed with retrograde labelling of Purkinje cells, after injections of tracers in the individual cerebellar nuclei of the cat (Courville and Faraco-Cantin, 1976; Voogd and Bigar6, 1980; Gibson et al., 1987). The Purkinje cells of the corticonuclear projection zones in the hemisphere follow the loops of the folial chain (Fig. 123, compare Fig. 98). Purkinje cells of the C1 and C3 zones can be retrogradely labelled from the anterior interposed nucleus. The C~ zone is broad in the ventral part of the anterior lobe and tapers more dorsally. The C3 zone is wide in the dorsal part of the anterior lobe, ventrally it is continuous with a narrow 'd2' zone 2 in the extreme lateral part of the anterior lobe. C1 and C3 are narrow in the ansiform lobule and reappear in the ansula and the paramedian lobule. They do not continue in the paraflocculus. The C2 zone was continuous from lobule III of the anterior lobe, through the entire hemisphere, into the flocculus. Its Purkinje cells were labelled from injections of the posterior interposed nucleus. Purkinje cells of the D zones occupied the extreme lateral part of the anterior lobe. A medial D1 and a lateral D 2 z o n e that projected to rostral and caudal parts of the lateral cerebellar nucleus, respectively, could be distinguished in the ansiform lobule, the paramedian lobule and the paraflocculus. The corticonuclear projection of the anterior lobe, the simple, ansiform and paramedian lobules was studied with anterograde transport of HRP by Dietrichs (198 l a) and Dietrichs and Walberg (1979a, 1980). Their observations on the projections of the A,B,C and D zones are essentially in accordance with the findings of Voogd and Bigar6 (1980). In their alternative nomenclature they shifted the zones one zone laterally, the presumed B zone projecting to the anterior interposed nucleus, and the C1 zone with the posterior interposed nucleus. This alternative nomenclature was discussed in the papers by Voogd and Bigar6 (1980), Haines et al. (1982) and Voogd et al. (1987a,b). The presence of C2, 2The term 'd 2' w a s applied to a narrow strip of climbing fiber-evoked potentials in the extreme lateral part of the anterior lobe by Ekerot and Larson (1979a, see Figs 171 and 175 ). The d 2 z o n e can be activated by the dorsal spino-olivo-cerebellar-climbing-fiber-pathand receives branches from climbing fibers which also innervate the lateral c3 zone (Ekerot and Larson, 1982).The use of the letter 'd' for this zone is misleading, because it neither projects to the lateral cerebellar nucleus or receives a projection from the principal olive. 181
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The cerebellum." chemoarchitecture and anatomy
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Fig. 124. Comparison of bands of AChE reaction product in the molecular layer of the anterior vermis of cat cerebellum and retrograde labelling of Purkinje cells in B and lateral A zones after an injection of HRP in the vestibular nuclei (A-C) and in the B and X zones after an injection in the lateral fastigial nucleus and the B compartment (D-F). Note different size of Purkinje cells in B and X zones. A = A zone; B = B zone; Deit = Deiters' nucleus; DV = descending vestibular nucleus; F = fastigial nucleus; IA = anterior interposed nucleus; MV = medial vestibular nucleus; X = X zone. I-V = lobules I-V. Voogd (1989). (
Dl and D2 zones in the paraflocculus of the cat is compatible with the anterograde tracing study of Dietrichs (1981 b). The corticonuclear projections of the electrophysiologically verified climbing fiber zones b, x, cl, c2, c3 and dl 3 w e r e determined for lobule V of the anterior lobe of the cat with anterograde tracing of [3H]leucine (Trott and Armstrong (1987a,b)). They were found to conform to the projections of the corresponding anatomical zones in the retrograde tracing studies of Voogd and Bigar6 (1980). The corticonuclear and -vestibular projection were correlated with the distribution of AChE for the anterior vermis of the cat by Brown and Graybiel (1983) and Voogd et al. (1991 a). Other studies of the projections of the anterior vermis to the fastigial nucleus and the vestibular nuclei, that have been known since Klimoff's (1899) study of the corticonuclear projection in the rabbit, did not distinguish between the zones in this region (Corvaja and Pompeiano, 1979; Dietrichs et al., 1983b; see Voogd 1964 and Brodal, 1974 for reviews of the older literature). Purkinje cells that project to the vestibular nuclei are located both in the A zone and in the B zone but not in the X zone (Fig. 124A-C). Purkinje cell axons of the A zone terminate in the fastigial nucleus, but some of these axons proceed to the vestibular nuclei, where they terminate at the border of Deiters' nucleus and the magnocellular medial vestibular nucleus (i.e. the ventral part of the lateral vestibular nucleus of Brodal and Pompeiano, 1957). The B zone projects to Deiters' nucleus (i.e. the dorsal part of the lateral vestibular nucleus). The projection of the A and B zones to different parts of the vestibular nuclei (Bigar6, 1980; Voogd and Bigar6, 1980; Voogd, 1989) was confirmed in the rabbit by Balaban (1984) and Epema (1990). These experiments substantiated the small projection of the A zone to Deiters' nucleus, as proposed by Andersson and Oscarsson (1978a). The wedge-shaped X zone in the dorsal part of the anterior lobe projects to the medial limb of the U-shaped nucleus at the junction of the fastigial and posterior interposed nuclei of the cat (Trott and Armstrong, 1987b). The Purkinje cells and their axons, when retrogradely labelled from this nucleus, are smaller than those of the A and B zones (Fig. 124D-F). The medial border of the retrogradely labelled B zone corresponds exactly with the lateral border of the parasagittal strip of high AChE-activity in the anterior vermis. The small Purkinje cells of the X-zone and the larger Purkinje cells with vestibular projections in the lateral A zone are located within the parasagittal AChE-positive strip. The B zone has a low content of AChE (Voogd, 1982). It was pointed out in a previous Section (5.3.) that the nucleocortical and corticonuclear projections in the cat are roughly reciprocal (Tolbert et al., 1978b; Gould, 1979; Dietrichs and Walberg, 1979a, 1980; Dietrichs and Walberg, 1985). Trott and Armstrong (1990) showed that nucleocortical projections to the electro-physiologically identified cl and c3 zones are scarce and that the major projection to the c2 zone of lobule V takes its origin from the posterior interposed nucleus.
30scarsson and co-workersadapted a modification of Voogd'snomenclature to indicate the electrophysiologically identified climbing fiber zones, using lower case letters instead of capitals (see Fig. 175). 183
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PHD
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PFL tI Fig. 125. White matter compartments C2, D1 and D2 in the paraflocculus of the cat in transverse, AChE-reacted sections. Note concentric arrangement of the compartments in the dorsal and the ventral paraflocculus. A caudalmost section; D = rostralmost section. ANS - ansiform lobule; brp = brachium pontis; C1-3 = C1-3 compartments; crest = restiform body; D - dentate nucleus; D (1,2)- D (1,2) compartments; F = fastigial nucleus; FLO = flocculus; IA - anterior interposed nucleus; IP = posterior interposed nucleus; PFLD - dorsal paraflocculus; PFLV- ventral paraflocculus; PMD = paramedian lobule. )
6.1.2. Compartments and corticonuclear projection zones in monkeys A l t h o u g h an analysis o f the c o m p a r t m e n t a l subdivision in the m o n k e y w o u l d be feasible f r o m serial HS, ggqvist-stained sections, the A C h E staining is m o r e distinct. A p r o m i n e n t c o n c e n t r a t i o n of A C h E - r i c h axons at the midline is flanked on each side of the anterior lobe by seven A C h E - r i c h strips, that delineate eight parasagittal c o m p a r t m e n t s in the cerebellar white m a t t e r (Fig. 127). Some of these c o m p a r t m e n t s are t o p o g r a p h i c a l l y related to certain cerebellar nuclei (Hess and Voogd, 1986; Voogd et al., 1987a,b). This is especially clear for two o f the c o m p a r t m e n t s , X and C2, b o t h of which have a high c o n t e n t of A C h E - p o s i t i v e fibres. The X c o m p a r t m e n t is n a r r o w a n d located in the white m a t t e r o f the vermis. It continues into the lateral, A C h E - r i c h b o r d e r z o n e o f the fastigial nucleus a n d the medial limb of the U - s h a p e d nucleus located between the caudal pole 184
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25
of the fastigial and the posterior interposed nuclei (Fig. 105). In some sections the two AChE-rich strips that border the compartment, fuse with its content into a single, broad AChE-positive band. In the ventral part of the anterior lobe the X compartment is narrow or even absent. The X compartment extends caudally into the lobules VI and in the lateral parts of the lobules VII and VIII. The X compartment separates the A and B compartments of the anterior vermis. The A compartment includes the fastigial nucleus. In the posterior vermis it becomes wider and is subdivided into a medial A1 and a lateral A 2 compartment. The B compartment is present in the anterior lobe and the simple lobule. Ventrally it empties into the space between the fastigial and the anterior interposed nucleus to continue into the lateral vestibular nucleus. Caudally it diverges far laterally at the junction of the lobules VI and VII, where it ends at the area devoid of cortex in the centre of the ansiform lobule. The C2 compartment is clearly related to the posterior interposed nucleus. Rostrally it is located dorsal to the anterior interposed nucleus, in the intermediate part of the hemisphere. From the posterior interposed nucleus the C2 compartment extends dorsolaterally into the ansiform lobule, caudally into the paramedian lobule and ventrolaterally into the paraflocculus and the flocculus (Fig. 148). In the anterior lobe C2 is located between the C~ and C3 compartments. Ventrally Cl and C3 fuse and are related to the 185
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anterior interposed nucleus. Both C 1 and C 3 diverge laterally in the central part of the ansiform lobule and extend into the paramedian lobule. C1 ends in the last folium of the paramedian lobule, C3 stops in the dorsal part of this lobule. A major, lateral expanse of the white matter is related to the dentate nucleus. In the dorsal paraflocculus and the petrosal lobule this dentate-related white matter is subdivided into the D1 and D2 compartments (Fig. 148). D 1 is located next dorsal to the C2 compartment. D1 is related to the medial dentate nucleus. D2 occupies the dorsal part of the paraflocculus and is related to the lateral dentate nucleus. In the anterior lobe and the paramedian lobule 186
The cerebellum." chemoarchitecture and anatomy
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Fig. 12 7. Distribution of acetylcholinesterase at the borders of white matter compartments in the anterior lobe of Macaca fascicularis A-C3 = A-C3 zones; b c - brachium conjunctivum; b p - brachium pontis; F L O = flocculus; I A - anterior interposed nucleus; m - m i d l i n e ; P F L V - ventral paraflocculus.
a narrow compartment, lateral to C3, may represent D 1. The compartmental subdivision of the white matter of the ventral paraflocculus and the flocculus will be discussed in the Section on the vestibulo-cerebellum (6.1.5.). Our interpretation of this AChE pattern in the primate cerebellum generally is supported by data on the corticonuclear projection in primates and subprimates (see Haines et al., 1982 and Haines and Dietrichs, 1991 for reviews of the literature). Results from experiments on Galago, using silver impregnation of degenerated axons, showed that at least six zones corresponding to the A,B,CI__3 and D zones of carnivores, could be identified in the anterior lobe (Haines, 1977a; Haines and Rubertone, 1979) (Fig. 128). Lesions of the B zone in lobule V were associated with an additional projection to the 187
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most medial portions of the posterior interposed nucleus, suggesting the presence of an X zone in the vermis of this species (see also Haines and Dietrichs, 1991). Similar observations were made for the corticonuclear projection of the anterior lobe of Saimiri sciurus, where the X zone extended from lobule IV into VI. Purkinje cell axons of the X zone in the medial part of the posterior interposed nucleus do not overlap with the more centrally located terminations of the C2 zone in this nucleus (Haines et al., 1982; Haines and Dietrichs, 1991). Separate D1 and D 2 z o n e s with projections to rostrodorsal and centroventral regions of the lateral cerebellar nucleus, were distinguished in the anterior lobe in Saimiri (Haines et al., 1982). The cortico-vestibular projection of the anterior vermis was studied by Voogd et al. (1991 a) in Macaca fascicularis. The disposition of these Purkinje cells in the lateral A zone and the B zone of the anterior lobe and the simple lobule was similar to the cat. Retrogradely labelled Purkinje cell axons were located in the lateral A and the B compartments that could be delineated in adjacent AChE-stained sections. The X zone and compartment did not contain Purkinje cells with vestibular projections. Only few observations in primates are available on the corticonuclear projection of the posterior lobe. Haines and Whitworth (1978) and Haines and Patrick (1981) studied the projection of the paramedian lobule and the paraflocculus in the tree shrew (Tupaia glis). They concluded that Cl_3 and a D zone, with a similar topography and corticonuclear projection as in the cat, were present in the paramedian lobule of the tree shrew. The C2 and the D zone continued into the paraflocculus, where the D zone could be subdivided into D1 and D2 zones on the basis of its differential projection to the lateral cerebellar nucleus. The organization of the posterior vermis in primates (Haines, 1975a,b) will be dealt with in the Sections on the vestibular cerebellum (6.1.5.) and the olivocerebellar projection (6.3.3.3.).
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Fig. 128. Diagrammatic representation of the corticonuclear projection of lobule V in Galago. There are at least six identifiable corticonuclear projection zones in the lobule V cortex. The vermis consists of zones A and B, the intermediate cortex of three zones C1 - C3 and the lateral cortex of a single D zone. f = flocculus; IC = intermediate cortex; LC = lateral cortex; lvn = lateral vestibular nucleus; 1-nia = lateral anterior interposed nucleus; m - nia = medial anterior interposed nucleus; m - nip = medial posterior interposed nucleus; nl = lateral cerebellar nucleus; nm = medial nucleus; vc - vermal cortex. Haines and R u b e r t o n e (1979)
188
The cerebellum." chemoarchitecture and anatomy
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The rough reciprocity of the corticonuclear and nucleocortical projections has been questioned in primates. In contrast to the observations in the cat Tolbert et al. (1978b), Tolbert and Bantli (1979) and Tolbert (1982) reported that all cerebellar nuclei projected to the vermal cortex in Macaca mulatta and that a major contingent came from the lateral cerebellar nucleus. Haines (1988) was unable to confirm these aberrant nucleocortical projections in Galago and suggested that some of Tolbert's injection sites in the lobules VI and VII of the caudal vermis may have extended in the neighbouring D zones. Haines (1989) found retrogradely labelled nucleocortical cells after injections of the C1, C3 and D zones to be relatively few and to be located in the corresponding anterior interposed and lateral cerebellar nuclei. The major nucleocortical projection in Saimiri takes its origin from the medial and posterior interposed nuclei, is bilateral and is directed at the A~ and C2 zones. This anisotropy in the nucleocortical projection in primates confirmed Trott and Armstrong's (1990) conclusions on the strong nucleocortical projection to the C2 zone in the cat. 6.1.3. Parasagittal zonation in the cerebellar cortex: Antigenic compartmentalization for Zebrin and other markers
The particulars of several Purkinje cell-specific markers that define parasagittal zones in adult rat cerebellum have been discussed in the first part of this chapter (see Section 3.1.2. for motilin, and the synthesizing enzyme of taurine (CSADase) and Section 3.1.8. for the zonal distribution of immunoreactive Purkinje cells in monkey and rat cerebellum, with the B.1 antibody of Ingram et al., 1985). CGRP, somatostatin and pseudocholinesterase (see Section 3.1.3.) are only present in zonally distributed bands of Purkinje cells during prenatal or early postnatal development. The complicated distribution of AChE was discussed in Sections 6.1.2. and 6.1.3. on the compartmental subdivision of the cerebellar white matter in cat and monkey. The distribution of AChE in the cerebellum of the rat is very similar (see Marani, 1986, Boegman et al., 1988; and Voogd, 1995 for reviews). Zebrin I and II (Section 3.1.8.) are the best-studied markers, that have been shown to be expressed by longitudinally organized subsets of Purkinje cells. Several other proteins and antigenic markers share the same or a very similar distribution as the Zebrins (the protein kinase C delta-isoform, Section 3.1.5.; the monoclonal antibody B30 of Stainier and Gilbert, 1989, Section 3.1.8.; the low affinity nerve growth factor receptor protein, Section 3.1.10.; and 5'-nucleotidase, Section 3.5.), whereas others only partially allign with the Zebrin pattern (HNK-1 antigen, Eisenman and Hawkes, 1993), or have a zonal distribution essentially complementary to the Zebrin pattern (cytochrome oxidase, Leclerc et al., 1990, P-path antigen, Leclerc et al., 1992, Section 3.1.8., and FAL in the Bergmann glia, Section 3.11.). Molecular markers of Purkinje cell heterogeneity include neurotransmitter receptors, such as the GABAB receptor (Section 3.7.2.), the muscarinic (m2) receptor (Section 3.10.2.), the dopamine D3 receptor (Section 3.8.) and the substance P receptor (Nakaya et al., 1994). It should be noted that the distribution of most markers, as identified in rodent species, may be different in other species or, as is the case for the muscarine m2 receptors, may be expressed in specific species only (Neustadt et al., 1988) (see Section 3.10.2.). An illustrative example of differential zonal distribution in closely related species was provided by Insel et al. (1994), who studied the distribution of vasopressin V~a receptors in the brain of different species of voles (microtine rodents). A complex distribution with alternating bands of high and low receptor density was observed in 189
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prairie voles. Instead, vasopressine Via receptors were confined to the nodulus in pine voles, and no receptors occurred in the cerebellar cortex of montane and meadow voles. The compartmentalization of motilin and taurine and the zonal patterns revealed by 5'-nucleotidase and Zebrin will be discussed in more detail. Chan-Palay (1984) described the distributions of Purkinje cells that react with antibodies against a conjugate of motilin (Chan-Palay et al., 1981; Nilaver et al., 1982) and against the synthesizing enzyme of taurine (L-cysteine sulfonic acid decarboxylase, CSADCase) (Chan-Palay et al., 1982a,b) (see also Magnusson et al., 1988, and Section 3.1.2.). Purkinje cells with motilin and/or GAD-like immunoreactivity together accounted for more than half of their total number. Purkinje cells that reacted with an antibody against motilin were most numerous in the flocculus and the paraflocculus. In the hemisphere they occurred in groups. They were fewer in the vermis, where they constituted a prominent midlineand two parasagittal bands in the lobules I-VI (Fig. 129). Motilin-immunoreactive Purkinje cells usually were larger than those containing only GAD. A majority of the Purkinje cells and many of the stellate and basket cells in the molecular layer reacted with the antibody against CSADC. These cells were distributed in a midline-band and 3 pairs of CSADC-positive and -negative bands on either side. The bands increased in width in the dorsal part of the anterior lobe (Fig. 130). In the vermis of the posterior lobe similar bands existed but were less distinct. Two CSADCpositive bands were present in the hemisphere. The flocculus and the paraflocculus contained the highest number of CSADC-positive Purkinje cells, but they were not zonally distributed. Chan-Palay's description of the zonal distribution of motilin and CSADC-immunoreactive Purkinje cells does not offer clues for a comparison with similar longitu-
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Fig. 129. Schematic drawing of the distribution ofmotilin-immunoreactive (M-i) Purkinje cells (open triangles) and glutamic acid decarboxylase-immunoreactive (GAD-i) Purkinje cells (filled circles) in a coronal section of rat cerebellum. M-i cells and GAD-i cells are both more concentrated in the flocculus and the paraflocculus than elsewhere. Both cell types occur in the vermis and participate in the formation of the sagittal microzones (arrows). M-i terminal axon projections in the deep cerebellar nuclei are heaviest in the dentate (D: left side) and GAD-i projections are heaviest in the lateral vestibular nucleus (LV: right side). ! = interposed nucleus; F = fastigial nucleus. Chan-Palay et al. (1981).
190
The cerebellum." chemoarchitecture and anatomy
Ch. I
III [anterior I. simple
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Fig. 130. Schematic summary of cysteine sulfinic acid decarboxylase (CSADCase)-positive sagittal microzones or bands in mouse cerebellum. The bands are clearest in the anterior lobe and the vermis, less sharply defined in the hemispheres (dense stipple), and most difficult to discern in the paraflocculus and flocculus (light stipple), because of intense CSADCase reactivity in most Purkinje cells. The dentate (D), interpositus (I), fastigial (F), and lateral vestibular nuclei (LVN) contain numerous CSADCase-positive cells. Chan-Palay et al. (1982b).
dinal patterns in the connections or in the distribution of other markers. The parasagittal zone of large, motilin-immunoreactive Purkinje cells may correspond to the B-zone, which, in the cat at least, contained larger Purkinje cells than the adjoining X zone. The majority of the terminals of the B-zone in Deiters' nucleus, however, contained GABA and did not react for motilin (Fig. 129). The distribution of 5'-nucleotidase (5"N) (Section 3.5.) in alternate longitudinal bands of high and low enzyme activity in the molecular layer of the cerebellar cortex of the mouse (Scott, 1963, 1964, 1965, 1967) was the first evidence for the biochemical compartmentalization of the cerebellar cortex. The pattern of 5'-N-positive and -negative zones is complete in the sense that it is present in all the lobules of vermis and hemisphere and unequivocal, because, in the mouse at least, the bands are clearly delineated (Marani, 1986). The 5'-N band pattern is very similar, if not identical, to the more recently described distribution of Purkinje cells in the rat, reacting with Purkinje cell-specific monoclonal antibodies to Zebrin-I (mabQ113) (Eisenman and Hawkes, 1989). The zonal distribution of 5'-N in the cerebellum of the mouse (see also Section 3.5.) was described in detail by Marani (1982a, 1986). A similar zonation of 5'-N was present in the cerebellum of the rat, the shrew (Marani, 1982a) and of Clethrionomys glarulus (Marani, 1982a). However, uniform and high levels of 5'-N without any indication of a longitudinal zonation characterize the molecular layer in some other rodents, the cat, some primates and in man (Scott, 1967; Marani, 1982a, 1986). The pattern in the rat is less distinct because a high background activity of 5'-N is present all over the molecular layer. This background activity disappears after long-standing lesions of the inferior olive (Marani, 1986). 191
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Fig. 131. Reconstructions of the zonal distribution of 5'-nucleotidase (5'-N) in the molecular layer of the cerebellum of the mouse. Numbers without prefix indicate the nomenclature for the Y-N-positive bands of Marani (1982); P-numbers on the left side refer to the nomenclature for correspondingZebrin I-positivebands of Hawkes and Leclerc (1987). ANT = anterior lobe; FLO = flocculus; PFL = paraflocculus; II-X = lobules II-X. Marani (1982a, 1986). (
A midline band flanked by six, symmetrically disposed 5'-N-positive bands can be recognized in mice (Figs 58A, 131, 132). The bands were indicated by Marani (1982a) with the numbers 1--7 for the anterior lobe and the simple lobule and 11-17 for the posterior lobe. The bands in the anterior and posterior lobe are not necessarily continuous. Staining in the 5'-N-positive bands is rather uniform both in transverse and sagittal sections and cannot be assigned to specific structures in the molecular layer. There is an abrupt change in enzyme activity at the lateral border of the 5'-N-positive bands towards the next 5"N-negative zone; enzyme activity falls off more gradually at their medial borders. The 5'-N-positive bands share the distinctness of their lateral borders with the AChE-postive bands in the cerebellum of the cat and with the borders of the small fiber compartments (the 'raphes') towards the next lateral large fiber (Purkinje cell fiber) compartment in the cerebellar white matter. 5'-N-positive bands are narrow in the ventral part of the anterior lobe. They increase in width in the dorsal parts of the anterior lobe and the simple lobule and even more so in the rest of the posterior lobe, where the 5'-N-negative zones are reduced to narrow slits (Fig. 131). Staining in the medial bands 1-5 of the anterior lobe is heavier than in the more lateral bands 6 and 7 of the hemisphere of the anterior lobe. In the posterior lobe the intensity of the staining differs for the different bands. Heavy staining is found in the bands 11 and 13 and much less reaction product is present in the intermediate band 12 (Fig. 58A). The epitopes recognized by Hawkes' family of monoclonal antibodies known as the 'anti-Zebrins' are localized on Purkinje cells (see Section 3.1.8.). Zonal patterns that are identical or very similar to Zebrin I and II have been described for the distribution of 5'-nucleotidase (see above), the p75 low affinity nerve growth factor receptor protein in the rat (Section 3.1.10., Fig. 38), protein kinase C delta (Fig. 133) (see Section 3.1.5.) and the B30 antibody of Stainier and Gilbert (1989) (see Section 3.1.8.). Immunoreactivity in mouse Purkinje cells for an antibody against H N K is partially congruent with the Zebrin negative Purkinje cells, but Zebrin+/HNK+ Purkinje cells also exist (Hawkes, 1992; Eisenman and Hawkes, 1993). The similarity between the Zebrin pattern and the transient zonal patterns in the development of the Purkinje cell specific marker L7 is discussed in Section 6.2. Complementary staining patterns were described for cytochrome oxidase in Saimiri sciureus and rat by Leclerc et al. (1990), for the distribution of P-path-immunoreactive (Edwards et al., 1989, see below) Purkinje cells in the mouse by Hawkes (1992), Leclerc et al. (1992) and Edwards et al. (1994) and for 3-fucosyl-N-acetyl-lactosamine (CDts) in mouse Bergmann glia (Fig. 94) (Bartsch and Mai, 1991; Marani and Mai, 1992). According to Eisenman and Hawkes (1989) the 5'-N and Zebrin I zonal patterns in mouse cerebellum are congruent (Fig. 135). There are also similarities with respect to the mode and the time scale of their development. 5'-N in neonatal mice is distributed uniformly in the molecular layer and the first signs of a banded distribution cannot be discerned before postnatal day 14 (Hess and Hess, 1986). Zebrin I development in rats 193
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 133. Distribution of protein kinase C delta-immunoreactive Purkinje cells in transverse section through the posterior lobe of rat cerebellum. This pattern is similar to the distribution of Zebrin. Bar - 0,5 mm. Chen and Hillman (1993a).
194
The cerebellum. chemoarchitecture and anatomy
Ch. I
also goes through a stage where all Purkinje cells express the epitope. The banded distribution appears relatively late, from postnatal day 15 onwards, when the immunoreactivity becomes suppressed in the future Zebrin I-negative zones (Leclerc et al., 1988). Differences between both patterns involve the distribution of the marker within the bands. Both for 5'-N and Zebrin I the intensity of the staining falls off in the more laterally located bands, but the sharp lateral borders and the differences in reactivity between the 5'-N-positive bands of the caudal vermis are not as clear with staining for Zebrin I. Moreover Zebrin I immunoreactivity extends into the Purkinje cell axons and compartments of Zebrin I-positive and -negative axons are present in the white matter that reflect the zonal distribution of the corresponding Purkinje cells. Zebrin I in rat cerebellum was compared to the distribution of AChE in the cerebellum of the rat (Boegman et al., 1988). These authors stressed the congruence of Zebrinpositive Purkinje cells with the accumulations of AChE in patches in the underlying granular layer. AChE in these patches is present in glomeruli, in certain Golgi cells and in other, unidentified components of the neuropil. Hawkes and Leclerc (1987) grouped the Zebrin I-positive Purkinje cells in a midline band (PI+) and seven symmetrically disposed parasagittal bands (P2+-PS+). Hawkes and Leclerc's (1987) numbering system for the Zebrin-positive and -negative bands in rat is indicated in Fig. 136. The P - bands of Zebrin-negative Purkinje cells bear the same number as the next medial P+ band. The pattern of Zebrin I-immunoreactive Purkinje cells is virtually identical in rat and mouse (Figs 139 and 140) (Eisenman and Hawkes, 1993). The numbering of 5'-nucleotidase-positive bands according to Marani (1986) and Hawkes and Leclerc's (1987) numbering system for the Zebrin-positive bands can be compared in Fig. 131 of the distribution of 5'-N in mouse cerebellum. The main features of the Zebrin pattern are the increase in width of the Zebrinpositive bands in the dorsal part of the anterior lobe, as compared to the ventral lobules I, II and III and the cortex in the bottom of the primary fissure (Figs. 139, 140, 143). The Zebrin-positive bands can be traced across the primary fissure in lobule VI that forms the rostral bank of the primary fissure. They also increase in width in dorsal lobule VI and fuse into an extensive, Zebrin-positive area that covers lobule VII and the adjoining ansiform lobule. Zebrin-negative bands reappear in caudal lobule VII and in the caudal folia of the Crus II and the paramedian lobule. The pattern is distinct in lobule VIII and the caudal copular portion of the paramedian lobule. Wide, Zebrinpositive separated by Zebrin-negative slits are present in lobule IX. Most Purkinje cells of lobule X are Zebrin-positive, although zonally distributed regions with higher and lower immunoreactivity can be recognized in the bottom of the postero-lateral fissure and in lobule X. The continuity of the Zebrin-positive zones is less clear than suggested by the published diagrams. Regions where the continuity of the zones cannot be assessed, include the lobule VII/ansiform lobule, where most Zebrin-positive Purkinje cells fuse into a single continuum, and the transitions between the lobules VII, VIII, IX and X in the bottom of the prepyramidal, secondary and posterolateral fissures. The P1 + band is narrow and consists of fused, bilateral portions. It is present in all lobules, fuses with the P2+ bands in the lobules VII and IX/X and is wider in lobule VIII. P2+ was identified in the anterior and posterior lobes, but its continuity cannot be established because it fuses with other P+ bands in lobule VII. P3+ is weakly immunoreactive in the anterior lobe, and looses its identity among the Zebrin-positive Purkinje cells of lobule VII. It reappears in caudal lobule VII and can be traced as a distinct band in lobule VIII. The apparent continuity of P3+ between the lobules VIII and IX may be false, a fusion of P3+ with P4+ into the P3+ band of lobule IX should 195
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J. Voogd, D. Jaarsma and E. Marani
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Fig. 134. Diagrammatic reconstruction of the Zebrin II/P-'path staining pattern of the mouse cerebellar vermis. Areas that are Zebrin II positive are shaded: areas that are P-path positive are unshaded; regions that are double labelled are solid black. I-X, cerebellar lobules. Inset: double labelling for Zebrin II and P-path may be limited to the lateral half of the P3+ in lobule VIII. Redrawn from Leclerc et al. (1992).
be considered. P4+ is distinct in the anterior lobe and the simple lobule, fuses with the other P+ bands at the transition of lobule VII and the ansiform lobule, and reappears as a wide, Zebrin-positive strip at the border of vermis and hemisphere at the level of the Crus II and the dorsal paramedian lobule. P5+, P6+ and P7+ are present in the hemisphere of the anterior and posterior lobe, but cannot be traced as individual bands across the Zebrin-positive ansiform lobule. One or more of these P+ bands may continue in the Zebrin-positive, lateral pole of lobule IX. The relations of the P5+/P7+ bands with the uniformly Zebrin-positive Purkinje cells of the paraflocculus and the flocculus have not been established. 196
The cerebellum." chemoarchitecture and anatomy
A
Fig. 135. Photomicrographs of the ventral surface of the uvula of the cerebellum of the mouse in adjacent sections reacted with Zebrin I antibody (A) and for the presence of 5'-nucleotidase (B). Note the identical pattern of staining in both even though the borders of the 5'-nucleotidase staining (B) are less distinct. Q 113 = mabQ113, 5'N - 5'-nucleotidase. Eisenman and Hawkes (1989).
Short strips of Zebrin-positive Purkinje cells have been noticed between the P1 +, P2+ a n d P3+ b a n d s in the a n t e r i o r lobe a n d the simple lobule. Two n a r r o w strips of Zebrin-positive Purkinje cells were identified in the ansiform lobule, between P4+ and P5+. They were considered as bifurcations of these bands, that were indicated as P4+ a n d P 5 b + with the additional strips as P 4 b + a n d P5a+. The n a r r o w stretches of Zebrinpositive Purkinje cells in the dorsal vermis of the anterior lobe a n d the simple lobule, were considered as 'satellite bands'. They are i n c o n s t a n t a n d n o t necessarily bilaterally symmetrical. D o r 6 et al. (1990) identified alternating P+ a n d P - zones in the cerebellum of the grey
Fig. 136. The reconstruction of parasagittal bands of Zebrin I (mabQ113-immunoreactive) Purkinje cells in the adult rat cerebellar cortex as seen from the anterior (a) and posterior (b). The band pattern is based upon the serial reconstruction of nine complete and five partial cerebella from sections cut in the horizontal plane and four complete reconstructions from sections cut coronally. Bands P1 + through P7+ are labelled. Hawkes and Leclerc (1987). 197
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J. Voogd, D. Jaarsma and E. Marani
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198
The cerebellum." chemoarchitecture and anatomy
Ch. I
opossum (Monodelphis domestica, Fig. 137). P l+ - P 3 + bands were identified in the anterior and posterior vermis. The precise identification of P2+ and P3+ was ambiguous in lobule V and different interpretations for the continuity of the reconstructed bands in the lobules VI-X were illustrated (Fig. 138). P4+ was located at the interface of vermis and hemisphere. It was located lateral to the area devoid of cortex at the border of lobule VII and the ansiform lobule, or was split in two Zebrin-positive bands, surrounding the cortexless area (compare Fig. 131 illustrating the 5'-N-positive band 15 in the cerebellum of the mouse) (Marani 1986). Zebrin I compartmentalization in Saimiri sciurus was studied by Leclerc et al. (1990). Both in the vermis and the hemispheres clusters of Zebrin I-immunoreactive Purkinje cells were separated by weakly stained Purkinje cell somata or unstained cells. Zebrinnegative bands, therefore, are less distinct than in rodents. P1 +, P2+ and P3+ bands are continuous from lobule to lobule and become narrower in the anterior lobe. P4+-P7+ bands were tentatively identified in the hemisphere, but not analysed in detail. A complementary histochemical zonation was detected for cytochrome oxidase, that was present in patches in the granular layer corresponding to the P - bands both in squirrel monkey and rat cerebellum. It is obvious from a comparison of the illustrations from the paper of Dor6 et al. (1990), showing the distribution of Zebrin I immunoreactivity in Purkinje cells and their axons and the zonation of AChE in monkey cerebellum, that the P2+ immunoreactivity in the anterior vermis corresponds to the X zone, and P 2 P2 P3
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Fig. 138. Three different interpretations of the adult Purkinje cell compartmentalization for Zebrin in the cerebellum of adult Monodelphis, illustrated in Fig. 136, are possible. A. The P2+ (dark grey) and P3 + (medium grey) bands are continuous and unbranched from lobule I to X. B. P2 + is continuous and unbranched, but a novel Zebrin II+ band is inserted between P1 and P2 in lobules VI to X (unshaded). C. P2+ bifurcates within lobule V to give two branches in lobules VI. Dor6 et al. (1990).
199
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Fig. 139. Drawings of three surface views of the mouse cerebellum, anterior, dorsal and posterior, showing the locations of the Zebrin+ bands of Purkinje cells. The Purkinje cell bands P1 +-P7 + are labelled in the dorsal view (for clarity, only the numerals have been used). Note that in the vermis of the posterior lobe the immunoreactive Purkinje cells form five to seven bands (posterior and dorsal views), whereas in lobules VII and VI all vermal Purkinje cells are immunoreactive (posterior and dorsal view). This pattern gradually changes in the anterior lobe to result in three to five very narrow immunoreactive bands (anterior view). In the hemispheres there are three major immunoreactive bands of Purkinje cells on either side (P5b +, P6 +, P7 +) plus two sub-bands in the paravermal area of the paramedian and ansiform lobules (P4b +, P5a +). Note too that the Purkinje cells are all Zebrin+ in the nodulus (lobule X, illustrated as indicated by arrows reflected out from the ventral surface of the cerebellum), the paraflocculus, and the flocculus. From Eisenman and Hawkes (1993).
200
The cerebellum." chemoarchitecture and anatomy
Ch. I
to the B zone. Our preliminary observations on the distribution of Zebrin I-immunoreactivity in the cerebellum of macaques and cats suggested that all Purkinje cell somata in these species were Zebrin-positive and that compartmentalization was less distinct than in the reports on other mammalian species. Bands of P-path immunoreactive Purkinje cells alternate with zebrin II immunoreactive neurons in the cerebellum of the mouse (Leclerc et al., 1992) (Fig. 134). In the P3+ band in the anterior vermis, lobule VII, VIII and dorsal IX, the P4+ band in the lobules V and VIII and the P2+ band in dorsal lobule IX the two epitopes are colocalized. The B1 monoclonal antibody of Ingrain et al. (1985) also detects a subset of Purkinje cells in monkey cerebellum, but their distribution did not correspond to the distribution of Zebrin I in the squirrel monkey (Leclerc et al., 1990) or to AChE as reported by Hess and Voogd (1986). 6.1.4. The corticonuclear projection of the cerebellum of the rat: Correlations with zebrin-antigenic compartmentalization
The corticonuclear projection of the cerebellum of the rat recently was reviewed by Buisseret-Delmas and Angaut (1993) and Voogd (1995). It was studied with anterograde degeneration methods (Goodman et al., 1963; Haines and Koletar, 1979; Umetani et al., 1986) and anterograde tracing with [3H]leucine (Armstrong and Schild, 1978a,b). One interesting feature of these studies is that they document a projection from the hemisphere to the dorsolateral protuberance of the fastigial nucleus, that has never been observed in carnivores or primates. According to the experiments of Armstrong and Schild (1978b) this projection originates from the Crus II and the adjoining paramedian lobule, with smaller contributions of the Crus I and the copula pyramidis. Umetani et al. (1986) limited the projection to the dorsolateral protuberance to the cortex of the medial hemisphere of the lobules between the primary and prepyramidal fissures. The cortex of the copula pyramidis, caudal to the prepyramidal fissure was found to project to the medial part of the anterior interposed nucleus. The localization of Purkinje cells with circumscribed projections to single cerebellar nuclei was investigated with anterograde transport of WGA-HRP (Buisseret-Delmas, 1988a,b; Buisseret-Delmas and Angaut, 1993). Buisseret-Delmas (1988a,b; Fig. 141) distinguished A and B zones in the anterior vermis on the basis of their projection to the fastigial and the dorsal part of the lateral vestibular nucleus, and their afferent climbing fiber projections from the caudal medial and dorsal accessory olives. She distinguished the portion of the medial hemisphere between the primary and prepyramidal fissures, that projects to the dorsolateral protuberance, in the A zone as the lateral extension of the A zone (Fig. 142). The X zone was distinguished from the A zone by Buisseret-Delmas et al. (1993) and found to project to an area located at the junction of the fastigial and posterior interposed nuclei that they indicated as the 'interstitial cell groups'. C1, C2 and C3 zones projected to the interposed nucleus, and received their climbing fibers from the rostral half of the dorsal accessory olive (C1 and C3) and the rostral medial accessory olive (C2). C1 and C3 are interrupted in the Crus I and projected to medial and lateral portions of the interposed nucleus, the projection of C2 occupied its intermediate one third. Voogd's (1964, 1969) original definitions of the C zones, with C1 and C3 projecting to the anterior interposed nucleus, and C2 to the posterior interposed nucleus, therefore, were not retained in this study. Three D zones were identified by Buisseret-Delmas and Angaut (1989b) in the hemisphere of the cerebellum of the rat (Fig.141). The Do zone is unique for the rat, and 201
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Fig. 140. Computer drawing of the reconstruction of the Zebrin Purkinje cells bands in the unfolded adult C57/B6 mouse cerebellum. The drawing was from immunostained 40/~m thick coronal frozen sections. The continuity of the bands has been determined as best as possible. On the left and bottom are the scales in millimeters. The two axes have different magnifications. On the right are marked the approximate boundaries of the vermal lobules. The flocculus and paraflocculus are not illustrated. One place where the data are ambiguous is within lobule V-VI, where a large number of short bands more caudally are dramatically reduced to just three at the rostral limit. It is not clear whether the P2 + or P3 + bands extend through the anterior lobe vermis (see also Fig. 139). The reconstruction data from coronal sections were not suitable to resolve the issue, so the cerebellum has also been reconstructed from horizontal sections. The upper inset panel shows the data from such a reconstruction, equivalent to the region indicated by a rectangle on the main drawing (scale in millimeters). The preferred interpretation is that the P2 + compartment does not extend far into the anterior lobe vermis, and that the first lateral Zebrin+ band in lobules I-IV is continuous with P3 + (as indicated by continuous lines in the upper inset panel and as shown in the main drawing). The alternative hypothesis, that the first lateral Zebrin + band in lobules I-IV is continuous with P2 +, is shown schematically in the lower inset panel. Eisenman and Hawkes (1993). (
projects to the dorsolateral hump and receives a projection from the medial half of the ventral leaf of the principal olive. The Do zone is present in the anterior lobe and the lobulus simplex, Crus II and the paramedian lobule. It is interrupted at the level of the Crus I. The D1 and D 2 z o n e s occupy the lateral border of the hemisphere. D~ extends uninterruptedly from lobule III through the copula pyramidis. O 2 extends beyond the copula into the lateral parts of the vermal lobules IX and X and into the paraflocculus. The corticonuclear relations of the D1 and D 2 z o n e s of the rat are reversed with respect to their namesakes in the cat. D1 projects to the dorsal, magnocellular part of the lateral cerebellar nucleus; D2 to the ventral, parvicellular part of this nucleus. A detailed analysis is available for the projections of the pyramis and the copula pyramidis (Umetani and Tabuchi, 1988) (Fig. 153) and lobule VIIb with the rostral paramedian lobule (Umetani, 1989) (Fig. 152). The C1 zone, with a projection to the anterior interposed nucleus, was lacking in the paramedian lobule and wide in the medial cortex of the copula. C2 was present in both lobules and C3 was limited to the rostral half of the paramedian lobule. D1 (corresponding to Do of Buisseret-Delmas and Angaut, 1989b) projected to the dorsolateral hump and D 2 (corresponding to both their D1 and D 2 zones) to the lateral cerebellar nucleus. The topography of the C zones in the rat, therefore, corresponds to the situation in cat and monkey, with the exception of the projection of a zone in the medial hemisphere to the dorsolateral protuberance of the fastigial nucleus, the presence of an additional Do zone and the reversal in the corticonuclear projection of D1 and D 2. This correspondence, however, does not explain the pattern of Zebrin-positive and -negative zones in the same region. The projections of the cortex to the fastigial and vestibular nuclei were correlated with the Zebrin immunoreactive Purkinje cell zones, using double staining with an anti-zebrin antibody and cobalt-stabilization of the retrograde labelled Purkinje cells on the same section. Purkinje cells in the A and B zones were retrogradely labelled from injections of WGA-HRP in Deiters' nucleus (Voogd et al., 1991b). In the anterior lobe and the simple lobule they were located in the zebrin-negative P1- and the lateral P2- zones (Fig. 143, left). The labelled Purkinje cells in P2- (i.e. in the B zone) were bordered on their medial side by P2+. Zebrin-positive 'satellite' bands bordered the labelled Purkinje cells in the lateral P1- zone on their medial side. WGA-HRP injections at the junction of the fastigial and posterior interposed nucleus labelled Purkinje cells of P2+ and the satellite bands between PI+ and P2+ (Fig. 143, right). Injections of the dorsolateral 203
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(D Fig. 142. Lateral extension of zone A in the posterior lobe with its projection to the dorsolateral protuberance (dlp) of the fastigial nucleus in the rat. The cumulative results of 8 injections of W G A - H R P in the posterior lobe are illustrated. The injection sites are represented as grey areas in B and the corresponding afferent and efferent connections are represented in black in A (inferior olive) and C (cerebellar nuclei), respectively. Dots indicate single labelled neurones in A and few sparsely labelled terminals in C. The lateral extension of the A zone receives a projection from the medial subnucleus c of the caudal medial accessory olive (MAO: A2-7). Buisseret-Delmas (1988a)
protuberance of the fastigial nucleus labelled Purkinje cells located between the primary and prepyramidal fissures overlapping with and located in between the P4b+ and P5a+ zones (Fig. 144). Zebrin-positive and -negative zones in the anterior vermis, therefore, approximately correspond with the corticonuclear projection zones A, X and B that were known from studies in other species than the rat. The projection of the Zebrinpositive Purkinje cells of the X (P2+) zone to the junction of the fastigial and posterior interposed nucleus is in accordance with the tendency of Zebrin-positive Purkinje cells to project to the caudal pole of the fastigial nucleus (Hawkes and Leclerc, 1986). The P4b+, P4b- and P5a+ zones corresponded with the zone projecting to the dorsolateral protuberance. In the simple lobule this zone was located immediately lateral to the B zone. As a consequence the fused P4b+ and P5a+ zones continue, rostral to the primary fissure, as the P3+ zone of the anterior lobe. The topography of the C and D zones in the rat has not yet been correlated with the Zebrin pattern, but it seems likely that C1, C3 and D (Do and D2 zones of Buisseret-Delmas and Angaut, 1989b), projecting to the anterior interposed and the rostral dentate nucleus, will prove to correspond to Zebrinnegative zones and that the C2 and D1 zones, that project to the posterior interposed and the caudal dentate, will be Zebrin-positive. Figure 145 depicts a diagram illustrating this hypothesis.
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Fig. 143. Comparison between Purkinje cells of the A and B zone (open circles) retrogradely labelled from the vestibular nuclei (left side) and retrograde labelling of Purkinje cells of the X zone after an injection of WGA-HRP in the transitional region of the fastigial and posterior interposed nucleus the rat (right side). Purkinje cells of the X zone occupy the Zebrin-positive P2+ zone; labelled Purkinje cells of the lateral A zone and the B zone are located in the Zebrin-negative P1- and P2- zones. Graphical reconstructions of transverse sections double labelled for HRP reaction product and Zebrin I immunocytochemistry.COP = copula pyramidis; CrI and II = crus I and II of the paramedian lobule; PMD = paramedian lobule; S1 = simple lobule; I-X = lobules I-X. Voogd et al. (1991b). (,
6.1.5. The corticovestibular and corticonuclear projections of the flocculus and the caudal vermis. Correlations with cytochemical zones and compartments Larsell (1934) subdivided the cerebellum in a somesthetic corpus cerebelli and the vestibulocerebellum. The compartmentalization of the corpus cerebelli was considered in the previous sections. The vestibulocerebellum of Larsell consists of the flocculus and the nodulus, the caudalmost lobules of the folial chains of vermis and hemisphere. They are separated from the corpus cerebelli by the posterolateral fissure, one of the earliest fissures to appear during ontogeny. In primitive mammals a narrow band of cortex along the attachment of the roof plate of the fourth ventricle interconnect the nodule and the flocculus. The modular organization and the characteristic afferent and efferent connections of the vestibulocerebellum extend, beyond the posterolateral fissure into the ventral part of lobule IX and in the adjacent cortex of the ventral paraflocculus which, therefore, should be included in it. The distinction between the corpus cerebelli and the vestibulo-cerebellum is clearly revealed by calretinin. The rather uniform staining of granule cells and parallel fibers in the corpus cerebelli suddenly stops at the borders of dorsal and ventral lobule IX and of the paraflocculus with the flocculus. In the vestibulocerebellum the unipolar brush cells are heavily stained on a lightly stained background (Floris et al., 1994) (Fig. 146). Different zonally distributed Purkinje cell markers, such as Zebrin I (Hawkes and Leclerc, 1987), motilin, taurine (Chan-Palay, 1984) and somatostatin (Villar et al., 1989) occur more uniformly in the flocculus, and the paraflocculus. Most Purkinje cells of the flocculus and the paraflocculus are Zebrin-positive and no banding has been observed with immunocytochemical methods. Compartmentally distributed differences in fiber size were never observed in the flocculus of carnivores, but compartments can be delineated in the white matter of the flocculus in rabbits and in old and new world monkeys, both with a myelin (the HS.ggqvist) stain and AChE histochemistry. In the rabbit (Van der Steen et al., 1991, 1994; Tan et al. 1992, 1995a,b,c) and the monkey (Macaca fascicularis, Hess and Voogd, 1986; Voogd et al. 1987a,b) 5, respectively 4 compartments can be recognized in the flocculus (Figs 147, 148 and 149). The medialmost compartment 4 of the rabbit flocculus is narrow and located at the border of the middle cerebellar peduncle (Fig. 147a,d). Compartments 1 and 3 are relatively rich in AChE and fuse in the dorsal and caudal white matter of the flocculus. The compartment 2 is poor in AChE and contains large Purkinje cell fibers in the myelin-stained sections. The fifth, most lateral compartment is the caudal extension of the C2 compartment of the paraflocculus. The compartments 2-5 continue, across the posterolateral fissure into the paraflocculus. In the first folium of the ventral paraflocculus of the rabbit (folium p of Yamamoto and Shimoyama, 1977), the compartments 1 and 3 enclose the dorsal tip of compartment 2 (Fig. 147b,c). Four compartments 207
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Ch. I
Fig. 144. Localization of retrogradely labelled Purkinje cells (open circles) from an injection of WGA-HRP in the dorsolateral protuberance of the medial cerebellar nucleus (left) and of anterograde transport of Phaseolus vulgaris leucaglutinin (PhaL) in climbing fibers (stripes) from the medial portion of the MAO (tectorecipient area), with respect to bands of Zebrin-I labelled Purkinje cells in the rat. Reconstructions from sections double-stained for Zebrin and WGA-HRP or PhaL. Numbering of Zebrin-immuno-reactive Purkinje cell zones according to Hawkes and Leclerc (1987). COP = copula pyramidis; CrI(II)= crus I(II) of the ansiform lobule; PMD =paramedian lobule; SI = simple lobule; I-X = lobules I-X. Voogd et al. (199 lb). (
extend over the entire ventral paraflocculus of Larsell (1970) in old and new world monkeys. The most lateral compartment continues as the C2 compartment in the petrosal lobule (Fig. 148b,c), the medial three compartments do not extend beyond the narrow junction of the cortex of the ventral paraflocculus with the petrosal cortex. The narrow, medialmost compartment 4 of the rabbit flocculus was not recognized in the primate cerebellum. The compartments 1, 2 and 3 of the monkey flocculus, correspond to their namesakes in the rabbit. Fibers from the white matter of the flocculus and the adjoining folia of the ventral paraflocculus collect in a compact bundle: the floccular peduncle. The peduncle is applied to the ventral surface of the lateral parvocellular cerebellar nucleus and arches over the restiform body (Fig. 147c,f). Its fibers surround the ventral limb of the lateral cerebellar nucleus as a capsule in rabbit and cat. Medium sized cells of the dorsal group y are located between the fibers of the peduncle in these species. Smaller cells in a dense AChE-positive neuropil accumulate, as the ventral group y, between the peduncle and the cochlear nuclei with the restiform body and, more medially, at the entrance of the peduncle in the vestibular nuclei. In monkeys the group y is compact and situated as an enlongated mass in the floccular peduncle. More caudally, small AChE-positive cells of Langer's (1985) basal interstitial nucleus extend into the lateral compartments of the flocculus and in the AChE-positive raphes between them. Some fibers of the compartments 1 and 2 of the rabbit flocculus arch through the dentate nucleus, to join the floccular peduncle at the lateral border of the vestibular nuclei. These 'arciform' fibers have been found in cat, rat, rabbit and Galago but seem to be absent in primates (Langer et al., 1985b). The white matter compartments of the flocculus do not simply continue as components of the floccular peduncle, but a reorganization takes place, that directs Purkinje cell axons from the compartments 2 and 4 to the medial vestibular nucleus and of the compartments 1 and 3 to the superior vestibular nucleus. The C2 compartment does not contribute to the floccular peduncle, but leads its fibers towards the posterior interposed nucleus. In monkeys the equivalent of compartment 1 of the rabbit extends in the roof of the fourth ventricle, along the basal interstitial nucleus of the cerebellum (Fig. 148). The corticonuclear and corticovestibular projections of the flocculus were studied by Dow (1936), Bernard (1987) in rat, Voogd (1964), Angaut and Brodal (1967), Sato et al., 1982a and b) in cat, Dow (1938), Langer et al. (1985b) in primates and Yamamoto and Shimoyama (1977), Yamamoto (1978), De Zeeuw et al. (1994a) and Tan et al. (1995c) in rabbit. The differential projection of the Purkinje cell zones of the flocculus was discovered by Yamamoto and Shimoyama (1977). The precise correspondence of the corticovestibular and group y projections of the rabbit flocculus with the ACHEcompartmentalization of its white matter and its functional correlates were discussed by Van der Steen et al. (1994), De Zeeuw et al. (1994a) and Tan et al. (1995a,b) (Fig. 188). A projection of the flocculus to the nucleus prepositus hypoglossi has been dis209
Ch. I
J. Voogd, D. Jaarsma and E. Marani
AXBGC~C~
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fMl P puted. This connection was described by Yamamoto (1978) in the rabbit, McCrea et al. (1979) and Yingcharoen and Rinvik (1983) in the cat, but denied by Sato (1982a) for the cat and by Epema (1990) for the rabbit. The projection is not present in subprimates (Haines, 1977a) or primates (Langer et al., 1985b). Compartmental subdivisions based upon myeloarchitectonic criteria or AChE-histo210
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 145. Diagram of the Zebrin-antigenic zones (left) and the corticonuclear projection (right) in the rat. The diagram of the corticonuclear projection is based upon data from Umetani et al. (1986); Umetani (1989), Bernard (1987); Buisseret-Delmas and Angaut (1989b, 1993) and Voogd et al. (1991b). There is a good correspondance between the corticonuclear projection zones A, X and B of the anterior vermis, and for the projection of the lateral extension of the A zone of Buisseret-Delmas (1988a) and the Zebrin pattern. For other zones this correspondance is conjectural or absent. The corticonuclear projection of the region between vermal lobule VIc and VII and the lateral A zone is not known. The corticonuclear projection zones are indicated with the same symbols as their target nuclei in the bottom diagram of the cerebellar nuclei. Regions projecting to the vestibular nuclei (a lateral strip of the A zone, the B zone and the caudal vermis) are indicated with open circles. 1-7 - Zebrin antigenic zones P1-P7" A - zone A; A~ - lateral extension of the zone of Buisseret-Delmas (1988a); B - zone B; C1-3 - zones C1-C3; CrI - crus I of the ansiform lobule; CrII - crus I! of the ansiform lobule; D - zone D; Do =one Do of Buisseret-Delmas and Angaut (1989); DLH - dorsolateral hump; DLP = dorsolateral protuberance of the medial nucleus; DMC - dorsomedial crest; FLO - flocculus; IA - anterior interposed nucleus; I P - posterior interposed nucleus; L - lateral cerebellar nucleus; MM = medial part of medial nucleus; N C - caudomedial part of the medial nucleus; P F L - paraflocculus; PMD = paramedian lobule; SI - simple lopule; tM/IP - transitional region of the medial nucleus and posterior interposed nucleus (interstitial cell groups of Buisseret-Delmas et al. 1993); X - zone X; I-X- lobules I-X. (
chemistry have been p r o p o s e d for the caudal vermis of the cat (Voogd, 1964, 1969) and the m o n k e y (Voogd et al., 1987a,b, Tan et al., 1995b). The caudal vermis projects to the fastigial nucleus, but also to the (posterior) interposed nucleus (Bigar6, 1980, cat; A r m s t r o n g and Schild, 1978a; Bernard, 1987; U m e t a n i and Tabuchi, 1988; Tabuchi et al., 1989, rat) a n d the ventral, parvocellular p o r t i o n o f the lateral cerebellar nucleus (Van R o s s u m , 1969; Wylie et al., 1994, rabbit; Haines, 1977a Galago; Bernard, 1987; Tabuchi et al., 1989, rat) a n d the vestibular nuclei including the g r o u p y. L o b u l e VIII of the caudal vermis gives rise to a small projection to the dorsal part of the lateral vestibular nucleus (i.e. Deiters' nucleus), but other corticovestibular connections o f the caudal vermis and the flocculus avoid Deiters' nucleus and terminate in other subdivisions of the vestibular nuclei. Corticovestibular fibers from the lobules X and IX of the caudal vermis were studied by D o w (1936), B e r n a r d (1987), Tabuchi et al. (1989) in rat, Voogd (1964), A n g a u t and Brodal (1967) in cat and D o w (1938), Haines (1977a) in primates. A detailed study of the corticonuclear and corticovestibular projections was m a d e of the ventral face of lobule X of the rabbit cerebellum by Wylie et al. (1994). Their findings are s u m m a r i z e d in Fig. 150 and can be correlated with the c o m p a r t m e n t a l A C h E - s u b d i v i s i o n of the white m a t t e r of lobule X by Tan et al. (1995b) and the a n a t o m i c a l and electrophysiological analysis of the olivocerebellar projection to this lobule by K a t a y a m a and N i s i m a r u (1988), B a l a b a n and H e n r y (1988) and K a n o et al. (1990, 1991). Corticovestibular fibers f r o m the flocculus and the lobules X and IX of the caudal vermis terminate in roughly c o m p l e m e n t a r y areas of the vestibular nuclei (Fig. 151) (Dow, 1936, 1938; Voogd, 1964; A n g a u t a n d Brodal, 1967; Haines, 1977a; L a n g e r et al. 1985b; Bernard, 1987). The caudal vermis o f the cat usually was considered to belong to the A zone, that projects to the fastigial nucleus. A c c o r d i n g to Bigar6 (1980) (Fig. 123) and Voogd and Bigar6 (1980) the entire lobule VII and the medial two-thirds of the lobules VIII, IX and X are connected with this nucleus. Courville and D i a k e w (1976) d e m o n s t r a t e d the sequential representation of the lobules VI-VIII in the fastigial nucleus of the cat with lobule VII projecting to the tail of the nucleus and lobules VI and VIII to m o r e r o s t r o d o r s a l and ventral parts respectively. A similar connection o f lobule VII to the
211
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Fig. 146. Parasagittal section of the rat cerebellar vermis immunostained with antiserum to calretinin. Folia are labelled with roman numerals according to Larsell. The pattern of immunoreactivity in the nodulus (lobule X) and portion of the ventral uvula (lobule IXd) differs from that in the other vermal folia. While in most of the vermis weak immunoreactivity is present in granule cell bodies within the granular layer and in parallel fibers within the molecular layer, in the folia X and IXd of the vestibulo-cerebellum the immunoreaction product is largely localized in unipolar brush cells and mossy fibers, MN, medial cerebellar nucleus. Bar = 1 mm. Floris et al. (1994).
tail of the fastigial nucleus was demonstrated by Yamada and Noda (1987) for the macaque monkey. Purkinje cells in the medial two-thirds of lobule IX of the cat also project to the medial and descending vestibular nuclei; those destined for the descending vestibular nucleus are concentrated near the midline and at the lateral border of the A zone (Matsushita and Wang, 1986). The lateral one-third of lobule IX is connected with the posterior interposed nucleus (Bigar6, 1980). A complicated pattern of Purkinje cell zones and patches was reported for the organization of the projection of lobule X to the superior, medial and descending vestibular nuclei in the cat (Shojaku et al., 1987). The zonal organization of the efferent connections of the caudal vermis in the rabbit is quite complex, with discrete zones in the lobules IX and X projecting to the fastigial, descending, superior and medial vestibular nuclei, and lateral zones connected to the interposed and different subdivisions of the lateral cerebellar nucleus (van Rossum, 212
The cerebellum: chemoarchitecture and anatomy
Ch. I
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Fig. 147. Photographs and diagrams of AChE-stained sections from rabbit flocculus. The compartments C2 and 1-4 of the flocculus white matter are indicated in the diagrams with different symbols, a-d. Rostralmost section, c-f. Caudalmost section. Arrows indicate the AChE-postive borders between the compartments, bp brachium pontis; C2 = C2 compartment; CO = cochlear nuclei; f (1-4, p) = folium 1-4 and folium p of the flocculus; L - lateral cerebellar nucleus; m = folium m of the flocculus; p f - floccular peduncle; PFLD/V = dorsal and ventral paraflocculus. Tan et al. (1995a).
213
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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$ Fig. 148. Compartmentation in AChE-stained transverse sections of the flocculus, the ventral paraflocculus and the petrosal lobule and the caudal hemisphere of Saimiri sciureus. Note AChE stained borders of the compartments and the differential staining in the molecular layer; AChE-positive cells of the basal interstitial nucleus of Langer are located along the borders of the compartments C2, 1 and 2 in (D) and (E). Group y is located within the floccular peduncle in (E). ANS = ansiform lobule; br.p = brachium pontis; c.rest = restiform body; C1-3 = C21-3 compartments; D = dentate nucleus; D1,2 = D1,2 compartments; F = fastgial nucleus; fis.post.lat = posterolateral fissure; FLOC = flocculus; IA = anterior interposed nucleus; IP = posterior interposed nucleus; lb.petr = petrosal lobule; PFLD = dorsal paraflocculus; PFLV = ventral paraflocculus. Courtesy of Dr. D.T. Hess.
1969). Some sort of zonal organization in the corticovestibular projection of these lobules in the rabbit also was reported by Balaban (1984), Epema et al. (1985) and Epema (1990). 214
The cerebellum." chemoarchitecture and anatomy
Ch. I
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More detailed, but still incomplete accounts of the corticonuclear projection of the caudal vermis are available for the rat. The terminal fields of the different lobules in the fastigial nucleus of the rat are organized in a circumferential manner (Armstrong et al., 1978a; Haines and Koletar, 1979) with more overlap between the lobules than has been reported for the cat (Courville and Diakew, 1976). PS.fillysaho et al. (1990) analysed the projections of the lobules VI-VIII to the fastigial nucleus in more detail in the rat. The terminal field of lobule Via is located in the middle subdivision of the fastigial nucleus, lobules VIb,c, VII and VII project both to the caudomedial and middle subdivisions, with the terminal field of lobule VIII reaching most rostrally. Terminations of these lobules in the dorsolateral protuberance are scarce or absent. A subdivision of lobule VII into a medial region, projecting to the caudomedial subdivision of the fastigial nucleus and of a lateral region projecting to the middle subdivision also was proposed by Umetani (1989) (Fig. 152). This medial region may correspond to the tecto-olivorecipient zone of Akaike (1986b, 1992) (Fig. 184). The lateral zone of lobule VII, that separates the tecto-olivo-recipient zone from the lateral extension of the A zone, with its projection to the dorsolateral protuberance of the fastigial nucleus may be identical to the X zone. 215
Ch. I
J. Voogd, D. Jaarsma and E. Marani C2
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Fig. 149. Diagrams of cerebellar cortex, showing the zonal configuration of the paraflocculus and the flocculus in rabbit, monkey and cat, based upon the compartmental subdivision of the paraflocculus and the flocculus in monkey and rabbit (Figs 147 and 148) and the olivocerebellar projection to the flocculus in cat (Voogd, 1964; Gerrits and Voogd, 1982) and rabbit (Tan et al., 1995a,b). Ca, D1 and D2 zones can be distinguished in the dorsal paraflocculus and the petrosal lobule of the monkey and in the dorsal paraflocculus and the rostral segment of the ventral paraflocculus of the cat. The C2 zone and a single D zone were distinguished in the paraflocculus (the petrosal lobule) of the rabbit cerebellum. The C2 zone, that extends over the entire cerebellum, occupies the lateral part of the flocculus in all species. Four zones 1-4 are present in the flocculus of the rabbit, three of these (1-3) are present in the primate flocculus. Of the 6 floccular climbing fiber (f) zones in the cat, fl corresponds to zone 4 of the rabbit, f2+3 to zone 3, f4 to zone 2 and f5+6 to zone 1. The zonal pattern of the flocculus proceeds for some distance on the paraflocculus. This transitional segment corresponds to folium p of the rabbit, to the ventral paraflocculus of the monkey and to the medial extension of the ventral paraflocculus (ME) of the cat. The lateral border of the hemisphere (heavy line) continues in the medial border of the flocculus. The inner surface of the cortex is shaded. Asterisks indicate the area without cortex in the center of the ansiform lobule. 1-4 = floccular zones 1-4 of rabbit and monkey; ANS = ansiform lobule; A N T anterior lobe; C 2 -~ zone C2; D1,2 = zones dl,2ME = medial extension of the ventral paraflocculus fl-6 = climbing fiber zones 1-6 of the flocculus of the cat - 9 F I P L - posterolateral fissure; FLO - flocculus; FP folium P (rabbit); P F L D = dorsal paraflocculus; PFLV = ventral paraflocculus; P M D - paramedian lobule; POST - posterior lobe.
Two strips of Purkinje cells were distinguished by Umetani and Tabuchi (1988) in the vermis of lobule VIII (Fig. 153). The medial zone projected to both subdivisions of the fastigial nucleus, the second zone to the posterior interposed and lateral vestibular nuclei. This region, therefore, may include equivalents of the X and B zones of the anterior lobe. It was bordered on its lateral side by the C1 zone. The zonal organization of the lobules IX and X was analysed by Bernard (1987) and Tabuchi et al. (1989) in the rat. According to Bernard (1987) the zonal arrangement in lobule IX is very similar to the pattern in cat and rabbit with a medial zone projecting to the fastigial nucleus and a middle zone connected with medial portions of the interposed nuclei, corresponding with the posterior interposed nucleus. The most lateral zone 216
The cerebellum." chemoarchitecture and anatomy
Ch.I
Olivocerebettar projection Katayoma gNisimaru '88 BOtQLan g Henry '88 Tan et at.'94
dorsal nodulus ventral, nodutus
Pcet[ axonaL tracing Wylie et al. '94
Fig. 150. Diagram of the afferent olivocerebellar projection according to Katayama and Nisimaru (1988) and the efferent projection of the zones of the nodulus to the vestibular and cerebellar nuclei according to Wylie et al. (1994) in the rabbit, fl = group fl of the medial accessory olive; DC = dorsal cap of Kooy; F = fastigial nucleus; IP = posterior interposed nucleus; MV = medial vestibular nucleus; P cell = Purkinje cell; SV = superior vestibular nucleus; VLO = ventrolateral outgrowth; I-VI = zones of rabbit nodulus, numbered according to Katayama and Nisimaru (1988). was connected with the ventral, parvicellular portion of the lateral cerebellar nucleus. The medial two zones shifted to a more lateral position in lobule X, where an additional zone, projecting to the medial vestibular nucleus, took up the most medial position. These zones also project in a differential manner, to the superior and descending vestibular nuclei and to the group y. The most lateral zone, that was connected with the lateral cerebellar nucleus, extended in lobule Xa and did not project to the vestibular nuclei. Although the Zebrin pattern in the caudal vermis is quite distinct and some correlations with the corticonuclear projection zones are obvious (Fig. 145), no precise comparisons with the Zebrin pattern have been made. Our knowledge of the cortico-vestibular and nuclear projection of the caudal vermis, therefore, is still deficient. Progress can be expected from anatomical and electrophysiological studies using the Zebrin pattern as a reference. 6.2. R E G I O N A L D I F F E R E N C E S IN T H E D E V E L O P M E N T O F T H E CEREBELLUM The development of the cerebellar cortex, the cerebellar nuclei and the precerebellar nuclei has been documented and reviewed in a series of papers on the rat by Altman (1975a,b,c) and Altman and Bayer (1985a,b, 1987a,b). Here we shall be concerned with 217
Ch. I
J. Voogd, D. Jaarsma and E. Marani
A
B
C
Flocculus
Nodulus
Uvula
Fig. 151. Diagram of the corticovestibular projections from flocculus, nodulus and uvula in Galago. Note complementarity between the projections of the flocculus and the caudal vermis, lvn = lateral vestibular nucleus; mvn = medial vestibular nucleus; spvn = spinal vestibular nucleus; svn= superior vestibular nucleus. Haines (1977a).
some aspects of regional differentiation in the development of the Purkinje cells of the cerebellar cortex. Several authors noticed that developing Purkinje cells are clustered into a number of parasagittal zones during early stages of cerebellar development, prior to the stage when the first fissures make their appearance. Purkinje cells and the cells of the cerebellar nuclei are generated in the ventricular layer (Jakob, 1928; Miale and Sidman, 1961), subsequently they migrate to the meningeal surface of the cerebellum where they settle in the cortical plate, deep to the external granular layer. Clustering of Purkinje cells in the cortical plate has been observed by a number of authors in mammals (Korneliussen, 1967, cetacea; Brown, 1985b; Brown et al., 1986, cat; Korneliussen, 1968b; Altman and Bayer, 1985b, rat; Hochstetter, 1929; Korneliussen, 1968c; Maat, 1978, 1981; Marani and Mai, 1992, man; Marani et al., 1986, rabbit; Kappel, 1981, monkey) and birds (Feirabend et al., 1976; Feirabend, 1983). Cell strands interconnect the Purkinje cell clusters with the cerebellar nuclei. The pattern that evolves from the position of the clusters in the cortical plate and their corticonuclear relations, is rather similar to the adult pattern of longitudinal corticonuclear projection zones (Korneliussen, 1967, 1968b; Kappel, 1981; Feirabend, 1983; Marani, 1986; Marani et al., 1986). The borders between the Purkinje cell clusters become indistinct when the fissures begin to develop and the 218
The cerebellum." chemoarchitecture and anatomy
Ch. I
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Fig. 152. Diagrams showing the topographic pattern of the projections from the various mediolateral levels of the tuber vermis (lobule VII) and the paramedian lobule to the cerebellar nuclear complex in the rat. A. Schematic diagram of the posterior surface of the cerebellum and subdivision of the tuber vermis and paramedian lobule, based on the topography of their projections. B. Schematic sagittal diagrams of the nuclear complex showing the terminal fields which receive projections from the individual subdivisions of the tuber vermis and paramedian lobule. AIN = anterior interposed nucleus; cm = caudomedial sub-division of the medial nucleus; Cop. pyr = copula pyramidis; D L H = dorsolateral hump; DLP = dorsolateral protuberance of the medial nucleus; LN - lateral cerebellar nucleus; LVN = lateral vestibular nucleus; m = medial nucleus; PIN = posterior interposed nucleus; Pml = paramedian lobule. Umetani (1989).
internal granular layer appears. Jacob (1928) and Hayashi (1924), were among the first to detect regional differences in corticogenesis in the human cerebellum (Fig. 154) and to correlate their corticogenetic zones with the corticonuclear projection to the different cerebellar nuclei (see Section 6.). A large time gap separates the stage when the borders between the clusters disappear, from the moment when the adult connections of the Purkinje cell have become established. Several methods have been employed to bridge this gap and to trace the developmental Purkinje cell patterns into adulthood. Altman and Bayer (1985b) in the rat, Brown (1985b) in the cat and Feirabend et al. (1985) in the chicken, using tritiated thymidine autoradiography, found evidence for differences in the time of birth between populations of Purkinje cells with a zonal distribution. According to Feirabend et al. (1985) some of the Purkinje cells of the lateral cerebellum and the cells of the lateral part of the central nuclei of the chicken are generated later than medially located cells. Zonal [3H]thymidine labelling of the Purkinje cells can be traced till after hatching. Several markers for adult Purkinje cells have been used to trace back their origin. Wassef and Sotelo (1984) studied the expression of cyclic GMP-dependent protein kinase (cGK) immunoreactivity during development of the rat (Fig. 155). They found a transient heterogeneity of the Purkinje cells for an antibody against cyclic GMP-dependent 219
Ch. I
J. Voogd, D. Jaarsma and E. Marani Tuber vermis
'
A Pyramis
ula
Paramedian Lobule
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127
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Fig. 153. Diagrams showing showing the projections from the various mediolateral projection-zones of the pyramis (lobule VIII) and the copula pyramidis to the cerebellar and vestibular nuclear complexes in the rat. A. Schematic diagram of the posterior surface of the cerebellum. The subdivision of the pyramis and copula are based on the topography of their efferent projections. B. Schematic sagittal diagrams of the cerebellar and vestibular nuclei showing the terminal areas which receive projections from the individual subdivisions of pyramis and copula pyramidis. Abbreviations: see Fig. 152. Umetani and Tabuchi (1988).
protein kinase (cGK) (De Camilli et al., 1984). Some clusters of Purkinje cells and their axons react with the antibody, while others are still negative. Some clusters could be recognized before their cells had completed their migration to the surface. Shortly after birth all Purkinje cells become cGK positive. Similar observations were made by Wassef et al. (1985) with two other Purkinje cell specific antibodies against calbindin-D28k (Legrand et al., 1983), and the Purkinje cell specific glycoprotein (PSG) (Langley et al., 1982) and by VanDaele et al. (1991) and Smeyne et al. (1991) for the transient zonal patterns in the development of the Purkinje cell-specific marker L7 (see Section 3.1.8.). Each antibody gave a different mosaic of positive and negative Purkinje cell clusters and this chemical heterogeneity disappeared shortly after birth, when all Purkinje cells became positive for the different markers. Some degree of heterogeneity is also present during the development of immunoreactivity for anti-parvalbumin in Purkinje cells of birds (Braun et al., 1986), but the early stages of development were not included in this study. Wassef et al. (1987) and Edwards et al. (1994) noticed that the surviving Purkinje cells in mice with mutations affecting their postnatal survival, are arranged in sagittal bands. This differential sensitivity of these Purkinje cells may be related to their chemical heterogeneity. Immunoreactivity for Zebrin (Hawkes and Leclerc, 1986) is not present before birth (Leclerc et al., 1988) and the reactivity for the enzyme 5'-nucleotidase, which is distributed in a similar zonal pattern in the molecular layer of the mouse also appears postnatally (Hess and Hess, 1986). The expression of the L7 gene in mouse Purkinje cells displays a similar develop220
The cerebellum: chemoarchitecture and anatomy
Ch. I
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caudal vermis Fig. 154. Regional variations in cerebellar corticogenesis. A. Diagram of the cerebellum of a two month human embryo. Unlabelled areas, small dots, large dots and black indicate successively more advanced stages in histogenesis. Redrawn from Jakob (1928). B. Subdivision of the mammalian cerebellum according to Jakob and }tayashi. Redrawn from Jakob (1928). A = medial corticogenetic zone of the vermis; B - lateral corticogenetic zone of the anterior vermis or pars intermedia; FLO = flocculus.
mental heterogeneity. It first appears in paired bands in the vermis and the flocculus, later in clusters in the hemispheres. Ultimately it is present in all Purkinje cells (Smeyne et al. (1991)). Oberdick et al. (1993) slowed down this process by manipulation of the promotor region (Fig. 156). The transient expression of L7 in Purkinje cell bands with high and low levels of expression, as shown in whole mounts of the mouse cerebellum, is very similar if not identical to the Zebrin pattern as documented for adult mice by Eisenman and Hawkes (1993) (Fig. 139). It is not known whether the developmental patterns for other general Purkinje cell markers also resemble the Zebrin pattern. Moreover, it was demonstrated by Oberdick et al. (1993) that the L7 pattern also develops in vitro and, therefore, is independent of the ingrowth or the presence of extracerebellar afferents. Similar observations were made by Leclerc et al. (1988) for the development of the Zebrin pattern in rats, after section of the extracerebellar afferents. These results strongly advocate a primary role for the Purkinje cells in the development of zonal patterns (Wassef et al., 1992c). Few signs have been noticed of longitudinal subdivisions in the development or in the adult granular layer. The zonal distribution of AChE in the development of this layer was discussed by Marani (1986). Monoclonal antibodies against Stage Specific Embryonic Antigen-1 (SSEA-1), also known as the X-hapten, Lacto-N-Fucopentax III or FAL (Fucosyl-N-Acetyl Lactosamine), showed a longitudinal subdivision in the mitotic external granular layer of the rabbit cerebellum. This pattern was present from embryological day E8-E9 till postnatal day P15-16 and consisted of alternating positive and negative strips. The positive staining for SSEA-1 antibodies is exclusively present at the cell membrane of cells in the external granular layer (Marani and Tetteroo, 1983, Marani, 221
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 155. Staining with cyclic 3',5'-guanosine monophosphate dependent protein kinase (cGK) antisera of sections of cerebellum of rat fetuses of embryonic day E17, E19 and a neonate (PO) cut in the frontal plane. A,B. E 17. Cluster I is composed of a medial sheet (arrow in B) lying against the germinative neuroepithelium. Close to the midline this sheet bends dorsally and reaches the cortex. The central cluster (CC) is located at the center of the hemicerebellum. C. E 19. In this section four of the five cGK-positive clusters I-V are present. The labelled fiber-like material, which tails the labelled clusters (* and o) indicates the migration pathw.ays followed by the Purkinje cells of the clusters I and III from the subventricular plate and the central cluster at E17 to their present, superficial position. D. PO rat pup. Fiber bundles linking the clusters I and III with the cerebellar nuclei intersect at the former position of the central cluster. It is suggested that the bundle from cluster III (*) terminates in the dorsolateral protuberance. In the adult this connection corresponds to the projection of the lateral extension of the A zone of Buisseret-Delmas (1988a, compare Figs. 142 and 144). Bar in A - 100 ~m, in B, C and D = 500 ~tm. Wassef and Sotelo (1984). (
1986; Marani et al., 1986). These results were confirmed with several antibodies against SSEA-1. In dissociated rabbit cerebellum the only cell type that was found to express this antigen was the granule cell (Marani and Tetteroo, 1983, Marani et al., 1983, 1986). These results demonstrate the presence of a longitudinal pattern in an intrinsic mitotic
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Fig. 156. Effects of promotor gene truncation on L7-1acZ banding pattern in mice. Cerebella were dissected free from the rest of the brain and stained in whole mount. Cerebella are viewed from posterior (POST) and anterior (ANY). Cerebella were taken from postnatal day 11 animals carrying 4 kb (top row), 500 bp (middle row) and 350 bp (bottom row) promoter constructs. The patterns are very similar to the Zebrin pattern in mouse cerebellum (compare Fig.139). Expression of L7 is absent or low in P1 +, P3 +, P5 + (indicated with PIN in bottom panels) and P7 + in the 500 and 350 bp constructs. In P4+ there is a strong expression of L7 in the 500 bp construct, and a weak expression in the 350 bp construct. Reversed levels of expression are observed for the region of P4b + and P5a +. In the 500 bp construct they are weakly stained (but the P4b + and P5a + bands are visible as separate strips), in the 350 bp construct there is a strong expression of L7 over the entire area of these bands indicated with FN in lower pannel). Oberdick et al. (1993), interpretations by the authors of this chapter.
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Fig. 157. Configuration of the inferior olive and organization of the olivocerebellar projection in the cat. Olivary subnuclei and their projection areas in the cerebellum and the cerebellar nuclei (upper diagram) are indicated with the same symbols. The unfolded inferior olive is illustrated in the bottom diagram. 1-4 = in diagram of the cerebellum: lobules 1-4 of Bolk; in diagram of the inferior olive: projections to lobules 1-4;fl = group beta; D = dorsal accessory olive; d.cap = dorsal cap; d.1. = dorsal leaf of the principal olive; dm.c.col. = dorsomedial cell column; 1= lateral; lob a = lobulus a of Bolk (X of Larsell); lob b = lobulus b of Bolk (IX of Larsell); lob c l = lobulus c l of Bolk (VIII of Larsell); lob c = lobulus c of Bolk (VI + VII of Larsell); lob.simpl. - lobulus simplex; M = medial accessory olive; m = medial; N.fast = fastigial nucleus; N.int = interposed nucleus; N.lat = lateral cerebellar nucleus; v.1. = ventral leaf of the principal olive; v.l.o. = ventrolateral outgrowth. Brodal (1940). (
layer of the cerebellum which may be related to the establishment of transient contacts between ingrowing afferent fibers or Purkinje cell dendrites and external granule cells. 6.3. T H E O R G A N I Z A T I O N O F T H E O L I V O C E R E B E L L A R P R O J E C T I O N The olivocerebellar projection has been studied mainly in the cat (see Brodal and Kawamura, 1980 and Voogd, 1982 for reviews). Fewer data are available for primates and sub-primates (Brodal and Brodal, 1981, 1982; Whitworth et al., 1983; Whitworth and Haines, 1986b). A more complete picture of the olivocerebellar projection in the rat is emerging (Buisseret-Delmas and Angaut, 1993; Ruigrok and Cella, 1995; Voogd, 1995). The evidence on the role of excitatory aminoacids as the transmitter in the olivocerebellar pathway was considered in Section 3.2.2. It was concluded that glutamate is the most likely transmitter of the climbing fibers. The configuration and ultrastructure of the inferior olive, the organization of the afferent connections of the olive, the olivocerebellar projection and the topographical distribution of peptidergic climbing fibers will be reviewed in the next sections.
6.3.1. Configuration and ultrastructure of the inferior olive The morphology and the subdivision of the inferior olive have been described in the comparative anatomical studies of Kooy (1917), Mar6schal (1934) and Whitworth and Haines (1986a). Brodal's (1940) subdivision of the inferior olive in the cat and his mode of representation of the olive as imagined unfolded in one plane (Fig. 157) have become generally accepted. Dorsal accessory (DAO), medial accessory (MAO) and principal (PO) subnuclei can be distinguished in most mammalian species (Figs 158 and 159). The dorsomedial subdivision of the caudal M A O was indicated as group beta by Brodal (1940). Three parallel, longitudinal cell columns, indicated from laterally to medially as a, b and c (c is the equivalent to the group beta) were distinguished in the caudal M A O of the macaque monkey (Bowman and Sladek, 1973). F o u r columns (a, b, c and beta) were delineated in the caudal M A O of the rat (Gwyn et al., 1977). This apparent discrepancy was solved by Frankfurter et al. (1977) and Ikeda et al. (1989), who showed that subnucleus b in squirrel and macaque monkeys can be subdivided into medial and lateral parts and that only the medialmost region of this subnucleus is connected with the oculomotor vermis (lobule VII) and, therefore, corresponds with subnucleus c in the rat (Hess, 1982b; Akaike, 1992). The rostral half of the M A O sometimes is indicated as the rostral extension of subnucleus a, but this does not serve a useful purpose, because its connections differ 225
Ch. I
J. Voogd, D. Jaarsma and E. Marani
substantially from the caudal half of the MAO. The dorsomedial cell column (DMCC) of the cat is located dorsomedial to the rostral half of the medial accessory olive. Bowman and Sladek (1973) identified their cell group g, which is connected to the medial tip of the ventral leaf of the PO, as the DMCC in the macaque monkey. The DMCC of the rat has been identified as a similar group, connected with the ventral leaf of the PO (Gwyn et al., 1977). However, since the DMCC is part of the MAO and forms a rostral continuation of the group beta in most species (Whitworth and Haines, 1986a),
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Diagrams of the inferior olivary complex of the rhesus monkey. Dorsal accessory olive is shown in black. Medial accessory olive and attached groups (nucleus fl and dorsomedial cell column) are hatched. Principle olive, dorsal cap, and ventrolateral outgrowth are left white. Subnuclei a, b, and c of the medial accessory olive according to the nomenclature of Bowman and Sladek (1973). A series of drawings of 13 equally spaced transverse sections through the olive in the upper panel is represented from rostrally (I) to caudally (XIII). In the lower panel the inferior olive is represented as imagined unfolded (see diagram at bottom of the Figure). In the drawings of the sections showing the principal olive, borders (broken lines) are indicated between the dorsal, lateral, and ventral lamella in accordance with Bowman and Sladek (1973). The solid line at the lateral convexity of the principal olive corresponds to the vertical line in the diagram of the unfolded olive, and is used for orientation. In this diagram broken lines in the medial accessory olive refer to borders betwcen regions which are not clearly separated. In the principal olive broken lines indicate the arbitrary separations between dorsal, lateral and ventral lamella shown in the drawings of transverse sections. The dorsomedial cell column is hidden behind the medial accessory olive (dotted outlines). Brodal and Brodal (1981). Fig. 159.
227
Ch. I
J. Voogd, D. Jaarsma and E. Marani
another cell group positioned directly dorsomedial to the MAO, has been identified as the DMCC in the rat (Azizi and Woodward, 1987; Bernard, 1987; Ruigrok and Voogd, 1990). Rostrally the DMCC of both sides seem to merge. The cell group connected to rostral leaf of the PO of the rat was indicated as the dorsomedial group (DM) (Fig. 158). A dorsal and a ventral leaf usually are distinguished in the PO: in the cat the ventral leaf is continuous with the medial pole of the dorsal accessory olive. In other mammals the DAO is continuous with the dorsal leaf and the ventral leaf ends as the DMCC in macaque monkeys and at the DM in rats. The ventral leaf of the PO caudally tapers into the ventrolateral outgrowth (VLO) that continues as the dorsal cap (DC) of Kooy (1917), located dorsal to the group beta. In dorsal view the DAO of the inferior olive of the cat and the monkey is boomerangshaped, with a narrow, medially directed, caudal tail. In the rat the caudal pole of the DAO consists of a dorsal and a ventral fold, that are continuous medially. The dorsal fold is only present at caudal levels, the ventral fold extends to the rostral tip of the DAO (Azizi and Woodward, 1987) (Fig. 158). Ovoid perikarya in Nissl-stained sections of the inferior olive of the cat are dispersed in clusters comprising up to eight neurons (Sotelo et al., 1974). The dendrites of typical Golgi-impregnated olivary neurons recurve towards the cell body (Fig. 160). This type of neuron, with a compact dendritic arbor, is the main cell type of the principal olive and the rostral pole of the MAO. Other subdivisions of the inferior olive contain a mixture of compact neurons and neurons with long, unramified dendrites. The latter type of neuron was mainly found in the caudal MAO, group beta and the DC (Scheibel and Scheibel, 1955; Scheibel et al., 1956; Ruigrok et al., 1990). Dendrites of both types of neurons give rise to long and thin branching spines (Bowman and King, 1973; Sotelo et al., 1974; Gwyn et al., 1977). The ultrastructure of the inferior olive was reviewed by De Zeeuw (1990) (Fig. 161). Glomeruli are characteristic features of the olivary neuropil (Nemecek and Wolff, 1969; Bowman and King, 1973; Sotelo et al., 1974; King et al. 1975; King, 1976; Gwyn et al., 1977; Rutherford and Gwyn, 1980; Bozhilova and Ovtscharoff, 1979). Glomeruli contain a core of dendritic spines contacted by axon terminals and surrounded by a glial capsule. On average, the glomeruli contain spines derived from six different neurons. Sometimes spines of the initial axonal segment are incorporated together with the dendritic spines in the same glomerulus (De Zeeuw et al., 1990a,b). Small gap junctions, with a heptalaminar structure including a 2 nm wide cellular space, were observed between spines in and outside glomeruli (Sotelo et al., 1974, 1986). De Zeeuw et al. (1990a) excluded the presence of gap junctions between elements of the same neuron. The importance of dendrodendritic coupling is further supported by the presence of dendritic lamellar bodies that can be associated with dendrodendritic gap junctions. These organelles are ubiquitously distributed in all olivary subdivisions and their density is higher in the olive than in any other brain area (De Zeeuw et al., 1995). Another form of aggregation of dendrites of olivary neurons is the dendritic thicket (Sotelo et al., 1974; 1986, Molinari, 1987; De Zeeuw et al., 1993). Thickets are formed by several dendrites in direct apposition with each other, but without any dendrodendritic membrane specializations. The extent of the electrotonic coupling of olivary neurons was studied by injecting lucifer yellow into single cells in slices of the guinea pig brain stem. Transfer of lucifer yellow via the gap junctions between dendrites, resulted in the labelling of aggregates consisting of up to 5 cells (Bernardo and Foster, 1986). The maximal spatial extent of electrotonic coupling in the inferior olive has not been determined, but may extend 228
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J. Voogd, D. Jaarsma and E. Marani
Fig. 161. GABAergic terminals derived from the cerebellar nuclei innervate dendritic spines (asterisks) in the glomeruli of the principal olive and medial accessory olive in the cat. The double labelled terminals can be recognized by the 15 nm gold particles (GABA) and the reaction products of the anterogradely transported WGA-HRP (large arrows). Note in B and C that cerebellar GABAergic terminals are directly apposed to dendritic spines coupled by gap junctions (small arrows). Open arrows indicate symmetric synapses. Scale bar in A = 0.31 r in B = 0.18 r in C = 0.35 r De Zeeuw et al. (1989b).
230
The cerebellum." chemoarchitecture and anatomy
Ch.I
beyond the classical morphological borders of the neuropil of the subdivisions of the inferior olive. De Zeeuw et al. (not yet published) demonstrated that synchrony between olivary neurons that project to the left and right Crus IIa of the rat in part can be explained by direct coupling through cells in the DMCC that bridge the left and right rostral DAO. Synchronization of activity in olivary neurons derived from different subdivisions is also supported by studies from Sj61und et al. (1980) and Lang et al. (1995) who induced a hypersynchronization of the climbing fiber discharge in the projections of particular olivary subnuclei by the administration of harmalin. Three types of axon terminals were recognized in the neuropil of the olive. It was found in aldehyde-fixed material postfixed in osmium that about half of the terminals contained spherical vesicles and were associated with asymmetrical synapses (Bowman and King, 1973; Mizuno et al., 1974; Sotelo et al., 1974; Gwyn et al., 1977; Rutherford and Gwyn, 1980). A second type contains pleomorphic vesicles and is associated with symmetrical synapses. A third type of terminal contains dense core vesicles. Most of the terminals that can be double labelled with an antegrade tracer from the cerebellar nuclei and an antibody against a conjugate of GABA belong to the second type, with pleomorphic vesicles and asymmetrical synapses (Angaut and Sotelo, 1989; De Zeeuw et al., 1988, 1989a and b, 1992, 1994b, Fig. 161). At present the cerebellar and vestibular nuclei, the nucleus prepositus hypoglossi, the parasolitary nucleus and the cuneate nucleus are the only known sources for this type of GABAergic terminal, and most of these terminals disappear after lesions of these nuclei (Nelson and Mugnaini, 1985; Nelson et al., 1986; Fredette and Mugnaini, 1991; De Zeeuw et al., 1993). Less frequently, GABAergic terminals with so-called crest-synapses can be observed (De Zeeuw et al., 1994b). These terminals occur relatively often in the DC and VLO and are derived from the area of group y and the ventral dentate nucleus (Fig. 162). Two other populations of GABA-containing terminals exist. One minor population contains clear, oval vesicles and sometimes was found apposed to the perikaryon. It was infrequently labelled from the cerebellum (De Zeeuw et al., 1989b). GABA-like immunoreactivity was also found in certain terminals of the granular type, that contained a large number of dense core vesicles. The granular terminals rarely made synaptic contacts. They could not be labelled from the cerebellum and they were always found outside the glomeruli, sometimes apposed to somata. The same type of granular terminal was labelled in the inferior olive with [3H]serotonin (Wiklund et al., 1981a) or antibodies to conjugates of serotonin (King et al., 1984). It seems likely, therefore, that serotonin and GABA are co-localized in a subpopulation of these terminals. All other reported non-cerebellar afferents terminate with boutons containing spherical vesicles (nuclei at the mesodiencephalic junction: King et al., 1978; Cintas et al., 1980; De Zeeuw et al., 1988, 1989a,b; spinal cord: King et al. 1976; Mizuno et al., 1976; Gwyn et al., 1983; Molinari and Starr, 1989; Molinari, 1988). Most of these terminals were apposed to distal dendrites or spines inside and outside glomeruli, relatively few were found to contact somata. Afferents from the spinal cord and the gracile nucleus never have been found in glomeruli in close contact with gap junctions (Molinari and Starr, 1989; Molinari, 1987, 1988; Molinari et al., 1990). The preferential axo-dendritic mode of termination of dorsal column afferents may be due to the relative paucity of spines on the long, radiating dendrites of the cell type that prevails in the spinal areas of the inferior olive. Axon collaterals from olivary neurons that terminate in the same and the contralateral inferior olive have only been observed in young kittens (Ramon y Cajal, 1911) and in cases of olivary hypertrophy in the cat (Ruigrok et al., 1990; De Zeeuw et al., 1990d). 231
Ch.I
J. Voogd, D. Jaarsma and E. Marani
Fig. 162. Double labelled terminals (GABA and anterogradely transported WGA-HRP) from the contralateral PrH terminate on distal and proximal portions of the neurons in the dorsal cap of the rat (A and B) and the rabbit (C). In A, a double labelled terminal (right) and a GABAergic large granular terminal (left) are apposed to dendritic spines (asterisks) coupled by a gap junction (small arrows). In B and C the terminals are apposed to somata. The large arrows indicate the WGA-HRP reaction products. The open arrows and the arrowhead indicate symmetric synapses and an asymmetric synapse, respectively. Scale bar in A - 0.24 r in B = 0.41 r in C = 0.39 r De Zeeuw et al. (1994).
Cerebellar, GABAergic and mesodiencephalic, non-GABAergic terminals contacted both spines inside glomeruli and dendritic shafts. The cerebellar (King et al., 1976; Angaut and Sotelo, 1987) and/or GAD or GABA-immunoreactive (Sotelo et al., 1986; Fredette and Mugnaini, 1991) terminals are associated with gap junctions inside the glomeruli. De Zeeuw et al. (1989a,b; 1990a and c) concluded that both GABAergic and 232
The cerebellum." chemoarchitecture and anatomy
Ch. I
non-GABAergic mesodiencephalic terminals in the PO and the rostral MAO of cat and rat innervate the same spines including those that are electrotonically coupled. However, the cerebellar GABAergic terminals, but not the mesodiencephalic terminals, showed a strong preference to be strategically located next to both dendrites coupled by a gap junction (Fig. 161). Most or all of the terminals contacting the somata of olivary neurons contained pleomorphic vesicles (Bowman and King, 1973; Gwyn et al., 1977; Rutherford and Gwyn, 1980; Sotelo et al., 1986). Moreover, large GABAergic granular terminals were observed next to the soma. The proportion of somatic GABAergic terminals, that could be labelled from the cerebellar nuclei was significantly lower than among the terminals in the entire neuropil (De Zeeuw et al., 1989b). The presence of a non-cerebellar, GABAergic innervation of the cerebellum was also suggested by Nelson and Mugnaini (1985) and Fredette and Mugnaini (1991), who showed that total cerebellectomy does not cause a total depletion of GAD-positive terminals in the olive of the rat, and by the electrophysiological studies of Andersson et al. (1988) and Weiss et al. (1990). The GABAergic innervation of the axon hillock appears to be of cerebellar origin (De Zeeuw et al., 1990b). Possible extracerebellar sources for a GABAergic innervation of the inferior olive include the raphe nuclei and the adjacent reticular formation (Bishop, 1984), the nucleus parasolitarius and the cuneate nucleus (Nelson and Mugnaini, 1989) and the intrinsic GABAergic neurons of the inferior olive. Intrinsic GABAergic neurons of the inferior olive seem to be rare. Mugnaini and Oertel (1985), Nelson and Mugnaini (1989) and Fredette et al. (1992) found only few GAD-positive cells in the inferior olive of the rat. These cells were usually small and provided with unbranched, ramifying dendrites. They were differently distributed in different species; in the cat most occur in the rostral tip of the MAO and in the dorsal fold of the DAO, in primates in the PO (Fredette et al., 1992). They were more numerous in the baboon, where they accounted for 5% of the neurons of the inferior olive (Walberg and Ottersen, 1989). Colchicine injections in rat, rabbit and cat enhanced their visibility and revealed a high density of GAD-positive neurons in the reticular formation directly dorsal to the inferior olive (Nelson and Mugnaini, 1989; Fredette et al., 1992). Large cells with dendrites penetrating in the neuropil of the olive, which resemble the cells of the reticular formation, were identified by Sotelo et al. (1974) in the cat. Similar, peri-olivary cells, containing glutaminase were illustrated by Kaneko et al. (1989) in the rat. Bishop (1984) and Bishop and King (1986) demonstrated that dendrites of intracellularly injected reticular neurons can contribute to the olivary glomeruli. 6.3.2. Afferent connections of the inferior olive
Afferent systems of the inferior olive have been reviewed by Brodal and Kawamura (1980) for the cat and by Martin et al. (1980) for the opossum. For the rat the tabulated summary of its afferent connections in the paper of Brown et al. (1977) and the review by Flumerfelt and Hryccyshyn (1985) are useful. Afferent systems of the inferior olive can be subdivided into three groups: (1) the GABAergic nucleo-olivary and vestibuloolivary projections; (2) the monoaminergic and cholinergic projections to the inferior olive; (3) the specific projections from the spinal cord, certain brain stem nuclei and the cerebral cortex will not be considered in this chapter. Their neurotransmitters are not known.
233
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J. Voogd, D. Jaarsma and E. Marani
6.3.2.1. The nucleo-olivary and vestibulo-olivary projections." The GABAergic afferents of the inferior olive The origin of the nucleo-olivary projection from the cerebellar nuclei (Section 5.2.) and their mode of termination (Section 6.3.1 .) has been considered before. In this section the topographical organization of the nucleo-olivary and the closely allied vestibulo-olivary projections will be reviewed. The nucleo-olivary projection is entirely GABAergic, the vestibulocerebellar projection only partially so. For the main part the nucleo-olivary and the olivocerebellar projections are reciprocally organized, in the sense that the cerebellar nuclei which are the target of a particular olivo-corticonuclear loop give rise to a nucleo-olivary projection to this particular olivary subnucleus. This reciprocity is not complete, because the olivary projections of some of the cerebellar nuclei terminate bilaterally in the olive. The olivary projection from Deiters' nucleus clearly reciprocates the olivocerebellar projection to the B zone, but for the vestibulo-olivary projections from other vestibular nuclei this reciprocity is less obvious. Nucleo-olivary fibers terminate in all parts of the inferior olive. Roughly the density of the GABAergic boutons in different subnuclei of the olive represents the density of the nucleo-olivary projection to these parts. The density of GAD immuno-reactivity is higher in the group beta of the rat, which also contains the largest reactive boutons, followed by the subnucleus c of the MAO, the DMCC, the medial and lateral poles of the DAO, the bend of the PO, the DC and the DM (Sotelo et al., 1986) (Fig. 166). Nelson and Mugnaini (1988) noticed that the dorsal fold of the rat DAO contains a higher immunoreactivity than the ventral fold and confirmed the difference in bouton-size between strongly immunoreactive and less reactive regions. Nelson et al. (1989) found the same distribution in a comparative study of GAD-immunoreactivity of the olive in rabbit, cat, rhesus monkey and man (Fig. 163). Activity is highest in the beta nucleus. The DAO contains several, differently stained regions, with high immunoreactivity in the rostromedial, and in the caudolateral parts of the nucleus. Staining in the caudal MAO is lowest in the central subnucleus b and higher in the lateral subnucleus a and the rostral portion of the MAO. Immunoreactivity of boutons in the DC (high in rabbit and monkey; low in rabbit and man) and the DMCC (high in rabbit and cat, and low in monkey) differs for different species. GAD-immunoreactive boutons disappear from the contralateral PO, the rostral MAO and the lateral half of the ventral fold of the DAO of the rat after chronic lesions of the cerebellar nuclei or the superior cerebellar peduncle in the rat. The dorsal fold of the DAO is depleted of GAD-positive boutons after lesions extending into the lateral vestibular nucleus (Fredette and Mugnaini, 1991). Additional destruction of the vestibular nuclei results in the disappearance of GAD from the group beta but not from the medial half of the ventral fold of the DAO and the caudal MAO (Nelson and Mugnaini, 1989). GAD-immunoreactive neurons in the parasolitary and cuneate nuclei could be labelled after injection of retrograde tracers in the inferior olive of the rat (Nelson and Mugnaini, 1989). These nuclei, therefore, provide additional GABAergic projections to the inferior olive. The projection of the ipsilateral parasolitary nucleus was located in the medial subnucleus c of the caudal MAO by these authors. Connections from the parasolitary nucleus (indicated as the lateral solitary nucleus) also were documented in earlier studies by Loewy and Burton (1978) and Molinari (1985) in the cat. The inhibitory connections from the cuneate nucleus in the rat are crossed and terminate in the medial DAO. A similar GABAergic cuneo-olivary pathway appears to be responsible 234
The cerebellum." chemoarchitecture and anatomy
Ch. I
caudal
INTENSITY 5-4
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rostral Fig. 163. Schematic generalized diagram of GAD immunostaining intensities in regions of the mammalian IO based upon studies in rabbit, cat and Rhesus monkey. Intensity was visually graded on a scale of 5-1, where 5 indicates the most intense staining. The beta nucleus and medial aspect of the rostral DAO have the highest intensity in the IO (black regions). Most of the IO is stained with intermediate intensities (hatched regions). Subnucleus b of the MAO (white region) is least intensely stained. Staining in the dorsal cap and the dorsomedial cell column (dotted regions) varied by species, aMAO = subnucleus a of the medial accessory olive; beta = subnuleus beta; bMAO = subnucleus b of the medial accessory olive; DAO = dorsal accessory olive; dc = dorsal cap; dmcc = dorsomedial cell column; PO = principal olive; vlo = ventrolateral outgrowth. Nelson et al. (1989).
for the i n h i b i t i o n o f n e u r o n s in the D A O on s t i m u l a t i o n o f the red n u c l e u s or the c o n t r a l a t e r a l r u b r o s p i n a l t r a c t in the cat (Weiss et al., 1990). T h e t o p o g r a p h y in the c e r e b e l l a r n u c l e o - o l i v a r y p r o j e c t i o n has b e e n s t u d i e d in different m a m m a l i a n species. T h e n u c l e o - o l i v a r y fibers f r o m the i n t e r p o s e d a n d l a t e r a l nuclei r u n in a s e p a r a t e tract, v e n t r a l to the b r a c h i u m c o n j u n c t i v u m in cat ( L e g e n d r e a n d C o u r v i l l e , 1987), rat ( C h o l l e y et al., 1989) a n d r a b b i t (Tan et al., 1995b). F a s t i g i o - o l i v a r y 235
Ch. I
J. Voogd, D. Jaarsma and E. Marani
and vestibulo-olivary fibers take other routes (Nelson and Mugnaini, 1989; Ruigrok and Voogd, 1990). It is generally assumed that the anterior- and posterior interposed nuclei project to the rostral DAO and MAO respectively and the lateral, dentate nucleus to the PO (cat: Tolbert et al. 1976b; Buisseret-Delmas and Batini, 1977; Dietrichs and Walberg, 1981, 1985, 1986; Courville et al., 1983a; monkey: Kalil, 1979; Asanuma et al., 1983; Gonzalo-Ruiz and Leichnetz, 1990; opossum: Martin et al., 1976, 1980; rat: Brown et al., 1977; Angaut and Cicirata, 1982; Haroian, 1982; Swenson and Castro, 1983 a and b; Nelson and Mugnaini, 1989; Ruigrok and Voogd, 1990). Points of discussion were, and still are the presence and the extent of the fastigio-olivary projection, the nucleo-olivary connections of subdivisions of the dentate nucleus and of certain cerebellar subnuclei in rat (dorsolateral protuberance of the fastigial nucleus, dorsolateral hump), the presence of ipsilateral nucleo-olivary projections and the relations of the cerebellar nucleo-olivary projection with the GABAergic and non-GABAergic vestibulo-olivary connections. A projection of the medial cerebellar nucleus to the caudal MAO was denied by Brown et al. (1977) and Haroian (1982) for the rat, but was found by Achenbach and Goodman (1968), Angaut and Cicerata (1982) and Swenson and Castro (1983a and b). In the cat negative findings were published by Graybiel et al. (1973), Tolbert et al., (1976b) and Courville et al. (1983a), but the connection was found to be present by
I
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Fig. 164. The nucleo-olivary projection in the rat. Data from Ruigrok and Voogd (1990). Upper and lower block diagrams represent the cerebellar and vestibular nuclei, and the subdivisions of the inferior olive respectively. According to Ruigrok and Voogd (1990) the cerebellar nuclei and their olivary target nuclei can be considered as a continuum, stretching from the rostral medial cerebellar nucleus, projecting to caudal MAO, to the lateral vestibular nucleus, projecting to the dorsal fold of the DAO. DL = dorsolateral protuberance of the medial cerebellar nucleus; DMC = dorsomedial cell column; IntA = anterior interposed nucleus; IntDL = dorsolateral hump; IntP = posterior interposed nucleus; IOD = dorsal accessory olive; IODM = dorsomedial cell column; IOM - medial accessory olive; IOP - principal olive; Lat = lateral cerebellar nucleus; LVe = lateral vestibular nucleus; M e d = medial cerebellar nucleus; VL = ventrolateral outgrowth.
236
The cerebellum." chemoarchitecture and anatomy
Ch. I
Sugimoto et al. (1980) and Dietrichs and Walberg (1985). In the monkey negative results were obtained in the anterograde tracer studies from the fastigial nucleus by Batton et al. (1977), Kalil (1979) and Asanuma et al. (1983). Ikeda et al. (1989), however, reported projections from the caudal part of the fastigial nucleus to medial portions of the caudal MAO in the macaque monkey. Ruigrok and Voogd (1990) in a recent study, using anterograde transport of Phasaeolus vulgaris lectin injected in the cerebellar nuclei of the rat, established projections from rostral regions of the fastigial nucleus to a lateral zone in the caudal MAO and of the caudal fastigial nucleus to the group beta (Fig. 164). The projection of the dorsolateral hump is located in a circumscribed area in the medial subnucleus c, that coincides with the tectal recipient zone of Akaike et al. (1992) that projects to the vermis of lobule VII and the lateral extension of the A zone in the hemisphere of the lobules VI and VII. The central portion of the caudal MAO (subnucleus b) lacks a nucleo-olivary projection. No terminations were found in the DC, which has been reported to receive fastigial projections by Angaut and Cicerata (1982), Swenson and Castro (1983a and b) in the rat and Dietrichs and Walberg (1985) in the cat. The results of experiments on nucleo-olivary projections from different parts of the dentate nucleus to the PO are conflicting. According to Beitz (1976) and Tolbert et al. (1976b) in the cat and Kalil (1979) in the monkey the dorso-ventral and medio-lateral relations are maintained in the nucleo-olivary projection of the dentate nucleus. Dietrichs and Walberg (1985) denied this and proposed a complicated rostro-caudal relationship between both structures. According to Angaut and Cicirata (1982) the medial dentate of the rat, including the dorsolateral hump, projects to the ventral leaf, and the lateral dentate to the dorsal leaf of the PO. Swenson and Castro (1983a and b) described a projection of the rostral dentate of the rat to the dorsal leaf and of the caudal dentate to the ventral leaf of the PO. Chan-Palay (1977) claimed that the projection in the monkey is reversed: caudal dentate projecting to the ventral PO. Some of her experiments, however, show an exclusive projection of the caudal dentate to the ventral leaf of the PO. Ruigrok and Voogd (1990) confirmed Angaut and Cicerata's (1982) topology for the rat (Fig. 164). They located the nucleo-olivary projection of the dorsolateral hump in the dorsomedial group (DM), the enlarged medial portion of the ventral leaf of the PO. The DMCC that was often confused with the DM of the rat, did not receive a nucleo-olivary projection. The nucleo-olivary projection is not completely crossed. Some fibers from the dorsolateral hump, IP and the ventromedial lateral nucleus recross at the level of the inferior olive, and terminate in the ipsilateral DM, rostral MAO and ventral leaf of the PO respectively. Ipsilateral labelling was sparse or absent in other parts of the inferior olive. Projections from the vestibular nuclei terminate in subnuclei that do not receive a nucleo-olivary projection, such as the DMCC, the group beta and the central region (subnucleus b) of the caudal MAO in cat (Fig. 165) (Saint-Cyr and Courville, 1979; Gerrits et al., 1985a), opossum (Martin et al., 1980) and rat (Nelson and Mugnaini, 1989). These projections are mainly crossed, and, in the rat at least, entirely or partially GABAergic. An ipsilateral projection from the superior vestibular nucleus to a lateral zone in the caudal MAO and the bend region of the PO in the cat was reported by Gerrits et al. (1985a). The uncrossed, GABAergic projection from the parasolitary nucleus to subnucleus c (Nelson and Mugnaini, 1989), and the bilateral projections from the nucleus prepositus hypoglossi to DC and VLO (Gerrits et al., 1985a; McCrea and Baker, 1985; De Zeeuw et al., 1993) should be included in the vestibulo-olivary projection. Terminations from the nucleus prepositus hypoglossi were located both in subnuclei dominated by descend237
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 165. The vestibulo-olivary projection in the cat. Localization of terminal fields with antegrade axonal transport of [3H]leucine. A. Contralateral projection from the superior vestibular nucleus. B. Ipsilateral projection from the medial vestibular nucleus. C. Bilateral projections from the nucleus prepositus hypoglossi. MAO = medial accessory olive; PO = principal olive. Gerrits et al. (1985a).
ing visual afferent connections (DC and VLO) and in the caudal MAO, where they overlap with vestibular afferents from the medial and the spinal vestibular nuclei. The prepositus hypoglossi nuclear projection to the DC in rat and rabbit is bilateral and deals mainly with the caudal DC. It contains GABAergic (De Zeeuw et al., 1993) and cholinergic (Barmack et al., 1991) components. Recently it was shown that these two neurotransmitters are co-localized in the same terminals (De Zeeuw et al, unpublished). The GABAergic projections from the nucleus prepositus hypoglossi to the VLO and 238
The cerebellum." chemoarchitecture and anatomy
Ch. I
subnucleus c are exclusively contralateral and much sparser. The VLO and the rostral DC receive their major GABAergic input from the ventral dentate nucleus and the adjacent group y in the rabbit (De Zeeuw et al., 1994b). 6.3.2.2. Monoaminergic and cholinergic projections A plexus of varicose catecholaminergic fibers has been mapped in the inferior olive of cat, monkey and in several rodents with histofluorescence methods (Fuxe, 1965a and b; Hoffman and Sladek, 1973; Sladek and Bowman, 1975; Sladek and Hoffman, 1980). The distribution of these varicose fibers is heterogeneous and displays marked species variations. The distribution of serotoninergic fibers is also heterogeneous and speciesdependent, but it differs from the distribution of the catecholamines (Wiklund et al., 1977, 1981a,b; Sladek and Hoffman, 1980; Takeuchi and Sano, 1983; Bishop and Ho, 1984; King et al., 1984; Par6 et al., 1987; Compoint and Buisseret-Delmas, 1988). Immunocytochemical methods, using antibodies against conjugates of serotonin, were applied in the investigations reported in the last five papers. The catecholaminergic innervation of the inferior olive of the rat is strongest for the dorsal leaf of the PO and the rostral MAO (Fig. 166) (Sladek and Bowman, 1975). For serotonin the most intensely innervated subnucleus of the inferior olive of the rat is the lateral portion of the rostral lamella of the DAO (Fuxe, 1965a,b; Takeuchi and Sano, 1983; Bishop and Ho, 1984; Par6 et al., 1987; Compoint and Buisseret-Delmas, 1988), the dorsal leaf of the DAO is spared (Fig. 166) (Takeuchi and Sano, 1983). For the MAO and the PO the descriptions differ. Takeuchi and Sano (1983) found a somewhat higher innervation of the rostral MAO. Bishop and Ho (1984) described a dense innervation by varicosities of the subnuclei a and b of the caudal MAO, moderate numbers of serotonin-like immunoreactive fibers in subnucleus c and the caudal PO and few elements in group beta and the DC (Fig. 166). Par6 et al. (1987) confirmed this distribution for the caudal MAO. Compoint and Buisseret-Delmas (1988) stressed the presence of an immunoreactive plexus around the caudal MAO, with few fibers penetrating the center of the nucleus. Both catecholaminergic and serotoninergic fibers predominate in the lateral part of the caudal MAO of the cat. At more rostral levels only the medial MAO and the DMCC are densely innervated by serotoninergic fibers. The catecholaminergic and serotoninergic innervation of the DAO are complementary, with a serotoninergic plexus occupying the lateral and caudal DAO with the exception of its most lateral border region (Wiklund et al., 1977; Takeuchi and Sano, 1983). A central column of the caudal MAO contains a dense plexus of serotoninergic fibers in monkey (Takeuchi and Sano, 1983) (Fig. 167) and opossum (King et al., 1984). Other parts of the MAO of the opossum, including subnucleus c and the rostral pole of the MAO are less densely innervated. Serotoninergic fibers also predominate in the lateral DAO of the monkey (Takeuchi and Sano, 1983) but a serotoninergic plexus is present in the entire DAO of the opossum (King et al., 1984). The distribution of serotoninergic fibers in the PO differs for the different species, with an innervation of the ventral leaf of the cat (Sladek and Bowman, 1975), both the ventral and the dorsal leaf in the monkey (Takeuchi and Sano, 1983) (Fig. 168), and the dorsal leaf of the PO in the opossum (King et al., 1984). Serotonin was localized in axons and their terminals at the ultrastructural level by Wiklund et al. (198 l a) in the DAO of the rat with high resolution autoradiography of [3H]serotonin and by King et al. (1984) and Compoint and Buisseret-Delmas (1988) with 239
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Fig. 166. The innervation of the inferior olive of the rat by catecholaminergic, serotoninergic, substance P, and gamma-aminobutyric acid decarboxylase (GAD)-immunoreactive fibers and the distribution of acetylcholinesterase (ACHE). a = subnucleus a of the medial accessory olive; b = subnucleus b of the medial accessory olive; beta = subnucleus beta; D A O = dorsal accessory olive; d c = dorsal cap; dl = dorsal eaf principal olive; D M = dorsomedial subnucleus; dmcc = dorsomedial cell column; MAO = medial accessory olive; PO = principal olive; vl = ventral leaf principal olive; vlo = ventrolateral outgrowth; XII = hypoglossal nerve.
preembedding immunocytochemistry in the same species. The label was located in thin axons and varicosities, containing large dense core vesicles and small, clear vesicles or tubulo-vesicular elements. Few terminals engage in synaptic contacts (Wiklund et al.: 5%; King et al.: 2%), mainly with dendritic shafts, and never with the spines located in the glomeruli. Configurations suggesting axo-somatic contacts were found in the subnucleus b of the caudal MAO in the opossum (King et al., 1984). Similar terminals in the rostral MAO of the cat, containing large dark core vesicles, displayed GABA-like immunoreactivity (De Zeeuw et al., 1989b). The possibility that certain, non-cerebellar GABAergic projections are derived from the raphe nuclei or the adjoining medial reticular formation (Bishop 1984), and that they are co-localized with serotonin still needs to be explored. According to Compoint and Buisseret-Delmas (1988) the serotoninergic innervation of the inferior olive of the rat, at least in part, is derived from cell groups surrounding it. The nucleus raphe obscurus and the reticular formation dorsal to the olive innervate the MAO, the reticular formation and cell groups lateral to the DAO innervate the latter subnucleus. Co-localization of serotonin and substance P, that display a similar distribution in the olive of the rat
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241
Ch. I
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Fig. 168. Schematic representation of the distribution of serotonin fibers in the inferior olivary complex of the cat (a, rostral; i, caudal). B = group beta; DA--DMC = dorsomedial cell column; DAO = dorsal accessory olive; DC = dorsal cap; MAO = medial accessory olive; PO = principal olive; POD - dorsal lamella of the principal olive; POV = ventral lamella of the principal olive. Takeuchi and Sano (1983).
(Bishop and Ho, 1984; Par6 et al., 1987) (Fig. 166), is another possibility (H6kfelt et al., 1978; Pelletier et al., 1981; Par6 et al., 1987). A differential distribution of AChE has been described in the neuropil of the inferior olive of cat (Marani et al., 1977) (Fig. 169), ferret, rabbit (Marani, 1986,), rat (Fig. 166), and the opossum (Martin et al., 1975). These distributions are fairly consistent as to the columnar distribution of the enzyme in the caudal MAO and the rostral DAO, the absence of AChE in the group beta and the presence of AChE in the DC. There are some points of resemblance with the distribution of serotonin in the DAO and the caudal MAO of the cat, but a causal relationship between the presence of AChE and the distribution of certain afferent or efferent (peptidergic) systems of the olive has not been established. A dense plexus of ChAT-immunoreactive fibers pervading the entire inferior olive of the cat, was reported by Kimura et al. (1981). Receptor binding using labelled ~bungarotoxin for nicotinic receptors (Hunt and Schmidt, 1978) and [3H]propyl-benzilyl choline for muscarinic receptors (Rotter et al., 1979a) was stronger in the PO and the MAO, than in the DAO of the rat. Wamsley et al. (1981) and Swanson et al. (1987) reported on the presence of muscarinic and nicotinic receptors in the olive, but did not distinguish between the subdivisions of the inferior olive. 6.3.3. The connections between the inferior olive and the cerebellum
When the olivocerebellar projection was studied in the cat with antegrade axonal transport of tritiated aminoacids it appeared that the labelled climbing fibers were arranged 242
Ch.I
The cerebellum." chemoarchitecture and anatomy
42
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Fig. 169. Diagram of the distribution of AChE in the inferior olive of the cat. A. Transverse sections, numbered from caudally to rostrally. B. Diagrams of the unfolded inferior olive. Black: heavy staining; stippled: medium staining; white: low staining. Abbreviations: DAO = dorsal accessory olive; dc = dorsal cap; dmcc = dorsomedial cell column; MAO = medial accessory olive; nfl = subnucleus beta; PO = principal olive; vlo = ventrolateral outgrowth. Marani et al. (1977).
in longitudinal strips (Courville, 1975) and that this zonal pattern closely resembled the zonal organization in the corticonuclear projection and the associated compartmental subdivision of the feline cerebellum (Voogd, 1969; Groenewegen and Voogd, 1977; 243
Ch.I
J. Voogd, D. Jaarsma and E. Marani
Groenewegen et al., 1979; Voogd and Bigar6, 1980). Electrophysiological studies of the olivocerebellar projection (Armstrong et al., 1973, 1974) and of spino-olivocerebellar climbing fiber paths (SOCPs) (Oscarsson, 1969, 1973, 1980) generally supported the zonal distribution of the climbing fibers. To express the similarity with the anatomical olivocerebellar climbing fiber zones Oscarsson and Sj61und (1977a) adopted a modification of Voogd's (1969) nomenclature to designate the zonal projections of the S O C P s . 4 Brodal and Kawamura (1980), who reviewed their extensive studies with retrograde labelling of the olivocerebellar pathway, similarly used Voogd's paradigm in the interpretation of their data. Information on the olivocerebellar projection in primates is less complete, but the available data suggest that its overall organization is similar to the cat. The principles underlying Voogd's nomenclature also have been applied to the olivocerebellar projection in the rat (Buisseret-Delmas, 1988a,b; Buisseret-Delmas and Angaut, 1993; Voogd, 1995) but in rodents other schemes have been proposed (Azizi and Woodward, 1987; Apps, 1990). The olivocerebellar projection to the anterior lobe, the hemisphere of the posterior lobe, the caudal vermis and the flocculus and the collateral projections to the cerebellar and vestibular nuclei will be reviewed. Some reports on the olivocerebellar projection in non-mammalian vertebrates are available. In the chicken the inferior olive consists of a dorsal and ventral laminae. The homologies of these laminae with the mammalian olive are complicated (see Furber, 1983; Arends and Voogd, 1989). The projection to the cerebellum is crossed and arranged in longitudinal zones (Freedman et al., 1977). A detailed topographical map of the relations between the olive and the cerebellum of the pigeon was published by Arends and Voogd (1989). Different lines of evidence on the myelo-architecture and the mossy and climbing fiber connections, therefore, support the longitudinal subdivision of the avian cerebellum. The inferior olive of the turtle is difficult to identify with histological staining methods. Ktinzle and Wiklund (1982) identified it on the basis of specific retrograde transport of D-[3H]aspartate. The olivocerebellar projection in the turtle also is organized in bands. Both in reptiles and birds the climbing fibers and the smooth parts of the Purkinje dendrites on which they terminate are limited to the deep parts of the molecular layer (compare the extent of the spiny branchlets in Purkinje cells of fish, birds and mammals in Fig. 12). 6.3.3.1. The olivocerebellar projection to the anterior lobe
The situation in the cat, where most data are available, will be considered first. The olivocerebellar projection to the anterior lobe in primates and the rat will be reviewed at the end of this section. Three broad zones, e.g. the vermis, the pars intermedia and the lateral zone or hemisphere proper, can be distinguished in the anterior lobe of the cat, in accordance with the classical description of Brodal (1940). The anterior vermis receives its climbing fibers from the caudal portions of the MAO and the DAO, the pars intermedia from the rostral MAO and DAO and the hemisphere from the PO. Voogd (1969), Armstrong et al. (1974) and Groenewegen and Voogd (1977) in the cat distinguished between a medial A zone, receiving fibers from the caudal half of the MAO and a lateral B zone which receives its climbing fibers from the caudal DAO (Fig. 119).
Zones defined by electrophysiological criteria usually are designated by lower case characters (i.e. the a, x, cl-3, d 1-2 zones; see Fig. 175).
4
244
The cerebellum." chemoarchitecture and anatomy
Ch. I
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Fig. 170. Laterality and somatotopical organization of the projection zones of the ventral funiculus spinoolivocerebellar climbing fiber path (VF-SOCP) in the cerebellum of the cat. A. Rostral aspect. B. Caudal aspect of the cerebellum. The position of the caudal border of the inferior colliculus (INF.COLL.) is indicated. Oscarsson and Sj61und (1977b).
Equivalent SOCP's terminating in the a and b zone of the anterior vermis, that could be activated from the isolated ventral and dorsal funiculus of the cord, were discovered by Oscarsson c.s. (see Oscarsson, 1969; 1973). The ventral funiculus (VF-) SOCP carries information from skin, joints and high and low threshold muscle afferents. The VFSOCP to the a zone is an ipsilateral pathway from the hindlimb. The VF-SOCP to the b zone is bilateral, the projection from the forelimbs was found to be located medially in the b (b~) zone, the hindlimbs in the more lateral b2 zone (Oscarsson and Sj61und, 1974, 1977a,b,c) (Figs 170 and 171). The a zone is also present as a wide, paramedian zone in lobule VIII; the b zone is restricted to the anterior vermis (Figs 170 and 183). The dorsal funiculus (DF-)SOCP synapses in the dorsal column nuclei and converges with the VF-SOCP at the level of the inferior olive. The DF-SOCP is activated from high threshold muscle afferents and from both tactile and nociceptive cutaneous afferents 245
Ch. I
J. Voogd, D. Jaarsma and E. Marani
A
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Fig. 171. Termination zones in the anterior lobe of the dorsal (A, DF-SOCPs), ventral (B, VF-SOCPs), dorsolateral (C, DLF-SOCPs) and lateral (D, LF-SOCP) spino-olivo-cerebellar paths. The diagrams represent those parts of lobules IV and V in the left half of the anterior lobe which are accessible at the cerebellar surface. Borders between zones a-d2 are indicated with lines. Areas activated from hindlimb and forelimb nerves through direct and indirect paths from the dorsal funiculus nuclei (DFN) or spinal cord to the inferior olive (IO) are indicated (see key). The DF-SOCP projection (from forelimb nerves) and the LF-SOCP projection to the c2 zone, and the VF-SOCP projection to the b zone are bilateral, whereas all other projections are ipsilateral. B-D based on Larson et al. 1969a, 1969b and Oscarsson and Sj61und 1977a, 1977b. Ekerot and Larson (1979a)
(Ekerot and Larson, 1979a; Ekerot et al., 1991a,b). Ipsilateral DF-SOCP's to the a zone and to the lateral b 2 zone, carry hindlimb information (Ekerot and Larson, 1979a; Ekerot and Larson, 1977, 1979a,b, 1982; see also Andersson and Erikson, 1981) de246
The cerebellum." chemoarchitecture and anatomy
A
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CAMPBELL & ARMSTRONG'85 Fig. 172. Origin from the border region of the rostral and caudal medial accessory olive of the olivocerebellar projection to the electrophysiologically identified x zone in the cat. a = a zone; b = b zone; fl = subnucleus beta; cl-c3 = cl-c3 zones; coll = inferior colliculus; d~, d2 = dl and d 2 zones; dmcc = dorsomedial cell column; M A O = medial accessory olive; ml = mldline; pvg = lateral border of vermis; x = x zone; IV, V = lobules IV and V. R e d r a w n from Campbell and A r m s t r o n g (1985).
scribed a third, 'x' zone in the anterior lobe of the cat, located between the a and b zones (Fig. 171). The x zone is not present in all lobules of the anterior lobe, but is restricted to the lobules IV and V. It receives an ipsilateral projection through the DF-SOCP from the forelimb, through climbing fibers that branch between the x zone and zone in the pars intermedia (Fig. 175). The VF-SOCP does not terminate in the x zone. The climbing fibers to the x zone take their origin from the junction of the caudal and rostral halves of the MAO (Fig. 172) (Campbell and Armstrong, 1985; Voogd, 1989). Olivocerebellar fibers terminating in the A, X and B zones and the Purkinje cell fibers from these zones occupy the corresponding white matter compartments (Figs 119 and 120) (Voogd, 1969; Groenewegen and Voogd, 1977; Voogd and Bigar6, 1980; Voogd, 1989). The projections of the Purkinje cells of the VF-SOCP-innervated x and b zones were verified by Andersson and Oscarsson (1978a) and Trott and Armstrong (1987a,b) and were found to correspond to the anatomical corticonuclear projection zones X and B. The border between the B zone and the most medial C1 zone of the pars intermedia may be difficult to assess. This border was not recognized with retrograde labelling by Brodal and Walberg (1977a), who considered the B and C1 zone as a single area innervated by the caudal and lateral DAO. However, the presence of separate B and C1 compartments in the white matter and the characteristic electro-physiological properties of the b and c~ zones of Oscarsson validate their distinction in cat cerebellum. The problem in defining the border between the caudal portion of the DAO, projecting to the B zone and the more rostral regions, projecting to the C1 and C3 zones was discussed by Brodal and Kawamura (1980) and Voogd (1989). There is agreement on the differential origin of the two projections, but a precise border has never been established. Collateral projections to the fastigial and Deiters' nucleus take their origin from the 247
Ch. I
J. Voogd, D. Jaarsma and E. Marani
NOCICEPTIVECLIMBINGFIBER INPUT P
DAO DAO
A
1"I
MAO
~ULILr~TIVE NON-NOCICEPTIVE NOT STUDIED
Fig. 173. A summary diagram of nociceptive and non-nociceptive cutaneous climbing fiber input to lobules IV and V of the cerebellum of the cat. Forked arrows show branching of olivary axons to innervate pairs of zones (Ekerot and Larson, 1982). PF, primary fissure; DAO, dorsal accessory olive; MAO, medial accessory olive. Garwicz (1992).
olivocerebellar fibers to the A and B zones respectively (Groenewegen and Voogd, 1977). Retrograde labelling after injections of the fastigial nucleus of the cat was found in cells of the caudal MAO, the group beta and the dorsal cap (Hoddevik et al., 1976; Courville et al., 1977; Ruggiero et al., 1977). The projection of the lateral part of the caudal MAO to the lateral fastigial nucleus (Dietrichs and Walberg, 1985) may include the collateral projection of the olivocerebellar fibers to the x zone, that was never studied separately. Somatotopical patterns in the anterior vermis are arranged as mediolaterally disposed subzones or microzones. Information on the somatotopy of the A zone is scarce. According to Oscarsson and Sj61und (1977a,c) and Ekerot and Larson (1979a,b) the a zone is dominated by the hindlimb. The DF-SOCP projection to the x zone is dominated by the forelimb but lacks a somatotopical organization (Ekerot and Larson, 1977, 1979b, 1982). The exclusive forelimb connections of the x zone are in accordance with the cervical and cuneate projections to the border region of the caudal and rostral halves of the MAO (Boesten and Voogd, 1975; Gerrits et al. 1984a) that contains the olivary neurons projecting to the x zone (Campbell and Armstrong, 1985). Climbing fibers to the x zone receive tactile input and can be strongly activated by noxious pinch of the 248
The cerebellum." chemoarchitecture and anatomy I
Ch. I
f
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skin (Fig. 175). The receptive fields for tactile and noxious stimulation coincide (Garwicz et al., 1992 in Garwicz, 1992). Climbing fibers to the b zone are responsive to cutaneous and deep stimuli but do not receive a nociceptive input (Figs 170, 171 and 175). Successively more rostral body segments (tail, hindlimb, thorax, forelimb, head) project to successively more medial microzones in the b zone (Anderson and Oscarsson, 1978b; Andersson and Eriksson, 1981). The climbing fibers innervating a microzone share the same receptive field. In the b zone each microzone has been shown to project to a separate set of neurons of Deiters' nucleus (Andersson and Oscarsson, 1978a). The microzones and the olivocorticonuclear micro-complexes to which they belong can be considered as the basic functional units of the cerebellum. Contrary to the cortical zones to which they belong, the microzones do not possess anatomical borders because they are physiological artefacts isolated from a somatotopical continuum. Climbing fiber projections from the contralateral sensorimotor cortex to the lateral part of the anterior vermis were described by Provini et al. (1967, 1968). They noticed a strong correspondance in the localization of the cortical and peripheral projections to this region. Andersson and Eriksson (1981) and Andersson and Nyquist (1983) concluded that the projections of the posterior sigmoid gyrus to the a, x and b zones were closely matched to those of the VF-SOCP with respect to somatotopical organization and laterality. The projections to the a and b zones were bilateral and those to the x zone were crossed. Andersson and Nyquist (1983) identified short latency projections from separate forelimb and hindlimb areas in the posterior sigmoid gyrus to zones in the pars intermedia (Fig. 174). The short latency projections to the b zone took their origin from a single area, intermediate between and overlapping with the hindlimb and forelimb areas. Responses to stimulation of the more medial portion of this area, adjoining the localization of the hindlimb were located in the lateral b 2 zone, responses from more laterally situated sites, located next to the forelimb area, were located in the 249
Ch. I
J. Voogd, D. Jaarsma and E. Marani
medial b 1 z o n e . The x zone only received a short latency projection from the forelimb area. The subdivision of the pars intermedia into three zones, with the C1 and C3 zones receiving climbing fibers from the rostral two thirds of the dorsal accessory olive with collateral projections to the anterior interposed nucleus and the C2 zone from the rostral portion of the MAO with collateral projections to the posterior interposed nucleus (Fig. 119) (Groenewegen et al., 1979), agrees with the organization of the corticonuclear projection from these zones. C2 does not reach as far ventrally as the C1 and C3 zones and is absent from the lobules I and III, where the C~ and C3 zones become contiguous (compare Fig. 170). This arrangement agrees with other, anterograde and retrograde studies of the anterior lobe in the cat (Armstrong et al., 1974; Brodal and Walberg, 1977a; Kawamura and Hashikawa 1979; Gibson et al., 1987). It is generally assumed that the three anatomical C zones are identical to their electrophysiological namesakes identified as the terminations of the different SOCP's. The rostral segments of the c~ and c3 zones receive a converging short latency input from the ipsilateral hind-limb through the VF- and the DF-SOCP (Oscarsson and Sj61und, 1974; Ekerot and Larson, 1979a). Ipsilateral forelimb components of the DF-SOCP and of a pathway ascending in the dorsolateral funiculus (DLF-SOCP) (Larson and Oscarsson and Larson et al., 1969) converge upon the rostral segments of these zones. A third zone, receiving forelimb input through the DF-SOCP, was identified by Ekerot and Larson (1979a,b) in the extreme lateral part of the anterior lobe and indicated as the d 2 zone. The Cl, c3 and d2 zones receive branches from a common set of climbing fibers (Ekerot and Larson, 1977, 1982). One group innervates the medial halves of the Cl and the c3 zone, another set branches between lateral c3 and the d 3 zone, a third system of climbing fibers, gives off branches to the x zone and the lateral c~ (or cx) (Campbell and Armstrong, 1985) zone (Figs 171 and 173). The cl, c3 and d 2 z o n e s belong to a collection of zones, that also includes the x zone, receiving short latency input through the DF-SOCP. Moreover, these zones are distinguished from the intercallated b, c2 and d 1 zones, because they are strongly activated by nociceptive stimuli (Ekerot et al., 1991a,b; Garwicz et al., 1992, in Garwicz, 1992) (Fig. 175). The innervation of these zones from the inferior olive is not uniform. The medial Cl, the c3 and the d 2 z o n e are supplied by the rostral DAO, but the x and cx zones from the MAO (Fig. 172) (Campbell and Armstrong, 1985; Apps et al., 1991; Trott and Apps, 1991). As a consequence the anatomical
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Fig. 177. Reconstructions of transverse sections through the anterior lobe from two experiments showing the
AChE banding pattern in the molecular layer, the responsive properties of the climbing fibers, and the locations of marking lesions. A. Transverse section through sublobules Vc to Vf showing a midline and bilateral lateral bands of AChE-positive activity. The area between the midline AChE band and the lateral border of the lateral band corresponds to zone A, as defined by Marani and Voogd (1977). All the units recorded within zone A were unresponsive to mechanical stimulation, except for one response that was located on the lateral edge of the lateral zone. Lateral to zone A, all the climbing fiber responses encountered represented areas of the extremities. B. Another experiment (similar to A except that the plane of the electrode penetrations and the histological section involved sublobules Vb to lobule III) where unresponsive units were encountered within zone A but lateral to the lateral AChE band; all of the climbing fiber responses were elicited by tactile stimulation. Robertson and Logan (1986).
and physiological nomenclatures for these zones no longer correspond. In an anatomical sense the cx (or lateral c~ zone), that receives climbing fibers from a caudomedial region of the rostral MAO, should be considered as part of the C2 zone. However, the physiological properties of the cx zone are indistinguishable from the (medial) c~ zone, which is innervated by the rostral DAO and represents the entire anatomical C1 (Fig. 173). A similar discrepancy may exist for the d 2 z o n e which, as the equivalent of the anatomical D 2 zone, supposedly receives climbing fibers from the dorsal leaf of the PO, but shares a climbing fiber projection with the c3 zone, that is innervated from the DAO. The intercallated c2 and d~ zones receive long latency projections through the DFSOCP, bilateral to the c2 zone, ipsilateral to d]. In addition a pathway in the lateral funiculus of the cord (LF-SOCP) (Larson et al., 1969b) provides a bilateral long-latency input to the c2 zone and the DLF-SOCP innervates the d] zone. Climbing fibers inner252
The cerebellum." chemoarchitecture and anatomy
Ch. I
vating the c3 and dl zones cannot be activated by nociceptive stimuli (Garwicz et al., 1992 in Garwicz, 1992) (Fig. 175). The multisynaptic pathways from the dorsal column to the inferior olive for these long-latency projections to c2 and dl, have not been identified. They may include connections from the dorsal column nuclei to the nuclei of the mesodiencephalic junction. The DF-SOCP projection to the c3 and presumably to the medial cl and d2 zones, is somatotopically organized. The rostro-caudal, hindlimb-forelimb organization of the pars intermedia has been known since Adrian (1943) and Snider and Stowell (1944). A corresponding rough topography in the projection of the lateral and rostromedial DAO to rostral and caudal segments of the C1 and C3 zones in the anterior lobe has been recognized with anatomical tracing methods (Fig. 119) (Brodal and Walberg, 1977a; Groenewegen et al., 1979; Gibson et al. 1987). The studies of Ekerot and Larson (1979b) and, especially, the mapping of nociceptive receptive fields of climbing fibers projecting to the anterior lobe by Ekerot et al. (1991a,b) showed a remarkable degree of somatotopical organization in the c3 zone (Fig. 176). The representation of the ipsilateral body half was double, with a mirror image of the sequence of receptive fields found in the medial and lateral c3 zones. A quite different, patchy and non-zonal type of somatotopical localization was described by Robertson (see Robertson, 1987 for a review), using natural stimulation in anaesthetized intact cats to record climbing fiber responses in the Purkinje cells. They found a relatively unresponsive area corresponding to the A zone, which was defined on the basis of AChE histochemistry (Fig. 177) (Robertson and Logan, 1986). A patch-like organization of the body representation exists throughout the anterior lobe hemisphere. Patches differ in size, isolated representations can be encountered within or adjacent to a patch involving a different area and adjacent patches seldom have representations of neighbouring skin areas. The somatotopical arrangement with forepaw in caudal and hindpaw in rostral parts of the anterior lobe is roughly maintained. The patchy representations in these areas resemble the 'fractured somatotopy' in mossy fiber systems (see Section 6.4.2.) and lack a definite sagittal zonal disposition. Branching of climbing fibers supplying the same sagittal zone, or a set of functionally similar zones, was first described by Armstrong et al. (1973). Systematic branching between the x and lateral c~, medial c~ and medial c3 and lateral c3 and d2 was described by Ekerot and Larson (1977, 1979a) and is illustrated in Figs 171,173 and 175. Branching between equivalent regions of the anterior lobe and the paramedian lobule has been anatomically substantiated by Brodal et al. (1980) and Rosina and Provini (1983) for the projections of the rostral DAO and MAO and for the PO of the cat, and by Eisenman and Goracci (1983), Payne et al. (1985); Wharton and Payne (1985) and Hrycyshyn et al. (1989) for the rat. Apps et al. (1991), who studied the branching between the x and cx zones of the cat cerebellum with double labelling methods, found only few doublelabelled neurons projecting to both zones in the border region of the rostral and caudal halves of the MAO. Branching to the medial c~, c3 and d2 zones was never studied with anatomical methods. Neurons supplying climbing fibers to the medial Cl zone are located in more lateral and caudal regions of the rostral DAO than those innervating the c3 zone (Trott and Apps, 1991). Neurons with branching axons should be located in the regions where the projections to c l and c3 overlap, and in the border region between the DAO and the ventral leaf of the PO, that innervates the D2 zone. It can be concluded that the anatomical and electrophysiological subdivisions of the pars intermedia of the anterior lobe of the cerebellum of the cat are not completely concordant (Fig. 173). Short-latency DF-SOCP innervated zones with cutaneous no253
Ch. I
J. Voogd, D. Jaarsma and E. Marani
ciceptive input alternate with zones lacking this input. This pattern extends into the vermis and the lateral zone. The short latency DF-SOCP zones are innervated from the MAO, rostral DAO and the PO. The lateral zone or hemisphere proper of the anterior lobe receives a projection from the principle olive. The distinction of this projection by Brodal (1940) was based on its absence in the cat, and its presence in the anterior lobe of the cerebellum of the rabbit, that extends further laterally. Projections of the PO to the extreme lateral part of the anterior lobe of the cat have been documented by Armstrong et al. (1974), Brodal and Walberg (1977a), Groenewegen et al. (1979) and Kawamura and Hashikawa (1979). Brodal and Kawamura (1980) discussed this projection and tentatively concluded that both the ventral and dorsal lamella of the PO projected to the anterior lobe, the dorsal lamella to the medial D1 zone and the ventral lamella to the lateral D 2 z o n e . It cannot be decided whether the dl and d 2 z o n e s identified in the electrophysiological studies of Ekerot and Larson (1979a, 1982) correspond to either D~ or the D2 zone. Some pertinent observations on the projection of the somatosensory cortex to the anterior lobe were made by Andersson and Nyquist (1983) (Fig. 174) in the cat. The c~, c2 and c3 zones receive short latency, somatotopically organized projections from the posterior sigmoid gyrus. The somatotopical organization of the c] and c3 zones is similar to that observed after peripheral stimulation, for the c2 zone no somatotopical organization was observed for the peripheral projections through the VF- and the DLF-SOCP. The corticocerebellar projection to the c2 zone is bilateral and the connections with the c~ and c3 zones are crossed, which is in accordance with the peripheral input to their zones. An exclusive projection to c2 was found to be present from the second somatosensory area. Short latency projections from the anterior sigmoid gyrus only terminated
dao
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\ C2 C~BA D2 Fig. 178. Semidiagrammatic illustration showing the zonal arrangement in lobules IV and V of Saimiri based on projections from specific subnuclei of IO to each zone. A = A zone; B - B zone; Cl_3 - C].3 zones; Dr, 2 = DI,2 zones; caudmao = caudal medial accessory olive; daom3 = medial/lateral part of the dorsal accessory olive; dlpo = dorsal leaf of the principal olive; rostmao - rostral medial accessory olive; vlpo - ventral leaf of the principal olive. Whitworth and Haines (1986b).
254
The cerebellum. chemoarchitecture and anatomy
Ch. I
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Fig. 179. Diagram of lamellar and zonal distribution of olivary afferents and efferents in the rat. The two lamellae (folds) of the dorsal accessory olive (DAO, 1 and 2) and the horizontal lamella of the medial accessory olive (MAC, 3) appear to receive afferents mainly from the spinal cord and dorsal column nuclei while projecting to anterior vermis and parts of intermediate cerebellum. The medial MAC (vertical lamella, 4) receives afferents from the vestibular and visual areas and projects to the posterior vermis as well as the flocculus. The rostral lamella of MAC and both lamellae of the principal olive (PC) receive projections from higher centers and send fibers to the lateral hemispheres. In the lower part of the figure, three drawings of the inferior olive demonstrate the lamellae corresponding to their sagittal zones of projection in the cerebellum. Azizi and Woodward (1987).
bilaterally in the dl zone. This zone could also be activated from parietal cortical areas. The d 2 z o n e was not investigated, it seems likely that its cortical afferentation is similar to c~ and c3, i.e. a crossed, somato-topically organized input from the posterior sigmoid gyrus. 255
Ch. I
J. Voogd, D. Jaarsma and E. Marani
The organization of the olivocerebellar projection to the anterior vermis in primates appears to be similar to the cat (Brodal and Brodal, 1981; Whitworth and Haines, 1986b) (Fig. 178). Olivocerebellar projections to the A, X and B zones were identified in a preliminary report on Macaca fascicularis by Voogd et al. (1987a,b, 1990). The projection to the A zone resembles the situation in the cat with respect to the presence of A1 and A2 subzones. Olivocerebellar projections to the X zone take their origin from intermediate levels of the MAO, that also project to the C2 zone. A collateral projection to Deiters' nucleus detaches from olivocerebellar fibers to the B zone. Projections from the DAO and the dorsal and ventral leaf to the hemisphere of the anterior lobe were described by Brodal and Brodal (1981) in macaque monkeys and C1, C 2 and C 3 and D zones were identified by Whitworth and Haines (1986) in the olivocerebellar projection to the anterior lobe in Saimiri sciureus. Studies of the olivocerebellar projection in the rat included the anterior lobe (ChanPalay et al., 1977; Furber and Watson, 1983; Campbell and Armstrong, 1983; Azizi and Woodward, 1987; Buisseret-Delmas, 1988a,b; Buisseret-Delmas and Angaut, 1989b, 1993; Buisseret-Delmas et al., 1993). Olivocerebellar projection zones in the cerebellum of the mouse were studied by Beyerl et al. (1982). Of these studies the paper of Azizi and Woodward (1987) is of interest because it introduced a new scheme for the identification of the zones (Fig. 179). Azizi and Woodward (1987) subdivided the MAO in a horizontal lamella (i.e. the subnuclei a and b of the caudal MAO), a vertical lamella (including the subnucleus c, the group beta and the dorsal cap) and a rostral lamella (corresponding to the rostral MAO). They distinguished a dorsal fold of the caudal DAO, which is joined laterally to the rest of the DAO, that was indicated as the ventral fold. The different lamellae and folds were found to project in a systematic manner to sagittal zones (Fig. 179). In the anterior lobe they distinguished projections from the horizontal lamella of the MAO to a medial vermal zone (corresponding to the A zone) and from the dorsal fold of the DAO to a lateral vermal zone (corresponding to B). The ventral fold projects to a single zone in the pars intermedia (corresponding to C~) and the rostral lamella of the MAO to a zone in the lateral cerebellum (corresponding to C2). The dorsal lamella of the PO projects to a medial zone in the hemisphere (D~) and the ventral lamella of the PO to the most lateral zone of the hemisphere (D2). With respect to the situation in the cat, therefore, the projections of the dorsal and ventral leaf of the PO to the D~ and D2 zones of the anterior lobe of the rat cerebellum are reversed. The projection of the DAO to the C3 zone was not identified. Buisseret-Delmas (1988a and b), Buisseret-Delmas and Angaut (1989b, 1993) and Buisseret (1993) using the same retrograde transport methods as Azizi and Woodward (1987), identified projections to the A, X, B and C1-C3 zones from the same subnuclei of the inferior olive as in the cat. They confirmed the projection of the dorsal fold of the caudal DAO to the B zone and traced projections to the C1 and C3 zones from the rostrolateral and rostromedial DAO respectively (Fig. 141). The main differences between their scheme lobe and the situation in the cat are the the presence in the anterior lobe of the rat cerebellum of an additional Do zone and the reversal in the projection of the dorsal and the ventral leaf of the PO to the D~ and D2 zones. The olivocerebellar projection to the pars intermedia is similar in rat and cat. The differences in the connections of the C~_3zones of rat cerebellum concern the corticonuclear projection of the zones of the pars intermedia (see Section 6.1.4.). The X zone was indentified in the lobules IV-VI of the cerebellum of the rat by Buisseret-Delmas et al. (1993) as a zone located lateral to the A zone, projecting to the junctional region of the fastigial and interposed nuclei (their 'interstitial cell groups'). 256
The cerebellum." chemoarchitecture and anatomy
Ch. I
rostral
BP
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1ram Fig. 180. Schematic illustration of the result of D-[3H]aspartate injection into lobules IV and V of the cerebellum of the rat. In the sketches of the cerebellar sections, retrogradely labelled axons and axon collaterals are indicated by lines and dots. Filled dots in the olives indicate the location of labelled cells. Retrograde labelling in cells of the inferior olive is also illustrated in more detail in the diagrams on the right. BP = brachium pontis; D A O = dorsal acessory olive; D N = Deiters' nucleus; F N - fastigial nucleus; LL = lateral lemniscus; LLV = ventral nucleus of the lateral lemniscusw; M A O - medial accessory olive; OI = inferior olive; OS = superior olive; PN - pontine nuclei; PO - principal olive; RB - restiform body; I-X lobules I-X. Wiklund et al. (1984).
It receives a projection from the subnuclei a, b and c of the caudal MAO. The origin of this projection in the rat appears to be more extensive than in the case of the X zone of the cat (compare Fig. 172) (Campbell and Armstrong, 1985). They also identified a CX zone in the lobules V and VI, medial to the C1, on the basis of its projection to the interstitial cell groups. It received its olivocerebellar projection from the group c at middle and rostral levels of the MAO. Buisseret-Delmas and Angaut (1989b, 1993) defined three D-zones in the lateral part of the anterior lobe on the basis of their corticonuclear projection to the dorsolateral hump (Do), the dorsal magnocellular part of the lateral cerebellar nucleus (D1) and the ventral parvicellular part of this nucleus (D2). They receive their olivocerebellar projections, respectively, from the DM group and the medial half of the ventral leaf of the PO (Do), the dorsal leaf of the PO (D,) and the lateral half of the ventral leaf of the PO (D2) (see Fig. 141). Several aspects of the olivocerebellar projection have been studied mainly in the rat. 257
Ch. I
J. Voogd, D. Jaarsma and E. Marani
AXBGGC~
D
I II Ill IV V Vlm Vlb Vlc Vll VIII IX
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::::::::::::::::::::::::::::::::::::::::::::::: :i:::::::::::::j.~ , ~ * . ~ .....:::~:~::~%:~.,
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Fig. 181. A comparison of the zonal distribution of Zebrin-immunoreactive Purkinje cells (left diagram) and the olivocerebellar projection (right diagram) in the rat. Diagram of the olivo-cerebellar projection is based on data from Furber and Watson (1983), Eisenman (1984), Azizi and Woodward (1987), Buisseret-Delmas and Angaut (1989b, 1993), Sugita et al. (1989), Apps (1990), Akaike (1992) and Ruigrok and Voogd (1992). Subnuclei of the inferior olive (bottom diagram) and their projection zones are indicated with the same symbols. With the exception of the olivocerebellar projection to the lateral extension of the A zone (A~) of Buisseret-Delmas (1988a) (Figs 141 and 144), the correspondance between the Zebrin zones and the olivocerebellar projection has not been verified experimentally. 1-7 = Zebrin antigenic zones P1 - P7; a = subnucleus a of the MAO; A = zone A; A~ = lateral extension of the A zone of Buisseret-Delmas (1988a); b = subnucleus b of the MAO; B = zone B; 13 = group beta; c = subnucleus c of the MAO; C1-3 = zones C1-C3; CrI = crus I of the ansiform lobule; CrlI = crus II of the ansiform lobule; D = zone D; Do = zone Do of Buisseret-Delmas and Angaut (1989b); D A O = dorsal accessory olive; dc = dorsal cap; dl = dorsal leaf of the PO; D M = dorsomedial subnucleus of the ventral leaf of the PO; dmcc = dorsomedial cell column; F L O = flocculus; M A O = medial accessory olive; P F L = paraflocculus; P M D = paramedian lobule; PO = pricipal olivary nucleus; SI = simple lobule; vl = ventral leaf of the PO; vlo = ventrolateral outgrowth; X = zone X; I-X = lobules I-X.
The zonal distribution of the olivocerebellar projection was first demonstrated by ChanPalay et al. (1977), using autoradiography of antegrade axonal transport of 35S-methion258
The cerebellum." chemoarchitecture and anatomy
Ch. I
ine. She advocated a bilateral projection of the olive, but this observation has never been confirmed. The labelling of climbing fibers in the molecular layer in her experiments always was discontinuous. The unlabelled spaces between the labelled climbing fiber strips were assumed to receive climbing fibers of extra-olivary origin. This argument subsequently was disproved by Campbell and Armstrong (1983), who showed that labelling of climbing fibers from the inferior olive in the molecular layer was continuous. The investigations of Wiklund et al. (1984), using selective retrograde transport of [3H]D-aspartate were done in rats. The injection sites in the cerebellar cortex were relatively large and defied a detailed analysis of the material in terms of zones. Their experiments clearly confirmed the presence of branching olivocerebellar fibers between the anterior lobe and lobule VIII of the caudal vermis and the paramedian lobule. Moreover, their experiments clearly showed the presence of collateral projections to the cerebellar nuclei and to Deiters' nucleus (Fig. 180). The zonal disposition of Zebrin-immunoreactive and non-immunoreactive Purkinje cells has not been compared in any detail to the olivocerebellar projection. According to Gravel et al. (1987) some of the borders between Zebrin-positive and negative zones coincide with borders of certain climbing fiber strips, but these strips were not further identified. Judging from the reported identity of some of the Zebrin-positive and -negative zones with certain corticonuclear and cortico-vestibular projection zones it seems likely, that the correspondence between the zonal organization in the olivocerebellar projection and the Zebrin pattern will be close (Fig. 181). 6.3.3.2. Olivocerebellar projection to the hemisphere of the posterior lobe According to Groenewegen et al. (1979) the olivocerebellar projection zones in the cat continue uninterruptedly from the anterior lobe across the primary fissure into the simple lobule, where they diverge laterally to enter the ansiform lobule (Fig. 119). The projection of the caudal MAO to the B zone cannot be traced beyond the border of the simple lobule (lobule VI) and lobule VII. The projections from the rostral DAO to the C~ and C3 zones in the ansiform lobule are narrow or interrupted, but reappear in the caudal folia of the crus II (the ansula). C3 is only present in the dorsal folial rosette of the paramedian lobule, C1 continues into the most ventral folia of this lobule. The projection of the rostral MAO to the C2 zone can be traced as an uninterrupted zone, through the medial ansiform lobule, and the centrolateral paramedian lobule into the paraflocculus, where it occupies a ventral position in the dorsal paraflocculus and a dorsal position in the ventral paraflocculus. The final segment of the C: zone innervates the caudo-medial portion of the flocculus (Gerrits and Voogd, 1982). The PO is connected with the D~ and the D2 zones of the entire hemisphere and sends collaterals to the lateral cerebellar nucleus. The antegrade [3-H]leucine tracing experiments of Groenewegen et al. (1979) did not allow for an analysis of the possible differential origin of the climbing fibers terminating in the D1 and D2 zones. The olivocerebellar connections of the simple and ansiform lobules were studied in detail by Rosina and Provini (1982; see Kotchabhakdi et al. 1978, for an earlier report). Their conclusions are in accordance with the scheme of Groenewegen et al. (1979), specifying the presence of a C~ zone in the central axis of the folial rosette, and the attenuation of the C3 zone in the lateral bend of the lobule. They found a projection of the ventral leaf of the PO to the medial D1 zone and of the dorsal leaf to the lateral D 2 zone of the ansiform lobule (Fig. 182). The olivocerebellar projection to the crus II and the paramedian lobule of the cat was 259
Ch. I
J. Voogd, D. Jaarsma and E. Marani
A
C
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Fig. 182. The olivocerebellar projection to the lobulus simplex and the ansiform lobule of the cat. A. Diagrammatic surface view of the cerebellar injected areas in the four different groups of folia of cerebellum. Thin lines indicate H R P injected areas in the medial crus I-simple lobule; dotted lines in the lateral crus I; thick lines in the lateral crus II; dashed lines in the intermedio-medial crus II. Arrows indicate the subdivisions of the ansiform lobule adopted in this study: (a) intercrural sulcus and border between crus I and lateral crus II; (b) border between lateral and intermediate crus II; (c) intracrural sulcus 2 and border between intermediate and medial crus II. B. Summarizing diagram of the olivary areas retrogradely labelled by H R P injections into the four different groups of folia, drawn on the schema of the IO imagined unfolded. The olivary areas corresponding to the different groups of cerebellar injected folia are marked by thin, dotted, thick and dashed lines as indicated in A. Four different hatchings mark the four olivary subdivisions (MAO, vl, dl and lateral bend, DAO) which project to the four different longitudinal strips (C2, D1, D2 and C1-C3) of the ansiform lobule, as reconstructed in C. C. Surface view of the localization of the longitudinal zones as reconstructed by these reported results (D1 and D2 zones) and by previous studies (C2 and C1-C3 zones). Abbreviations: CrI, II = crus I, II of the ansiform lobule; C1-3 = C1-3 zones; D1,2 = D1,2 zones; D A O = dorsal accessory olive; dl = dorsal leaf of the PO; 1 = lateral; L.pm = paramedian lobule; L.sim = simple lobule; m = medial; M A O = medial accessory olive; Pfl.d = dorsal para-flocculus, PO = principal olive; vl = ventral leaf of PO. Rosina and Provini (1982).
the subject of series of reports, that reflect the changing views of the main actors in the course of time (Brodal and Courville, 1973; Courville et al., 1973; Brodal et al., 1975; Brodal and Walberg, 1977b; Walberg and Brodal, 1979). Brodal and Kawamura (1980), reviewing this work, concluded that C~, C2 and D zones are present in the paramedian lobule. They were unable to identify C3 in the dorsal lobules and stated that the dorsal and ventral leaf of the PO project to narrow D1 and D 2 z o n e s respectively. A reverse origin of the projection of the ventral and the dorsal leaf of the PO to the D~ and D 2 was advocated by Groenewegen et al. (1979) and substantiated by Rosina and Provini (1982) for the ansiform lobule. Electrophysiological studies of the configuration of the termination of climbing fibers in the paramedian lobule are scarce. Trott and Apps 260
The cerebellum." chemoarchitecture and anatomy
d
c3
Ch. I
C2 ci
V
|
iv, VII PMD
DOOO
ol
VIII
| Fig. 183. Activation of climbing fibers projecting to the caudal paramedian lobule (PM) in (B) by stimulation of collaterals terminating in the c~, c2, c3 and d zones of the anterior lobe indicated in (A). Redwawn from Oscarsson and Sjolund (1977b).
(1993) identified c~, c 2 and c3 zones in the rostral paramedian lobule on the basis of their electrophysiologically defined climbing fiber input and their projections to the cerebellar nuclei. They concluded that the presence of the c3 zone was variable. Oscarsson and Sj61und (1977b) delimited an ipsilateral hindlimb VF-SOCP projection to cl and c3 zones in the pars copularis of the paramedian lobule: the zones fuse into a wide area occupying almost the entire ventralmost paramedian folium. Stimulation of the c1_3and the d zones in the anterior lobe activated climbing fibers in the corresponding zones of the paramedian lobule (Fig. 183). Jeneskog (1974) recorded climbing fiber evoked potentials in the c~ and d zones of the anterior lobe and the paramedian lobule, after stimulation in and around the red nucleus. Rubral and spino-olivary pathways converge upon these zones: the DLF-SOCP terminates in the d zone, and the hindlimb and forelimb components of the DF-SOCP in the ventral and dorsal portions of the Cl zone of the paramedian lobule (Jeneskog, 198 l a). Tentatively they identified a C3 zone in the dorsolateral paramedian lobule and located the C2 zone in the central region of this lobule after mesencephalic stimulation. The total picture of the olivocerebellar projection to the paramedian lobule is less complete, but still very similar to the anterior lobe, with fusion of the C~ and C3 zones in its ventralmost part. The discontinuity of the C3 zone in the dorsolateral and ventrolateral parts of the lobule allows the C2 zone to leave the paramedian lobule to enter the paraflocculus. 'The paraflocculus is probably the most enigmatic cerebellar lobule from a functional point of view' (Brodal and Kawamura, 1980), but the pattern of its climbing fiber 261
Ch. I
J. Voogd, D. Jaarsma and E. Marani
afferents is simple and straightforward, with concentric projections of the rostral MAO to the C2 zone along its medial border and of the PO to the D zones along its periphery (Fig. 119) (see Brodal and Kawamura, 1980, for a discussion of the results of retrograde tracing studies from the paraflocculus). The main problem in the zonal structure of the paraflocculus is posed by the presence of a distinct subdivision of the D compartment and clear evidence for the projection of D~ and D 2 z o n e s to different parts of the dentate nucleus and the lack of evidence on a corresponding differentiation in the olivocerebellar projection from the PO. An educated quess can be made on the basis of the generally acknowledged nucleo-olivary projections of the rostromedial dentate to the dorsal leaf and the caudolateral dentate to the ventral leaf of the PO. Because the D~ zone projects caudolaterally and the D 2 z o n e of the paraflocculus rostromedially in the dentate, it seems likely that the D~ zone is innervated by the ventral leaf and the D 2 z o n e by the dorsal leaf of the PO. The olivocerebellar connections of the hemisphere of primates have been studied in the classical studies of Holmes and Stewart (1908), that were reviewed and updated by Jansen and Brodal (1958) (see also Voogd et al., 1990). Information on monkeys is limited to the retrograde transport experiments of Brodal and Brodal (1981, 1982) with injections of HRP in the simple, ansiform and paramedian lobules, in macaques, that suggest that C~, C2, C3 and D zones are present in these lobules. The diagram of Buisseret-Delmas and Angaut (1993) of the organization of the olivocerebellar projection in the rat (Fig. 141), appears very similar to the situation in the cat, but closer inspection learns that there are several important differences. One difference concerns the presence in the hemisphere of the posterior lobe of an additional zone, which has never been observed in the cerebellum of either cat or primates, located in the medial region of the hemisphere of the simple lobule, the ansiform lobule and the rostral folium of the paramedian lobule. This zone projects to the dorsolateral protuberance of the fastigial nucleus. It is absent from the anterior lobe and the copula pyramidis (see Section 6.1.4.). The olivocerebellar projection from the medial subnucleus c to this zone in the hemisphere (the lateral extension of the A zone of Buisseret-Delmas (1988a)) was recognized with retrograde transport from small WGA-HRP injections in this area (Fig. 142). She considered it to belong to and and to be continuous with the vermal A zone. The projection of the caudal MAO to the hemisphere was not noticed by Campbell and Armstrong (1983) and was not accounted for in Azizi and Woodward's (1987)
Fig. 184. The tecto-recipient zones in the vermis (lobule VII) and the hemisphere (paramedian lobule, Crus II and simple lobule) of rat cerebellum. Note their absence from anterior lobe, Crus I and copula pyramidis. Compare Fig. 142. ANT = anterior lobe; COP = copula pyramidis; CrI(II) = crus I(II) of ansiform lobule; PMD = paramedian lobule; SI = simple lobule. IV-IX = lobules IV-IX. Redrawn from Akaike (1992).
262
The cerebellum." chemoarchitecture and anatomy
Ch. I
scheme of the olivocerebellar projection in the rat. Akaike (1986bc, 1987, 1989, 1992) identified a similar zone with stimulation of the ipsilateral superior colliculus. It receives climbing fibers from the rostral subnucleus c (the tectorecipient zone of the MAO), that branch between medial VIb and the crus II (Fig. 184). It differs from the lateral extension of the A zone of Buisseret-Delmas (1988a) because it is absent from the crus I and from the rostral folia of the paramedian lobule. According to Akaike (1986c) it is separated from a similar zone in the vermis of lobule VII that also receives a climbing fiber input from the tectum, by a narrow non-tectal zone which straddles the paramedian sulcus. The tectal zone of the hemisphere is bordered on its lateral side by a zone receiving climbing fiber input from the ipsilateral wishker area (Akaike, 1989). The olivary neurons projecting to the tectal zone of lobule VII are also located in the tectorecipient area of subnucleus c, medial to the neurons projecting to the tectal zone in the hemisphere (Akaike, 1986a). The two populations are completely separated and do not collateralize between vermis and hemisphere (Akaike, 1986a,b,c, 1992). The position of the lateral extension of zone A in the hemisphere of the simple lobule differs from Buisseret-Delmas' (1988b) diagram, because it is located lateral and not medial to the B zone (see Section 6.1.4.). This peculiar position was confirmed in an experiment with an injection of the antegrade tracer Phaseolus vulgaris lectin in the rostral subnucleus c of the rat (Fig. 144) (Voogd, unpublished), with climbing fiber labelling in this zone and a collateral projection to the dorsolateral protuberance of the fastigial nucleus. To make the picture complete, it should be remembered (Section 6.3.2.1.) that the rostral subnucleus c of the caudal MAO is the target of the nucleoolivary projection of the dorsolateral protuberance (Fig. 164) (Ruigrok and Voogd, 1990). Counterstaining with the Zebrin I antibody showed that the termination of the climbing fibers coincides with the area of the Zebrin immunoreactive zones P4b and 5a extending from the simple lobule into the paramedian lobule. C1, C2, C3 and D zones receiving climbing fibers from the rostral DAO and MAO and the PO presumably are present in more lateral parts of the hemisphere of the simple lobule. The crus I of the ansiform lobule seems to lack a tectal response zone (Akaike, 1986b, 1987, 1992), and B, C~ and C3 zones are not represented in the ansiform lobule (Furber and Watson, 1983; Buisseret-Delmas, 1988a,b). The lateral extension of the A zone (tectal response zone of Akaike) and the C1, C2 and C3 zones reappear in the crus II and the dorsal folia of the paramedian lobule (Furber and Watson, 1983; Buisseret-Delmas, 1988a,b). The lateral extension of the A zone (Buisseret-Delmas, 1988a) and the C3 zone are absent from the copula pyramidis, that contains representations of the C1, C2 and D zones (Fig. 185) (Eisenman, 1981; Azizi and Woodward, 1987; Apps, 1990). Multiple D zones with projections from the PO are present in the entire hemisphere. According to Azizi and Woodward (1987) and Buisseret-Delmas and Angaut (1989b) the medial D~ zone (zone 6 of Azizi and Woodward, 1987) and the lateral D2 zone (zone 7 of Azizi and Woodward), receive their climbing fibers from the dorsal and the lateral half of the ventral leaf of the PO respectively (Figs 141 and 179). The dorsolateral hump sends a nucleo-olivary projection to the dorsomedial subnucleus (DM) of the olive, that is attached to the ventral leaf of the PO and has often been confused with the DMCC (Azizi and Woodward, 1987; Ruigrok and Voogd, 1990). Olivocerebellar projections from this subnucleus to the hemisphere of the simple lobule, the crus II, the paramedian lobule and the copula, but not to the crus I, have been noticed by Furber and Watson (1983) and Buisseret-Delmas and Angaut (1989b, 1993: their Do zone) (Fig. 141). The projection of the DM to the Do zone, therefore, is limited to the same lobules as the projection of the DAO to the C~ and C3 zones. Little is known about the olivocerebellar 263
Ch. I
J. Voogd, D. Jaarsma and E. Marani
B8 a
9b c
drn
9
drn 7.
caudal Fig. 185. The olivocerebellar projection to the pyramis and the uvula (lobules 8 and 9) of the rat cerebellum. A,B. Olivocerebellar projection zones of the lobules 8 and 9. C. Origin of these projections, indicated in diagrams of transverse sections through the inferior olive, a = subnucleus a of the medial accessory olive; b = subnucleus b of the medial accessory olive; beta = group beta; c = subnucleus c of the medial accessory olive; d - dorsal accessory olive; dm = dorsomedial subnucleus; rn - medial accessory olive; pr = principal olive; 8 and 0 = lobules VIII and IX of Larsell. Relabelled and reproduced from Eisenman (1984).
projection to the paraflocculus of the rat. According to Furber and Watson (1983) and Azizi and Woodward (1987) and Buisseret-Delmas and Angaut (1993) it receives climbing fibers from the rostral MAO and the PO. There is a good correspondence between the organization of the olivocerebellar projection in the rat and the cat. The main exceptions are the presence of the lateral extension of the A zone (or tectal response zone) and the Do zone in the hemisphere of 264
The cerebellum." chemoarchitecture and anatomy
Ch. I
the rat cerebellar hemisphere, that seem to be absent in the cat. The interruption of C1 and C3 zones in the crus I and the presence of a C3 zone in the crus II and the paramedian lobule, and its absence in the copula are in accordance with the findings in the cat. Projections of the dorsal and the ventral leaf of the PO to the D1 and D2 zone respectively, are still controversial in the cat, but seem to be well established for the rat. With the exception of the olivocerebellar projection to the lateral extension of the A zone, which coincided with the area of the P4b/P5a Zebrin immunoreactive Purkinje cell zones (Fig. 144), no direct comparisons of the olivocerebellar projection with the Zebrin pattern are available. However, in view of the similarity of the corticonuclear projection zones to the zonal arrangement in the olivocerebellar projection (see Section 6.1.4.), it seems likely that the P5b+ zone corresponds to C: and that the Zebrin-negative zones that border upon P5 correspond to the C~ and C3 zones (Fig. 181). 6.3.3.3. The olivocerebellar projection to the caudal vermis and the flocculus
In all species the zonal arrangement of the olivocerebellar projection to the vermis of the simple lobule (lobule VI) is very similar to the anterior lobe. Caudal MAO and DAO projections to A and B zones were traced to lobule VI in the cat (Groenewegen and Voogd) (1977) (Fig. 119) and the rat (Buisseret-Delmas and Angaut, 1993) (Fig. 141). The olivocerebellar projection to the rest of the caudal vermis differs substantially from that to the anterior lobe, with characteristic patterns for each of the lobules VII-X. Collateral projections to the fastigial nucleus have been observed with injections of antegrade tracers in the MAO and its subnuclei and labelling of (sub)zones in the caudal vermis (Groenewegen and Voogd, 1977), but these collaterals have not been systematically studied. Brodal and Kawamura (1980) and Sugita et al. (1989) noticed that the cells of the inferior olive projecting to the A zone of the lobules VI-VIII in cat and rat respectively, were arranged along the periphery of the caudal MAO, surrounding a central region projecting to the A zone of the anterior lobe (Fig. 186). The differences between their diagrams concern the origin of the projection to lobule VI, and the involvement of the group beta, which provides a sparse projection to lobule VII of the cat, (Hoddevik et al., 1976) but projects heavily to the lobules VI and VIII of the rat (Sugita et al., 1989; Apps, 1990). The A zone of lobule VI of the cat receives a single projection from a cell column in the medial caudal MAO, located lateral to the cells projecting to lobule VII (Fig. 186). The situation in the rat is more complicated with two labelled foci in the lobules Via and b after injections of WGA-HRP. One is located in the group beta, the other is a column situated along the lateral margin of the caudal MAO and extending along the border with the rostral half of this nucleus. The localization of the latter column is very similar to the cells projecting to the X zone of the anterior lobe in the cat (Campbell and Armstrong, 1985), and may correspond to the olivary neurons projecting to the X zone in the rat (Buisseret-Delmas et al., 1993). Retrograde labelling in the subnucleus c appears with injections of the caudal lobule VIC of the rat (Sugita et al., 1989). The existence of multiple sites in the caudal MAO that project to lobule VI of the rat is in accordance with the subdivision of this lobule in multiple corticonuclear projection zones and Zebrin-positive and -negative Purkinje cell strips (see Sections 5.6.1.3. and 6.1.4.). Laterally the A zone is bordered by the projection of the caudal DAO (cat: Hoddevik et al., 1976) or the dorsal fold of the DAO (rat: Buisseret-Delmas, 1988a; Azizi and Woodward, 1987) to the B zone. The B zone ends at the border of lobule VI and lobule VII in the cat, and at the VIa/VIb border in the rat. 265
Ch. I
J. Voogd, D. Jaarsma and E. Marani
A.i
-
t
MEDIAL ACC.OLIVE rostral
dm.c.col. X~r )tIE
~I[ X[
Villi Vlr VI V IV caudal
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Fig. 186. The projection of the medial accessory olive to the vermis based upon retrograde tracing experiments in the cat. A ( 1 , 2 ) - A(1,2) zone; dm.c.col. = dorsomedial cell column; 1= lateral; m = medial; nucl. fl = subnucleus beta; I - X - lobules I-X. Brodal and Kawamura (1980).
According to Hoddevik et al. (1976) the main projection to lobule VII in the cat takes its origin from the rostromedial portion of the caudal MAO. This region receives afferents from the contralateral superior colliculus (Weber et al., 1978; Saint Cyr and Courville, 1982) and projects in a topical manner to the lobules VI and VII, with the rostral superior colliculus being located in the medial part of these lobules and the caudal colliculus more laterally (Kyuhou and Matsuzaki, 199 l a,b). An equivalent projection to lobule VII (the vermal visual area) was traced in macaque monkeys from the medial, Z-shaped portion of subnucleus b of the caudal MAO (Frankfurter et al., 1977; Brodal and Brodal, 1981; Yamada and Noda, 1987; Ikeda et al., 1989). The olivocerebellar projection to lobule VII of the rat differs from the adjoining lobules VI and VIII in being derived from a single cell column in the medial MAO (Sugita et al., 1989). This projection to lobule VII of the rat of the tectal response zone of the subnucleus c of the caudal MAO has been studied by Hess (1982b) and in a series of publications of Akaike (see Akaike, 1992 for a review). The neurons projecting to 266
The cerebellum." chemoarchitecture and anatomy
Ch. I
lobule VII are located in the caudal half of the tectorecipient zone of the caudal MAO, caudal to and intermingled with cells projecting to the lateral extension of the A zone in the medial hemisphere (see also the previous Section 6.3.3.2.). There is no climbing fiber branching between lobule VII and this zone in the hemisphere. The climbing fiber responses on stimulation of the superior colliculus occupy the medial two thirds of lobule VII (Akaike, 1985, 1986b,c, 1992). A similar, medial localization of the climbing fiber evoked potentials on tectal stimulation was reported in the earlier study of Jeneskog (1983) in the cat. The nature and the precise origin of the climbing fiber afferents to the lateral, non-tectal zone of lobule VII are not known. The area may be related to the X-zone or to a climbing fiber zone in more caudal lobules receiving afferents from the group beta. In the rat this non-tectal zone bridges the paramedian sulcus and consists of an 1 mm wide strip of cortex between the vermal and hemispheral zones supplied by the neurons of the tectorecipient area of subnucleus c of the caudal MAO. The relatively simple schemes of the olivocerebellar projection to lobule VIII based on studies in the cat by Hoddevik et al. (1976), Groenewegen and Voogd (1977) and Brodal and Kawamura (1980) now have evolved into the more complicated patterns defined by Eisenman (1981, 1984) and Apps (1990) for the rat. These patterns are of special interest because they bear a strong resemblance to the distribution of Zebrinpositive and -negative zones in lobule VIII of the rat (Hawkes and Leclerc, 1987) (see also Section 6.1.3.) and to the compartmentalization of this lobule in AChE-stained material of macaque monkeys (Hess and Voogd, 1986). Eisenman (1981) found two strips of climbing fibers in lobule VIII innervated by subnucleus a of the caudal MAO that were separated by a projection from the group beta. Climbing fibers terminating at the junction of lobule VIII with the copula pyramidis originated from the middle portion of the lateral DAO. Apps' (1990) observations were rather similar. The two strips receiving projections from subnucleus a constitute isolated regions, because projections from subnucleus a (and b: Sugita et al., 1989; Apps, 1990) are absent from lobule VII and scarce in IX. The group beta projection to lobule VIII, shifts medially in lobule IX, where it occupies a position next to the midline (Fig. 185) (Eisenman, 1984). This situation is reminiscent of the presence of distinct Zebrin-negative P 1 - and P 2 - strips, separated by P2+ in lobule VIII of the rat that disappear or become much narrower in the adjacent lobules VII and IX. The presence of an X zone in lobule VIII of the rat was postulated by Buisseret-Delmas et al. (1993). The olivocerebellar projection to lobule IX has been most extensively studied in cat and rabbit, data on rat and primates are less complete. Groenewegen and Voogd (1977) and Groenewegen et al. (1979) distinguished three zones in lobule IX of the cat: a medial zone, receiving a projection from the caudal MAO and/or the group beta, an intermediate zone receiving a projection from the DMCC and a lateral zone corresponding to C2, with climbing fibers from the rostral MAO. Similar patterns in the olivocerebellar projection to the uvula were reported by Brodal (1976) and Brodal and Kawamura (1980). Sato and Barmack (1985) in the rabbit and Kanda et al. (1989) in cat, distinguished projections of the group beta to two medial zones from the projection of the caudal MAO to a slightly more lateral area, and located a narrow strip receiving a projection from the ventral leaf of the PO between the lateral DMCC and rostral MAO (C2) zones (Fig. 187). Presumably, this pattern is shared by the rat (Eisenman, 1984; Bernard, 1987; Apps, 1990). Buisseret-Delmas et al. (1993) considered the projections from the DMCC and subnucleus c of the MAO to lobule IX of the rat to belong to the X and CX zones. Correlations of the olivocerebellar projection with the Zebrin pattern of lobule IX have not yet been verified. Retrograde labelling from lobule IX in the same 267
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subnuclei of the inferior olive was observed in the opossum (Linauts and Martin, 1978), in primates (Brodal and Brodal, 1981, 1982) and in subprimates (Whitworth et al., 1983). Zonal projections of the inferior olive to the nodulus (lobule X) and the flocculus have been substantiated in different species. Purkinje cells of these lobules are uniformly Zebrin-positive in rat, mouse, opossum, squirrel monkey (see Section 6.1.3.) and rabbit (Jaarsma unpublished observations). A compartmental subdivision of the white matter of the nodulus and the flocculus was demonstrated with AChE-histochemistry in the rabbit and the monkey (Section 6.1.5.) and correlated with the olivocerebellar projection to these lobules (Voogd et al., 1987a,b; Tan et al., 1995a,b). The pattern in the olivocerebellar projection to lobule X is quite distinct from lobule 268
The cerebellum." chemoarchitecture and anatomy
Ch. I
IX. The zones provided with climbing fibers from the group beta, the DMCC and the rostral MAO continue for some distance over the cortex of the nodule, but they are replaced by climbing fibers from the dorsal cap (DC) and the ventrolateral outgrowth (VLO). A large proportion of the climbing fibers from the latter two subnuclei are branches from climbing fibers which also terminate in the flocculus (Takeda et al., 1989a,b; Maekawa et al., 1989). Projections from rostral MAO, DMCC, group beta and DC, but not from the VLO were traced in the rabbit with HRP injections of lobule X and ventral IX by Alley et al. (1975). Zonally organized projections to lobule X from DC and VLO were demonstrated with anterograde axonal transport by Groenewegen and Voogd (1977) and Kawamura and Hashikawa (1979) in the cat. Retrograde labelling in these subnuclei after injections of retrograde tracers in lobule X was confirmed by Whitworth et al. (1983) for Galago and by Walberg et al. (1987) for the cat. Katayama and Nisimaru (1988) based their description of the olivocerebellar projection to lobule X on retrograde axonal transport experiments in the rabbit (Fig. 150). The projection from the group beta occupies the same medial position as in lobule IX. The lateral zones that are innervated from the DMCC and the rostral MAO, only extend over the dorsal surface of lobule X. The intermediate region contains a central zone, receiving a projection from the VLO, flanked by two strips innervated from the DC. The results of the anterograde and retrograde tracing experiments of Balaban and Henry (1988), also in the rabbit, were rather similar. They found the VLO zone to extend on the dorsal surface of lobule X and the ventral lobule IX and the two DC zones to be restricted to its ventral surface. These observations on the extent of the DC and VLO innervated zones are in complete accordance with the complex spike recordings in nodule Purkinje cells of the rabbit of Kano et al. (1990). The projections from the DC to lobule X and the flocculus were traced with parvalbumin-immunohistochemistry in rat pups by Wassef et al. (1992a,b). She showed that the cells of the DC (but not the cells of the VLO) transiently express immunoreactivity for antibodies against parvalbumin from birth till the 15th postnatal day. During this period two bundles of parvalbumin-immunoreactive climbing fibers were present in lobule X, corresponding to the medial and lateral DC innervated zones described in the rabbit. Olivocerebellar projections from the DC and the rostral one third of the MAO were first traced to the flocculus by Alley et al. (1975) with retrograde transport of HRP in the rabbit. The origin of the olivocerebellar projection to the flocculus was subsequently confirmed, using the same method, by Hoddevik and Brodal (1977), Yamamoto (1979) and Tan et al. (1995b) in the rabbit, Gerrits and Voogd (1982), Gould (1980) and Sato et al. (1983a) in the cat, Brodal and Brodal (1982) and Langer et al. (1985a) in macaque monkeys, Whitworth et al. (1983) in Galago and Blanks et al. (1983) and Bernard (1987) in the rat. The zonal organization of the olivocerebellar projection to the flocculus is similar in all species that were studied. Paired climbing fiber zones innervated by the caudal DC (the zones 2 and 4 of the rabbit flocculus) interleave with two zones innervated by the rostral DC and the VLO (zones 1 and 3) (Fig. 188). A fifth, lateral C2 zone is innervated by the rostral MAO. In the rabbit these zones correspond with the 5 corticonuclear and corticovestibular projection zones and the climbing fibers innervating a zone occupy the same white matter compartment as the axons of the Purkinje cells of this zone (Tan et al. 1995a,b,c) (Figs 147, 149 and 188) (see Section 6.1.5.). This arrangement is of special interest because it has been implicated in the spatial organization of eye movement control. Briefly the caudal DC of the rabbit transmits information about contralateral 269
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Fig. 188. Diagram of the origin of the climbing fiber afferents and the projections of the Purkinje cells of the five Purkinje cell zones of the flocculus of the rabbit. The zones and the corresponding white matter compartments containing their Purkinje cell axons and climbing fiber afferents are indicated with the same symbols. 1-4 = compartments and zones 1-4; DC = dorsal cap; fl-4 = folia 1-4 of the rabbit flocculus; fm = medial folium of the flocculus; fp = folium p of the ventral paraflocculus; Ip = posterior interposed nucleus; M A O = medial accessory olive; MV = medial vestibular nucleus; SV = superior vestibular nucleus; VLO = ventrolateral outgrowth; Y = group y. From Tan et al. (1995b,c).
movements of the surround around a vertical axis. Cells in the rostral DC and the VLO are excited by movements around an oblique horizontal axis that is oriented roughly parallel to the axis of the ipsilateral anterior semicircular canal (see Van Der Steen et al., 1994 for a review). Groenewegen and Voogd (1977) first described the projection of the caudal DC to a central zone in the cat flocculus flanked by two zones innervated by the rostral DC/VLO (zones 2 and 4). This pattern was extended by Gerrits and Voogd (1982) in tracing studies in the cat. The 7 floccular zones distinguished by these authors correspond to the 5 zones of the rabbit flocculus, because two of them are double (Fig. 149). Moreover, Gerrits and Voogd (1982) noticed that the zonal pattern in the olivo270
The cerebellum." chemoarchitecture and anatomy
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caudal Fig. 189. Diagram of the projection from the inferior olive to the flocculus and the ventral paraflocculus in the rat. The medial accessory olive (MAO) and the principal (PO) are drawn as diagrams of the unfolded inferior olive; the cortex of the flocculus and the ventral paraflocculus are unfolded. Different climbing fiber zones are indicated with different symbols. The climbing fiber zones of the rat flocculus corresponds to those in the rabbit (Fig. 188). F D corresponds to zone 1 of the rabbit; FE to zone 2; F D ' to zone 3 and F E ' to zone 4. F D and F D ' continue as zone D of the paraflocculus. C2 - C2 zone; caud -- caudal; d c = dorsal cap; FD, F D ' = F D ( ' ) zone (projections of vlo and PO); FE, F E ' = FE ( ' ) zone (projections of dc); F L O d and v = dorsal and ventral surface of the flocculus; M A O = medial accessory olive; PO = principal olive; rost = rostral; vlo - ventrolateral outgrowth; V P F L = ventral paraflocculus. Ruigrok et al. (1992)
cerebellar projection extends beyond the posterolateral fissure in the ventral paraflocculus (the 'ME': the medial extension of the ventral paraflocculus) (Fig. 149). In the anterograde axonal tracing studies of Tan et al. (1995b) in the rabbit the olivocerebellar projection was matched with the white matter compartmentalization in adjacent AChE-stained sections and, consequently, with the corticovestibular and corticonuclear projection of the correspooding zones (Fig. 188) (see Section 6.1.5.). The medial and central compartments 4 and 2 contained fibers from the caudal DC and the more laterally located compartments 3 and 1 conducted fibers from the rostral DC and the VLO to corresponding zones in the molecular layer. The fifth, most lateral compartment contained fibers of the rostral MAO to the Cz zone. The rostral DC and VLO 271
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Fig. 190. Distribution of C R F and CGRP-immunoreactive climbing fibers in P7 mouse cerebellum (A-C) and in neurons of the inferior olive (D-F). CRF-immunoreactive climbing fibers and neurons are indicated by dots, CGRP-immunoreactive climbing fibers and neurons by open circles, a, b and c = subnuclei a, b and c of the medial accessory olive; beta + group beta C1, C2 = Crus I and II; D A O - dorsal accessory olive; dc = dorsal cap; E G L . M L = external granular layer and molecular layer; F -- flocculus; I G L = internal granular layer; PF = paraflocculus; M A O = medial accessory olive; PO = principal olive; PL - Purkinje cell layer. Redrawn from Yamano and Tohyama (1993). (
projections extended, across the posterolateral fissure in folium P of the ventral paraflocculus, where the compartments 1 and 3 fuse around the tip of compartment 2, containing the olivocerebellar fibers of the caudal DC. The olivocerebellar projection to the flocculus of primates was not yet studied in detail. Zone 2 of the flocculus of macaques receives a projection from the caudal dorsal cap and, therefore, corresponds to zone 2 of the rabbit flocculus (Fig. 149). Recently Ruigrok et al. (1992) analysed the olivocerebellar projection to the flocculus and the adjacent paraflocculus in the rat with anterograde axonal tracing with the lectin Phaseolus vulgaris and retrograde transport of WGA-HRP (Fig. 189). Their results confirmed and extended the observations in other species, discussed in the previous paragraphs (Fig. 149). Two pairs of interdigitating zones, innervated by the caudal DC and the rostral DC/VLO, and a lateral C2 zone could be distinguished in the rat. The distal segments of these zones crossed the posterolateral fissure and were found to receive climbing fibers from more rostromedial levels of the DC (the FE/FE' zones, corresponding to zones 2 and 4 of the rabbit) and the ventral leaf of the PO (the FD'/FD zones, corresponding to zones 1 and 3). The rostral shift in the origin of climbing fibers directed to more distal (rostral) segments of a set of continuous, olivocerebellar projection zones is in accordance with Yamamoto's (1979) observations on the projection of intermediate levels of the DC and of the PO to the folium P of the ventral paraflocculus of the rabbit, and with Gerrits and Voogd's (1982) distinction of a lateral and rostral shift in the projection of successively more rostral parts of the DC and the VLO/PO to flocculus and ventral paraflocculus of the cat. Collateral projections of the olivocerebellar pathways from the DC and the VLO to the medial and superior vestibular nuclei were described by Balaban (1984), but have been denied by Groenewegen and Voogd (1977), Gerrits and Voogd (1982) and Ruigrok et al. (1991). Ruigrok et al. (1992) observed a strong projection to the parvocellular lateral cerebellar nucleus concomitantly with the climbing fiber labelling in the FD'/D zones. The anatomical and electrophysiological studies of Takeda and Maekawa (1989a and b), Maekawa et al. (1989), Kusonoki et al. (1990) and Kano et al. (1990, 1991) in the rabbit have shown that 36% of the climbing fibers innervating the flocculus and 64% of the climbing fibers in the nodule that could be driven by optokinetic stimuli, could be activated by collaterals terminating in the complementary lobule of the vestibulocerebellum. Retrograde labelling from flocculus and nodule with different fluorescent tracers resulted in double labelling of 9-27% of the DC neurons and 12-48% of the cells of the VLO. There was no distinct spatial segregation of the cells projecting to the two lobules.
273
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Fig. 191. Schematic line drawings of the unfolded opossum cerebellum modified after Larsell and Jansen (1972). The broken lines indicate the boundaries of the corticonuclear zones A-D after Klinkhachorn et al. (1984a). The distribution of the three types of enkephalinergic axons is indicated by the frequency and size of the symbols: the beaded axons by asterisks (C), the mossy fibers by dots (A), and the climbing fibers by triangles (B). I-X indicate vermal lobules; CR I, II, crura I and II, F, flocculus; LS, lobulus simplex; PFL, paraflocculus; PML, paramedian lobule. D. Distribution of enkephalinergic axons in a horizontal section through the cerebellar nuclei. D, dentate nucleus, F, fastigial nucleus; IPA, anterior interposed nucleus; IPP, posterior interposed nucleus. From King et al. (1987). (
6.3.4. The distribution of peptides and calcium binding proteins in climbing fibers and cells of the inferior olive A transient immunoreactivity in subpopulations of climbing fibers during the maturation of the cerebellum was described for somatostatin and CGRP. Somatostatin-immunoreactive climbing fibers are located in midsagittal and parasagittal bands in the vermis of lobules VI-VIII, some of which remain present in the flocculus and paraflocculus of adult rats (Vincent et al., 1985; Villar et al., 1989) (see Section 3.1.3., Fig. 20). Neuronal labelling in the inferior olive was limited to the MAO. Transient immunoreactivity for CGRP in immature climbing fibers, surrounding the somata of Purkinje cells, and in cells of the inferior olive was noticed in the early postnatal period in rats and mice (Kubota et al., 1987, 1988; Morara et al., 1989, 1992; Rosina et al., 1992; Chedotal and Sotelo, 1992; Yamano and Tohyama, 1993, 1994). These immunoreactive climbing fibers define longitudinal bands, first next to the midline, later in paravermal regions, the hemisphere and in the flocculus. Chedotal and Sotelo (1992) used the expression of CGRP immunoreactivity during prenatal development in the rat to trace olivocerebellar fibers to the cerebellar anlage in the fetal rat at El7. They identified the transient CGRP-immunoreactive climbing fibers in early postnatal stages bands as the A, B and C3 zones. Yamano and Tohyama (1994) noticed in early postnatal mice, that the bands of CGRP-immunoreactive climbing fibers alternate with CRF-immunoreactive climbing fibers (Fig. 190A-C). In postnatal mice CGRP disappears from the climbing fibers and the number of climbing fibers expressing CRF increases. Immunoreactivity for CGRP and CRF was never observed in the same neurons of the inferior olive. Both CGRP and CRF containing neurons occur in the group fl, subnucleus c of the MAO, the dorsal fold of the DAO, and in the dorsal cap (Fig. 190D-F), but the two cell populations occupy slightly different regions of these subnuclei. Their results on the localization of alternating bands of CRF and CGRP-immunoreactive climbing fibers in lobule X resemble the identification of parvalbumin-immunoreactive climbing fibers in two dorsal cap-innervated stripes in this lobule of the immature rat cerebellum by Wassef et al. (1992a,b) (see also Section 6.3.2.1.). The results of Yamano and Tohyama (1994), however, are difficult to interpret in terms of olivocerebellar projection, because their definition of the olivary subnuclei in the mouse is not precise enough. Enkephalin (ENK)-like immunoreactivity in subpopulations of climbing fibers and cells of the inferior olive has only been found in the opossum (King et al., 1986a, 1987) but not in other adult species. A more wide-spread distribution of corticotrophin releasing factor (CRF)-like immunoreactivity in certain climbing fibers has been observed in the cerebellum of the opossum (Cummings et al., 1989) and several other species including rat and mouse (Cummings et al., 1983; Van den Dungen et al., 1987), rabbit (Errico 275
Ch. I
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and Barmack, 1993), cat and sheep (Kitahama et al., 1988; Cummings et al., 1988, 1989), and Saimiri sciurus and Macaca fascicularis (Foote and Cha, 1988; Cha and Foote, 1988). The distribution of CRF, ENK and CCK in the cerebellum and the precerebellar nuclei, the species-differences and the co-localization of these peptides, recently was reviewed by King et al. (1992). Climbing fibers containing ENK are located in patches, that constitute discontinuous 276
The cerebellum." chemoarchitecture and anatomy
Ch. I
midline and parasagittal bands in the vermis of opossum cerebellum (Figs 191B and 192) (King et al., 1986a, 1987). The position of the parasagittal band in the anterior lobe approximately corresponds to the border of the A and B zones, as determined by Klinkhachorn et al. (1984a and b) from the corticonuclear projection in this species. This description also would fit a localization in the X zone, which has not yet been described in the opossum. The presence of immunoreactive somata in the lateral cell group a of the caudal MAO (Walker et al., 1988; King et al., 1989) is in accordance with such an enkephalinergic climbing fiber projection to the X-zone. ENK-immunoreactive climbing or mossy fibers have not been observed in the cerebellum of rat, cat or primates. However, opiate receptor binding is present over the molecular and to a lesser extent over the granular layer of rat cerebellum (Zajac and Meunier, 1980; Robson et al., 1984). A detailed description of the localization of CRF in climbing fibers is available for the opossum (Cummings et al., 1989). CRF is present in all lobules but the numbers of reactive climbing fibers and of CRF-like immunoreactivity in individual fibers show local differences. CRF-immunoreactive climbing fibers are concentrated in midline and parasagittal bands in the vermis (Fig. 193), the density of the CRF climbing fiber innervation is high for the vermis of the lobules VI-VII and lobule X and the flocculus. Concentrations of less reactive climbing fibers occur in the hemisphere. CRF-containing climbing fibers are concentrated in the flocculus and the adjacent part of the paraflocculus. The density of CRF-immunoreactive climbing fibers and the density of CRF receptors, determined by radioligand binding of [125I]-Tyr-ovine CRF, show a close correspondence for this region (Cummings et al., 1989). The distribution of CRF in neurons of the inferior olive of the opossum was determined with immunohistochemistry and in-situ hybridization (Cummings et al., 1989). All subdivisions of the olive contain CRF-positive neurons, but the number of labelled neurons and their staining intensity varies within and among these subnuclei. Staining levels were consistently lower in subnucleus a of the caudal MAO, when compared to the subnuclei b and c (which includes the neurons of the less immunoreactive group beta). Cells in the dorsal cap and the rostral MAO stained intensely with both methods. Rostral DAO, especially its lateral part, contains more labelled cells than medial and caudal regions of this subnucleus. Cell bodies in the PO reveal less CRF immunoreactivity, but the caudal PO contains strongly immunoreactive neurons. In the opossum CRF-like and ENK-like immunoreactivity co-exist in climbing fibers in midsagittal and parasagittal bands at the border of the lobules VII and VIII, from here they shift to a position in the base of the paramedian lobule, laterally in lobule X and ventrally in the flocculus. CRF is more widely distributed in the climbing fibers and single-labelled and double-labelled climbing fibers co-exist in these foci. CRF-like immunoreactive neurons are double-labelled for ENK in the subnuclei a and c of the caudal MAO and in the dorsal cap (Cummings and King, 1990). This pattern corresponds with the known projections of the periphery of the caudal MAO (subnuclei a, c and group beta) to the caudal vermis and of the dorsal cap to a lateral zone in lobule X and to the flocculus and the adjacent paraflocculus (see Section 6.3.3.3.). A few CCK-labelled climbing fibers are located in the same region of the caudal vermis of the opossum as the ENK/CRF (double) labelled fibers. They may originate from CCKpositive neurons in subnucleus c, where most cells are also irnmunoreactive for CRF (King and Bishop, 1990). The widespread distribution of CRF-containing climbing fibers in the cat resembles the situation in the opossum (Cummings et al., 1988; Cummings, 1989). Some of the CRF in climbing fibers is concentrated in bands. Bands are prominent in midline and 277
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 193. Camera lucida drawings of 60/lm transverse sections (A,B) through the opossum cerebellum that illustrate the distribution of corticotrophin releasing factor immunoreactive (CRF-IR) climbing and mossy fibers. Stripes in the molecular layer represent climbing fibers; the dots represent mossy fibers. The density and intensity of each symbol represent the relative number of fibers and staining intensity for respective fibers within each region. Roman numerals I-X indicate cerebellar lobules. PML, paramedian lobule~ LS, lobus simplex; CRII, crus II; PF[, para-flocculus; F, flocculus; DN, deep nuclei; x7. C. Camera lucida drawing of a 60 pm sagittal section of vermal lobule X. M, molecular layer; P, Purkinje cell layer; G, granule cell layer; pc, Purkinje cell; mf, mossy fibers; CF, climbing fibers. Note greater density of mossy fibers at the ventral medial aspects of folium. Bar in A,B = 1 ram, in C = 70 pm. Cummings et al. (1989). <.-
paramedian regions of the anterior lobe, in the intermediate part of the anterior lobe and the simple lobule, in the caudal Crus II, the paramedian lobule and the caudal vermis (Fig. 194). The distribution of CRF-containing climbing fibers in the flocculus and the adjacent ventral paraflocculus is heterogeneous, CRF-like immunoreactivity is less prominent in the rest of the paraflocculus. When we compared the immunoreactivity to the distribution of AChE in the anterior lobe, the paramedian band of CRF-containing climbing fibers appears to coincide with the lateral border of the paramedian AChE-positive band in the molecular layer, i.e. with the X-zone, and/or the lateral A-zone. The same position was found for the paramedian band of CRF-containing climbing fibers in the opossum. The two prominent bands of CRF-containing climbing fibers in the intermediate zone probably are located within the C1 and the C2 zones, the C3 zone seems to be less immunoreactive. CRF-containing cell bodies in the inferior olive of the cat occur in all subdivisions, with a columnar distribution in medial and lateral parts of the caudal MAO, CRF-positive cells in the groups beta, the dorsal cap and the ventromedial cell column, and a predominant localization in caudal and lateral D A O (Kitahama et al., 1988; Cummings, 1989). All cells of the inferior olive of the monkey express some degree of CRF-immunoreactivity (Cha and Foote, 1988), activity is highest in the caudal and central MAO. CRFcontaining climbing fibers in the cerebellar cortex are present in most of the cortex. A high and uniform distribution is present in lobule IX, dorsal VIII and in the ventral paramedian lobule (the authors identified this lobule as belonging to the paraflocculus) and as midline paramedian and mid-hemispheral bands in the A and C2 zones of the anterior lobe (Figs 195 and 196). The localization in the C2 zone would be in accordance with the situation in the cat. The zonal distribution in the ventral anterior lobe is more complicated. CRF-receptor binding, using [~2sI]-ovine C R F was higher over the molecular layer (Millan et al., 1986). In the human brain immunoreactivity was present in the great majority of the neurons of all subdivisions of the inferior olive and in climbing fibers in the molecular layer of the anterior vermis (Powers el al., 1987). Originally immunoreactivity with an antibody against C R F could not be detected in rat cerebellum (Swanson et al., 1982). C R F immunoreactive fibers in the molecular layer were observed by Olschowka et al. (1982), Cummings et al. (1983), Merchenthaler (1984), Sakanaka et al. (1987) and Van den Dungen et al. (1987, 1988). Palkovits et al. (1987) identified these fibers as climbing fibers with light and EM-immunohistochemical methods. They found CRF-immunoreactive neurons in all subdivisions of the inferior olive with a predominance in the PO. C R F m R N A was transcribed by the cells of the inferior olive and depleted at'ter a lesion of the contralateral olivocerebellar tract. C R F receptor binding, using [I~25]-ovine CRF, was highest over the granular layer of rat cerebellum (DeSouza et al., 1985). It is not possible to correlate the distribution of immunoreactive cells in the adult 279
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inferior olive with the olivocerebellar projection more precisely without double labelling experiments of olivocerebellar fibers with CRF-immunocytochemistry. It seems likely, however, that CRF-immunoreactivity is not confined to certain subsystems and that gradients in the different subdivisions of the inferior olive are responsible for the banded 281
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Fig. 196. Dark-field photomicrograph of a sagittal section through the vermis of the cerebellum of the squirrel monkey. Note that some areas of the molecular layer (M) contain dense CRF-immuno-reactive axons while others do not. However, intensely immuno-reactive axons are evident in the Purkinje cell layer (P) even in those regions in which such axons are not evident in the molecular layer. Occasional labelled axons are evident in the granular layer (G) and in the white matter (W). Scale bar = 100/lm. Cha and Foote (1988).
distribution in the cortex. Interestingly, the a m o u n t of C R F - l i k e i m m u n o r e a c t i v i t y in cells of the inferior olive increases in pontine cats ( K i t a h a m a et al., 1988). C R F messenger R N A m e a s u r e d with a [35S]-labelled oligonucleotide probe increased in neurons of the caudal dorsal cap of rabbits after long-lasting optokinetic stimulation of the contralateral eye ( B a r m a c k and Young, 1990) (Fig. 197). By itself C R F has little or no effect on n e u r o n a l activity, but it potentiates the excitatory effects of g l u t a m a t e and aspartate, the putative n e u r o t r a n s m i t t e r s of the climbing fibers on the Purkinje cells (Bishop, 1990; Bishop and K e r r 1992). K i t a h a m a et al. (1988) also raised the question whether the C R F in precerebellar nuclei is under endocrinological control, but they were unable to observe effects of a d r e n a l e c t o m y or hypophysectomy. Some calcium-binding proteins occur in subpopulations of climbing fibers. Transient labelling with antibodies against p a r v a l b u m i n was observed in neurons of the dorsal cap
Fig. 197. Optokinetically induced increase in corticotrophin releasing factor (CRF) mRNA in caudal dorsal cap of the inferior olive of the rabbit revealed by darkfield photomicrograph of an emulsion-coated brain-stem section. The rabbit received 37 hr of binocular optokinetic stimulation in the posterior to anterior direction with respect to the left eye, causing a 360% increase in levels of CRF mRNA in the right dorsal cap. A,B. Bright-field and dark-field views of the same tissue section are shown. The finer spatial resolution of the emulsion demonstrates clustering of silver grains over individual olivary neurons. Scale bar = 200 r (Barmack and Young, 1990) 282
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and in climbing fibers in the two DC-innervated zones in lobule X and in a single zone in the rat flocculus by Wassef et al. (1992a,b) in rat pups. Calretinin-immunoreactive climbing fibers were observed in the cerebellum of the chicken (Rogers, 1987) and the rat (Floris et al., 1994). The precise topography of these climbing fibers has not yet been reported. 9 ~.-.
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6.4. MOSSY FIBER SYSTEMS
6.4.1. Concentric and discontinuous, lobular arrangements of mossy fiber systems Mossy fibers take their origin from many different sites in the brain stem and the spinal cord. The topographical organization of mossy fiber systems differs considerably from the climbing fiber projection. Mossy fiber systems enter the cerebellum through the restiform body and the middle cerebellar peduncle, lateral to and along the brachium conjunctivum. The main orientation of the stem fibers of the mossy fibers is transverse. These stem fibers are known as semicircular fibers, which constitute a layer situated rostral and dorsal to the cerebellar nuclei and peripheral to the olivocerebellar fibers, which are immediately apposed to the surface of the cerebellar nuclei and their efferent tracts (Fig. 198). Medially the layer of semicircular fibers continues into the cerebellar commissure, that consists of a central part, containing the decussation of the uncinate tract and the more peripherally located decussation of the mossy fibers (Voogd, 1964). Most mossy fiber systems are partially crossed. Secondary vestibulocerebellar and ventral spinocerebellar fibers, that enter the cerebellum in a ventral and medial position, occupy a ventral and rostral position in the cerebellar commissure; pontocerebellar fibers that enter the cerebellum laterally and caudally, occupy a dorsal and caudal position in the commissure. These fibers are located in the dorsal white matter of the anterior lobe, in the medullary ray of the lobules VI and VII and in the base of lobule IX. On their way towards the commissure the pontocerebellar fibers come to the meningeal surface, in the bottom of the intercrural fissure, where cortex is absent in the center of the ansiform lobule and in the interparafloccular sulcus in the center of the parafloccular loop, where the cortex is also interrupted. Mossy fiber systems within the cerebellum display a concentric arrangement: vestibulocerebellar fibers are located most centrally, and terminate in the cortex in the bottom of the fissures; spino-, cuneo-, and reticulocerebellar fibers extend more peripherally and
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pontocerebellar fibers cover the apex of the lobules. An electrophysiological corrolary of this concentric arrangement is the observation of superficial and deep terminations of exteroceptive and proprioceptive components of the cuneocerebellar tract respectively (Ekerot and Larson, 1972). As a consequence mossy fiber projections are discontinuous in the direction across the folium: being absent at the base or apex of the folium, and reappearing in the next (Jasmin and Courville, 1987b; Tolbert et al., 1993). Another consequence is that the distribution of mossy fibers is mainly transverse and lobular, with the restriction that these systems generally populate several adjacent lobules and that the borders between different mossy fiber systems do not necessarily correspond to the bottom of the fissures. The presence of a concentric arrangement in their distribution does not preclude a large degree of overlap between different mossy fiber systems. The question of the presence of overlap in their terminations is complicated by the termination of some mossy fiber systems in alternating longitudinal strips. The origin and distribution of primary and secondary vestibulocerebellar mossy fibers has been studied repeatedly. Vestibular root fibers enter the cerebellum from the superior vestibular nucleus, as part of the ascending branch of the vestibular root (Mannen et al., 1982; Sato et al., 1989). The development of the primary vestibulocerebellar projections was studied in rat embryos, where the root fibers can be distinguished by their parvalbumin-immunoreactivity (Morris et al., 1988). The literature on the projection of the vestibular nerve was reviewed in the study of Gerrits et al. (1989) on the primary vestibulocerebellar projection in the rabbit. The evidence on the origin and distribution of secondary vestibulocerebellar projections was reviewed for the cat by Brodal (1974), Kotchaphakdi and Walberg (1978), Batini et al. (1978, lobules VI and VII), Matsushita and Okado (1981, lobules I and II), Sato et al. (1983b, flocculus), Magras and Voogd (1985) and Blanks (1990, flocculus), for the monkey by Brodal and Brodal (1985), Langer et al. (1985a, ftocculus) and Frankfurter et al. (1977, lobule VII), for the rat by Rubertone et al. (1995) and for the rabbit by Thunnissen et al. (1989), Epema et al. (1990) and Tan and Gerrits (1992). The general conclusions on the distribution of the vestibulocerebellar projection are depicted in Figs 199 and 200, taken from the papers on the rabbit from the last three authors. Both primary and secondary vestibulocerebellar fibers terminate in lobule X and ventral lobule IX, in the lobule I and II and in the cortex in the depth of the vermal fissures. This projection is mostly ipsilateral for the fibers of the vestibular root and bilateral for the secondary vestibulocerebellar projection. The secondary vestibulo-cerebellar projection to the hemisphere is restricted to the flocculus and the adjacent cortex of the ventral paraflocculus (Fig. 203). A primary vestibulocerebellar projection to the flocculus is absent in the rabbit (Gerrits et al., 1989). Vestibulocerebellar mossy fibers take their origin from neurons in all vestibular nuclei, with the exception of the Deiters' nucleus and a sparse projection from the magnocellular medial vestibular nucleus (Figs 200 and 201). The distribution of neurons projecting to either flocculus or caudal vermis or to both is rather similar and is bilaterally symmetrical. Most neurons were found in the medial, superior and descending vestibular nuclei in this order. Neurons projecting to lobules IX and X, to the flocculus and to both parts of the cerebellum occur in a ratio of 12:4:1 (Epema et al., 1990). A statistical preference was found for the superior vestibular nucleus for a projection to the contralateral flocculus (Tan and Gerrits, 1992). Widespread projections of the nucleus prepositus hypoglossi and neighbouring perihypoglossal nuclei terminate bilaterally with an ipsilateral predominance in the vermis, the flocculus and the paraflocculus and in the cerebellar nuclei (see McCrea and 285
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Fig. 199. Primary and secondary vestibulocerebellar mossy fiber projections in the rabbit, determined with antegrade axonal transport of [3H]leucine and WGA-HRP. Upper panels: sagittal sections; lower panels: transverse sections through the caudal vermis. K196: ipsilateral distribution of fibers of the vestibular nerve. Gerrits et al., (1989); C2098: bilateral distribution of fibers from the medial vestibular nucleus (MV); K82: bilateral distributions of fibers from the superior vestibular nucleus (SV; Thunnissen et al., 1989). Dense termination in the sagittal sections is indicated with heavy hatching, scattered labelled mossy fiber rosettes with light hatching and dots. Note similarity in the distribution of primary and secondary vestibulocerebellar projections. (
Baker, 1985 and Roste, 1989 for reviews). Barmack et al. (1992b) noticed a particularly strong projection of the nucleus prepositus hypoglossi to the ventral paraflocculus (folium p) in the rabbit (Figs 84 and 201). Some cerebellum-projecting neurons of the nucleus prepositus hypoglossi and the caudal parts of the vestibular nuclear complex contain acetylcholine and/or C R F (see Section 3.10.1. and 6.4.5.). Anatomical classifications of the spinocerebellar tracts are based on their level and nuclei of origin, their decussation within the spinal cord, their position in the lateral funiculus, their entrance route into the cerebellum and their lobular and zonal distribution. These different criteria are not necessarily correlated. Oscarsson (1973) in his comprehensive review of the functional organization of spinocerebellar paths, distinguished the classical dorsal and ventral spinocerebellar tracts and added a third tract, the rostral spinocerebellar tract (Oscarsson and Uddenberg 1964), that originates from the cervical cord. Moreover, Oscarsson (1973) included the cuneocerebellar tract as one of the direct spinocerebellar pathways. The dorsal spinocerebellar and cuneocerebellar tracts are ipsilaterally ascending and terminating pathways that transmit information about external events from the lower and upper extremity respectively. Both contain proprioceptive and exteroceptive components. The ventral spinocerebellar and rostral spinocerebellar tracts are hindlimb and forelimb tracts, that convey information to the cerebellum about interneurons mediating flexor reflex afferents and related motor centers in the spinal cord. These tracts terminate bilaterally in the cerebellum. A fifth direct spinocerebellar pathway arises from the central cervical nucleus (Wiksten, 1979b). Although Oscarsson's criteria were functional rather than anatomical and the great flow of new information on the origin and termination of these tracts had not yet started, his classification is still useful. The dorsal spinocerebellar tract takes its origin from Clarke's column and from a group of neurons in Rexed's (1954) dorsal laminae IV-V! (see Yaginuma and Matsushita, 1987; Matsushita and Hosoya, 1979; Matsushita et al., 1979; and Grant and Xu, 1988, and Xu and Grant, 1994, for complete references on rat and cat). It terminates, mainly ipsilaterally, in nine strips in the vermis, the pars intermedia and the extreme lateral part of the lobules III-V of the anterior lobe (Fig. 206C), bilaterally in lobule VIII of the caudal vermis and in parts of the paramedian lobule. The cuneocerebellar projection takes its origin from the external cuneate nucleus and the internal cuneate and gracile nuclei (Gordon and Seed, 1961). The exteroceptive and proprioceptive components of the cuneocerebellar tract synapse in the internal- and external cuneate nuclei respectively (Cooke et al., 1971). The differential termination of exteroceptive and proprioceptive mossy fibers in the apical and basal part of the folia is known from an electrophysiological analysis of the two components of this tract (Ekerot and Larson, 1972). These proximo-distal differences are reflected to some degree in the antegrade tracer studies with injections of the external and internal cuneate nucleus. The projection of the internal cuneate, moreover, is predominantly uncrossed 287
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Fig. 200. Origin of secondary vestibulocerebellar mossy fibers in the rabbit. A-C. Transverse sections through the vestibular nuclei. D. Dorsal view of a reconstruction of the vestibular nuclei. E-G. Transverse sections with retrogradely labelled cells, from injections of fast blue in the lobules IX and X (dots), of nuclear yellow in the flocculus (open circles) and from both injection sites (astarisks). H. Distribution of cells retrogradely labelled from the flocculus. I. Distribution of cells retrogradely labelled from nodulus and uvula, bc = brachium conjunctivum; CO = cochlear nulei; cr = restiform body; d = dorsal part of group y; dfb = direct fastgiobulbar tract; DV - descending vestibular nucleus; gVII = genu of facial nerve; IN = iinterstitial vestibular nucleus; LV = lateral vestibular nucleus; MV = medial vestibular nucleus; MVc = caudal part of medial vestibular nucleus; MVmc = magnocellular medial vestibular nuleus; MVpc = parvocellular medial vestibular nucleus; nV = spinal root of trigeminal nerve; NVpar = parabrachial cerebellar nucleus; PH = nucleus prepositus hypoglossi; S = nucleus of the solitary tract; SV = superior vestibular nucleus; tu = uncinate tract; v = ventral part of group Y; VI = nucleus of the 6th nerve; X = group X of the vestibular nuclei; Y = group y. Bar = 0,6 ram. Epema et al. (1990). (
and directed to the hemisphere (Fig. 202) (Gerrits et al., 1985a; Jasmin and Courville, 1987a,b). The cuneocerebellar tract is distributed to the anterior lobe and the lobules VI, VIII and the paramedian lobule of the posterior lobe (Grant, 1962). The distribution of cuneocerebellar fibers in the hemisphere is complementary to the distribution of the dorsal spinocerebellar tract; in the vermis both projections overlap. This overlap may be related to the predominantly mediolateral organization of somatotopical projections to the anterior vermis and the existence of rostro-caudal somatotopical gradients in the hemisphere in spino-olivo-cerebellar climbing fiber paths (Oscarsson, 1973). Somatotopy in the dorsal spinocerebellar tract was discussed by Xu and Grant (1988) and for the cuneocerebellar projection by Jasmin and Courville (1987b) and Hummelsheim et al. (1985). The ventral spinocerebellar tract is a composite pathway that contains crossed components from lower sacrococcygeal segments, from lumbar spinal border cells and from different cell groups in the lumbar intermediate zone (Matsushita and Hosoya, 1979; Matsushita et al., 1979; Grant et al., 1982; Matsushita and Ikeda, 1980; Matsushita and Yaginuma, 1989 and Yaginuma and Matsushita, 1989). The lower lumbar and sacrococcygeal component terminates preferentially in the apical part of the rostralmost lobules I and II of the anterior lobe. Spinal border cells project to a more extensive area, including the lobules I-V (Fig. 206D). The terminations of the spinal border cells are mainly ipsilateral to their origin, i.e. the fibers recross in the cerebellar commissure. They are distributed to the apical part of the lobules II-V of the anterior lobe (Yaginuma and Matsushita, 1986, Xu and Grant, 1990). The rostral spinocerebellar tract takes its origin from cell groups in Rexed's (1954) laminae VI, VII and VIII of the intermediate zone and a cell group in lamina V of the dorsal horn. The fibers from lamina VIII cross within the cord, the others ascend in the ipsilateral lateral funiculus (Petras, 1977; Petras and Cummings, 1977; Snyder et al., 1978; Matsushita et al., 1978, 1979; Wiksten and Grant, 1980, 1986). The rostral spinocerebellar tract terminates more dorsally than the dorsal and ventral spinocerebellar tract, in the lobules IV, V of the anterior lobe and in the lobules VI, VIII and the paramedian lobule. Their distribution is bilateral, but mainly ipsilateral. Spinocerebellar fibers from lower cervical segments terminate mainly in the vermis of the simple lobule (Matsushita et al., 1985; Matsushita and Ikeda, 1987). The central cervical-cerebellar projection is crossed, with a bilateral distribution in the lobules of the anterior lobe, the bottom of the primary fissure and lobule VIII (Fig. 206A) (Wiksten, 1979a,b; see Matsushita and Tanami, 1987 for a survey of the literature). The termination of the central cervical fibers overlaps with the secondary vestibu289
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locerebellar projection (Matsushita and Wang, 1987) (Fig. 206B) rather than with other spinocerebellar tracts. An important collateral projection to the cerebellar nuclei takes its origin from the central cervical nucleus (Matsushita and Yaginuma, 1995). The central cervical nucleus itself is a site for convergence of vestibulospinal and propriospinal input from neck muscles (Hirai et al., 1978, 1984; Hirai, 1987). Systematic and complete studies with antegradely transported axonal markers on the 290
The cerebellum." chemoarchitecture and anatomy
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distribution of mossy fibers from the paramedian and lateral reticular nuclei are few. A rough idea of their lobular distribution can be gained from the reviews and the papers on retrograde axonal transport of Somana and Walberg (1978), Dietrichs and Walberg (1979b), Gould (1980), Qvist (1989a) and Ruigrok and Cella (1995). The trigeminocerebellar projection recently was studied and reviewed by Ikeda and Matsushita (1992). The basal pontine nuclei, the nucleus reticularis tegmenti pontis and the adjacent paramedian pontine reticular formation are connected with the cerebellum through the middle cerebellar peduncle. Both the nucleus reticularis tegmenti pontis and the paramedian pontine reticular formation send their fibers through the midline raphe into the pes pontis (fibrae rectae), where they deflect laterally to occupy the deep stratum of the middle cerebellar peduncle. The paramedian pontine reticular formation of the cat projects bilaterally to lobule VII and the caudal part of lobule VI and to the ansiform lobule, i.e. to the visual areas of the cerebellum involved in control of saccades (Gerrits and Voogd, 1986; Yamada and Noda, 1987). Fibers of the reticular nucleus of the pons distribute bilaterally, with ipsilateral predominance, to all lobules of the cerebellum, with the exception of the lobules I and X and the dorsal paraflocculus (Kawamura and Hashikawa, 1981; Gerrits and Voogd, 1986). This projection includes the flocculus and the adjacent part of the ventral paraflocculus (Fig. 203) (Gerrits and Voogd, 1989) and collateral projections to the cerebellar nuclei (see Section 5.6.). The projection through the middle cerebellar peduncle of rostral and caudal parts of the pes pontis to the cerebellum is reversed (Von Bechterew, 1885; Spitzer and Karplus, 1907; Voogd, 1964; Voogd et al., 1990). Fibers originating in the ventral and superficial layers of the pes pontis travel in superficial layers of the middle cerebellar peduncle and terminate preferentially in caudal and ventrolateral parts of the cerebellum. Von Bechterew (1885) was able to distinguish this pathway (his 'cerebral', i.e. rostral system) because it acquires its myelin rather late. One of its main constituent is the corticopontocerebellar projection from the visual cortex to the caudal vermis and the paraflocculus. Fibers from caudal and central portions of the pes pontis occupy deeper layers of the peduncle and distribute to more rostral parts of the cerebellum. This pathway corresponds to the early myelinating 'spinal' (i.e. caudal) component of the middle cerebellar peduncle of Von Bechterew (1885). It conveys the cortico-pontocerebellar projection to the anterior lobe. Attempts to analyse the pontocerebellar projection in more detail have shown that the organization of this pathway is extremely complicated. Injections of retrograde tracers in single lobules usually result in bilateral labelling of multiple cell columns or shells. The afferent projections from the cerebral cortex and other sources showed the same degree of dispersion. Our knowledge of informationflow in different cortico-pontocerebellar subsystems, therefore, remains incomplete. The cortical and non-cortical afferents and the efferent connections of the pons were studied and reviewed by Brodal and Jansen (1946), Mower et al. (1979), Rosina and Provini (1980, 1984), R Brodal (1968a,b, 1971, 1972, 1978a,b, 1982, 1987), Gerrits and Voogd (1986, 1987, 1989), Keizer et al. (1984), Ugolini and Kuypers (1986), Gerrits and Voogd (1989), Aas (1989), Bjaalie et al. (1991), R Brodal and Bjaalie (1987, 1992) and Nikundiwe et al. (1994) for the cat; Eisenman (1980), Eisenman and Noback (1980), Azizi et al. (1981, 1985), Anderson and Flumerfelt (1984), Angaut et al. (1985), Kosinski et al. (1986), Leggen et al. (1989), Wells et al. (1989), Mihailoff et al. (1989), Mihailoff (1993), Voogd (1995) and Ruigrok and Cella (1995) for the rat; Mihailoff et al. (1980) for the opossum; R Brodal (1980, 1982), Langer et al. (1985a), Schmahmann
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Fig. 202. Cuneocerebellar projection to ipsilateral cerebellum in the cat. Left side: diagrams from antegrade axonal transport of [3H]leucine in transverse sections. Borders of compartments in adjacent Hfiggqvist-stained sections are indicated on the right. Abbreviations: A(1-3) - A(1-3)compartment; 'A' = concentration of mossy fibers in A compartment; B = B compartment; bp = brachium pontis; 'C1 + C 2 ' = concentration of mossy fibers in C1 and C2 compartments; C1-3 = C1-3 compartment; 'C3' - concentration of mossy fibers in C3 compartment; cr = restiform body; D = Dcompartment; ' D ' = concentration of mossy fibers in D compartment; F = fastigial nucleus; F L - flocculus; HVI - hemisphere of lobule VI (simple lobule); IA = anterior interposed nucleus; L = dentate nucleus; PAR = paramedian lobule; P F L D = dorsal paraflocculus; PFLV = ventral paraflocculus; X = X compartment; 'X/B' --- concentration of mossy fibers in border region of X and B compartments. Gerrits et al. (1985b). (
and Pandya (1989, 1993), Fries (1990), Glickstein et al. (1985, 1994), Stein and Glickstein (1992) and Yhielert and Thier (1993) for the monkey. 6.4.2. Zonal arrangement in the termination of mossy fibers: Correlations with cytochemical maps
During their course as semicircular fibers mossy fibers branch extensively. Some branches are distributed to the anterior and the posterior lobe. This type of branching, which was studied by Heckroth and Eisenman (1988), may explain the mirrored somatotopy which is present in the two lobes. The branches in the medullary rays are of a small calibre. In the granular layer the mossy fibers branch preferentially in a direction across the long axis of the folium (Scheibel, 1977), i.e. with the same orientation as the climbing fibers (Fig. 6). The termination of entire mossy fiber systems in the granular layer often consist of a number of parallel longitudinal arrays of mossy fiber rosettes (van Rossum, 1969). These strips are not as sharply delimited as the terminal zones of the climbing fibers in the molecular layer. Discrete strips often are visible only in the periphery of the projection field; in the center the strips coalesce into a single field. Corresponding concentrations of stem fibers are present in the cerebellar white matter. The termination of mossy fibers in longitudinal strips was first observed and illustrated for the termination of the spinocerebellar fibers in the rabbit (Voogd, 1967; Van Rossum, 1969). It was also reported for the spinocerebellar projections in Tupaia glis and the ferret (Voogd, 1969), the cat (Voogd, 1969 and the series of papers of Matsushita c.s., cited above), Trichosuris vulpecula (Watson et al., 1976), the Virginia opossum (Hazzlet et al., 1971) and the rat (Gravel and Hawkes, 1990; Tolbert et al., 1993). A zonal pattern is also characteristic for the termination of the cuneocerebellar tract in the cat (Voogd, 1969; Gerrits et al., 1985b; Jasmin and Courville, 1987a and b) and rat (Ji and Hawkes, 1994), projection of the lateral reticular nucleus both in cat (Ktinzle, 1975; Russchen, 1976) and rat (Chan-Palay et al., 1977) and in the secondary vestibulocerebellar projections to lobule IX and the anterior lobe (Epema et al., 1985; Matsushita and Wang, 1987) (Fig. 206B). Zonation has not been observed in the distribution of the primary and secondary vestibulocerebellar root fibers to lobule X and the flocculus and was only observed in some parts of the vermis for the projections from the basal pontine nuclei and the reticular nucleus of the pons in the cat and the tree shrew (Voogd, 1969; Gerrits and Voogd, 1986; Kawamura and Hashikawa, 1981). The zonal distribution of spinocerebellar and pontocerebellar fibers in Tupaia glis (Voogd, 1969) is illustrated in Figs 204 and 205, made from silver impregnated sections with large lesions of the pes pontis and low cervical cordotomies. The zonal pattern is striking in the tree shrew and the zonation in the spinocerebellar projection is generally similar to that in other species. A precise comparison of the spinocerebellar and ponto293
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Fig. 203. Mossy fiber projections to the flocculus and the adjacent paraflocculus in the cat. Based on antegrade tracing experiments with tritiated leucine. Notice the lack of basal pontine and reticulopontine projections to the flocculus and their presence in the medial extension (ME) and the caudal lobules (PFLVc) of the ventral paraflocculus in the upper three diagrams. Vestibulo-cerebellar fibers in lower two diagrams terminate both in the flocculus and the ME. A, AP - stereotactic planes; DV = descending vestibular nucleus; FL = flocculus; LV = lateral vestibular nucleus; ME = medial extension of the ventral paraflocculus; MV = medial vestibular nucleus; N P - nuclei pontis = NRTP = nucleus reticularis tegmenti pontis; P F L D - - dorsal paraflocculus; PFLV(c) = (caudal folium of the) ventral paraflocculus; SV - superior vestibular nucleus. Gerrits and Voogd (1989). (
cerebellar projections is not possible, because the experiments were done in different animals. It is not known whether they interdigitate or overlap. More detailed observations on the zonation in the spinocerebellar and trigeminocerebellar projection were reported by Matsushita c.s. for different spinocerebellar pathways in the cat. They plotted the mossy fiber rosettes on reconstructions of the surface of the individual folia. Data on the distribution of mossy fibers on the dorsal (caudal) surface of lobule IV are assembled in Fig. 206. Spinocerebellar fibers from the central cervical nucleus (Fig. 206A) (Matsushita and Tanami, 1987) and the medial vestibular nucleus (Fig. 206B) (Matsushita and Wang, 1987) terminate in three, presumably overlapping zones 1-3 in the bottom of the lobule. Zone 3 is stated to be located at the border of the zones A and B. This interpretation is supported by our map of the distribution of AChE in rostral lobule IV (Fig. 206G). Spinocerebellar fibers from the cervical enlargement are located in the same three zones, in more apical parts of the lobule (Matsushita et al., 1985; Matsushita and Ikeda, 1987). Spinocerebellar fibers from the thoracic cord distribute to the midline and to three parasagittal zones, numbered 2-4. Zone 4 is situated in the medial B zone, a number of patches are present in the lower portion of the hemisphere (Fig. 206C) (Yaginuma and Matsushita, 1987). Spinal border cells project to more apical parts of the hemisphere, with the most medial zone 1 being located at the border of the B and C1 zone (Fig. 206D) (Yaginuma and Matsushita, 1986; Matsushita and Yaginuma, 1989). Fibers from the lower lumbar cord distributed widely over vermis and hemisphere in a distinct zonal pattern (Yaginuma and Matsushita, 1989). The termination of lower lumbar, sacral and coccygeal fibers in the apical parts of the lobules is mostly restricted to a single band around two mm. from the midline, i.e. overlapping the B zone (Matsushita, 1988). Similar plots from the cuneocerebellar (Fig. 206E) and the basal pontocerebellar projection (Fig. 206F) were reproduced from Gerrits (1985). Cuneocerebellar fibers terminate in bands in the vermis and the hemisphere, the gap that separates them presumably corresponds to the B zone. Pontocerebellar fibers are mainly restricted to the apical hemisphere, i.e. the C and D zones, and are scarce in the vermis. The mossy fibers in lobule IV, therefore, belong to different pathways, that terminate in transversely oriented projection fields with a different baso-apical distribution. Vestibulo-, spino- and cuneocerebellar fields can be subdivided into rostro-caudally oriented concentrations of mossy fiber rosettes. The different fields and their concentrations of terminals partially overlap. Correlations between the terminations of mossy fibers and cytochemical maps are rare. Gerrits et al. (1985b) mapped the localization of cuneocerebellar fibers with respect to the borders of white matter compartments in adjacent, H~iggqvist-stained sections (Fig. 202). The projections of low thoracic-lumbar cord to the cerebellum of the rat were compared to the localizations of Zebrin l-immunoreactive Purkinje cells in surface maps of all relevant cerebellar lobules by Gravel and Hawkes (1990). These authors noticed 295
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Fig. 204. Diagrams of the distribution of degenerated, silver impregnated spinocerebellar and pontocerebellar fibers after lesions of the cervical cord and the pes pontis with the nucleus reticularis tegmenti pontis in sagittal (upper panels), transverse (middle panels) and horizontal sections (lower panels) through the cerebellum of Tupaia glis. Note zonal distribution in the vermis and pars intermedia and complementarity of the two projections to the cortex and to the cerebellar nuclei illustrated in middle and lower panels. ANS = antiform lobule; cr = restiform body; fl = primary fissure; FLO = flocculus; ia = anterior interposed nucleus; i p posterior interposed nucleus; L = lateral cerebellar nucleus; m = medial cerebellar nucleus ; PFL = paraflocculus; SI = simple lobule; I-X = lobules I-X. Voogd, unpublished.
a detailed correspondence between the zonal distribution of spinocerebellar fibers in the rat with that reported for other species. The P1 + Zebrin band in the anterior lobe usually overlaps with a spinocerebellar cluster, a second cluster is located under P 1-, few terminals underlie P2+ and a third concentration of mossy fiber rosettes coincides with 296
The cerebellum." chemoarchitecture and anatomy
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P 2 - (i.e. the B zone) extending into P3+ (Fig. 207). Matsushita et al. (1991), who mapped fibers from the cervical cord in Zebrin-I stained sections of rat cerebellum found less correspondence of the concentrations of rosettes with the borders of the immunoreactive Purkinje cell zones. Ji and Hawkes (1994) showed that cuneocerebellar mossy fiber terminals are located between the concentrations of lumbar spinocerebellar mossy fiber rosettes in P1 +, P1- and P2- of lobules II and III of the rat cerebellum (Fig. 207). A close correspondence between multiple patches of mossy fibers with vibrissal receptive fields and the Zebrin-negative P 1-, P2- and P3- zones of lobule IX of the rat cerebellum, was observed by Chockkan and Hawkes (1994). An organization of the spinocerebellar projection in medio-laterally oriented bands, located mainly near the junctions of the lobules I-II, II-III and III-IV (i.e. in the bottom 297
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of the fissures), separated by terminal-free areas and the presence of anterior-posteriorly aligned regions within these bands with high and lower densities of the terminals, was shown in computer reconstructions of the unfolded cerebellar cortex of the rat by Tolbert et al. (1993). It was implied by these authors that the anterior-posteriorly oriented zonation in the spinocerebellar projection was less distinct than had been suggested by previous authors. We would agree that some of the published diagrams of the zonation in the spinocerebellar projection exaggarate the sharpness of this projection. In reality the localization is more diffuse, especially in the bottom of the fissures. Parasagittal focussing in mossy fiber systems is most pronounced in the white matter, in the granular layer the mossy fibers disperse.
6.4.3. The somatotopical organization in mossy fiber pathways The somatotopical organization and the convergence in somatosensory mossy fiber paths has been investigated in great detail in the micromapping studies in the rat and other species using natural stimulation of Welker and his collaborators (see Welker, 1987 299
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of the fractured somatotopy of the mossy fiber projections in the cerebellum of the rat. Patches with similar receptive fields are indicated with abbreviations for the stimulation sites on the head and the extremities. Redrawn from Welker (1987). Cr = crown; El - eyelids; Fbp = furry buccal pad; F L = forelimb and hand; G = gingiva; H L = hindlimb; I, I] = crus I and II; Li = lower incisor; L1 -- lower lip; Lob.ant. = anterior lobe; lob.sim = lobulus simplex; N = nose; Nk = neck; P = pinna; P F L = paraflocculus; P M L = paramedian lobule; PY = pyramis; Rh = rhinarium; Ui = upper incisor; U1 = upper lip; UV = uvula.
300
The cerebellum." chemoarchitecture and anatomy
Ch. I
for a review). They observed a mosaic of small (0.1-0.3 m m 2) columnar modules or patches in the granular layer that receive short latency somatosensory projections from receptive fields located within a specific body structure (Fig. 208). Receptive fields from specific body structures often are represented in multiple patches on different folia and this distribution is not necessarily longitudinal. Topographical continuities in the periphery are not maintained in the projection to the patches in the granular layer. The arrangement of adjacent patches is asomatotopic and the mossy fiber sources of adjacent patches are disjunctive. This type of fractured somatotopy is found in parts of the hemisphere (Crus I and II, paramedian lobule) and vermis (lobules VIII and IX) in rat, opossum and cat; it is folia-specific and highly reproducible. Branching was found of axon collaterals from single cells in the spinal trigeminal nucleus between pairs of patches with the same receptive fields located on the same or on a different folium, or between patches located in vermis and hemisphere (Woolston et al., 1981). Projections from the somatosensory cortex and the superior colliculus, moreover, were found to conform to the patchy mosaic of projections of the periphery to the granular layer of the rat cerebellar hemisphere (Bower et al., 1981; Kassel, 1980). Welker's observations raise interesting questions about the sites of convergence of mossy fiber pathways mediating peripheral, cortical and tectal information, about the significance of a detailed somatotopical localization (fractured, or longitudinal) in the first link of a mossy fiber-parallel fiber pathway, which would get blurred or even lost in the transvere projections of the parallel fibers and, finally, about the relationship between fractured somatotopy and longitudinal zonation in corticonuclear and climbing fiber pathways and even in the mossy fiber systems themselves. Convergence in mossy fiber pathways may occur at precerebellar level. Convergence of tecto- and corticocerebellar pathways takes place in the pontine nuclei; a direct tectocerebellar pathway does not exist. It is not clear how much of the convergence between peripheral and cortical pathways takes place at a precerebellar level. For certain climbing fiber pathways from the cerebral cortex and the spinal cord it has been shown that this convergence occurs in the sensory relay nuclei (Andersson, 1984). The terminations of certain spinocerebellar tracts certainly overlap with the pontocerebellar projection in the apex and in the hemispheral portions of the lobules (Figs 205 and 206) and the same probably holds for the trigeminocerebellar projection (Jasmin and Courville, 1987a). Systematic convergence of peripheral and cortico-pontine input on the same glomeruli or on the same group of granule cells, therefore, is possible, but has never been studied at the necessary level of precision with anatomical methods. Electrophysiological investigations of somatotopic localization in the granular layer, be it longitudinal zonal (Ekerot and Larson, 1980) or fractured (Bower and Woolston, 1983) both adduced evidence that the same somatotopical pattern is transmitted to the overlying Purkinje cells. These authors found no evidence for lateral spread along the parallel fibers. Llinas (1982) (see also Welker, 1987) explained the preferential connection of granule cells with the Purkinje cells overlying them by the greater greater number of parallel-Purkinje cell synapses on the ascending part of the parallel fiber, but this leaves the function of the long sidebranches of the parallel fiber unexplained. The presence of multiple representations of the same body part in the granular layer is in accordance with the description of mossy fibers as mainly transversely oriented, bilaterally distributed semicircular fibers, which give off collaterals during their course. Multiple representations of the same body part are also present as microzones in different longitudinally oriented climbing fiber systems. Cerebral and peripheral input has been found to converge on to these microzones (Andersson and Eriksson, 1981; 301
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Andersson and Nyqvist, 1983) (see Section 6.3.3.1., Fig. 176). Since zonal borders were never determined in Welker's studies, it is not known whether the mossy fiber patches and the climbing fiber-microzones correspond. A crude, somatotopically arranged convergence of certain climbing and mossy fiber paths from the peripheral nerves and the somatomotor cortex to the cerebellum has been noticed by several authors (Provini et al., 1967, 1968; Ekerot and Larson, 1973, 1980). A topographical correspondence also has been noticed for mossy and climbing fibers containing the same peptides (see next Section 6.3.4.). The convergence of mossy and climbing fibers, that share the same somatotopical and neurochemical properties, on to the same regions of the cerebellar cortex, is remarkable because they reach their targets by quite different routes. This convergence may be explained by the presence of a common developmental factor that determines the distribution of these afferents over the cortex (see Section 6.2.). 6.4.4. Collateral projections of mossy fiber systems to the cerebellar nuclei. The nuclear projection of the red nucleus
Projections from mossy fiber systems to the cerebellar nuclei (see also Section 5.6.) were traced from the spinal cord, the lateral reticular nucleus and the nucleus reticularis tegmenti pontis and adjacent parts of the pontine nuclei. Projections from the vestibular nuclei are still disputed. No collateral projections to the nuclei were traced from the cuneocerebellar system. A special position is taken by the projection from the red nucleus to the interposed nucleus. Evidence for a collateral origin from mossy fibers terminating in the cerebellar cortex was provided by Qvist (1989a and b) with retrograde double-labelling from the cortex and the cerebellar nuclei and by Shinoda et al. (1992) with intraaxonal labelling for the collateral projections from the lateral reticular nucleus, the reticulo-tegmental nucleus and the pontine nuclei of the cat. Spinal projections to the cerebellar nuclei have been reported by several authors in cat (Szentagothai in Eccles et al. 1967; Voogd, 1969; Matsushita and Ikeda, 1970; Ikeda and Matsushita, 1973; Robertson et al., 1983), from the cervical enlargement and the central cervical nucleus in the rat (Matsushita and Yaginuma, 1990, 1995) and Tupaia (Fig.204). They terminate bilaterally in both interposed nuclei and in the fastigial nucleus. The projection from the central cervical nucleus in the rat terminates mainly contralaterally in the rostral fastigial nucleus and mainly in central portions of the anterior and posterior interposed nuclei, excluding the caudal fastigial nucleus, the dorsolateral protuberance, the dorsolateral hump, the lateral cerebellar nucleus and the nucleus of Deiters. The collateral projections from the lateral reticular nucleus terminate in the same regions of the cerebellar nuclei as the direct spinocerebellar projections. According to Matsushita and Ikeda (1976) they are absent from the lateral cerebellar nucleus in the cat, but according to Dietrichs (1983b) certain parts of the lateral nucleus receive lateral reticular afferents. A weak projection of the lateral reticular nucleus to the lateral vestibular nucleus that was described by Dietrichs and Walberg (1979a) in the cat, recently was confirmed in the rat (Ruigrok et al., 1995). Collateral projections from the pontine nuclei were mostly traced from the nucleus reticularis tegmenti pontis. Smaller contributions from the dorsolateral and medial pontine nuclei were found by Gerrits and Voogd (1987) in the cat and Mihailoff (1993) in the rat. Their termination is mostly in the lateral part of the posterior interposed nucleus and in the lateral cerebellar nucleus. The caudal pole of the fastigial nucleus receives a projection in cat and Tupaia (Fig.204). It appears as though the collateral 302
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projections of spinocerebellar and reticulotegmental pontine mossy fibers are found in parts of the nuclei that receive a projection of the Iobules that are strongly innervated by these fibers. This topic was illustrated and discussed by Gerrits and Voogd (1987) for the connections of the nucleus reticularis tegmenti pontis with the cerebellar nuclei and the paraflocculus of the cat. The complementarity in the projections of the spinal cord and the pontine nuclei (including the nucleus reticularis tegmenti pontis) in Tupaia is illustrated in Fig. 204. A connection from the red nucleus to the cerebellar nuclei of the cat was described with the retrograde cell degeneration method by Brodal and Gogstad (1954). This connection was confirmed with retrograde tracers in the rat (Huisman et al., 1983; Naus et al., 1985). Huisman et al. (1983) using fluorescent double-labelling techniques, showed that the rubrocerebellar fibers arise as collaterals from the rubrospinal tract. Rubrocerebellar fibers were found to terminate in the anterior interposed nucleus with axonal degeneration methods in the cat (Courville, 1968; Courville and Brodal, 1966). Walberg and Dietrichs (1986) suggested that the rubronuclear projection is either small or absent. Rubrocerebellar fibers were found to terminate in intermediate parts of the cerebellar cortex (Dietrichs and Walberg, 1983). Retrograde degeneration or labelling of rubral cells in previous studies was explained by the interruption of fibers of passage by large lesions or injections of the cerebellar nuclei. 6.4.5. The chemoarchitecture of mossy fibers It is generally assumed that the distribution of mossy fiber rosettes over the cerebellar cortex is uniform and that all mossy fibers use glutamate as a neurotransmitter (Raymond et al., 1984; Beitz et al., 1986; Clements et al., 1986, 1987; Kaneko et al., 1987, 1989). Intense immunoreactivity, using antibodies against phosphate-activated glutaminase or conjugates of glutamate was present in many neurons of the precerebellar nuclei of the rat, including the basal and reticular tegmental pontine nuclei, the vestibular ganglion, the medial and superior vestibular nuclei and the groups x, y and f, the nucleus prepositus hypoglossi, the external cuneate nucleus, the lateral reticular nucleus and the paramedian reticular formation (Beitz et al., 1986; Kaneko et al., 1989). There is strong evidence, however, for more heterogeneity because certain mossy fibers have been shown to be cholinergic and others to contain several neuroactive peptides. Serotonin has been demonstrated in certain mossy fibers with high resolution autoradiography after topical or ventricular infusion of [3H]serotonin in rodents (Chan-Palay, 1975; Beaudet and Sotelo, 1981). Authors using immunocytochemical methods for serotonin in rat, opossum and cat, however, concluded that varicose serotoninergic fibers distribute throughout the cerebellar cortex, but that few if any terminate as mossy fibers (Bishop and Ho, 1985; Bishop et al., 1985; Takeuchi et al., 1982). The presence of ChAT-immunoreactive mossy fibers in the lobules X and IX of the caudal vermis and the flocculus in several mammalian species was discussed in Section 3.10.1. ChAT-positive mossy fiber rosettes were most numerous in the caudal vermis of the rat, the rosettes were large in X and smaller in ventral lobule IX and the lobules I-III of the anterior lobe (Fig. 83). The ChAT-positive mossy fiber innervation of the flocculus was restricted to the ventral folium and the ventral half of the dorsal folium of this lobule. It was less dense than the innervation of lobule X, but a particularly dense plexus of thin, beaded fibers, which may detach from the mossy fibers, was present in this lobule. ChAT-immunoreactive mossy fibers innervating the lobules I, II, IX and X of the rabbit, were most numerous in the banks of the precentral and posterolateral 303
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fissures. The flocculus receives a sparse innervation, but a heavy concentration of ChAT-positive mossy fibers is present in the ventral paraflocculus of the rabbit. ChAT activity measurements (Fig. 84) revealed that the highest ChAT activity of any cerebellar region in rat, rabbit, cat or monkey ever measured was found for folium 2 of the ventral paraflocculus of the rabbit (Barmack et al., 1992a). The origin of these cholinergic mossy fibers was traced from the caudal medial vestibular nucleus and the nucleus prepositus hypoglossi, with double-labelling of retrogradely transported HRP from injections in the caudal vermis and the flocculus and ChAT immunohistochemistry, both in rat and rabbit (Fig. 201) (Barmack et al., 1992b). Single and double-labelled ChAT-immunoreactive neurons were primarily found in the caudal part of the medial vestibular nucleus, the nucleus prepositus hypoglossi and the vestibular efferent nucleus. They were absent from the superior nucleus. Single HRP labelled cells were present in all vestibular nuclei with the exception of the lateral vestibular nucleus. The cholinergic projection to the ventral paraflocculus of the rabbitis exclusively derived from the nucleus prepositus hypoglossi. In other brain stem nuclei only a few neurons in the lateral reticular nucleus were double labelled for HRP and ChAT following injections in the lobules X and IX in rat and rabbit. Several peptides (enkephalin-ENK, CCK, CRF, calcitonin gene-related peptide, CGRP) have been localized in mossy fibers. When they are present these mossy fibers occur ubiquitously, but they display a preferential localization in certain lobules and, for ENK-CCK and CRF, at least, in midline and parasagittal bands in vermis and paravermis. These concentrations of mossy fiber rosettes usually are aligned with the climbing fiber bands containing the same peptide (Figs 191-195). ENK-, CRF- and CCK-like immunoreactivity in mossy fibers has been reported in the opossum (King et al., 1986a,b, 1987; Cummings and King, 1990; Cummings et al., 1989; King and Bishop, 1990). With respect to their distribution the three peptides differ. Enkephalin containing mossy fibers are limited to the vermis and the the flocculonodular lobe. CRF-containing fibers are more numerous; the vermis of lobule VI and the flocculonodular lobe are densely CRF-innervated. Sagittal and parasagittal concentrations of mossy fibers containing CRF-like immunoreactivity are particularly evident in the anterior lobe and lobules VII-IX. Scattered mossy fibers are present in the hemispheres. CRF and ENK-like immuno-reactivity co-exists in some mossy fibers (and climbing fibers) in the midsagittal and parasagittal bands in the caudal vermis and in the flocculus (Cummings and King, 1990). Numerous CCK-containing mossy fibers are present in all lobules of the opossum cerebellum, with indications of a zonal distribution in the vermis of lobule III and in parts of the caudal vermis (Fig. 193). Enkephalinergic mossy fibers have also been reported in the rat, where they are universally distributed, with a preference for the vermis (Schulman et al., 1981). CRF also occurs in mossy fibers in cat and sheep (Cummings et al., 1988; Cummings, 1989), rat (Van den Dungen et al., 1988) and rabbit (Errico and Barmack, 1993). The distribution of CRF-immunoreactive mossy fibers in the cerebellum of the cat is very similar to the opossum, with concentrations of mossy fibers underlying the stained bands of immunoreactive climbing fibers in vermis and pars intermedia, and heavy labelling in the flocculonodular lobe (Cummings, 1989) (Fig. 194). The localization of CGRP-immunoreactive mossy fibers over the cerebellum of the cat differs substantially from that of the other peptides. They are present in the paraflocculus, the paramedian and ansiform lobules and in the pars intermedia of the simple lobule and the anterior lobe. In the anterior vermis they are located in the apices of the lobules. 304
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In the posterior vermis they are concentrated in the lobules VII and VIII and in the deep part of the granular layer of dorsal lobule IX. They are absent from ventral IX, X and the flocculus. Some CRF-positive mossy fibers can be labelled in monkeys (Saimiri sciureus and Macacafasciculara, Foote and Cha, 1988) but they seem to be less frequent than in other mammalian species. Some of these CRF-immunoreactive rosettes may originate as collaterals from the climbing fibers in these species. Neurons in several precerebellar nuclei which are known to give rise to mossy fibers are immunoreactive for one or more of these peptides. CRF-like immunoreactivity was located in the medial and descending vestibular nuclei (including groups f and x), the nucleus prepositus hypoglossi, the lateral reticular nucleus and other parts of the medullary reticular formation and the solitary tract nucleus in rat (Olschowka et al., 1982; Sakanaka et al., 1987; Van den Dungen et al., 1987, 1988), opossum (Cummings et al., 1989; Cummings and King, 1990) and cat (Cummings, 1989). These authors also found such cells in the external cuneate nucleus of the cat (but not in the opossum) and in the spinal trigeminal nucleus, the locus coeruleus and the raphe nuclei of both species. The profusion of CRF-immunoreactive neurons that could be retrogradely double-labelled from injections in the caudal vermis of the rabbit cerebellum was stressed by Errico and Barmack (1993). Some of these nuclei also contain CCK-like immunoreactive neurons in the opossum and ENK-like immunoreactive cells both in rat and opossum (i.e. the nucleus prepositus hypoglossi, the medial vestibular nucleus, the lateral reticular nucleus, parts of the medullary reticular formation and certain raphe nuclei (Finley et al., 1981; Williams and Dockray, 1983; King et al. 1987; Walker et al., 1988; Cummings and King, 1990). ENK- and CCK immunoreactive neurons could be double-labelled with injections of HRP in the cerebellum of the opossum (Walker et al., 1988; King and Bishop, 1990). CCK/HRP double-labelled neurons also were present in the medial vestibular and prepositus hypoglossi nucleus. Co-existence of these peptides in single neurons of these nuclei has also been demonstrated for ENK with CRF (Cummings and King, 1990) CGRP-containing neurons were double-labelled with HRP from injections in the cerebellum of the cat in the lateral reticular nucleus, the external cuneate nucleus, the descending vestibular nucleus and in the lateral and ventral divisions of the basilar pons (Bishop, 1992). The origin of a major contingent of CGRP-immunoreactive mossy fibers from the pontine nuclei may explain their preferential distribution to the cerebellar hemisphere.
7. P O S T S C R I P T
7.1. BIOCHEMICAL CORRELATES OF CELL TYPES AND FIBER SYSTEMS The chemical neuroanatomy of the cerebellum offers many examples of cell types, grisea and their connections, that can be recognized on the basis of the expression of particular biochemical markers. However, its significance extends far beyond mere recognition of what was already known. In fact, previous unrecognized or neglected cell types, such as the unipolar brush cell (Section 3.6.2.) and the glycinergic interneuron of the cerebellar nuclei (Section 5.3.), and new connections like the multilayer plexus of monoaminergic and cholinergic afferents (Sections 3.8., 3.9. and 3.10.), have been uncovered by virtue of their biochemical properties. Existing cell populations could be split. In 305
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particular, the Purkinje cells can be subdivided into multiple, biochemically distinct subpopulations, distinguished by their contents of certain neuropeptides (Sections 3.1.2. and 3.1.3.) protein kinase C subtypes (PKC~: Section 3.1.5.), receptors (GABAB receptors, muscarinic receptors, and possibly substance P and vasopressin receptors: Sections 3.7.2., 3.10.2. and 6.1.3.) or other biochemical markers (Section 3.1.8.). Several of these biochemically distinct subpopulations are located in longitudinal zones and/or are restricted to certain lobules of the cerebellum. Purkinje cells proved to be particularly rich in markers related to the inositol phosphate-second messenger pathways and the control of intracellular CaZ+-concentration (Section 3.1.4.). Some markers such as cyclic GMPdependent protein kinase, L7 and the InsP3-recptor protein have proven useful to outline the development, the somatodendritic and axonal extent and the ultrastructure of the Purkinje cells (Sections 3.1.4., 3.1.8., 6.2.). Specific markers for granule cells are less numerous. Granule cells, however, uniquely express specific types of glutamate and GABA receptor sububits (see Sections 3.3. and 3.7.). Granule cells are heterogeneous in their expression of calretinin and a zonal distribution has been reported for nitric oxide synthase (Section 3.4.) in granule cells. Among the GABAergic interneurons of the cerebellar cortex, the Golgi cells and the Lugaro cells are biochemically distinct from the basket and stellate cells, in that basket and stellate cells both react with antibodies against calmodulin (Section 3.6.1.), and contain nitric oxide synthase (Section 3.4.). Golgi cells and Lugaro cells both have been identified with type-specific antibodies (Section 3.6.2.). Sub-populations of Golgi cells were distinguished on the basis of their enkephalin- and somatostatin-like immunoreactivities, the co-localization of GABA with glycine-like immunoreactivity (Section 3.6.2.) and the presence of the metabotropic glutamate receptor subunits mGluR5 and mGluR2/3 in (different?) populations of Golgi cells (Section 3.3.2.). Biochemical markers may be helpful in future studies to characterize subtypes of Golgi cells. One factor that hinders such a correlation is our insufficient knowledge of their axonal trajectories of the Golgi cells. Clues for a morphological taxonomy of the Golgi cells are embodied in the finding of a mainly transverse orientation of Golgi cell axons in the direction of the long axis of the folium by De Zeeuw et al. (1994c) and the discovery of the candelabra cell in the cerebellar cortex as a new type of (Golgi?) cell with a transversely oriented axon, distributed to the molecular layer by Lain6 and Axelrad (1994) (Section 2). The distinction of the small, GABAergic neurons of the cerebellar nuclei that give rise to the nucleo-olivary projections, from the large, non-GABAergic relay cells (Mugnaini and Oertel, 1981) (Section 5.2.) certainly represents one of the most consistent correlations between a type of neuron with a specific projection, and its neurotransmitter. However, the dichotomy of the neurons of the cerebellar nuclei into GABAergic and non-GABAergic neurons was complicated by the discovery of small interneurons that co-localize GABA and glycine, by reports on the presence of GABAergic nucleocortical projections (Section 5.3.) and the occurrence of both excitatory aminoacid neurotransmitters as well as acetylcholine, glycine and cholecystokinin in large neurons of the cerebellar nuclei (Section 5.4.). No specific markers are available for mossy and climbing fibers. However, subsets of mossy and climbing fibers can be distinguished by their immunoreactivity towards antibodies against selected neuropeptides (Sections 6.3.4. and 6.4.4.). A subset of secondary vestibulo-cerebellar mossy fibers, taking their origin from the medial and spinal vestibular nuclei and the nucleus prepositus hypoglossi, reacts with antibodies against choline-acetyltransferase (Sections 3.10.1. and 6.4.1 .). 306
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7.2. NEUROTRANSMITTERS AND THEIR RECEPTORS One of the goals of chemical neuroanatomy is to uncover the chemical nature of signalling pathways. As in other parts of the brain the amino acids glutamate and GABA are likely to be the predominant neurotransmitters in the excitatory (mossy, parallel and climbing fiber) and inhibitory (intrinsic cortical and corticonuclear) pathways of the cerebellum respectively (Sections 3.2.1 .-3.2.3.). The characteristics of the excitatory and inhibitory responses of glutamate and GABA in part depend on the type of receptor expressed post-synaptically. Molecular biology has unraveled a great variety of ionotropic and metabotropic glutamate and GABA receptor types, and recent studies have shown that each cell type is provided with a characteristic set of glutamate and GABA receptor types (Tables 2 and 3). In particular granule cells express a large array of glutamate and GABA A receptor subunits. The functional significance of this is puzzling in view of the single type of excitotory mossy fiber and inhibitory Golgi cell input to the granule cells. Studies with transgenic mice may be useful in unravelling the specific roles of particular subunits in the cerebellar circuitry. Diffusely projecting extracerebellar afferents that contain monoamines (noradrenalin, serotonin, histamine and, possibly, dopamine, Sections 3.8. and 5.7.) and acetylcholine (Sections 3.10.1. and 3.10.2.) and their receptors have been identified. They terminate in a multilayer plexus in the cortex and in the cerebellar nuclei. It is generally assumed that these pathways play a modulatory role in the cerebellar circuitry, but their cellular targets are still largely unknown. Their functional importance was highlighted in papers on the cellular physiology of serotonin (e.g. Bishop and Kerr, 1992) and in the studies of the modulation of compensatory eye movements by adrenergic or cholinergic agonists and antagonists by Pompeiano and Collewijn and co-workers (Pompeiano et al., 1991; Tan and Collewijn, 1991, 1992a,b; Tan et al., 1991,1992,1993a,b; Van Neerven et al., 1991; Collewijn et al., 1992). 7.3. LOBULES AND ZONES The stock in trade of the classical neuroanatomist includes the subdivision of the cerebellum in the vestibulocerebellum and the largely somesthetic corpus cerebelli, the modular organization of the cerebellum and the subdivision of the cerebellar nuclei and the zonal and lobular patterns in the termination of mossy and climbing fibers. The distinction of the vestibulocerebellum as a specific subdivision of the cerebellum received support from studies of the transient biochemical properties of its primary vestibular mossy fibers (Morris et al., 1988) (Section 6.4.1.) and its climbing fiber afferents from the dorsal cap (Wassef et al., 1992a,b) (Sections 6.1.5. and 6.2.) during early stages of their development. Early observations on the high context of the vestibulocerebellum of acetylcholinesterase (ACHE) and cholinacetyltransferase (CHAT) were confirmed and extended by the preferential termination of ChAT-immunoreactive mossy fibers in the vestibulocerebellum of different mammalian species (Barmack et al., 1992a,b) (Sections 3.10.1. and 6.4.1.). Calretinin-immunoreactivity differentiates the adult vestibulocerebellum from other lobules by the strong staining of the unipolar brush cells that prevail in the vestibulo-cerebellum, and the relatively low immunoreactivity in granule cells and parallel fibers (Section 6.1.5.). The distribution of calretinin-immunoreactivity also emphasized the extension of the vestibulocerebellum, beyond the posterolateral fissure, into the ventral uvula and the paraflocculus. The compartmental organization of the white matter of the cerebellum as an expres307
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sion of the distribution of the Purkinje cells in longitudinal zones, the precise correspondence in the parasagittal arrangement of the olivocerebellar and the cortico-nuclear projections and the near perfect correlation of the anatomical zones with their electrophysiological counterparts have been known for several decades (Voogd, 1964, 1969; Oscarsson, 1969; Armstrong et al., 1973; Groenewegen and Voogd, 1977; Groenewegen et al., 1979; Voogd and Bigar6, 1980). It was concluded that the output of the cerebellum is organized as a series of modules that incorporate one or more Purkinje cell zones, their cerebellar or vestibular target nuclei, their climbing afferents and the reciprocally organized nucleo-olivary connections. Still, the discovery by Hawkes et al. (1985) (Sections 3.1.8. and 6.1.3.) in the rat, of a longitudinal zonal pattern in the distribution of Zebrinpositive and -negative Purkinje cells, with its perspective of a biochemical diversity of the modules, came as a surprise. As a pattern the zonal distribution of Zebrin was not new; it was preceded by the discovery of the 5'-nucleotidase patttern in the molecular layer of the mouse cerebellum by Scott (1964) (the identity of the two patterns was shown by Eisenman and Hawkes 1989) (see Section 6.1.3.) and of the AChE-positive and -negative zones in the molecular layer of the cerebellum of the cat (Marani and Voogd, 1977) (Sections 3.10.3. and 6.1.1 .). The great impact of the discovery of Zebrin on studies of the cerebellum was due to the unique localization of the Zebrin epitopes in the Purkinje cells, the distinctness of, and the facility in demonstrating the Zebrin pattern in different species with antibodies against Zebrin I and, later, against Zebrin II (Dor6 et al., 1990). The Zebrin pattern is positively or negatively correlated with the distribution of other Purkinje cell markers, discussed by Hawkes (1992) and Leclerc et al. (1992), and in Sections 3.1.8. and 6.1.3. of this chapter. Notably, the distributions of PKC~ (Chen and Hillman, 1993a) (Section 6.1.3.) and of nerve growth factor receptor protein (Section 3.1.10.) in the Purkinje cells conform to the Zebrin pattern. The distribution of certain substances in the Bergmann glia appears to be linked to the Zebrin pattern: this may be true for 5'-nucleotidase (Section 3.5.) and has been established for 3-fucosyl-acetyllactosamine (Bartsch and Mai, 1991) (Section 3.11.) that occurs preferentially in the Bergmann glia of the Zebrin-negative zones. Does the Zebrin pattern result from the interdigitation of two sets of Purkinje cells that differ in their biochemical properties and in their afferent and efferent connections? The truth, probably, is less simple. Purkinje cells of the A and B zones of rat cerebellum, that project to the lateral vestibular nucleus, are uniformly Zebrin-negative and are delimited by Zebrin-positive bands and satellite bands (Fig. 143). Other zones, that can be defined by their corticonuclear and olivocerebellar connections, such as the lateral extension of the A zone of Buisseret-Delmas (1988a), include both Zebrin-positive and Zebrin-negative regions (Fig. 144). Morever, uniformly Zebrin-positive lobules, like lobule VII, the nodulus, the flocculus and the paraflocculus, contain a complex zonal substructure (Sections 6.1.4., 6.1.5. and 6.3.3.3.). The recent maps of the corticonuclear and olivocerebellar connections of the cerebellum of the rat (Buisseret-Delmas and Angaut, 1993) are fairly accurate and based on the same principles as applied in the cat by Groenewegen and Voogd (1977), Groenewegen et al. (1979) and Voogd and Bigar6 (1980). The Figures 145 and 181 that compare the zonal organization of these connections with the Zebrin pattern are still largely hypothetical. There is no doubt, however, that the two patterns are correlated, but there is no simple 1:1 relationship. The borders between corticonuclear and olivocerebellar projection zones often are located within, rather than in between, the Zebrinpositive and -negative zones. The observation that the cerebellar midline divides the 308
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Zebrin-positive P l+ zone in independent left and right subzones (Hawkes and Leclerc, 1987) and the subzonation resulting from the partial overlap of different markers, discussed by Hawkes (1992) and Leclerc et al. (1992) should be taken into account for a more detailed comparison of the two patterns. Do cerebellar modules differ in other respects than in the biochemical properties of their Purkinje cells? True; they differ in the efferent connections of their target nuclei, but their internal organization seems to be very similar. Differences have been reported for the organization of the nucleocortical projections from the different nuclei (Section 5.3.) and it has been claimed that all nucleocortical projections to the C1 and C2 zones of the pars intermedia of the cat take their origin from the posterior interposed nucleus, i.e. from the target nucleus of C2 zone (Trott and Armstrong, 1990). The nucleo-olivary projections from the posterior interposed nucleus to the rostral medial accessory olive is dense (Nelson et al. 1989), and the amount of electrotonic coupling in this olivary subnucleus is the highest in the olive (Llinas and Yarom, 1981). Moreover, a recurrent excitatory loop connects the posterior interposed nucleus, via the Darkschewitsch nucleus and the medial tegmental tract, with the rostral medial acccessory olive (Ruigrok and Voogd, 1995). The C2-nucleus interpositus posterior module, therefore, may be an example of a special type of module, characterized by well-regulated mass action, tight interconnections and a relative lack of internal specialization. The C2 zone, moreover, lacks a somatotopic organization (Section 6.3.3.1.). The C2 module differs in most of these respects from the C1/C3-nucleus interpositus anterior module. Nucleocortical projections from this nucleus are rare or absent (Trott and Armstrong, 1990), the nucleoolivary projection from the anterior interposed nucleus to the dorsal accessory olive (Nelson et al., 1989; Ruigrok and Voogd, 1990) and the degree of electrotonic coupling of the neurons of the subdivision of the inferior olive are rather weak. Moreover, it lacks a recurrent nucleo-mesencephalo-olivary pathway. Both the olivo-cerebellar pathway from the dorsal accessory olive to the C~ and C3 (and d2) zones and the cortico-nuclear projection of these zones to the anterior interposed nucleus display a detailed somatotopical (microzonal) organization (Section 6.3.3.1.). The search for differences in the internal organization of the modules and their biochemical and electrophysiological correlates, therefore, should continue. 7.4. THE ROLE OF BIOCHEMICALLY DEFINED SYSTEMS IN CEREBELLAR MOTOR CONTROL Important contributions of the chemical anatomy of the cerebellum concern our understanding of its role in motor control. The perpendicular arrangement of the parallel fibers and the Purkinje cell zones and microzones is one of the fundamental structural properties of the cerebellum. This arrangement optimalizes the chance that any given mossy fiber input of the cerebellum may interact, through the parallel fibers, with the Purkinje cells or the climbing fibers of a particular zone or microzone. This 'suggests that a mossy fiber input to a restricted part of (a) zone can influence a number of microzones via the parallel fibres. If the strength of the parallel fiber-Purkinje cell transmission is modified separately in each microzone, specific combinations of microzones may be selected for each mossy fiber input. This modification would be performed by the climbing fibers system, which has been shown to exert both short-term (Ebner and Bloedel, 1984) and long-term (Ito et al., 1982; Ekerot and Kano, 1985, 1989) effects on the parallel fiber-Purkinje cell transmission. (...) The propagation of activity along the parallel fibers from (one) zone into the (next) zone would result in output units 309
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involving parts of both zones. Probably spread of activity across the borders of the sagittal zones is a general feature. This would allow far more complex synergies as, for example, combinations of distal and postural muscles' (Garwicz and Andersson, 1992, p. 621). The processes responsible for the selection of the appropriate output for a particular mossy fiber-parallel fiber input may function at the level of the synapses of the mossy fibers with granule cells, unipolar brush cells, or Golgi cells and at the level of the parallel fiber-Purkinje cell synapse. Several systems may be involved in this selection process. Nitric oxide, that is synthetized by subsets of granule cells (Schilling et al., 1994; Hawkes and Turner, 1994) and in stellate and basket cells (Bredt et al., 1990) (Section 3.4.) has been shown to be involved in the production of long-term depression (LTD) of transmission in synapses of the parallel fibers with the Purkinje cell dendritic spines (see Section 3.1.6.), but may also function at the level of the granular layer. Parallel fiber-Purkinje cell transmission also can be blocked by adenosine, acting on the A1adenosine receptors on the parallel fibers (Section 3.5.). The production of adenosine and the levels of the enzyme 5'-nucleotidase in the molecular layer that degrades adenosine monophosphate to adenosine, both are under the influence of the climbing fibers, and, in certain species, 5'-nucleotidase is distributed in bands. An important piece of information on the selection process concerns the heterogeneous distribution at the synapses between the parallel fibers and the Purkinje cell dendritic spines of the ionotropic glutamate receptors, that are blocked during LTD, and of the metabotropic glutamate receptors that elicit this reaction. Somogyi and co-workers have shown that immunoreactivity for the mGluR1 unit of the metabotropic receptor was never associated with the postsynaptic density of the synapse, but was localized at perisynaptic and extrasynaptic sites. This localization is in marked contrast with the ionotropic receptor subunits that are primarily located at the postsynaptic membrane (see Sections 3.3.1. and 3.3.2.). Induction of LTD could be inhibited by in-vitro immunoinactivation of the mGluR1 subunit (Shigemoto et al., 1994) and LTD could not be elicited in transgenic mice that lack this subunit (Aiba et al., 1994). A major drawback of many studies of the chemical neuroanatomy is that they were conducted in only one species, the rat. There is extensive evidence for species differences in the distribution of the synthetizing enzyme of acetylcholine (CHAT), muscarinic cholinergic receptors and acetylcholinesterase (see Section 3.10.), and there is reason to assume that a similar interspecies variability exists for other transmitter systems. The expression of Zebrin by certain subpopulations of Purkinje cells, and the zonal patterns in the distribution of 5'-nucleotidase, only occur in certain species. It is a fortunate coincidence for the experimental neuroscientist that the Zebrin zonal pattern is expressed in rats, but in other species like the cat or macaque monkeys all Purkinje cells are Zebrin-immunoreactive. Many species-differences in the chemical neuroanatomy of the cerebellum may be due to the selectivity of the antibodies employed in the immunocytochemical techniques, but other differences may be real and may reflect true variations in structure or in the transmission and second messenger systems of the cerebellum.
8. ACKNOWLEDGEMENTS The authors wish to express their gratitude to the many scientists and publishers who gave permission to reproduce illustrations from their publication and made the original 310
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prints available to us. Dr. Greet Schalekamp, Dr. Tom Ruigrok and Dr. Doug Hess allowed the use of some of their unpublished material. The secretarial assistance of Edith Klink, the photography of Eddie Dalm and the artwork of Karin Voogd are gratefully acknowledged. Figures 1 and 4 were redrawn from Ramon y Cajal (1911) by Philip Wilson FMAA, AIMI.
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369
The cerebellum: chemoarchitecture and anatomy J. VOOGD, D. JAARSMA AND E. MARANI
......... but the Spirits inhabiting the Cerebel perform unperceivedly and silently their Work of Nature without our Knowledge or Care. Thomas Willis. Of the Anatomy of the Brain. Englished by Samual Pordage, Esquire, London. Printed for Dring, Harper, Leigh and Martyn, 1681. Facsimile Edition, McGill University Press, Montreal, 1965. p. 111.
1. INTRODUCTION During the last 150 years the morphology of the cerebellum attracted numerous histologists. Its relatively simple structure, with its three-layered cortex and clearly defined afferent and efferent connections made it one of the favourite sites in the brain to test out new hypotheses on the connectivity, the development and chemical interaction in nervous tissue. We have attempted to review present knowledge about the external and internal morphology of the cerebellum and to relate the 'classical' topography of the cerebellum to the more recently discovered chemical specificity of its neurons and afferent and efferent pathways. Not all what is new in the histochemistry of the cerebellum is relevant to a better understanding of its chemoarchitecture. This review, therefore, does not pretend to be complete. It is focussed on afferent and intrinsic connections of the cerebellum. The efferent connections of the cerebellum to the brain stem and the spinal cord have not been systematically covered.
2. CYTOLOGY OF THE CEREBELLAR CORTEX A complete description of the histology of the cerebellar cortex was given by Ramon y Cajal (1911) (Figs 1 and 4). More recently the anatomy of the cortex including its ultrastructure was reviewed by Braitenberg and Atwood (1958), Eccles et al. (1967), Fox et al. (1967), Mugnaini (1972), and Palay and Chan-Palay (1974). Three layers are distinguished in the cortex (Fig. 3). The granular layer borders on the central white matter of the cerebellum. The Purkinje cell layer contains the cell bodies of the Purkinje cells, that are arranged in a single row. The perikarya of the Bergmann glia (the Golgi epithelial cells) are intercallated between the larger Purkinje cells (Fig. 9A). The molecular layer has a low cell content. It contains the dendritic arbors of the Purkinje cells and the Bergmann glial fibers, which run up to the pial surface where they constitute the external glial limiting membrane. The morphology of the cerebellar cortex can be characterized as a lattice: '... it can only be represented in two planes perpendicular to each other and having definite relations to the longitudinal and transversal axes of the
Handbook of Chemical Neuroanatomy, Vo112. Integrated Systems of the CNS, Part IH L.W. Swanson, A. Bj6rklund and T. H6kfelt, editors 9 1996 Elsevier Science B.V. All rights reserved.
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Fig. 1. Cerebellar cortical circuits. Top. Diagram showing the main mossy fiber-granule cell-Purkinje cell circuit and the innervation of the granule cells by the axonal plexus of the Golgi cell. A: mossy fiber; a: granule cell; B: Purkinje cell axon; b: parallel fiber; c: Golgi cell; d: Purkinje cell. Bottom. Similar diagram showing the main cortical circuit and the connection of the basket cell with the Purkinje cell somata. A: mossy fiber; a: granule cell; B: Purkinje cell axon; b: basket cell; C: climbing fiber; c: Purkinje cell soma. Redrawn from Ramon y Cajal (1911).
The cerebellum." chemoarchitecture and anatomy
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A Fig. 2. D i a g r a m s of the cerebellar circuit. Inhibitory neurons are indicated in black. A. M a i n circuit. B. Cortical interneurons and recurrent pathways. Abbreviations: B = basket cell; cf = climbing fiber; G = Golgi cell; G R = granule cell; IO = inferior olive; m f = mossy fiber; nc = nucleocortical axons; no = nucleo-olivary axons; pcc = recurrent Purkinje cell axon collaterals; P cell = Purkinje cell; P C N = precerebellar nuclei; p f - parallel fiber; pi = pinceau of basket cell axons; S = stellate cell; U B C = unipolar brush cell; 1 = extracerebellar mossy fiber; 2 - nucleo-cortical mossy fiber; 3 - mossy fiber collateral of uni-polar brush cell.
animal. The whole three dimensional structure, therefore, cannot be obtained by rotation but by translation in two directions, thus producing a lattice' (Braitenberg and Atwood, 1958, p. 1). The elements of the main cerebellar circuit were discovered by Ramon y Cajal (1888, 1911). The electrophysiological properties of the circuit were established by Eccles et al. (1967). The main circuit (Figs 1 and 2) consists of the mossy fiber afferent system, that terminates on the granule cells; the granule cell axons that ascend to the molecular layer and bifurcate into parallel fibers, that run in the long axis of the folium and terminate on the Purkinje cells and the projection of the Purkinje cells to the cerebellar or vestibular nuclei. Each Purkinje cell is innervated by a single climbing fiber (Ramon y Cajal, 1911; Eccles et al., 1966a) that takes its origin from the contralateral inferior olive. The synaptic connections of mossy fibers, parallel fibers and climbing fibers are excitatory. The Purkinje cells are inhibitory and use gamma aminobutyric acid (GABA) as a transmitter (Ito and Yoshida, 1964). Small interneurons of the cerebellar cortex (stellate, basket and Golgi cells) receive a parallel fiber input and constitute inhibitory feed back and feed forward loops terminating on the granule cells and the Purkinje cells (Figs 1, 2 and 4). The main determinant of the firing rate of Purkinje cells is the mossy fiberparallel fiber system. Excitatory coupling between climbing fibers and Purkinje cells is very strong, but the frequency of the complex spikes evoked in Purkinje cells by the climbing fiber is too low to contribute significantly to its firing rate. The function of the climbing fibers, therefore, is one of the main problems in cerebellar neurobiology. Purkinje cells project to the cerebellar or the vestibular nuclei, where their axons terminate with inhibitory synapses. The cerebellar nuclei receive their excitatory drive from collaterals of the mossy and the climbing fibers.
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Fig. 3. A. Nissl-stained section of the cerebellar cortex of the cat. G = Golgi cell; Gr = granule cells; P = Purkinje cell, asterisks: protoplasmatic islands of Held. Bar = 20 r Purkinje (1837).
B. diagram of the cerebeUar cortex of
Granule cells are small neurons located in cell nests in the granular layer. Cell-free spaces in the granular layer, that are known as the protoplasmatic islands of Held, contain the terminals of the mossy fibers (Fig. 3A, asterisks). Mossy fibers originate from many different sites in the spinal cord and the brain stem and constitute the main afferent system of the cerebellar cortex. Mossy fibers are myelinated fibers that branch extensively within the cerebellar white matter and the granular layer. They terminate with large irregular swellings (the mossy fiber rosettes, Figs 1, 5 and 6) that are located along or at the end of the axon. Each rosette forms the center of a complex synapse (cerebellar glomerulus) between the mossy fiber rosette, the dendrites of several granule cells and the terminals of one type of short axon (Golgi) cell of the cerebellar cortex. More than one mossy fiber rosette may be present within a protoplasmatic island. Granule cells possess 3-4 short dendrites, terminating in claw-like excrescenses (Fig.7). The thin, unmyelinated axon ascends towards the molecular layer, where it bifurcates in the form of a T. The two branches, that are known as the parallel fiber, pursue a straight course in the long axis of the folia, parallel to the thousands of other parallel fibers that constitute the bulk of the molecular layer. Parallel fibers synapse with dendrites of Purkinje cells and short axon cells in the molecular layer. Both the ascending portion of the granule cell axon and the parallel fiber are beaded. These varicosities probably correspond to the synaptic sites (Fig. 7D-E). Parallel fibers are very long. In monkeys their length varied between 0.8 and 5 mm. (Fox and Barnard, 1957). Maximal lengths of parallel fibers of 4.6-5.0 mm were reported for the rat (Brand et al., 1976; Schild, 1980; Mugnaini, 1983). The mean length
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Fig. 4. Semidiagrammatic parasagittal section through a folium of the mammalian cerebellum, based on data
from Golgi-stained material9 A: molecular layer; B: granular layer; C: white matter; a: Purkinje cell; b: basket cells of the lower molecular layer; d: terminal basket formation of the basket cell axon; e: superficial stellate cells; f: Golgi cell; g: granule cells with their ascending axons; h: mossy fibers; i: the bifurcation of the granule cell axons; j: epithelial glial cell; m: astrocyte of the granular layer; n: climbing fiber; o: branching point of Purkinje cell recurrent axon collaterals. Redrawn from Ramon y Cajal (1911).
of parallel fibers of 4.4 mm, measured after microinjections of biocytin in the granular layer in the rat (Pichitpornchai et al., 1994) is close to the mean length of these fibers of 5 mm, estimated with stereological techniques by Harvey and Napper (1988). The two branches of the parallel fiber are of equal length (Pichitpornchai et al., 1994). Shorter parallel fibers are located at the base of the molecular layer (mean branch length 2.08 mm), they become progressively longer as they approach the pial surface (mean branch length 2.35 mm: Pichitpornchai et al., 1994). Parallel fibers in the superficial molecular layer are of a smaller calibre than deep parallel fibers (Fox and Barnard, 1957, monkey). A similar increase in size of the parallel fibers from superficial to deep laminae of the molecular layer was noticed by Pichitpornchai et al. (1994) in the rat. They also observed proximo-distal tapering of parallel fibers. Van der Want et al. (1985a,b) observed corresponding differences in synaptic size in superficial and deep layers of the molecular layer in the cat. The size and the spacing of the varicosities along the parallal fibers was found to be correlated with their caliber. The mean interval between two varicosities was 5.2 ~tm for the parallel fibers, 4.02 ~tm for the ascending axon of the granule cell (Pichitpornchai et al., 1994). The lamination in the molecular layer may be the expression of a deep to superficial gradient in the development of the parallel fibers
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Fig. 5. Mossy fiber rosettes in the granular layer. Left. Mossy fiber rosettes from neurons of the lateral reticular nucleus, labelled with antegrade transport of Phasaeolus vulgaris lectin. Bar = 25/lm. Right: Mossy fibers, Golgi impregnation. Cajal (1911). Abbreviations: a = large, terminal rosettes; b = rosettes 'en passage'; c = small rosette 'en passage'; G = granular layer; M - molecular layer; W = white matter. Courtesy of Dr. T.J.H. Ruigrok.
(Pellegrino and Altman, 1979). A population of thick, short parallel fibers was noticed by Pitchitpornchai et al. (1994) in the deep parts of the molecular layer. Deep lying parallel fibers may be myelinated and are one of the constituents of the supraganglionic plexus located above the Purkinje cells (Mugnaini, 1972). The mossy fiber-parallel fiber-Purkinje cell pathway is characterized by a large divergence. Each mossy fiber terminates on a great number of granule cells and each granule cell contacts hundreds of Purkinje cells along its parallel fiber. An average parallel fiber with a length of 6 mm forms approximately 1100 boutons (Brand et al., 1976). A portion of the molecular layer 6 mm wide contains approximately 750 Purkinje cell dendritic sheets (Brand and Mugnaini, 1976). This number is somewhat lower than the number of available boutons, when a parallel fiber would synapse once with each Purkinje cell it meets on its way (Brand et al., 1976). It is higher than the estimate of Napper and Harvey (1988b) in the rat that 15% of the boutons on parallel fibers synapse with non-Purkinje cells and that the rest synapses once with half of the Purkinje cell dendritic sheets it meets on its way. The granule cell/Purkinje cell ratio was estimated at 274/1 by Harvey and Napper (1988) and at 350-500/1 for different lobules of rat vermis by Drfige et al. (1986). Napper and Harvey (1988) concluded that there are some 175.000 parallel fiber synapses on a single Purkinje cell of the rat. Fox et al. (1967) arrived at a number of 120.000 in monkeys. The actual strength of the convergence of individual mossy fibers to Purkinje cells depends on the distribution of their mossy fiber rosettes. Electrophysiological studies of Bower and Woolston (1983) in the rat demonstrated that Purkinje cells are most responsive to mossy fiber input that reaches the granule cells located immediately below them. Llinas (1982) explained this strong radial connectivity by the greater number of
The cerebellum." chemoarchitecture and anatomy
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Fig. 6. Drawing of horizontal section through rat cerebellum showing orientation of mossy fibers. A. Elliptical segment or stripe of mossy fiber terminals in the medial portion of the anterior lobe showing the strong caudal-rostral organization of the terminal neuropil. Note the small cluster of granule cell bodies at the open arrow. B. Single mossy fiber from the next adjacent section showing the almost linear caudal-rostral pattern of the related terminals and small groups of parallel fibers (pf). a: View of rat cerebellum from the above showing approximate position of the field illustrated (note square and arrow), b: Medial sagittal section through cerebellum showing approximate location and plane of section. Abbreviations: fp = fissura prima; Isim = lobulus simplex; crI = crus I; fsp = fissura superior posterior; fpl = fissura posterolateralis; pf - parallel fiber. Golgi modification; 21-day-old rat. Scheibel (1977).
synapses with Purkinje cells on the ascending portion of the parallel fiber. However, according to Napper and Harvey (1988) the synapses on ascending portions of parallel fibers would account for only 3% of the total number of synapses of these fibers. Pichitpornchai et al. (1994), who observed a closer spacing of varicosities on the ascending axon and the proximal branches of the parallel fibers than on their distal branches, concluded that parallel fibers will exert a graded synaptic influence on their target Purkinje cells, with the most powerful influence occurring on cells located around the proximal regions of the fibers where they bifurcate. Mossy fiber terminal branches in the granular layer are oriented longitudinally, in the same plane as the Purkinje cells (Scheibel, 1977), (Fig. 6) (see also Section 6.4.2.). Mossy fibers, therefore, preferentially activate longitudinally oriented patches of Purkinje cells. Different types of mossy fiber rosettes were described by Brodal and Drablos (1963) with the Glees and Rheumont-Lhermitte silver impregnations and the Golgi method in rat and cat. Highly branching mossy fibers terminating in small, relatively simple rosettes, located along or at the end of the fiber, occur in all parts of the cerebellum. Large rosettes, consisting of aggregations of larger and smaller argyrophilic particles, interconnected by fiber fragments occur exclusively in nodulus and adjoining uvula, lingula and flocculus. The dendritic tree of the Purkinje cell is flattened in a plane perpendicular to the long
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The cerebellum." chemoarchitecture and anatomy
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Fig. 7. Granule cells and parallel fibers after an injection of biocytin in lobule X of the cerebellum of the rat. A. Biocytin labelled granule cell. B. Golgi-impregnated granule cells and parallel fibers in transverse section. Cajal (1911). C. biocytin injection site in granular layer and labelled parallel fibers in molecular layer. D. bifurcation site of labelled parallel fibers. E. labelled varicose parallel fibers. Abbreviations: A: molecular layer; B: granular layer; C: white matter; a: granule cell axon; b: bifurcation of granule cell axon; d: Purkinje cell; f: Purkinje cell axon; g: granular layer; I: injection site; m: molecular layer. Bars in A = 12/~m, in C = 500 /~m, in D and E - 50/~m. Courtesy of Dr. T.J.H. Ruigrok. (
axis of the folia (Figs 8 and 9). The soma and the proximal dendrites of the Purkinje cell are relatively smooth, the distal dendrites (spiny branchlets) bear long-necked spines (Fox and Barnard, 1957). When the parallel fibers traverse the Purkinje cells they terminate with boutons en passage on the spines of their spiny branchlets. Climbing fibers terminate on short, stubby spines on the proximal dendrites of the Purkinje cells (Larramendi and Victor, 1967; Palay and Chan-Palay, 1974) (Figs 10, 11 and 14). The axon of the Purkinje cell is myelinated (Fig. 9) and gives rise to recurrent collaterals (Bishop, 1982, 1988; Bishop and O'Donoghue, 1986; Bishop et al., 1987; O'Donoghue and Bishop, 1990). The collaterals form a plexus of beaded axons, mainly at the level of the Purkinje cell layer (Fig. 8a and b). They terminate on neighbouring 9
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Fig. 8. Purkinje cell of rat cerebellum. Intracellular injection with lucifer yellow and staining with anti-lucifer yellow antibody. PAP method, cresyl violet counterstained. Note plexus of beaded axon collaterals in A and B and spiny branchlets in C. Courtesy of Dr. T.J.H. Ruigrok. Bars in B = 50 ~m, in C = 5/~m. Abbreviations: a = Purkinje cell axon; cp = collateral plexus.
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 9. Purkinje cell in sagittal section. H~iggqvist stain. A. The small, densely stained nuclei in the Purkinje cell layer belong to the Bergmann glial cells. B. Initial segment of Purkinje cell myelinated axon (A) surrounded by pinceau of terminal basket cell axons. Abbreviations: A = Purkinje cell axon; B = Bergmann glial fiber; D = Purkinje cell dendrite. Bar - 25 pm.
10
The cerebellum: chemoarchitecture and anatomy
Ch. I
s
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Fig. 10. Synapses on mouse Purkinje cell. Climbing fibers terminate on short, stubby spines of proximal dendrites (Ds), parallel fibers terminate on spiny branchlets (Bs). Stellate cell and basket cell axons terminate on proximal dendrites and soma. Larramendi and Victor (1967).
Purkinje cells. The recurrent collaterals extend into the molecular layer where they contact basket cells (O'Donoghue et al., 1989). The whole collateral arborization is oriented perpendicular to the long axis of the folia, i.e. in the same plane as the dendritic tree of the Purkinje cell. In the cat it measures 300-700 #m in the sagittal and 100-400 #m in the transverse direction (Bishop, 1988). The width of the arborization and its penetration in the molecular and granular layers varies for different parts of the cerebellum. Recurrent collaterals of Purkinje cell axons are constituents of the infra- and supraganglionic plexus. The main Purkinje cell axon enters and traverses the white matter to terminate on cells of the cerebellar or the vestibular nuclei. Climbing fibers (Fig. 14) innervate the Purkinje cells, each Purkinje cell receiving only one climbing fiber (Ramon y Cajal, 1911). The olivocerebellar parent fibers of the climbing fibers branch extensively in the cerebellar white matter. For the adult rat the ratio of climbing fiber innervated Purkinje cells to neurons of the inferior olive is approximately 10:1 (Schild, 1970; Delhaye-Bouchaud et al., 1985). During their development the Purkinje cells receive more than one climbing fiber, it is not known how these supernumary climbing fibers are eliminated. Branching of olivocerebellar fibers occurs 11
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J. Voogd, D. Jaarsma and E. Marani
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Fig. 11. Diagram of the interaction of Purkinje cell dendrite with a climbing fiber and several parallel fibers. A proximal Purkinje cell dendrite (pd) shows stubby thorns contacted by a climbing fiber (cf), whereas parallel fibers (pf) synapse on spines protruding from a spiny branchlet (sb). Rossi et al. (1991).
in the parasagittal plane (Armstrong et al., 1973; Brodal et al., 1980; Rosina and Provini, 1983). This is one of the reasons for the longitudinal, strip-like organization of the olivocerebellar projection (see Section 6.3.3.). Transverse branching is limited to climbing fibers terminating in certain longitudinal strips (Ekerot and Larson, 1982). Climbing fibers take their origin from the contralateral inferior olive. For a long time the origin of the climbing fibers remained obscure. Their ultrastructure and their mode of termination were first recognized by Larramendi and Victor (1967) (Figs 10 and 11) in the mouse as beaded fibers, with boutons en-passage, filled with rounded vesicles terminating on short spines on Purkinje cell proximal dendrites. The clear intervesicular axoplasm distinguishes climbing fibers from the neurofilamentous basket cell axons. Earlier Scheibel and Scheibel (1954) had reviewed Ramon y Cajal's (1888) original description of the morphology of the climbing fiber. They concluded that climbing fibers emit collaterals in the granular and molecular layer, that terminate in glomeruli, on somata of Golgi, basket and stellate cells and on neighbouring Purkinje cells. Szentagothai and Rajkovits (1959) subsequently identified climbing fibers in axonal degeneration studies from their 'Scheibel-collaterals' and concluded that the climbing fibers originate from the inferior olive. Hamori and Szentagothai (1966b) described the climbing fibers as packed with neurofilaments and making synaptic contacts with few vesicles on the smooth parts of the dendrites. They probably mistook ascending collaterals of basket cell axons for the climbing fibers. The origin of the climbing fibers from the inferior olive was finally settled by Desclin (1974), who observed their degeneration with axonal silver impregnation methods after lesioning the inferior olive of the rat with 3-acetylpyridin (3-AP) administrated intra-peritoneally. In an exhaustive analysis of the normal light- and ultrastructural morphology of the climbing fiber, Palay and Chan-Palay (1974) observed the existence of climbing fiber glomeruli and synapses with Golgi cells in the granular layer and synaptic contacts of climbing fiber tendrils with basket and stellate cells. Desclin and Colin (1980) were unable to confirm these types of collateral contacts, outside the Purkinje cells, in an 12
The cerebellum." chemoarchitecture and anatomy
Ch. I
//
Fig. 12. Purkinje cells from the cerebellum of, from left to right, birds (Gallus domesticus, Feirabend, 1983); mammals (cat, Cajal, 1911) and fish (Gnathonemus petersii, Nieuwenhuys, 1969). Note different length, orientation and position in the molecular layer of the spiny dendritic branchlets.
ultrastructural study of the cerebellar cortex of 3-AP-treated rats. O'Donoghue et al. (1989) found intracellularly stained basket cells of the cat to lack climbing or mossy fiber terminals on their somata. During postnatal maturation of the cerebellum of the mouse, Mason and Gregory (1984) found many axons that combine the morphology and synaptic connections of both climbing and mossy fibers. These combination fibers are rare in the adult. Purkinje cell dendritic trees in the molecular layer remain oriented perpendicular to the parallel fibers irrespective of the changes in direction of the folial chain. Their dendrites share this orientation with the climbing fibers terminating on them. This type of spatial organization is found in all vertebrates and is the main condition which determines the morphology of the cerebellum. Purkinje cells in fish and amphibians are not arranged in a monolayer, but can be clustered in specific parts of the cortex, reminiscent of the clustering of the Purkinje cells during early stages of cerebellar development in all vertebrates (Nieuwenhuys, 1967). Purkinje cells in lower vertebrates differ from the mammalian type by the disposition of their smooth, proximal branches and their spine-loaden terminal branches in the molecular layer (Fig. 12). In fish the proximal smooth branches are found at the same level as the somata of the Purkinje cells, and the distal spiny branchlets extend as straight spikes into the molecular layer. This condition was extensively studied by Nieuwenhuys and Nicholson in the cerebellum of mormyrid fish (Nieuwenhuys and Nicholson, 1969a,b). As a consequence the climbing fibers, that synapse with the smooth proximal part of the dendrites, do not 'climb' 13
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into dendrites within the molecular layer, but terminate at the same level as the perikarya of the Purkinje cells (Kaiserman-Abramof and Palay, 1969). In reptiles and birds the smooth, proximal dendrites with their climbing fiber terminals do not extend beyond the lower third of the molecular layer (Mugnaini, 1972; Freedman et al., 1977; Kfinzle 1985). Only in mammals the smooth branches and the climbing fiber arborizations reach the pial surface (Fig. 12). Cerebellar nuclei that contain the target cells of the Purkinje cell axons have been described in species of all vertebrate classes. In some species of fish target cells of the Purkinje cell axons are also located within the cortex among the Purkinje cells (the 'eurodendroid' cells of Nieuwenhuys et al., 1974). The cells of the 'fourth cortical layer' in some aquatic mammals, that are located below the granule cells in the white matter, can be considered as displaced cerebellar nuclear cells (Ogawa, 1934). Interneurons in the cerebellar cortex are inhibitory and constitute various feed-back and feed-forward circuits between parallel fibers, granule cells and Purkinje cells (Figs 1, 2 and 4). Their dendrites are located in the molecular layer, where they are contacted by parallel fibers. Golgi cells are most numerous in the upper part of the granular layer. Some of their dendrites ramify in the granular layer, where they are contacted by mossy fiber terminals in the glomeruli. The dendritic tree of Golgi cells is not oriented in a specific plane. Recently it was shown by De Zeeuw et al. (1994c) that axons of Golgi cells course for some distance in the supra- or infraganglionic plexus in the direction of the long axis of the folia, before they branch into a dense telodendrion in the granular layer. Their terminals are located at the periphery of the glomeruli, where they synapse with granule cell dendrites (Fox et al., 1967). Their ratio was estimated in the rat as 4-6 Golgi cells for each Purkinje cell. The number of Golgi cells is about three times higher in lobule X than in other lobules (Drfige et al., 1986). However, unipolar brush cells (see below and Section 3.6.3.) may have been mistaken for Golgi cells by these authors.
Fig. 13. Orthogonal arrangement of basket cell axons (thick horizonal fibers oriented in the plane of the Purkinje cells in A and B) and parallel fibers (thin, vertical fibers in A and B). A. Drawing from Golgiimpregnated section, Cajal (1911). B. Bodian-stained section of rat cerebellar cortex. Abbreviations: A and B = stellate cells; C = basket cell axon; E = pericellular basket; F = Purkinje cell dendritic tree; G = climbing fiber; Pb = pericellular baskets. Bar = 100/~m.
14
The cerebellum." chemoarchitecture and anatomy
Ch.I
Fig. 14. Phaseolus vulgaris lectin-labelled climbing fibers of rat cerebellum. A. Sagittal section. B. Coronal section. Abbreviations: G = granular layer; M = molecular layer; P = Zebrin- labelled Purkinje cells. Bar = 100/lm. Courtesy of Dr. T.J.H. Ruigrok.
Apart from the parallel fiber boutons on the dendrites, the soma of Golgi cells is contacted by Purkinje cell recurrent collaterals (Hamori and Szentagothai, 1966a, 1968, Palay and Chan Palay, 1974). Mossy- and climbing fiber terminals on Golgi cells, that were mentioned by several authors (Hamori and Szentagothai, 1966a, 1968, Palay and Chan Palay, 1974) have not yet been confirmed in experimental axonal tracing studies. Myelinated fibers, indicated as mossy and climbing fibers, and recurrent collaterals of Purkinje cell axons, terminate on Golgi cell somata with large, crenelated synapses ('synapse en marron': Palay and Chan-Palay, 1974). The synapse en marron recently was identified by Mugnaini and Floris (1994) as a synapse of the mossy fibers with the unipolar brush cells of the cerebellar cortex. Stellate cells are located in the entire molecular layer, basket cells constitute a special population located in its lower one third. Dendrites of stellate and basket cells are oriented in a direction perpendicular to the long axis of the folium. Axons of stellate cells terminate on Purkinje cell dendrites. The basket cell axon increases in thickness after it emerges from its soma (Figs 1 and 13). It runs, perpendicular to the long axis of the folium, above the perikarya of the Purkinje cells and gives off descending and ascending collaterals. The descending collaterals branch and surround and synapse with the somata of Purkinje cells. The axons of these pericellular baskets of the Purkinje cell terminate in a periaxonal plexus (the pinceau) surrounding the initial segment of the Purkinje cell axon. Ascending collaterals of the basket cell axon terminate on the smooth surface of the proximal dendrites of Purkinje cells. O'Donoghue et al. (1989) who studied the connections of intracellularly stained basket cells and Purkinje cells in the cat concluded that each basket cell soma received input from recurrent collaterals from a single Purkinje cell. Other afferents of the basket cell include parallel fibers, climbing fibers and stellate and basket cell axons (Palay and Chan Palay, 1974). The infra- and supraganglionic plexus, are located on either side of the layer of Purkinje cell somata. They contain myelinated Purkinje cell collaterals. Most myelinated fibers in the supraganglionic layer are oriented in the long axis of the folia and, therefore, represent myelinated granule cell axons or, possibly, axons of candelabrum cells (Lain6 and Axelrad, 1994) or Golgi cells (De Zeeuw et al. 1994c). In silver impregnations these axons are distinctly smaller than the basket cell axons, that cross them at right angles 15
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J. Voogd, D. Jaarsma and E. Marani
(Fig. 13). The so-called multi-layer fibers also contribute to the plexus surrounding the Purkinje cells. These fibers were traced from several sources, including the noradrenergic, serotoninergic and cholinergic cell groups of the brain stem. They ramify in all layers of the cortex, but constitute the densest plexus at the level of the Purkinje cells. They are discussed more fully in Sections 3.8., 3.9. and 3.10.1. Several other neuronal cell types have been identified in the cerebellar cortex. The Lugaro cell is a relatively rare fusiform neuron, located just below the Purkinje cell layer (Lugaro, 1894; Fox, 1959; Palay and Chan-Palay, 1974). Its dendrites stretch out along the boundary of the granular and the Purkinje cell layer, the destination of its axon is not known. Lugaro cells can be discriminated from Golgi cells immunocytochemically with specific antibodies (Section 3.6.2., Fig. 67). The candelabrum cell has been recognized in Golgi-impregnated sections from rat cerebellum by Lain6 and Axelrad (1994). The neuron is rather frequently encountered in all lobules of the cerebellum. Its medium sized perikaryon is sqeezed in between the somata of Purkinje cells. Its dendritic tree is somewhat flattened and mainly extends in the parasagittal plane. One or two dendrites course through the molecular layer, dividing into few, slightly oblique branches, that are covered with irregularly distributed spines. A few slender dendrites branch in the upper granular layer. The axon courses in the direction of the long axis of the folium in the Purkinje ceil - or the supraganglionic layer, and gives off terminal, beaded branches that ascend in the molecular layer at regular parasagittal intervals. The chemical anatomy of the candelabrum cell has not yet been studied (Section 3.6.2). The unipolar brush cells were first identified in the rat by Altman and Bayer (1977) as the 'pale cells' of the granular layer. These cells are intermediate in size between the granule and the Golgi cells, and possess a typical, pale nucleus. They are concentrated in the nodulus, the ventral uvula, the flocculus and parts of the paraflocculus. They are born after the Purkinje cells, but before the stellate, basket and granule cells. The cells were sporadically recognized as monodendritic neurons in a number of immunocytochemical studies (see Section 3.6.3), but have been characterized with Golgi impregnation and electron microscopic methods only recently (Floris et al., 1994; Mugnaini and Floris, 1994; Mugnaini et al., 1994). The name 'unipolar brush cell' was given by Mugnaini and colleagues (Mugnaini and Floris, 1994) after the tip of the stubby dendrite, that forms a tightly packed group of branchlets resembling a paint brush (Fig. 68). The soma of unipolar brush cells is spherical to oval and carries thin appendages. The axon only can be impregnated for a short distance, suggesting that its distal, unimpregnated part is myelinated. Side branches of the axon terminate in rosette-like formations in the granular layer (Fig. 2) (Berthi6 and Axelrad, 1994; Floris et al., 1994; Rossi et al., 1995), the main stem of the axon may enter the white matter. Unipolar brush cells are innervated by one or two mossy fiber rosettes, in the form of particularly extensive contacts. Mossy fibers end on the perikaryon as well as on the dendritic brush (Mugnaini et al., 1994). These large synapses correspond to the 'synapse en marron' of Palay and Chan-Palay (1974), originally identified as a mossy fiber-Golgi cell synapse (see also Monteiro et al., 1986). Unipolar brush cells also receive symmetrical synapses from boutons containing pleomorphic vesicles, presumably originating from Golgi cells or Purkinje cell recurrent axons. Some of the dendritic branchlets may be presynaptic to dendrites of other cells in the granular layer (Floris et al., 1994). Pale cells, monodendritic and unipolar brush cells are all more frequent in the vestibulocerebellum. The chemical identity of the unipolar brush cell will be discussed in Section 3.6.3.
16
The cerebellum." chemoarchitecture and anatomy
Ch. I
3. CHEMICAL ANATOMY OF THE CEREBELLAR CORTEX By virtue of its laminated and relatively simple structure the cerebellar cortex has served as the playground for every student who wanted to test a histochemical reaction or a new antibody on the brain. From this large body of data we have selected those which are important for the understanding of the morphology and the connections of the cerebellum. The localization in the cerebellar cortex of neurotransmitters, peptides, second-messenger systems, calcium-binding proteins and other biochemical markers is reviewed separately for each cell type of the cortex and for the mossy and climbing fibers. Glutamate and GABA receptors, nitric oxide, adenosine, the monoamine afferent systems and receptors, the hypothalamo cerebellar and histaminergic afferents and the cholinergic systems and acetylcholinesterase are discussed in separate sections. The chemoarchitecture of the cerebellar cortex has been reviewed by Schulman (1983), Nieuwenhuys (1985) and Oertel (1993). 3.1. PURKINJE CELLS
3.1.1. Gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD) and the GABA-transporters in Purkinje cells Purkinje cells use gamma-aminobutyric acid (GABA) as their main neurotransmitter and exert a postsynaptic inhibitory effect on cells of the cerebellar and vestibular nuclei (Ito and Yoshida, 1964; Obata et al., 1967; Obata, 1969, 1976; Obata and Takeda, 1969). GABA in rabbit Purkinje cells was first demonstrated using a histochemical method, demonstrating the conversion of GABA into succinic acid (Van Gelder, 1965). In selective uptake studies of [3H]GABA in cerebellar slices, only a low activity was present over the Purkinje cells (H6kfelt and Ljungdahl, 1970, 1971; Schon and Iversen, 1972). Minimal uptake of [3H]GABA was also observed for Purkinje cell axon terminals (Storm-Mathisen, 1975). All Purkinje cell somata of the cerebellum of the rat and their primary and secondary dendrites were immunoreactive for antisera against glutamic acid decarboxylase (GAD), the synthesizing enzyme of GABA (Fig. 62D,E). Varicose fibers and terminals in the cerebellar nuclei were densely stained (Saito et al., 1974; McLaughlin et al., 1974; Oertel et al., 1981b; Perez de la Mora et al., 1981; Somogyi et al., 1985). Immunoreactivity in Purkinje cell somata was generally found to be weak, or to be dependent on blocking of axonal transport by colchicine (Ribak et al., 1978). Strong immunoreactivity in Purkinje cell somata was, however, reported by Mugnaini and Oertel (1985) with an anti-GAD antiserum produced by Oertel et al. (1981 a). The presence of GAD mRNA in Purkinje cells has been demonstrated with in situ hybridization histochemistry in rodents and primates resulting in dense labelling over somata of Purkinje cells (Wuenschell et al., 1986; Julien et al., 1987; Ferraguti et al., 1990; Herrero et al., 1993). Two forms of GAD with apparent molecular weights in the range of 59-67 kDa, that differ by 2-4 kDa, were identified by Chang and Gottlieb (1988) and Martin et al. (1991). In situ hybridization histochemistry with probes for the high molecular weight form, GAD67, and the low molecular weight form, GAD65, showed a prevalent localization of GAD67 over GAD65 in Purkinje cell bodies of rat cerebellum. The reverse localization was reported for Golgi cells (Esclapez et al., 1993; Feldblum et al., 1993). A differential distribution of GAD67 and GAD65 in Purkinje cells was found in immunocytochemical studies with specific antibodies for GAD67 and GAD65. Antibody K2, which is specifc 17
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for GAD67 , strongly immunoreacted with Purkinje cell perikarya, their proximal dendrites and their axon terminals in rat cerebellum (Kaufman et al., 1991; Moffett et al., 1994). The monoclonal antibody GAD-6, which is specific for GAD65 (Chang and 18
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 15. Localization of GABA-like immunoreactivity in semithin sagittal sections of rat (A,B) and chick (C,D) cerebellum. A. Low magnification of cell bodies and neuronal processes reacting with monoclonal anti-GABA antibodies. B. Higher magnification of an area partially included in the frame in A. Note strong immunoreactivity in stellate cell bodies (open arrow), in Golgi neurons (thick arrow), in the basket terminals surrounding Purkinje cell bodies, in puncta at glomeruli (dotted line) and in axons in the white matter. Immunoreactivity in Purkinje cells is weak. C and D. Two typical patterns of GABA-like immunoreactivity observed in Vibratome slices of the chick cerebellum. C: Intensely (thick arrow) and weakly (open arrow) immunoreactive Purkinje cells together with the staining in their dendritic arborization (thick arrowhead). D. Basket terminals around two weakly stained Purkinje cells (open arrowheads). Molecular layer (MO); Purkinje cell layer (P); granular cell layer (GL); white matter (WM). Bar in A = 100 r bar in B, C and D = 25 r (Matute and Streit, (1986). (
Gottlieb, 1988), i m m u n o r e a c t e d with axon terminals of Purkinje cells, but p o o r l y imm u n o s t a i n e d the p e r i k a r y a of Purkinje cells ( K a u f m a n et al., 1991). Antibodies against conjugates of G A B A were first applied to d e m o n s t r a t e specific G A B A - l i k e i m m u n o r e a c t i v i t y in Purkinje cells by S t o r m - M a t h i s e n et al. (1983). Imm u n o r e a c t i v i t y of the cell b o d y and the dendrites with antibodies against conjugates of G A B A was generally f o u n d to be weak or absent, but strong in the a x o n and the myelinated axon collaterals in the infraganglionic, but especially in the supra-ganglionic plexus, and in their terminals in rat (Ottersen and S t o r m - M a t h i s e n , 1984a,b, 1987; Ottersen et al., 1987; M a d s e n et al., 1985; Sdgudla et al., 1985; G a b b o t t et al., 1986; M a t u t e and Streit, 1986; S o m o g y i et al., 1986; A o k i et al., 1986) cat (Somogyi et al., 1985) and m o u s e ( T a k a y a m a , 1994). Staining in Purkinje cell s o m a t a in the chicken was stronger t h a n in m a m m a l s ( M a t u t e and Streit, 1986) (Figs 15, 62, 63). Several G A B A t r a n s p o r t e r proteins, that are active in the high-affinity u p t a k e of GABA
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Fig. 16. Schematic diagram to illustrate the concept of dynamic interrelationships between taurine, motilin, and gamma-aminobutyric acid (GABA) in a single neuron. A neuron with both substances in coexistence may have fluctuating levels of one or both substances depending upon parameters of rhythm, time, and physiologcal demands for one or another mediator during specific types or phases of activity. Chan-Palay (1984). 19
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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The cerebellum." chemoarchitecture and anatomy
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Fig. 17. Immunoreactive staining with taurine (Tau2) antibody in rat cerebellum fixed with 4% paraformaldehyde. A. Tau2-immunoreactive staining on a coronal section though lobule 6 of the vermis. Purkinje cells and dendrites exhibiting Tau-LI were separated by bands of unstained Purkinje cells. M, molecular layer; P, Purkinje cell layer; G, granule cell layer; W, white matter; P", Purkinje cell layer out of plane focus. Location, bregma = 11,5 mm. B. High-power photomicrograph of area indicated in A demonstrating taurine-like immunoreactivity in Purkinje cells (short solid arrows) and dendrites (long solid arrows) separated by unstained Purkinje cells (open arrows). C. Tau2-immunoreactive staining in a horizontal section through lobule 3 of the vermis. D. Adjacent section to (C) demonstrating that taurine-like immunoreactivity was completely absorbed by incubation of Tau2 (1 : 40) with original antigen, taurine conjugated to KLH using glutaraldehydeborohydride. Bars in A, C, and I) = 100/.tm, in B = 50 r Magnusson et al. (1988).
GABA, have been cloned (GAT1-4: Guastella et al., 1990; Lopez-Corcuera et al., 1992; Borden et al., 1992; Liu et al., 1993; and GAT-B: Clark et al., 1992). All transporters occur in brain tissue. The regional distribution of GAT1 was studied by Rattray and Priestly (1993) with in situ hybridization in rat cerebellum. GAT1 mRNA is not expressed by Purkinje cells, but strongly by Bergmann glial cells. GAT-2 may be confined to glia (Liu et al., 1993), but detailed studies of their localization have not been published thus far. 3.1.2. Motilin and taurine in Purkinje cells
Certain inconsistencies in the results on the localization of GABA in Purkinje cells were discussed by Chan-Palay (1984). She concluded that GABA is present in varying amounts in different Purkinje cells and that it may co-exist with other neuroactive substances, notably with motilin and taurine, that also produce an inhibitory action on postsynaptic cells (Fig. 16). The presence of motilin in Purkinje cells was demonstrated with an antibody directed against conjugates of motilin (Chan-Palay et al., 1981; Nilaver et al., 1982). More than half of the Purkinje cells of the rat are immunoreactive for this antibody and in human cerebellum their proportion was even higher (Nilaver et al., 1982). Chan-Palay et al. (1981) found coexistence of GAD and motilin in 10-20% of the Purkinje cells of the rat. The presence of motilin in Purkinje cells has, however, been disputed by Lange (1986), who was unable to demonstrate the presence of motilin using radioimmuno-assay and reversed phase HPLC in extracts of rat cerebellum. Only one of Lange's anti-motilin antibodies, all of which had been demonstrated to be effective in demonstrating motilin-like immunoreactivity in rat duodenum, was found to immunoreact with Purkinje cells in immunocytochemical studies with rat cerebellum. Taurine has been proposed as a neurotransmitter in certain fiber systems. In the guinea pig cerebellum it was found to exert a hyperpolarising effect on Purkinje cell dendrites and was proposed as a neurotransmitter in stellate cell-Purkinje cell synapses (Okamoto et al., 1983). [3H]Taurine was found to accumulate in Purkinje cells. Immunocytochemical studies with antibodies specific for cysteine-sulfonic acid decarboxylase (CSADCase), the enzyme involved in taurine synthesis, by Chan-Palay et al. (1982a,b), showed that CSADCase immunoreactivity was present in most, but not all the Purkinje cells of rat cerebellum, and was more prominent in the main dendritic arbor than in the perikarya and the axon. CSADCase, motilin and GAD-like immunoreactivities were found to co-exist in Purkinje cells located near the midline. In contrast to the observations of Chan-Palay et al. (1982a,b), Almarghini et al. (1991) found CSADCase immunoreactivity to be localized in Bergmann glia and interfascicular oligodendrocytes and to be absent from Purkinje and stellate cells. Most authors who used antisera against conjugates of taurine to localize taurine-like 21
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immunoreactivity found staining in all Purkinje cells of the cerebellum of the rat (Madsen et al., 1985; Campistron et al., 1986b; Tomida and Kimura, 1987; Ida et al., 1987; Ottersen et al., 1988b; Ottersen, 1988, 1989). Magnusson et al. (1988) recognized a zonal distribution of taurine-immunoreactivity similar to the zonal labelling of CSADCase immunoreactivity observed by Chan-Palay et al. (1982a,b) (Fig. 130) in paraformaldehyde-fixed brain tissue (Fig. 17), but not in glutaraldehyde-fixed tissue. Analysis of semithin sections and immunogold electron microscopy indicated that taurine-immunoreactivity is selectively enriched in the somata, proximal and distal dendrites and axon terminals of the Purkinje cells (Fig. 18). Stellate and basket cell somata and their axon terminals are only weakly immunolabelled (Ottersen et al., 1988b; Ottersen 1988, 22
The cerebellum." chemoarchitecture and anatomy
Ch.I
Fig. 18. Photomicrographs showing the distribution of taurine-like immunoreactivity in the rat cerebellum, and the results of different control experiments. A,B. Semithin (0.5 r sagittal sections through vermis posterior treated with taurine (Tau) antiserum 20 diluted 1:3000 and subsequently processed according to the peroxidase-antiperoxidase procedure. A. Intense labelling of the somata, dendrites (small arrowheads), and axons (crossed arrow) of the Purkinje cells. The neurons (large arrowheads) and glial processes (small arrows) of the molecular layer appear immunonegative. Small asterisks indicate pial surface, large asterisk indicates Purkinje cell enlarged in B. B. Note staining of Purkinje cell dendritic spines (large arrow heads). Inset: Semithin test section mounted on the same slide as the tissue section shown in A and B and incubated in the same drops of sera. The test section contains brain protein-glutaraldehyde conjugates of different amino acids, separated by brain tissue that appears as darkly stained zones. Code: 1, GABA; 2, glutamate; 3, taurine; 4, glycine; 5, none (i.e., no amino acid in the reaction mixture); 6, aspartate; 7, glutamine (only small part of section represented in this particular view). The taurine conjugate is selectively stained. C. Transverse semithin section through nucleus interpositus anterior showing intense staining of axons (arrows) and nerve terminal-like dots, some of which (large arrowheads) appear to contact unstained cell bodies (asterisks). Same procedure as in A. D. Thin layer chromatograms (5 mm width) of soluble brain extracts fixed with glutaraldehyde and subsequently stained with an antiserum against glutamate (left strip) or taurine (right strip). The taurine antiserum reveals a single spot which has comigrated with free taurine and which is separate from the spot labelled by the glutamate antiserum. E, T and G indicate the application sites of the brain extract, taurine and glutamate, respectively. E. Adjacent section to that shown in A and accompanying test section (inset) incubated with the taurine antiserum after preabsorption with glutaraldehyde-taurine complexes (200/tM with respect to taurine). There is virtually no staining. Abbreviations: MO, molecular layer; GC, granule cell layer. Bars 25/~m. Ottersen (1988). (
1989). Following hypo-osmotic stress there is a transient shift of taurine from the Purkinje cells to the B e r g m a n n glial c o m p a r t m e n t , where taurine-immunoreactivity now becomes a p p a r e n t (Nagelhus et al., 1993).
3.1.3. Calcitonin gene-related peptide (CGRP), acetylcholinesterase (ACHE), somatostatin and tyrosine hydroxylase in Purkinje cells Some neuroactive substances have been reported to be present in Purkinje cells only for a certain period during development. Calcitonin gene-related peptide ( C G R P ) is almost undetectable in adult rat cerebellum, but a transient immunoreactivity in i m m a t u r e Purkinje cells in rat cerebellum has been detected with antisera against C G R P ( K u b o t o et al., 1987, 1988; Chedotal and Sotelo, 1992). In adult rats, however, C G R P - l i k e immunoreactivity, co-localized with G A D immunoreactivity, can be detected in m a n y Purkinje cells near injections of colchicine (Kawai et al., 1985, 1987). C G R P receptors measured autoradiographically with [125I]CGRP as the ligand, are a b u n d a n t in adult rat and h u m a n cerebellum. [125I]CGRP-binding is dense over the molecular and Purkinje cell layers and low over the granular layer and the cerebellar nuclei (Inagaki et al., 1986). Binding to the molecular layer occurs in a pattern of longitudinal stripes (Kruger et al., 1988) and increases after intraperitoneal administration of harmalin (Rosina et al., 1990, 1992). Choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) have been found to be transiently expressed in Purkinje cells during development: Purkinje cells in certain parts of the i m m a t u r e rat and guinea pig cerebellum, including the lobules IX and X of the caudal vermis, display a transient reactivity for A C h E (Csillik et al., 1963, 1964; A l t m a n and Das, 1970; Odutola, 1970; Brown et al., 1986). The authors suggested that this transient A C h E activity in Purkinje cells is due to a transient cholinoceptive stage, when they are contacted by cholinergic mossy fiber afferents. A similar, transient expression of C h A T was observed in Purkinje cells of the rat vestibulocerebellum (Gould and Butcher, 1987). Pseudo-cholinesterase was localized in adult Purkinje cells of the 23
Ch. I
J. Voogd, D. Jaarsma and E. Marani
lobules IX and X (Robertson et al., 1991). These cells are arranged in multiple, sagittal bands (Gorenstein et al., 1987). Robertson et al. (1991) were unable to confirm the transient staining with AChE in rat Purkinje cells. Somatostatin was located in rat Purkinje cells using polyclonal and monoclonal antibodies against conjugates of somatostatin (Johansson et al., 1984; Vincent et al., 1985; Villar et al., 1989). Reactive Purkinje cells were especially numerous in parts of the vermis and paraflocculus and flocculus during early postnatal stages, but mostly disappeared later on (Figs 19 and 20). In part of the vermis they were located in bands. In the adult rat Purkinje cells can be stained on the ventral aspect of the paraflocculus (Gonzalez et al., 1988) and in the vermis, after interventricular administration of colchicine (Villar et al., 1989). Somatostatin-like immunoreactivity was also observed in climbing fibers, that were correlated with the patches of immunoreactive Purkinje cells and, more diffusely distributed, in Golgi cells (Villar et al., 1989). The presence of somatostatin in adult rat Purkinje cells of the paraflocculus was confirmed with nonradioactive in situ hybridization for somatostatin mRNA (Kiyama and Emson, 1990). Specific binding of iodinated agonists of somatostatin was studied in rat, using ligands for short, 14 amino-acid ([125I]SS-14) and long forms ([125I]SS-28). Binding in the cerebellar cortex was found to be low, but strong binding of both ligands was observed over the cerebellar nuclei (Uhl et al., 1985). Binding to somatostatin receptors in the human cerebellar cortex was higher. Different distribution patterns were noted among the patients studied, with higher densities over the granular layer (Laquerriere et al., 1994). Leroux et al. (1985) and Gonzalez et al. (1988) failed to demonstrate specific binding over the cerebellar nuclei of the rat of a different SS-14 ligand, but confirmed binding of SS-28 (Leroux et :al., 1985). Binding of an octopeptide somatostatin analogue was reported to be almost absent in rat cerebellum (Reubi and Maurer, 1985) and low over the cerebellar cortex of the human cerebellum, with intermediate values in the molecular layer (Reubi et al., 1986). Tyrosine hydroxylase, the synthesizing enzyme of dopamine, is expressed by Purkinje cells of the ventral vermis (lobules I and X) and the hemisphere (ansiform lobule, paraflocculus) of rat cerebellum (Takada et al., 1993). Expression of tyrosine hydroxylase by Purkinje cells is increased in the mutant tottering and leaner mice (Austin et al., 1992). 3.1.4. The localization of the IP3 receptor and the intracellular calcium stores of Purkinje cells
The phosphoinositide system is a second messenger system coupled to metabotropic, G protein-linked receptors (see Ross et al. (1990), Mayer and Miller (1990), Ferris and Snyder (1992) and Berridge (1993), for reviews). Receptor-mediated hydrolysis of phosphatidylinositol (PIP2) is catalyzed by phospholipase C and leads to the formation of inositol-l,4,5-triphosphate (IP3) and diacylglycerol (DAG), two second messengers that function in a bifurcating signal pathway. Other inositol phosphates (inositol 1,3,4,5tetrakiphosphate, IP4; inositol 1,3,4,5,5-pentakiphosphate, IPs; and inositol hexakiphosphate, IP6) have been localized in rat cerebellum (Vallejo et al., 1987; Theibert et al., 1987, 1991). Phosphorylation of IP 3 by the enzyme IP 3 3-kinase leads to the formation of IP4. IP3, through activation of IP3 receptors, causes Ca 2+ mobilization from intracellular sources, whereas DAG, together with Ca 2+, activates the enzyme protein kinase C that phosphorylates regulatory proteins. The localization of phospholipase C, IP 3 receptors and protein kinase C has been extensively studied in Purkinje cells. 24
The cerebellum: chemoarchitecture and anatomy
Ch. I
B
C
f
Fig. 19. Schematic illustration of the zonal distribution of somatostatin immunoreactive Purkinje cells at
different levels of the cerebellum of a 21 day old rat. Drawings have been made from frontal, cresyl-violet stained sections. Each dot represents 2-5 cells. Abbreviations: 5-9, cerebellar lobules V-IX; 4V, 4th ventricle; COP, copula pyramis; CR2, crus 2, ansiform lobule; FL, flocculus; PFL, paraflocculus; PM, primary fissure; SF, secondary fissure. Villar et al. (1989).
IP 3 3-kinase, the enzyme that produces IP 4 from IP3, was exclusively localized in Purkinje cells of the rat using immunohistochemistry (Mailleux et al., 1991 a, Mizuguchi et al., 1992) and in situ hybridization in rat and human cerebellum (Mailleux et al., 1991 b, 1992). Immunoreactivity was present in Purkinje cell dendrites more than in the perikarya. Intense immunolabelling of the dendritic spines was observed in the rat (Yamada et al., 1992; Go et al., 1993) (Fig. 21) but a specific role of IP 4 in Purkinje cell dendritic spines has not been disclosed. A similar localization in Purkinje cell dendritic spines was described for the mGluR1 subunit of the metabotropic glutamate receptor (Section 3.3.2., Fig. 52). Different isoenzymes of the phospholipase C (PLC) family, belonging to three major groups (fl, ~ and d), have been identified (Rhee et al., 1989; Rhee and Choi, 1992). PLC-fll, PLC-y and PLC-~ have been localized with in situ hybridization in the brain of the rat. Moderate activity was found for PLC-fll in the granular layer and strong activity in Purkinje cells and granule cells for PLCT'. The activity of PLC-d is low and may be localized in glial cells (Choi et al., 1989). PLC-A m R N A that was localized in rat Purkinje cells by Ross et al. (1989b), probably codes for a thiol-protein disulphide oxido-reductase and not for a PLC (Berridge, 1993). The IP 3 receptor has been found to be identical to the Purkinje cell-specific P400 25
Ch. I
26
-
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J. Voogd, D. Jaarsma and E. Marani
The cerebellum." chemoarchitecture and anatomy
Ch. I
protein (Mignery et al., 1989) (Fig. 22). The P400 protein was originally isolated by Mallet et al. (1976) as a Purkinje cell-specific protein, that was reduced in homozygous Purkinje cell-deficient (pcd, Mullen et al., 1976) and 'staggerer' (Sidman et al. 1962) mice, but relatively enriched in the cerebella of 'reeler' and 'weaver' mutant mice, with a loss of granule cells (Mikoshiba et al., 1979). Immunocytochemical studies with a monoclonal antibody specific for P400 protein, indicated that the protein was localized in somata, dendrites and axons of Purkinje cells in rodents (Maeda et al., 1988; Nakanishi et al., 1991; Rodrigo et al., 1993). The development of Purkinje cells could be traced with P400-immunostaining of staged cerebella of mouse embryos (Maeda et al., 1989) (Fig. 24). At the ultrastructural level it was identified on the plasma-membranes and the endoplasmatic reticulum, including the subsurface cisterns (Maeda et al., 1989). Notably Purkinje cells of'staggerer' mice, that are defective in synaptic contacts of parallel fibers and lack dendritic spines, do not express P400-immunoreactivity, whereas P400-immunoreactivity was found at 'normal' levels in ectopic Purkinje cells of 'reeler' cerebellum (Mariani et al., 1977; Mikoshiba et al., 1980; Maeda et al., 1989) (Fig. 23), and in the few remaining Purkinje cells of 'pcd' mutant mice. Cloning of the P400 protein cDNA revealed that it was identical to the IP 3 receptor protein, as well as the Purkinje cell-specific PCPP-260 protein isolated by Walaas et al.
Fig. 21. IP3-3-kinase immunoreactivity in the rat cerebellum. Electron micrograph showing intense immunoreactivity in the dendritic spines of Purkinje cells. Bar 2 r Yamada et al. (1992).
27
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 22. Localization of inositol 1,4,5-triphosphate receptor with PCD6 antibody in frozen sections of rat cerebellum by immunofluorescence. Sagittal section of the cerebellar cortex. Small arrows in the granule cell layer (GL) point to segments of immunoreactive axons which represent recurrent collaterals of Purkinje cell axons. Mignery et al. (1989).
(1986) and the PDC6 protein of Nordquist et al. (1988). The localization of P400 (= IP3 receptor) mRNA in Purkinje cells was confirmed by in situ hybridization (Furuichi et al., 1989) (Fig. 25). The IP3 receptor was purified from rat cerebellum as a protein with a molecular weight of 260 kDa (Supattapone et al., 1988). The primary structure of the mouse IP3 receptor protein, and its partial homology to the skeletal muscle ryanodine receptor were elucidated by Mignery et al. (1990). The IP3 receptor is composed of four identical subunits of a molecular weight of 320 kDa, and forms a calcium-permeable channel (Maeda et al., 1991). Three additional cDNAs encoding for the IP3 receptor, 28
The cerebellum." chemoarchitecture and anatomy
Ch. I
named IP~R-II, III and IV, were identified by Sfidhof et al. (1991) and Ross et al. (1992), but were not found to be expressed at significant levels by Purkinje cells. The presence of the IP~ receptor in Purkinje cells was confirmed immunocytochemically. In immunocytochemical studies with gold-conjugates, that allow precise ultrastructural localization of the immunoreactivity, it was shown that gold particles were located on membranes of the endoplasmatic reticulum in somata, dendrites, dendritic spines and axons of the Purkinje cells (Mignery et al., 1989; Ross et al., 1989a; Sharp et al., 1993a,b) (Fig. 26). Immunolabelling predominated in the smooth-surfaced endoplasmatic reticulum, including the subsurface cisterns, but was also found on portions of the perinuclear and rough endoplasmatic reticulum, and on the cis-cisternae, but not the intermediate and trans-cisternae, of the Golgi apparatus. IP~-receptor immunoreactivity was also observed in a subpopulation of spherical or elongated, membrane-bound structures, named calciosomes (Volpe et al., 1989), that are present throughout the cytoplasm of the Purkinje cells (Volpe and Villa, 1991; Nori et al., 1993). Strong immunoreactivity for the IP~ receptor was found on stacks of flattened cisternae of the endoplasmatic reticulum (Otsu et al., 1990; Satoh et al., 1990; Takei et al., 1992, 1994). The labelling on the cisternal stacks was mostly located in the spaces between the cisternae and between the cisternae and the plasmalemma or mitochondria (Satoh et al., 1990; Takei et al., 1992, 1994). It should be noted that the amount of cisternal stacks in Purkinje cells may depend on the conditions of perfusion fixation. The presence of cisternal stacks in healthy Purkinje cells, therefore, has been disputed (Takei et al., 1994).
9
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Fig. 23. Section of reeler m u t a n t m o u s e cerebellum stained with m o n o c l o n a l a n t i b o d y 4C11 against the P400 protein. N o t e stained Purkinje cells in the cortex (CX) and in the central mass o f dislocated cells (DP). Bar = 200/~m. M a e d a et al. (1989).
29
Ch. I
J. Voogd, D. Jaarsma and E. Marani
9 ~~
-~A~
~
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., t.
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Fig. 24. Sagittal sections of mouse cerebella of various ages stained with a monoclonal antibody (4C 11) against the P400 protein.The samples were from (A) postnatal day O (PO), (B) P3, (C) P5, (D) P7, (E) P10, (F) P15, and (G) P20 cerebellum. A section from a P20 old mouse cerebral cortex did not react with same antibody (H). Magnification 100• Maeda et al. (1989).
30
The cerebellum." chemoarchitecture and anatomy
Ch. I
Nevertheless the formation of stacks of cisternae of the endoplasmatic reticulum could be induced by overexpression of IP3 receptors in fibroblasts, which indicates that cisternal stacks may exist as special organelles related to the IP3 receptor (Takei et al., 1994). The localization of the IP3 receptor has been compared to the localization of other luminal or membrane components of the endoplasmatic reticulum related to Ca 2+ homeostasis. The membrane pump CaZ+-ATPase, immunolabelled with antibodies against cardiac CaZ+-ATPase, was found to be located in regular cisternae of the endoplasmatic reticulum, the lateral tips of cisternae of the Golgi complex and in calciosomes of Purkinje cells (Kaprielian, 1989; Michelangeli et al., 1991; Villa et al., 1991; Takei et al., 1992) (Fig. 27). Distal axons of Purkinje cells, however, lacked CaZ+-ATPase immunoreactivity (Takei et al., 1992). Appreciable levels of calsequestrin, the main intraluminal calcium-binding protein of muscle, were present in Purkinje cells of the chicken. Calsequestrin-immunoreactivity was present over the lumen (Villa et al., 1991) and membranes (Takei et al., 1992) of stacked and isolated cisternae of the endoplasmatic reticulum and in a subpopulation of calciosomes (Volpe et al., 1988; Volpe and Villa, 1991). Mammalian Purkinje cells do not have calsequestrin but, instead, express calretic-
d
b
9
~..
ML PL
]cL " ;7-'.-11
Fig. 25. Localization of the P400-specific mRNA by in situ hybridization, a. Autoradiograph of a sagittal section of mouse cerebellum, b. Higher magnification of a. ML, molecular layer; PL, Purkinje cell layer; GL, granular layer. Furuichi et al. (1989).
31
Ch. I
~.
J. Voogd, D. Jaarsma and E. Marani
:.5.. ,~ -.
Fig. 26. Electron-microscope immunocytochemical localization of InsP3 receptor in Purkinje cells of rat cerebellum using pre-embedding avidin-biotin labelling and InsP3 receptor antiserum. A, B. Nuclear membrane and some, but not all, portions of endoplasmic reticulum (ER) are labelled. C. Higher magnification of the dendritic pole of a labelled Purkinje cell. Note the unlabelled ER very close to labelled ER. D. Labelled portions of endoplasmic reticulum (L-ER) immediately subjacent to an unlabelled presynaptic terminal (U-T). Cell membrane indicated with triangles in (D) and (E). E. InsP3 receptor antiserum. Labelled portions of ER near plasma membrane, but not directly subjacent to presynaptic terminal. F. Preimmune serum. No specific label is present, even though the section is very close to the surface of the vibratome section. Abbreviations: L-ER, labelled endoplasmic reticulum; L-G, labelled Golgi apparatus; L-NM, labelled nuclear membrane; U-ER, unlabelled endoplasmic reticulum; U-G, unlabeled Golgi apparatus; U-M, unlabelled mitochondrion; U-NM, unlabelled nuclear membrane. Scale bars for all panels 1 r (Ross et al., 1989a).
ulin (Treves et al., 1990). Calreticulin-immunoreactvity was located in stacks of rough and smooth endoplasmatic reticulum in rat Purkinje cells (Nori et al., 1993). Calsequestrin and calreticulin are not exclusively present in Purkinje cells, but also in other cell types of the cerebellar cortex. 3.1.5. Protein kinase C in Purkinje cells
Protein kinase C (PKC) plays an important role in the control of several cellular processes, such as the short-term modulation of membrane excitability and transmitter release, positive or negative interaction with the conductance through various ion channels and the regulation of gene expression and cell proliferation (Shearman et al., 1989, 1991; Farago and Nishizuka, 1990; Nishizuka et al., 1991). PKC, that through phosphorylates multiple target protein including neurotransmitter receptors, and has been implicated in long-term depression (LTD) of glutamate sensitivity of Purkinje cells (Cr~pel and Krupa, 1988). Breakdown of PIP2 by phospholipase C (see Section 3.1.4) in Purkinje 32
The cerebellum." chemoarchitecture and anatomy
Ch. I
a
b
C
Fig. 27. Immunofluorescence localization of the cerebellar Ca2+-ATPase in a transverse cryosection of adult chicken cerebellum. CaS/CI-IgG localizes the Ca2+-ATPase to the Purkinje cell bodies in the Purkinje layer (b), and the dendritic trees in the molecular layer (a). Very faint immunofluorescence was detected in the granule cell layer (c). Bar 50/~m. Kaprielian et al. (1989).
cells can activate PKC through the production of DAG and the mobilization of C a 2+ from the endoplasmatic reticulum. Alternative routes for the production of DAG and the mobilization of C a 2+ from extracellular sources are available (Nishizuka et al., 1991). Three isoenzymes of PKC have been distinguished on the basis of the analysis of the sequence homology of complementary DNA clones from different sources. The PKCtypes I, II and III of Huang et al. (1987a,b) are the products of the 7', fl and ~ genes respectively (Ono et al., 1987; Nishizuka, 1988). The PKC fl isoenzyme occurs in two forms, flI and flII, generated through alternative splicing (Ono et al., 1987; Nishizuka, 1988; Saito et al., 1989; Shimohama et al., 1990; Farago and Nishizuka, 1990). PKC ~, fl and 7' are calcium-dependent forms. In addition, calcium-independent isoenzymes of PKC have been identified: ~, e, e' and ( (Ono et al., 1988). Non-specific antibodies against PKC were found to strongly immunostain Purkinje cell perikarya, dendrites and axons (Mochly-Rosen et al., 1987; Kitano et al., 1987; Saito et al., 1988). Immunocytochemical studies with subtype specific antibodies and in situ hybridisation histochemistry have shown that several PKC subtypes are located in Purkinje cells (Figs 28 and 29, Table 1) (Brandt et al., 1987; Huang et al., 1987a,b, 1988, 1991; Ase et al., 1988; Hashimoto et al., 1988; Hidaka et al., 1988; Kose et al., 1988; Shimohama et al., 1990; Wetsel et al., 1992; Chen and Hillman, 1993a; Garcia et al., 1993; Merchenthaler et al., 1993). PKC~' immunoreactivity occurs at high levels in both the somatodendritic and axonal domains of Purkinje cells, and is absent from other cell types of the cerebellar cortex. Immunoreactivity for PKC ~ is also present in Purkinje 33
Ch. I
J. Voogd, D. Jaarsma and E. Marani
cells. PKC d-immunoreactive Purkinje cells are distributed in immunopositive and immunonegative columns (Fig. 133) (Chen and Hillman, 1993a). According to Wetsel et al. (1992) Purkinje cells were stained with antisera against PKC e, but Chen and Hillman's (1993a) found Purkinje cells to be unlabelled for PKC e. PKC/6 and e' were not located in Purkinje cells (Table 1). 3.1.6. cGMP; cGMP-dependent protein kinase and nitric oxide synthase in Purkinje cells Purkinje cells are the only cerebellar cell type containing cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGK) (Walter et al., 1981; Walter, 1984; Lohmann et al., 1981; De Camilli et al., 1984; Wassef and Sotelo, 1984). cGK-immunoreactivity is present throughout the entire Purkinje cell, including its dendrites and its axon (Fig. 30). During development Purkinje cells display a heterogeneity in their expression of immunoreactivity for cGK (Wassef and Sotelo, 1984, rat; Levitt et al., 1984, monkey) (see Section 6.2.). A 23 kD protein, which is likely to be a substrate of cGK was found to be concentrated in Purkinje cells (Walter, 1984; Nairn and Greengard, 1983). Immunoreactivity for guanylate cyclase, the synthesizing enzyme of cGMP, was
Fig. 28. Developmental expression of protein kinase C (PKC) isoenzymes in rat cerebellum. Immunofluorescent staining of cerebellar cortex by antibodies specific for PKC 1, corresponding to PKC~" (panels A, B and C), PKCfl (panels D, E and F) and PKC~ (panels G, H and I). Sagittal sections of cerebellum of 1-week-old (A, D and G), 2-week-old (B, E and H) and 3-week-old (C, F and I) rats were used. PKCz- antibody stained mainly the Purkinje cell bodies and dendrites throughout the development. PKCfl antibody stained the cerebellar granule cells in the external germinal layer (EGL) of the 1- and 2-week-old rats and mainly the granular layer of the 3-week-old rats. PKC~ antibody stained both granule cells and Purkinje cells throughout the development. Huang et al. (1991).
34
The cerebellum." chemoarchitecture and anatomy
Ch. I
....
.,
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Fig. 29. Immunostaining for different isozymes of PKC in the rat cerebellum. PKC0~ (A) is present in Purkinje cells (P). The dendrites of these cells can be followed as far as the top of the molecular layer (m). The granular layer (g) and the white matter (w) are not stained. To avoid crowding, the abbreviations for layers of the cerebellum are indicated only in (A); however, the layer of Purkinje cells (P) is indicated in each figure for orientation. PKCfl (B) and PKCflI I (C) are present only in cells of the granular layer. PKCg is present in Purkinje cells and Bergmann glial cells in the molecular layer (D). Not only the perikarya but also the dendrites of Purkinje cells in the molecular layer and their axons in the granular layer are immunopositive. The antiserum for PKC~ stained Purkinje cells (E) and presently unidentified cells below the unstained Purkinje cells (F). The dorsally located folia contain mainly unstained Purkinje cells. Their axonal origin is surrounded by immunopositive cells. In the basal folia, the Purkinje cells are immunostained. PKCe is present in Purkinje cell (G), whereas PKCe' is present in cells in the molecular and granular layers and in the nerve fibers surrounding the unstained Purkinje cells (H). Antiserum against PKC~" stained only Purkinje cells in the cerebellum (I). Bar 100 •m. Wetsel et al. (1992).
35
Ch. I
J. Voogd, D. Jaarsma and E. Marani
localized in Purkinje cells, but does also occur in other cell types of the cerebellar cortex (Zwiller et al., 1981; Ariano et al., 1982; Nakane et al., 1983; Poegge and Luppa, 1988). cGMP was, however, found to be absent from rat Purkinje cells, using antibodies against conjugates of cGMP in combination with sodium nitroprusside-stimulation of cGMP synthesis. Prominent cGMP-immunoreactivity within the molecular layer was detected in Bergmann glial cells (Fig. 56) (Berkelmans et al., 1989; De Vente et al., 1989, 1990). Soluble guanylate cyclase is activated by nitric oxide (NO) (see Section 3.4). NO has been implicated in the generation of long term depression (LTD) of parallel fibermediated EPSP's in Purkinje cells. LTD can be prevented by the application of haemoglobin that absorbs NO, or by the inhibition of NO synthesis (CrGpel and Jaillard, 1990; Shibuki and Okada, 1991; Ito, 1991). However, nitric oxide synthase, the synthesizing enzyme of NO, appears to be absent from the Purkinje cell (Section 3.4.).
3.1.7. Calcium-binding proteins in Purkinje cells Calbindin-D28K, parvalbumin and calmodulin are cytosolic, calcium-binding proteins of the EF-hand family (see Baimbridge et al., 1992 and Andressen et al., 1993 for reviews), that are present in high amounts in Purkinje cells. Calretinin is a calciumbinding protein closely related to calbindin-D28K, that is absent from the Purkinje cells, but present in other neurons and in afferent mossy and climbing fibers of the cerebellar cortex (Rogers, 1989; Arai et al., 1991; RGsibois and Rogers, 1992; Floris et al., 1994). One of the calcium-binding proteins, the 28 kDa vitamin-D-dependent calcium-binding protein (calbindin-D28K), occurs in most, if not all, Purkinje cells in rat and chicken cerebellum (Lawson, 1981; Roth et al., 1981; Jande et al., 1981a,b; Baimbridge and Miller, 1982; Legrand et al., 1983; Schneeberger et al., 1985; Kosaka et al., 1993; Amenta et al., 1994). Its presence in soma, dendrites and axon was demonstrated with polyclonal and monoclonal antibodies raised against calbindin-D28K (Fig. 31A). Its exclusive presence in the cerebellum in Purkinje cells was confirmed with in situ hybridization with cDNA probes in rat and mouse (Nordquist et al., 1988; Iacopino et al., 1990; Abe et al., 1992a; Kadowaki et al., 1993). According to Garcia-Seguera et al. (1984) only 74% of the rat Purkinje cells was immunoreactive for a polyclonal antibody raised against chick duodenal calbindin-28K. This antibody also stained Golgi cells in the granular layer in rat and human cerebellum (Fournet et al., 1986). Developmental gradients in the expression of immunoreactivity for calbindin-28K by Purkinje cells were studied by Legrand et al. (1983) and Wassef et al. (1985) (see Section 6.2.). TABLE
1. Immunoreactivities o f P K C in cerebellar neurons
Isoenzymes
P u r k i n j e cells
Basket &
G r a n u l e cells
Cerebellar nuclei
+
++
+
s t e l l a t e cells Alpha
++
Beta
-
+
++
+
Gamma
+++
-
-
-
Delta
+++
+++
-
-
Epsilon
-
+
++
++
Zeta
++
+
++
++
Chen and Hillman (1993a)
36
The cerebellum." chemoarchitecture and anatomy
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P 21
Fig. 30. A. Frontal section through the cerebellum and attached brainstem of an adult rat. All the Purkinje cells are stained by cyclic 3',5'-guanosine monophosphate-dependent protein kinase (cGK) antiserum, including their dendrites in the molecular layer and their axon terminals in the deep nuclei and in the brainstem (arrow). Bar = 1 mm. B. Higher magnification of the neurons indicated by an arrow head in A. Like a few other isolated labelled cells found in variable locations, these cells are considered as ectopic Purkinje cells. Bar = 50/lm. C. cGK immunoreactive neuron in the cerebellum of 1 day-old rat. This ectopic Purkinje cell is located in the white matter and its appearance mimics that of 1-day-old Purkinje cells as visualized in Golgi impregnated material. Bar = 25 ~m. Wassef and Sotelo (1984).
Calmodulin-immunoreactivity was observed both in Purkinje cells and in cells of the cerebellar nuclei of the rat (Lin et al., 1980; Means and Dedman, 1980; Seto-Oshima et al., 1983, 1984). During postnatal development calmodulin-immunoreactivity was transiently present in the inner part of the external germinative layer and in fibers in the white matter of P3-P11 rat pups (Seto-Oshima et al., 1984). Polyclonal antibodies against parvalbumin stain all Purkinje cells and stellate and basket cells in the molecular layer of rat and avian cerebellum (Figs 31B and 32) (Celio and Heizmann, 1981; Heizmann, 1984; Braun et al., 1986; Endo et al., 1985; Schneeberger et al., 1985; Seto-Oshima et al., 1983; Rogers, 1989; Kosaka et al., 1993). The localization of parvalbumin in Purkinje, stellate and basket cells was confirmed in the rat with non-radioactive in situ hybridization (Kadowaki et al., 1993). Parvalbumin 37
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. .
Fig. 31. A. Calbindin-D28k immunoreactivity. B. Parvalbumin-immunoreactivity in rat cerebellar cortex. Purkinje cells react with both antibodies; arrows in B indicate parvalbumin- immunoreactive stellate and basket cells. Bar - 50/zm. Courtesy of Dr. M.P.A. Schalekamp.
supposedly occurs in GABAergic neurons (Celio and Heizmann, 1981) and/or neurons with characteristically high firing rates (Karmy et al., 1991). Karmy et al. (1991) studied the co-localization of parvalbumin and cytochrome oxidase, as an indicator of metabolic activity, in many regions of the brain. They found only weak immunoreactivity with antibodies against cytochrome oxidase in parvalbumin immunoreactive Purkinje cells of the rat. A developmentally regulated polypeptide (PEP-19), that is a presumptive neuronspecific calcium binding protein, was identified in adult and neonatal rat cerebellum and its amino acid sequence was determined (Ziai et al., 1986). PEP-19-like immunoreactivity is expressed by Purkinje cells and by the cartwheel cells of the dorsal cochlear nucleus of the mouse (Mugnaini et al., 1987). Berrebi et al. (1991) drew attention to the expression of PEP-19, CaBP and other Purkinje cell markers (cerebellin, L7: see below) by bipolar cells and other neurons of the retina.
3.1.8. Other specific biochemical markers for Purkinje cells Several polypeptides, that are present in all Purkinje cells, but not in other cells of the cerebellum, have been mentioned in the previous sections of this chapter. They include the IP3 receptor (identical to the P400 protein and to the PCPP-260 protein of Walaas et al., 1986) (see Section 3.1.4), IP3-3-kinase (Section 3.1.4), cGMP-dependent protein kinase (Section 3.1.5), PEP-19 and calbindin-D28K (Section 3.1.7). Two other 38
The cerebellum." chemoarchitecture and anatomy
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proteins, cerebellin and L-7 that occur in all Purkinje cells, are dealt with in this section. Other proteins only occur in certain subpopulations of Purkinje cells. Zebrin I and II (Hawkes et al., 1985) are the prototypes of this group. The restriction of the Zebrins to a subpopulation of Purkinje cells is the more remarkable because they are originally present in all Purkinje cells of rat neonates (Leclerc et al., 1988). The developmental histories of cGMP-dependent protein kinase, calbindin-D28K and L-7 are quite different, in that these proteins make their first appearance in subpopulations of fetal Purkinje cells and only in later stages become expressed by all Purkinje cells of the cerebellum (Wassef and Sotelo, 1984; Smeyne et al., 1991) (Section 6.2.). Purkinje cell-specific markers include several glyco- and phosphoproteins, peptides, antigenic determinants that have not been identified or determinants that Purkinje cells share with other, non-cerebellar cell types. One of the first sera specific for rat Purkinje cells was obtained, using immunohistochemical screening, by Woodhams et al. (1979), but the antigen corresponding to this antibody has not been identified. Reeber et al. (1981) isolated a Purkinje cell specific 24 kDa glycoprotein from rat, that was present (Reeber et al., 1981) throughout the whole somatodendritic extent of the Purkinje cells, associated with the plasma membrane, as well as with the rough endoplasmatic reticulum and polysomes, the cytoplasmic side of the nuclear envelope and subsurface cisterns (Langley et al., 1982). Visinine, a soluble, 24 kDa protein, isolated from chicken retina, was found to be an exclusive marker for Purkinje cells in rat cerebellum (Yoshida et al., 1985). Specific staining of Purkinje cells was also found with monoclonal antibodies directed against human T cells (Garson et al., 1982), against certain cytoplasmic antigens in Purkinje cells (Weber and Schachner, 1982) and against antigenic determinants on trypanosomes (Wood et al., 1982). One of the antibodies (UCHT 1), isolated by Garson et al. (1982) is remarkable because its antigen is not present in Purkinje cells from 'staggerer' mutant mice (Caddy et al., 1982), a property the UCHT 1 antigen shares with the IP3 receptor protein (Section 3.1.4). One group of Purkinje cell-specific markers, the cerebellins, has been studied in more detail. A Purkinje cell-specific hexadecapeptide called 'cerebellin' and its metabolite, des-Serl-cerebellin were isolated and sequenced by Slemmon et al. (1984). Cerebellin immunoreactivity as studied with polyclonal antibodies in rat was found in soma and dendrites of nearly all Purkinje cells, but was absent beyond the initial axon segment (Slemmon et al., 1984). Cerebellin-immunoreactivity could also be demonstrated in cerebella of different species, including human and chick (Morgan et al., 1988), and in cartwheel cells and basal dendrites of pyramidal neurons of the dorsal cochlear nucleus (Fig. 33) (Mugnaini and Morgan, 1987). Cerebellin differs from most other markerproteins of Purkinje cells in being absent from other sites in the CNS, including the retina (Berrebi et al., 1991). Slemmon et al. (1988) and Morgan et al. (1988) concluded from an analysis of cerebellin immunoreactivity in Purkinje cells of different mutant mice with a varying loss of the granule cells, that the amount of cerebellin is correlated with the formation and the number of parallel fiber-Purkinje cell synapses. L-7 is a protein specific for Purkinje cells. Labelling with polyclonal antibodies against predicted L-7 sequences was present in somata, including the nucleus, in dendrites and dendritic spines, and in axon and axon terminals of Purkinje cells. All Purkinje cells, but no other types of cerebellar neurons appeared to be labelled (Berrebi and Mugnaini, 1992). The expression of the L-7 gene by all adult Purkinje cells of the rat cerebellum was reported by Nordquist et al. (1988, their PCD5 clone), Oberdick et al. (1990) Vandaele et al. (1991, their Purkinje cell protein-2) and Smeyne et al. (1991). According to Oberdick et al. (1990) and Berrebi et al. (1991) the L-7 gene is also expressed by retinal 39
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J. Voogd, D. Jaarsma and E. Marani
Fig. 32. Parvalbumin immunoreactivity in the developing cerebellar cortex of the zebra finch. A. Incubation day D 16: Clusters of labelled Purkinje cells of varying staining intensity. Stained Purkinje cells axons are seen in the internal granular layer (IGL). Note the areas containing unstained or only slightly stained cells and the dot-like staining pattern in the external granular layer (EGL). B. Adult: The dendrites of the Purkinje cells have reached the cerebellar surface and are now fully branched. Between them many immuno-stained basket and stellate cells are visible. Parvalbumin immunoreactivity in Purkinje cell axons is no longer visible except for a few fragments lying in the internal granular layer (IGL). The layer of Purkinje cells is still interrupted by parvalbumin immunonegative areas. Calibration bar in A - 50/lm, in B = 100/~m. Braun et al. (1986).
bipolar cells. The initial expression of the L-7 gene by zonally distributed Purkinje cells during prenatal and early postnatal development was studied by Vandaele et al. (1991), Smeyne et al. (1991) and Oberdick et al. (1993) (see also Section 6.2.). 40
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Several other markers are only present in zonally distributed subpopulations of Purkinje cells (see also Section 6.1.3.). The monoclonal antibody B1 of Ingram et al. (1985) was raised against rat embryonic forebrain membranes. Purkinje cells in broad parasagittal bands, alternating with B 1-negative zones, were immunoreactive in the cerebellum of Macaca fascicularis. Other neurons in the molecular layer and cells of the cerebellar nuclei were also stained by this antibody. A similar pattern of B l-immunoreactivity was present in the cerebellum of the rat. The monoclonal antibody mabQ 113 was developed, specified and used in anatomical studies by Hawkes et al. (1985), Hawkes and Leclerc (1986, 1987), Hawkes and Gravel (1991), Hawkes (1992) and Leclerc et al. (1992). It is directed against a 120 Kda protein (Zebrin I); the function of this protein is still unknown. A specific subpopulation of Purkinje cells displays immunoreactivity for Zebrin I in their dendrites, soma, axon and axon terminals. Zebrin I-positive and negative Purkinje cells are distributed in parasagittal bands (Fig. 34) (see also Section 6.1.3.). Ultrastructurally Zebrin I-immunoreactivity in rat Purkinje cells is localized in the cytosol. It is absent from membrane-bound organelles such as the mitochondria and the synaptic vesicles. In large dendrites reaction product is associated with microtubuli, in spines it is located at the postsynaptic densities. An antibody raised against the cerebellum of the weakly electric fish Apteronotus (anti-Zebrin II: Brochu et al., 1990) recognizes the same Purkinje cells as anti-Zebrin I in the cerebellum of the rat, and is effective in staining these neurons in a large number of other species such as the opossum (Fig. 137). The epitope of the Zebrin II antibody is associated with a 36 kDa polypeptide identified as the glycolytic enzyme aldolase C. In situ hybridization of Zebrin II mRNA showed a strong signal in mouse Purkinje cells with normal regional heterogeneity (Hawkes, 1992; Ahn et al., 1994). Rat Purkinje cells containing low affinity nerve growth factor receptor protein (Sotelo and Wassef, 1991; Dusart et al., 1994) (see Section 3.1.10 and Fig. 38C,D), PKC delta (see Section 3.1.10 and Fig. 133), or the monoclonal antibody B30 of Stainier and Gilbert (1989), that recognizes two minor gangliosides, show the same distribution as Zebrin-stained Purkinje cells. Although the distribution of the enzyme 5'-nucleotidase in the molecular layer of rat and mouse cerebellum (Scott, 1963; Marani, 1982a,b) is identical to that of the Zebrins (Eisenman and Hawkes, 1993) (Fig. 135), it may be located in Bergmann glial and not in Purkinje cells (see Section 3.5.). Several proteins are distributed in more or less complementary patterns, either in Zebrin-negative Purkinje cells (Ppath, HNK, cytochrome oxidase) or in Bergmann glia (3a-fucosyl-N-acetyl lactosamine [FAL], glycogen phosphorylase). The antibody P-path is directed against acetylated gangliosides (Edwards et al., 1989, 1994; Leclerc et al., 1992) and reacts with Zebrin-negative Purkinje cells in mouse cerebellum (Fig. 134). The localization of cytochrome oxidase was described by Hess and Voogd (1986), Leclerc et al. (1990) and Harley and Biejalew (1992) in the cerebellum of macaques, the squirrel monkey and the rat. The localization of HNK was studied by Eisenman and Hawkes (1993) in the mouse. The FAL-epitope (Fig. 94; Bartsch and Mai, 1991) and the enzyme glycan phosphorylase (Marani and Boekee, 1973; Harley and Bielajew, 1992) have been located in subsets of mouse Bergmann glial cells, that are distributed in a complementary manner with respect to the Zebrin pattern. Gangliosides are glycolipids, concentrated in the outer layer of neural plasma membranes. Biochemical analysis showed a correlation between the selective degeneration of Purkinje cells in pcd and nervous mutant mice with the loss of the ganglioside GT~A. GT~A was more concentrated and the ganglioside GD~A was diminished in weaver mutant mice with a selective loss of the granule cells (Seyfried et al., 1983, 1987; Marani 41
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J. Voogd, D. Jaarsma and E. Marani
Fig. 33. Light photomicrographs showing cerebellin immunoreactivity in rat cerebellum (A-C) and the dorsal cochlear nucleus (D-F) in parasagittal sections. A. Cerebellar hemisphere with part of the underlying dorsal cochlear nucleus (DCoN, arrowhead). CN, cerebellar nuclei. B. Immunostaining in DCoN. The cell bodies of cartwheel neurons in the superficial layers (layers 1 and 2) of the DCoN and the plexus in the deeper region (layer 3) predominate. The plexus is most dense in the upper portion of the deep region, which may correspond to layer 3 of the feline nuclei, a zone that contains the basal dendritic arbors of the bipolar pyramid neurons, one of which is indicated by an arrow. C. Immunoreaction product is present in Purkinje cell body and main dendrites. D. In the axon, immunostaining is restricted to the initial axon segment (arrowhead). E. Three subependymal displaced Purkinje cells in DCoN. Smaller cell bodies of several cartwheel neurons (arrowheads) are also shown. F. Portion of the ventral cochlear nucleus in which immunostaining is restricted to rare cartwheel cell bodies (arrowheads) displaced in the superficial granular layer. Bars in A and B = 0.5/~m, in C-F = 50 r Mugnaini et al. (1987).
a n d M a i , 1992). A n o t h e r g a n g l i o s i d e , GD3, was localized in i m m a t u r e P u r k i n j e cells o f the rat, u s i n g a m o n o c l o n a l a n t i b o d y (Fig. 35). I m m u n o r e a c t i v i t y d i s a p p e a r e d f r o m the cell b o d y in the adult, b u t r e m a i n e d p r e s e n t in the m o l e c u l a r layer ( R e y n o l d s a n d Wilkin,
42
The cerebellum." chemoarchitecture and anatomy
Ch. I
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Fig. 34. 50/lm horizontal sections through the cerebellar cortex of the rat at postnatal day 25 to show the distribution of mabQ113 (Zebrin I) immunoreactivity. A. The peroxidase reaction product is confined exclusively to a subset of Purkinje cells that are distributed symmetrically into parasagittal compartments in both the vermis and hemispheres. Labelling of the bands of Zebrin I-immunoreactive Purkinje cells P l+ to P7+ according to Hawkes and Leclerc (1987). Scale bar = 500/~m. B. A higher-power view of P5 + and P6 + of the posterior lobe hemisphere, in the lobules bordering the intercrural fissure. Immunoreactivity is seen to extend throughout the Purkinje cell, and no other cell types in the cerebellum are stained. Scale bar = 200/zm. C. In addition to the regular band display, additional narrow 'satellite' bands are also common. The arrowheads indicate two such satellites in the posterior lobe vermis. Scale bar = 100/lm. Leclerc et al. (1988).
1988). Levine et al. (1986), who used another monoclonal antibody against GD3, found immunoreactivity of reactive astrocytes in mouse mutants, but failed to observe a reaction within the Purkinje cells. These different results probably are due to differences in fixation (Reynolds and Wilkin, 1988). 3.1.9. Cytoskeleton and metabolism of Purkinje cells The DNA content of mature Purkinje cells is high. Feulgen-DNA or propidiumiodideDNA reveal hyperdiploid values (Bernocchi, 1986; Bernocchi et al., 1986). Purkinje cells stand out by their high content of enzymes, mostly dehydrogenases (Adams, 1965). Their content of the glycolytic enzyme enolase is low (Pelc et al., 1986; Vinores et al., 43
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J. Voogd, D. Jaarsma and E. Marani
1984). However, Purkinje cells of the human cerebellum stand out from other nerve cells by their high content of aldolase-C (Royds et al., 1987). Purkinje cells do not react with antibodies against the phosphorylated forms of the 70, 150 and 200 kDa neurofilament proteins (Pelc et al., 1986; Matus et al., 1979; Marc et al., 1986; Langley et al., 1988). The phosphorylated form of the 200 kDa protein is present in axons in the granular layer, that were identified as Purkinje cell axons by Marc et al. (1986) and as mossy fibers by Langley et al. (1988), both in the rat (Fig. 36). The non-phosphorylated form of the neurofilament proteins was found to be present in the entire Purkinje cell with the exception of distal dendrites. According to Marc et al. (1986) the protein is present as filamentous aggregates. Langley et al. (1988) stated that a monoclonal antibody against the non-phosphorylated form of the 200 kDa protein is present in soma and dendrites as patches of diffuse immunoreactivity without a filamentous substructure. In Friedreich's ataxia neurofilament, mainly the phosphorylated form, is expressed by human Purkinje cells within their soma and dendrites (Marani, unpublished results) (Fig. 37). The process of endocytosis in Purkinje cell has been studied in relation to synaptogenesis of the Purkinje cell dendrites. Glycoproteins located on the parallel fiber are also pinocytosed into the Purkinje cell. Lysosomal action degradates these glycoproteins. In this process alpha-D-massosidase plays an important role, which is selectively present in the Purkinje cell dendrites (Dontenwill et al., 1983). Other glycoproteins, like K+Na+ATP-ase are not taken up, indicating a receptor-mediated recognition of some glycans of the glycoproteins. The specificity of the pinocytosis for certain molecules suggests that this recognition is the preliminary event in the establishment of Purkinje cell synapses.
3.1.10. Nerve growth factor and nerve growth factor-receptor protein in Purkinje cells Nerve growth factor-like immunoreactivity was present in Purkinje cell somata and dendrites, with dense labelling in the paraflocculus, and in neurons of the cerebellar nuclei and the lateral vestibular nucleus of rat cerebellum (Nishio et al., 1994). All but a few of the Purkinje cells of the adult rat cerebellum stain with an antiserum against basic fibroblast growth factor. Staining was observed in all cellular compartments (Matsuda et al., 1992). P75 nerve growth factor-receptor protein (NGF-R) is present in developing and adult Purkinje cells. Yan and Johnson (1988) and Cohen-Cory et al. (1989) described and reviewed the development of NGF-R in rat cerebellum. Low affinity NGF-R immunoreactivity has been demonstrated with species-specific monoclonal antibodies in Purkinje cells of adult rats (Pioro and Cuello, 1988, 1990; Pioro et al., 1991; Fusco et al., 1991; Dusart et al., 1994), monkey and human brain (Mufson et al., 1991). Immunoreactivity was present in the somata, dendrites and the proximal axon of the Purkinje cells. Additional immunoreactivity in granule cells was reported by Vega et al. (1994), using Bouin's fixative. NGF-R mRNA is expressed during early development in neurons of the rat external granular layer and in Purkinje cells. It peaks at postnatal day 10 and declines afterwards (Cohen-Cory et al., 1989; Lu et al., 1989) but also can be demonstrated in Purkinje cell somata in adult rodents (Fig. 38) (Koh et al., 1989) and primates (Mufson et al., 1991). NGF-R immunoreactivity was found to be highest in the flocculonodular lobe (Pioro and Cuello, 1988, 1990; Fusco et al., 1991). A distribution with strong expression in the flocculonodular lobe, the ventral parts of the anterior lobe and the lobules VII, VIII and 44
The cerebellum." chemoarchitecture and anatomy
Ch.I
Fig. 35. Double-immunofluorescent staining of 20-day rat cerebellar sections with antibodies to GD 3 ganglioside and glial acidic fibrillary protein (GFAP). Purkinje cell dendrites are intensely GD3-immunoreactive (A) but do not extend to the pial surface, unlike the Bergmann glial fibers (B), which project brightly GFAPimmunoreactive end-feet onto the pial membrane. Scale bar is 35 ~tm. Reynolds and Wilkin (1988).
45
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Immunocytochemical staining patterns of two monoclonal anti-bodies directed against nonphosphorylated and phosphorylated neurofilaments were studied in the cerebellum of developing normal rats. A. Non-phosphorylated neurofilaments on postnatal day 11. B. Day 21. Basket cell axons form a characteristic brush-like plexus around the initial segment of the Purkinje cell axon. C. Phosphorylated neurofilaments on postnatal day 13. D. Postnatal day 21. Stained filaments are restricted to Purkinje cell and basket cell axons and are absent from the Purkinje cell cytoplasm. Calibration bars in A and C 30/lm, in B and D 10 ~tm. Marc et al. (1986).
Fig. 36.
46
The cerebellum." chemoarchitecture and anatomy
Ch. I
IX of the caudal vermis and low activity in the hemisphere, was described by Mufson et al. (1991) for primates and man. The administration of colchicine results in the expression of N G F - R in most cerebellar Purkinje cells (Pioro and Cuello, 1988, 1990; Pioro et al., 1991). Koh et al. (1989) and Fusco et al. (1991) found N G F - R mRNA expression and NGF-R immunoreactivity in adult rat~ to be present in alternating Purkinje cell zones of strong and weak activity (Fig. 38C,D). This zonal pattern was also observed by Pioro and Cuello (1990). Its correspondence to the pattern of mabQ113 (Zebrin) immunoreactive zones (Hawkes and Leclerc, 1987) was noticed by Sotelo and Wassef (1991) and verified by Dusart et al. (1994) in adult rats. Lesions of the white matter, or knife cuts isolating the dorsal portion of the vermis of the rat cerebellum induces NGF-R immunoreactivity in previously unstained Purkinje cells (MartinezMurillo et al., 1993; Dusart et al., 1994).
3.1.11. Immunoreactivity of Purkinje cells in paraneoplastic diseases Specific forms of immunoreactivity of Purkinje cells have been discovered in human paraneoplastic conditions. Subacute cortical cerebellar degeneration in man may be associated with several types of carcinoma (see Vecht et al., 1991 for review). It has been most frequently observed in association with ovarian or endometrial carcinoma, but it also occurs as a rare sequal of small-celled bronchial carcinoma. It is generally characterized by a diffuse or patchy loss of Purkinje cells; granule cells also can be affected (Brain et al., 1951; McDonald, 1961; Brain and Wilkinson, 1965; Schmid and Riede, 1974; Steven et al., 1982). Strong labelling of Purkinje cells and weak staining of the granular layer was observed in sections of human cerebellum with a serum of patient with cerebellar degeneration with Hodgkin's disease using the indirect fluorescent staining procedure (Trotter et al., 1976). Sera of patients with carcinoma of the ovary were found to react with human Purkinje cells and neurons of the cerebellar nuclei using the same method (Greenlee and Sun, 1985). Jaeckle et al. (1985) distinguished a granular cytoplasmic and a diffuse form
9
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Fig. 37. Expression of phosphorylated neurofilament localization in normal human cerebellar cortex (A) and in Friedreich's disease (B). Note the strong positivity of the white matter and the molecular layers in a case of Friedreich's ataxia. No expression was found in the normal folium that was Nissl counterstained to demonstrate the granular and Purkinje cell layer. M = molecular layer, P = Purkinje cell layer, G = granular layer, F = fiber layer. Marani, unpublished.
47
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Fig. 38. A. Nerve growth factor-R (NGF-R) transcripts are localized within Purkinje cells in the paraflocculus of rat cerebellum. B. NGF-R immunocytochemistry shows the perikarya of the Purkinje cells as well as the dense staining of the molecular layer, where the dendritic trees of the Purkinje cells arborize. Arrows in C and D point to parasagittal zones of intense labelling interdigitated with weaker labelling. Bar = 90 r Koh et al. (1989).
of immunoreactivity of human Purkinje cells with sera from patients with cerebellar degeneration suffering from ovarian or breast cancer. The diffuse form of Purkinje cell staining also was observed at higher concentrations with some sera of normal controls. Moreover the diffuse staining is not restricted to Purkinje cells, but also involves stellate, basket and some granule cells (Andersson et al., 1988). Cunningham et al. (1986) further analysed the sera causing granular deposits in the Purkinje cell cytoplasm with immunoblotting of extracts of human Purkinje cells. This so-called anti-Yo serum recognizes a 62 kDa and a 34 kDa protein. Antibodies raised against both proteins react with Purkinje cells in tissue sections (Fig. 39). The strongest reaction was observed for the antibody against the 62 kDa protein. The specificity of this reaction and the presence of anti-Yo immunoreactivity in tumor tissue was demonstrated by Furneaux et al. (1990). The 34 kDa antigen was found to correspond to the c D N A sequence of a clone recognized from a cerebellar expression library by a serum from a patient with paraneoplastic cerebellar degeneration (Dropcho et al., 1987; Furneaux et al. 1989). Other forms of immunoreactivity, with different cerebellar epitopes and a different localization of the immunoreactivity have been described (Tanaka et al., 1986; Smith et al., 1988; Rodriguez et al., 1988; Tsukamoto et al., 1989; Szabo et al., 1991). Differences in the localization of the immunoreactivity and in the characterization of the epitopes may be due to the use of rat cerebellum instead of human cerebellum in testing the sera by Tanaka et al. (1986), Smith et al., (1988) and Tsukamoto et al. (1989). Szabo 48
The cerebellum." chemoarchitecture and anatomy
Ch. I
et al. (1991) isolated the (NuD) neuronal antigen recognized by sera from patients with paraneoplastic encephalomyelitis associated with small-celled bronchus carcinoma. This serum, also designated as anti-Hu, reacts with nuclei of neurons in the CNS, including the cerebellum (Andersson et al., 1988). 3.2. EXCITATORY PATHWAYS The cerebellar cortex is innervated by two types of excitatory afferents, the mossy and climbing fibers, and an intrinsic excitatory fiber system, the parallel fibers. An additional excitatory intrinsic pathway may be formed by unipolar brush cells, that give rise to mossy fiber-like fibers. The excitatory amino acid glutamate is the most likely neurotransmitter candidate for these pathways. An inherent problem in the localization of glutamate as a neurotransmitter is that there is no unequivocal marker for glutamatergic neurons and fibers since glutamate also participates in several metabolic pathways of nerve cells (Van den Berg and Garfinkel, 1971; Fonnum, 1984; Erecinska and Silver, 1990). The identification of glutamatergic pathways, therefore, is based upon a combination of anatomical, biochemical and physiological techniques (Fonnum, 1984). Immunocytochemistry with antibodies against glutamate (Storm-Mathisen et al., 1983) and physiological studies have proven to be particularly fruitful in the identification of glutamate as the neurotransmitter of the cerebellar excitatory pathways. These methods, however, do not totally exclude the possibility that other excitatory amino acids, such as aspartate or homocysteate, also participate as excitatory neurotransmitters. This holds in particular for the climbing fibers that have been frequently proposed to use aspartate as their primary neurotransmitter (see below). A major problem with 'nonglutamate' excitatory neurotransmitter candidates is that, as yet, no vesicular uptake
Fig. 39. Immunofluorescence of rat Purkinje cells with anti-Yo serum of a patient suffering from a cerebellar syndrome with ovarian carcinoma. Courtesy Dr. Ch. J. Vecht and Dr. J.W.B. Moll, Department of Neurology, Erasmus University Medical Center, Rotterdam.
49
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system has been isolated for these compounds. Instead, glutamate has been shown to accumulate in synaptic vesicles by a proton-driven vesicle transporter. This vesicle transporter is highly specific for glutamate, and in contrast to the cytoplasma membrane 50
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 40. Immunostaining in rat cerebellar cortex produced by anti-glutamate(Glu) mAb 2D7 (A,C,C',E and F) or by 'anti-GABA' mAb 3A12 (B and D). A, B (overview) and C, D (details) are from a pair of consecutive semithin sections. C and C' are enlargements of areas indicated in A and C, respectively. A and B. Note drastic difference in labelling patterns obtained with the two antibodies, gr, granule cell layer; P, Purkinje cell layer; mol, molecular layer. C. Frame indicating part of area shown enlarged in C'. C and D. Complementary labelling in stellate cells (stars), Golgi cell (arrows), pinceau formed by basket cell terminals (double arrow heads). Mossy fiber terminal-like structures in C (arrow head) fit into glomerular arrangements outlined by dots in D (arrow head). C'. Densely packed puncta probably represent parallel fiber terminals in molecular layer. E. Numerous strongly stained patches (arrow heads) and some fibers (arrow) are reminiscent of mossy fiber terminals. Granule cells with unlabelled nuclei appear less immunoreactive than those in C. F. Large mossy fiber terminal with several synaptic contacts (arrows) shows higher surface density of gold granules (EM immunogold procedure) than another terminal nearby (stars). Bars 100 ~tm in B, D and E, 1 r in F. Liu et al. (1989). (
transporter, does not transport aspartate (reviewed by Nicholls and Atwell, 1990; Jahr and Lester, 1992). 3.2.1. Mossy fibers Glutamate-like immunoreactivity in mossy fibers
Although subpopulations of mossy fibers may be peptidergic or cholinergic (see Sections 3.10. and 6.4.5.), it is now generally accepted that most if not all of the mossy fibers use L-glutamate as their principal neurotransmitter. The glutamatergic nature of mossy fibers has been evidenced with immunocytochemistry with antibodies against glutamateglutaraldehyde (Storm-Mathisen et al., 1983) or carbodiimide-glutamate conjugates (Madl et al., 1986). The rationale of this method is that glutamate, although ubiquiteously present throughout the neuronal cytoplasm at relatively high concentrations (~ 10 mM; Van den Berg and Garfinkel, 1971; Nichols and Attwell, 1990), is particularly enriched in glutamatergic nerve terminals, because of the presence of synaptic vesicles that concentrate glutamate to at least 60 mM. When electron microscopic post-embedding immunogold protocols are employed, quantitative and statistical analysis of the distribution of immunolabelling can be performed (e.g. see Ottersen, 1989). Glutamate immunoreactivity is widely distributed throughout the granular layer, but is enriched over mossy fiber rosettes in rat (Figs 40 and 41) (Ottersen and Storm-Mathisen, 1984a,b, 1987; Ottersen et al., 1987, 1990; Liu et al., 1989; Ji et al., 1991), cat (Somogyi et al., 1986) and monkey (Zhang et al., 1990). Mossy fiber rosettes contained significant higher levels of immunoreactivity than Golgi cell terminals and granule cell dendrites. Enriched glutamate-like immunoreactivity was also demonstrated in anterogradely horseradish peroxidase-wheat germ agglutinin (WGA-HRP) labelled spinocerebellar mossy fiber terminals. Notably, the density of glutamate-like immunoreactivity showed a strong positive correlation with the density of synaptic vesicles in these mossy fiber terminals (Ji et al., 1991). The anterogradely labelled mossy fiber terminals had a similar density of glutamate-like immunoreactivity as other mossy fiber rosettes. Mossy fiber terminals were not enriched in aspartate- or GABA-like immunoreactivities (Ji et al., 1991; Zhang et al., 1990). Data from physiological studies including recent patch-clamp studies are in line with the assumption that glutamate is the neurotransmitter of mossy fibers (Garthwaite and Brodbelt, 1989, 1990; Silver et al., 1992; D'Angelo et al., 1993; Rossi et al., 1995). 51
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Glutamine and glutaminase
Ottersen et al. (1992) quantified the compartmentalization of glutamate and glutamine in the cerebellar cortex of the rat, using post-embedding immunogold immunocytochemistry. They found the highest ratios of glutamate/glutamine in parallel fibers, high ratios in mossy and climbing fibers, low ratios in Purkinje and granule cells and in basket cell and Golgi cell terminals and the lowest ratios in Bergmann glia and astrocytes. This distribution is in accordance with uptake of glutamate from the extracellular space by glial cells, and its conversion into glutamine by the enzyme glutamine synthase, that is exclusively present in glia (Van den Berg and Garfinkel, 1971; Norenberg and MartinezHernandez, 1979; Fonnum, 1984; Erecinska and Silver, 1990). The glutaminase-glutamine loop is closed by diffusion of glutamine into neurons, that contain glutaminase, the enzyme that catalyzes the hydrolytic cleavage of glutamine to form glutamate. Wenthold et al. (1986) and Kaneko et al. (1987, 1989) used antibodies against glutaminase as an alternative approach to determine the cellular localization of glutamate. In the granular layer glutaminase-like immunoreactivity was present in granule cell somata (Wenthold et al., 1986) and in in small clusters, that probably represent mossy fiber rosettes (Fig. 42e) (Wenthold et al., 1986; Kaneko, 1987, 1989). Intense glutaminase-like immunoreactivity was also detected in several precerebellar nuclei, that give rise to mossy fibers, such as the pontine nuclei, the reticular nucleus of the pons, the lateral reticular nucleus, the vestibular nuclei and the external cuneate nucleus (Fig. 42a-d). Neurons in some of these nuclei have also been shown to react with antibodies against conjugates of glutamate (Beitz et al., 1986; Clements et al., 1986; Raymond et al., 1984). Glutamate transporters
The major mechanism by which synaptically released glutamate is inactivated is by highaffinity, sodium-dependent transport (Fonnum, 1984; Nicholls and Attwell, 1990). The sodium-dependent glutamate transporters are present in both neurons and astroglial cells, and have been assumed to be enriched on nerve terminals of glutamatergic axons. [3H]D-aspartate, a metabolically inert substrate of the glutamate transporter with very low affinity for glutamate receptors, has been widely used to locate glutamate or aspartate using fiber systems in the brain (Fonnum, 1984). Autoradiographic studies on cryostate sections indicate that [3H]D-aspartate binding sites are particularly enriched in the molecular layer, but are also present in the granular layer (Greenamyre et al., 1990; Anderson et al., 1990). Studies in cerebellar slices, however, show that [3H]Daspartate is not taken up by mossy fiber terminals (Garthwaite and Garthwaite, 1988). Accordingly, [3H]D-aspartate is not retrogradely transported by mossy fibers, allthough it is efficiently transported by climbing fibers (Wiklund et al., 1984). Three high-affinity sodium-dependent glutamate transporters have been cloned in rat: GLT-1 (Pines et al., 1992; Tanaka, 1993), EAAC1 (Kanai and Hediger, 1992), and GLAST (Storck et al., 1992). Recently, also four subtypes of human glutamate transporters, EAAT1-EAAT4, have been cloned with similar properties as their rat counterparts (Arriza et al., 1994; Fairman et al., 1994). In situ hybridisation and immunocytochemistry showed a differential distribution of the three transporters throughout the cerebellum. GLT1 is concentrated in the Bergmann glial fibers, but also occurs in the glial processes of the granular layer and in the cerebellar nuclei (Danbolt et al., 1992; Rothstein et al., 1994). High levels of GLAST are present in Bergmann glial fibers, but it is essentially absent from the granule cell layer. In the cerebellar nuclei it is mostly 52
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Fig. 41. Electron micrographs of serial sections through a glomerulus in the granular layer of the cat cerebellar cortex. The section shown in (A) was reacted with antiserum to glutamate (GLU), the section in (B) with antiserum to GABA. The electron-dense gold particles show immunoreactive sites. For GLU the highest density of gold appears to be over the mossy fiber terminal (mt) and the lowest over glial processes and Golgi cell terminals (1-3). This was confirmed by statistical comparison of the populations. The same Golgi cell terminals are strongly reacting for GABA, while other processes have only a low surface density of gold. Scale (A and B) 0.5/Ira. Somogyi et al. (1986).
53
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I
Fig. 42. Phosphate-activated glutaminase-like immunoreactivity (PAG-LI) in the precerebellar nuclei and the cerebellar cortex of the rat. Intensely labelled neuronal somata are seen in the pontine tegmental reticular nucleus of Bechterew (a), pontine nuclei (b), external cuneate nucleus (c), and lateral reticular nucleus of the medulla oblongata (d). Small clusters of grains, possible axon terminals, with PAG-LI are seen in the granular layer of the cerebellar cortex (e). Fine grains with PAG-LI are densely distributed, but no cell bodies are seen in the inferior olivary nucleus (f). CM, cerebellar medulla; G, granular layer; M, molecular layer; ML, medial lemniscus; P, pontine longitudinal fibers; Py, pyramidal tract; R, raphe. Scale bar 200 pm in a-d, f, 50 pm in e. Kaneko et al. (1987).
54
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associated with neurons (Rothstein et al., 1994). EAAC1 has been exclusively localized in neurons, with high densities in Purkinje cells and granule cells. Interestingly, immunocytochemical data show that EAAC1 is enriched in axon terminals of Purkinje cells, indicating that EAAC1 is not selective for glutamatergic nerve terminals. In accordance with the biochemical data, there was no immunocytochemical evidence for the presence of EAAC 1 or one of the other glutamate transporter proteins in mossy fiber terminals. Taken together the above data indicate that mossy fiber terminals are not provided with high-affinity glutamate transporters. Consequently, glutamate released by mossy fibers is likely to be predominantly cleared through glial cells (Wilkin et al., 1982; Garthwaite and Garthwaite, 1988). However, since glial processes do not enter the glomeruli (e.g. see Palay and Chan-Palay, 1974), an exclusive glial uptake implies that 'mossy fiber glutamate' molecules have to travel throughout extracellular space of the glomeruli before being inactivated. The clearance of 'mossy fiber glutamate' may be particularly slow at the giant mossy fiber-unipolar brush cell synapses, that may extend over 12-40 ,um2 with multiple clusters of presynaptic vesicles apposed to continuous regions of postsynaptic densities (Mugnaini and Floris, 1994). In fact, unusually long excitatory postsynaptic responses have been observed in unipolar brush cells following mossy fiber stimulation, consistent with a slow clearance of synaptically released glutamate (Rossi et al., 1995). 3.2.2. Climbing fibers Aspartate and glutamate
Several observations have led to the assumption that L-aspartate is the principal neurotransmitter of climbing fibers. (1) Destruction of the inferior olive in the rat with 3-acetylpyridine resulted in a small decrease in cerebellar aspartate concentration in total tissue homogenate (Nadi et al., 1977) and synaptosomal fractions (Rea et al., 1980). However, these observations were not confirmed by Perry et al. (1976). (2) It was demonstrated that after 3-acetylpyridine treatment Ca2+-dependent and K+-induced release of aspartate was significantly decreased (Toggenburger et al., 1983). Glutamate release was more dramatically decreased (e.g. see Cu6nod et al., 1989). (3) It was observed that climbing fibers but not mossy fibers in rat (Wiklund et al., 1984) and monkey (Matute et al., 1987) retrogradely transported [3H]D-aspartate. These experiments, however, only showed that climbing fibers are provided with high-affinity sodium-dependent glutamate transporter protein, and did not give information about the kind of transmitter used by the climbing fibers (see 3.2.1.). It should be noted that high affinity glutamate transporters have not yet been located at synapses of climbing fibers in immunocytochemical studies with antibodies against high-affinity glutamate transporters, although this possibility is still open since a detailed electron microscopical analysis of the cerebellar molecular layer has not yet been done (Rothstein et al., 1994). (4) Physiological studies suggested that the distal region of the Purkinje cell dendrites was relatively less sensitive towards aspartate as compared to glutamate than the proximal dendrites (Cr6pel et al., 1982). Since climbing fibers chiefly innervate the proximal two-thirds of the Purkinje cell dendritic tree (Palay and Chan-Palay, 1974), these data would be consistent with the proposal that aspartate is a climbing fiber transmitter, whereas glutamate is the transmitter of the parallel fibers (see Cu6nod et al., 1989). Voltage-clamp studies of Purkinje cells in slices, however, suggest that climbing fibers 55
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Fig. 43. Photomicrographs of consecutive semithin sections from rat inferior olive stained with antisera to aspartate, glutamate and GABA, respectively. All neurons (arrows) in this field are labelled for aspartate and glutamate, but unlabelled for GABA. Glial cells (arrowheads; identity established on the basis of electron microscopic analysis of adjacent sections) contain little or no GABA and glutamate immunoreactivities, but are moderately stained with the aspartate antiserum. Asterisks indicate fiber bundles. Scale bar = 50 r Insets show test sections incubated together with the respective tissue sections. The test antigens are GABA (1), glutamate (2), taurine (3), glycine (4), 'none' (5), aspartate (6), and glutamate (7). Note selective staining of the respective amino acid conjugates. Zhang et al. (1990). (
and parallel fibers activate the same type of glutamate receptors (Llano et al., 1991). Summarizing, one may conclude that the case for aspartate as the principal neurotransmitter of climbing fibers is far from being conclusive. Zhang et al. (1990), who compared glutamate- and aspartate-like immunoreactivities in the neurons of the inferior olive and climbing fibers in rat and baboon (Papaio anubis), showed that glutamate and aspartate-like immunoreactivities were co-localized in all neurons of the inferior olive, with a slightly heavier staining in the principal olive (Fig. 43). Significant glutamate-like, but little aspartate-like labelling, however, was recognized over climbing fiber profiles and, therefore, it was concluded that glutamate and not aspartate is the most likely transmitter of the climbing fibers (see also Zhang and Ottersen, 1993). It was also concluded that the presence of aspartate-like immunoreactivity in cell bodies is an unreliable indicator of transmitter identity.
Homocysteate Cu6nod et al. (1989) reported on the results of a series of experiments on K+-induced release of different transmitters by the cerebellum of the rat, after previous destruction of the inferior olive by 3-acetylpyridine. Release of aspartate was found to be decreased compared to the controls, with the main decrease occurring in the hemisphere. Values for the vermis were only slightly lower than in normal rats. This difference might be explained by a relative sparing of neurons in the caudal inferior olive, that project to the vermis. Decreased values after 3-acetylpyridine treatment were also found for adenosine (see Section 3.5) and for homocysteic acid. For the release of these substances no differences were noticed between vermis and hemisphere. Homocysteic acid was originally considered as a transmitter of the climbing fibers (Grandes et al., 1989), but proved to be located in Bergmann glia (Figs 44 and 45) (Cu6nod et al., 1990; Grandes et al., 1991). Climbing fibers, therefore, interact with Bergmann glia, both in the release of homocysteic acid and in 5'-nucleotidase-regulated adenosine release (see Section 3.5). Immunocytochemical studies have shown that subpopulations of climbing fibers may use peptides as a neurotransmitter, including somatostatine, corticotrophin-releasing factor and enkephalin. Their distribution and characteristics will be discussed in Section 6.3.4.
3.2.3. Granule cells and parallel fibers In early studies it was found that in 'staggerer', 'weaver' and 'reeler' mutant mice which have almost complete or partial loss of their granule cells (McBride et al., 1976a; Hudson et al., 1976) and in rats or mice that lost their granule cells by a viral infection or 57
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postnatal X-irradiation (McBride et al., 1976b; Rohde et al., 1979), glutamate was depleted. However, the interpretation of this finding is not immediately clear, because mossy fiber terminals and inhibitory interneurons of the cerebellar cortex also may have been affected. Furthermore, it proved difficult to exclude aspartate as a transmitter of granule cells (Rohde et al., 1979; Roffler-Tarlov and Turey, 1982). Also the demonstration of Garthwaite and Garthwaite (1985) that granule cells in slices accumulate [3H]Daspartate did not provide conclusive evidence about the nature of the neurotransmitter used by parallel fibers. Immunocytochemical studies strongly support glutamate as the neurotransmitter of the parallel fibers. Thus, glutamate-like immunoreactivity but no other amino acids were enriched over parallel fiber terminals in rat (Ottersen and Storm-Mathisen, 1984a,b, 1987; Ottersen et al., 1987, 1990; Liu et al., 1989) (Fig. 40), cat (Somogyi et al., 1986) and monkey (Zhang et al., 1990). Also electrophysiological experiments are in favour of glutamate as the neurotransmitter at the parallel fiber-Purkinje cell synapse (Barbour, 1993 and references therein).
~
[!2s .
Fig. 44. Immunocytochemical localization of homocysteate (HCA) in Bergmann glia with polyclonal antiHCA antibodies. A. Test system mimicking immunocytochemical procedure. Conjugates are assembled in 'sandwich' construction with tissue as spacer and contain the following compounds (from top to bottom): HCA, Glu (glutamate), Asp (aspartate), Tau (taurine), Gly (glycine), GABA (~,-aminobutyric acid), L-Ala (L-alanine), fl-Ala (fl-alanine), Htau (homotaurine), Hypotau (hypotaurine), Gline (glutamine), Ca (cysteate), CSA (cysteine sulphinate), HCSA (homocysteine sulphinate), Cys (cysteine), Cyt (cystine), Met (methionine), carnosine, Hcys (homocysteine), cystathionine, gluta-thione, homocarnosine, y-Glu-Glu (y-glutamyl glutamate), fl-L-Asp-Gly (fl-L-aspartyl glycine), no AA (no amino acid conjugated to glutaraldehyde-treated rat brain protein). B. Pattern of HCA-like immunoreactivity in low-power view of rat cerebellar cortex in semithin section. Double arrow: fibrous, radially oriented immunoreactive element. Arrowheads, stained varicosities in association with Purkinje cell dendrites. C. Pattern of HCA-like immunoreactivity in rat cerebellar section pretreated with 3-acetylpyridine 10 days previously and degeneration of the inferior olive. No changes in the distribution of HCA are apparent. Bars 50/lm. Grandes et al. (1991).
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Fig. 45. Immunocytochemical localization of homocysteate (HCA) with polyclonal anti-HCA antibodies. A. Staining pattern in section close to that in Fig. 44B at higher magnification. Cell (asterisk) and capillary (circle) used as landmarks in A and B. B and C. Electron micrographs from ultrathin section immediately preceding semithin section in A. The HCA-immunoreactive varicosities indicated with arrows in (A) were identified as parts of the glial sheath surrounding Purkinje cell dendrites (d) in B and C. Bars: 10/lm in A, 5/lm in B, 1 j~m in C. Grandes et al. (1991).
Specific markers for granule cells are few. Seyfried et al. (1983), concluded from biochemical analysis in 'weaver' mutant mice that the ganglioside GDIA was more concentrated in granule cells. Webb and Woodhams (1984) developed three monoclonal 59
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antibodies (G-l-3; 7-8D2 and 8-20-1), that recognize cell surface antigens expressed by rat granule cells and their axons (see also Reynolds and Wilkin, 1988). Calcium-binding proteins, with the exception of calretinin (Rogers, 1989; Arai et al., 1991; Kadowaki et al., 1993; Floris et al., 1994) have not been localized in granule cells. Proteine kinase C (PKC) e, flI and II, e and ~"are expressed by rat granule cells (Ase et al., 1988; Wetsel et al., 1992; Chen and Hillman, 1993) (Table 1). 3.3. LOCALIZATION OF GLUTAMATE RECEPTORS
3.3.1. lonotropic glutamate receptors Glutamate activates two main classes of glutamate receptors, the ionotropic and metabotropic glutamate receptors. The ionotropic receptors are receptor/channel complexes that can be categorized into three groups according to their differential sensitivity to agonist ligands, as ~-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors, formerly known as quisqualate receptors, kainate receptors, and N-methylD-aspartate (NMDA) receptors (Monaghan et al., 1989; Mayer and Miller, 1990; Westbrook, 1994). The non-NMDA (AMPA and kainate) receptors display rapid kinetics. They are typically inhibited by 7-cyano-7-nitroquinoxaline-2,3-dione (CNQX), are permeable to monovalent cations (Na+, K+), but mostly impermeant to Ca 2+, and have been implicated in fast excitatory synaptic transmission (Mayer and Westbrook, 1987; Jahr and Lester, 1992). NMDA receptor channels, instead, have relatively slow kinetics, are also permeable to Ca 2+ ions, and are typically inhibited by D-2-amino-5-phosphonovalerate (APV). NMDA receptors are characterized by a voltage-dependent channel block by MgZ+-ions. They are dependent on, and are equipped with a coagonist site for glycine. Apart from their role in excitatory synaptic transmission, NMDA receptors have been implicated in synaptic plasticity and in developmental processes like cell migration and synaps formation (Collingridge and Singer, 1990). AMPA receptors
AMPA receptors have been autoradiographically labelled with [3H]AMPA and the antagonist [3H]CNQX: [3H]AMPA binding is moderately high over the rodent (Rainbow et al., 1984b; Monaghan et al., 1984; Nielsen et al., 1990; Garcia-Ladona et al., 1991; Makowiec et al., 1991) and human (Jansen et al., 1990) cerebellum, and is higher over the molecular than over the granular layer. [3H]CNQX binding sites are preferentially localized over the molecular layer, but cerebellar [3H]CNQX binding is relatively higher than [3H]AMPA binding, when the two are compared to binding levels of both ligands in other brain areas (e.g. see Fig. 6 in Nielsen et al., 1990). This difference is not due to [3H]CNQX binding to kainate receptors since these receptors are preferentially localized in the granular layer. Both [3H]AMPA and [3H]CNQX binding in the molecular layer was decreased in Purkinje cell deficient (pcd) mutant mice, but strongly upregulated in granuloprival mice (Makowiec et al., 1991). These observations favour a primary localization of AMPA receptors on Purkinje cells and an upregulation of the number of AMPA receptors on Purkinje cells as a consequence of deafferentation (Makowiec et al., 1991). Originally, AMPA receptors were assessed as quisqualate-sensitive [3H]glutamate binding sites (Cha et al., 1988, and references therein). Quisqualate-sensitive [3H]glutamate binding is strongly increased by the presence of CaC12, and is relatively high in the cerebellar molecular layer. CaC12-dependent quisqualate-sensitive [3H]glutamate bind60
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ing over the molecular layer, however, is largely insensitive to AMPA (Cha et al., 1988). These sites most likely correspond to the quisqualate-sensitive metabotropic glutamate receptors (Young et al., 1991), that have been recently demonstrated to be expressed at high levels by Purkinje cells (see Section 3.3.2.). Kainate receptors
High-affinity [3H]kainate binding sites predominate in the granular layer in rat (Monaghan and Cotman, 1982; Olson et al., 1987; Cambray-Deakin et al., 1990; Bahn et al., 1994) and man (Jansen et al., 1990). Low to moderate levels of [3H]kainate binding occur in the rat cerebellar nuclei. [3H]Kainate binding is not affected in Purkinje cell deficient (pcd) or 'nervous' mutant mice, but is decreased in granuloprival mice (Griesser et al., 1982). This decrease concerns the granular but not the molecular layer (Olson, 1987; Makowiec et al., 1991). Henke et al. (1981) noted a high level of low-affinity [3H]kainate binding sites in the molecular layer of pigeon cerebellum. Similar [3H]kainate binding sites were also labelled in the chicken cerebellum (Henley and Barnard, 1990), in fish (Maler and Monaghan, 1991) and in amphibian cerebellum, although in the amphibian kainate-binding sites seems to have somewhat different pharmacological and functional properties (reviewed in Henley, 1994). The chicken kainate binding sites could also be labelled by [3H]CNQX (Henley and Barnard, 1990). Several non-mammalian vertebrate kainate-binding proteins have been purified and cloned. These proteins display some homology towards mammalian ionotropic AMPA and kainate receptor subunits (see below), but are smaller (40-50 kDa instead of 100 kDa), and do not form functional receptors channels (reviewed by Hollman and Heinemann, 1994; Henley, 1994). In situ hybridisation and immunocytochemistry has shown that avian kainate-binding protein is localized in Bergmann glia (Fig. 95) (Somogyi et al., 1990; Gregor et al., 1992 and others). Somogyi et al. (1990) showed that immunostaining with a monoclonal antibody (IX-50) against chicken kainate-binding protein, was also localized in Bergmann glia in the cerebellum of fish. Frog kainatebinding protein, however, is widely distributed throughout the frog brain. High receptor densities were found in cerebellum, but their cellular distribution has not yet been reported (Dechesne et al., 1990; Wenthold et al., 1990). N M D A receptors
The distribution of NMDA receptors has been autoradiographically determined as NMDA-replaceable [3H]glutamate binding sites. In rat (Greenamyre et al., 1985; Monaghan and Cotman, 1985) and human cerebellum (Jansen et al., 1990), moderate densities of binding sites are found over the granular layer and in the cerebellar nuclei, whereas binding over the molecular layer is low. Olson et al. (1987) and Makowiec et al. (1991) reported that NMDA-sensitive [3H]glutamate binding is unchanged in Purkinje cell deficient (pcd) mutant mice, but that the density of binding sites is considerable reduced over the granular layer in granuloprival mice. These data suggest that NMDAbinding sites are absent on Purkinje cell dendrites and, instead, are present on granule cells and perhaps on stellate, basket and Golgi cells. Using different ligands including the competitive antagonist [3H]-2-carboxypiperazine-4-yl-propyl-l-phosphonic acid ([3H]CPP), [3H]glycine that specifically binds to the glycine coagonist site of NMDA receptors, and the non-competetive channel blockers [3H]MKS01 and [3H]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP), it was found that the pharmacological properties of NMDA receptors in the cerebellar 61
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cortex were different from those in other brain areas (reviewed in Monaghan and Anderson, 1991). Notably, cerebellar NMDA receptors label poorly with the noncompetetive channel blockers [3H]MK801 and [3H]TCP (Maragos et al., 1988; Monaghan and Anderson, 1991). Monaghan and coworkers recognized at least 4 pharmacologically distinct NMDA receptor types throughout the brain and recently demonstrated that their pharmacological heterogeneity reflects differences in subunit composition (see below; Buller et al., 1994). They identified two populations of NMDA receptors in the cerebellar cortex. One population of 'antagonist-prefering' sites, that can be labelled by [3H]CPP, is present throughout the brain, and represents NMDA receptors containing NR2A subunits. The second population consists of the 'cerebellar-like' sites, where competitive antagonists and the agonist homoquinolinate are relatively ineffective in displacing the NMDA-sensitive [3H]glutamate binding. They reflect the presence of NR2C subunit, that is uniquely expressed by cerebellar granule cells (Buller et al., 1994). The low level of [3H]MK801 and [3H]TCP binding in the cerebellum remains to be explained. Distribution of subunits
Like other classes of ionotropic receptors functional glutamate receptor channel complexes are multimeric proteins. Recent molecular cloning studies have revealed families of AMPA (GluR1-GluR4, also named GluRA-GluRD), kainate (GluR5-GluR7, and KA1 and KA2), NMDA (NR1, named ~'1 in mice, and NR2A-NR2D, named el-e4 in mice) and orphan (~1 and ~2) glutamate receptor subunits (reviewed in Nakanishi, 1992; Sommer and Seeburg, 1992; Hollman and Heinemann, 1994). The diversity of glutamate receptor subunits is further increased through alternative splicing that primarily involves the AMPA receptor subunits GluR1-GluR4, each of which exists in two versions, i.e. flip or flop, and the NR 1 subunit, that has eight splice variants. Combinatorial expression studies have demonstrated that the subunits aggregate into functional receptor channels in the homomeric as well as the heteromeric configuration. Thus multiple functionally distinct forms of each receptor type can be formed through different combinations of subunits (see below). In situ hybridisation (KeinS.nen et al., 1990; Monyer et al., 1991, 1994; Araki et al., 1993; Sato et al., 1993; Wisden and Seeburg, 1993; Akazawa et al., 1994; Laurie and Seeburg, 1994; Watanabe et al., 1994; and others) and immunocytochemical studies with antibodies for specific subunits (Martin et al., 1992, 1993; Petralia and Wenthold, 1992; Brose et al., 1993; Baude et al., 1994; Nusser et al., 1994; Petralia et al., 1994a,b,c; Jaarsma et al., 1995b) have shown that subunits are heterogeneously distributed throughout the cerebellum, each cell type expressing a characteristic set of subunits (see Table 2). AMPA subunits
AMPA receptor subunits are not only expressed by cerebellar neurons, but also by Bergmann glia, that express high levels of GluR 1 (GluRA) and GluR4 (GluRD) mRNA (Table 2, Fig. 46). GluR1 subunit mRNA is also expressed by Purkinje cells but not by other cerebellar cells (Kein~inen et al., 1990; Monyer et al., 1991; Sato et al., 1993). GluR4 mRNA in addition to Bergmann glial cells, is produced by granule cells and neurons of the deep nuclei. Granule cells express a GluR4 splice variant exclusively found in the cerebellum, GluR4c, consisting of GluR4 with the flop module and a truncated C-terminus (Gallo et al., 1992). GluR2 (GluRB) mRNA is found over the granular and molecular layers, in Purkinje cells, and in cells of the deep nuclei (Fig. 46). 62
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GluR3 (GluRC) mRNA is not expressed in granule cells, but occurs in Golgi cells, stellate and basket cells, Purkinje cells and cells in the deep nuclei (Fig. 46) (KeinS.nen et al., 1990; Monyer et al., 1991; Sato et al., 1993). The distribution of AMPA subunits has been studied immunocytochemically with antibodies specific for GluR1, GluR2/3, and GluR4 (Martin et al., 1992; Petralia and Wenthold, 1992; Baude et al., 1994; Nusser et al., 1994; reviewed in Jaarsma et al., 1995b). The processes of the Bergmann glia are densely immunostained for GluR1 and GluR4 (Fig. 47) confirming in situ hybridisation. Electron microscopy showed that GluR1 immunoreactivity was localized throughout the cytoplasma membrane (Baude et al., 1994). Dense immunostaining was associated with the processes of Bergmann fibers ensheathing PC spines and the attached synaptic varicosities of parallel fibers and climbing fibers (Fig. 48). This indicates that AMPA receptors on Bergmann glia may be activated by glutamate released by parallel fibers or climbing fibers, allthough there is no clue as yet of the functional role of glial cell activation (see discussion Baude et al., 1994, but also Mfiller et al., 1992). Purkinje cells are weakly-to-moderately immunopositive for GluR1. Dense GluR1 immunolabelling was found at the post-synaptic membrane specialisations of the dendritic spines of Purkinje cells, facing parallel and climbing fiber boutons (Fig. 48). The post-synaptic membranes of the parallel fiber-Purkinje cell and the climbing fiberPurkinje cell synapses are also stongly immunoreactive for GluR2/3 (Nusser et al., 1994; Jaarsma et al., 1995b). The GluR2/3 antibodies immunoreact with all cerebellar neurons. The perikarya and dendritic arbors of Purkinje cells densely immunostain, whereas
TABLE Type
AMPA
Kainate
NMDA
orphan
2.
Distribution of glutamate receptor subunit mRNAs in rat cerebellum Subunit
Cell t y p e PC
GrC
GoC
GluR1
+ flip
-
-
-
+ + flip
-
GluR2
+ + flip/flop
+ flip
+
+
-
++
GluR3
+ flip
-
++
++
-
+
GluR4
-
+ 4c-flop
-
-
+ + flip
+
GluR5 GluR6
+ -
. ++
GluR7
-
-
-
+
KA1
+
.
.
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glia; D C N ,
deep cerebellar nuclei.
B a s e d o n d a t a f r o m K e i n ~ n e n et al., 1990; M o n y e r et al., 1991, 1994; L a m b o l e z et al., 1992; A r a k i et al., 1993; L o m e l i et al., 1992, 1993; S a t o et al., 1993; W i s d e n a n d S e e b u r g , 1993; A k a z a w a S e e b u r g , 1994; W a t a n a b e
et al., 1994; L a u r i e a n d
et al., 1994.
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The cerebellum." chemoarchitecture and anatomy
Ch. I
5',.2 uletl
Fig. 47. Sagittal sections of the rat cerebellar cortex immuno-labelled with antibodies to GluR1 (a), GluR2/3 (b,e), and GluR4 (c,d). As, astrocyte-like cells; BG, Bergmann glial processes; Go, Golgi cell; Gr, granular layer; L, Lugaro cell; Mo, molecular layer; Pj, Purkinje cell body; WM, white matter; small arrow, Purkinje cell dendrite; asterisks, Bergmann glial cell body; arrow head, basket/stellate cell. Petralia and Wenthold (1992).
light-to-moderate staining neurons occur in basket/stellate cells, Golgi cells and granule cells (Fig. 47) (Martin et al., 1992, 1993; Petralia and Wenthold, 1992; Jaarsma et al., 1995b). Unipolar brush cells are also strongly GluR2/3-immunopositive (Jaarsma et al., 1995b). Dense and moderate GluR2/3-staining was found in the perikarya and neuropil of the deep nuclei, respectively. Using electronmicroscopic immunogold protocols, that allow precise ultrastructural localization of the immunoreaction product, Nusser et al. (1994) obtained stong proof that GluR2/3 immunoreactivity is associated with postsynaptic membrane specialisations of excitatory synapses in the cerebellar cortex (Fig. 49B, C, F). Their data indicate that GluR2/3 immunoreactivity is considerably stronger at parallel fiber-Purkinje cell, climbing fiber-Purkinje cell and parallel fiber-stellate cell synapses than at mossy fiber-granule cell synapses (compare Figs 49B and C with F). Conventional peroxidase-DAB (3,3'-diaminobenzidine tetrahydrochloride)-immuno65
Ch. I
J. Voogd, D. Jaarsma and E. Marani
electron microscopy also indicates that GluR2/3 immunoreactivity is relatively weak at the mossy fiber-granule synapses (Jaarsma et al., 1995b). The post-synaptic membranes of the giant mossy fiber-unipolar brush cell synapses are, however, strongly GluR2/3 immunopositive (Jaarsma et al., 1995b). The GluR4 antibodies, in addition to the Bergmann glia, moderately immunostain the granular layer, and the neuropil and perikarya in the the deep nuclei (Fig. 47). It was originally assumed that GluR4-immunostaining in the granular layer was associated with granule cells (Martin et al., 1992, 1993; Petralia and Wenthold, 1992), but electron microscopy showed that GluR4 immunoreactivity is localized in the astroglia (Jaarsma et al., 1995b). Thus granular layer astroglia like Bergmann glia express AMPA receptor subunits, but unlike the Bergmann glia, do not have GluR1. The absence of GluR4-immunoreactivity in granule cells can be explained by the fact that granule cells primarily express an atypical form of GluR4, GluR4c (see above), that is not recognized by the GluR4 antibodies currently available. AMPA receptors are believed to mediate most of the fast excitatory neurotransmission in the brain, including the cerebellum. Concordantly the types of AMPA receptor subunits expressed by a cell largely determine the characteristics of the fast excitatory responses (see Jonas and Spruston, 1994). The GluR2 subunit dominate the AMPA receptor channel behavior, in that homomeric GluR2 channels as well as heteromeric
Fig. 48. Electron micrograph of the synaptic distribution of immunoreactivity for the GluR 1 subunit of the AMPA receptor in rat cerebellum as detected by an antibody against the carboxy-terminal (intracellular) region of GluR1. A. A spine (s) emerging from a Purkinje cell dendrite (Pd) establishes an immunopositive type 1 synapse (solid arrows) with a parallel fiber terminal (pft). Intra-cellular immunoreactivity is present inside Bergmann glial cell processes along dendritic elements (e.g., open arrow). B. The peroxidase reaction end-product labels the postsynaptic density (psd) at the intracellular face of the postsynaptic membrane (pom) and not the synaptic cleft between the presyaptic (pem) and postsynaptic (pom) membranes. Scale bars in A = 0.5 r in B = 0.1 r Baude et al. (1994).
66
The cerebellum." chemoarchitecture and anatomy
Ch. I
channels formed with the participation of GluR2 show the properties of 'typical' AMPA receptors, i.e. linear current-voltage relations and a relative impermeability to Ca 2+. Homo- or heteromeric channels without GluR2, instead, display inward rectification and are permeable to Ca 2+ and other divalent cations (for references see Sommer and Seeburg, 1992; and Hollman and Heinemann, 1994). These channels usually are not found in neuronal cells, concordant with the notion that most neuronal cells express GluR2, but are present in Bergmann glial cells (Mfiller et al., 1992; but see also Burnashev et al., 1992). In accordance with the presence of high levels of GluR 1 and GluR4 but absence of GluR2 in these cells. By combining two powerful methods, i.e. the patch-clamp technique to characterize the properties of native receptor channels in single cells in brain slices, followed by single cell PCR-amplification methods to analyse the mRNA contents of the respective cells semiquantitatively, Jonas et al. (1994) recently showed that the CaZ+-permeability of native AMPA receptor channels in cerebral cortical cells is related to the relative abundance of GluR2 subunit mRNA in the respective cells. Thus inhibitory interneurons of the cerebral cortex have low GluR2/non-GluR2 ratios (-- 0.3) and highly Ca 2+permeable AMPA receptors (which, however, display linear current-voltage relations unlike 'Bergmann-glial' AMPA receptors), whereas pyramidal cells, which have a relatively high GluR2/non-GluR2 mRNA ratio (-- 3), contain CaZ+-impermeable AMPA receptors. PCR-amplification analysis of AMPA subunit m R N A of Purkinje cells, indicates that GluR2 mRNAs are more abundant than GluR1 and GluR3 mRNAs (Lambolez et al., 1992), implying that Purkinje cells express weakly CaZ+-permeable AMPA receptors. Also in (pooled) granule cells GluR2 mRNA is more abundant than non-GluR2 (GluR4) mRNA. PCR-amplification analysis has not yet been done for other cerebellar cells. One might speculate that the basket, stellate and Golgi cells express CaZ+-permeable AMPA receptor like the inhibitory interneurons of the cerebral cortex, since according to in situ hybridisation data they seem to express relative high levels of GIuR3 compared to GluR2 (see Table 2). AMPA receptors made from different subunits may have different desensitization kinetics. Desensitization is particularly fast for AMPA receptors formed with GluR3flop or GluR4-flop (Mosbacher et al., 1994). Granule cells produce GluR4-flop and GluR2 (Table 2), and therefore are likely to have fast (submillisecond) desensitizing AMPA receptors. This could explain the very fast decay kinetics of non-NMDA component of the excitatory post-synaptic currents (EPSCs) at the mossy fiber-granule cell synapses (Silver et al., 1992; Rossi et al., 1995). If this holds true this would imply that the length of the excitatory responses at the mossy fiber-granule cell synapses is largely controlled by the desensitization properties of the AMPA receptors and does not depend upon the time course of transmitter removal, that may be relatively slow at these synapses (Jonas and Spruston, 1994) (see Section 3.2.1.). Purkinje cells express GluRl-flip, GluR2-flip and -flop, and GluR3-flip mRNA (Lambolez et al., 1992) that form AMPA receptors with desensitization time constants 3-5 times slower than 'GluR4-flop-GluR2' channels (Mosbacher et al., 1994). Concordantly, AMPA receptors in Purkinje cells appear to have relatively slow desensitization kinetics (Barbour et al., 1994). Also the decay phases of AMPA receptor-mediated EPSCs in Purkinje cells after parallel fiber or climbing fiber activation, have slow time constants (Perkel et al., 1990; Llano et al., 1991; Barbour et al., 1994). Interestingly stellate/basket cells, that express AMPA receptors with the same desensitization kinetics as Purkinje cell AMPA receptors, showed much faster decaying parallel fibers EPSCs (Barbour et al., 1994). Barbour et al. (1994) concluded that glutamate is rapidly cleared 67
Ch. I
J. Voogd, D. Jaarsma and E. Marani
at the parallel fiber-stellate/basket cell synapses, resulting in rapid deactivation of postsynaptic AMPA receptors, whereas synaptically released glutamate seems to be present during a prolonged time at Purkinje cell synapses. According to H/~usser (1994) EPSCs of climbing fiber-Purkinje cell synapses have decay time constants that are slower than parallel fiber-evoked EPSCs in Purkinje cells, which may be explained by the fact that clearance of glutamate at climbing fiber synapses is slower due to their larger size.
68
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 49. Electron micrographs showing the subsynaptic segregation of GluR2/3 AMPA receptor subunits (GluR B/C/4c) and the metabotropic mGluRl~ glutamate receptor (mGluR1; see Section 3.3.2) as revealed by post-embedding immunogold labelling. A and B. Consecutive sections of the same synaptic junctions showing that immunoparticles for mGluRl~ (double arrows in A) are concentrated at the edge, whereas immunoparticles for GluR2/3 (arrows in B) are concentrated in the main body of synaptic junctions established by parallel (pft) and climbing (cft) fiber terminals with spines (s) of Purkinje cell dendrites (Pd). Note that mGluRlcz is often localized extrasynaptically (double arrow heads in A). C. Immunoreactivity for GluR2/3 (arrows) was always very strong on basket and stellate (Stc) cells. D and E. Double immunolabelling of mGluRl~ (large particles, double arrows) and GluR2/3 (small particles, arrows) immunoreactivity in the synapses on spines (s) of Purkinje cells, confirming synaptic segregated subsynaptic localization of mGluR1 and GluR2/3. The synapse in E is from Triton treated material. F. Generally a lower density of immunoparticles for GluR2/3 (arrows) has been found in synapses between mossy fiber terminals (mt) and granule cell dendrites (d) than in the parallel fiber synapses (compare to B and C). Scale bars = 0.1 r in A,B,D,E, 0.2 r in C,F. Nusser et al. (1994). (
Kainate subunits
Kainate receptor subunits are most prominent in granule cells, which express high amounts of both GluR6 and KA2 mRNA (Fig. 50) (Wisden and Seeburg, 1993). Purkinje cells express moderate levels of GluR5 and low levels of KA1 mRNAs; basket and stellate cells express GluR7 mRNA, and neurons of the deep cerebellar nuclei produce GluR7 and KA2 mRNAs (Table 2, Fig. 50) (Wisden and Seeburg, 1993). The high level of kainate receptor mRNA expression by granule cells is in accordance with the preferential binding of [3H]kainate over the granular layer (see above, but also Bahn et al., 1994). Immunocytochemical studies with antibodies specific for GluR6 and GluR7 show that dense GluR6/7-immunostaining occurred over the granule cell layer, where it was associated with granule cell perikarya and dendrites (Petralia et al., 1994c; Jaarsma et al., 1995b). The post-synaptic membranes of the mossy fiber-granule cell synapses were strongly immunoreactive for GluR6/7 (Jaarsma et al., 1995b). Stellate and basket cells and cells in the deep cerebellar nuclei were also immunoreactive for GluR6/7. KA2 immunoreactivity was relatively low in the cerebellar cortex and was concentrated over the glomeruli and neurons in the deep nuclei (Petralia et al., 1994c; Jaarsma et al., 1995b). Recombinant expression studies have shown that GluR5 and GluR6 form glutamategated channels in the homomeric configuration as well as in the heteromeric configuration with KA1 and KA2, whereas KA1 and KA2 do not assemble into functional receptor channel complexes (reviewed in Wisden and Seeburg, 1993). Thus functional kainate receptors can be formed in several cerebellar cells, in particular in granule cells expressing significant levels of GluR6 and KA2. Unequivocal physiological evidence for the presence of kainate receptors in the cerebellum (as well as in other brain areas) is, however, still lacking (see discussion Wisden and Seeburg, 1993). It should be noted that kainate in spite of its low affinity for AMPA receptors, potently activates AMPA receptors, and that excitatory responses evoked by kainate in brain tissue are generally mediated through AMPA receptors. Recombinant kainate receptors have been demonstated to desensitize very rapidly, which in part may explain why kainate receptor responses have not been detected (Wisden and Seeburg, 1993). Another possibility is that kainate receptor responses are masked by AMPA receptors, which are assumed to be present in much higher concentrations in neurons (Wenthold et al., 1994 and references therein). Autoradiographic (see above) and immunocytochemical studies (Jaarsma et al., 1995b), however, suggest that kainate receptors predominate over AMPA recep69
Ch. I
~LuR-5
J. Voogd, D. Jaarsma and E. Marani
,~ .~LuR ::6
B .3LuR 7
C Mol
Gr P "If ;:' l"'.s,"''~,;,"\ T (
'
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Fig. 50. Distribution of GluR5 (A), GluR6 (B), GluR7 (C), KA-1 (D), KA-2 (E) of subunits in RNAs of high-affinity kainate receptor mRNAs in coronal sections at level of the cerebellum of the rat. Gr, granular layer; LC, locus coeruleus; Mol, molecular layer; P, Purkinje cell layer; Po, pontine nuclei. Scale bars: 2.3 mm. Wisden and Seeburg (1993).
tors in granule cells, and may significantly contribute to excitatory neurotransmission at the mossy fiber-granule cell synapses. N M D A subunits Functional NMDA receptors are believed to be generated as heteromeric assemblies of NR1 subunits with members of the NR2 subunit family. The pharmacological and kinetic heterogeneity of NMDA receptors seems to be primarily dependent upon the type of NR2 subunit (Monyer et al., 1992; Meguro et al., 1992; Nakanishi, 1992; Buller et al., 1994), although NR1 diversity generated through alternative splicing may also contribute to NMDA receptor heterogeneity (Buller et al., 1994; Hollman and Heineman, 1994). Essentially all cerebellar neurons seem to express significant levels of NR1 mRNA (Table 2, Fig. 51E) (Moriyoshi et al., 1991). The main splice variants produced in the cerebellum are NR 1-2 (with 3'-end deletion 1) and to a lesser extent NR1-4 (with 3'-end deletions 1 and 2; Laurie and Seeburg, 1994). There is a remarkable difference between the Purkinje cells and the other cells of the cerebellar cortex, in that Purkinje cells express high levels of the NRI-a forms (without 5'-insertion), whereas in the other cells the N 1-b splice variants (with 5'-insertion) predominate (Laurie and Seeburg, 1994). NR2 subunit mRNAs are heterogeneously expressed throughout cerebellar neurons (Table 2, Fig. 51) and show pronounced changes during development (Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1994). NR2 subunit mRNAs are most prominent in granule cells, that express high levels of NR2C mRNA and moderate levels of NR2A mRNAs in adult rodent cerebellum (Fig. 51). Interestingly, whereas NR2A mRNA expression in rodent granule cells begins early postnatally, NR2C first appears in later stages (postnatal day 10-11 in rat) in post-migratory cells of the internal granular layer. It apparently replaces NR2B, which is transiently expressed by cerebellar granule cells (Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1994). The expression of NR2C starts in granule cells of the caudal vermis (lobules VIII-X) and subsequently extends throughout the whole cerebellar cortex by postnatal day 13 (see Fig. 3N and O in Watanabe et al., 1994 and Fig. 7 in Akazawa et al., 1994). This pattern is compat-
70
The cerebellum." chemoarchitecture and anatomy
Ch. I
ible with the sequence of maturation of the granule cells (Altman, 1972). According to Akazawa et al. (1994) and Watanabe et al. (1994), but not Monyer et al. (1994), NR2C mRNA is also expressed in the external granular layer during the first postnatal days. NMDA receptors have been demonstrated to be critically involved in granule cell migration (Komuro and Rakic, 1993; Rossi et al., 1993). Since NR2B is transiently expressed by granule cells during the period of migration, one may speculate that receptors made with NR2B and NR1 (and possibly NR2A) may act as 'migration receptors'. The presence of multiple NMDA receptors in granule cells is consistent with the presence of multiple NR2 subunits and has recently been demonstrated with patchclamp methods (Farrant et al., 1994): Pre-migratory and migratory granule cells were shown to express NMDA receptor channels with conductancy properties of recombinant NMDA receptors formed by co-expression of NR 1 and NR2A or NR2B. Mature post-migratory cells, in addition, express 'low-conductance' NMDA receptor channels, which have the properties of NMDA receptors with NR2C (Monyer et al., 1994). Basket, stellate cells, Golgi cells and neurons in the cerebellar nuclei express NR2D mRNAs (Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1994). Neurons of the cerebellar nuclei also produce NR2A mRNA, but it is not clear whether NR2A and NR2D producing cells reflect distinct neuronal populations. It should be noted that NMDA receptors composed of NR1 and NR2D have very slow deactivation kinetics (roll = 4.8 s) (Monyer et al., 1994) and, therefore, may modulate the cell activity during many seconds even when the receptor channel has been briefly activated by glutamate (see discussion Monyer et al., 1994). Quinlan and Davies (1985) have provided indirect physiological evidence for the presence of NMDA receptors in stellate and basket cells, by showing that NMDA may induce inhibition of Purkinje cells. Also neurons of the deep cerebellar nuclei have been shown to display prominent NMDA responses in cerebellar slice cultures (Audinat et al., 1990).
,Sg~,'
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Fig. 51. Bright-field micrographs showing cellular distributions of the NMDA receptor channel subunit mRNAs in the cerebellar cortex of the adult mouse: (A) el (mouse homologue of NR2A) mRNA; (B) e2 (NR2B); (C) e3 (NR2C); (D) e4 (NR2D); and (E) ~'1 (NR1). Each photograph in the figure was taken from lobule V of the cerebellar vermis, and the expression patterns of the respective subunit mRNAs are identical to those in remaining regions of the cerebellum. Sections were counter-stained with toluidine blue. Arrows indicate cell bodies of the Purkinje cells. Gr, granular layer; Mol, molecular layer. Scale bar = 50 r Watanabe et al. (1994).
71
Ch. I
J. Voogd, D. Jaarsma and E. Marani
The presence of NMDA receptors on Purkinje cells has been disputed. Some studies have supported the presence of NMDA-receptors on Purkinje cells (Sekiguchi et al., 1987), but in most studies no evidence of NMDA-receptors on Purkinje cells has been found (e.g. Audinat et al., 1990; Perkel et al., 1990; Llano et al., 1991; Farrant and Cull-Candy, 1991). Studies of Krupa and Cr6pel (1990) and Rosenmund et al. (1992) have indicated that NMDA receptors are present on most Purkinje cells during early post-natal life, but disappear with age. Both in situ hybridisation in rat and mouse and immunocytochemical studies in rat have shown that the NR1 subunit is expressed at high levels by Purkinje cells (Brose et al., 1993; Akazawa et al., 1994; Monyer et al., 1994; Petralia et al., 1994a; Watanabe et al., 1994). Petralia et al. (1994a) further demonstrated that NRl-immunoreactivity occur at the post-synaptic membrane specialisations in Purkinje cell spines. With respect to the NR2 subunits, Akazawa et al. (1994) found that rat Purkinje cells may express NR2D mRNA until post-natal day 8 and thereafter express low levels of NR2A mRNA. Accordingly Petralia et al. (1994b) observed that Purkinje cells display a low level of NR2A/B immunoreactivity, also in the post-synaptic densities of Purkinje cell dendritic spines, indicating that low levels of 'NR1-NR2A' receptors may be present at parallel fiber or climbing fiber synapses. Watanabe et al. (1994), however, found that mouse Purkinje cells only express low levels of NR2B (indicated as e2, which is the mouse homolog of NR2B) until one day postnatally, but not at any later stage, whereas according to Monyer et al. (1994) Purkinje cells do not produce any NR2 mRNA at any age. One may conclude from the in situ hybridisation and immunocytochemical data, that in spite of the presence of high levels of NR1 subunit, Purkinje cells both during development and in adulthood are likely to express none or only low amounts of functional NMDA receptors, which is in line with the aut0radiographic data. NR1 subunits can also form receptor-channel complexes in the homomeric configuration, but these channels produce very small currents and are, therefore, unlikely to contribute significantly to the excitatory actions of glutamate in Purkinje cells (Moriyoshi et al., 1991). Orphan receptors
51 and 52 are two related subunits isolated by homology screening. 51 is not produced in the rodent cerebellum, but 52 is selectively expressed by Purkinje cells (Araki et al., 1993; Lomeli et al., 1993). The subunit protein is distributed throughout the somatodendritic domain of the Purkinje Cells, similar to other glutamate receptor subunits (Araki et al., 1993). The function of S1 and 52 is not yet understood. The subunit protein does not bind glutamate receptor agonists and does not aggregate into functional receptors (Lomeli et al., 1993).
3.3.2. Metabotropic glutamate receptors Metabotropic glutamate receptors are coupled to G-proteins and modulate intracellular second messenger systems. The metabotropic glutamate receptors consist of at least seven subtypes that can be subdivided into three subgroups on the basis of sequence homology, agonist selectivity, and second messenger system (Nakanishi, 1992; Tanabe et al., 1992): (1) mGluR1 and mGluR5, that are coupled primarily to activation of phosphoinositide hydrolysis and are activated by quisqualate (QA) and 1S,3R-aminocyclopentane dicarboxylate (1S,3R-ACPD); (2) mGluR2 and mGluR3, that are coupled to inhibition of the cAMP cascade, are sensitive to pertussis toxin, and are activated by 72
The cerebellum." chemoarchitecture and anatomy
Ch. I
1S,3R-ACPD, but are insensitive to QA; and (3) mGluR4, mGluR6 and mGluR7, which are also coupled to inhibition of the cAMP cascade, and are potently activated by L-2-amino-4-phosphonobutyrate (L-AP4), but are insensitive to QA and 1S,3R-ACPD. Metabotropic glutamate receptors have been implicated in multiple neuronal processes including modulation of transmitter release, plasticity phenomena such as long term potentiation and long term depression, and other long term changes of neuronal functions (see Schoepp, 1994 for a review). With the exception of mGluR6 that is expressed only in retina, all metabotropic receptors are expressed in the cerebellum. mGluR1 mRNA is expressed to some extent by most cerebellar neurons (Shigemoto et al., 1992), but is found at very high levels in Purkinje cells. Immunocytochemistry shows that the mGluR1 protein is localized in the spines of Purkinje cell dendrites (Fig. 52) (Martin et al., 1992; Baude et al., 1993; Shigemoto et al., 1994). Dense mGluR1 immunostaining is also associated with the brushes of unipolar brush cells (Jaarsma, Mugnaini, Shigemoto et al., in preparation). Interestingly, mGluR1 immunostaining is not associated with the post-synaptic region of the giant mossy fiber-unipolar brush cell synapses, but instead, occurs at very high levels in spiny appendages and small branchlets that emanate from the dendritic stem that do not have synaptic specialisations (see Mugnaini et al., 1994). Recently workers from Somogyi's group (Baude et al., 1993; Nusser et al., 1994) demonstrated, with immunogold techniques, that mGluR1 immunoreactivity in Purkinje cell spines (as well as in other neurons) was never localized to the postsynaptic membrane specialisations of the synapses, but was associated with perisynaptic and extrasynaptic regions. This is in marked contrast with ionotropic glutamate receptor subunits that are primarily located at the postsynaptic membrane (Fig. 49) (Nusser et al., 1994). It has been proposed that, as a consequence of its peri-and extrasynaptic localization, mGluR1 is only activated during high frequency stimuli, because low frequency stimuli may not release enough glutamate to reach the perisynaptic receptors at significant concentrations (Baude et al., 1993; Nusser et al., 1994). It was originally reported by Kano and Kato (1987) that a QA/transAPCD-sensitive glutamate receptor is critically involved in the induction of long term depression (LTD) of parallel-fiber-Purkinje cell synapses, a cerebellar paradigm of synaptic plasticity that is induced following repetitive stimulation of parallel fibers in conjunction with climbing fiber input (Ito, 1989; Linden and Connor, 1993). Recently strong evidence has been obtained that mGluR1 plays a major role in cerebellar LTD: (1) the induction of LTD could be inhibited with antiserum that inactivated mGluR1 in an in vitro model of LTD (Shigemoto et al., 1994); and (2) LTD could not be induced in a mutant mouse lacking mGluR1 (Aiba et al., 1994). In these animals the anatomy of the cerebellum was not overtly disturbed. The Purkinje cells showed some minor morphological alterations, but had normal excitatory responses upon parallel fiber and climbing activation. Interestingly, the animals showed characteristic cerebellar symptoms such as ataxic gait and intention tremor, which suggest that mGluR1, possibly through its role in LTD, is important in cerebellar function. The mGluR5 receptor is selectively localized to a subpopulation of Golgi cells with the receptor protein localized throughout the somato-dendritic domain of the cells (Abe et al., 1992b; Shigemoto et al., 1993). Also mGluR2 and mGluR3 mRNA's are selectively expressed by Golgi cells (Ohishi et al., 1993, 1994), although mGluR3 may also occur in glial cells (Ohishi et al., 1994; Tanabe et al., 1993). Using an antibody selective for mGluR2 and mGluR3, Ohishi et al. (1994) found that mGluR2/3 immunoreactivity was strongest in Golgi axon terminals in the glomeruli (Figs 53 and 54). The Golgi axon terminals are not in close contact with mossy fibers, but the distance between mossy fiber 73
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 52. A. Photomicrograph of semithin 3/lm thick plastic section of the nodulus of rat cerebellar cortex immunostained with an antibody against the carboxyterminus of the metabotropic glutamate receptor, mGluRl~z (antibody A52) (Shigemoto et al. 1994). Immunoreaction product in the molecular layer (ml) has a punctate distribution. Very little staining occur in the perikarya and primary dendrites of Purkinje cells (PC). In the granular layer moderate and dense immunoreactivity is localized to the perikarya and 'brushes' (open arrows) of unipolar brush cells (asterisks in cell nucleus), respectively. Bar = 20/lm. B. Electron micrograph of the molecular layer showing that puncta within the cerebellar molecular layer correspond to mGluRlctimmunoreactive spines (arrows) of Purkinje cells. Curved arrow point to an immunoreactive spine branching from an unlabelled Purkinje cell dendrite (PCd). pf, parallel fiber terminal. Bar = 0.5/lm. Courtesy of Jaarsma, Dino, Mugnaini, Ohishi and Shigemoto.
terminals and Golgi axon terminals is usually less than 1 ~tm, and it is possible that glutamate released from mossy fibers may diffuse into the intercellular space to activate mGluR2/3 on the Golgi cell axons (see Section 3.2.1.). mGluR2/3 in Golgi axon terminals may be involved in the regulation of inhibitory neurotransmitter release, which would imply that mossy fibers may directly influence inhibitory neurotransmission on granule cell dendrites (e.g. see discussion Ohishi et al., 1994). Both mGluR5 and mGluR2/3 antibodies immunostain subpopulations of Golgi cells (Shigemoto et al., 1993; Ohishi et al., 1994). mGluR2/3 immunoreactive Golgi cells constitute three-quarters of the total population of Golgi cells (defined as GABApositive, parvalbumin-negative cells of the granular layer), whereas only a small population of Golgi cells appears mGluR5 positive. Large mGluR2/3-positive Golgi cells were frequently encountered in the Purkinje cell layer and the superficial part of the granular layer (Fig. 53), and at least in part may represent the candelabrum cells as described by Lain6 and Axelrad (1994, see section 2). In contrast, large mGluR5-positive immunoreactive Golgi cells were mostly found deeper in the granular layer. This indicates that mGluR2/3 and mGluR5 positive Golgi cells represent different subpopulations of Golgi cells. It remains to be determined whether mGluR2/3 and mGluR5 positive cells are entirely exclusive or overlapping populations, and whether yet another 74
The cerebellum." chemoarchitecture and anatomy
Ch. I
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Fig. 53. a. Immunocytochemicallocalization of mGluR2/3 in a parasagittal section through the vermis of rat cerebellum. The most intense immunoreactivity is seen in the granular layer. Staining in the molecular layer is associated with Golgi cell dendrites. B. Drawing of the section shown in (a) showing presumed Golgi cell bodies with mGluR2/3 immunoreactivity (closed circles) and those without mGlu2/3 immunoreactivity (open circles). G, granular layer; M, molecular layer; P, Purkinje cell layer; W, white matter. Bar = 500 #m. Ohishi et al. (1994).
subpopulation exists that is both mGluR5 and mGluR2/3 negative. It is important to realize that mGluR2/3 as well as mGluR5 immunoreactivity is detected in both small and large Golgi cells, and that, therefore, the segregation of Golgi cells into mGluR2/3 respectively mGluR5-positive and negative cells does not correspond to previous classifications which were based on size (e.g see Palay and Chan-Palay, 1974; but also section 3.6.2.). The L-AP4-sensitive mGluR, m G l u R 4 is expressed at high levels by granule cells (Kristensen et al., 1993; Tanabe et al., 1993), whereas mGluR7 m R N A is produced by Purkinje cells (Okamoto et al., 1994; Saugstadt et al., 1994). Physiological data indicate that the L-AP4 sensitive mGluRs are predominantly located presynaptically, where they may act as autoreceptors to regulate glutamate release. Studies in turtle suggest the presence of a L-AP4-sensitive presynaptic glutamate receptor at the parallel fiber-Purkinje cell synapse (Larson-Prior et al., 1989). Thus, possibly, mGluR4 is located presynaptically on parallel fiber boutons. The ultrastructural localisation of m G l u R 4 and m G l u R 7 remained to be determined at the time of writing this manuscript. 75
Ch. I
J. Voogd, D. Jaarsma and E. Marani
3.4. NITRIC OXIDE: THE CEREBELLAR LOCALIZATION OF NITRIC OXIDE SYNTHASE, GUANYLATE CYCLASE AND CYCLIC GMP Nitric oxide (NO) (see Dawson et al., 1992 and Vincent and Hope, 1992 for reviews) has gained importance as an intracellular and diffusible intercellular messenger in the cerebellum, since the demonstration by Garthwaite et al. (1988, 1989) that N-methyl-Daspartate (NMDA) receptor activation caused an increase in cyclic guanosine 3',5'monophosphate (cyclic GMP) in the cerebellum by stimulating the release of a diffusible messenger with properties similar to a endothelium-derived relaxing factor which was identified as NO. They considered granule cells as the main source of NO and glial cells as the main target for the activation of soluble guanylate cyclase by NO and the production of cyclic GMP. The enzyme nitric oxide synthase (NOS), that produces NO and citrullin from arginine, occurs as several isoenzymes (Knowles et al., 1989). Type I NOS is a constitutive, calcium and calmodulin-dependent enzyme, present in neurons and, possibly, in glia. Type II NOS is calcium-independent and can be induced in macrophages and glial cells by exposure to bacterial lipopolysaccharide (Galea et al., 1992; Murphy et al., 1993). Type III NOS is the endothelial iso-enzyme. NOS-I, II and III are produced by different genes (Bredt et al., 1991; Lamas et al., 1992; Xie et al., 1992; Lowenstein et al., 1992; Lyons et al. 1992; Ogura et al., 1993). NOS displays NADPH-dependent diaphorase
Fig. 54. Ultrastructural localization of mGluR2/3 immunoreactivity in the granular layer of rat cerebellar cortex. Dense immunoreaction products accumulate in axon terminals of Golgi cells, which often make synaptic contacts (curved arrows) with possible granule cell dendrites around a mossy fiber terminal (MT) in the cerebellar glomerulus. Bar = 0.5 r Ohishi et al. (1994).
76
The cerebellum." chemoarchitecture and anatomy
Ch. I
activity and can be demonstrated in aldehyde-fixed tissue by NADPH-dependent reduction of tetrazolium salts to visible formazans (Hope et al., 1991). NOS-I has been localized with antisera to the purified enzyme (Bredt et al., 1990) and by in situ hybridization to NOS-I mRNA (Bredt et al., 1991) in basket cells and in granule cells and their axons, where NOS-I is co-localized with NADPH diaphorase (Bredt et al., 1991; Vincent and Kimura, 1992; Schmidt et al., 1992; Schilling et al., 1994). NADPH-diaphorase-positive granule cells are distributed in a symmetrical pattern of heavily stained clusters, separated by granule cells that were stained weakly, or not at all (Fig. 55) (Schilling et al., 1994). The NADPH-diaphorase-positive granule cell clusters were correlated with the Zebrin pattern in the overlying molecular layer by Hawkes and Turner (1994). A sparse axonal network and a few cells were stained in the cerebellar nuclei (Vincent and Kimura, 1992). Schmidt et al. (1992) also found weak NOS-I immunoreactivity in Bergmann glia and astrocytes where it co-localized with NADPH-diaphorase. NOS-II was expressed by astrocytes in lipopolysaccharide-stimulated cultures. These cells also double-label for NADPH-diaphorase (Galea et al., 1992). Guanylate cyclase, the enzyme responsible for the synthesis of cyclic GMP from guanosine triphosphate, was localized with immunofluorescence in Purkinje, granule stellate and Golgi cells and in oligodendrocytes, astroglia and Bergmann glial fibers of the cerebellar cortex of the rat (Zwiller et al., 1981). The localization in Purkinje and granule cells and in astrocytes was confirmed by Ariano et al. (1982), Nakane et al. (1983) and Schmidt et al. (1992). Bergmann glia and small cells in the molecular and granular layers were weakly stained. Expression of the soluble guanylyl cyclase mRNA in rat cerebellum was moderate in Purkinje, basket, stellate and Golgi cells, weak in granule cells, but could not be demonstrated in glial cells (Matsuoka et al., 1992, see also Burgunder and Cheung, 1994). Cyclic GMP was located with immunohistochemical methods in Bergmann glia (Cumming et al., 1977, 1979; Chan-Palay and Palay, 1979; Ariano et al., 1982) and in a subpopulation of stellate and basket cells (Chan-Palay and Palay, 1979). Its preferential localization in Bergmann glia and cerebellar astrocytes was stressed by Berkelmans et al. (1989) and De Vente et al. (1989, 1990), using antibodies against conjugates of cyclic GMP and activation of cyclic GMP by sodium nitroprusside in slices of rat cerebellum. They observed a patchy distribution of the reactive Bergmann glia in the molecular layer (Fig. 56). Purkinje and granular cells remained unstained. Immunoreactive varicose (mossy?) fibers and astrocytes and/or Golgi cells were observed in the granular layer. Owing to the differential localizations of NOS, guanylate cyclase and cyclic GMP, the cellular basis for the actions of cerebellar NO remains difficult to establish. Basket and stellate cells appear to be the only cell types that can be stimulated by NMDA receptors (Quinlan and Davis, 1985; Hussain et al., 1991) that contain both NOS-I, guanylate cyclase and cyclic GMP. It has been suggested that carbon monoxide (CO) is an activator of soluble guanylyl cyclase in Purkinje cells. Heme oxygenase-2, which degrades heme to biliverdin and releases carbon monoxide in the process, was shown to be co-localized with guanyl cyclase in rat Purkinje and granule cells with in situ hybridization histochemistry (Verma et al., 1993). 3.5. ADENOSINE, 5'-NUCLEOTIDASE AND ADENOSINE DESAMINASE Adenosine-like immunoreactivity was found in rat Purkinje cells, using polyclonal anti77
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 55. Coronal section through the copula pyramidis (lobule VIII). In the adult rat granule cells in the lateral tip of the copula pyramidis show strongly reduced staining intensity for NADPH-diaphorase, in contrast to the medial copula, where a cluster of heavily stained granule cells can be seen. g, granular layer; m, molecular layer. Scale bar = 200/lm. Schilling et al. (1994).
sera against a conjugate of the adenosine derivative laevulinic acid (Braas et al., 1986). Staining was present in the cell soma outside the nucleus, extending in the dendrites. Weaker staining was observed in the granular layer. Adenosine is co-released with adenosine triphosphate (ATP) and certain neurotransmitters (Richardson and Brown, 1987). High affinity uptake sites for adenosine are present in all layers of the cerebellar cortex (Marangos et al., 1982; Nagy et al., 1985; Biss6rbe et al., 1985). Steady state concentrations of adenosine are maintained through the activities of only three enzymes, 5'-nucleotidase (5'-N), adenosine kinase and adenosine deaminase. Adenosine kinase and adenosine deaminase were located mainly in the soluble fractions of rat cerebellar homogenates, whereas 5'-N was present in subcellular fractions (Philips and Newsholme, 1979), mainly in the synaptosomal fraction (Marani, 1977). Adenosine deaminase-immunoreactivity in rat cerebellum was present with one out of five polyclonal sera prepared by Nagy et al. (1988). Staining was present in most Purkinje cells with a variation in intensity. Staining was observed in the Purkinje cell axons and terminals in the cerebellar and vestibular nuclei. The localization of 5'-N will be discussed below. Adenosine blocks the parallel fiber-induced simple spike discharge in Purkinje cells (Kostopoulos et al., 1975) but not the climbing fiber-mediated synaptic transmission (Kocsis et al., 1984). The effect of adenosine is presynaptic and is mediated by A1adenosine receptors that are located on parallel fibers. A 1-adenosine receptors are coupled to pertussis toxin-sensitive G proteins and inhibit adenyl cyclase. Activation of A 1-adenosine receptors decreases transmitter release from the terminals (Dolphin and Prestwich, 1985, see Fredholm and Dunwiddie, 1988, for a review). The presence of Al-adenosine receptors on parallel fibers was demonstrated autoradiographically by 78
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 56. cGMP-immunostaining of adult rat cerebellum. A. Section of a cerebellar slice that was incubated with cyclic GMP antiserum, in the presence of 1 mM isobutyl-methylxanthineto inhibit phosphodiesterase activity and 10 r nitroprusside and post-fixed in paraformaldehyde. B. Same areas of the same section as shown in (A) after removal of cGMP-immunostaining using the methods of Tramu et al. (1978) and reincubation of the section with glial fibrillary acidic protein-antiserum. Note presence of cGMP-immunoreactivity in Bergmann glial fibers and in thin varicose fibers (arrows in A) and in astrocytes or Golgi cells (arrow head in A). Bars = 100 r De Vente et al. (1989).
Goodman and Snyder (1982) and Goodman et al. (1983) using specific binding of [3H]cyclohexyladenosine ([3H]CHA) and Weber et al. (1990), using the antagonist [3H]DPCPX (Fig. 57). Binding was highest over the molecular layer, with lower concentrations in the granular layer. Binding was absent in the granuloprival cerebellum of 'weaver' mice (Goodman et al., 1983; Wojcik and Neff, 1982, 1983). Al-adenosine receptors were present over the entire molecular layer; no bands of high activity, corresponding to the 5'-N pattern, were observed (Fastbom et al., 1987). Adenosine is released in a Ca2+-dependent manner by K + stimulation from rat cerebellar slices (Cu6nod et al., 1989; Do et al., 1990). The stimulated release of adenosine was decreased by 60-70% in vermis and hemisphere, in slices from 3-acetylpyridine-treated rats, which may indicate that the released adenosine, at least in part, is released by climbing fibers. The 'climbing fiber-dependent' adenosine release, however, occurs with some time delay after the K + stimulus. Adenosine, therefore, has been proposed to be derived from extracellular degradation of released nucleotides by ectonucleotidases. Inhibition of 5'-nucleotidase (5'-N) by ~,fl-methylene-ADP and GMP, indeed, decreased stimulated adenosine release by 50-60%. 5'-Nucleotidase (5'-N) is an integral glycoprotein of the cellular plasma membrane in a wide range of animal cells. Its functional role is still unclear. 'Possibilities .... include recovery of purines and pyrimidines from the extracellular space, the extracellular formation of neuromodular adenosine from released nucleotidases and non-enzymatic functions related to the interaction of 5'-nucleotidase with compartments of the cytoskeleton and extracellular matrix' (Schoen et al., 1987). 5'-N catalyses the production of adenosine by the hydrolytic cleavage of 5'-nucleotide monophosphates (i.e. adenosine5'-monophosphate). The development of 5'-N in the cerebellum was studied by Schoen et al. (1987, 1988, 1990). 5'-N in the molecular layer of mouse cerebellum is distributed in positive and negative parasagittal bands (Scott, 1963). The distribution of cerebellar 5'-N has been reviewed by Marani (1986). Its zonal distribution in mice is very similar to the distribution of the m a b Q l l 3 (Zebrin)-positive dendrites of Purkinje cells in the molecular layer (Marani, 1986; Eisenman and Hawkes, 1989) (Figs 58A, 130, 131, 135) (Section 6.1.4.). The 79
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to a section of rat cerebellum. Left. Photomicrograph of a pyrorine Y-stained section of rat cerebellum showing the molecular layer (ML), the Purkinje cell perikarya (PC), the granule cell layer (GL), and some white matter ( W M ) . R i g h t . Darkfield photomicrograph of the tissue section incubated with 0.8 nM [3H]DPCPX and apposed to nuclear emulsion-coated coverslips. The silver grains are from the same area shown on the left. Note the high density of A~ adenosine receptors in the molecular layer and moderate labelling in the granule cell layer. The white matter and the Purkinje cell bodies showed background levels of labelling. Bar = 5 0 / l m . Weber et al. ( 1 9 9 0 ) .
reaction product in the histochemical procedure of Scott (1964, 1965, 1967) is uniformly distributed within the bands of high 5'-N activity in the molecular layer. In the hemisphere the staining in some of the 5'-N bands is less uniform and assumes the aspect of radially disposed striations. Hess and Hess (1986) tentatively identified these striations as the processes of the Bergmann glia. These authors found 5'-N in the molecular layer of Purkinje cell-deficient mice (pcd and nr strains) to be reduced and the residual enzyme activity to be localized in approximately the same position as the surviving Purkinje cells. This would imply that the expression of 5'-N in Bergmann glia is regulated by the adjoining Purkinje cells. Marani's electron microscopic enzyme-histochemical studies (Marani, 1981, 1982a,b, 1986), favoured a localization of the enzyme in the subsurface cisterns and the spine apparatus of the spines of the Purkinje cell dendrites and within boutons of parallel fibers (Fig. 58B-D). A non-zonal distribution of 5'-N in the somata of Purkinje cells and other large cells of the cerebellar cortex was observed when different substrates for the enzyme histochemical reaction for 5'-N were used (Scott, 1967; Marani, 1982a and b, 1986; Hess and Hess, 1986). According to Marani this represents a rest-activity of non-specific phosphatases, that disappears when the appropriate inhibitors are used. The presence of 5'-N in parallel fibers was disputed by Hess et al. (1983), who showed that 5'-N remains at significant levels in the molecular layer in agranular 'weaver' 80
The cerebellum." chemoarchitecture and anatomy
Ch. I
cerebellum. According to Kreutzberg et al. (1978) 5'-N is predominantly associated with glial membranes. Schoen et al. (1987, 1988), used monoclonal and polyclonal sera directed against rat liver 5'-N in the localization of cerebellar 5'-N in addition to the enzyme-histochemical techniques. They found the enzyme to be situated at the outer border of the plasma membranes of Bergmann glial fibers in the molecular layer, astroglial endfeet around blood vessels and glial processes surrounding Purkinje and granule cells (Fig. 59). They were unable to confirm Marani's observations of an intracellular localization of the enzyme. The study of Schoen et al. (1987) was done in rats, which do not have the longitudinal band pattern of 5'-N with their antibody directed against this enzyme. Balaban et al. (1984) observed an increase of cerebellar 5'-N in the P2 (synaptosome) fraction after climbing fiber activation with harmaline in rats (Fig. 60). Harmaline synchronizes the discharge in climbing fibers from certain parts of the inferior olive and induces a rhythmic tremor (Sj61und et al., 1977, 1980). Two different climbing fiber induced effects, therefore, may be involved in adenosine-mediated blockade of transmission in parallel fiber-Purkinje cell synapses: an increased release of nucleotides and an increase of cerebellar 5'-N. Loss of climbing fiber-induced 5'-N and/or adenosinemediated blockade of transmission in the parallel fiber-Purkinje cell synapses (see Marani, 1986) would explain the long-term increase of simple spike activity that occurs when complex spikes are suppressed by destruction or inactivation of the inferior olive in rats (Colin et al., 1980; Montarolo, 1982). Bloedel and Lou (1987), however, observed a short-term facilitation of transmission in the mossy fiber-parallel fiber-Purkinje cell pathway on stimulation of climbing fibers in the cat. This difference may be due to species-dependent differences in 5'-N mediated formation of adenosine or to a facilitation at the level of mossy fiber-granule cell synapse. If the formation of adenosine is largely dependent on the degradation of nucleotides by 5'-N, the zonal distribution of this enzyme in different species and of the climbing fibers which promote their release would be of crucial importance (see Marani (1986) and Section 6.1.4.). 3.6. INTERNEURONS OF THE CEREBELLAR CORTEX Stellate, basket and Golgi cells are inhibitory (Eccles et al., 1964a, 1966a,b,c,d, 1967). It was against this background that Uchizono (1965) (see also Uchizono, 1969 for a review) formulated and tested his hypothesis that excitatory and inhibitory axon terminals in aldehyde fixed tissue can be distinguished by the shape of their synaptic vesicles (Fig. 61). Inhibitory boutons contain flattened vesicles (F-type boutons) and excitatory boutons contain spherical vesicles (S-type boutons). Earlier Gray (1959) distinguished two types of synaptic junction, which were also supposed to represent the excitatory and inhibitory synapse (Landis and Reese, 1974). Gray type 1 junctions are characterized by a widening of the synaptic cleft that contains dense material and a distinct asymmetry caused by the presence of a dense undercoating of the postsynaptic membrane. It was considered to be excitatory. The thickening of the pre- and postsynaptic membranes in the Gray type 2 junction is symmetrical and the cleft is narrow; this type was supposed to be inhibitory. According to Uchizono (1969) there is an excellent correlation in the cerebellar cortex of the cat of S-type boutons with Gray's type 1 synaptic junctions and of F-types with a synapse of Gray's type 2. For the excitatory connections of the mossy and climbing fibers and for the parallel fiber-Purkinje cell synapse the correlation with S-type terminals and Gray 1 synaptic junctions still is valid. For the terminals of the inhibitory interneurons of the cerebellar cortex (Golgi cells: pleomorphic vesicles, synap81
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 58. Light and electron micrographs of incubations for 5-nucleotidase according to Scott (1967). A. Detail of the light microscopic location of 5'-nucleotidase in uvula (IX) and pyramis (VIII). B. Electron microscopic location of 5'-nucleotidase reaction products in the subsurface cisternae of a Purkinje cell dendrite. C. Electron microscopic localization of 5'-nucleotidase in the spine apparatus of Purkinje cell dendritic spines (asterisks). D. Localization of reaction product in a parallel fiber bouton, synapsing on a Purkinje cell dendritic spine. Bars in A = 1 mm, in B,C = 0.5 ~tm, in D = 0.25 ~tm. Marani (1977).
82
The cerebellum. chemoarchitecture and anatomy
Ch.I
Fig. 59. 5'-Nucleotidase immunohistochemical staining of rat cerebellum. A. Immunofluorescence. B. PAPmethod. Enzyme activity is predominantly found within the molecular layer on Bergmann glial fibers (long arrows). Purkinje cells are surrounded by fine rims of reaction product (small arrows). Within the granular layer 5'-nucleotidase activity is diffusely scattered between granule cells (arrow heads). Vibratome sections. C. Longitudinally sectioned Bergmann glia cell processes (B) of the molecular layer of rat cerebellum. Fine DAB reaction product is located on adjacent membranes of these processes (arrows). Bars in A,B = 50 ~tm, in C = 0.5/lm. Schoen et al. (1987).
tic junction resembles Gray type 1; basket cells: ellipsoid, irregular and spherical vesicles, Gray type 2; stellate cells: flattened vesicles, Gray type 2) there is a greater variation in morphology (Palay and Chan-Palay, 1974). Cell bodies and terminals of the Golgi, basket and stellate cells can be labelled with selective uptake of [3H]GABA (H6kfelt and Ljungdahl, 1970, 1971; Schon and Iversen, 1972), immunostaining with antibodies against GAD (Saito et al., 1974; McLaughlin et al., 1974; Oertel et al., 1981b; Mugnaini and Oertel, 1985) and in situ hybridization for G A D 6 5 and G A D 6 7 (Wuenschell et al., 1986; Julien et al., 1987; Esclapez et al., 1993; Feldblum et al., 1993). They are also immunostained with antibodies against conjugates 83
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Fig. 60. Harmaline-induced changes in 5'-nucleotidase (5'-N) activity of cerebellar fractions in rats with an intact inferior olive (vehicle injection on day 1) or a 3-acetyl-pyridine (3-AP) olivectomy. Data are represented as means + S.E. for 8 animals in each group. The changes in 5'-N levels in each fraction (CH-crude homogenate, P 1, P2, P3 and $3) are shown for intact and 3-AP olivoectomized animals that served as either controls (C) or received harmaline injections (H) 45 min prior to decapitation. Harmaline evoked an increase in 5'-N activity in the CH and P2 fraction of rats with an intact olive; it evoked a decrease in the activity after 3-AP olivectomy. No significant effects appear in the P1, P3 or $3 fractions in either intact or 3-AP olivectomized animals. Balaban et al. (1984).
of GABA (Figs. 15, 40, 62 and 63) (Ottersen and Storm-Mathisen, 1984a,b; Somogyi et al., 1985; Aoki et al., 1986; Gabbott et al., 1986; Matute and Streit, 1986; Ottersen et al., 1987). 3.6.1. Stellate and basket cells Stellate cells are located in the entire, and basket cells in the deep part of the molecular layer. The dendritic arborizations of both cell types are flattened in a plane perpendicular to the long axis of the folium. Both receive synapses from parallel fibers on their dendrites. The axon of the stellate cell terminates on shafts of dendrites from Purkinje, basket, Golgi and stellate cells in the molecular layer. The immunoreactivity of stellate cells for antibodies against conjugates of taurine (Madsen et al., 1985; Magnusson et al., 1988; Ottersen etal. 1988b) is low. This is in contrast with the selective uptake by stellate and basket cells of [3H]taurine and the immunoreactivity of these cells with antibodies against CSADS, the synthesizing enzyme of taurine (Chan-Palay et al., 1982a,b) (see Section 3.1.2.). The localization of CSADS in basket and stellate cells has, however, been disputed, since Almerghini et al. (1991) found CSADS-immunoreactivity to be exclusively localized in glial cells and not in neurons of the cerebellar cortex. Basket axons have been studied extensively (Palay and Chan-Palay, 1974). They extend in a direction across the axis of the folium (Fig. 13) and terminate with ascending 84
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Fig. 61. Size and shape analysis of synaptic vesicles in S-type and F-type synapse. Diameters of both major and minor axis in each synaptic vesicle in both types of synapses were measured. Ordinate shows the length of the major axis, while the abscissa that of the minor axis of vesicles in each type of synapse. Diameters of vesicles in S-type synapse (white) are distributed around the 45-degree line between ordinate and abscissa, while those in F-type synapse (black) of both white and black circles indicates the relative frequency of occurrence. Elongation index ratio of average length of major versus minor axis of vesicles in S-type synapse was about 1.2, while that of F-type synapse was about 1.7. Uchizono (1965).
branches on the primary dendrites of Purkinje cells and constitute the baskets surrounding the cell body that end in the pinceau around the initial part of the axon of the Purkinje cells. GABA-like immunoreactivity was present in boutons of stellate and basket cell axons on Purkinje cell dendritic shafts, in basket cell terminals on dendrites of stellate cells and on Purkinje cell somata (but not in all of them) and in some of the axons of the pinceau (Gabbott et al., 1986). No specific neurochemical properties seem to distinguish the basket cells from the stellate cells. According to Somogyi et al., (1986) GABA-like immunoreactivity is weaker in stellate cells than in basket and Golgi cells. Basket and stellate cells are immunoreactive for antibodies against parvalbumin, like the Purkinje cells (Fig. 31B). No reactivity for these antibodies or m R N A probes for parvalbumin was mentioned for the Golgi cells (Celio and Heizmann, 1981; Heizmann, 1984; Schneeberger et al., 1985; Endo et al., 1985; Braun et al., 1986; Kadowaki et al., 1993; Kosaka et al., 1993). Calretinin immunoreactivity is present in a subpopulation of stellate and basket cells in the cerebellum of the chicken, where it is co-localized with parvalbumin in some of the cells (Rogers, 1989). Immunoreactivity with antibodies against PKC ~, fl, g, e and possibly ~"is present in stellate and basket cells (Fig. 29, Table 1). The localization in basket and stellate cells of nitric oxide synthase, guanylyl cyclase and cyclic GMP has been reviewed in Section 3.4.
3.6.2. Golgi cells and Lugaro cells Golgi cells are located in the granular layer, and have been roughly subdivided into large Golgi cells, which are located in the superficial part of the granular layer and small Golgi cells located more deeply. The dendrites of both types extend into the molecular layer, where they are not confined to a single plane. The axons branch repeatedly to form a 85
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Fig. 62. Cerebellar cortex of cat reacted by the unlabelled antibody enzyme method. A and B. Serial semithin (1/lm) sections reacted under postembedding conditions with an anti-GABA serum (A) or with the same serum after solid phase absorption (B). Basket cells (BC) and Golgi cells (GC) are strongly immunoreactive, Purkinje cells (PC) and stellate cells (SC) reacted less strongly. GABA-immunoreactive terminals are present in all layers, but the terminals of basket cells around the Purkinje perikarya and in the pinceau (p) and the Golgi cell terminals in the glomeruli (gl) are especially strongly reacting. C-E. Preembedding demonstration of GABA and GAD in vibratome sections. The distribution of amino acid and its synthesizing enzyme are very similar, but perikarya of basket, stellate, and Golgi cells, and basket cell axons (ba) stain stronger for the amino acid than for G A D in animals not treated with colchicine. F and G. Electron micrographs of glomeruli demonstrating GABA (F) and GAD (G) in the terminals (asterisks) of Golgi cells. The dendritic digits of granule cells (diamonds) receive synapses (arrows) from the immunoreactive terminals as well as from the mossy fiber terminals (mft). Bar in A-E - 50/~m, in F and G = 0.5 r Somogyi et al. (1985).
86
The cerebellum." chemoarchitecture and anatomy
Ch. I
dense plexus in the granular layer. The terminals participate in the formation of the glomeruli where they make synaptic contact with the granule cell dendrites. Golgi cells in the upper molecular layer in rat and cat are selectively recognized by a monoclonal antibody (rat-303, Hockfield, 1987) (Figs. 66 and 69B). Uptake studies have shown that [3H]GABA and [3H]glycine uptake result in similar patterns of axonal labelling in the granular layer, in circular deposits resembling the periphery of the glomeruli, whereas no [3H]glycine labelling was found over the pericellular baskets of the Purkinje cells (Wilkin et al., 1981a). Concordantly it was shown that a large proportion of the Golgi cells, in addition to GABA, was also immunoreactive for antibodies against conjugates of glycine (Ottersen et al., 1987, 1988a; Campistron et al., 1986a; Takayama, 1994). In a high percentage of these glycine containing Golgi cells (40%) glycine-like and GABA-like immunoreactivity co-exist (Ottersen and StormMathisen, 1987). GABA and glycine-like immunoreactivity co-exist in most Golgi cell
~:.
.. ~!
Fig. 63. Photomicrographs of semithin (0.5 r tissue and test sections incubated with GABA antiserum 26 diluted 1:100 (A) or glycine antiserum 31 diluted 1:60 (B). Three of the four Golgi neurons that are glycine immunoreactive (thick arrows) are also stained with the GABA antiserum in the adjacent section; the fourth glycine-positive neuron (crossed arrow) is virtually immunonegative for GABA. Most if not all glomeruli (arrowheads) show GABA-like-immunoreactive as well as glycine-like-immunoreactive positive Golgi cell terminals. The molecular layer contains no glycine-like immunoreactive positive structures except for a few fibrous processes (small arrows in B). The terminals of the basket and stellate cells and their respective cell bodies (double arrowhead) are glycine immunonegative, but GABA immunopositive. Asterisks, Purkinje cell bodies. Other abbreviations: MO and GC, molecular and granule cell layers, WM, white matter. Bar = 50 r Ottersen et al. (1988a).
87
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J. Voogd, D. Jaarsma and E. Marani
Fig. 64. Electron micrograph showing GABA-LI in a glomerulus in the granular layer of the cerebellar cortex (rat). After incubation in rabbit primary antiserum, the section was treated with sheep anti-rabbit immunoglobulins bound to colloidal gold particles (16 nm). Axon terminals of the inhibitory Golgi cell (GO) show a high density of gold particles, whereas the densities of such particles over the mossy fibre boutons (MF) and granule cell dendritic digits (GC) are close to back-ground level. Scale bar = 0.5 ~tm. Ottersen and StormMathisen (1987).
terminals located at the periphery of the glomeruli (Somogyi et al., 1986; Ottersen et al., 1987) (Fig. 63) (Ottersen et al., 1988a) (compare Figs 41 and 65). A similar localization of GABA-like and glycine-like immunoreactivity was found in the rat and the baboon (Ottersen et al., 1987) (Fig. 65). Some displaced Golgi cells were present in the molecular layer of the baboon and many fibers running in the supraganglionic plexus in the direction of the long axis of the folium displayed glycine-like immunoreactivity. In summary the majority of the Golgi cells may use glycine in addition to GABA. Golgi cells in the rat stain strongly for acetylcholinesterase (Brown and Palay, 1972; 88
The cerebellum." chemoarchitecture and anatomy
Ch. I
Altman and Das, 1970). They share this property with a group of displaced Golgi cells located in the lower molecular layer in the rabbit (Ramon y Cajal, 1911; Spa~;ek, 1973). A subpopulation of Golgi cells in the cat and man, but not in rat or rabbit, is immunoreactive for choline acetyltransferase (see Section 3.10.1., Fig. 86). Certain Golgi cells in rats were found to be immunostained with antibodies against conjugates of somatostatin (Johansson et al., 1984; Vincent et al., 1985; Villar et al., 1989) (Fig. 20) or enkephalin (Schulman et al., 1981; Ibuki et al., 1988). Calcium binding proteins have not been found in Golgi cells, with the exception of a single antibody against calbindin-D28k, that stains Purkinje cells and Golgi cells in rat and human cerebellum (Garcia-Segura et al., 1984; Fournet et al., 1986), and the presence of calretinin in some Golgi cells (Arai et al., 1991; Floris et al., 1994). Of the PKC subtypes only PKC e' has been localized in Golgi cells (Wetsel et al., 1992). None of the immunoreactive subpopulations of Golgi cells seem to correspond exclusively to one of the anatomical subtypes distinguished by Palay and Chan-Palay (1974). Similarly, Golgi cell heterogeneity due to differential expression of metabotropic glutamate receptors, does not correspond to any anatomical subdivision (see Section 3.3.2.). It remains to be elucidated to what extent the different immunocytochemical markers overlap. Lugaro cells are fusiform cells located below the Purkinje cell layer, with dendrites arising from opposite poles of the cell and somata extending for long distances beneath the Purkinje cell layer. Lugaro cells are chemically distinct from Golgi cells in that they are selectively recognized by two monoclonal antibodies (cat 301 and 304, Sahin and Hockfield, 1990) (Fig. 67). Like Golgi cells they are immunoreactive for antibodies against GABA (Aoki et al., 1986). Ottersen et al. (1988a) included the Lugaro cells with the Golgi cells and found GABA-like and glycine-like immunoreactivity to be colocalized in Lugaro cells. Lugaro cells are assumed to primarily innervate the granule cells (Palay and Chan-Palay, 1974), although according to Fox (1959) their axon may enter the molecular layer. 3.6.3. Unipolar brush cells
Unipolar brush cells often have been interpreted as small Golgi cells. They are, however, non-GABAergic and non-glycinergic (Aoki et al., 1986; Mugnaini et al., 1994). Since they give rise to mossy fiber rosette-like terminals in the granular layer they are likely to be glutamatergic (Berthie and Axelrad, 1994; Rossi et al., 1995) (Section 3.2.1.). Unipolar brush cells can be distinguished from other granular layer neurons by a number of immunocytochemical markers (reviewed by Mugnaini and Floris, 1994). Following their original characterization as pale cells by Altman and Bayer (1977), they were first recognized by Hockfield (1987) using a monoclonal antibody against spinal cord gray matter, Rat-302 (Fig. 69). A study of Harris et al. (1993) showed that Rat-302 is directed against high molecular weight neurofilament protein, and that unipolar brush cells are strongly immunostained by several different antibodies against high molecular weight neurofilament protein. Unipolar brush cells are essentially unstained and moderately stained with antibodies against middle and low molecular neurofilament protein, respectively. Cozzi et al. (1989) and Munoz (1990) identified unipolar brush cells in the rat and human cerebellum, respectively, on the basis of their immunoreactivity to antisera against proteins of the secretogranin (or chromogranin) family. Unipolar brush have a relatively high density of large dense core vesicles, which in conjunction with the 89
Ch. I
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J. Voogd, D. Jaarsma and E. Marani
/111
,,
t
.,~i,~!,;,i~..i.!.,;.,!. ..... ~i~
-!
;i,~~!ii~.......... i,il %!::i: i:ii!i~;:~!~:i~i!'i:i;: !!il
Fig. 65. Details from sagittal vibratome section of baboon cerebellum (vermis) treated with the glycine antiserum diluted 1:250. A. One of the immunopositive Golgi cells shows a dendrite (small arrowheads) extending into the molecular layer, and a thinner process (axon?) which seems to engage in a glomerulus-like structure (large arrowhead). Note the presence of thick and thin immunoreactive fibers (thin arrows) in the deep part of the molecular layer, which also contains scattered immunoreactive neurons (thick arrow). B. Radial fibrous processes (small arrows) interpreted as Bergmann glia, show weak glycine-like immunoreactivity. Large arrow points at an immunostained Golgi cell. Asterisks indicates immunonegative Purkinje cell. C. Immunopositive Golgi-like neurons (arrows) occur in or just below the layer of the unstained Purkinje cells (asterisks). Large arrowhead indicates a stained Golgi cell slightly outside the plane of focus. Inset: Arrowhead, fusiform cell with horizontal dendrites situated directly beneath the Purkinje cells (asterisk). G, granular layer; M, molecular layer. Bar = 100 ~tm. Ottersen et al. (1987).
presence of chromogranins might indicate that they are neurosecretory of some kind of peptide (see discussion Mugnaini et al., 1994). Particularly dense immunostaining in unipolar brush cells was obtained with antibodies against calretinin (R6sibois and 90
The cerebellum." chemoarchitecture and anatomy
Ch. I .~i/./~.~...:~.!;:.~:,~.i:ii~;..:.~i:~:;~i~il;~.:ili~~:~:.i.~i~.:i:.:i.i.~:..~..i:,i:Zi:i~i,iii~' i;~.~;;.i:i~;i:i;~.~i,:;i~i~,i:i~i:. ..:"": ii~!!.! !~ ;i:~../.iii:~iI i~:i!i~:":::':;~"~"!
Fig. 66. A. Monoclonal antibody Rat-303 recognizes neurons in the granule cell layer (g), but not in the Purkinje cell (p) or molecular (m) layers of the rat cerebellar cortex. Scale bar = 100/~m. B. The morphology of Rat-303-positive neurons matches that described for Golgi II cells: a large ceil body emitting relatively stout dendrites from many points over the cell circumference (I3). Scale bar = 10 ~m. Hockfield (1987). R o g e r s , 1992; B r a a k a n d B r a a k , 1993; Floris et al., 1994). C a l r e t i n i n - i m m u n o r e a c t i v i t y in u n i p o l a r b r u s h cells was u s e d for a d e t a i l e d c h a r a c t e r i z a t i o n o f this cell type, a n d to
Fig. 67. Lugaro cells are molecularly distinct from Purkinje cells. In the pairs illustrated in A and B, the same fields were photographed under fluorescent optics separately for FITC (anti-calbinding, A) and Texas Red (Cat-301 in B). The blood vessel (triangle) passing through the field can be used to align the photographs. The cell type-specific antibody Cat-301 recognizes Lugaro, but not Purkinje cells in cat cerebellum, while anticalbindin recognizes Purkinje, but not Lugaro cells. Scale bar = 50/lrn. Sahin and Hockfield (1990). 91
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 68. Immuno-electron micrograph of an unipolar brush cell stained with antiserum to calretinin. The micrograph was obtained near the surface of the immunoreacted slice, as indicated by open areas in the tissue and over the cell nucleus (stars). The unipolar cell body has an irregular contour. The nucleus (N) shows a deep indentation (T) and the cytoplasm contains an array of ringlet subunits (R). The short dendrite forms an extensive synapse (arrows) with a calretinin-negative mossy fiber ending (mf). At the aspect opposite the synapse the dendrite contains numerous mitochondria. Granule cells (CG) are immunonegative. Bar = 1/~m. Floris et al. (1994).
92
The cerebellum." chemoarchitecture and anatomy
Ch. I
study its distribution in the cerebella of different species (Fig. 68) (Floris et al., 1994; Dino and Mugnaini, in preparation). 3.7. LOCALIZATION OF GABA RECEPTORS A N D GLYCINE RECEPTORS 3.7.1.
GABA A
receptors
GABA receptors can be divided in two main classes, GABAA and GABA~ (Bowery et al., 1980; Barnard et al., 1987; Sieghart, 1989; Sivilotti and Nistri, 1993; Mody et al., 1994). G A B A A receptors are ionotropic receptors that gate chloride channels. They are inhibited by bicuculline and picrotoxin and activated by muscimol. G A B A A receptors are the target of for a variety of drugs including benzodiazepine tranquilizers, and barbiturates. Benzodiazepines bind to a modulatory site that facilitates G A B A A receptor function. Different types of G A B A A receptors can be pharmacologically distinguished based on their differential sensitivity to benzodiazepine ligands and barbiturates (Doble and Martin, 1992). Originally the distribution of G A B A A receptors was studied autoradiographically with [ 3 H ] G A B A in the presence of the baclofen and the absence CaC12 or with [ 3 H ] m u s cimol. G A B A A receptors in rat and mouse cerebellum were found to be highly concentrated in the granular layer and relatively low in the Purkinje cell and molecular layers
iii!i!i......
iiii!i!, . . . . . . . .%i~i~?ii,i':,iil;i:,i:;i,:~ ..:~.:~:~;-~: .~::.
.........~.............. i~. :~+~s~ , i~ ~.:~:~
k:~iiii~
~.:~:~,
!',i~;:,i~i?i;i;i?:'iii~
....::....:::.~:: .?.::,i,~....::.:...:;~::~,~.~u:~;~:.u:~:::;-;...: ::~N~r:~;~-::":~:.:~
~ ; : , i~: i:~i ~~' !,~' i: ~!i i'~'~"~i:.~: ;:
:9 .:......,,L:,...::...... . ~-..:.::.;id:
i~. ..~................ .;:~:.,.~.,..:....~: ............. .. ~........ :,:~,.:..,. 9
". . . . . . . . .
9
............========...=....... ==== ~ . - . . .
:...:". ' ::~::~~ '. i:
~
i
Fig. 69. A. Monoclonal antibody Rat-302 recognizes a subset of neurons, later identified as unipolar brush cells, restricted to the granular layer of the flocculus and the vermis of rat cerebellum (arrows), whereas in other areas of the cerebellum no positive cells are found. B. In contrast, antibody Rat-303 recognizes Golgi-II cells in the granular layer (g) in the entire cerebellum. C. Rat-302 also recognizes Purkinje cells outside the caudal vermis and the flocculus. D. Rat-302 positive cells in the vermis. E and F. Unipolar brush cells recognized by Rat-302 have a round cell body and short dendrites ending in a spray of appendages (arrows). g, granular layer; m, molecular layer; p, Purkinje cell layer. Scale bars: 500 p m in A and B, 50 p m in C and D, 10 p m in E and F. Hockfield (1987).
93
Ch. I
J. Voogd, D. Jaarsma and E. Marani
(Fig. 70) (Palacios et al., 1980, 1981a; Kingsbury et al., 1980; Bowery et al., 1987; Rotter et al., 1988; Wilkin et al., 1981b). Binding to benzodiazepine receptors of [3H]flunitrazepam (Young and Kuhar, 1979, 1980; Vaccarino et al., 1985) or [3H]Ro15-1788 (Schoch et al., 1985), that would be expected to follow the pattern of [3H]muscimol binding to GABAA receptors, with the highest values in the granular layer, actually is reversed, with higher binding over the molecular layer. The inverse partial benzodiazepine agonist [3H]Ro 15-4513, however, exhibit higher binding to the granular layer than to the molecular layer (Sieghart et al., 1987). [3H]Ro15-4513 binding sites in the cerebellar granular layer differ from [3H]Ro15-4513 binding sites in the molecular layer and other brain regions in that they are insensitive to diazepam and other benzodiazepine agonists (see below). Yet another type of GABAA receptor is labelled with the antagonists [3H]bicuculline methochloride and [3H]SR95531, which preferentially bind to the low-affinity GABAA sites, and which bind at low density to the cerebellum (Bristow and Martin, 1988). Autoradiographic studies with mutant mice have revealed different changes in [3H]flunitrazepam and [3H]muscimol binding: [3H]flunitrazepam binding in the molecular layer in 'Purkinje cell degeneration' (pcd) mutant mice was decreased at 45 days, after the degeneration of the Purkinje cells. A further decrease was observed at 300 days, concomitant with the loss of the granule cells. Benzodiazepine receptors, therefore, may be located on Purkinje cells, and on parallel fibers in the molecular layer (Vaccarino et al., 1985; Kahle et al., 1990). An increase of [3H]flunitrazepam binding was found in 'weaver' cerebellum, with a loss of the granule cells (Chang et al., 1980; Kahle et al., 1990). Fry et al. (1985) found [3H]flunitrazepam binding to homogenates and tissue sections of the cerebellum of 'lurcher' mutant mice, with loss of their Purkinje cells and most granule cells, to be unchanged, whereas [3H]muscimol binding was reduced. Rotter et al. (1988)concluded that the decrease in [3H]muscimol binding in 'weaver', 'staggerer' and 45 days 'pcd' mutant mice was associated with a loss of granule cells and granule cell-Purkinje cell contacts. No [3H]muscimol labelling was observed over the deep Purkinje cell clusters in 'reeler' mutants, suggesting that these sites are absent on Purkinje cells. The electron microscopical studies of Chan-Palay (1978) and Chan-Palay and Palay (1978) in the rat indicate that [3H]muscimol binding to GABAA receptors is found on the plasma membrane of Purkinje cell somata, primary dendrites and initial axonal segment, and is also present on basket cells, their axons and the pinceau formation, on stellate cells and on dendrites of Golgi and granule cells. GABA A receptor subunits
To date five subunit classes of GABAA receptors, and several isoforms of each class have been cloned: ~1-~6, fll-fl3, 7'1-7'3, ~, and el and e2 (reviewed in Wisden and Seeburg, 1992; DeLorey and Olsen, 1992; Doble and Martin, 1992). GABAA receptors are constructed as hetero-oligomeric (presumably pentameric) assemblies of subunits. The ~-subunits in particular determine the different affinities of benzodiazepine ligands (Doble and Martin, 1992; Wisden and Seeburg, 1992). The y subunits are required for benzodiazepine-sensitivity, which indicates that the benzodiazepine binding site probably resides at the interface between the ~- and y-subunits. The fl-subunits are an essential structural component of the GABAA receptors, since without fl subunits, recombinant GABAA receptors are poorly expressed. The distribution of the distinct subunit mRNAs throughout the rodent cerebellum has been investigated in many studies and has been reviewed by Laurie et al., (1992) and Persohn et al. (1992) (Table 3). Most cerebellar neurons, including basket/stellate cells, Purkinje cells, and neurons of the deep nuclei 94
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 70. Bright-field (A) and dark-field (B) photomicrographs of a cerebellar folium of rat cerebellum labelled with [3H]muscimol to demonstrate the GABAA receptors. Note the clustering of grains over the area corresponding to the glomeruli of the granule cell layer and the low level of autoradiographic grains over the molecular layer (m) and the Purkinje cell layer (arrows). W, white matter. Bar = 100 r Palacios et al. (1981a).
express ~1, f12 and the 72 subunit mRNAs (Fig. 71, Table 3). Many types of subunits are produced by granule cells, which express c~l, ~z6,f12, f13, ?'2, fi mRNA and possibly also ill, cz4 and 7"3 mRNA (Fig. 71). No data are available on the subunit mRNAs expressed by Golgi cells. Bergmann glial cells express distinct levels of ~z2, 7'1, and possibly fll m R N A (Fig. 72, Table 3). The distribution of G A B A A receptor subunits has also been studied immunocytochemically with subunit specific antibodies. The monoclonal antibodies bd-24, which is selective for the ~zl subunit (but does not immunoreact with the rat antigen) and bd-17, which reacts with f12 and f13, preferentially stain the granular layer in rat, cat and monkey (Fig. 73) (Richards et al., 1987; Somogyi et al., 1989; Baude et al., 1992), and conform to the distribution of [3H]muscimol binding. A similar pattern was obtained with the monoclonal antibody 62-3G1 of De Bias et al. (1988), which also react with f12 and f13, and with several antibodies specific for the cz1 subunit (Meinecke et al., 1989; Gutidrrez et al., 1994 and references therein). Immunostaining in the granular layer prevails in the glomeruli, but granule cell membranes are also lightly stained (Fig. 73). Golgi cells were found to be unstained. Immunostaining in the molecular layer is compatible with a localization on Purkinje cell dendrites and stellate and basket cell somata. Purkinje cell somata and the basket cell-pinceau formations are lightly stained or unstained. In the deep nuclei, the majority of the cell bodies and also the dendrites were outlined by immunoreactivity. Electron microscopy by Somogyi et al. (1989) 95
Ch. I
J. Voogd, D. Jaarsma and E. Marani
TABLE
3.
Distribution of GABAA receptor subunit mRNAs in rat cerebellum
Subunit
Cell type PC
GrC
al
++
a2
.
a3
-
o~4
.
.
~5
.
.
a6
-
fll
.
.
++ -
yl ?'2 y3
Based
-
not detected; stellate
on data
+
-
.
.
.
or +
+
-
-
. . -
.
DCN
or +
.
-
.
or +
++
+
-
+
+
-
-
+
-
-
-
+
-
++
+
+
-
++
-
-
-
-
-
-
++
-
_
_
or +
or +
+, positive;
++, strongly
cells; Bg, Bergmann
glia; DCN,
from
Bg
.
++
f13
Symbols:
. -
f12
cells; StC,
++ .
BC/StC
Laurie
et al., 1992; Persohn
positive; deep
PC, Purkinje
cerebellar
cells; GrC,
granule
cells; BC, basket
nuclei.
et al., 1992.
showed that both bd-24 (al) and bd-17 (,82/3) immunoreactivity occurs at the synapses established by Golgi cells with granule cell dendrites. Immunoreactivity, however, is also present at non-synaptic sites throughout the surface of the granule cell including the somatic membrane which does not receive synapses, but is absent from the postsynaptic membrane specialisations of mossy fiber-granule cell synapses. No immunoreactivity is present in the Bergmann glia and granular layer astroglia. In Purkinje cells, the synaptic junctions formed by basket cells are often immunopositive. Immunoreactivity was also found along most of the dendritic surface including the dendritic spines. No bd-24 and bd-17 immunoreactivity could be observed at the parallel fibers synapses (Somogyi et al., 1989). A different subcellular localization than that of the a l and fl2/3 subunits was observed for the a6 subunit: Baude et al. (1992) showed that a6 immunoreactivity, which was confined to the glomeruli in the granular layer, was detectable only at the postsynaptic membranes facing Golgi cell boutons, and did not occur on extrasynaptic membranes. g-subunit immunoreactivity, which was also specifically localized in the granular layer, was found in both the glomeruli and on granule cell bodies (Benke et al., 1991a). Immunocytochemical studies with antibodies specific for the y2 subunit (Benke et al., 1991 b) or for each of the two splice variants, ~ ' 2 L ( o n g ) and ) " 2 S ( h o r t ) (Guti6rrez et al., 1994), showed that 7'2 immunostaining is most abundant over the molecular layer, moderately dense over the deep nuclei, and low over the granular layer. This distribution is similar to the distribution of [3H]flunitrazepam binding sites, which is consistent with the idea that the 7' subunit is required for the binding of [3H]flunitrazepam and other benzodiazepine agonists, y2L and y2s immunoreactivities seem to be similarly distributed, but their exact cellular localisation remains to be described. The subunit compositions of functional GABAA receptors in the different cerebellar cells have been discussed by Laurie et al. (1992) and Pershon et al. (1992). Purkinje cells are likely to contain alf127'2 GABAA receptor/channel complexes, and possibly also 96
The cerebellum. chemoarchitecture and anatomy
Ch. I
~6
,13 ilili-l~1ill :lllll!llll ::l ll l li:lii:~l:l"
i........
~li
Fig. 71. Bright-field photomicrographs showing cellular distribution of mRNAs of ~1, ~6, f12, f13, y2 and GABAA receptor subunits in the cerebellum of the rat. Arrows indicate examples of labelled stellate/basket cells; arrowheads delineate Purkinje cells. Gr, granule cells; Mol, molecular layer; P, Purkinje cells. Scale bar = 50 r Laurie et al. (1992).
~lf13y2 or otlfl2fl3y2 receptors. These receptors would have a B E 1 subtype of benzodiazepine site, which is consistent with the observations that BE1, but not B E 2 benzodiazepine receptors, are found at high levels in the cerebellum (Doble and Martin, 1992). ~ l f 1 2 y 2 G A B A A Receptors are also likely to be present in basket/stellate cells and neurons of the deep nuclei. Granule cells producing at least six different subunits may have multiple subtypes of G A B A A receptors. The high level of [ 3 H ] m u s c i m o l and [ 3 H ] R o 15-4513 binding in combination with a low level of [3H]flunitrazepam binding to the granular layer can be explained by the presence of o~6(fl2and/orfl3)y2 receptors, which exhibit benzodiazepine agonist-insensitive Ro 15-4513 binding as well as high-affinity [3H]muscimol binding. It has been recently demonstrated that an alcohol-non-tolerant rat line, which is highly susceptible to impairment of postural reflexes by benzodiazepine agonists, has a point 97
Ch. I
J. Voogd, D. Jaarsma and E. Marani
i~}ii!::i~!i'iiiiili~ii]i!ii!~ili!iiiiiii::ili~ii::i~tliii:t ) 'ill
i
~t:, ....................... i~ii:~'~!:::.i";i::., " ,:~ ......
,
.........
~9
9
..............
!
J......................................................... :.~ :'':
~:':::::::::..t........:.. ~
..... ~:::
i,~,, .....
9.................
....
,., ,:::,,~:, ....... ~:,,,,~
~
" ......
~:,s
':;:::
::::,.::~:~
" " " ~Ji:"!~ . ~
~
~
'~
9
Fig. 72. ~2 (A) and 7'1 (B and C) GABAA subunit mRNAs are localized in Bergmann glial cells in rat cerebellum. A and B are low-power dark-field, C is high-power bright field of the image in (B)./3, putative Bergmann glia; Gr, granule cells; Mol, molecular layer; P, Purkinje cells. Arrowheads in A and B indicate 'halo' of silver grains along the boundary of the granule cell/molecular layers. In a high-power bright-field view of the 7'1 probe autoradiographic signal (C), Purkinje cells (arrow heads) and granule cells appear to be unlabelled, whereas other small cells (arrows) in the Purkinje layer have clusters of silver grains over them. There is also a density of grains higher than background over the molecular layer in areas having no cell bodies. Scale bars in A and B = 100 ~m, in C = 35/lm. Laurie et al. (1992).
98
The cerebellum." chemoarchitecture and anatomy
....~
.
. ...~,~.,-~c"q ~ .:.~..-
. ,~,-"
Ch. I
..,~.~
.+.<
......
.~.
Fig. 73. Low-power dark-field (A) and microscopic brigh-field (B, Normarski optics) images of the distribution of immunoreactivity in the rat cerebellum using mAb bd-17 against the fl2/3 GABA A receptor subunits. Immunostaining is very intense over the granular layer (PMgr), moderately dense over the molecular layer (PMmo) and the deep nuclei and absent in the white matter (my). In B, note the intense staining of the glomeruli in the granular layer, and virtually absence of staining in Purkinje cells (Pc). Bars 1 mm in A, 50/~m in B. Richards et al. (1987).
99
Ch. I
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mutation in the ~6 subunit, which dramatically increases the sensitivity of ~6fl27'2 receptors for benzodiazepine agonists (Korpi et al., 1993). Since ~6 subunits are exclusively expressed by cerebellar granule cells these data indicate that the increased susceptibility to benzodiazepine impairment of postural relexes is mediated at the level of the granule cells, and imply that ~6 subunit containing G A B A a receptors are important in the cerebellar circuits controlling movements (Korpi et al., 1993). Immunoprecipitation indicate that ~6 mostly colocalize with ~1, f12 or/33, and 7'2 (Khan et al., 1994). It has also been proposed that ~6 assemble into GABAA receptor complexes without ~1, and into receptors without 7'2, but with the g subunit (see Laurie et al., 1992). Granule cells may also make G A B A A receptors without the e6 subunit. Mfiller et al. (1994) characterized the properties of GABAA receptors in Bergmann glial cells in developing rat cerebellum. Significant benzodiazepine-insensitive GABAA receptor conductancies were detected in Bergmann glial cells between post-natal days 7 and 10, but were small or undetectable in the adult. It was also observed that Bergmann glial cells in addition to c~l-immunoreactivity, transiently express g-subunit immunoreactivity, indicating that the transient GABAA responses are mediated by a GABAA receptor composed of ~1 and ~ subunits. In view of the in situ hybridisation data also fll and yl may contribute to the Bergmann glial GABAA receptor (Laurie et al., 1992; Pershon et al., 1992).
3.7.2. GABAB receptors GABAB receptors are insensitive to the GABAA-antagonists bicuculline or picrotoxin, and are selectively activated by baclofen. GABAB receptors are believed to be G-protein coupled receptors similar to the metabotropic glutamate receptors. Their molecular structure remains, however, to be elucidated (Bowery, 1993). GABAB receptors are mostly coupled to adenylate cyclase, and exert their inhibitory action through the activation of potassium channels or inhibition of CaZ+-channels (Bowery, 1993). Ca 2+sensitive binding of [3H]GABA, with suppression of GABA A binding by isoguvacine, or binding of [3H]baclofen were used to study the distribution of GABAB receptors. GABAB sites are present at high density in the molecular layer (Wilkin et al., 1981b; Gehlert et al., 1985; Bowery et al., 1985, 1987), where they are distributed in a pattern of alternating, parasagittal zones of high and low [3H]GABA binding (Albin and Gilman, 1988; Turgeon and Albin, 1993). GABAB receptor binding in the granular layer is very low, but above background (Turgeon and Albin, 1993). A low amount of GABAB receptor binding was found over the cerebellar nuclei in adult rats. The cerebellar nuclei, however, transiently express a very high density of GABAB receptors during development (Turgeon and Albin, 1993). The cellular localization of GABAB receptor binding in the molecular layer is controversial (reviewed in Turgeon and Albin, 1993). Wojcik and Neff (1984) reported that GABAB induced inhibition of adenylate cyclase was reduced in 'weaver' mutant mice with a loss of their granule cells. It was not affected by Purkinje cell loss in the appropriate mutants or by climbing fiber deafferentation by 3-acetylpyridine in rats. Kato and Fukuda (1985) found [3H]baclofen binding to GABAB receptors to be decreased in homogenates of rat cerebellum, after destruction of the inferior olive with 3-acetylpyridine. They concluded that GABAB receptors were located on climbing fibers and that the residual binding could be associated with granule cells. They explained the apparent discrepancy with the results of Wojcik and Neff (1984) by assuming that the high affinity binding sites, which were mainly affected in their experiments, were not 100
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coupled to adenylate cyclase and, therefore, would have escaped to be noticed in the experiments of these authors. Bowery et al. (1987), who analyzed GABAB binding in several strains of mutant mice, however, advocated that the majority of GABAB receptors had a postsynaptic localization on Purkinje cell dendrites. Turgeon and Albin (1993), who analyzed GABAB receptor binding in rats with methyl azoxymethanol lesions of granule cells, or with 3-acetylpiridine lesions, and in stumbler mutant mice lacking Purkinje cell dendrites, also concluded that GABAB sites are primarily located on Purkinje cell dendrites. Finally, a recent immunoelectron microscopic study with monoclonal antibodies to L-baclofen indicate that GABAB receptors are found on both Purkinje cell dendrites and parallel fibers (Martinelli et al., 1992).
3.7.3. Glycine receptors Glycine-mediated inhibition of neuronal activity results from activation of the glycine receptor, a ligand-gated chloride channel (Betz, 1991). The distribution of this receptor has been autoradiographically studied with the antagonist [3H]strychnine. These studies indicate that the glycine receptor is almost absent from the cerebellum (Zarbin et al., 1981). The cerebellum, however, displays a significant number of [3H]glycine binding sites mainly distributed over the granular layer (Bristow et al., 1986), but these sites are assumed to represent the glycine coagonist sites of NMDA receptors (see Section 3.3.1 .). The glycine receptor has been demonstrated to be a pentameric protein composed of ligand binding ~ and structural fl subunits (Langosch et al., 1990). Variants of the ligand-binding ~ subunit (~1, ~2, ~3) have been cloned, that modify the pharmacological and the physiological properties of the glycine receptors. In situ hybridisation has revealed a hybridisation signal of the probe for the/3 subunit in all layers of the cerebellar cortex and the cerebellar nuclei, with a particular strong signal in the Purkinje cell and granular layers (Malosio et al., 1991). The ~l-subunit mRNA is expressed at low level in the cerebellar nuclei, and by 'rare single cells' in the granular layer, whereas ~3 mRNA appears to be selectively expressed by cerebellar granule cells. The ~2-subunit mRNA is not produced in the adult cerebellar cortex (Malosio et al., 1991). A monoclonal antibody selective for the N-terminal sequence of the ~ subunits (mAb2b), immunostained sparse puncta on cell somata in both the granular and molecular layer (Kirsch and Betz, 1993). Another monoclonal antibody, mAb4a, which bind to ~ and fl subunits, in addition to sparse puncta in the granular and molecular layer also produced diffuse labelling over Purkinje cell perikarya and occasional cell bodies in the granular layer (Kirsch and Betz, 1993). The punctate labelling is believed to reflect subunit staining at post-synaptic membrane specialisations (Kirsch and Betz, 1993). The diffuse labelling, instead, probably represents the fl subunit. Gephyrin is a 93 kDa peripheral membrane protein that co-purifies with glycine receptors. It has been proposed to anchor the glycine receptor to sub-synaptic tubulin (Betz, 1991). In situ hybridisation (Kirsch et al., 1993) and immunocytochemistry (Araki et al., 1988; Kirsch and Betz, 1993) show that gephyrin is widely distributed throughout the brain, including the cerebellum. Gephyrin mRNA is expressed by Purkinje cells and granule cells. Punctae of gephyrin immunostaining were concentrated over the molecular layer, and on cell bodies in the granular layer, but were virtually absent on Purkinje cell somata. The functional implications of the presence of glycine receptor subunits and gephyrin in the cerebellum are poorly understood. Granule cells express 0~3, the fl subunit and gephyrin and, therefore, have all the ingredients to produce functional glycine receptors. 101
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These receptors could be involved in the inhibitory actions of Golgi cells, in view of the fact that a subpopulation of Golgi cells may be glycinergic (see above). However, the presence of glycine receptors in granule cells is difficult to reconcile with the absence of any strychnine labelling in the cerebellum (Zarbin et al., 1981; see discussion Malosio et al., 1991). Gephyrin and/or the fl subunit are also produced by cells that do not express ~ subunits, such as the Purkinje cells. It has been postulated that gephyrin and/or the fl-subunit participate in other transmitter-gated ion channels, like the GABAAreceptors (see Malosio et al., 1991; Kirsch et al., 1993). For instance, Triller al. (1987) showed that gephyrin-immunoreactive postsynaptic specialisation face GABAergic synapses in the cerebellar cortex (Triller et al., 1987). Chen and Hillman (1993b) demonstrated that gephyrin-immunoreactive postsynaptic membrane specialisations (labelled with antibody R7A) faced a subpopulation of glutamate decarboxylase (GAD) immunoreactive boutons in the cerebellar nuclei. Since glycine often colocalizes with GABA it is possible that GABAergic boutons facing gephyrin-immunoreactive postsynaptic membrane specialisations, are also glycinergic. 3.8. M O N O A M I N E R G I C A F F E R E N T SYSTEMS AND RECEPTORS Several monoaminergic pathways terminate in the cerebellum (And6n and Ungerstedt, 1967). Their terminations in the cerebellar nuclei and the inferior olive will be considered elsewhere (Sections 5.2. and 6.3.2.2.). The monoaminergic fibers are primarily serotoninergic and noradrenergic (NA). The serotoninergic and noradrenergic fibers are present in all three layers of the cerebellar cortex. Their morphology differs from the classical mossy and climbing fiber afferents. Serotonin
Serotoninergic fibers in the cerebellar cortex were first demonstrated with the histofluorescence method, and were mainly recognized as transverse fibers in the molecular layer (H6kfelt and Fuxe, 1969). In studies using specific uptake, [3H]serotonin accumulated in small calibre, varicose axons. The varicosities were filled with minute, 15-25 nm round or flattened agranular vesicles and contained some large granular vesicles with a diameter of 50-90 nm (Beaudet and Sotelo, 1981). Synaptic contacts of the varicosities were few, most of them being present in the molecular layer. These, and other studies
A
B
Fig. 74. Camera lucida drawing of serotonin immunostainingin Lobule X of the cerebellum of the rat (A) and
the paramedian lobule (PML) (B). Sagittal sections. Grl = granular layer; ML = molecularlayer; WM = white matter. Calibration bar = 200/lm. Bishop and Ho (1985). 102
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Fig. 75. Photomicrographs of serotonin immunoreactive fibers in selected regions of rat cerebellar cortex. A. Dark-field photomicrograph. B. High-power bright-field photomicrographs illustrating the beaded nature of the immunoreactive fibers (arrows). Vermal lobule X. Abbreviations: see Fig. 74. Calibration bar = 200 tim. Bishop and Ho (1985).
using specific uptake of [3H]serotonin also reported labelling over mossy fiber rosettes (Chan-Palay, 1975; Bloom et al., 1971). The presence of serotoninergic mossy fibers could, however, not be confirmed immunocytochemically with antibodies directed against conjugates of serotonin in rat, cat and opossum (Takeuchi et al., 1982; Bishop et al., 1985; Kerr and Bishop, 1991). Serotonin-like immunoreactivity was present in randomly oriented fibers in the molecular layer in the rat. Long transverse fibers were present in superficial strata of the molecular layer in the rat (Takeuchi et al., 1982) or in deep parts of the molecular layer in the opossum (Bishop et al., 1985), radially oriented fibers were most numerous in the vermis of rat (Figs 74 and 75) (Bishop and Ho, 1985). The granular layer contains a loose serotoninergic plexus. A layer of fine, beaded serotonin-containing fibers is present below the Purkinje cells in rat and opossum (Bishop and Ho, 1985; Bishop et al., 1985). The Purkinje cell layer only contains passing fibers. In the caudal vermis of the oppossum the serotoninergic innervation of the cortex is concentrated in midsagittal and parasagittal bands. In the cat serotonin-immunoreactive fibers are dense in the granular and Purkinje cell layers with only a few fibers in the molecular layer (Kerr and Bishop, 1991). These serotoninergic axons and varicosities have a uniform distribution throughout all lobules of the cerebellum with the exception of lobule X were the fiber density is low (Kerr and Bishop, 1991). A dense plexus of serotoninergic fibers is also found in all of the deep nuclei of the cat. In addition, there appear to be serotonin positive cell bodies in the deep nuclei (Kerr and Bishop, 1991) (Section 5.7.). Double-labelling experiments in rat (Bishop and Ho, 1985) (Fig. 76), opossum (Walker et al., 1988) and cat (Kerr and Bishop, 1991) revealed that the serotoninergic innervation of the cerebellum originates from the reticular formation. In the rat these neurons are distributed over the medullary and the pontine reticular formation (nucleus reticularis gigantocellularis, nucleus reticularis paragigantocellularis and nucleus pontis oralis). The origin of these fibers from the gigantocellular reticular formation and from a few neurons located in the medullary pyramids is more restricted in the opossum. Kerr and Bishop (1991) found that the serotoninergic projection to the cerebellar cortex of the cat shows some degree of topographical organization: Serotoninergic fibers in the anterior vermis arise from neurons located within the paramedian reticular nucleus, the 103
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lateral reticular nucleus and the lateral tegmental field, whereas fibers to the caudal vermis and the paramedian lobules exclusively derive from the lateral reticular nucleus. The hemispheres receive serotoninergic input from cells in the lateral tegmental field, the periolivary reticular formation and the paramedian reticular formation. Allthough retrogradely labelled cells were found in the raphe, in no case these cells were serotoninergic (Kerr and Bishop, 1991). Serotonin receptors
Physiological studies indicate that serotonin may play a modulatory role in the cerebellar circuitry (see Gardette and Cr6pel, 1993; Kerr and Bishop, 1992; and references therein). Serotonin interacts with a particularly large number of functionally and pharmacologically diverse receptor types. The serotonin receptors have been placed into five subgroups, 5HT1, 5HT 2, 5HT3, 5HT4 and 5HT5 (Tecott and Julius, 1993). The 5HT3 receptors are ionotropic receptors that mediate rapid excitatory responses in neurons, whereas the other serotonin receptor types are coupled to G-proteins, through which they activate second messenger cascades. The 5HT1 receptors comprise a heterogeneous group with at least six subtypes, named 5HT1A-5HT1F receptors. Ligand binding autoradiography with [3H]serotonin, which preferentially labels 5HT1 receptors, shows that a low level of [3H]serotonin binding occurs over the cerebellar cortex with the highest density over the molecular layer (Pazos and Palacios, 1985). The deep nuclei presented a higher density of [3H]serotonin binding with a lateral-to-medial decrease in receptor density. The cerebellar [3H]serotonin binding sites have the pharmacological properties of 5HT1B receptors (Pazos and Palacios, 1985). More recent autoradiographic studies with ligands specific for the 5HT1B and the 5HT1D receptors, which are closely related to 5HT1B receptors, indicate that the cerebellar cortex has 5HT1B, but no 5HT1D receptors (Bruinvels et al., 1993). The presence of 5HT1B receptors in rat cerebellum was confirmed by in situ hybridisation studies showing that 5HT1B receptor mRNA is expressed at high levels by Purkinje cells, but also by cells in the molecular layer, and in the deep cerebellar nuclei (Appel et al., 1990; Voigt et al., 1991; Maroteaux et al., 1992). In view of the findings that 5HT1B mRNA levels are relatively high in Purkinje cells, whereas receptor binding is low in the molecular and Purkinje cell layers, but relatively high in the deep nuclei, one is tempted to speculate that the 5HT1B receptor is localized presynaptically on Purkinje cell axon terminals. The ultrastructural localization of the 5HT1B receptors remains, however, to be determined. No 5HT1A receptors were found in the rat cerebellum with receptor autoradiography or in situ hybridisation (Pompeiano et al., 1992). Using immunocytochemistry with an antibody specific for the 5HT1A receptor, Matthiesen et al. (1993), however, found that the 5HT1A receptor occurs in Purkinje cells of immature, but not in adult cerebellum. The 5HTlc and 5HT 2 receptors were found at low concentrations in the deep nuclei in autoradiographic studies (Pazos et al., 1985; Molineaux et al., 1989). Since 5HT 2 and 5HTlc are structurally related they have been recently renamed as 5HTzA and 5HTzc receptors, respectively. In situ hybridisation studies have shown that mRNAs of both receptor types are expressed in the deep cerebellar nuclei (Pompeiano et al., 1994). The other known serotonin receptors (e.g. 5HT3 and 5HT4) seem to be essentially absent from rodent cerebellum (Tecott and Julius, 1993; Domenech et al., 1994). Summarizing, the known serotonin receptors are expressed at low levels or are not expressed at all in the cerebellum, with the exception of the deep nuclei which express significant levels of different types of serotonin receptors.
104
The cerebellum." chemoarchitecture and anatomy
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Fig. 76. Schematic drawings showing the distribution of labelled neurons in the medullary and pontine reticular formation in the rat, after injections of H R P in the vermis and adjacent parts of the hemisphere. Filled triangles represent neurons retrogradely labelled with H R P alone; closed circles represent neurons immunolabelled for serotonin alone; and asterisks represent double-labelled neurons. Each symbol represents one neuron in each population. Abbreviations: BC = brachium conjunctivum; BP = brachium pontis; CP = cerebral peduncle; D R = dorsal raphe nucleus; EC = external cuneate nucleus; ICP = inferior cerebellar peduncle; IN = interpeduncular nucleus; IOC = inferior olive; M R = nucleus raphe magnus; MV = medial vestibular nucleus; PG = nucleus paragigantocellularis; PH = nucleus pre-positus hypoglossi; PO = nucleus pontis oralis; R G = gigantocellular reticular nucleus; R M = nucleus raphe magnus; RO = nucleus raphe oralis; RP = nucleus raphe pallidus; SV = superior vestibular nucleus; V = nucleus of spinal tract of the trigeminal nerve; VII = nucleus of facial nerve. Redrawn from Bishop and Ho (1985).
Noradrenalin
The noradrenergic (NA) innervation of the cerebellum is mostly directed at the cortex. In the original studies of H6kfelt and Fuxe (1969) with a histochemical fluorescence method for the demonstration of catecholamines (Falck et al., 1962), NA containing fibers were found to be present as a randomly oriented plexus in all three layers of the cortex of rat cerebellum. The density of the NA innervation was lower in the flocculonodular lobe and the uvula than in the corpus cerebelli. A similar plexus was illustrated in rat cerebellum by Grzanna et al. (1989), using an antibody against a conjugate of NA (Geffard et al., 1986), and by Fritschy and Grzanna (1989) with an antibody against dopamine-fl-hydroxylase (Fig. 77A), the synthetic enzyme that converts dopamine to 105
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NA. A heavy plexiform innervation of the granular layer by thin varicose axons and parallel fiber-like fluorescence in the molecular layer was also found in chicken cerebellum. The parent fibers in the cerebellar white matter were concentrated in two parasagittal bundles (Mugnaini and Dahl, 1975). Axons containing dark core vesicles, which were labelled by specific uptake of [3H]NA terminated on Purkinje cell proximal dendrites and spines in the molecular layer (Bloom et al., 1971; Yamamoto et al., 1977). The inhibitory action of NA on Purkinje cells and the transduction of this response by cyclic AMP were reported in the subsequent papers of Hoffer et al. (1971) and Siggins et al. (1971). Purkinje cells therefore seem to be the main target of the NA innervation (Felten et al., 1986; see also Llano and Gerschenfeld, 1993). The ultrastructure of NA fibers of the cerebellar cortex and other parts of the rat CNS was analyzed with pre-embedding dopamine-fl-hydroxylase immunohistochemistry by Olschowka et al. (1981). Immunoreaction product was present in the axoplasm, associated with smooth endoplasmatic reticulum, Golgi apparatus, synaptic and large dense core vesicles and the outer membranes of mitochondria. Large varicosities were interconnected by narrow intervaricose axon segments. Varicosities, filled with clear, round synaptic vesicles and large dark-core vesicles, made asymmetric contacts with dendrites, but never with somata or axons. More than 50% of the labelled varicosities in the cerebellum made synaptic contacts: most of them with dendritic shafts, fewer on spines. Kimoto et al. (1981) used the histofluorescence method for light microscopy and potassium permanganate fixation in their electron microscopic studies of NA in rat cerebellum. They found NA-containing nerve terminals making contact with granule cell dendrites and secondary and spiny branchlets of the Purkinje cell dendritic tree. The terminals contained a large number of small dark core vesicles and a couple of larger ones. No synaptic contacts with the Purkinje cell somata were observed. Triarhoe and Ghetti (1986) are of the opinion that these results should be reconsidered because a distinction between NA and serotonin is not possible with this fixation method. The cerebellar NA fibers in the rat take their origin from the dorsal part of the locus coeruleus (group A6 of Dahlstr6m and Fuxe, 1964) and the A4 groups in the roof of the fourth ventricle. Single NA neurons innervate both the cerebellum and the forebrain (see also Steindler, 1981). The fibers enter the cerebellum close to the fourth ventricle. According to Pickel et al. (1973) the main entrance route is through the superior cerebellar peduncle. Pasquier et al. (1980) localized the cells of origin of the NAprojections to the cerebellum in the rat with specific retrograde transport of dopaminefl-hydroxylase. They traced the main projection from the entire caudal pole of the locus coeruleus and smaller contributions from the nucleus subcoeruleus and the cell groups A7 and A5; all projections are bilateral. The cerebellar projections in the cat take their origin from NA-containing cells located around the superior cerebellar peduncle, and the dorsolateral part of the locus coeruleus (Chu and Bloom, 1974; Somana and Walberg, 1978; see also Dietrichs, 1985, 1988). Grzanna et al. (1989), Fritschy and Grzanna (1989) and Grzanna and Fritschy (1991) reported a selective loss of the terminal portions of NA axons originating from the locus coeruleus in rats treated with the neurotoxin DSP (N-(2-chloroethyl-N-ethyl-2 bromobenzylamine)). DSP affects the NA innervation of the cerebellum, the cerebral cortex, the pontine nuclei, the inferior olive, the vestibular and cochlear nuclei, the sensory nuclei of the trigeminal nerve and the dorsal horn but spares the NA axonal plexus in the basal forebrain, the hypothalamus and most of the brain stem and the cord. After 24 hours loss of NA-staining is most-pronounced in the molecular layer, but with longer survival times most NA fibers have disappeared (Fig. 77B). 106
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 77. Effects of DSP-4 (50 mg/kg) on the noradrenaline innervation of the cerebellar cortex of the rat. A. Control section incubated with an antibody against dopamine-fl-hydroxylase. B. Section from a DSP-4-treated rat. Parasagittal plane; dark-field photomicrographs. Magnification 90x. Fritschy and Grzanna (1989).
Adrenergic receptors Adrenergic receptors can be subdivided in ~l,0t2 and fll,fl2 receptors on the basis of p h a r m a c o l o g i c a l criteria (Bylund and Pritchard, 1983; M i n n e m a n et al., 1981). M o s t 107
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Fig. 78. A. x28 and B. x42 power photomicrographs of autoradiographs of ]2Siodocyanopindol (~2SlCYP) binding to adrenergic beta receptors in rat cerebellar cortex illustrating the high density of silver grains in the molecular layer and 'patches' of increased receptor density over the Purkinje cell layer, m, molecular layer; g, granule cell layer. (C) shows the patches at x 140 magnification with dark-field illumination. Sutin and Minneman (1985). (
receptors in the cerebellum are of the fl type, low levels of specific binding of the ~2 agonist [3H]para-aminoclonidine are associated with the granular layer in rat cerebellum (Unnerstall et al., 1984). Binding assays with the fl-adrenergic antagonist [3H]dihydroalprenolol in homogenates of cat cerebellum showed that the receptors are mainly of the f12 type and that they are evenly distributed over the vermis, the hemisphere and the cerebellar nuclei (Pompeiano et al., 1989). Elevated binding to the molecular layer was found in autoradiographs of tissue sections of rat cerebellum with ligands which bind non-selectively to fl receptors (Palacios and Kuhar, 1980). The same pattern emerged when either fll or f12 receptors were eliminated by specific inhibitors, with high levels of f12 binding in the molecular layer and much lower levels over the Purkinje and the granule cells. The overall contribution of fll receptor binding was low (Rainbow et al., 1984a). Beta adrenergic receptor binding was evenly distributed over the molecular layer, but at the level of the Purkinje cell somata the ligand is bound in irregular patches (Fig. 78) (Sutin and Minneman, 1985). These patches are most prominent in vermis and paravermal zones of the lobules I-IX and are less frequent in the medial hemisphere. Incubations with specific fll and f12 antagonists showed that the receptors in these patches are of both subtypes. 109
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Polyclonal antibodies which recognize domains of the f12 adrenergic receptor other than the NA-binding site, immunoreact with distal segments and dendritic spines of rat Purkinje cells. Immunoreactivity is present throughout the dendritic cytoplasm and accumulates at the dendritic membrane, opposite synaptic clefts (Strader et al., 1983). Dopamine
Dopaminergic projections to the cerebellum are still controversial. Low levels of dopamine (DA) supposedly represent a precursor of noradrenaline. DA-like immunoreactivity, as demonstrated with an antibody against a conjugate of DA, was present in fibers with irregularly spaced varicosities in all layers of the cerebellar cortex of the rat (Panagopoulos et al., 1991). Radially oriented DA fibers were abundant in the molecular layer. These fibers may take their origin from the A10 dopaminergic cell group of Dahlstr6m and Fuxe (1964) in the medial tegmental area. Cerebellar DA levels in rat cerebellum decreased by 50% after lesions of the A10 area (Kizer et al., 1976) and connections of the A10 area with the cerebellar nuclei and the granular and Purkinje cell layers were demonstrated with antegrade and retrograde axonal transport methods by Simon et al. (1979). Recently Ikai et al. (1992) reported a dopaminergic projection of the A10 group in the rat to restricted portions of the cerebellar hemisphere (Figs 79A,B and 80). Projections of the A10 group to the lateral cerebellar nucleus, however, appeared to be non-dopaminergic, as demonstrated immunocytochemically with an antibody against tyrosine hydroxylase, the enzyme involved in the conversion of tyrosine to DA. Fibers and terminals in the cortex were most numerous in the granule and Purkinje cell layer and scarce in the molecular layer. Their distribution differs, therefore, from the DA fibers described by Panagopoulos et al. (1991), which predominate in the molecular layer. Dopamine receptors
Five different receptors for dopamine have been distinguished, D~-Ds, which are often grouped as Dl-like (D1 and Ds) and Dz-like (D2, D3, and D4) receptors with high affinities for [3H]SCH23390 and spiperone, respectively (Grandy and Civelli, 1992). The D~ receptor is considered to be stimulatory, whereas the D 2 receptor is either inhibitory or unlinked to adenylate cyclase. Autoradiographic studies using [3H]SCH23390 as the ligand show that D~ receptor binding in the cerebellar cortex is low (Camps et al., 1990; Mansour et al., 1992). According to Camps et al. (1990), who compared the distribution of D~ and D 2 receptors in the cerebellum of rat, mouse, guinea pig, cat, monkey and man, Dl-binding was only detected in the Purkinje cell and molecular layer in rat and cat. Dz-receptors, labelled with [3H]CV 205-502, were only found at significant levels in rat cerebellum, specifically localized in the molecular layer of lobule X and IXc. In lobule IX high activity is distributed in sagittal and parasagittal columns. Although similar labelling has been repeatedly found with other 'D2-1igands' it was recently shown by both autoradiography and in situ hybridisation that the dopamine receptors in the rat nodulus and ventral uvula are of the D3-type. Thus Bouthenet et al. (1991) noticed a very low D 2 signal over the granular layer of all cerebellar lobules, and a high expression of D3 mRNA by Purkinje cells in lobule X and IXc. Autoradiographic studies with D3-preferring ligands, [3H]quinpirole (Gehlert, 1993; Levant et al., 1993) and 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin (L6vesque et al., 1992) showed a similar type of labelling in rat cerebellum (Fig. 81). The functional role of the cerebellar D3 receptor is puzzling in view of the lack of significant dopaminergic innervation. It is 110
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Fig. 79. Photomicrograph of camera lucida drawing (inset) of anterogradely labelled axons and terminal boutons in the crus I ansiform lobule of rat cerebellum after cholera toxin (b fragment) injection into the contralateral ventral tegmental area. Arrows point to 'rosettes' characteristic of mossy fiber endings in the granular layer (G). R Purkinje cell layer; WM, white matter. Scale bar = 40/xm. Ikai et al. (1992).
notable that substance P receptors have a very similar distribution in rat cerebellum (Nakaya et al., 1994). 3.9. H Y P O T H A L A M O CEREBELLAR CONNECTIONS AND HISTAMINERGIC PROJECTIONS Hypothalamo cerebellar fibers were demonstrated with different anterograde axonal transport techniques in squirrel monkey, cat and rat (Dietrichs, 1984; Dietrichs and Haines, 1985; Haines et al., 1984, 1985, 1986). Most fibers enter the cerebellum bilaterally with an ipsilateral dominance from the central grey, passing medial to the superior cerebellar peduncle. They enter the granular layer, where they branch. Fibers passing around the Purkinje cell somata enter the molecular layer, where most of them assume a longitudinal course in the long axis of the folia (Fig. 82). Few labelled mossy fibers and no labelled climbing fibers were seen. These 'multilayer' hypothalamo cerebellar fibers are more frequent in the cortex of the flocculus and the vermis, but are also present in parts of the hemisphere. Some fibers project to the cerebellar nuclei (Dietrichs and Haines, 1985; Haines et al., 1990). The projection takes its origin from cells in the posterior, lateral and dorsal hypothalamic areas, the lateral mammillary nucleus and the periventricular nucleus (Dietrichs and Zheng, 1984; Dietrichs, 1984; Haines and Dietrichs, 1984, 1987; Dietrichs et al., 1985b). Immunocytochemical studies have demonstrated the existence of histaminergic neurons, which are concentrated in the tuberomammillary nucleus of the posterior hypothalamus, and which give rise to fibers to almost all parts of the brain (reviewed by Wada 111
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F1 Fig. 80. Schematic diagram of the unfolded rat cerebellar cortex, showing the distribution of fibers and terminals (dots) arising from the ventral tegmental area. CP, opula pyramis; Crus I, crus I ansiform lobule; Crus II, crus II ansiform lobule; F1, flocculus; PFI, paraflocculus; Pm, paramedian lobule; Simplex, simple lobule. Ikai et al. (1992).
et al., 1991). A low and low to moderate density of histamine-immunopositive fibers has been observed in rat (Inagaki et al., 1988) and guinea pig cerebellum (Airaksinen and Panula, 1988), respectively. Like the hypothalamo cerebellar fibers, the histaminergic fibers were sparsely distributed to all cortical layers, and were more frequent in the vermis and the flocculus than in other lobules. A moderate density of histaminergic fibers occurred in the cerebellar nuclei and the inferior olive. Three subtypes of histamine receptors have been pharmacologically identified in the brain: H1, H2 and H3 receptors. H1 receptors are coupled to phospholipase C, through which the inositol 1,4,5-trisphosphate-Ca 2+ and diacylglycerol-protein kinase C cascades are activated. Receptor autoradiographic studies with [3H]mepyramine and [125I]iodobolpyramine show that essentially no H~ receptors occur in the rat and human cerebellar cortex (Palacios et al., 1981b), whereas high densities are found over the molecular layer of guinea pig and mouse cerebellum (Palacios et al., 1981b; Rotter and Frostholm, 1986; Bouthenet et al., 1988). Moderate densities were found in mouse and guinea pig deep cerebellar nuclei (Rotter and Frostholm, 1986; Bouthenet et al., 1988). H1 receptor density over the molecular layer was greatly decreased in Purkinje cell deficient mice, whereas in reeler mice, which contain malpositioned Purkinje cells, high H~ receptor density was found over regions with heterotopically located Purkinje cells. No change in H1 receptor density was found in weaver mouse cerebellum, which is almost devoid of cerebellar granule cells. These data indicate that H~ receptors are predominantly localized on Purkinje cell dendrites (Rotter and Frostholm, 1986). 112
The cerebellum." chemoarchitecture and anatomy
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Fig. 81. Bright-field autoradiographs showing [3H]quinpirole binding to dopamine D 3 receptors to coronal sections of the caudal cerebellum of rat. Note high-density of binding sites over the molecular layer (m) of the nodulus (X). Also note stripes of increased [3H]quinpirole binding over molecular layer of the uvula (IX). g, granular layer; IO, inferior olive; MVe-medial vestibular nucleus; 12, hypoglossal nucleus. Gehlert (1993).
H 2 receptors, which are coupled to adenylate cyclase were also found in the molecular layer of guinea pig cerebellum (Ruat et al., 1990). H3 receptors are autoreceptors involved in the inhibition of the release of histamine. A low level of H3 receptors were found to be homogeneously distributed throughout the rat cerebellar cortex (Arrang et al., 1987; Pollard et al., 1993). 3.10. CHOLINERGIC SYSTEMS AND ACETYLCHOLINESTERASE (ACHE) IN THE CEREBELLUM 3.10.1. Distribution of choline acetyltransferase Biochemical measurement of distinct levels of acetylcholine (McIntosh, 1941; Kfisa et al., 1982) and its biosynthetic enzyme, choline acetyltransferase (CHAT) in cerebellar tissue (K/tsa and Silver, 1969; Salvaterra and Foders, 1979; Hayashi, 1987; and others) indicated the presence of a cholinergic innervation in the cerebellum. ChAT activity varies among different lobules with the highest levels in the nodulus and ventral uvula. Following deafferentation of the cerebellar cortex, ChAT activity is considerably de113
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Fig. 82. A. Semidiagrammatic representation showing the correlation between the patterns of individually wheat germ agglutinin (WGA)-HRP labelled hypothalamo-cerebellar fibers and the diffuse patterns seen just above and below the Purkinje cell layer. B. Darkfield microphotograph of the termination of WGA-HRPlabelled hypothalamo-cerebellar fibers. Note the clusters of label just above (solid arrows) and below (open arrows) the Purkinje cell layer. GL, granular layer; ML, molecular layer. Squirrel monkey. Scale bar = 75 ~m for B. Haines et al. (1986)
114
The cerebellum." chemoarchitecture and anatomy
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creased, indicating that the cholinergic innervation is mostly of extracerebellar origin (K/tsa and Silver, 1969; Asin et al., 1984). Acetylcholine and ChAT were found in the glomerular fraction consistent with the view that acetylcholine may be the transmitter in a subpopulation of mossy fibers (Isr~iel and Whittaker, 1965; Bal/tzs et al., 1975). Early anatomical studies have employed acetylcholinesterase (ACHE) histochemistry. However, AChE activity is disproportionally high in the cerebellum (reviewed in Silver, 1974), and its widespread distribution in non-cholinergic cells and fiber systems precludes its use as a marker for cholinergic neurons. More reliable methods to unravel the anatomy of the cerebellar cholinergic system became available with the development of antibodies specific for CHAT. Early immunocytochemical studies revealed a subpopulation of ChAT-positive mossy fibers in rabbit and man (Kan et al., 1978, 1980). However, subsequent studies with monoclonal antibodies, failed to demonstrate ChAT positive staining in the cerebellum (Armstrong et al., 1983; Houser et al., 1983). Yet more recent studies by Ojima et al. (1989) in rat, by Illing (1990) in cat, by Barmack et al. (1992a) in rat, rabbit, cat, and monkey, and by DeLacalle et al. (1993) in man, demonstrated the presence of at least four types of cholinergic innervation in the cerebellum: (1) a subpopulation of ChAT-positive mossy fibers which primarily innervate the nodulus and the ventral uvula of the vermis; (2) a sparse plexus of thin beaded fibers which is present in all layers; (3) thin ChAT-positive fibers that innervate the cerebellar nuclei; and (4) a still controversial subpopulation of cholinergic Golgi cells. In addition, Ikeda et al. (1991) reported some typical granule cells with their bifurcating parallel fibers to be ChAT-immunoreactive in the cat, but this finding was not confirmed by others. Ojima et al. (1989) showed that a significant number of ChAT-positive mossy fiber rosettes was present throughout most vermal lobules, but that the density in the nodulus and ventral uvula was approximately ten times that of other vermal lobules (Fig. 85A). ChAT-positive mossy fiber rosettes were also enriched in the flocculus and the ventral paraflocculus. The distribution of ChAT-positive rosettes in rat observed by Barmack et al. (1992a) was essentially similar, although they suggested that the ChAT-positive rosettes were also enriched in lobules I, II and III (Fig. 83). They also demonstrated that ChAT-positive rosettes were concentrated in the nodulus and the ventral uvula of rabbit (Fig. 84), cat and monkey, and that the distribution of ChAT-positive profiles correlated with the distribution of ChAT activity. Large and small mossy fiber rosettes and transitional forms with the plexus of beaded fibers were distinguished in different species (Barmack et al., 1992a). Illing (1990) noted a 'wealth of ChAT-immunoreactive mossy fibers' that were slightly enriched in the ventral folia I to III, IX and X. ChAT-positive mossy fibers were also observed in human cerebellum, but their spatial distribution remains to be described (Kan et al., 1980; DeLacalle et al., 1993). By combining retrograde tracing and ChAT-immunocytochemistry Barmack et al. (1992b) demonstrated that the ChAT-positive mossy fibers in the nodulus, ventral uvula and the flocculus are likely to be secondary vestibular afferents. After injections of horseradish peroxidase in the lobules X and IX in rat and rabbit, neurons in the caudal medial vestibular nucleus and the nucleus prepositus hypoglossi were double labelled (Fig. 198). The flocculus and the paraflocculus of the rabbit receive a small, cholinergic projection, mainly from ChAT-positive neurons of the nucleus prepositus hypoglossi (Barmack et al., 1992b). Csillik et al. (1964) already drew the conclusion from the preferential distribution of AChE-positive glomeruli in the 'archi-cerebellum', that they represent terminals of a cholinergic system of secondary vestibulo cerebellar mossy fibers. Ikeda et al. (1991) noticed that in cat the number ChAT-positive mossy fibers 115
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Fig. 83. Choline-acetyltransferase (ChAT)-like immunoreactivity in the rat uvula-nodulus. A,B. Photomicrographs taken of lobule Xb. These photomicrographs demonstrate varieties of ChAT-positive mossy fiber rosettes in the granule cell layer and in the molecular layer of the rat nodulus. C. Illustration of ChAT-positive staining of mossy fiber rosettes and fine fibers innervating both the granule cell layer and molecular layer of the nodulus (lobule Xb). D. Illustration of three kinds of ChAT-positive mossy fiber rosettes found in the rat cerebellum: (1) Large grape-like terminals found with highest density in the uvula-nodulus (lobules IXb, Xa,b), (2) smaller mossy fiber rosettes that are characteristic of both the uvula (lobule IXa) and the anterior vermis (lobule I), (3) finely beaded ChAT-positive fibers that are found in both the granule cell layer and molecular layer throughout the cerebellum. Primary antibody: rat-~pig-ChAT. Barmack et al. (1992a). (
were reduced following excitotoxic and electrolytic lesions in the cerebellar nuclei (see below). The distribution of ChAT-positive mossy fibers roughly corresponds to that of unipolar brush cells (see Section 3.6.3.), which raises the question whether these mossy fibers innervate these cells. Electron microscopic analysis of ChAT-immunoreactivity in the nodulus showed that a minority (10-20%) of ChAT-immunoreactive mossy fiber terminals synapse on brush cell profiles, and that a minority (10-30%) of the mossy fiber terminals contact unipolar brush cells that are immunoreactive for ChAT (Jaarsma, 1995c). Barmack et al. (1992a) noted a dense population of small ChAT-positive mossy fiber-like terminals in the tip of lobules IXa,b of rat cerebellum that are significantly smaller than the other ChAT positive rosettes (Fig. 83). This type of labelling was not described by Ojima et al. (1989) but could be observed in their Fig. 4. The source and characteristics of this peculiar population of terminals is still unknown. Ojima et al. (1989), Illing (1990), Ikeda et al. (1991), Barmack et al. (1992a), and DeLacalle et al. (1993) observed thin ChAT-positive beaded fibers which were distinct from mossy fiber profiles. According to Ojima et al. (1989) these fibers in rat were most frequent immediately beneath or within the Purkinje cell layer. A substantial number of varicose fibers could also be identified in the molecular layer (Fig. 85B). Illing (1990) in cat noted that thin beaded fibers in the molecular layer mostly course at right angles from the Purkinje cell layer to the surface of the cerebellar cortex, whereas in the granular layer these fibers have an irregular course. The source of the sparse plexus of 'non-mossy' ChAT-immunoreactive fibers is presently unknown. It has been proposed that they originate in the ponto-mesencephalotegmental cholinergic complex (Illing, 1990), but at least for rat this seems to be untrue, since not a single retrogradely labelled, cholinergic neuron was found in the pontomesencephalic tegmentum after tracer injections in the cerebellar cortex (Woolf and Butcher, 1989). The cerebellar nuclei, instead, receive afferents from tegmental cholinergic neurons (see next paragraph). Ojima et al. (1989) showed that all cerebellar nuclei in rat are innervated by CHATimmunoreactive fibers. The density of these fibers varies between the different nuclei. Moderately dense innervation was found in most of the medial nucleus and in the magnocellular part of the lateral nucleus, whereas only a few ChAT-immunoreactive fibers invade the ventromedial parvicellular portion of the lateral nucleus and most of the interposed. Also in the cerebellar nuclei of man a moderate density of ChAT-positive fibers has been observed (DeLacalle et al., 1993). Both in rat and man these fibers did not form pericellular networks. DeLacalle et al. (1993) and Ikeda et al. (1991) also found 117
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sporadic ChAT-positive neurons in the cerebellar nuclei. As demonstrated by Woolf and Butcher (1989) with retrograde tracing, at least a portion of the cholinergic fibers in the cerebellar nuclei are likely to originate from the pedunculopontine tegmental nucleus. The projections are bilateral, but most prominently ipsilateral (Woolf and Butcher, 1989). Cerebellar Golgi cells display strong AChE activity (Shute and Lewis, 1965; Brown and Palay, 1972 and others), but there has been no evidence of ChAT-immunoreactive Golgi cells in the rat (Ojima et al., 1989; Barmack et al., 1992a; Jaarsma et al., 1995c). 118
The cerebellum." chemoarchitecture and anatomy
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ChAT-positive Golgi cells have been found in cat (Illing, 1990; Ikeda et al., 1991) and in man (DeLacalle et al., 1993), although Barmack et al. (1992a) did not see them in their cat and monkey material. Illing (1990) found that the CHAT119
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Distribution and density of choline-acetyltransferase (ChAT)-immunoreactive Golgi cells (dots) in two subsequent 100 pm thick parasagittal sections through the vermis of the cerebellar of the cat. Roman numbers indicate lobules according to Larsell; pcs, pedunculus cerebellaris superior. Scale bar - 2 mm. B and C. Drawings of immunoreactive Golgi cells from 100 pm thick cerebellar sections. The uppermost cell is from the hemisphere, the lower one from the dorsal vermis. The arrows point to processes thought to be axons. ML, molecular layer; GL, granular layer. The border of gray to white matter is marked by a dashed line. Illing (1990). 120
The cerebellum." chemoarchitecture and anatomy
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positive Golgi cells mostly are positioned close to or in the Purkinje cell layer of both the vermal and hemispheral lobules (Fig 86). Also in man, ChAT-positive Golgi cells seem to be preferentially located beneath the Purkinje cell layer, but they were found to have a heterogeneous lobular distribution, with higher densities in the flocculonodular lobules (DeLacalle et al., 1993). In cat the ChAT positive Golgi cells have been estimated to represent only 5% of the total Golgi cell population (Illing, 1990). Ikeda et al. (1991) and DeLacalle et al. (1993) found ChAT immunoreactive neurons in the cerebellar nuclei in cat and man, respectively. According to Ikeda et al. (1991) cholinergic neurons in the cerebellar nuclei project to the cerebellar cortex in the form of mossy fibers, and to the thalamus and the red nucleus (see Section 5.2.).
3.10.2. Cholinergic receptors Nicotine receptors Both nicotine and muscarine receptors have been detected in the cerebellum. Nicotine receptors are cation-gating ion channel complexes and consist of five subunits, like the nicotine receptors in skeletal muscles. The subunit composition of neuronal nicotine receptor is found to be different from the muscle receptor, which is made up of four types of subunits (o~fle~) with two copies of the alpha subunit per receptor. A number of putative nicotine subunits are expressed in the mammalian brain, including at least five alpha subunits, named cz2, cz3, cz4, cz5, and cz7 and three subunits homologous to the muscle beta subunit, /32, /33 and/34 (Deneris et al., 1989; Duvoisin et al., 1989; Wada et al., 1989, 1990; Seguela et al., 1993). The subunits ~3, ~4, and/32 are expressed in rat cerebellum, the granular layer displaying low levels of cz3, cz4 and/32 mRNA, and Purkinje cells expressing high levels of/32 (Wada et al., 1989). Most Purkinje cells do not express any of the alpha subunits, but a few Purkinje cells display strong hybridisation signals with the cz2, ~3 or ~4 probes. Cells in the cerebellar nuclei express moderate levels of cz4 and/32 mRNA. A recent study with antibodies against the/32 subunit showed that the intensity of fl2-immunostaining was high in the perikarya and main dendritic arbors of Purkinje cells, low in granule cells and moderate in neurons of the cerebellar nuclei, thus confirming the results from in situ hybridisation (Hill et al., 1993). Swanson et al. (1987) using a iodinated monoclonal antibody to chicken neuronal acetylcholine receptors ([125I],mAB270) that is assumed to specifically immunoreact with the fl2-subunit of rat, obtained a somewhat different immunodistribution offl2-subunits, with labelling concentrated in the granular layer. Functional channels gated by nicotinic agonists are formed by certain combinations of alpha and beta subunits (Deneris et al., 1991). Receptors made of cz4 and f12 subunits are most common in the brain, and are believed to represent high-affinity [3H]acetylcholine binding sites, (that can be labelled in the presence of atropine to block muscarinic sites), and the [3H]nicotine binding sites of the brain (Clarke, 1993). Accordingly in rat, high-affinity [3H]nicotine binding sites are present at low density in the granular layer and the cerebellar nuclei, which are the structures expressing both cz4 and //2 subunits, but are essentially absent in the Purkinje cell and molecular layer, where ~4 subunits are lacking (Clarke et al., 1985). It should be noted that Purkinje cells, in spite of the fact that they express high levels of fl2 subunit, may lack functional nicotine receptors, because of the absence (as far as known) of alpha subunits. The cz7 subunit is different from the other neuronal nicotine receptor subunits in forming functional channels in the homomeric configuration (S6guala et al., 1993) that, unlike other nicotine receptors of the brain, are sensitive to ~-bungarotoxin (czBTX).
121
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The distribution of ~7-mRNA throughout the brain roughly correspond to that of [125I]~BTX binding sites. It is therefore assumed that nicotine receptors made up of ~7 subunits represent the major portion of brain ~BTX binding sites (Clarke et al., 1985; S6gu61a et al., 1993). The level of [125I]~BTX binding in most of the rat cerebellum is low. Patches of intense labelling were, however, found over glomeruli in the lobules I, IXd, X and the flocculus (Fig. 87; Hunt and Schmidt, 1978; Frostholm and Rotter, 1986). Frostholm and Rotter (1986) postulated that these [125I]0~BTX binding sites were located presynaptically on mossy fibers, based on the observation that developmental 122
The cerebellum." chemoarchitecture and anatomy
Ch. I
appearance of the binding sites corresponds to the appearance of the mossy fibers. Notably, the distribution of [125I]~BTX binding sites is similar to that of ChAT-positive mossy fibers rosettes, which raises the possibility that cerebellar '~BTX-receptors' are localized presynaptically on the subpopulation of cholinergic mossy fibers. The distribution of ~7 mRNA has yet to be studied systematically in the vestibulo-cerebellum, but data from in situ hybridisation presently available suggest that no ~7-mRNA is expressed in the cerebellum, which is consistent with the idea of a presynaptic localisation of '~7-receptors' (S6gu61a et al., 1993). Recently an immunocytochemical study on the distribution of the ~7 protein has been reported, in which three different monoclonal antibodies raised against chicken ~7 were employed (Dominguez del Toro et al., 1994). The results of this study, however, are puzzling since immunolabelling in the cerebellar cortex was mainly localized in Purkinje cells, which disagrees both with the distribution of ~7-mRNA and [125I]~BTX binding sites. Muscarine receptors
Muscarine receptors are coupled to G-proteins and may activate various second messenger cascades. At least four subtypes (M1, M2, M3 and M4) could be distinguished according to pharmacological criteria (e.g. see Waelbroeck et al., 1990), whereas molecular cloning has revealed five distinct subtypes (ml-m5; Hulme et al., 1990). Membrane binding and autoradiographic studies have shown that cerebellar muscarine receptors have the pharmacological properties of the cardiac (M2)-type (Mash and Potter, 1986; Spencer et al., 1986; Waelbroeck et al., 1987, 1990; Araujo et al., 1991; Aubert et al., 1992). Concordantly, the cerebellum has been demonstrated to primarily express m2-mRNA (Vilar6 et al., 1992, 1993) and m2 receptor protein (Levey et al., 1991; Li et al., 1991). It has been proposed that also m3 (respectively M3) receptors may be expressed in the cerebellum, because granule cells in culture express m3 mRNA (Fukamauchi et al., 1991). Immunochemical and pharmacological studies, however, indicate that m3 receptors represent less than 10% of the cerebellar muscarine receptor population (Waelbroeck et al., 1990; Wall et al., 1991). Moreover, no high-affinity binding sites for [3H]4-DAMP, a ligand with preference for M3 receptors, were found in the cerebellum (Araujo et al., 1991). Early autoradiographic studies on the topographic distribution of muscarine receptors in rat cerebellum with [3H] propylbenzilylcholine mustard as the ligand showed that muscarine receptors were confined to the molecular layer of the nodulus and the ventral uvula (Rotter et al., 1979a,b). Subsequent autoradiographic studies in rat with the more potent antagonist [3H]quinuclinidyl benzylate ([3H]QNB, Neustadt et al., 1988) and with M2-specific ligands [3H]oxotremorine (Spencer et al., 1986; Vilar6 et al., 1992), and [3H]AF-DX384 (Aubert et al., 1992), all gave identical results. Muscarine receptors were found to be localized throughout the whole cerebellar cortex, with the highest densities in the molecular layer of the nodulus and the ventral uvula (Fig. 88). The amount of muscarine receptors in the other lobules was relatively low, with higher densities in the granular and Purkinje cell layers than in the molecular layer (Fig. 88). Muscarine receptors also occur in the cerebellar nuclei (Spencer et al., 1986; Neustadt et al., 1988; Vilar6 et al., 1992) and in some parts of the white matter. In the caudal folia of the uvula (lobule IXb) muscarine receptors appear to be distributed in five parasagittal columns of high receptor density that traverse the granular layer (Fig. 88D, Neustadt et al., 1988). In situ hybridisation shows that m2 mRNA in rat cerebellum is localized in the granular layer as well as in the deep nuclei (Vilar6 et al., 1992). In accordance with the autoradiographic data the highest signal is present in the nodulus and ventral uvula. 123
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 88. Bright-field photograph showing cresyl-violet staining (A and C) and corresponding dark-field photomicrographs showing the distribution of muscarine receptors using [3H]QNB binding (B and D, respectively) in the caudal cerebellum of the rat. A and B. Coronal section through the nodulus (X) and the rostral uvula (IX) showing high density of [3H]QNB binding over the molecular layer (m) of lobule X and ventral region of lobule IX. Also note higher binding densities at the level of the Purkinje cell layer (P) in most lobules. C and D. Coronal section at the level of the dorso-caudal uvula (IXa,b). Note five parasagittal columns (large arrows in D) of high receptor density that traverse the granular layer (g); w, white matter. Scale bars = 1 mm. Neustadt et al. (1988).
Since in this region the mRNA signal is in the granule cell layer, whereas receptor binding is high over the molecular layer (Fig. 89), one is tempted to speculate that the muscarine receptors are associated with parallel fibers. In fact, analysis of sections immunostained by Levey et al. (1991) with a polyclonal antibody specific for the m2 receptor, showed that parallel fibers are immunostained (Jaarsma et al., 1995a). Parallel fiber staining was seen in all lobules. It remains to be determined whether the amount of parallel fiber staining is most prominent in the nodulus and ventral uvula, concordant with the density distribution of receptor binding. In addition to parallel fibers the m2-antibody also immunostained Golgi cells and some mossy fiber rosettes (Jaarsma et al., 1995a). Neustadt et al. (1988) using receptor autoradiography noted that the topographic distribution of muscarine receptors in rabbit and guinea pig cerebellum is characterized by the presence of parasagittal columns of very high receptor density over the molecular layer (Fig. 90). In rabbit the bands of high muscarine receptor density were most prominent in the anterior lobe (lobules I-V), in Crus I and II, and in the flocculus and the ventral paraflocculus. Their distribution only partially corresponded to the Zebrinpositive zones (Jaarsma et al., 1995a). The receptors in the parasagittal bands were 124
The cerebellum." chemoarchitecture and anatomy
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Fig. 89. Comparison between the distribution of m2 muscarine receptor mRNA (A) and [3H]oxotremorine-M binding to muscarine M2 receptor sites (B) in approximately equivalent coronal sections of rat brain at the level of the caudal cerebellum. Note that m2 mRNAs in the ventral uvula (IX) and the nodulus are localized in the granular layer, whereas M2 receptor binding sites are in the molecular layer (m). 12, hypoglossalnucleus. Scale bar - 3 ram. Vilar6 et al. (1992).
located in Purkinje cell dendrites as demonstrated immunocytochemically with a monoclonal antibody specific for the m2 receptor protein. A second characteristic feature of the distribution of rnuscarine receptor in rabbit cerebellum, is the presence of a stripe of very high receptor density immediately above the Purkinje cell layer in the nodulus and the ventral uvula (Fig. 90B) (Neustadt et al., 1988). Immunocytochemistry showed that the receptors in this transverse stripe were localized on a population of densely packed parallel fibers (Jaarsma et al., 1995a). In addition to these parallel fibers and Purkinje cell dendrites, also Golgi cells, that in rabbit in part are located in the molecular layer (Spa~ek et al., 1973), and a subpopulation of mossy fiber rosettes are immunostained with m2-receptor antibody (Jaarsma et al., 1995a). Autoradiographic data from human (Cortes et al., 1987) and cat cerebellum (D. Jaarsma, unpublished observations), and immunocytochemical data from monkey (Jaarsma and Levey, unpublished) indicate that the distribution of muscarine receptors in the cerebellum of these species is different from that in rodents and rabbit. The level of ligand binding was very low with slightly higher receptor densities over the granular layer. There is no increased receptor density in the nodulus and the ventral uvula. The overall distribution in monkey was similar. Only Golgi cells and a subpopulation of mossy fibers rosettes, (mainly located in the vermal lobules III-VI), were immunostained (Jaarsma and Levey, unpublished). Summarizing it appears that the cellular and regional distribution of muscarine receptors in the cerebellum is different between different species. Golgi cells and subpopulations of mossy fibers seem to express muscarine receptors most constantly. An interesting aspect about the presence of muscarine receptors in parallel fibers in rat and rabbit, is that the lobular distribution of m2-containing parallel fibers, is the same as that of ChAT-positive mossy fiber rosettes (see above). This raises the possibility that muscarine m2 receptor are specifically expressed by those granule cells that are innervated by cholinergic mossy fibers. If this proves to be true, this would imply that there 125
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is some regulatory interaction between the ChAT-positive mossy fibers and granule cells through which the expression of muscarine m2-receptor is regulated. Although lightmicroscopic analysis suggest that the muscarinic receptors are absent in granule cell dendrites at the synapses with ChAT-positive mossy fibers, this possibility can not be excluded without electron microscopic examination. The function of muscarine receptors in the parallel fibers is puzzling since there seems to be little cholinergic innervation in the molecular layer (see above). 126
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Physiological responses to acetylcholine Only a few physiological studies on the actions of acetylcholine in the cerebellar cortex have been reported and the results of these studies were often contradictory: Iontophoretic application of acetylcholine has been observed to mildly excite Purkinje cells via a muscarinic (Crawford et al., 1966) or a nicotic (McCance and Phillis, 1968) mechanism, or to inhibit them via a mixed muscarinic and nicotinic mechanism. For interneurons no effect of acetylcholine was found by Crawford et al. (1966), whereas De la Garza et al. (1987) found a strong nicotinic excitatory effect. Cr6pel and Dhanjal (1982) reported slow depolarisations of Purkinje cells, accompanied by an increase in membrane resistance, with application of high doses of acetylcholine in slices of the nodulus and the ventral uvula of the rat's cerebellum, that was mediated through muscarine receptors.
3.10.3. Acetylcholinesterase Based on their substrate specificity cholinesterases can be subdivided into the acetylcholinesterases (AChE's) and the butyryl or pseudocholinesterases. Several isoenzymes of AChE have been detected in brain tissue. Three to four isoenzymes of AChE can be distinguished in developing brain (Henderson, 1977). In the chicken the faster migrating forms predominate in embryos. Their composition changes progressively into the slowly migrating forms of the adult tissues. Pseudocholinesterase can be distinguished from the acetylcholinesterases by specific inhibitors (iso-OMPA or ethopropazine) and by using butyrylcholiniodide as a substrate, which is split predominantly, though not exclusively by pseudocholinesterase. Most other esterases can be inhibited using eserine (Marani et al., 1977). Butyrylcholinesterase has also been localized with immunocytochemical methods (Barth and Ghandour, 1983). AChE-staining in the molecular layer of the mammalian cerebellum is generally lower than in the granular layer, but great variations occur and the pattern is reversed in the human and the avian cerebellum (Friede and Fleming, 1964). AChE-staining generally is higher in the vestibulocerebellum than in other parts of the cerebellum. The reactivity in the glomeruli in rat (Csillik et al., 1964; Brown and Palay, 1972 and many others) and rhesus monkey (Robertson and Roman, 1989) follows this general pattern, but the distribution of AChE-positive Golgi cells is more uniform (Brown and Palay, 1972). In the cerebellar white matter of monkey cerebellum AChE-rich fibers are distributed in parasagittal compartments, that are aligned with concentrations of strongly ACHEreactive regions in the granule cell layer and narrower, AChE-rich 'spikes' in the molecular layer (Hess and Voogd, 1986; Marani, 1986). Purkinje cells in certain parts of rat and guinea pig cerebellum, including the lobules IX and X of the caudal vermis, display a transient reactivity for ACHE, which disappears later. AChE was localized in adult Purkinje cells of the lobules IX and X (Robertson et al., 1991); these cells are arranged in multiple, sagittal bands (Gorenstein et al., 1987). Robertson et al. (1991), however, were unable to confirm the transient staining with AChE in rat Purkinje cells. AChE-positive displaced Golgi cells (Ramon y Cajal, 1911) are present in the lower one third of the entire molecular layer of the rabbit cerebellum (Spa~ek et al., 1973) and strong AChE-staining is present in this stratum in the vermis and certain lobules of the hemisphere in the same species (Tan et al., 1995a). An AChE-band pattern was detected in the molecular layer of the vermis of the anterior and posterior lobes in 2-4 month old cats (Marani and Voogd, 1977) (Fig. 120). 127
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J. Voogd, D. Jaarsma and E. Marani
Reaction product in transverse sections was present in narrow striations reaching from the AChE-negative Purkinje cell somata to the pial surface; in sagittal sections the distribution of AChE was more uniform. The same banding pattern could be visualized in adult cats, with AChE-histochemistry on aldehyde-fixed tissue (Brown and Graybiel, 1983). No such banding was present in the cerebellum of rats or primates (Marani, 1986). Spikes of strong AChE-activity sometimes extend from heavily stained portions of the granular layer into the molecular layer, at certain preferred localizations (Hess and Voogd, 1986). The ultrastructural localisation of AChE has methodological problems, because of uncontrolled diffusion of reaction product (e.g. see discussion Brown and Palay, 1972). In the molecular layer AChE-reaction product was located in subsurface cisterns of Purkinje cell dendrites and in the spine apparatus of their dendritic spines and, extracellularly, associated with the dendritic plasma membrane and around parallel fibers (Marani, 1982a, 1986). AChE was never found in the synaptic cleft of parallel fiberPurkinje cell synapses, lntra-axonal deposits in parallel fibers were considered as an artefact. Neurons in deep parts of the molecular layer that may represent basket or stellate cells, contain AChE-reaction product in their rough endoplasmatic reticulum. AChE in cat molecular layer, therefore, is associated with neurons and not with glial cells. AChE in the molecular layer of the rat is preferentially located in Bergmann glia (Friede and Fleming, 1964; Barth and Ghandour, 1983; Gorenstein et al., 1987; Robertson et al., 1991). Brown (1985a) demonstrated an increase in AChE-staining in the cat molecular layer, 2-6 weeks after lesions of the inferior olive. Upregulation of AChE was found in the projection zones of the lesioned olivary neurons. These plastic changes are reminiscent of the re-appearance of neonatal AChE in Purkinje cells after the transection of their axons (Phillis, 1968). AChE-staining in the granular layer is distributed in 'An alternating fine mosaic of stained and unstained spots, with regional differences in staining intensity' that represent AChE-positive Golgi cells and moderately to strongly stained glomeruli. Granule cell somata remain unstained in the rat cerebellar cortex (Altman and Das, 1970). The presence of AChE in glomeruli has been generally acknowledged (Gerebtzoff, 1959; Csillik et al., 1964; Shute and Lewis, 1965). AChE-reaction product in rat cerebellum is located outside the mossy fiber terminal, between the axonal plasma membrane and the dendrites of the granule cells and the Golgi cell terminals. AChE in Golgi cells is associated with the cisterns of the rough endoplasmatic reticulum and the perinuclear cisterns. Granule cells, Purkinje cells and glia are AChE-negative (Brown and Palay, 1972). The preferential staining of the borders of white matter compartments (the 'raphes', Voogd, 1964) for AChE (Hess and Voogd, 1986; Marani, 1986; Voogd, 1995; Fig. 126) and its use in topographical analysis of the corticonuclear and olivocerebellar projections will be considered in Sections 6.1.1., 6.1.2., 6.1.5. and 6.3.3. Several authors have attempted to devise other functions for AChE than the hydrolytic cleavage of acetylcholine; their proposals were reviewed by Greenfield (1984) and Appleyard and Jahnsen (1992). 3.11. NEUROGLIA The morphology of the neuroglia in the cerebellar cortex was reviewed by Palay and Chan-Palay (1974) and Ghandour et al. (1980). Three forms were distinguished, the Bergmann glial fibers in the molecular layer that take their origin from Golgi epithelial cells, located in the Purkinje cell layer, the astroglia and the oligodendroglia. The 128
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 91. Bergmann glial fibers in the rat cerebella of various ages. PAP immunocytochemistry with anti-GFAP antiserum. The ages of the rats are 3, 5, 8 days and 30 months in A, B, C and D, respectively. EG, external granule cell layer; M, molecular layer; G, granule cell layer. Scale bars = 20/lm. De Blas and Cherwinski (1985).
enzyme histochemistry of cerebellar glia was studied by Sotelo (1967). Neuroglia has its own metabolism, that is qualitatively different from that of neurons. The glial anaerobic glycolysis is high in neuronal neuroglia and interfascicular neuroglia. The aerobic glycolysis is secondary and much weaker than in nerve cells. The hexose129
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J. Voogd, D. Jaarsma and E. Marani
Fig. 92. Micrograph of adult cerebellar cortex, incubation with vimentin antibody V9 counterstaining with cresylviolet. Sagittal unfixed cryostat section. Bergmann glial fibers (arrowheads) and large astrocytes (arrows) are seen in the molecular and granular layer, respectively, pc, Purkinje cell; ml, molecular layer; gl, granular layer. Bar = 100 pm. Roeling and Feirabend (1988)
monophosphate shunt is also important in glia. In oligodendroglia and astroglia different enzyme patterns were found. The main difference concerns glycogen, which is metabolised almost exclusively in astroglia (Sotelo 1967). Bergmann glia and astroglia are immunoreactive for anti-glial fibrillary acidic protein (GFAP) (Fig. 91). The development and the adult configuration of the Bergmann glia has been studied, using antibodies against GFAP (Bignami and Dahl, 1974; De Blas, 1984; De Blas and Cherwinsky, 1985; Levitt and Rakic, 1980; Gr~iber and Kreutzberg, 1985; Pelc et al., 1986). Bergmann glia can also be demonstrated with anti-vimentin (Dupouey et al., 1985; Roeling and Feirabend, 1988) (Fig. 92) and with cell-specific monoclonal antibodies (De Blas, 1984; De Blas and Cherwinsky, 1985; Edwards et al., 1986). Butyrylcholinesterase has been localized in Bergmann glia and in glial cells in the granular layer using an immunocytochemical method in the rat (Barth and Ghandour, 1983). Carbonic anhydrase is a specific marker for oligodendrocytes (Cammer, 1984). The distribution of oligodendrocytes in the cerebellar cortex of the mouse was studied with immunostaining for carbonic anhydrase II by Ghandour et al. (1980, 1981). Occasionally oligodendrocytes are present in the molecular layer, they are more common in the granular layer and abundant in the white matter, sn-Glycerol-3-phosphate-dehydrogenase (GPDH) in mice is present in adult oligodendrocytes (De Vellis et al., 1977), but also in Bergmann glia (Fisher et al., 1981) (Fig. 93). The expression of GPDH in Bergmann fibers is dependent on the adjoining Purkinje cells (Fisher, 1983). A similar interdependency may exist for 5'-N in Bergmann glia (Hess and Hess, 1986) (see Section 130
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 93. Sagittal section through the cerebellum of a 25-day-old BALB/cBy mouse, with anti-sn-glycerol-3 phosphate dehydrogenase (GPDH) serum (1:10,000) viewed with a dark-field condenser. The molecular layer is densely stained although clear areas representing non-staining cell bodies and processes are numerous. Intensely stained cell bodies of the Bergmann glia are located between large unstained Purkinje cell somata, at the boundary between the molecular and granular layers. Magnification x l00. Fisher et al. (1981). 3.5.), for t a u r i n e t h a t shifts f r o m P u r k i n j e cells to B e r g m a n n glia u n d e r c o n d i t i o n s of h y p o - o s m o t i c stress ( N a g e l h u s et al., 1993) (see Section 3.1.2.) a n d Z e b r i n I, t h a t a p p e a r s in B e r g m a n n glia after lesions of the cerebellum ( D u s a r t et al., 1994).
Fig. 94. A and B. Distribution of 3-fucosyl-N-acetyl-lactosamine (FAL)-immunoreactive Bergmann glial cells in adult mouse cerebellum. Note zonal distribution of immunoreactive neuroglia in molecular layer (MOL) in A. GCL, granular layer. Bars in A = 800/~m, in B = 100 ~tm. Courtesy of Dr. J.K. Mai, Department of Neuro-anatomy, Heinrich Heine University, Dfisseldorf. 131
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J. Voogd, D. Jaarsma and E. Marani
Fig. 95. Chicken cerebellum, 1 day old, 1-/lm thick sections. Post-embedding immunocytochemistry with antibody IX-50 against a putative kainate receptor shows immunoreactivity outlining Purkinje cells (Pc), their main ascending dendrites (arrows) as well as their spiny branchlets. Immunoreactivity is associated with Bergmann glial cells (BG) which lie beneath and amongst the Purkinje cells. Scale bar = 20/~m. Somogyi et al. (1990).
Several substances are distributed in zonally distributed Bergmann glial cells. This may be the case for 5'-N, and has been observed for 3-fucosyl-acetyl-lactosamine (FAL) immunoreactivity in mouse cerebellum (Fig. 94) (Bartsch and Mai, 1991; Marani and Mai, 1992). The protein kinase types ~, flII, y and ( have been localized in Bergmann glia (Shimohama et al., 1990; Hidaka et al., 1988; Wetsel et al., 1992). Bergmann glial cells 132
The cerebellum." chemoarchitecture and anatomy
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contain both nitric oxide synthase and guanylcyclase and are the main cerebellar store for cyclic GMP (see Section 3.4.). As mentioned before also homocysteic acid (Cu6nod et al., 1990; Grandes et al., 1991; Tschopp et al., 1992) (Figs 44 and 45) and 5'nucleotidase (Kreutzberg et al., 1978 and Fig. 59; see, however, Marani 1986 and Fig. 58) are associated with Bergmann glia. Climbing fiber-induced release of homocysteic acid and adenosine, therefore, involve this glial compartment (see Sections 3.2.2. and 3.5.). Bergmann glial cells and granular layer astroglia are provided with glutamate receptors and, therefore, may be actively involved in the cerebellar neural transmission (Fig. 95) (see Section 3.3.1.).
4. GROSS ANATOMY OF THE MAMMALIAN CEREBELLUM The gross anatomy of the cerebellum is the morphology of its cortical sheet. The cerebellum in mammals, birds and some reptiles is subdivided by transverse fissures of varying depth in lobes, lobules and folia. Two paramedian sulci demarcate the vermis from the hemispheres in the mammalian cerebellum. The present nomenclature for the mammalian cerebellum evolved from older purely descriptive studies of the human cerebellum (reviewed by Glickstein, 1987), elaborate comparative anatomical studies of the adult cerebellum (Elliot Smith, 1903; Bolk, 1906; Riley, 1928, reviewed by Larsell 1970; Larsell and Jansen, 1972) and comparative embryological investigations of the development of the folial pattern (Stroud, 1895; Bradley 1903, 1904; Larsell, 1947, 1952, 1953, 1954, 1970). The cerebellum of the rat served as the prototype for Larsell's subdivision of the mammalian cerebellum in 10 lobules (Larsell, 1952, 1970) (Fig. 96).
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Larsell extensively studied its adult morphology and the development of its folial pattern. More recent accounts of the cerebellum of the rat (Voogd, 1995) (Fig. 97) and the mouse (Marani and Voogd, 1979) are available. The cerebellum of the cat was described by Larsell (1953, 1970) and Voogd (1964) (Fig. 98). The gross morphology of the cerebellum in different species of macaques was described by Larsell (1953, 1970), and by Madigan and Carpenter (1971) for Macaca rhesus. The anatomy of the cerebellum of macaques is very similar to other primates (Macaca fascicularis, Fig. 99) (Larsell, 1970: Cercocebus sp. Cebus sp., Ateles, Saimiri sciurus) and subprimates (Haines, 1969). It is possible to recognize the main subdivisions in nearly all mammalian cerebella by inspection of the branching pattern of the arbor vitae in a midsagittal section of the vermis (Fig. 98) and the characteristic convolutions of the folia of the hemisphere. For the lobules of the vermis either the classical nomenclature of the human cerebellum or Larsell's (1952, 1970) numbering system can be applied. The anterior lobe consists of lingula (lobule I), the central lobule (II, III), and the culmen (IV, V). The base of the lingula is continuous with or embedded in the anterior medullary velum. The subdivision of the anterior lobe by the preculminate fissure into the lobules I-III and the lobules IV-V is of prime relevance to its connectivity. It is usually deep and its walls are subfoliated. The anterior lobe is separated from the posterior lobe by the deep primary fissure. The posterior lobe vermis can be subdivided in most mammals in the declive (VI), folium and tuber (VII A and VII B), the pyramis (VIII), the uvula (IX) and the nodule (X). The secondary and posterolateral fissures in between the lobules VIII, IX and X usually are quite distinct. The prepyramidal fissure separating lobule VII and VIII is less obvious in some species and the borders between the lobules VI, VII A and VII B often are difficult to recognize. For the lobules of the hemisphere of the mammalian cerebellum both Bolk's (1906) descriptive terms and Larsell's numerals can be used. In Larsell's nomenclature the hemispheral lobules bear de prefix H to the number of the vermal lobule with which they are continuous. Bolk's (1906) and Larsell's (1952, 1970) nomenclature for the hemisphere are not readily interchangeable because they are based on conflicting views on the morphology of the mammalian cerebellum (Fig. 96). Bolk (1906; see Glickstein and Voogd, 1995, for a recent discussion of Bolk's views) stressed the difference between vermis and hemisphere that should be considered as independent folial chains, which have a tendency to local longitudinal expansion. For the cat the folial chains are illustrated in Fig. 98. In the anterior lobe the folial chains of vermis and hemisphere develop in parallel. The inter- and most of the intralobular fissures continue uninterruptedly from the vermis into the hemispheres and a paramedian sulcus is either absent or shallow. The same situation prevails in the region of the posterior lobe immediately behind the primary fissure. Bolk coined the name 'lobulus simplex' to express the continuity of vermis and hemisphere in this part of the cerebellum. The vermal portion of the simple lobule corresponds to the declive (Larsell's lobule VI). Caudal to the lobulus simplex the folial chain of the hemisphere deviates from the vermis and forms a series of loops. The first loop is the ansiform lobule, that can be subdivided in the Crus I and the Crus II. The cortex in the center of the ansiform lobule, in between folium vermis (lobule VIIA of Larsell) and the hemisphere, usually is interrupted (Figs 97 and 99). This interruption may involve all layers of the cortex, with white matter coming to the surface, or only affect the parallel fibers of the molecular layer. At the level of the paramedian lobule the folial chains of vermis and hemisphere are aligned and the cortex between them usually is continuous. The cortex of the pyramis (Larsell's lobule VIII) 135
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continues into the caudal part of the paramedian lobule as the copula puramidis. The paraflocculus and the flocculus are the caudal segments of the folial chain of the hemisphere. The cortex between the paraflocculus and flocculus and the caudal vermis 136
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(uvula and nodulus: lobules IX and X of Larsell) usually is completely interrupted. White matter invades the centre of the successive loops of the folial chain of the hemisphere and separates the successive segments of the flocculus and the paraflocculus. The dorsal and ventral segments of the paraflocculus are designated as the dorsal and ventral paraflocculus and the portion of the paraflocculus located in the subarcuate fossa of the petrosal bone is known as the petrosal lobule. This nomenclature is purely descriptive and these terms do not necessarily refer to identical segments of the paraflocculus in different species (see Fig. 97e for the topology in the rat and Fig. 149 for a comparison of rabbit, monkey and cat). The border between the paraflocculus and the flocculus is the posterolateral fissure. This fissure is one of the first to develop in the hemisphere. Its localization was established by Larsell (1970) for many mammalian species. Larsell tried to verify the relations between vermis and hemisphere by tracing the development of the transverse fissures. He attached great importance to the confluence of the vermal and hemispheral parts of fissures which, in the adult, would indicate the transverse continuity of the lobules of vermis and hemisphere. Larsell's nomenclature for the cerebellar hemisphere is rather unpractical and the significance of some of his many subdivisions (significance in the sense that a portion of the hemisphere has something in common with the vermal lobule to which it belongs) is questionable.
5. THE CEREBELLAR NUCLEI The (central) cerebellar nuclei and the lateral vestibular nucleus of Deiters receive the axons of the Purkinje cells of the cerebellar cortex and serve as the main output stations of the cerebellum. The vermis and the flocculus also project to other vestibular nuclei, but here the Purkinje cell axons compete with vestibular root fibers, intrinsic and commissural vestibular connections and projections from the medial cerebellar nucleus and, therefore, are not the dominant afferent system. Large numbers of Purkinje cells axons converge upon the cerebellar nuclei. The Purkinje cell/dentate nuclear cell convergence ratio is about 14:1 in the rhesus monkey and about 30:1 in the rat (Chan-Palay, 1977). It was estimated from Golgi impregnated Purkinje cell axons of the rat that between 20-50 perikarya of central nuclear neurons are included within the conical terminal field of a single axon (Chan-Palay, 1977), but the actual number of central nuclear neurons contacted by a single axon must be far greater. A considerable overlap in the corticonuclear projection of adjacent cortical areas was observed in axonal tracing studies (Courville and Diakew, 1976; Armstrong and Schild, 1978a,b; Haines and Koletar, 1979; Haines et al., 1982). Convergence and overlap are greatest in the rostrocaudal dimension where the entire length of the cortical sheet is compressed in the small volume of the cerebellar nuclei. Convergence is much less in the transverse direction and overlap may be even absent at the borders of neighbouring longitudinal zones where the Purkinje cells project to different nuclei (Voogd, 1964; Voogd and Bigar6, 1980). The convergence of the Purkinje cells from apex and base of a lobule onto the same cerebellar nuclear neurons is usually taken for granted. This type of convergence is of potential interest, because it combines the output of Purkinje cells that are under the influence of quite different mossy fiber-parallel fiber systems: corticopontine and exteroceptive systems at the apex and vestibular and proprioceptive systems at the base of the lobules (see also Section 6.4.2.). The subdivision of the cerebellar nuclei is closely related to the longitudinal, zonal 138
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Fig. 100. Midsagittal sections through the cerebellum of man (A), rhesus monkey (B), rat (C) and cat (D).
organization of the corticonuclear and olivocerebellar projections. In this view the cerebellar nuclei are the output stations of corticonuclear modules and their independent olivocerebellar control systems. Certain cytochemical correlates of this longitudinal organization exist, but there are no signs of a corresponding cytochemical differentiation in the output of the cerebellar nuclei. The main input to the cerebellar nuclei from the Purkinje cells is GABAergic and inhibitory (Sections 3.1.1. and 5.5.). The nuclear cells receive their excitatory drive from collaterals from afferent mossy and climbing fiber systems (Section 5.6.). Collaterals of olivocerebellar fibers are organized according to the same longitudinal principle as their climbing fiber terminals, i.e. collaterals terminate in the cerebellar nucleus that receives axons from Purkinje cells that receive climbing fibers from the same parent axons (Groenewegen and Voogd, 1977). Mossy fiber input to the cerebellar nuclei is more diffuse, i.e. not limited to a single nucleus, and more selective, i.e. not present in all mossy fiber systems. Mossy fibers, therefore, are diverse in origin and dissimilar with respect to their collateral projections to the cerebellar nuclei, but as yet neurochemical correlates of this diversity are lacking. The monoaminergic input to the central nuclei 139
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J. Voogd, D. Jaarsma and E. Marani
is diffuse and independent of the mossy and climbing fiber collaterals. It is mainly represented by serotonin in a varicose, all pervading plexus (Section 5.7.). Neurons of the cerebellar nuclei are a mixed population of cells of all shapes and sizes. Several authors noticed a binominal distribution for cell size in the cerebellar nuclei. According to Courville and Cooper (1970) all four central nuclei of the monkey contain cells of all sizes. They noticed a peak in the size distribution for the lateral and interposed nuclei at 22 ~tm cell diameter. Palkovits et al. (1977) distinguished two populations of neurons, one with small and one with large nuclei, in the fastigial and posterior interposed nuclei and less clearly so in the lateral nucleus of the cat. Chan-Palay (1977) classified the cells of the lateral cerebellar nucleus in approximately equal groups of small and large neurons, with a separation at 180 t i m 2 surface area for the rat and 270 t i m 2 for the monkey. These binominal distributions can be explained by the presence of a population of small, GABAergic neurons and a non-GABAergic population of putative glutamatergic cells of different sizes, as recently reported by Batini et al. (1992) for the rat cerebellar nuclei (Section 5.1.3.) (Fig. 110). The targets of the projections of individual cerebellar nuclei in the brain stem and the cord differ, but some of their projections i.e. to the thalamus, show a remarkable degree of overlap. As yet there exists no corresponding differentiation in the chemoarchitecture of the cerebellar nuclei. The only system for which the neurotransmitter is known is the nucleo olivary projection that takes its origin from the population of small, GABAergic neurons (Mugnaini and Oertel, 1985). GABA also may be present in the intrinsic connections of the cerebellar nuclei and in their nucleocortical projection (Section 5.3.). 5.1. SUBDIVISION OF THE CEREBELLAR NUCLEI The cerebellar nuclei with their efferent tracts border on the ventricular surface of the cerebellum. Ventrolaterally they are continuous with the vestibular nuclei. They are surrounded by a sheet of afferent fibers from the restiform body and the middle cerebellar peduncle. This sheet is perforated by the bundles of Purkinje cell axons that terminate in the cerebellar nuclei or continue through or along these nuclei to the vestibular nuclei. The cerebellar nuclei of mammals can be subdivided according to Brunner (1919) into three, medio-laterally arranged nuclei or according to Weidenreich (1899) and Ogawa (1935) into a rostrolateral and a caudomedial nuclear group. Cytoarchitectonic criteria can be used to subdivide the cerebellar nuclei, but the presence of fiber bundles, and the disposition of their efferents in the nuclear hilus and in their efferent tracts are especially important in this respect. Brunner's (1919) mediolateral subdivision into the medial (fastigial), lateral (dentate) and interposed nuclei is based on the contours of the cerebellar nuclear mass. The three nuclei are part of a continuum and the nuclear borders therefore remain arbitrary. Although Brunner's concept of the central nuclei as a single mass has been disproved, his names for the nuclei have been retained. Weidenreich's (1899) comparative studies in mammals had already shown that fiber bundles subdivide the nuclei into a caudomedial group, that includes the medial nucleus and the caudal part of the Brunner's interposed nucleus (the nucleus interpositus posterior of Ogawa, 1935) and a rostrolateral group that is composed of the lateral cerebellar nucleus and the rostral part of the interposed nucleus (the nucleus interpositus anterior). The nuclei within each group are interconnected by cell bridges. The subdivision of Weidenreich-Ogawa can be readily appreciated in lagomorpha (Ono and Kato, 1938; Snider 1940) in carnivores (Flood and 140
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Jansen, 1961; Voogd 1964) and primates (Courville and Cooper, 1970; Chan-Palay, 1977). In rodents and insectivores the separation between the two nuclear groups is less distinct and the connections between the nuclei of each group are more extensive (Korneliussen, 1968a; Ohkawa 1957). The subdivision of Weidenreich-Ogawa received strong support from the localization of the fibers in the superior cerebellar peduncle. A small medial and a large lateral portion can be distinguished in this pathway in most mammals at its exit from the central nuclei (see* in Fig. 102). Experiments in cat (Verhaart, 1956; Voogd, 1964) and rat (Haroian et al., 1981) have shown that the medial part of the superior cerebellar peduncle takes its origin from the nuclei of the caudomedial group, mainly from the ipsilateral posterior interposed nucleus, and the lateral portion from the ipsilateral anterior interposed and lateral cerebellar nucleus. The efferent connections of the cerebellar nuclei were reviewed by Voogd et al. (1990), Ruigrok and Cella (1995), Voogd (1995) and Glickstein and Voogd (1995). The efferent connections of the lateral and interposed nuclei are summarized in Fig. 101. They give rise to nucleo olivary fibers which, at their exit from the nuclei, occupy a more ventral and medial position than the main bundle of the superior cerebellar peduncle (Legendre and Courville, 1987). After its decussation at the border of met- and mesencephalon, the superior cerebellar peduncle splits in ascending and descending branches. An uncrossed descending branch that detaches from the peduncle prior to its decussation, is only present in rodents (Ramon y Cajal, 1911). The main target of the crossed descending branch is the nucleus reticularis tegmenti pontis. This nucleus gives rise to a recurrent 141
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Fig. 102. A. Course of the direct fastigiobulbar tracts (left) and the uncinate tract (u) (right) in the stereotaxic planes P6-P8 in the cat. bc, brachium conjunctivum; bp, brachium pontis; CO, cochlear nuclei; cr, restiform body; dfb, direct fastigiobulbar tract; drV, spinal tract of the trigeminal nerve; DV, spinal vestibular nucleus; F, fastigial nucleus; FLO, flocculus; gVII, genu of the facial nerve; IA, anterior interposed nucleus; L, lateral cerebellar nucleus; LV, lateral vestibular nucleus; mlf, medial longitudinal fasciculus; MV, medial vestibular nucleus; sad, dorsal acoustic striae; SV, superior vestibular nucleus; vma, anterior medullaryvelum; vsc, ventral spinocerebellar tract. Modified from Voogd (1964). B. Hfiggqvist-stainedsection at the level of P7. Note the uncinate tract (u) arching over the brachium conjunctivum and the medial course of the direct fastigiobulbar tract (dfb). bc, brachium conjunctivum; cr, restiform body; dfb, direct fastigiobulbar tract; IA, anterior interposed nucleus; oc, olivocerebellar fibers; SV, superior vestibular nucleus; u, uncinate tract; vsc, ventral spinocerebellar tract; asterisk, medial one-third of the brachium conjunctivum, containing fibers from the posterior interposed nucleus. Voogd et al. (1990) (
mossy fiber pathway to the cerebellar cortex, with a strong collateral projection to the cerebellar nuclei. The ascending branch terminates in the red nucleus, in nuclei at the mesodiencephalic junction, including Darkschewitsch nucleus, that give rise to the medial and central tegmental tracts, which terminate in the inferior olive. The ascending branch terminates in the thalamus. Not all the nuclei contribute equally to each connection. The lateral and the anterior interposed nuclei project heavily to the nucleus reticularis tegmenti pontis and the red nucleus, whereas the posterior interposed nucleus is preferentially connected with the nuclei of the mesodiencephalic junction. The fastigial nucleus gives rise to fibers of the uncinate tract, that decussate within the cerebellum, and to the ipsilateral fastigiobulbar tract, that passes medial to the superior cerebellar peduncle in the lateral wall of the fourth ventricle (Fig. 102) (Voogd, 1964; Batton et al., 1977). The uncinate tract gives rise to a small, ascending bundle, that terminates in the central grey, the mesencephalic tegmentum and the thalamus. The majority of the uncinate- and direct fastigiobulbar tract fibers terminate in the vestibular nuclei and the reticular formation of pons and medulla oblongata. Regions containing predominantly small cells have been distinguished in the ventral parts of the cerebellar nuclei of cat (Flood and Jansen, 1961; Voogd, 1964), rat (Korneliussen, 1968a; Beitz and Chan-Palay, 1979; Voogd, 1995), several subprimates (Haines, 1977b) and primates (Courville and Cooper, 1970). These parvicellular regions are not well-defined and difficult to compare in different species. Even where they were designated as subnuclei (Flood and Jansen, 1961) these subdivisions had no clear functional basis. Data on the connections of the small cells are scanty and conflicting. Haines (1977b) proposed direct and indirect projections of the parvicellular medial and lateral cerebellar nuclei to the oculomotor nuclei. Itoh and Mizuno (1979) found a projection of the parvicellular dentate in the cat to the pulvinar and Mugnaini and Oertel (1985) noticed a concentration of small, GABAergic, nucleoolivary neurons in the ventral parvicellular part of the lateral nucleus of the rat. The group y of Brodal and Pompeiano (1957) and the basal interstitial nucleus of Langer (1985) are two nuclei which are located within the cerebellum, outside the 'classical' cerebellar nuclei. The group y was defined as a small-celled subgroup of the vestibular nuclei of the cat, that caps the restiform body dorsally. Ventrolaterally it is in contact with the dorsal cochlear nucleus. Dorsally scattered cells form strands extending to the dentate nucleus. Medially it is continuous with the small cells of the caudal pole of the superior vestibular nucleus, located dorsolateral to Deiters' nucleus also known as the group 1 of Brodal and Pompeiano (1957). In transverse sections the cells of the y group are fusiform because they are located between the fibers of the floccular peduncle (Voogd, 1964). 143
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144
The cerebellum." chemoarchitecture and anatomy
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Fig. 104. Two transverse, AChE-incubated sections through the cerebellar nuclei of the cat. A. Rostral section. B. Caudal section. Note medium-sized cells of dorsal group y in floccular peduncle and strongly AChE positive ventral group y along dorsal border of restiform body in (A); U-shaped nucleus between IP and F in (B). cr = restiform body; F = fastigial nucleus; flo+y = floccular peduncle with group y; IA = anterior interposed nucleus; IP - posterior interposed nucleus; IP/F = U-shaped nucleus between F and IP; L = lateral cerebellar nucleus; sad = stria acoustica dorsalis.
Graybiel and Hartweg (1974) found retrograde labelling in the cells of the y group of the cat after injection of retrograde tracers in the oculomotor nucleus. Matters have been further complicated by the introduction of the term 'infracerebellar nucleus' for the y group by Gacek (1977, 1979)l, and the distinction of ventral and dorsal divisions in the group y (Kevetter and Perachio, 1986; Highstein and Reisine, 1979; Highstein and 145
Ch. I
J. Voogd, D. Jaarsma and E. Marani
Fig. 105. The cerebellar nuclei of Macaca fascicularis. Upper diagram is a graphical reconstruction of the cerebellar nuclei in a dorsal view. Levels of the transverse sections are indicated. The U-shaped, transitional nucleus located between the fastigial (F) and posterior inter-posed nucleus (IP) is indicated with double hatching, bc = brachium conjunctivum; BIN = basal interstitial nucleus of Langer; cr = restiform body; DV = descending vestibular nucleus; F = fastigial nucleus; IA - anterior interposed nucleus; IP = posterior interposed nucleus; L = lateral cerebellar nucleus; LV = lateral vestibular nucleus (Deiters'); MV = medial vestibular nuleus; SV = superior vestibular nucleus; Y = group y; asterisk = medial one-third of the brachium conjunctivum. )
McCrea, 1988). A more realistic approach would be to consider the cells of the y group as the bed-nucleus of the floccular peduncle, and to realize that a distinction between the group y and the superior vestibular nucleus, that receives a major part of the fibers of the floccular peduncle, remains arbitrary (Tan et al., 1995a). The basal interstitial nucleus was described by Langer (1985) in the monkey as a broadly distributed interstitial population of neurons in the white matter ventral to the cerebellar nuclei and extending from the white matter of the nodulus, into the peduncle of the flocculus. The nucleus was distinguished from the group y in the monkey. It is reciprocally connected with the flocculus. It seems likely from their descriptions that the group y of Brodal and Pompeiano corresponds to the portion of Langer's basal interstitial nucleus that lies embedded in the floccular peduncle. The 'group y' of the monkey can be considered as the enlarged, caudal pole of the superior vestibular nucleus (corresponding to the group 1 of Brodal and Pompeiano, 1957, in the cat). Some features of the cerebellar nuclei of the cat, the monkey, and the rat shall be discussed in the next sections. The cerebellar nuclei of birds were described by Feirabend (1983) and Arends and Zeigler (1991 a,b). 5.1.1. The cerebellar nuclei of the cat (Figs 103 and 104) The subdivision of Weidenreich-Ogawa can be applied to the central nuclei of the cat. This is not amazing, because Ogawa's (1935) description of the central nuclei of pinnepedia is based on earlier, unpublished, material from the cerebellum of cat and dog. Moreover the parasagittal organization of the corticonuclear and olivocerebellar projections, that provided important clues for the subdivision of the central nuclei, was first and most extensively studied in the cat (Hohman, 1929; Voogd, 1964, 1969; Courville et al., 1974; Groenewegen and Voogd, 1977). Large cells are prominent in the rostral part of the medial nucleus, small cells predominate in its ventromedial and caudal parts (Flood and Jansen, 1961). The lateral border of the medial nucleus is flush with the AChE-positive raphe which forms the lateral border of the medial A-compartment of the anterior vermis. AChE is concentrated in the lateral and ventral parts of the medial nucleus and in the neuropil of cell groups scattered in between the medial and the anterior interposed nucleus. Caudally these AChE-positive clusters coalesce in a U-shaped nucleus located at the transition of the medial and the posterior interposed nucleus (Fig. 104B). The medial limb of the U forms the lateral border zone of the fastigial nucleus, the lateral limb usually is included with
1 Gacek (1977, 1979) used the term infracerebellar nucleus for the cells embedded in the fasciculus angularis (i.e. the floccular peduncle) as it arches over the restiform body and applied the term 'y group' to the cells of the group 1 of Brodal and Pompeiano (1957).
146
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the posterior interposed nucleus. The large cells in this area are the target of the Purkinje cells in the X-zone of the anterior vermis (Trott and Armstrong, 1987a,b). The cells in the lateral limb of the U, that extend rostrally as the AChE-positive cell groups medial to the anterior interposed nucleus, may provide the target for the 'C ~' zone, the lateral portion of the Cl-zone, that shares its afferent, climbing fiber input with the X-zone (Ekerot and Larson, 1982; Campbell and Armstrong, 1985; Trott and Armstrong, 1987a) (Section 6.3.3.1., Fig. 174). The cells of this area project to the spinal cord (Matsushita and Hosoya, 1979) and can be double-labelled from the cord and the thalamus (Bharos et al., 1981). Apart from the medial limb of the U-shaped subnucleus, the medial nucleus at this level consists of a dorsally directed tail that extends into the white matter of the posterior lobe vermis. It receives Purkinje cell axons from the vermal visual area of lobule VII (Voogd, 1964; Courville and Diakew, 1976). It constitutes one of the targets of the collateral projection from the nucleus reticularis tegmenti pontis (Gerrits and Voogd, 1987). Other parts of the medial nucleus receive their mossy fiber collaterals from the lateral reticular nucleus (Ktinzle, 1975; Russchen et al., 1976) and the spinal cord (Matsushita and Ikeda, 1970). The ventral, parvicellular part of the medial nucleus is located at the base of the lobules IX and X. The posterior and anterior interposed and the lateral cerebellar nucleus are clearly delimited. The border between the anterior and posterior interposed nucleus is not located in a frontal plane, but passes obliquely forward, from caudolaterally to rostromedially (Fig. 103). The posterior interposed nucleus, therefore, extends far rostrally, medial to the anterior interposed nucleus. The caudal pole of the anterior interposed nucleus is located far laterally, adjacent to and merging with the lateral nucleus. AChE is concentrated along the borders of the nuclei and in the ventrolateral portions of the lateral and posterior interposed nuclei. Elongated, AChE-positive cells in the floccular peduncle, ventral to the lateral nucleus, belong to cell group y of the vestibular nuclei of Brodal and Pompeiano (1957) (Fig. 104A). The medium-sized cells of group 1 which are located dorsolateral to Deiters' nucleus, should be distinguished from the group y. They constitute a caudal extension of the superior vestibular nucleus and display strong AChE activity.
5.1.2. The eerebellar nuclei of primates The cerebellar nuclei in primates were described by Courville and Cooper (1970) and Chan-Palay (1977, Macaca mulatta), Haines (1971, Galago), and Haines and Dietrichs (1991, Saimiri sciurus). The cerebellar nuclei of the human cerebellum were reviewed by Larsell and Jansen (1972) and Voogd et al. (1990). The four nuclei of the subdivision of Weidenreich-Ogawa can be recognized in macaque fascicularis and their topograph-
Fig. 106. Horizontal (A) and transverse (B,C) AChE-stained sections through the cerebellar nuclei of Macaca fascicularis. Note connection of IA and dorsal pole of dentate nucleus in (B) and of medial lamella of the dentate nucleus and border region of IA and IP in (A), U-shaped nucleus located between F and IP in (B) and (C) and strong AChE-reactivity in this nucleus in (A); localization of medial limb of this U-shaped nucleus in X compartment (B and C); extension of AChE-positive C2 compartment in border region of 1A and IP in B; interstitial nucleus of Langer in (B) and large group y in (C). C2 = C2 compartment; F = fastigial nucleus; IA = anterior interposed nucleus; IP = posterior interposed nucleus; IP/F = U-shaped nucleus between F and IP; L = lateral cerebellar nucleus; Lp = parvocellular part of lateral cerebellar nucleus; y = group y; X = X compartment. 148
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of a core and a shell; the neuropil of the shell is connected with the medial limb of the dentate nucleus. The anterior interposed nucleus consists of medial and lateral portions, separated by a notch in the dorsal surface. The caudal pole of the anterior interposed nucleus merges with the medial limb of the dentate nucleus. The dentate nucleus displays ventrolaterally and caudally directed bulges. The hilus of the dentate nucleus is directed rostromedially, caudally it is closed by the medial limb of the dentate nucleus. In its caudal portion the structure of the medial limb is compact, with AChE-positive cell bodies and neuropil concentrated along its borders, similar to the rest of the dentate nucleus. Rostrally where the medial limb surrounds and merges with the anterior interposed nucleus, its structure is loose and AChE-staining less prominent, A subdivision of the dentate nucleus of the monkey in the two parts that were distinguished in the human dentate nucleus, i.e. in the rostromedial neodentatum with large neurons and the caudolateral palaeodentatum, with densely packed, smaller neurons (Gans, 1924; Demol6, 1927a,b) is not feasible and is not warranted by Chan-Palay's (1977) detailed cytological analysis of this nucleus. The so-called group y is large and compact and occupies the rostral part of the floccular peduncle. Medially it is continuous with the caudal pole of the superior vestibular nucleus. The small AChE-positive cells of Langer's (1985) basal interstitial nucleus appear more caudally and extend from the flocculus into the roof of the fourth ventricle, ventral to and in between the dentate and posterior interposed nuclei. 5.1.3. The cerebellar nuclei of the rat
The cerebellar nuclei of the rat were described by Goodman et al. (1963), Korneliussen (1968a) and Voogd (1995). They are very similar to the nuclei of the cerebellum of the mouse, illustrated by Marani (1982a). The morphology and the cytology of the lateral and the medial nucleus of the rat were analysed in Chan-Palay's (1977) monograph on the dentate nucleus and in the paper of Beitz and Chan-Palay (1979). The cerebellar nuclei of the rat are difficult to subdivide according to the scheme of WeidenreichOgawa. They constitute, more or less, a single mass, with a number of protrusions, that were first named by Goodman et al. (1963) as the dorsolateral protuberance of the medial nucleus, and the dorsomedial crest and the dorsolateral hump, two excrescences of the interposito-dentate complex (Figs 107 and 108). Similar subnuclei were distinguished in the cerebellum of the mouse (Marani, 1986). The connections of the cerebellar nuclei in the rat differ in some respects from carnivores and primates. The medial nucleus was subdivided by Korneliussen (1968a) into a caudoventral parvicellular part, the magnocellular dorsolateral protuberance and a 'middle' part that contains cells of intermediate size. The dorsolateral protuberance is situated more laterally. It projects into the white matter of the posterior lobe and receives Purkinje cell fibers from a medial zone of the lobules VI and VII of the posterior lobe hemisphere (Goodman et al., 1963; Armstrong and Schild, 1978a,b; Buisseret-Delmas, 1988a,b) (see also Section 6.1.4.). The posterior interposed nucleus is best distinguished in horizontal sections. A cellfree zone separates it from the anterior interposed and lateral nuclei. Medially the posterior interposed nucleus is continuous with the fastigial nucleus. Cells at the junction of these two nuclei project to the spinal cord (Matsushita and Hosoya, 1978 and Bentivoglio and Kuypers, 1982). This region was distinguished as the interstitial cell group, the target nucleus of the X zone, by Buisseret-Delmas et al. (1993). At the junction of the anterior interposed and lateral nuclei the dorsolateral hump 151
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forms a distinct ridge on the rostral aspect of these nuclei. A more caudally located cluster of large neurons usually is indicated by the same name. Some authors included the dorsolateral hump with the anterior interposed nucleus (Goodman et al. 1963; Voogd, 1995), others delegated it to the lateral nucleus (Chan-Palay, 1977; Angaut and Cicirata, 1982), sometimes it was considered as a separate subdivision, peculiar to rodents. It gives rise to the uncrossed descending limb of the superior cerebellar peduncle (Woodson and Angaut, 1984; Bentivoglio and Molinari, 1986; Rubertone et al., 1990), that detaches from the superior cerebellar peduncle before its decussation. The uncrossed descending limb terminates in the lateral reticular formation (Ramon y Cajal, 1911; Mehler, 1967, 1969; Achenbach and Goodman, 1968; Faull, 1978; Chan-Palay, 1977). The ventromedial part of the lateral nucleus consists of smaller cells. Those in the region of the hilus correspond to the small intrinsic neurons described by Chan-Palay (1977), the densely packed small cells located ventral to the hilus may be identical to the concentration of GABAergic nucleo-olivary cells that was illustrated in this region by Mugnaini and Oertel (1985). AChE staining does not distinguish the parvicellular part of the lateral nucleus, but the medium-sized cells and the neuropil of the group y, located ventral to it display a high AChE activity. In a rostral direction the group y (Brodal and Pompeiano, 1957) becomes continuous with the superior vestibular nucleus). Deiters' nucleus with its large AChE-positive perikarya in an unstained neuropil is wedged in between the AChE-rich areas of the group y and the medial vestibular nucleus and reaches far dorsally into the hilus region of the central nuclei. Purkinje cell fibers enter Deiters' nucleus as perforating fibers, passing in between the dorsolateral protuberance and the anterior interposed nucleus, and through the middle part of the medial nucleus. More rostrally, where Deiters' nucleus has disappeared, the AChE-rich neuropil of the superior and medial vestibular nuclei meet at the oblique border between the two nuclei. 5.2. THE GABAERGIC NUCLEO-OLIVARY PROJECTION NEURONS OF THE CEREBELLAR NUCLEI Cell size has generally been considered of less importance for the subdivision of the central nuclei, than the presence of local differences in cell density resulting from sheets or bundles of fibers between and around the nuclei. Interest in the question whether different cell-types can be distinguished in the cerebellar nuclei on the basis of size, dendritic and axonal morphology and neurotransmitter content was renewed since the observation that the projection of the cerebellar nuclei to the inferior olive arises from a population of small GABAergic neurons. The nucleo-olivary projection was discovered with anterograde tracing with tritiated amino acids by Graybiel et al. (1973) and with retrograde tracing by Gould and Graybiel (1976) and Tolbert et al. (1976b), all in the cat. The nucleo-olivary and olivo nuclear projections are reciprocally organized (see Section 6.3.3.). Tolbert's (Tolbert et al., 1978a) and Courville and Cooper's (1970) quantitative analysis clearly showed that all sizes of neurons were present in all central nuclei in monkey and cat. Histograms of the soma diameter of the nuclear neurons projecting to the thalamus and the cerebellar cortex are very similar to the overall size distribution of these neurons. The cells in the cerebellar nuclei of the cat that project to the inferior olive, however, constitute a population of small, spindle shaped neurons (Fig. 109). This 154
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Fig. 109. Histograms of the maximum soma diameters of neurons of the cerebellar nuclei of the cat retrogradely labelled with HRP from injection sites in A the thalamus (VL), B the inferior olive (IO), and C the cerebellar cortex (CbS). D is a histogram of the overall size distribution of neurons in the cerebellar nuclei. Note that cerebellothalamic and nucleocortical projections arise from a heterogeneous population of neurons whose diameters correspond to the size spectrum of neurons present in the deep nuclei, whereas the cerebello-olivary pathway arises from a specific population of small neurons. Tolbert et al. (1978a).
population of small cells was recognized in retrograde tracer experiments in opossum (Martin et al., 1976, 1980) cat (Tolbert et al., 1976b; Tolbert et al., 1978a; Bharos et al., 1981; Legendre and Courville, 1987), rat (Angaut and Cicirata, 1982; Hess, 1982b; Brown et al., 1977; Bentivoglio and Kuypers, 1982; Buisseret et al., 1989; Swenson and Castro, 1983b) and monkey (Chan-Palay, 1977). The small cells were depleted in the cerebellar nuclei of lurcher mutant mice, probably due to the degeneration of the inferior olive (Heckroth, 1994). According to Tolbert et al. (1978a) the nucleo-olivary neurons in the cat are concentrated in the ventral parts of the dentate and posterior interposed nuclei, they are scarce in the fastigial nucleus. Concentrations of these small neurons were also reported in the rostral and caudal poles and the hilar portion of the dentate nucleus and the lateral parts of the interposed nucleus in the rat (Brown et al., 1977; Chan-Palay, 1977). A more diffuse distribution of these cells was noticed in experiments with retrograde tracing of Martin et al. (1976) in the dentate and interposed nuclei of the opossum and in double labelling studies with fluorescent dyes in rat (Bentivoglio and Kuypers, 1982) and cat (Bharos et al., 1981). Neurons of the cerebellar nuclei and the lateral cerebellar nucleus of rat and cat that react with antibodies against GAD or conjugates of GABA, generally are small (Mugnaini and OerteI, 1981, 1985; Houser et al., 1984; Gabbott et al., 1986; Kumoi et al., 155
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J. Voogd, D. Jaarsma and E. Marani
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Fig. 110. Diameter distributions of GABA-like immunoreactive (GABA-IR) and glutamate-like immunoreactive (Glu-IR) neurons in the nucleus medialis (NM), the nucleus interpositus (NI) and the nucleus lateralis (NL) of rat cerebellum. The populations of GABA-IR (A) and Glu-IR (B) neurons were clearly peaked and similar in size in all three nuclei. In C the spectra from the three nuclei are averaged for both populations and plotted together. The size range of the GABA and Glu overlap is the same as for cells positively identified as colocalizing GABA and Glu. Abscissae: diameter of the neurons in p m (class interval 2.5/lm). Ordinates: percentage of neurons in each diameter class. Batini et al. (1992).
1987, 1988; Buisseret-Delmas et al., 1989; Walberg et al., 1990; Takayama, 1994; Moffett et al., 1994). Batini et al. (1992), who measured cell diameters of GABA and single labelled glutamate-immunoreactive neurons, showed that they represent two populations with little overlap in all cerebellar nuclei of the rat (Figs 110 and 111). Consistent with glutamate being a metabolic precursor for GABA, most of the GABAergic neurons co-localized glutamate. GAD or GABA-like immunoreactive cells are scattered through all parts of the cerebellar nuclei, but have been reported to be more sparse in the fastigial nucleus, where they are concentrated in its ventral portion and to be preferentially located in the hilus and in the ventral parvocellular subnucleus of the lateral cerebellar nucleus of the rat (Mugnaini and Oertel 1985; Buisseret-Delmas et al. 1989). These small 156
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 111. Typical distribution of glutamate-like and GABA-like immunoreactive neurons in the nucleus lateralis of rat cerebellum. Consecutive sections immunostained for glutamate (A) and GABA (B) are shown as mirror images. Note the difference in size of the stained neurons. Batini et al. (1992).
neurons proved to be the origin of the GABAergic, nucleo-olivary pathway in the rat (Nelson et al. 1984) and the rabbit (Nelson and Mugnaini, 1989). De Zeeuw et al. (1988, 1989b) demonstrated that all the terminals in the inferior olive that could be traced from the cerebellar nuclei by anterograde transport of WGA-HRP could be labelled with a polyclonal antibody against GABA. The nucleo-olivary projection, therefore, seems to be entirely GABAergic. Some of the small GABAergic perikarya of the cerebellar nuclei and the lateral vestibular nucleus also contain glycine (Ottersen et al., 1987; Walberg et al., 1990; Chen and Hillman, 1993b; Takayama, 1994), but glycine could not be detected in terminals of the nucleo-olivary pathway in the rat (De Zeeuw, unpublished observations). Projections to certain subdivisions of the inferior olive also take their origin from the vestibular nuclei (Saint Cyr and Courville, 1979; Gerrits et al. 1985a) that contains a population of small GABAergic neurons in all its subnuclei (Kumoi et al., 1987, guinea pig). Some of these connections are also GABAergic (Nelson et al., 1986), but it is not known whether all vestibulo-olivary connections use this neurotransmitter. One of the main sources for the vestibulo-olivary projection is the nucleus prepositus hypoglossi (Saint Cyr and Courville, 1979; McCrea and Baker, 1985; Gerrits et al., 1985a). Both GABA (de Zeeuw et al., 1993) and acetylcholine (Barmack et al., 1991) have been identified as neurotransmitters in this pathway. At present it seems unlikely that the small nucleo-olivary neurons possess collaterals that terminate in other targets. Tolbert et al. (1978a), however, found many of these neurons in the cat to be antidromically activated from the thalamus and the cerebellar cortex in addition to the inferior olive. Ban and Ohno (1977) also produced electrophysiological evidence for collateralization of nucleo-olivary cells to the red nucleus or more rostral structures. Bharos et al. (1981) and Bentivoglio and Kuypers (1982), were unable 157
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to double label these small neurons, after combined injections of one fluorescent tracer in the caudal medulla, in a region including the inferior olive, and a second injection with another fluorescent tracer in either thalamus with tectum or the spinal cord. These results were corroborated for the combination of thalamus and inferior olive by Legendre and Courville (1987) for the cat and the red nucleus and the inferior olive by Teune et al. (1995) for the rat. The existence of GABAergic projections of the cerebellar nuclei to other precerebellar relay nuclei is not excluded by these experiments. Projections from GABAergic neurons of the lateral cerebellar nucleus of the rat to the basal pontine nuclei were demonstrated by Border et al. (1986). 5.3. NUCLEOCORTICAL AND INTRINSIC NEURONS OF THE CEREBELLAR NUCLEI Nucleocortical neurons occasionally have been described in Golgi material (Ramon y Cajal, 1911) and were considered as the origin of the climbing fibers by Carrea et al. (1947). Their existence was confirmed by retrograde transport of HRP injected in various parts of the cerebellar cortex in the cat (Gould and Graybiel, 1976; Tolbert et al., 1976a, 1978b; Dietrichs, 1981a,b, 1983a; Dietrichs and Walberg, 1979a, 1980; Trott and Armstrong, 1990), in primates (Tolbert et al., 1977, 1978b; Tolbert and Bantli, 1979; Haines, 1978b, 1988; Haines and Pearson, 1979), and in the rat (Chan-Palay, 1977; Hess, 1982a; Buisseret-Delmas and Angaut, 1988, 1989a). The nucleocortical projection was reviewed by Tolbert (1982). The size of the nucleocortical cells in the cat follows the same frequency distribution as the central nuclear cells as a whole and as the neurons that could be retrogradely labelled from the thalamus (Fig. 109). Single neurons, intracellularly stained with HRP, with axons leaving the central nuclei in the superior cerebellar peduncle, were shown to emit collaterals, that could be traced to the cerebellar cortex (McCrea et al., 1978). Antidromic invasion and collision experiments (Tolbert et al., 1977, 1978a) also favoured a collateral origin of the nucleocortical fibers from projection neurons. There is some evidence for GABA or acetylcholine as the neurotransmitter of the nucleocortical fibers, but the majority, probably, uses glutamate or aspartate, in accordance with their collateral origin from large relay cells of the cerebellar nuclei. 20% of the nucleocortical neurons in the medial cerebellar nucleus that were WGA-HRP labelled from the cerebellar cortex of the rat, reacted with an antibody raised against GABA (Angaut et al., 1988). Most if not all nucleocortical neurons in the rat were found to be immunoreactive for an antibody to a conjugate of GABA in the experiments of Batini et al. (1989). She labelled GABAergic cells of all sizes from the cortex, although fewer retrogradely labelled cells were present among the smaller GABAergic neurons, most of which presumably project to the inferior olive. The proportion and the size distribution of nucleocortical neurons of the rat labelled with the same antibody to a conjugate of GABA were reported to be different in a later publication of the same authors (Batini et al., 1992). Again, the retrogradely labelled nucleocortical neurons were of all sizes with a peak around a diameter of 20-25 r With almost 50%, the population of single labelled glutamate-immunoreactive nucleocortical neurons far exceeded the GABAergic population. The glutamate-immunoreactive neurons were also larger and peaked at a diameter of 20-25 r The GABAergic-nucleocortical cells may be identical to the small nucleocortical neurons that were identified in the posterior interposed nucleus and along the boundaries of the lateral nucleus of the rat by retrograde transport of an antibody to GAD 158
The cerebellum." chemoarchitecture and anatomy
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(Chan-Palay et al., 1979). Antegrade transport of an antibody to GAD in nucleocortical fibers resulted in labelling of a small number of mossy fiber rosettes in the cortex (Chan Palay et al., 1979). Antegrade transport in nucleocortical fibers of other, non-specific tracers also resulted in the labelling of mossy fiber rosettes (Kultas-Illinsky et al., 1979; Tolbert et al., 1980). The morphology of these nucleocortical mossy fiber rosettes was not distinctive and they never displayed any of the features of GABAergic terminals, such as pleomorphic vesicles and symmetrical synapses. H~mori and Tak~cs (1989) and H~.mori et al. (1990) distinguished 4 types of mossy fiber rosettes in the cerebellar cortex of rat and cat with immunohistochemistry of glutamate and GABA and deafferentation of the cerebellum. Two of their types of mossy fiber rosettes, i.e. large, GABA-immunoreactive rosettes, with round or pleomorphic synaptic vesicles that accounted for 3% of all mossy fiber rosettes in the cat and small rosettes with small, pleomorphic synaptic vesicles, that reacted with an antibody to a conjugate of glutamate and accounted for less than 10% of the mossy fiber rosettes in the rat, presumably represented the endings of nucleocortical fibers. Mossy fibers of extracerebellar origin terminated as large, glutamate-immunoreactive rosettes; a fourth type of small, GABAergic rosettes with small synaptic vesicles was of intracortical origin. According to Ikeda et al. (1991) the cholinergic, ChAT-immunoreactive afferent fibers in the cortex of the cerebellum of the cat, that include mossy fibers, disappear after electrolytic or kainate lesions of the cerebellar nuclei. Different types of nucleocortical neurons have been distinguished on the basis of their localization with respect to the corticonuclear projection. Most nucleocortical cells were considered to be of a reciprocal type since they were located within an area receiving Purkinje cell projections from the same region of the cortex from which they can be retrogradely labelled (Dietrichs, 1981a,b, 1983a; Dietrichs and Walberg, 1979a, 1980; Tolbert, 1982; Buisseret-Delmas and Angaut, 1988, 1989a; Haines, 1988). Other nucleocortical neurons were found outside the corticonuclear projection area, either ipsilaterally (the non-reciprocal neurons of Buisseret-Delmas and Angaut, 1988, 1989a, see also Hess, 1982a, Dietrichs and Walberg, 1979a, 1980) or even contralaterally (symmetrical neurons: Buisseret-Delmas and Angaut, 1988, 1989a rat; Tolbert et al., 1978b cat and monkey; Haines, 1978a,b; Haines and Pearson, 1979, treeshrew; Haines, 1988, Galago; Dietrichs, 1983a; Dietrichs and Walberg, 1980, cat). Non-reciprocal and contralateral neurons in cat and rat were most numerous in the fastigial and posterior interposed nuclei. Contralateral neurons in the posterior interposed nucleus of Galago were small and fusiform and resembled the nucleo-olivary neurons (Haines, 1988). Non-reciprocal connections were the rule in primates where they originated mainly from the ventral part of the dentate nucleus. Reciprocity in general was more noticeable in cat and rat. A more restricted origin of the sparse nucleocortical projection to the electrophysiologically identified C~ and C2 zones and the strong bilateral projection to the C2 zone of the paravermal cortex of the cat from the posterior interposed and fastigial nuclei was advocated by Trott and Armstrong (1990). The existence of a rest group of neurons that remains unaffected by large lesions of the efferent cerebellar pathways in the kitten has been claimed as evidence in favour of the presence of intrinsic or nucleocortical neurons in the central nuclei (Jansen and Jansen 1955). Many of these neurons were found to be large and to be located in the posterior interposed nucleus. Intrinsic neurons of the cerebellar nuclei have been observed in Golgi preparations of the rat by Chan-Palay (1973a, 1977) as small multipolar neurons in the dentate nucleus. The terminals of these intrinsic, inhibitory neurons on the soma and dendrites of cerebellar nuclear cells were tentatively identified as small 159
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boutons with few elliptical vesicles in a light matrix (E-type, Chan-Palay, 1973b, 1977). Wassef et al. (1986), however, were unable to distinguish between these E-types and Purkinje cell terminals (Chan-Palay's A-type) in the cerebellar nuclei of mutant Pcd mice. Some 15% of the small and large GAD-positive terminals in the cerebellar nuclei of these mutants were spared, although these mice lost 99% of their Purkinje cells. Intrinsic GABAergic connections of the nuclei should have been spared in these animals, but the possibility that these 15% represent sprouting from another GABAergic cell population cannot be excluded. Non-GABAergic intrinsic connections through boutons with spherical vesicles carrying Gray-type I synapses were postulated by Chan-Palay (D-type, 1973b); no other evidence is available on this system. Strong evidence for the presence of a population of small glycinergic interneurons in the cerebellar nuclei of the rat was supplied by Chen and Hillman (1993b). Glycine immunoreactivity was restricted to a population of small neurons throughout the cerebellar nuclei and was present in boutons outlining somata of large relay cells. Intense glycine receptor immunoreactivity was observed in these large cells, opposite the glycinergic terminals. Many, but not all of these small, glycinergic neurons colocalize GABA. Since glycine-immunoreactivity was not observed in nucleo-olivary terminals (De Zeeuw, unpublished observations) it seems likely that these glycine and glycine/GABA containing neurons are interneurons. Rather similar observations were made for calretinin in a subpopulation of small nuclear cells, that give rise to a dense plexus in the cerebellar nuclei of the rat (Floris et al., 1994). 5.4. NON-GABAergic PROJECTION N E U R O N S OF THE CEREBELLAR NUCLEI
Glutamate and aspartate have been suggested as the neurotransmitter of the nonGABAergic projection neurons. Immunoreactivity with an antibody to a conjugate of glutamate was found in neurons of all sizes in all cerebellar nuclei of the rat (Batini et al., 1992) (Fig. 110) and with antibodies to conjugates of aspartate in large neurons of the cerebellar nuclei of the rat (Kumoi et al., 1988; Chen and Hillman, 1993b). A reaction product of glutamate with carbodiimide (gamma-Glu-Glu) has been localized with immunocytochemistry in neurons of all sizes in all the cerebellar nuclei of carbodiimide-perfused rats (Monaghan et al., 1986, see also Madl et al., 1986, 1987). Moderate, glutaminase (GLNase)-like immunoreactivity was present in scattered small cells of the cerebellar nuclei of the rat (Kaneko et al., 1989). Cells reacting with an antibody against aspartate aminotransferase (AATase), that catalyses the conversion of glutamate to aspartate, are more numerous than the GLNase and gamma-Glu-Glucontaining cells, but not all cerebellar nuclear neurons were found to contain AATase (Monaghan et al., 1986). Since AATase-like immunoreactivity has been found in Purkinje, basket and stellate cells of the cerebellar cortex, this enzyme apparently is also present in GABAergic neurons. Monaghan et al. (1986) tentatively concluded that three types of neurons can be distinguished in the cerebellar nuclei of the rat. The first group includes putative glutamatergic neurons containing GLNase- and gamma-Glu-Glu-like immunoreactivity, the second group corresponds to the GABAergic neurons that contain AATase-like activity and the third group are the neurons that do not react with any of the three antibodies and whose neurotransmitters remain to be determined. They could not exclude the presence of aspartate-containing neurons in the cerebellar nuclei because elevated levels of AATase and GLNase can be present both in aspartatergic as well as in glutamatergic neurons. The large cells of Deiters' nucleus in guinea pig (Kumoi 160
The cerebellum." chemoarchitecture and anatomy
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Fig. 112. Retrograde transport of [3H]choline (A,B) to perikarya (arrow-heads) in medial part of nucleus interpositus (Int) or in lateral cerebellar nucleus (LatC) and of wheat germ agglutinin-coupled HRP (E,F), but absence of retrogradely labelled elements in corresponding regions after application of D-[3H]aspartate to the red nucleus (C,D). Rat, bright-field (A,E) as well as dark-field (B,F) illumination. Cresyl violet counterstaining. Bars = 0.5 mm. Bernays et al. (1988).
et al., 1987) and cat (Walberg et al., 1990), reacted with antibodies raised against conjugates of aspartate. The same giant cells in the cat also stained with antibodies to conjugates of glutamate (Walberg et al., 1990). The presence of excitatory amino acid transmitters in the projection neurons of the cerebellar nuclei was also investigated by retrograde transport of D-[3H]aspartate in the cerebellorubral pathway, but no such transport was observed in the rat (Bernays et al., 1988 (Fig. 112C,D)). Interruption of this pathway, however, resulted in a decrease of high affinity glutamate uptake in the caudal, magnocellular portion of the red nucleus (Nieoullon et al., 1984), which could be explained by a loss of cerebellorubral, glutamate containing terminals. High affinity uptake of glutamate in other cerebellar target nuclei 161
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162
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The cerebellum." chemoarchitecture and anatomy
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Fig. 113. A. Darkfield micrograph of a frontal section through the cerebellar white matter and nuclei of an experiment with an injection of Phaseolus vulgaris leucaglutinin into part of the principal nucleus of the inferior olive and the rostral dorsal accessory olive in the rat. Labelled axons can be seen in the restiform body (cr). Bundles of labelled axons directed toward the cerebellar hemispheres seem continuous with innervated areas of the lateral cerebellar nucleus (L), the dorsolateral hump (DLH) and the anterior interposed nucleus (NIA). At this low magnification, the plexuses of nerve terminals in the nuclei appear as 'clouds' of fine dots representing individual varicosities. In the lateral cerebellar nucleus, the sector receiving labelled terminals is sharply demarcated from neighbouring tissue that does not receive labelled innervation, suggesting a detailed topographical organization of the PO projection to the lateral cerebellar nucleus. Bar = 250/tm. B. Photomontage of olivary axon in the border of the white matter and interposed nuclei of rat cerebellum which gives off a thin collateral toward an area of innervation. The branching point is indicated by an arrow. The thin collateral follows a tortous course (for a short distance it is obscured by an underlying thicker axon) and demonstrates several varicosities that may be the sites of synaptic interaction. Bar = 20/Lm. C. Darkfield micrograph of a sector of the lateral cerebellar nucleus in a sagittally sectioned specimen. Thicker olivocerebellar axons (arrow) run in the overlying white matter and some traverse the neuropil in the right part of the micrograph. These arriving olivary axons seem to give rise to the dense plexus of thin varicose terminal fibers, which extend over most of the illustrated neuropil. Bar = 20/tm. Van der Want et al. (1989a). (
such as the rostral parvocellular portion of the red nucleus and the ventrolateral nucleus of the thalamus, however, shows an increase rather than a decrease after lesions of the cerebellorubral and thalamic pathway. Several explanations were offered for this phenomenon, one of which involves the loss of a cholinergic, cerebellar efferent pathway that would facilitate glutamate release from corticothalamic or corticorubral terminals in these nuclei (Nieoullon and Dusticier, 1981; Nieoullon et al., 1984). The existence of cholinergic efferents among the projections of the cerebellar nuclei received support from observations by the same authors of a temporary decrease of ChAT activity in the red nucleus, especially in its rostral part, after lesions of the cerebellorubral fibers (Nieoullon and Dusticier, 1981). Moreover specific retrograde transport of [3H]choline in the cerebellorubral pathway to large perikarya in the anterior half of the lateral cerebellar nucleus of the rat was reported by Bernays et al. (1988) (Fig. 112) and to cerebellar nuclei in the cat by Stanton and Orr (1985). Evidence for the existence of cholinergic neurons in the cerebellar nuclei is controversial; ChAT was found to be absent in these cells (Kimura et al., 1981, cat; Armstrong et al., 1983, rat), but to be present in the large cells of Deiters' nucleus (Kimura et al., 1981). Ikeda et al. (1991) reported the presence of ChAT-immunoreactive neurons in the cerebellar nuclei of the cat; these cells give rise to both thalamic projections and nucleocortical collaterals. One group of small neurons in the rhesus monkey, which was described by Langer (1985) under the name of the basal interstitial nucleus (see Section 5.1 .), displays uniform and strong AChE activity. These small cells lie dispersed in the white matter of the flocculus and the nodulus and ventral to the dentate and the posterior interposed nucleus, in the roof of the fourth ventricle. The presence of strongly AChE-positive but ChAT-negative small cells in the white matter of the flocculus of the rat, that may correspond to Langer's interstitial nucleus, was noticed by Komei et al. (1983). The interstitial nucleus should be distinguished from the group y (Brodal and Pompeiano, 1957), which is a lateral extension of the superior vestibular nucleus, located ventral to the dentate nucleus. The cells of group y are slightly larger AChE-positive neurons. In accordance with other parts of the superior vestibular nucleus, group y contains a small number of GAD-positive cells, but most of its cells display aspartatelike immunoreactivity (Kumoi et al., 1987). 163
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Reports on the presence of other transmitters or transmitter-related substances have been published. Some neurons in the medial and posterior interposed nucleus of the opossum were immunoreactive with antibodies to CCK (King and Bishop, 1990, Fig. 116). Some somatostatin-like immunoreactive neurons were present in the medial part of the fastigial nucleus of the rat (Vincent et al., 1985). Occasional cells labelled with antibodies against conjugates of taurine were observed by Ottersen et al. (1988b) in rat cerebellum. Glycine-immunoreactivity was observed in large neurons of the ventral fastigial nucleus of the rat (Chen and Hillman, 1993b). PKC subtypes in neurons of the cerebellar nuclei have been specified by Kose et al. (1988), Shimohama et al. (1990), Huang et al. (1991), Merchenthaler et al. (1993), Garcia et al. (1993) and Chen and Hillman (1993a) (Table 1). 5.5. A F F E R E N T CONNECTIONS OF THE CEREBELLAR NUCLEI: P U R K I N J E CELL AXONS The Purkinje cell axons constitute the main afferent system of the cerebellar nuclei. Other afferents include collaterals of mossy and climbing fibers, a projection from the contralateral red nucleus (see Section 5.6. and 6.4.4.) and certain monoaminergic systems (see Section 3.8.). The terminals of the Purkinje cell axons in the cerebellar nuclei and the lateral vestibular nucleus contain pleomorphic synaptic vesicles, most of which are elliptical and are provided with Gray type II or an intermediate type of synapse. These terminals cover most of the soma and the large dendrites, extend on the spines and the small dendrites and are found as axo-axonal synapses on the initial segment. These terminals first were identified by antegrade axonal transport of [3H]-GABA in the rat (McGeer et al., 1975) and tritiated leucine in the cat (Walberg et al., 1976). Most of the boutons lining the surface of cells of the cerebellar nuclei contain GAD or GABA-like immunoreactivity (Saito et al., 1974; Wassef et al., 1986; Fonnum et al., 1970; Fonnum and Walberg, 1973; Houser et al., 1984; Roffler-Tarlov et al., 1979; Kumoi et al., 1987; Hawkes and Leclerc, 1986). Most if not all Purkinje cells are GABAergic (Chan-Palay, 1984; Mugnaini and Oertel, 1985), but with respect to other substances they constitute a heterogeneous population (see Sections 3.1.2. and 3.1.3.). The possibility of a differential distribution in the cerebellar nuclei of the terminals of chemically different populations of Purkinje cells has been studied for taurine and for the Purkinje cell specific antibody mabQ 113 (anti-Zebrin). For taurine the results were conflicting. The distribution of taurine in parasagittal bands of Purkinje cells in the cerebellar cortex was based upon the presence in these cells of the synthesizing enzyme of taurine CSADase (Chan-Palay et al., 1982a,b). No such a differential distribution was noticed, however, for taurine-like immunoreactivity in Purkinje cells (Madsen et al., 1985; Ottersen and Storm-Mathisen, 1987) or for taurine-containing terminals in the cerebellar nuclei (Ottersen et al., 1988b). Zebrin-immunoreactive and non-immunoreactive Purkinje cells are distributed in parallel longitudinal bands in the cortex of rat cerebellum (Hawkes et al., 1985) and distribute their axons to different parts of the cerebellar nuclei (Hawkes and Leclerc, 1986). Light microscopical observations showed that all Zebrin positive boutons on the soma and dendrites of large central nuclear cells contained GAD, and that most GADpositive boutons on individual cells either were Zebrin-positive or -negative. The two populations of Purkinje cells, therefore, terminate on different central nuclear cells. Zebrin-positive Purkinje cells of the vermis projected to the caudal part and a majority of Zebrin-negative Purkinje cells to the rostral part of the fastigial nucleus of the rat. 164
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Hawkes and Leclerc (1986) estimated that Zebrin-positive terminals made up the great majority of the GAD-positive boutons on the class of Zebrin receptive neurons. This suggests that intrinsic GABAergic terminals are few and that extracerebellar and intrinsic non-GABAergic connections account for at most 20% of the terminals on these cells. GABA receptors in the cerebellar nuclei were not systematically studied (see Section 3.7.). 5.6. EXTRACEREBELLAR AFFERENTS OF THE CEREBELLAR NUCLEI: COLLATERALS OF MOSSY AND CLIMBING FIBERS Extracerebellar afferents of the cerebellar nuclei consists of the collaterals of mossy and climbing fiber systems, that provide their excitatory drive (Eccles et al., 1967), the cholinergic (Section 3.10.), and the monoaminergic afferent systems. The existence of extracerebellar afferents to the central nuclei is known from antegrade axonal tracing experiments and was verified in ultrastructural studies. Injection of [3H]-leucine or other antegrade tracers in the inferior olive in the cat and the rat resulted in labelling over climbing fiber strips in the molecular layer and over parts of the cerebellar nuclei and the lateral vestibular nucleus (Courville, 1975; Groenewegen and Voogd, 1977; Groenewegen et al., 1979; Kawamura and Hashikawa, 1979; Balaban, 1984, 1988; Van der Want and Voogd, 1987; Van der Want et al., 1989a and b). The presence of projections of the inferior olive to the vestibular nuclei outside the lateral vestibular nucleus was denied by Groenewegen and Voogd (1977), but advocated by Balaban (1984, 1985, 1988) in experiments on rabbits. When the projection of the inferior olive to the fastigial nucleus was analysed with ultrastructural autoradiography of [3H]-leucine in the cat, the labelled boutons were predominantly found on small, distal dendrites, but never on somata. These terminals contain spherical vesicles and occasional dense core vesicles in an electron-lucent matrix and were provided with asymmetrical synapses (Van der Want and Voogd, 1987) (Fig. 115). The origin of these terminals as collaterals from olivocerebellar fibers terminating as climbing fibers in the cortex was first suggested by electrophysiological studies (Eccles et al., 1967) and by the observation of a strict topographical relation between the olivocerebellar climbing fiber zones and the termination of olivocerebellar fibers in the cerebellar nuclei (Groenewegen and Voogd, 1977). Direct proof of a collateral origin of olivonuclear fibers was provided by the retrograde transport to the central nuclei of [3H]-D-aspartate, injected in the cerebellar cortex of rats (Wiklund et al., 1984); the double-labelling studies in the cat with fluorogold implants in the cerebellar nuclei combined with injections of rhodamine-spheres in the cortex (Qvist, 1989b) and the observations of collaterals in antegrade tracing with the lectin from Phaseolus vulgaris in the rat (Van der Want et al., 1989a,b) (Fig. 113). Collaterals from olivocerebellar fibers generally terminate in the particular cerebellar nucleus that receives the axons of the Purkinje cells innervated by the same set of olivocerebellar fibers. It is not known whether all subdivisions of the inferior olive project to the cerebellar nuclei. The topographical distribution of the olivonuclear projections is reviewed in section 6.3.3. Some of the nuclei in the brainstem and the spinal cord, that give rise to mossy fibers terminating in the granular layer, also project to the cerebellar nuclei. Antegrade and retrograde axonal tracing experiments demonstrated projections in rat and cat from the spinal cord (Szentagothai in Eccles et al., 1967; Voogd, 1969; Matsushita and Ikeda, 1970; Robertson et al., 1983; Ikeda and Matsushita, 1973; Matsushita and Yaginuma, 1990, 1995), the lateral reticular nucleus and from the nucleus reticularis tegmenti pontis 165
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J. Voogd, D. Jaarsma and E. Marani
Fig. 114. EM autoradiogram of a spiny dendrite in the fastigial nucleus of the cat. A labelled mossy fiber terminal, originating from an injection of tritiated leucine into the nucleus reticularis tegmenti pontis, is densely filled with uniform spherical vesicles. Boutons with flattened vesicles form synaptic contacts on the same dendrite. Cat. Van der Want et al. (1987).
and adjacent regions of the pontine nuclei (Ktinzle, 1975; Russchen et al., 1976; ChanPalay et al., 1977; Matsushita and Ikeda, 1976; Eller and Chan-Palay, 1976; Martin et al., 1977; Ruggiero et al., 1977; Hoddevik, 1978; Dietrichs and Walberg, 1979a, 1987; Dietrichs, 1983b; Brodal et al., 1986; Gerrits and Voogd, 1987; Qvist, 1989a,b; Shinoda et al., 1992; Mihailoff, 1993). Other mossy fiber systems, such as the cuneocerebellar tract and the basal pontine nuclei provide only few or no collaterals to the cerebellar nuclei. Their distribution is reviewed in Section 6.4.4. Terminals in the fastigial nucleus of the cat originating from the reticular and the vestibular nuclei ranged widely in size and formed asymmetric synapses with small and large dendrites but not with somata. These boutons contained clear, spherical vesicles, and they occurred in two types, that differed in the aggregation of their vesicles (ChanPalay, 1977) which are present in equal numbers among the terminals from all sources (Van der Want et al., 1987) (Fig. 114). Morphologically these boutons are indistinguishable from the olivonuclear boutons that were described in Van der Want's companion study (Van der Want and Voogd, 1987) (Fig. 115). The differences in distribution between the climbing fiber and the different types of mossy fiber terminals in the rat and monkey lateral cerebellar nucleus reported by Chan-Palay (1973a, 1977), that were based on the similarity in morphology of these terminals in the cerebellar cortex and the nuclei, were not confirmed. According to the ultrastructural degeneration studies of Ikeda and Matsushita (1973) spinocerebellar fibers terminate both on dendrites and on somata. 166
The cerebellum." chemoarchitecture and anatomy
Ch. I
5.7. EXTRACEREBELLAR A F F E R E N T S OF THE CEREBELLAR NUCLEI: SEROTONINERGIC, N O R A D R E N E R G I C , D O P A M I N E R G I C AND PEPTIDERGIC PROJECTIONS Serotonin-like immunoreactivity resides in a fine network of varicose fibers in the neuropil of all cerebellar nuclei. This plexus is most dense in the hilar region of the lateral cerebellar nucleus of the rat (Takeuchi et al., 1982), among the small, intrinsic neurons of this region (Chan-Palay, 1977) and in the caudal and dorsal regions of the central nuclei of the opossum (Fig. 117) (Bishop et al., 1985). The noradrenergic innervation of the cerebellar nuclei was studied with histochemical fluorescence methods by H6kfelt and Fuxe (1969) in the rat and Landis et al. (1975) in mice and by Mugnaini and Dahl (1975) in the chicken and with selective uptake of [3H]noradrenalin (Chan-Palay, 1977) and dopamine-fl-hydroxylase immocytochemistry (Pasquier et al., 1980) in rats. The network of varicose noradrenergic fibers in the cerebellar nuclei is much less dense than the serotoninergic plexus and in the chicken a noradrenergic innervation of the central nuclei even is completely lacking (Mugnaini and Dahl, 1975). The possibility should be considered that this plexus mainly consists of passing fibers on their way to the cortex (Sachs et al., 1973). A heterogeneous population of terminals, containing large dense-core vesicles with a diameter of 900 A, were found to be labelled in the cerebellar nuclei after intraventricular infusions of 3H-serotonin in the rat. Few of these boutons show synaptic specializations (Chan-Palay, 1975, 1977). The ultrastructural morphology of nor-
Fig. 115. EM autoradiogram showing two climbing fiber boutons with spherical and pleomorphic vesicles, labelled from an injection with tritated leucine into the inferior olive make synaptic contact with a dendrite of a neuron of the fastigial nucleus of the cat. Van der Want and Voogd (1987).
167
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adrenergic terminals in the cerebellar nuclei has not been studied. Ultrastructural studies of the central nuclei using antibodies against conjugates of serotonin or noradrenalin have not yet been reported. Neurons of the ventral tegmental area of the rat project to the cerebellar nuclei. These neurons do not contain tyrosine-hydroxylase and this projection, therefore, is nondopaminergic, contrary to the projection of the dopaminergic A10 group located in the same area, to the cerebellar cortex of the rat (Ikai et al., 1992). Peptides, such as enkephalin, corticotropin-releasing factor (CRF) and cholecystokinin (CCK) that occur in both mossy and climbing fibers(see Sections 6.3.2.2. and 6.4.3.) have been demonstrated in the cerebellar nuclei of several mammals. The distribution of these peptides in a plexus of beaded fibers in these nuclei is very similar the plexiform distribution of serotonin and noradrenalin, substances that do not occur in climbing or mossy fibers. Enkephalin-like activity was concentrated in fibers along the lateral and dorsal borders of the dentate nucleus and in the rostral part of the fastigial nucleus and in varicosities in all parts of the cerebellar nuclei of the opossum (King et al., 1987) (Fig. 188). Enkephalin-like activity in cat and rat was found in some mossy fiber rosettes, but not in climbing fibers (King et al., 1987); the cerebellar nuclei have not yet been studied in these species. CRF-like immunoreactivity was present in a diffuse plexus of beaded fibers in all cerebellar nuclei of the opossum (Cummings et al., 1989) and in a similar plexus with concentrations in the anterior interposed nucleus and the ventral dentate in the cat (Cummings, 1989). CCK-like immunoreactivity was present in a similar plexus 168
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J. Voogd, D. Jaarsma and E. Marani
of varicose fibers in the cerebellar nuclei of the opossum (King et al., 1986b; King and Bishop, 1990).
6. EFFERENT AND AFFERENT CONNECTIONS OF THE CEREBELLAR CORTEX: CORTICONUCLEAR, OLIVOCEREBELLAR AND M O S S Y FIBER C O N N E C T I O N S AND CYTOCHEMICAL MAPS
Morphological, embryological, physiological and cytochemical studies have revealed longitudinal, zonal patterns in the cerebellar cortex. The cytoarchitecture of the cerebellar cortex is uniform, without clear signs of a regional differentiation. Differences in the size of granule and Purkinje cells between vermis and hemisphere, with larger and less densely packed cells in the vermis, and smaller more densely packed cells in the hemisphere, were noticed by Lange (1982) and Drfige et al. (1986) for human and rat cerebellum. Local differences also exist for the Golgi and/or the unipolar brush cells, which are more closely packed in the flocculus and the vermis of the posterior lobe, than in the anterior vermis and the hemisphere. Maps showing local differences in density of the Purkinje cells have been published for the cerebellum of the turtle Pseudomys scripta elegans and the lizard Varanus exanthematicus (Gerrits and Voogd, 1973; Bangma et al., 1983). The cerebellum of these species is rather simple and consists of a single leaf. Three zones with a different density of the Purkinje cells, that also differ in their projection to the cerebellar and vestibular nuclei were distinguished. Similar density maps are not available for the mammalian cerebellum, probably because the curvature of the cortex makes this technically difficult. There are indications in the mammalian cerebellum for medio-lateral, zonally distributed differences in the size of the Purkinje cells (Chan-Palay et al. 1981; Voogd, 1989) and an even better case can be made for the existence of systematic differences in the size of the fibers taking their origin from different Purkinje cell zones (Fig. 118) (Voogd, 1964, 1967, 1969; Voogd and Bigar6, 1980). The mediolateral compartmentalization of the Purkinje cells and their axons is related to the zonal organization of the corticonuclear projection and was first recognized by Klimoff (1899). He also concluded from his Marchi experiments in the rabbit that the corticonuclear projections are uncrossed, each hemivermis projecting to the medial cerebellar nucleus and to the vestibular nuclei, and the hemisphere to the dentate/interposed complex. The differential projection of the left and right hemivermis to the medial nuclei of either side implies that a sudden transition in the corticonuclear projection, in this case across the midline, is perfectly compatible with the uniform structure of the adult cortex. During early, fetal stages of cerebellar development the cortex is not uniform and discontinuities in the anlage of the Purkinje cell layer are present that subdivide it into a number of bilateral symmetrical parasagittal zones or clusters (see Section 6.2.). The first to demonstrate the paired origin and the zonal pattern in the development of the cortex were Hayashi (1924) and Jakob (1928) who distinguished an intermediate zone (the 'pars intermedia', Zwischenstiick or B convolution) located between the anterior vermis and the hemisphere in the 3-4 month human fetus (Fig. 154). The anlage of the anterior vermis was paired and consisted of the two A convolutions. Its histogenesis was more advanced than in the pars intermedia. Development of the cortex in the hemisphere lagged far behind. Caudally the triangular intermediate zone extended into the posterior lobe in the region of the paramedian sulcus where it degenerated and disappeared. The dentate nucleus developed in the hemisphere, the emboliform nucleus 170
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 118. Myeloarchitectonic compartments of the ferret cerebellum. In A the thin fibers at the border of the A and B compartments (R) are shown. In B the small Purkinje cell fibers of the C2 compartment are flanked by larger Purkinje cell fibers of C, and C3. H~iggqvist stain. Bar = 100/lm. Voogd (1969). (anterior interposed nucleus, Hayashi 1924) and the globose nucleus (posterior interposed nucleus, Jakob, 1928) belonged to the pars intermedia and the medial nuclei occupied the white matter of the vermis. Jansen and Brodal (1940, 1942) studied the corticonuclear projection of the corpus cerebelli with the Marchi method in cat, rabbit and macaque. They confirmed the uncrossed projection of each hemivermis to the medial nucleus and the vestibular nuclei. In the hemisphere they distinguished an intermediate zone, that projects to Brunner's (1919) interpositus nucleus and a lateral zone, that is connected with the lateral nucleus. They noticed the correspondence between their three-zonal arrangement in the corti171
Ch. I
J. Voogd, D. Jaarsma and E. Marani
conuclear projection of the anterior lobe and the corticogenetic zones of Hayashi and Jakob, but at the level of the cortex the exact borders of the three zones of Jansen and Brodal remained arbitrary. The paramedian sulcus, which is the border between vermis and pars intermedia, is often absent or indistinct and the border between the intermediate and lateral zones in Jansen and Brodal's scheme depends upon the arbitrary border between the interpositus and lateral nuclei. Moreover the longitudinal topology of their zones was defective. In the anterior lobe the orientation of the three zones was approximately perpendicular to the interlobular fissures, but when they were extrapolated to include the posterior lobe, the intermediate and lateral zones did not follow the curved axis of the folial chain of the hemisphere, and their common border cut through the centers of the folial loops of the ansiform lobule and the paraflocculus. Brodal's (1940; see also Jansen and Brodal, 1958) earlier studies of the olivocerebellar projection supported the distinction of the three zones in the anterior lobe. In the cat the dorsal accessory olive was found to project to the vermis and to the entire hemisphere of the anterior lobe. An additional projection of the rostral pole of the principal olive to the extreme lateral part of the anterior lobe could be observed in rabbit, monkey and man, but not in the cat. In the anterior lobe of rabbit and man, therefore, three zones can be distinguished on each side of the midline: the (hemi)vermis and the intermediate zone, that receive fibers from the dorsal accessory olive, and the lateral zone, that receives a projection of the principal olive and is absent in the cat. On the basis of these observations Jansen and Brodal (1940) called 'attention to the striking conformity in the arrangement of the corticonuclear and olivocerebellar projections, both systems apparently being arranged according to principles entirely different from those prevailing within the spinocerebellar and vestibulocerebellar projections'. The similarity in the organization of the olivocerebellar and corticonuclear projections remained one of the central concepts in the anatomy of the cerebellum that received ample support from later studies on longitudinal zonation. At the level of the cortex the borders between zones containing Purkinje cells that project to different target nuclei usually cannot be distinguished, but in the white matter the Purkinje axons from these zones collect in parasagittally oriented sheets that appear as white matter compartments in transverse sections. The sheets or compartments containing the Purkinje cell axons remain separated by narrow spaces. Voogd (1964, 1969) in cat and ferret, Marani (1982a, 1986) in the mouse and Feirabend and Voogd (1986) in the chicken traced these fiber compartments in myelin-stained (Hfiggqvist's method, 1936; Voogd and Feirabend, 1981) sections throughout the cerebellum. Large caliber fibers, that were identified as the axons of Purkinje cells, with an admixture of smaller fibers were present within the compartments and small myelinated fibers accumulate at their borders (Fig. 118). These borders usually are continuous with the borders between the subdivisions of the cerebellar nuclei and this configuration, therefore, made it possible to predict the longitudinal zonal organization in the corticonuclear projection (Figs 119 and 120). Corticonuclear projection zones are continuous from lobule to lobule and they are oriented perpendicular to the long axis of the folia: they follow the curved axis of the folial chains of vermis and hemisphere. The large caliber fibers within the compartments of the white matter could be identified as Purkinje cell axons with axonal tracing methods and immunohistochemistry with Purkinje cell-specific antibodies. They appear as discrete bundles, separated by narrow gaps with antibodies against cyclic GMP-dependent protein kinase (De Camilli et al., 1984) and calbindin-D28K (Paxinos, unpublished observations) in the rat and other species, with anti-Zebrin in cats and macaques (where Zebrin reacts with all Purkinje 172
The cerebellum." chemoarchitecture and anatomy
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of the zonal arrangement of the corticonuclear and olivocerebellar projection in the cat. The cerebellar nuclei are represented by circles. The diagram of the flattened inferior olive at the bottom of the figure is constructed according to Brodal (1940: Fig. 157). regions that are interconnected are indicated with the same symbols. The topography in the connections of the C] and C3 zones is indicated with open and filled diamonds. A - A zone; A N S I = ansiform lobule; B = cellgroup beta; B = B zone; C1-3 = C1-3 zones; D = D zones; D A O = dorsal accessory olive; dc = dorsal cap; dl = dorsal leaf of the PO; dmcc = dorsomedial cell column; F = fastigial nucleus; F L O C = flocculus; IA = anterior interposed nucleus; IP = posterior interposed nucleus; L = lateral cerebellar nucleus; LV - lateral vestibular nucleus; M A O = medial accessory olive; M E - medial extension of the ventral paraflocculus; P F L D - dorsal paraflocculus; PFLV = ventral paraflocculus; P M D = paramedian lobule; PO = principal nucleus of the inferior olive; SI - simple lobule; vest = vestibular nuclei; VI-X = lobules VI-X; vl - ventral leaf of the PO; vlo - ventrolateral outgrowth; X = X zone. Modified from Groenewegen et al. (1979).
173
Ch.I
J. Voogd, D. Jaarsma and E. Marani
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The cerebellum." chemoarchitecture and anatomy
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Fig. 120. Compartments in the white matter of the cerebellum of the cat. Drawings and reconstructions from H~iggqvist and AChE-stained sections. Compartments are indicated with different symbols. A-D. Graphical reconstructions of the rostral aspect of the anterior lobe (A) and the posterior lobe (B), the dorsal aspect (C) and the caudal aspect (D) of the cerebellum. Compare Fig. 98. E-G. Transverse sections. A = A compartment; ANS = ansiform lobule; A N T = anterior lobe; B = B compartment; C1-3 = C1-3 compartments; cr = restiform body; D(1,2) = D(1,2) compartments; F = fastigial lateral cerebellar nucleus; P F L = paraflocculus; P M D = paramedian lobule; SI = simple lobule; vest - vestibular nuclei; X - X compartment; III-IX = lobules III-IX. (.
cells) and with parvalbumin in the immature avian cerebellum (Braun et al., 1986) (Fig. 32). Monoamino oxidase in the white matter of the cerebellum of the chicken, is also restricted to the large caliber fiber areas (Feirabend, 1983; Marani, 1981). Systematic differences in the caliber of the Purkinje cell fibers were noticed in myeloarchitectonic studies in cat and ferret (Voogd, 1964, 1969; Voogd and Bigar6, 1980) and retrograde tracing of Purkinje cell axons (Voogd et al., 1991a). The small fibers located in the gaps between the bundles of Purkinje cell axons were called 'raphes' when they were positively stained with the H~iggqvist method (Fig.118; Voogd, 1964) and with AChE-histochemistry (Figs 122 and 127) (Hess and Voogd, 1986). Within the medullary core of the monkey cerebellum Hess and Voogd (1986) described fibers that stain densely for AChE and which are distributed in longitudinally oriented sheets, that appear as stripes in cross-sections (Fig. 127). These stripes in the white matter were aligned with AChE-rich bands, containing concentrations of reactive glomeruli in the granular layer, and narrow AChE-rich stripes in the molecular layer. A prominent midline band of AChE-rich axons flanked on each side by 5 or 6 ACHErich bands delineated a number of compartments in the cerebellar white matter. Within the white matter and the granular layer, cytochrome oxidase-rich axons and glomeruli were distributed in a longitudinally banded pattern, topographically identical to, but of lower contrast than, the banded distribution of ACHE. Both in the monkey and the cat the number and the disposition of the AChE-rich zones in the cerebellar white matter corresponded exactly with the raphe-like concentrations of small fibers at the borders of large fiber compartments in myelin-stained sections (Fig. 122). The highest amount of AChE was found in dense strips at the borders of the compartments; within the compartments the reactivity of the fibers for AChE differed. Compartments that contained large axons usually displayed very little AChE activity whereas the activity of AChE was high in compartments with smaller fibers. An explanation for the entire banded distribution of AChE in the molecular and granular layers and in the white matter as yet is not available, but it is clear that this distribution faithfully reflects the basic plan of the longitudinal zonation of the mammalian cerebellum. The possibility that zonally distributed differences in size and connections of the Purkinje cells are correlated with specific chemical properties of these cells was first raised by Marani (Marani and Voogd, 1977; Marani, 1981, 1982a; Marani, 1986) on the basis of the distribution of 5'-nucleotidase and acetylcholinesterase in the molecular layer and by Chan-Palay (1984) who reported a restricted distribution of certain peptides in subsets of Purkinje cells. More recently a complete pattern of alternating zones of immunoreactive and non-immunoreactive Purkinje cells was described by Hawkes and Leclerc (1986, 1987) with a Purkinje cell-specific antibody (anti Zebrin-I) in the rat and by Brochu et al. (1990) with anti-Zebrin II in the rat and other species (see Section 6.1.3.). The distribution of the Zebrin-positive Purkinje cells was very similar to the distribution of the enzyme 5'-nucleotidase in the molecular layer of certain rodents (Eisenman and Hawkes, 1989). 175
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J. Voogd, D. Jaarsma and E. Marani
The concept of the similarity in the organization of the corticonuclear and olivocerebellar projections, that dates from the work of Jansen and Brodal, received strong support from the observation that the distribution of the olivocerebellar fibers from certain subdivisions of the inferior olive was congruent with the parasagittal compartmentalization of the white matter (Voogd, 1969). The zonal termination of the climbing fibers could be visualized in autoradiograms of the olivocerebellar fibers using anterograde transport of [3H]leucine (Courville et al., 1974; Groenewegen and Voogd, 1977; Groenewegen et al., 1979). The olivocerebellar fibers located within a compartment were found to terminate both as climbing fibers on the Purkinje cells of the corresponding zone and on the cells of their cerebellar or vestibular target nucleus. The corticonuclear projection zones were experimentally verified in the antegrade degeneration and transport studies of Haines in primates and subprimates (see Haines et al., 1982 for a review) and Dietrichs in the cat (Dietrichs 1981a,b, 1983a; Dietrichs and Walberg, 1979a, 1980; Dietrichs et al., 1983b), and by retrograde labelling of the Purkinje cells and their axons from their target nuclei (Courville and Faraco-Cantin, 1976; Voogd and Bigar6, 1980; Balaban, 1984). A similar longitudinal pattern emerged from the electrophysiological studies of Oscarsson (1969, 1973, 1979, 1980; Oscarsson and Sj61und, 1974, 1977a,b,c; Andersson and Oscarsson, 1978a; Ekerot and Larson, 1979a,b; Ekerot et al., 1987, 1991a,b; Andersson and Eriksson 1981; Andersson and Nyqvist, 1983; Garwicz, 1992) and Armstrong et al. (1974) of the distribution of the climbing fibers in the cerebellar cortex. The electrophysiological identification of these climbing fiber zones rests on their laterality, their topography and on differences in latency between the different spino-olivocerebellar climbing fiber paths. These studies greatly extended our knowledge on the branching of the climbing fibers (Armstrong et al., 1973; Ekerot and Larson, 1982) and on the internal topography of the zones and lead to the distinction of the 'micro zone' as the smallest somatotopically defined unit engaged in the processing of information (Andersson and Oscarsson, 1978b; Garwicz, 1992). Direct proof of the congruence of the corticonuclear and olivocerebellar projections was obtained in the antegrade tracing studies of the Purkinje cells in electrophysiologically identified climbing fiber zones by Trott and Armstrong (1987a,b). The olivocerebellar and nucleo-olivary projections are reciprocally organized in the sense that a cerebellar target nucleus of one or more particular Purkinje cell zones is connected with the subdivision of the inferior olive that provides climbing fibers to the Purkinje cells of the same zone and a collateral projection to the target nucleus (see Ruigrok and Voogd, 1990, for a review of the literature). The zonal organization of the cerebellar cortex and the compartmental subdivision of the cerebellar white matter, therefore, are the manifestations of the modular organization of the output systems of the cerebellum. A module consists of a Purkinje cell zone, or a set of zones, its cerebellar or vestibular target nucleus with its GABAergic nucleo-olivary projection and its nonGABAergic output system and a sustaining olivocerebellar projection. Is the number of modules constant among the mammalian species and are they correlated with the different forms of chemoarchitectonic zonation in the cortex and the white matter? These questions only can be answered after a more thorough review of the corticonuclear projection (Sections 6.1.1., 6.1.2., 6.1.4. and 6.1.5.), the chemoarchitectonic evidence on zonation (Section 6.1.3.) and the olivocerebellar and nucleocerebellar projections (Section 6.3.) in different mammalian species. The existence of such a basic plan has been questioned (Boegman et al., 1988), not so much because the different maps for parasagittal zonation are mutually exclusive, but mainly because most 176
The cerebellum." chemoarchitecture and anatomy
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of the studies on the topography of afferent and efferent connections were conducted in carnivores and primates, whereas the immunocytochemical studies, that revealed a finer grain in the parasagittal organization were mostly restricted to the rat. Evidence on the corticonuclear projection in cat and primates and its correlation with white matter compartments, mostly dating from the 1960s to the early '80s, will be discussed in Sections 6.1.1. and 6.1.2. The compartmentalization of the cerebellar cortex for Zebrin (Hawkes et al., 1985) and other markers, such as 5'-Nucleotidase (Scott, 1963), that was mostly studied in rodents, will be considered next (Section 6.1.3.). Correlations of the corticonuclear projection in the rat with these cytochemical maps are reviewed in Section 6.1.4. and efferent connections of the vestibulocerebellum in Section 6.1.5. Some regional differences in the development of the cerebellum, mainly concerning the transient, chemical heterogeneity of the Purkinje cells, are considered in Section 6.2. The inferior olive and the large body of anatomical and electrophysiological evidence on the olivocerebellar projection, that has contributed so much to the ideas on the zonal organization of the cerebellar cortex, are discussed in Section 6.3. 6.1. COMPARTMENTS AND CORTICONUCLEAR PROJECTION ZONES: CORRELATIONS WITH CYTOCHEMICAL MAPS 6.1.1. Corticonuclear projection zones in the cat: Correlation with white matter compartments and cytochemical zones
In the cerebellum of the cat at least 8 zones have been defined on the basis of the corticonuclear and olivocerebellar projections (Fig. 119). The corresponding white matter compartments have been delineated in myelin-stained or AChE-stained sections in ferrets (Voogd, 1967, 1969), cats (Voogd, 1964, 1989) and monkeys (Hess and Voogd, 1986; Voogd et al., 1987a,b; Voogd and Hess, 1989). AChE in the anterior vermis of the cerebellum of young cats is present in bands in the molecular layer (Marani and Voogd, 1977; Voogd and Bigar6, 1980) (Fig. 121). An AChE-positive band at the midline is separated by an AChE-negative area from a strongly reactive, paramedian band. At the lateral, sharp border of the paramedian band a narrow AChE-negative band is found, that merges into the hemisphere, which is uniformly AChE-positive. The bands diverge in the dorsal part of the anterior lobe and in the simple lobule. AChE in the molecular layer is present in parallel striations. The general direction of these striations corresponds to the orientation of the Purkinje cells and to the direction of the cortical zones. A similar, but less distinct pattern of AChE positive and negative zones is present in the lobules VIII-X of the caudal vermis. The AChE-stained material illustrated in this chapter differs from Marani and Voogd's (1977) original illustrations because it is derived from aldehyde-fixed tissue from the adult animal (Brown and Graybiel, 1983). Sections prepared in this way display the borders between the compartments in the white matter in addition to the banded pattern in the molecular layer. Three compartments can be distinguished on both sides of the midline in the region of the vermis of the anterior lobe: a medial A compartment, a lateral B compartment and a wedge-shaped X compartment in between A and B (Figs 120 and 122). Compartments A and B are present in all lobules of the anterior lobe, but an X compartment is only present in its dorsal part (lobules IV and V). Ventrally the fused AChE-positive borders of the X compartment continue in the lateral border of the fastigial nucleus. The fibers of the B compartment pass lateral to the fastigial nucleus and medial to the interposed nuclei to enter Deiters' lateral vestibular nucleus from dorsally. It is clear from a comparison of 177
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J. Voogd, D. Jaarsma and E. Marani
the AChE-positive borders between the compartments and the zonal distribution of AChE in the molecular layer, that the medial border of the B compartment is exactly in register with the lateral border of the first paramedian band of high AChE activity in the molecular layer. Consequently, the cortical X zone corresponds to the lateral part of this first paramedian band of AChE-activity, but its border towards the A zone cannot be recognized in the distribution of AChE in the molecular layer. The B zone corresponds to the second, AChE-negative strip of the molecular layer. Previously, the correspondence between the localization of AChE in the molecular layer and the corticonuclear projection has been studied by Brown and Graybiel (1983), but they failed to recognize the X zone. The molecular layer of the hemisphere of the anterior lobe is uniformly ACHEpositive. In the white matter of the hemisphere of the anterior lobe C1,C2,C3 and one or more D compartments can be delineated in AChE or HS.ggqvist-stained material (Figs 120 and 122). The C2 compartment is present in the dorsal part of the anterior lobe (lobules III, IV and V). More ventrally the C1 and C3 compartments are contiguous. The anterior interposed nucleus is located within the fused C1 and C3 compartments, the C2 compartment can be traced into the more caudally located posterior interposed nucleus. In H/iggqvist-stained sections of the cerebellum the fibers of the A,B,C1 and C3 compartments were larger than those of the X and C2 compartments. Staining for AChE generally is more intense in C2 and X than in the large fiber compartments. The AChE-banding pattern in the molecular layer of the anterior vermis and the white matter compartments of the anterior lobe continue, across the primary fissure, into the posterior lobe. A wide A compartment, flanked by diverging X and B compartments is present in the vermis of the simple lobule (Figs 119 and 120). The B compartment ends at the area without cortex in the center of the ansiform lobule, where it abuts on the pontocerebellar fibers of the cerebellar commissure that reach the surface at this point. It is not clear whether the X compartment continues from the simple lobule into lobule
Fig. 121. Zonal distribution of A C h E in the cerebellum of the cat. A. Whole mount preparation of the anterior lobe. B. Transverse section through the anterior lobe. H E M = hemisphere; m = midline AChE-positive band; L = parasagittal AChE-positive band; arrows = lateral border of L. Marani (1986).
178
The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 122. Photographs of white matter of the anterior lobe of the cerebellum of the cat. A. Borders of compartments and midline (m) are delineated by AChE-reactive raphes. The lateral border of the X compartment is in line with the lateral border of the parasagittal AChE-positive strip in the molecular layer; the molecular layer over the B compartment is AChE-negative. B. H~iggqvist-stain. Compartments A-D are delineated by dark stripes containing small calibre fibers. Same level as Fig. 120E.
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CORTICO~/#cSTT'## LAR ~ CORpTdCjONcUC~EAR Ve~#Y" CAUDAL ANTERIOR Fig. 123. D i a g r a m of the corticonuclear projection in the cat. Based on retrograde labelling of Purkinje cells and their axons after injections of H R P in their target nuclei. A = A zone; A = Anterior interposed nucleus; A N S = ansiform lobule; B = B zone; C1-C3 = C1-C3 zones; C R I I = crus II of the ansiform lobule; D1-D2 = D1-D2 zones; DV = descendng vestibular nucleus; F - fastigial nucleus; F L O C = flocculus; IP = posterior interposed nucleus; L = lateral cerebellar nucleus; LOB POST = posterior lobe; LOB A N T = anterior lobe; LV = Deiters' nucleus; MVmc = magnocellular medial vestibular nucleus; MVpc = parvicellular medial vestibular nucleus; P F L = paraflocculus; P F L D = dorsal paraflocculus; PFLV = ventral paraflocculus; P M E D = paramedian lobule; SV = superior vestibular nucleus; I-X = lobules I-X. Bigar6 (1980).
VII. Usually the entire white matter of lobule VII is considered to belong to the A compartment. 180
The cerebellum: chemoarchitecture and anatomy
Ch. I
C1,C2,C3 and D compartments can be distinguished in the hemisphere of the simple lobule. C1 and C3 compartments are narrow or interrupted in the white matter of the ansiform lobule. A medial C2 and more laterally located D1 and D 2 compartments can be recognized in the white matter of this lobule. The complete set of compartments is present again in the white matter of the caudalmost folial rosette of the Crus II of the ansiform lobule (the ansula). From here these compartments pass into the paramedian lobule. C3 is present in the dorsal part of paramedian lobule only, and ends at the border with the pars copularis. C~ continues into the ventral most folia of the paramedian lobule as the most medial compartment of the hemisphere. C2 and the narrow, laterally located D1 and D2 compartments continue from the paramedian lobule into the dorsal paraflocculus (Figs 119 and 120). The dorsal and ventral paraflocculus contain C2, D1 and D 2 compartments. Concentrically arranged AChE-positive raphes, that stain strongly for ACHE, separate the compartments of the dorsal and ventral paraflocculus in the rostral part of the parafloccular loop (Figs 125 and 126). The rostral (dorsomedial) dentate nucleus is located within the D 2 compartment, the caudal (ventrolateral) dentate within the D~ compartment and the C2 compartment enters the lateral pole of the posterior interposed nucleus. Distinct compartments cannot be delineated in the white matter of the flocculus of the cat. The zonation in the corticonuclear projection that could be predicted from the disposition of the white matter compartments, was confirmed with retrograde labelling of Purkinje cells, after injections of tracers in the individual cerebellar nuclei of the cat (Courville and Faraco-Cantin, 1976; Voogd and Bigar6, 1980; Gibson et al., 1987). The Purkinje cells of the corticonuclear projection zones in the hemisphere follow the loops of the folial chain (Fig. 123, compare Fig. 98). Purkinje cells of the C1 and C3 zones can be retrogradely labelled from the anterior interposed nucleus. The C~ zone is broad in the ventral part of the anterior lobe and tapers more dorsally. The C3 zone is wide in the dorsal part of the anterior lobe, ventrally it is continuous with a narrow 'd2' zone 2 in the extreme lateral part of the anterior lobe. C1 and C3 are narrow in the ansiform lobule and reappear in the ansula and the paramedian lobule. They do not continue in the paraflocculus. The C2 zone was continuous from lobule III of the anterior lobe, through the entire hemisphere, into the flocculus. Its Purkinje cells were labelled from injections of the posterior interposed nucleus. Purkinje cells of the D zones occupied the extreme lateral part of the anterior lobe. A medial D1 and a lateral D 2 z o n e that projected to rostral and caudal parts of the lateral cerebellar nucleus, respectively, could be distinguished in the ansiform lobule, the paramedian lobule and the paraflocculus. The corticonuclear projection of the anterior lobe, the simple, ansiform and paramedian lobules was studied with anterograde transport of HRP by Dietrichs (198 l a) and Dietrichs and Walberg (1979a, 1980). Their observations on the projections of the A,B,C and D zones are essentially in accordance with the findings of Voogd and Bigar6 (1980). In their alternative nomenclature they shifted the zones one zone laterally, the presumed B zone projecting to the anterior interposed nucleus, and the C1 zone with the posterior interposed nucleus. This alternative nomenclature was discussed in the papers by Voogd and Bigar6 (1980), Haines et al. (1982) and Voogd et al. (1987a,b). The presence of C2, 2The term 'd 2' w a s applied to a narrow strip of climbing fiber-evoked potentials in the extreme lateral part of the anterior lobe by Ekerot and Larson (1979a, see Figs 171 and 175 ). The d 2 z o n e can be activated by the dorsal spino-olivo-cerebellar-climbing-fiber-pathand receives branches from climbing fibers which also innervate the lateral c3 zone (Ekerot and Larson, 1982).The use of the letter 'd' for this zone is misleading, because it neither projects to the lateral cerebellar nucleus or receives a projection from the principal olive. 181
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J. Voogd, D. Jaarsma and E. Marani
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Fig. 124. Comparison of bands of AChE reaction product in the molecular layer of the anterior vermis of cat cerebellum and retrograde labelling of Purkinje cells in B and lateral A zones after an injection of HRP in the vestibular nuclei (A-C) and in the B and X zones after an injection in the lateral fastigial nucleus and the B compartment (D-F). Note different size of Purkinje cells in B and X zones. A = A zone; B = B zone; Deit = Deiters' nucleus; DV = descending vestibular nucleus; F = fastigial nucleus; IA = anterior interposed nucleus; MV = medial vestibular nucleus; X = X zone. I-V = lobules I-V. Voogd (1989). (
Dl and D2 zones in the paraflocculus of the cat is compatible with the anterograde tracing study of Dietrichs (1981 b). The corticonuclear projections of the electrophysiologically verified climbing fiber zones b, x, cl, c2, c3 and dl 3 w e r e determined for lobule V of the anterior lobe of the cat with anterograde tracing of [3H]leucine (Trott and Armstrong (1987a,b)). They were found to conform to the projections of the corresponding anatomical zones in the retrograde tracing studies of Voogd and Bigar6 (1980). The corticonuclear and -vestibular projection were correlated with the distribution of AChE for the anterior vermis of the cat by Brown and Graybiel (1983) and Voogd et al. (1991 a). Other studies of the projections of the anterior vermis to the fastigial nucleus and the vestibular nuclei, that have been known since Klimoff's (1899) study of the corticonuclear projection in the rabbit, did not distinguish between the zones in this region (Corvaja and Pompeiano, 1979; Dietrichs et al., 1983b; see Voogd 1964 and Brodal, 1974 for reviews of the older literature). Purkinje cells that project to the vestibular nuclei are located both in the A zone and in the B zone but not in the X zone (Fig. 124A-C). Purkinje cell axons of the A zone terminate in the fastigial nucleus, but some of these axons proceed to the vestibular nuclei, where they terminate at the border of Deiters' nucleus and the magnocellular medial vestibular nucleus (i.e. the ventral part of the lateral vestibular nucleus of Brodal and Pompeiano, 1957). The B zone projects to Deiters' nucleus (i.e. the dorsal part of the lateral vestibular nucleus). The projection of the A and B zones to different parts of the vestibular nuclei (Bigar6, 1980; Voogd and Bigar6, 1980; Voogd, 1989) was confirmed in the rabbit by Balaban (1984) and Epema (1990). These experiments substantiated the small projection of the A zone to Deiters' nucleus, as proposed by Andersson and Oscarsson (1978a). The wedge-shaped X zone in the dorsal part of the anterior lobe projects to the medial limb of the U-shaped nucleus at the junction of the fastigial and posterior interposed nuclei of the cat (Trott and Armstrong, 1987b). The Purkinje cells and their axons, when retrogradely labelled from this nucleus, are smaller than those of the A and B zones (Fig. 124D-F). The medial border of the retrogradely labelled B zone corresponds exactly with the lateral border of the parasagittal strip of high AChE-activity in the anterior vermis. The small Purkinje cells of the X-zone and the larger Purkinje cells with vestibular projections in the lateral A zone are located within the parasagittal AChE-positive strip. The B zone has a low content of AChE (Voogd, 1982). It was pointed out in a previous Section (5.3.) that the nucleocortical and corticonuclear projections in the cat are roughly reciprocal (Tolbert et al., 1978b; Gould, 1979; Dietrichs and Walberg, 1979a, 1980; Dietrichs and Walberg, 1985). Trott and Armstrong (1990) showed that nucleocortical projections to the electro-physiologically identified cl and c3 zones are scarce and that the major projection to the c2 zone of lobule V takes its origin from the posterior interposed nucleus.
30scarsson and co-workersadapted a modification of Voogd'snomenclature to indicate the electrophysiologically identified climbing fiber zones, using lower case letters instead of capitals (see Fig. 175). 183
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PHD
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PFL tI Fig. 125. White matter compartments C2, D1 and D2 in the paraflocculus of the cat in transverse, AChE-reacted sections. Note concentric arrangement of the compartments in the dorsal and the ventral paraflocculus. A caudalmost section; D = rostralmost section. ANS - ansiform lobule; brp = brachium pontis; C1-3 = C1-3 compartments; crest = restiform body; D - dentate nucleus; D (1,2)- D (1,2) compartments; F = fastigial nucleus; FLO = flocculus; IA - anterior interposed nucleus; IP = posterior interposed nucleus; PFLD - dorsal paraflocculus; PFLV- ventral paraflocculus; PMD = paramedian lobule. )
6.1.2. Compartments and corticonuclear projection zones in monkeys A l t h o u g h an analysis o f the c o m p a r t m e n t a l subdivision in the m o n k e y w o u l d be feasible f r o m serial HS, ggqvist-stained sections, the A C h E staining is m o r e distinct. A p r o m i n e n t c o n c e n t r a t i o n of A C h E - r i c h axons at the midline is flanked on each side of the anterior lobe by seven A C h E - r i c h strips, that delineate eight parasagittal c o m p a r t m e n t s in the cerebellar white m a t t e r (Fig. 127). Some of these c o m p a r t m e n t s are t o p o g r a p h i c a l l y related to certain cerebellar nuclei (Hess and Voogd, 1986; Voogd et al., 1987a,b). This is especially clear for two o f the c o m p a r t m e n t s , X and C2, b o t h of which have a high c o n t e n t of A C h E - p o s i t i v e fibres. The X c o m p a r t m e n t is n a r r o w a n d located in the white m a t t e r o f the vermis. It continues into the lateral, A C h E - r i c h b o r d e r z o n e o f the fastigial nucleus a n d the medial limb of the U - s h a p e d nucleus located between the caudal pole 184
The cerebellum." chemoarchitecture and anatomy
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25
of the fastigial and the posterior interposed nuclei (Fig. 105). In some sections the two AChE-rich strips that border the compartment, fuse with its content into a single, broad AChE-positive band. In the ventral part of the anterior lobe the X compartment is narrow or even absent. The X compartment extends caudally into the lobules VI and in the lateral parts of the lobules VII and VIII. The X compartment separates the A and B compartments of the anterior vermis. The A compartment includes the fastigial nucleus. In the posterior vermis it becomes wider and is subdivided into a medial A1 and a lateral A 2 compartment. The B compartment is present in the anterior lobe and the simple lobule. Ventrally it empties into the space between the fastigial and the anterior interposed nucleus to continue into the lateral vestibular nucleus. Caudally it diverges far laterally at the junction of the lobules VI and VII, where it ends at the area devoid of cortex in the centre of the ansiform lobule. The C2 compartment is clearly related to the posterior interposed nucleus. Rostrally it is located dorsal to the anterior interposed nucleus, in the intermediate part of the hemisphere. From the posterior interposed nucleus the C2 compartment extends dorsolaterally into the ansiform lobule, caudally into the paramedian lobule and ventrolaterally into the paraflocculus and the flocculus (Fig. 148). In the anterior lobe C2 is located between the C~ and C3 compartments. Ventrally Cl and C3 fuse and are related to the 185
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J. Voogd, D. Jaarsma and E. Marani
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anterior interposed nucleus. Both C 1 and C 3 diverge laterally in the central part of the ansiform lobule and extend into the paramedian lobule. C1 ends in the last folium of the paramedian lobule, C3 stops in the dorsal part of this lobule. A major, lateral expanse of the white matter is related to the dentate nucleus. In the dorsal paraflocculus and the petrosal lobule this dentate-related white matter is subdivided into the D1 and D2 compartments (Fig. 148). D 1 is located next dorsal to the C2 compartment. D1 is related to the medial dentate nucleus. D2 occupies the dorsal part of the paraflocculus and is related to the lateral dentate nucleus. In the anterior lobe and the paramedian lobule 186
The cerebellum." chemoarchitecture and anatomy
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a narrow compartment, lateral to C3, may represent D 1. The compartmental subdivision of the white matter of the ventral paraflocculus and the flocculus will be discussed in the Section on the vestibulo-cerebellum (6.1.5.). Our interpretation of this AChE pattern in the primate cerebellum generally is supported by data on the corticonuclear projection in primates and subprimates (see Haines et al., 1982 and Haines and Dietrichs, 1991 for reviews of the literature). Results from experiments on Galago, using silver impregnation of degenerated axons, showed that at least six zones corresponding to the A,B,CI__3 and D zones of carnivores, could be identified in the anterior lobe (Haines, 1977a; Haines and Rubertone, 1979) (Fig. 128). Lesions of the B zone in lobule V were associated with an additional projection to the 187
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J. Voogd, D. Jaarsma and E. Marani
most medial portions of the posterior interposed nucleus, suggesting the presence of an X zone in the vermis of this species (see also Haines and Dietrichs, 1991). Similar observations were made for the corticonuclear projection of the anterior lobe of Saimiri sciurus, where the X zone extended from lobule IV into VI. Purkinje cell axons of the X zone in the medial part of the posterior interposed nucleus do not overlap with the more centrally located terminations of the C2 zone in this nucleus (Haines et al., 1982; Haines and Dietrichs, 1991). Separate D1 and D 2 z o n e s with projections to rostrodorsal and centroventral regions of the lateral cerebellar nucleus, were distinguished in the anterior lobe in Saimiri (Haines et al., 1982). The cortico-vestibular projection of the anterior vermis was studied by Voogd et al. (1991 a) in Macaca fascicularis. The disposition of these Purkinje cells in the lateral A zone and the B zone of the anterior lobe and the simple lobule was similar to the cat. Retrogradely labelled Purkinje cell axons were located in the lateral A and the B compartments that could be delineated in adjacent AChE-stained sections. The X zone and compartment did not contain Purkinje cells with vestibular projections. Only few observations in primates are available on the corticonuclear projection of the posterior lobe. Haines and Whitworth (1978) and Haines and Patrick (1981) studied the projection of the paramedian lobule and the paraflocculus in the tree shrew (Tupaia glis). They concluded that Cl_3 and a D zone, with a similar topography and corticonuclear projection as in the cat, were present in the paramedian lobule of the tree shrew. The C2 and the D zone continued into the paraflocculus, where the D zone could be subdivided into D1 and D2 zones on the basis of its differential projection to the lateral cerebellar nucleus. The organization of the posterior vermis in primates (Haines, 1975a,b) will be dealt with in the Sections on the vestibular cerebellum (6.1.5.) and the olivocerebellar projection (6.3.3.3.).
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Fig. 128. Diagrammatic representation of the corticonuclear projection of lobule V in Galago. There are at least six identifiable corticonuclear projection zones in the lobule V cortex. The vermis consists of zones A and B, the intermediate cortex of three zones C1 - C3 and the lateral cortex of a single D zone. f = flocculus; IC = intermediate cortex; LC = lateral cortex; lvn = lateral vestibular nucleus; 1-nia = lateral anterior interposed nucleus; m - nia = medial anterior interposed nucleus; m - nip = medial posterior interposed nucleus; nl = lateral cerebellar nucleus; nm = medial nucleus; vc - vermal cortex. Haines and R u b e r t o n e (1979)
188
The cerebellum." chemoarchitecture and anatomy
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The rough reciprocity of the corticonuclear and nucleocortical projections has been questioned in primates. In contrast to the observations in the cat Tolbert et al. (1978b), Tolbert and Bantli (1979) and Tolbert (1982) reported that all cerebellar nuclei projected to the vermal cortex in Macaca mulatta and that a major contingent came from the lateral cerebellar nucleus. Haines (1988) was unable to confirm these aberrant nucleocortical projections in Galago and suggested that some of Tolbert's injection sites in the lobules VI and VII of the caudal vermis may have extended in the neighbouring D zones. Haines (1989) found retrogradely labelled nucleocortical cells after injections of the C1, C3 and D zones to be relatively few and to be located in the corresponding anterior interposed and lateral cerebellar nuclei. The major nucleocortical projection in Saimiri takes its origin from the medial and posterior interposed nuclei, is bilateral and is directed at the A~ and C2 zones. This anisotropy in the nucleocortical projection in primates confirmed Trott and Armstrong's (1990) conclusions on the strong nucleocortical projection to the C2 zone in the cat. 6.1.3. Parasagittal zonation in the cerebellar cortex: Antigenic compartmentalization for Zebrin and other markers
The particulars of several Purkinje cell-specific markers that define parasagittal zones in adult rat cerebellum have been discussed in the first part of this chapter (see Section 3.1.2. for motilin, and the synthesizing enzyme of taurine (CSADase) and Section 3.1.8. for the zonal distribution of immunoreactive Purkinje cells in monkey and rat cerebellum, with the B.1 antibody of Ingram et al., 1985). CGRP, somatostatin and pseudocholinesterase (see Section 3.1.3.) are only present in zonally distributed bands of Purkinje cells during prenatal or early postnatal development. The complicated distribution of AChE was discussed in Sections 6.1.2. and 6.1.3. on the compartmental subdivision of the cerebellar white matter in cat and monkey. The distribution of AChE in the cerebellum of the rat is very similar (see Marani, 1986, Boegman et al., 1988; and Voogd, 1995 for reviews). Zebrin I and II (Section 3.1.8.) are the best-studied markers, that have been shown to be expressed by longitudinally organized subsets of Purkinje cells. Several other proteins and antigenic markers share the same or a very similar distribution as the Zebrins (the protein kinase C delta-isoform, Section 3.1.5.; the monoclonal antibody B30 of Stainier and Gilbert, 1989, Section 3.1.8.; the low affinity nerve growth factor receptor protein, Section 3.1.10.; and 5'-nucleotidase, Section 3.5.), whereas others only partially allign with the Zebrin pattern (HNK-1 antigen, Eisenman and Hawkes, 1993), or have a zonal distribution essentially complementary to the Zebrin pattern (cytochrome oxidase, Leclerc et al., 1990, P-path antigen, Leclerc et al., 1992, Section 3.1.8., and FAL in the Bergmann glia, Section 3.11.). Molecular markers of Purkinje cell heterogeneity include neurotransmitter receptors, such as the GABAB receptor (Section 3.7.2.), the muscarinic (m2) receptor (Section 3.10.2.), the dopamine D3 receptor (Section 3.8.) and the substance P receptor (Nakaya et al., 1994). It should be noted that the distribution of most markers, as identified in rodent species, may be different in other species or, as is the case for the muscarine m2 receptors, may be expressed in specific species only (Neustadt et al., 1988) (see Section 3.10.2.). An illustrative example of differential zonal distribution in closely related species was provided by Insel et al. (1994), who studied the distribution of vasopressin V~a receptors in the brain of different species of voles (microtine rodents). A complex distribution with alternating bands of high and low receptor density was observed in 189
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prairie voles. Instead, vasopressine Via receptors were confined to the nodulus in pine voles, and no receptors occurred in the cerebellar cortex of montane and meadow voles. The compartmentalization of motilin and taurine and the zonal patterns revealed by 5'-nucleotidase and Zebrin will be discussed in more detail. Chan-Palay (1984) described the distributions of Purkinje cells that react with antibodies against a conjugate of motilin (Chan-Palay et al., 1981; Nilaver et al., 1982) and against the synthesizing enzyme of taurine (L-cysteine sulfonic acid decarboxylase, CSADCase) (Chan-Palay et al., 1982a,b) (see also Magnusson et al., 1988, and Section 3.1.2.). Purkinje cells with motilin and/or GAD-like immunoreactivity together accounted for more than half of their total number. Purkinje cells that reacted with an antibody against motilin were most numerous in the flocculus and the paraflocculus. In the hemisphere they occurred in groups. They were fewer in the vermis, where they constituted a prominent midlineand two parasagittal bands in the lobules I-VI (Fig. 129). Motilin-immunoreactive Purkinje cells usually were larger than those containing only GAD. A majority of the Purkinje cells and many of the stellate and basket cells in the molecular layer reacted with the antibody against CSADC. These cells were distributed in a midline-band and 3 pairs of CSADC-positive and -negative bands on either side. The bands increased in width in the dorsal part of the anterior lobe (Fig. 130). In the vermis of the posterior lobe similar bands existed but were less distinct. Two CSADCpositive bands were present in the hemisphere. The flocculus and the paraflocculus contained the highest number of CSADC-positive Purkinje cells, but they were not zonally distributed. Chan-Palay's description of the zonal distribution of motilin and CSADC-immunoreactive Purkinje cells does not offer clues for a comparison with similar longitu-
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Fig. 129. Schematic drawing of the distribution ofmotilin-immunoreactive (M-i) Purkinje cells (open triangles) and glutamic acid decarboxylase-immunoreactive (GAD-i) Purkinje cells (filled circles) in a coronal section of rat cerebellum. M-i cells and GAD-i cells are both more concentrated in the flocculus and the paraflocculus than elsewhere. Both cell types occur in the vermis and participate in the formation of the sagittal microzones (arrows). M-i terminal axon projections in the deep cerebellar nuclei are heaviest in the dentate (D: left side) and GAD-i projections are heaviest in the lateral vestibular nucleus (LV: right side). ! = interposed nucleus; F = fastigial nucleus. Chan-Palay et al. (1981).
190
The cerebellum." chemoarchitecture and anatomy
Ch. I
III [anterior I. simple
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Fig. 130. Schematic summary of cysteine sulfinic acid decarboxylase (CSADCase)-positive sagittal microzones or bands in mouse cerebellum. The bands are clearest in the anterior lobe and the vermis, less sharply defined in the hemispheres (dense stipple), and most difficult to discern in the paraflocculus and flocculus (light stipple), because of intense CSADCase reactivity in most Purkinje cells. The dentate (D), interpositus (I), fastigial (F), and lateral vestibular nuclei (LVN) contain numerous CSADCase-positive cells. Chan-Palay et al. (1982b).
dinal patterns in the connections or in the distribution of other markers. The parasagittal zone of large, motilin-immunoreactive Purkinje cells may correspond to the B-zone, which, in the cat at least, contained larger Purkinje cells than the adjoining X zone. The majority of the terminals of the B-zone in Deiters' nucleus, however, contained GABA and did not react for motilin (Fig. 129). The distribution of 5'-nucleotidase (5"N) (Section 3.5.) in alternate longitudinal bands of high and low enzyme activity in the molecular layer of the cerebellar cortex of the mouse (Scott, 1963, 1964, 1965, 1967) was the first evidence for the biochemical compartmentalization of the cerebellar cortex. The pattern of 5'-N-positive and -negative zones is complete in the sense that it is present in all the lobules of vermis and hemisphere and unequivocal, because, in the mouse at least, the bands are clearly delineated (Marani, 1986). The 5'-N band pattern is very similar, if not identical, to the more recently described distribution of Purkinje cells in the rat, reacting with Purkinje cell-specific monoclonal antibodies to Zebrin-I (mabQ113) (Eisenman and Hawkes, 1989). The zonal distribution of 5'-N in the cerebellum of the mouse (see also Section 3.5.) was described in detail by Marani (1982a, 1986). A similar zonation of 5'-N was present in the cerebellum of the rat, the shrew (Marani, 1982a) and of Clethrionomys glarulus (Marani, 1982a). However, uniform and high levels of 5'-N without any indication of a longitudinal zonation characterize the molecular layer in some other rodents, the cat, some primates and in man (Scott, 1967; Marani, 1982a, 1986). The pattern in the rat is less distinct because a high background activity of 5'-N is present all over the molecular layer. This background activity disappears after long-standing lesions of the inferior olive (Marani, 1986). 191
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Fig. 131. Reconstructions of the zonal distribution of 5'-nucleotidase (5'-N) in the molecular layer of the cerebellum of the mouse. Numbers without prefix indicate the nomenclature for the Y-N-positive bands of Marani (1982); P-numbers on the left side refer to the nomenclature for correspondingZebrin I-positivebands of Hawkes and Leclerc (1987). ANT = anterior lobe; FLO = flocculus; PFL = paraflocculus; II-X = lobules II-X. Marani (1982a, 1986). (
A midline band flanked by six, symmetrically disposed 5'-N-positive bands can be recognized in mice (Figs 58A, 131, 132). The bands were indicated by Marani (1982a) with the numbers 1--7 for the anterior lobe and the simple lobule and 11-17 for the posterior lobe. The bands in the anterior and posterior lobe are not necessarily continuous. Staining in the 5'-N-positive bands is rather uniform both in transverse and sagittal sections and cannot be assigned to specific structures in the molecular layer. There is an abrupt change in enzyme activity at the lateral border of the 5'-N-positive bands towards the next 5"N-negative zone; enzyme activity falls off more gradually at their medial borders. The 5'-N-positive bands share the distinctness of their lateral borders with the AChE-postive bands in the cerebellum of the cat and with the borders of the small fiber compartments (the 'raphes') towards the next lateral large fiber (Purkinje cell fiber) compartment in the cerebellar white matter. 5'-N-positive bands are narrow in the ventral part of the anterior lobe. They increase in width in the dorsal parts of the anterior lobe and the simple lobule and even more so in the rest of the posterior lobe, where the 5'-N-negative zones are reduced to narrow slits (Fig. 131). Staining in the medial bands 1-5 of the anterior lobe is heavier than in the more lateral bands 6 and 7 of the hemisphere of the anterior lobe. In the posterior lobe the intensity of the staining differs for the different bands. Heavy staining is found in the bands 11 and 13 and much less reaction product is present in the intermediate band 12 (Fig. 58A). The epitopes recognized by Hawkes' family of monoclonal antibodies known as the 'anti-Zebrins' are localized on Purkinje cells (see Section 3.1.8.). Zonal patterns that are identical or very similar to Zebrin I and II have been described for the distribution of 5'-nucleotidase (see above), the p75 low affinity nerve growth factor receptor protein in the rat (Section 3.1.10., Fig. 38), protein kinase C delta (Fig. 133) (see Section 3.1.5.) and the B30 antibody of Stainier and Gilbert (1989) (see Section 3.1.8.). Immunoreactivity in mouse Purkinje cells for an antibody against H N K is partially congruent with the Zebrin negative Purkinje cells, but Zebrin+/HNK+ Purkinje cells also exist (Hawkes, 1992; Eisenman and Hawkes, 1993). The similarity between the Zebrin pattern and the transient zonal patterns in the development of the Purkinje cell specific marker L7 is discussed in Section 6.2. Complementary staining patterns were described for cytochrome oxidase in Saimiri sciureus and rat by Leclerc et al. (1990), for the distribution of P-path-immunoreactive (Edwards et al., 1989, see below) Purkinje cells in the mouse by Hawkes (1992), Leclerc et al. (1992) and Edwards et al. (1994) and for 3-fucosyl-N-acetyl-lactosamine (CDts) in mouse Bergmann glia (Fig. 94) (Bartsch and Mai, 1991; Marani and Mai, 1992). According to Eisenman and Hawkes (1989) the 5'-N and Zebrin I zonal patterns in mouse cerebellum are congruent (Fig. 135). There are also similarities with respect to the mode and the time scale of their development. 5'-N in neonatal mice is distributed uniformly in the molecular layer and the first signs of a banded distribution cannot be discerned before postnatal day 14 (Hess and Hess, 1986). Zebrin I development in rats 193
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J. Voogd, D. Jaarsma and E. Marani
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Fig. 133. Distribution of protein kinase C delta-immunoreactive Purkinje cells in transverse section through the posterior lobe of rat cerebellum. This pattern is similar to the distribution of Zebrin. Bar - 0,5 mm. Chen and Hillman (1993a).
194
The cerebellum. chemoarchitecture and anatomy
Ch. I
also goes through a stage where all Purkinje cells express the epitope. The banded distribution appears relatively late, from postnatal day 15 onwards, when the immunoreactivity becomes suppressed in the future Zebrin I-negative zones (Leclerc et al., 1988). Differences between both patterns involve the distribution of the marker within the bands. Both for 5'-N and Zebrin I the intensity of the staining falls off in the more laterally located bands, but the sharp lateral borders and the differences in reactivity between the 5'-N-positive bands of the caudal vermis are not as clear with staining for Zebrin I. Moreover Zebrin I immunoreactivity extends into the Purkinje cell axons and compartments of Zebrin I-positive and -negative axons are present in the white matter that reflect the zonal distribution of the corresponding Purkinje cells. Zebrin I in rat cerebellum was compared to the distribution of AChE in the cerebellum of the rat (Boegman et al., 1988). These authors stressed the congruence of Zebrinpositive Purkinje cells with the accumulations of AChE in patches in the underlying granular layer. AChE in these patches is present in glomeruli, in certain Golgi cells and in other, unidentified components of the neuropil. Hawkes and Leclerc (1987) grouped the Zebrin I-positive Purkinje cells in a midline band (PI+) and seven symmetrically disposed parasagittal bands (P2+-PS+). Hawkes and Leclerc's (1987) numbering system for the Zebrin-positive and -negative bands in rat is indicated in Fig. 136. The P - bands of Zebrin-negative Purkinje cells bear the same number as the next medial P+ band. The pattern of Zebrin I-immunoreactive Purkinje cells is virtually identical in rat and mouse (Figs 139 and 140) (Eisenman and Hawkes, 1993). The numbering of 5'-nucleotidase-positive bands according to Marani (1986) and Hawkes and Leclerc's (1987) numbering system for the Zebrin-positive bands can be compared in Fig. 131 of the distribution of 5'-N in mouse cerebellum. The main features of the Zebrin pattern are the increase in width of the Zebrinpositive bands in the dorsal part of the anterior lobe, as compared to the ventral lobules I, II and III and the cortex in the bottom of the primary fissure (Figs. 139, 140, 143). The Zebrin-positive bands can be traced across the primary fissure in lobule VI that forms the rostral bank of the primary fissure. They also increase in width in dorsal lobule VI and fuse into an extensive, Zebrin-positive area that covers lobule VII and the adjoining ansiform lobule. Zebrin-negative bands reappear in caudal lobule VII and in the caudal folia of the Crus II and the paramedian lobule. The pattern is distinct in lobule VIII and the caudal copular portion of the paramedian lobule. Wide, Zebrinpositive separated by Zebrin-negative slits are present in lobule IX. Most Purkinje cells of lobule X are Zebrin-positive, although zonally distributed regions with higher and lower immunoreactivity can be recognized in the bottom of the postero-lateral fissure and in lobule X. The continuity of the Zebrin-positive zones is less clear than suggested by the published diagrams. Regions where the continuity of the zones cannot be assessed, include the lobule VII/ansiform lobule, where most Zebrin-positive Purkinje cells fuse into a single continuum, and the transitions between the lobules VII, VIII, IX and X in the bottom of the prepyramidal, secondary and posterolateral fissures. The P1 + band is narrow and consists of fused, bilateral portions. It is present in all lobules, fuses with the P2+ bands in the lobules VII and IX/X and is wider in lobule VIII. P2+ was identified in the anterior and posterior lobes, but its continuity cannot be established because it fuses with other P+ bands in lobule VII. P3+ is weakly immunoreactive in the anterior lobe, and looses its identity among the Zebrin-positive Purkinje cells of lobule VII. It reappears in caudal lobule VII and can be traced as a distinct band in lobule VIII. The apparent continuity of P3+ between the lobules VIII and IX may be false, a fusion of P3+ with P4+ into the P3+ band of lobule IX should 195
Ch. I
J. Voogd, D. Jaarsma and E. Marani
P~
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Fig. 134. Diagrammatic reconstruction of the Zebrin II/P-'path staining pattern of the mouse cerebellar vermis. Areas that are Zebrin II positive are shaded: areas that are P-path positive are unshaded; regions that are double labelled are solid black. I-X, cerebellar lobules. Inset: double labelling for Zebrin II and P-path may be limited to the lateral half of the P3+ in lobule VIII. Redrawn from Leclerc et al. (1992).
be considered. P4+ is distinct in the anterior lobe and the simple lobule, fuses with the other P+ bands at the transition of lobule VII and the ansiform lobule, and reappears as a wide, Zebrin-positive strip at the border of vermis and hemisphere at the level of the Crus II and the dorsal paramedian lobule. P5+, P6+ and P7+ are present in the hemisphere of the anterior and posterior lobe, but cannot be traced as individual bands across the Zebrin-positive ansiform lobule. One or more of these P+ bands may continue in the Zebrin-positive, lateral pole of lobule IX. The relations of the P5+/P7+ bands with the uniformly Zebrin-positive Purkinje cells of the paraflocculus and the flocculus have not been established. 196
The cerebellum." chemoarchitecture and anatomy
A
Fig. 135. Photomicrographs of the ventral surface of the uvula of the cerebellum of the mouse in adjacent sections reacted with Zebrin I antibody (A) and for the presence of 5'-nucleotidase (B). Note the identical pattern of staining in both even though the borders of the 5'-nucleotidase staining (B) are less distinct. Q 113 = mabQ113, 5'N - 5'-nucleotidase. Eisenman and Hawkes (1989).
Short strips of Zebrin-positive Purkinje cells have been noticed between the P1 +, P2+ a n d P3+ b a n d s in the a n t e r i o r lobe a n d the simple lobule. Two n a r r o w strips of Zebrin-positive Purkinje cells were identified in the ansiform lobule, between P4+ and P5+. They were considered as bifurcations of these bands, that were indicated as P4+ a n d P 5 b + with the additional strips as P 4 b + a n d P5a+. The n a r r o w stretches of Zebrinpositive Purkinje cells in the dorsal vermis of the anterior lobe a n d the simple lobule, were considered as 'satellite bands'. They are i n c o n s t a n t a n d n o t necessarily bilaterally symmetrical. D o r 6 et al. (1990) identified alternating P+ a n d P - zones in the cerebellum of the grey
Fig. 136. The reconstruction of parasagittal bands of Zebrin I (mabQ113-immunoreactive) Purkinje cells in the adult rat cerebellar cortex as seen from the anterior (a) and posterior (b). The band pattern is based upon the serial reconstruction of nine complete and five partial cerebella from sections cut in the horizontal plane and four complete reconstructions from sections cut coronally. Bands P1 + through P7+ are labelled. Hawkes and Leclerc (1987). 197
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J. Voogd, D. Jaarsma and E. Marani
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Fig. 137. Four frontal sections taken at different levels through the cerebellum of the adult Monodelphis immunoperoxidase stained by using antiZebrin II to reveal the compartmentalization of the vermis. The lobules are labelled with Roman numerals. The scale bar - 1,500/2m. Dot6 et al. (1990).
198
The cerebellum." chemoarchitecture and anatomy
Ch. I
opossum (Monodelphis domestica, Fig. 137). P l+ - P 3 + bands were identified in the anterior and posterior vermis. The precise identification of P2+ and P3+ was ambiguous in lobule V and different interpretations for the continuity of the reconstructed bands in the lobules VI-X were illustrated (Fig. 138). P4+ was located at the interface of vermis and hemisphere. It was located lateral to the area devoid of cortex at the border of lobule VII and the ansiform lobule, or was split in two Zebrin-positive bands, surrounding the cortexless area (compare Fig. 131 illustrating the 5'-N-positive band 15 in the cerebellum of the mouse) (Marani 1986). Zebrin I compartmentalization in Saimiri sciurus was studied by Leclerc et al. (1990). Both in the vermis and the hemispheres clusters of Zebrin I-immunoreactive Purkinje cells were separated by weakly stained Purkinje cell somata or unstained cells. Zebrinnegative bands, therefore, are less distinct than in rodents. P1 +, P2+ and P3+ bands are continuous from lobule to lobule and become narrower in the anterior lobe. P4+-P7+ bands were tentatively identified in the hemisphere, but not analysed in detail. A complementary histochemical zonation was detected for cytochrome oxidase, that was present in patches in the granular layer corresponding to the P - bands both in squirrel monkey and rat cerebellum. It is obvious from a comparison of the illustrations from the paper of Dor6 et al. (1990), showing the distribution of Zebrin I immunoreactivity in Purkinje cells and their axons and the zonation of AChE in monkey cerebellum, that the P2+ immunoreactivity in the anterior vermis corresponds to the X zone, and P 2 P2 P3
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Fig. 138. Three different interpretations of the adult Purkinje cell compartmentalization for Zebrin in the cerebellum of adult Monodelphis, illustrated in Fig. 136, are possible. A. The P2+ (dark grey) and P3 + (medium grey) bands are continuous and unbranched from lobule I to X. B. P2 + is continuous and unbranched, but a novel Zebrin II+ band is inserted between P1 and P2 in lobules VI to X (unshaded). C. P2+ bifurcates within lobule V to give two branches in lobules VI. Dor6 et al. (1990).
199
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Fig. 139. Drawings of three surface views of the mouse cerebellum, anterior, dorsal and posterior, showing the locations of the Zebrin+ bands of Purkinje cells. The Purkinje cell bands P1 +-P7 + are labelled in the dorsal view (for clarity, only the numerals have been used). Note that in the vermis of the posterior lobe the immunoreactive Purkinje cells form five to seven bands (posterior and dorsal views), whereas in lobules VII and VI all vermal Purkinje cells are immunoreactive (posterior and dorsal view). This pattern gradually changes in the anterior lobe to result in three to five very narrow immunoreactive bands (anterior view). In the hemispheres there are three major immunoreactive bands of Purkinje cells on either side (P5b +, P6 +, P7 +) plus two sub-bands in the paravermal area of the paramedian and ansiform lobules (P4b +, P5a +). Note too that the Purkinje cells are all Zebrin+ in the nodulus (lobule X, illustrated as indicated by arrows reflected out from the ventral surface of the cerebellum), the paraflocculus, and the flocculus. From Eisenman and Hawkes (1993).
200
The cerebellum." chemoarchitecture and anatomy
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to the B zone. Our preliminary observations on the distribution of Zebrin I-immunoreactivity in the cerebellum of macaques and cats suggested that all Purkinje cell somata in these species were Zebrin-positive and that compartmentalization was less distinct than in the reports on other mammalian species. Bands of P-path immunoreactive Purkinje cells alternate with zebrin II immunoreactive neurons in the cerebellum of the mouse (Leclerc et al., 1992) (Fig. 134). In the P3+ band in the anterior vermis, lobule VII, VIII and dorsal IX, the P4+ band in the lobules V and VIII and the P2+ band in dorsal lobule IX the two epitopes are colocalized. The B1 monoclonal antibody of Ingrain et al. (1985) also detects a subset of Purkinje cells in monkey cerebellum, but their distribution did not correspond to the distribution of Zebrin I in the squirrel monkey (Leclerc et al., 1990) or to AChE as reported by Hess and Voogd (1986). 6.1.4. The corticonuclear projection of the cerebellum of the rat: Correlations with zebrin-antigenic compartmentalization
The corticonuclear projection of the cerebellum of the rat recently was reviewed by Buisseret-Delmas and Angaut (1993) and Voogd (1995). It was studied with anterograde degeneration methods (Goodman et al., 1963; Haines and Koletar, 1979; Umetani et al., 1986) and anterograde tracing with [3H]leucine (Armstrong and Schild, 1978a,b). One interesting feature of these studies is that they document a projection from the hemisphere to the dorsolateral protuberance of the fastigial nucleus, that has never been observed in carnivores or primates. According to the experiments of Armstrong and Schild (1978b) this projection originates from the Crus II and the adjoining paramedian lobule, with smaller contributions of the Crus I and the copula pyramidis. Umetani et al. (1986) limited the projection to the dorsolateral protuberance to the cortex of the medial hemisphere of the lobules between the primary and prepyramidal fissures. The cortex of the copula pyramidis, caudal to the prepyramidal fissure was found to project to the medial part of the anterior interposed nucleus. The localization of Purkinje cells with circumscribed projections to single cerebellar nuclei was investigated with anterograde transport of WGA-HRP (Buisseret-Delmas, 1988a,b; Buisseret-Delmas and Angaut, 1993). Buisseret-Delmas (1988a,b; Fig. 141) distinguished A and B zones in the anterior vermis on the basis of their projection to the fastigial and the dorsal part of the lateral vestibular nucleus, and their afferent climbing fiber projections from the caudal medial and dorsal accessory olives. She distinguished the portion of the medial hemisphere between the primary and prepyramidal fissures, that projects to the dorsolateral protuberance, in the A zone as the lateral extension of the A zone (Fig. 142). The X zone was distinguished from the A zone by Buisseret-Delmas et al. (1993) and found to project to an area located at the junction of the fastigial and posterior interposed nuclei that they indicated as the 'interstitial cell groups'. C1, C2 and C3 zones projected to the interposed nucleus, and received their climbing fibers from the rostral half of the dorsal accessory olive (C1 and C3) and the rostral medial accessory olive (C2). C1 and C3 are interrupted in the Crus I and projected to medial and lateral portions of the interposed nucleus, the projection of C2 occupied its intermediate one third. Voogd's (1964, 1969) original definitions of the C zones, with C1 and C3 projecting to the anterior interposed nucleus, and C2 to the posterior interposed nucleus, therefore, were not retained in this study. Three D zones were identified by Buisseret-Delmas and Angaut (1989b) in the hemisphere of the cerebellum of the rat (Fig.141). The Do zone is unique for the rat, and 201
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Fig. 140. Computer drawing of the reconstruction of the Zebrin Purkinje cells bands in the unfolded adult C57/B6 mouse cerebellum. The drawing was from immunostained 40/~m thick coronal frozen sections. The continuity of the bands has been determined as best as possible. On the left and bottom are the scales in millimeters. The two axes have different magnifications. On the right are marked the approximate boundaries of the vermal lobules. The flocculus and paraflocculus are not illustrated. One place where the data are ambiguous is within lobule V-VI, where a large number of short bands more caudally are dramatically reduced to just three at the rostral limit. It is not clear whether the P2 + or P3 + bands extend through the anterior lobe vermis (see also Fig. 139). The reconstruction data from coronal sections were not suitable to resolve the issue, so the cerebellum has also been reconstructed from horizontal sections. The upper inset panel shows the data from such a reconstruction, equivalent to the region indicated by a rectangle on the main drawing (scale in millimeters). The preferred interpretation is that the P2 + compartment does not extend far into the anterior lobe vermis, and that the first lateral Zebrin+ band in lobules I-IV is continuous with P3 + (as indicated by continuous lines in the upper inset panel and as shown in the main drawing). The alternative hypothesis, that the first lateral Zebrin + band in lobules I-IV is continuous with P2 +, is shown schematically in the lower inset panel. Eisenman and Hawkes (1993). (
projects to the dorsolateral hump and receives a projection from the medial half of the ventral leaf of the principal olive. The Do zone is present in the anterior lobe and the lobulus simplex, Crus II and the paramedian lobule. It is interrupted at the level of the Crus I. The D1 and D 2 z o n e s occupy the lateral border of the hemisphere. D~ extends uninterruptedly from lobule III through the copula pyramidis. O 2 extends beyond the copula into the lateral parts of the vermal lobules IX and X and into the paraflocculus. The corticonuclear relations of the D1 and D 2 z o n e s of the rat are reversed with respect to their namesakes in the cat. D1 projects to the dorsal, magnocellular part of the lateral cerebellar nucleus; D2 to the ventral, parvicellular part of this nucleus. A detailed analysis is available for the projections of the pyramis and the copula pyramidis (Umetani and Tabuchi, 1988) (Fig. 153) and lobule VIIb with the rostral paramedian lobule (Umetani, 1989) (Fig. 152). The C1 zone, with a projection to the anterior interposed nucleus, was lacking in the paramedian lobule and wide in the medial cortex of the copula. C2 was present in both lobules and C3 was limited to the rostral half of the paramedian lobule. D1 (corresponding to Do of Buisseret-Delmas and Angaut, 1989b) projected to the dorsolateral hump and D 2 (corresponding to both their D1 and D 2 zones) to the lateral cerebellar nucleus. The topography of the C zones in the rat, therefore, corresponds to the situation in cat and monkey, with the exception of the projection of a zone in the medial hemisphere to the dorsolateral protuberance of the fastigial nucleus, the presence of an additional Do zone and the reversal in the corticonuclear projection of D1 and D 2. This correspondence, however, does not explain the pattern of Zebrin-positive and -negative zones in the same region. The projections of the cortex to the fastigial and vestibular nuclei were correlated with the Zebrin immunoreactive Purkinje cell zones, using double staining with an anti-zebrin antibody and cobalt-stabilization of the retrograde labelled Purkinje cells on the same section. Purkinje cells in the A and B zones were retrogradely labelled from injections of WGA-HRP in Deiters' nucleus (Voogd et al., 1991b). In the anterior lobe and the simple lobule they were located in the zebrin-negative P1- and the lateral P2- zones (Fig. 143, left). The labelled Purkinje cells in P2- (i.e. in the B zone) were bordered on their medial side by P2+. Zebrin-positive 'satellite' bands bordered the labelled Purkinje cells in the lateral P1- zone on their medial side. WGA-HRP injections at the junction of the fastigial and posterior interposed nucleus labelled Purkinje cells of P2+ and the satellite bands between PI+ and P2+ (Fig. 143, right). Injections of the dorsolateral 203
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protuberance of the fastigial nucleus labelled Purkinje cells located between the primary and prepyramidal fissures overlapping with and located in between the P4b+ and P5a+ zones (Fig. 144). Zebrin-positive and -negative zones in the anterior vermis, therefore, approximately correspond with the corticonuclear projection zones A, X and B that were known from studies in other species than the rat. The projection of the Zebrinpositive Purkinje cells of the X (P2+) zone to the junction of the fastigial and posterior interposed nucleus is in accordance with the tendency of Zebrin-positive Purkinje cells to project to the caudal pole of the fastigial nucleus (Hawkes and Leclerc, 1986). The P4b+, P4b- and P5a+ zones corresponded with the zone projecting to the dorsolateral protuberance. In the simple lobule this zone was located immediately lateral to the B zone. As a consequence the fused P4b+ and P5a+ zones continue, rostral to the primary fissure, as the P3+ zone of the anterior lobe. The topography of the C and D zones in the rat has not yet been correlated with the Zebrin pattern, but it seems likely that C1, C3 and D (Do and D2 zones of Buisseret-Delmas and Angaut, 1989b), projecting to the anterior interposed and the rostral dentate nucleus, will prove to correspond to Zebrinnegative zones and that the C2 and D1 zones, that project to the posterior interposed and the caudal dentate, will be Zebrin-positive. Figure 145 depicts a diagram illustrating this hypothesis.
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Fig. 143. Comparison between Purkinje cells of the A and B zone (open circles) retrogradely labelled from the vestibular nuclei (left side) and retrograde labelling of Purkinje cells of the X zone after an injection of WGA-HRP in the transitional region of the fastigial and posterior interposed nucleus the rat (right side). Purkinje cells of the X zone occupy the Zebrin-positive P2+ zone; labelled Purkinje cells of the lateral A zone and the B zone are located in the Zebrin-negative P1- and P2- zones. Graphical reconstructions of transverse sections double labelled for HRP reaction product and Zebrin I immunocytochemistry.COP = copula pyramidis; CrI and II = crus I and II of the paramedian lobule; PMD = paramedian lobule; S1 = simple lobule; I-X = lobules I-X. Voogd et al. (1991b). (,
6.1.5. The corticovestibular and corticonuclear projections of the flocculus and the caudal vermis. Correlations with cytochemical zones and compartments Larsell (1934) subdivided the cerebellum in a somesthetic corpus cerebelli and the vestibulocerebellum. The compartmentalization of the corpus cerebelli was considered in the previous sections. The vestibulocerebellum of Larsell consists of the flocculus and the nodulus, the caudalmost lobules of the folial chains of vermis and hemisphere. They are separated from the corpus cerebelli by the posterolateral fissure, one of the earliest fissures to appear during ontogeny. In primitive mammals a narrow band of cortex along the attachment of the roof plate of the fourth ventricle interconnect the nodule and the flocculus. The modular organization and the characteristic afferent and efferent connections of the vestibulocerebellum extend, beyond the posterolateral fissure into the ventral part of lobule IX and in the adjacent cortex of the ventral paraflocculus which, therefore, should be included in it. The distinction between the corpus cerebelli and the vestibulo-cerebellum is clearly revealed by calretinin. The rather uniform staining of granule cells and parallel fibers in the corpus cerebelli suddenly stops at the borders of dorsal and ventral lobule IX and of the paraflocculus with the flocculus. In the vestibulocerebellum the unipolar brush cells are heavily stained on a lightly stained background (Floris et al., 1994) (Fig. 146). Different zonally distributed Purkinje cell markers, such as Zebrin I (Hawkes and Leclerc, 1987), motilin, taurine (Chan-Palay, 1984) and somatostatin (Villar et al., 1989) occur more uniformly in the flocculus, and the paraflocculus. Most Purkinje cells of the flocculus and the paraflocculus are Zebrin-positive and no banding has been observed with immunocytochemical methods. Compartmentally distributed differences in fiber size were never observed in the flocculus of carnivores, but compartments can be delineated in the white matter of the flocculus in rabbits and in old and new world monkeys, both with a myelin (the HS.ggqvist) stain and AChE histochemistry. In the rabbit (Van der Steen et al., 1991, 1994; Tan et al. 1992, 1995a,b,c) and the monkey (Macaca fascicularis, Hess and Voogd, 1986; Voogd et al. 1987a,b) 5, respectively 4 compartments can be recognized in the flocculus (Figs 147, 148 and 149). The medialmost compartment 4 of the rabbit flocculus is narrow and located at the border of the middle cerebellar peduncle (Fig. 147a,d). Compartments 1 and 3 are relatively rich in AChE and fuse in the dorsal and caudal white matter of the flocculus. The compartment 2 is poor in AChE and contains large Purkinje cell fibers in the myelin-stained sections. The fifth, most lateral compartment is the caudal extension of the C2 compartment of the paraflocculus. The compartments 2-5 continue, across the posterolateral fissure into the paraflocculus. In the first folium of the ventral paraflocculus of the rabbit (folium p of Yamamoto and Shimoyama, 1977), the compartments 1 and 3 enclose the dorsal tip of compartment 2 (Fig. 147b,c). Four compartments 207
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The cerebellum." chemoarchitecture and anatomy
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Fig. 144. Localization of retrogradely labelled Purkinje cells (open circles) from an injection of WGA-HRP in the dorsolateral protuberance of the medial cerebellar nucleus (left) and of anterograde transport of Phaseolus vulgaris leucaglutinin (PhaL) in climbing fibers (stripes) from the medial portion of the MAO (tectorecipient area), with respect to bands of Zebrin-I labelled Purkinje cells in the rat. Reconstructions from sections double-stained for Zebrin and WGA-HRP or PhaL. Numbering of Zebrin-immuno-reactive Purkinje cell zones according to Hawkes and Leclerc (1987). COP = copula pyramidis; CrI(II)= crus I(II) of the ansiform lobule; PMD =paramedian lobule; SI = simple lobule; I-X = lobules I-X. Voogd et al. (199 lb). (
extend over the entire ventral paraflocculus of Larsell (1970) in old and new world monkeys. The most lateral compartment continues as the C2 compartment in the petrosal lobule (Fig. 148b,c), the medial three compartments do not extend beyond the narrow junction of the cortex of the ventral paraflocculus with the petrosal cortex. The narrow, medialmost compartment 4 of the rabbit flocculus was not recognized in the primate cerebellum. The compartments 1, 2 and 3 of the monkey flocculus, correspond to their namesakes in the rabbit. Fibers from the white matter of the flocculus and the adjoining folia of the ventral paraflocculus collect in a compact bundle: the floccular peduncle. The peduncle is applied to the ventral surface of the lateral parvocellular cerebellar nucleus and arches over the restiform body (Fig. 147c,f). Its fibers surround the ventral limb of the lateral cerebellar nucleus as a capsule in rabbit and cat. Medium sized cells of the dorsal group y are located between the fibers of the peduncle in these species. Smaller cells in a dense AChE-positive neuropil accumulate, as the ventral group y, between the peduncle and the cochlear nuclei with the restiform body and, more medially, at the entrance of the peduncle in the vestibular nuclei. In monkeys the group y is compact and situated as an enlongated mass in the floccular peduncle. More caudally, small AChE-positive cells of Langer's (1985) basal interstitial nucleus extend into the lateral compartments of the flocculus and in the AChE-positive raphes between them. Some fibers of the compartments 1 and 2 of the rabbit flocculus arch through the dentate nucleus, to join the floccular peduncle at the lateral border of the vestibular nuclei. These 'arciform' fibers have been found in cat, rat, rabbit and Galago but seem to be absent in primates (Langer et al., 1985b). The white matter compartments of the flocculus do not simply continue as components of the floccular peduncle, but a reorganization takes place, that directs Purkinje cell axons from the compartments 2 and 4 to the medial vestibular nucleus and of the compartments 1 and 3 to the superior vestibular nucleus. The C2 compartment does not contribute to the floccular peduncle, but leads its fibers towards the posterior interposed nucleus. In monkeys the equivalent of compartment 1 of the rabbit extends in the roof of the fourth ventricle, along the basal interstitial nucleus of the cerebellum (Fig. 148). The corticonuclear and corticovestibular projections of the flocculus were studied by Dow (1936), Bernard (1987) in rat, Voogd (1964), Angaut and Brodal (1967), Sato et al., 1982a and b) in cat, Dow (1938), Langer et al. (1985b) in primates and Yamamoto and Shimoyama (1977), Yamamoto (1978), De Zeeuw et al. (1994a) and Tan et al. (1995c) in rabbit. The differential projection of the Purkinje cell zones of the flocculus was discovered by Yamamoto and Shimoyama (1977). The precise correspondence of the corticovestibular and group y projections of the rabbit flocculus with the ACHEcompartmentalization of its white matter and its functional correlates were discussed by Van der Steen et al. (1994), De Zeeuw et al. (1994a) and Tan et al. (1995a,b) (Fig. 188). A projection of the flocculus to the nucleus prepositus hypoglossi has been dis209
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The cerebellum." chemoarchitecture and anatomy
Ch. I
Fig. 145. Diagram of the Zebrin-antigenic zones (left) and the corticonuclear projection (right) in the rat. The diagram of the corticonuclear projection is based upon data from Umetani et al. (1986); Umetani (1989), Bernard (1987); Buisseret-Delmas and Angaut (1989b, 1993) and Voogd et al. (1991b). There is a good correspondance between the corticonuclear projection zones A, X and B of the anterior vermis, and for the projection of the lateral extension of the A zone of Buisseret-Delmas (1988a) and the Zebrin pattern. For other zones this correspondance is conjectural or absent. The corticonuclear projection of the region between vermal lobule VIc and VII and the lateral A zone is not known. The corticonuclear projection zones are indicated with the same symbols as their target nuclei in the bottom diagram of the cerebellar nuclei. Regions projecting to the vestibular nuclei (a lateral strip of the A zone, the B zone and the caudal vermis) are indicated with open circles. 1-7 - Zebrin antigenic zones P1-P7" A - zone A; A~ - lateral extension of the zone of Buisseret-Delmas (1988a); B - zone B; C1-3 - zones C1-C3; CrI - crus I of the ansiform lobule; CrII - crus I! of the ansiform lobule; D - zone D; Do =one Do of Buisseret-Delmas and Angaut (1989); DLH - dorsolateral hump; DLP = dorsolateral protuberance of the medial nucleus; DMC - dorsomedial crest; FLO - flocculus; IA - anterior interposed nucleus; I P - posterior interposed nucleus; L - lateral cerebellar nucleus; MM = medial part of medial nucleus; N C - caudomedial part of the medial nucleus; P F L - paraflocculus; PMD = paramedian lobule; SI - simple lopule; tM/IP - transitional region of the medial nucleus and posterior interposed nucleus (interstitial cell groups of Buisseret-Delmas et al. 1993); X - zone X; I-X- lobules I-X. (
chemistry have been p r o p o s e d for the caudal vermis of the cat (Voogd, 1964, 1969) and the m o n k e y (Voogd et al., 1987a,b, Tan et al., 1995b). The caudal vermis projects to the fastigial nucleus, but also to the (posterior) interposed nucleus (Bigar6, 1980, cat; A r m s t r o n g and Schild, 1978a; Bernard, 1987; U m e t a n i and Tabuchi, 1988; Tabuchi et al., 1989, rat) a n d the ventral, parvocellular p o r t i o n o f the lateral cerebellar nucleus (Van R o s s u m , 1969; Wylie et al., 1994, rabbit; Haines, 1977a Galago; Bernard, 1987; Tabuchi et al., 1989, rat) a n d the vestibular nuclei including the g r o u p y. L o b u l e VIII of the caudal vermis gives rise to a small projection to the dorsal part of the lateral vestibular nucleus (i.e. Deiters' nucleus), but other corticovestibular connections o f the caudal vermis and the flocculus avoid Deiters' nucleus and terminate in other subdivisions of the vestibular nuclei. Corticovestibular fibers from the lobules X and IX of the caudal vermis were studied by D o w (1936), B e r n a r d (1987), Tabuchi et al. (1989) in rat, Voogd (1964), A n g a u t and Brodal (1967) in cat and D o w (1938), Haines (1977a) in primates. A detailed study of the corticonuclear and corticovestibular projections was m a d e of the ventral face of lobule X of the rabbit cerebellum by Wylie et al. (1994). Their findings are s u m m a r i z e d in Fig. 150 and can be correlated with the c o m p a r t m e n t a l A C h E - s u b d i v i s i o n of the white m a t t e r of lobule X by Tan et al. (1995b) and the a n a t o m i c a l and electrophysiological analysis of the olivocerebellar projection to this lobule by K a t a y a m a and N i s i m a r u (1988), B a l a b a n and H e n r y (1988) and K a n o et al. (1990, 1991). Corticovestibular fibers f r o m the flocculus and the lobules X and IX of the caudal vermis terminate in roughly c o m p l e m e n t a r y areas of the vestibular nuclei (Fig. 151) (Dow, 1936, 1938; Voogd, 1964; A n g a u t a n d Brodal, 1967; Haines, 1977a; L a n g e r et al. 1985b; Bernard, 1987). The caudal vermis o f the cat usually was considered to belong to the A zone, that projects to the fastigial nucleus. A c c o r d i n g to Bigar6 (1980) (Fig. 123) and Voogd and Bigar6 (1980) the entire lobule VII and the medial two-thirds of the lobules VIII, IX and X are connected with this nucleus. Courville and D i a k e w (1976) d e m o n s t r a t e d the sequential representation of the lobules VI-VIII in the fastigial nucleus of the cat with lobule VII projecting to the tail of the nucleus and lobules VI and VIII to m o r e r o s t r o d o r s a l and ventral parts respectively. A similar connection o f lobule VII to the
211
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tail of the fastigial nucleus was demonstrated by Yamada and Noda (1987) for the macaque monkey. Purkinje cells in the medial two-thirds of lobule IX of the cat also project to the medial and descending vestibular nuclei; those destined for the descending vestibular nucleus are concentrated near the midline and at the lateral border of the A zone (Matsushita and Wang, 1986). The lateral one-third of lobule IX is connected with the posterior interposed nucleus (Bigar6, 1980). A complicated pattern of Purkinje cell zones and patches was reported for the organization of the projection of lobule X to the superior, medial and descending vestibular nuclei in the cat (Shojaku et al., 1987). The zonal organization of the efferent connections of the caudal vermis in the rabbit is quite complex, with discrete zones in the lobules IX and X projecting to the fastigial, descending, superior and medial vestibular nuclei, and lateral zones connected to the interposed and different subdivisions of the lateral cerebellar nucleus (van Rossum, 212
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213
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1969). Some sort of zonal organization in the corticovestibular projection of these lobules in the rabbit also was reported by Balaban (1984), Epema et al. (1985) and Epema (1990). 214
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More detailed, but still incomplete accounts of the corticonuclear projection of the caudal vermis are available for the rat. The terminal fields of the different lobules in the fastigial nucleus of the rat are organized in a circumferential manner (Armstrong et al., 1978a; Haines and Koletar, 1979) with more overlap between the lobules than has been reported for the cat (Courville and Diakew, 1976). PS.fillysaho et al. (1990) analysed the projections of the lobules VI-VIII to the fastigial nucleus in more detail in the rat. The terminal field of lobule Via is located in the middle subdivision of the fastigial nucleus, lobules VIb,c, VII and VII project both to the caudomedial and middle subdivisions, with the terminal field of lobule VIII reaching most rostrally. Terminations of these lobules in the dorsolateral protuberance are scarce or absent. A subdivision of lobule VII into a medial region, projecting to the caudomedial subdivision of the fastigial nucleus and of a lateral region projecting to the middle subdivision also was proposed by Umetani (1989) (Fig. 152). This medial region may correspond to the tecto-olivorecipient zone of Akaike (1986b, 1992) (Fig. 184). The lateral zone of lobule VII, that separates the tecto-olivo-recipient zone from the lateral extension of the A zone, with its projection to the dorsolateral protuberance of the fastigial nucleus may be identical to the X zone. 215
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Fig. 149. Diagrams of cerebellar cortex, showing the zonal configuration of the paraflocculus and the flocculus in rabbit, monkey and cat, based upon the compartmental subdivision of the paraflocculus and the flocculus in monkey and rabbit (Figs 147 and 148) and the olivocerebellar projection to the flocculus in cat (Voogd, 1964; Gerrits and Voogd, 1982) and rabbit (Tan et al., 1995a,b). Ca, D1 and D2 zones can be distinguished in the dorsal paraflocculus and the petrosal lobule of the monkey and in the dorsal paraflocculus and the rostral segment of the ventral paraflocculus of the cat. The C2 zone and a single D zone were distinguished in the paraflocculus (the petrosal lobule) of the rabbit cerebellum. The C2 zone, that extends over the entire cerebellum, occupies the lateral part of the flocculus in all species. Four zones 1-4 are present in the flocculus of the rabbit, three of these (1-3) are present in the primate flocculus. Of the 6 floccular climbing fiber (f) zones in the cat, fl corresponds to zone 4 of the rabbit, f2+3 to zone 3, f4 to zone 2 and f5+6 to zone 1. The zonal pattern of the flocculus proceeds for some distance on the paraflocculus. This transitional segment corresponds to folium p of the rabbit, to the ventral paraflocculus of the monkey and to the medial extension of the ventral paraflocculus (ME) of the cat. The lateral border of the hemisphere (heavy line) continues in the medial border of the flocculus. The inner surface of the cortex is shaded. Asterisks indicate the area without cortex in the center of the ansiform lobule. 1-4 = floccular zones 1-4 of rabbit and monkey; ANS = ansiform lobule; A N T anterior lobe; C 2 -~ zone C2; D1,2 = zones dl,2ME = medial extension of the ventral paraflocculus fl-6 = climbing fiber zones 1-6 of the flocculus of the cat - 9 F I P L - posterolateral fissure; FLO - flocculus; FP folium P (rabbit); P F L D = dorsal paraflocculus; PFLV = ventral paraflocculus; P M D - paramedian lobule; POST - posterior lobe.
Two strips of Purkinje cells were distinguished by Umetani and Tabuchi (1988) in the vermis of lobule VIII (Fig. 153). The medial zone projected to both subdivisions of the fastigial nucleus, the second zone to the posterior interposed and lateral vestibular nuclei. This region, therefore, may include equivalents of the X and B zones of the anterior lobe. It was bordered on its lateral side by the C1 zone. The zonal organization of the lobules IX and X was analysed by Bernard (1987) and Tabuchi et al. (1989) in the rat. According to Bernard (1987) the zonal arrangement in lobule IX is very similar to the pattern in cat and rabbit with a medial zone projecting to the fastigial nucleus and a middle zone connected with medial portions of the interposed nuclei, corresponding with the posterior interposed nucleus. The most lateral zone 216
The cerebellum." chemoarchitecture and anatomy
Ch.I
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dorsal nodulus ventral, nodutus
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Fig. 150. Diagram of the afferent olivocerebellar projection according to Katayama and Nisimaru (1988) and the efferent projection of the zones of the nodulus to the vestibular and cerebellar nuclei according to Wylie et al. (1994) in the rabbit, fl = group fl of the medial accessory olive; DC = dorsal cap of Kooy; F = fastigial nucleus; IP = posterior interposed nucleus; MV = medial vestibular nucleus; P cell = Purkinje cell; SV = superior vestibular nucleus; VLO = ventrolateral outgrowth; I-VI = zones of rabbit nodulus, numbered according to Katayama and Nisimaru (1988). was connected with the ventral, parvicellular portion of the lateral cerebellar nucleus. The medial two zones shifted to a more lateral position in lobule X, where an additional zone, projecting to the medial vestibular nucleus, took up the most medial position. These zones also project in a differential manner, to the superior and descending vestibular nuclei and to the group y. The most lateral zone, that was connected with the lateral cerebellar nucleus, extended in lobule Xa and did not project to the vestibular nuclei. Although the Zebrin pattern in the caudal vermis is quite distinct and some correlations with the corticonuclear projection zones are obvious (Fig. 145), no precise comparisons with the Zebrin pattern have been made. Our knowledge of the cortico-vestibular and nuclear projection of the caudal vermis, therefore, is still deficient. Progress can be expected from anatomical and electrophysiological studies using the Zebrin pattern as a reference. 6.2. R E G I O N A L D I F F E R E N C E S IN T H E D E V E L O P M E N T O F T H E CEREBELLUM The development of the cerebellar cortex, the cerebellar nuclei and the precerebellar nuclei has been documented and reviewed in a series of papers on the rat by Altman (1975a,b,c) and Altman and Bayer (1985a,b, 1987a,b). Here we shall be concerned with 217
Ch. I
J. Voogd, D. Jaarsma and E. Marani
A
B
C
Flocculus
Nodulus
Uvula
Fig. 151. Diagram of the corticovestibular projections from flocculus, nodulus and uvula in Galago. Note complementarity between the projections of the flocculus and the caudal vermis, lvn = lateral vestibular nucleus; mvn = medial vestibular nucleus; spvn = spinal vestibular nucleus; svn= superior vestibular nucleus. Haines (1977a).
some aspects of regional differentiation in the development of the Purkinje cells of the cerebellar cortex. Several authors noticed that developing Purkinje cells are clustered into a number of parasagittal zones during early stages of cerebellar development, prior to the stage when the first fissures make their appearance. Purkinje cells and the cells of the cerebellar nuclei are generated in the ventricular layer (Jakob, 1928; Miale and Sidman, 1961), subsequently they migrate to the meningeal surface of the cerebellum where they settle in the cortical plate, deep to the external granular layer. Clustering of Purkinje cells in the cortical plate has been observed by a number of authors in mammals (Korneliussen, 1967, cetacea; Brown, 1985b; Brown et al., 1986, cat; Korneliussen, 1968b; Altman and Bayer, 1985b, rat; Hochstetter, 1929; Korneliussen, 1968c; Maat, 1978, 1981; Marani and Mai, 1992, man; Marani et al., 1986, rabbit; Kappel, 1981, monkey) and birds (Feirabend et al., 1976; Feirabend, 1983). Cell strands interconnect the Purkinje cell clusters with the cerebellar nuclei. The pattern that evolves from the position of the clusters in the cortical plate and their corticonuclear relations, is rather similar to the adult pattern of longitudinal corticonuclear projection zones (Korneliussen, 1967, 1968b; Kappel, 1981; Feirabend, 1983; Marani, 1986; Marani et al., 1986). The borders between the Purkinje cell clusters become indistinct when the fissures begin to develop and the 218
The cerebellum." chemoarchitecture and anatomy
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Fig. 152. Diagrams showing the topographic pattern of the projections from the various mediolateral levels of the tuber vermis (lobule VII) and the paramedian lobule to the cerebellar nuclear complex in the rat. A. Schematic diagram of the posterior surface of the cerebellum and subdivision of the tuber vermis and paramedian lobule, based on the topography of their projections. B. Schematic sagittal diagrams of the nuclear complex showing the terminal fields which receive projections from the individual subdivisions of the tuber vermis and paramedian lobule. AIN = anterior interposed nucleus; cm = caudomedial sub-division of the medial nucleus; Cop. pyr = copula pyramidis; D L H = dorsolateral hump; DLP = dorsolateral protuberance of the medial nucleus; LN - lateral cerebellar nucleus; LVN = lateral vestibular nucleus; m = medial nucleus; PIN = posterior interposed nucleus; Pml = paramedian lobule. Umetani (1989).
internal granular layer appears. Jacob (1928) and Hayashi (1924), were among the first to detect regional differences in corticogenesis in the human cerebellum (Fig. 154) and to correlate their corticogenetic zones with the corticonuclear projection to the different cerebellar nuclei (see Section 6.). A large time gap separates the stage when the borders between the clusters disappear, from the moment when the adult connections of the Purkinje cell have become established. Several methods have been employed to bridge this gap and to trace the developmental Purkinje cell patterns into adulthood. Altman and Bayer (1985b) in the rat, Brown (1985b) in the cat and Feirabend et al. (1985) in the chicken, using tritiated thymidine autoradiography, found evidence for differences in the time of birth between populations of Purkinje cells with a zonal distribution. According to Feirabend et al. (1985) some of the Purkinje cells of the lateral cerebellum and the cells of the lateral part of the central nuclei of the chicken are generated later than medially located cells. Zonal [3H]thymidine labelling of the Purkinje cells can be traced till after hatching. Several markers for adult Purkinje cells have been used to trace back their origin. Wassef and Sotelo (1984) studied the expression of cyclic GMP-dependent protein kinase (cGK) immunoreactivity during development of the rat (Fig. 155). They found a transient heterogeneity of the Purkinje cells for an antibody against cyclic GMP-dependent 219
Ch. I
J. Voogd, D. Jaarsma and E. Marani Tuber vermis
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ula
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Fig. 153. Diagrams showing showing the projections from the various mediolateral projection-zones of the pyramis (lobule VIII) and the copula pyramidis to the cerebellar and vestibular nuclear complexes in the rat. A. Schematic diagram of the posterior surface of the cerebellum. The subdivision of the pyramis and copula are based on the topography of their efferent projections. B. Schematic sagittal diagrams of the cerebellar and vestibular nuclei showing the terminal areas which receive projections from the individual subdivisions of pyramis and copula pyramidis. Abbreviations: see Fig. 152. Umetani and Tabuchi (1988).
protein kinase (cGK) (De Camilli et al., 1984). Some clusters of Purkinje cells and their axons react with the antibody, while others are still negative. Some clusters could be recognized before their cells had completed their migration to the surface. Shortly after birth all Purkinje cells become cGK positive. Similar observations were made by Wassef et al. (1985) with two other Purkinje cell specific antibodies against calbindin-D28k (Legrand et al., 1983), and the Purkinje cell specific glycoprotein (PSG) (Langley et al., 1982) and by VanDaele et al. (1991) and Smeyne et al. (1991) for the transient zonal patterns in the development of the Purkinje cell-specific marker L7 (see Section 3.1.8.). Each antibody gave a different mosaic of positive and negative Purkinje cell clusters and this chemical heterogeneity disappeared shortly after birth, when all Purkinje cells became positive for the different markers. Some degree of heterogeneity is also present during the development of immunoreactivity for anti-parvalbumin in Purkinje cells of birds (Braun et al., 1986), but the early stages of development were not included in this study. Wassef et al. (1987) and Edwards et al. (1994) noticed that the surviving Purkinje cells in mice with mutations affecting their postnatal survival, are arranged in sagittal bands. This differential sensitivity of these Purkinje cells may be related to their chemical heterogeneity. Immunoreactivity for Zebrin (Hawkes and Leclerc, 1986) is not present before birth (Leclerc et al., 1988) and the reactivity for the enzyme 5'-nucleotidase, which is distributed in a similar zonal pattern in the molecular layer of the mouse also appears postnatally (Hess and Hess, 1986). The expression of the L7 gene in mouse Purkinje cells displays a similar develop220
The cerebellum: chemoarchitecture and anatomy
Ch. I
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caudal vermis Fig. 154. Regional variations in cerebellar corticogenesis. A. Diagram of the cerebellum of a two month human embryo. Unlabelled areas, small dots, large dots and black indicate successively more advanced stages in histogenesis. Redrawn from Jakob (1928). B. Subdivision of the mammalian cerebellum according to Jakob and }tayashi. Redrawn from Jakob (1928). A = medial corticogenetic zone of the vermis; B - lateral corticogenetic zone of the anterior vermis or pars intermedia; FLO = flocculus.
mental heterogeneity. It first appears in paired bands in the vermis and the flocculus, later in clusters in the hemispheres. Ultimately it is present in all Purkinje cells (Smeyne et al. (1991)). Oberdick et al. (1993) slowed down this process by manipulation of the promotor region (Fig. 156). The transient expression of L7 in Purkinje cell bands with high and low levels of expression, as shown in whole mounts of the mouse cerebellum, is very similar if not identical to the Zebrin pattern as documented for adult mice by Eisenman and Hawkes (1993) (Fig. 139). It is not known whether the developmental patterns for other general Purkinje cell markers also resemble the Zebrin pattern. Moreover, it was demonstrated by Oberdick et al. (1993) that the L7 pattern also develops in vitro and, therefore, is independent of the ingrowth or the presence of extracerebellar afferents. Similar observations were made by Leclerc et al. (1988) for the development of the Zebrin pattern in rats, after section of the extracerebellar afferents. These results strongly advocate a primary role for the Purkinje cells in the development of zonal patterns (Wassef et al., 1992c). Few signs have been noticed of longitudinal subdivisions in the development or in the adult granular layer. The zonal distribution of AChE in the development of this layer was discussed by Marani (1986). Monoclonal antibodies against Stage Specific Embryonic Antigen-1 (SSEA-1), also known as the X-hapten, Lacto-N-Fucopentax III or FAL (Fucosyl-N-Acetyl Lactosamine), showed a longitudinal subdivision in the mitotic external granular layer of the rabbit cerebellum. This pattern was present from embryological day E8-E9 till postnatal day P15-16 and consisted of alternating positive and negative strips. The positive staining for SSEA-1 antibodies is exclusively present at the cell membrane of cells in the external granular layer (Marani and Tetteroo, 1983, Marani, 221
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The cerebellum." chemoarchitecture and anatomy
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Fig. 155. Staining with cyclic 3',5'-guanosine monophosphate dependent protein kinase (cGK) antisera of sections of cerebellum of rat fetuses of embryonic day E17, E19 and a neonate (PO) cut in the frontal plane. A,B. E 17. Cluster I is composed of a medial sheet (arrow in B) lying against the germinative neuroepithelium. Close to the midline this sheet bends dorsally and reaches the cortex. The central cluster (CC) is located at the center of the hemicerebellum. C. E 19. In this section four of the five cGK-positive clusters I-V are present. The labelled fiber-like material, which tails the labelled clusters (* and o) indicates the migration pathw.ays followed by the Purkinje cells of the clusters I and III from the subventricular plate and the central cluster at E17 to their present, superficial position. D. PO rat pup. Fiber bundles linking the clusters I and III with the cerebellar nuclei intersect at the former position of the central cluster. It is suggested that the bundle from cluster III (*) terminates in the dorsolateral protuberance. In the adult this connection corresponds to the projection of the lateral extension of the A zone of Buisseret-Delmas (1988a, compare Figs. 142 and 144). Bar in A - 100 ~m, in B, C and D = 500 ~tm. Wassef and Sotelo (1984). (
1986; Marani et al., 1986). These results were confirmed with several antibodies against SSEA-1. In dissociated rabbit cerebellum the only cell type that was found to express this antigen was the granule cell (Marani and Tetteroo, 1983, Marani et al., 1983, 1986). These results demonstrate the presence of a longitudinal pattern in an intrinsic mitotic
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Fig. 156. Effects of promotor gene truncation on L7-1acZ banding pattern in mice. Cerebella were dissected free from the rest of the brain and stained in whole mount. Cerebella are viewed from posterior (POST) and anterior (ANY). Cerebella were taken from postnatal day 11 animals carrying 4 kb (top row), 500 bp (middle row) and 350 bp (bottom row) promoter constructs. The patterns are very similar to the Zebrin pattern in mouse cerebellum (compare Fig.139). Expression of L7 is absent or low in P1 +, P3 +, P5 + (indicated with PIN in bottom panels) and P7 + in the 500 and 350 bp constructs. In P4+ there is a strong expression of L7 in the 500 bp construct, and a weak expression in the 350 bp construct. Reversed levels of expression are observed for the region of P4b + and P5a +. In the 500 bp construct they are weakly stained (but the P4b + and P5a + bands are visible as separate strips), in the 350 bp construct there is a strong expression of L7 over the entire area of these bands indicated with FN in lower pannel). Oberdick et al. (1993), interpretations by the authors of this chapter.
223
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J. Voogd, D. Jaarsma and E. Marani
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Ch. I
Fig. 157. Configuration of the inferior olive and organization of the olivocerebellar projection in the cat. Olivary subnuclei and their projection areas in the cerebellum and the cerebellar nuclei (upper diagram) are indicated with the same symbols. The unfolded inferior olive is illustrated in the bottom diagram. 1-4 = in diagram of the cerebellum: lobules 1-4 of Bolk; in diagram of the inferior olive: projections to lobules 1-4;fl = group beta; D = dorsal accessory olive; d.cap = dorsal cap; d.1. = dorsal leaf of the principal olive; dm.c.col. = dorsomedial cell column; 1= lateral; lob a = lobulus a of Bolk (X of Larsell); lob b = lobulus b of Bolk (IX of Larsell); lob c l = lobulus c l of Bolk (VIII of Larsell); lob c = lobulus c of Bolk (VI + VII of Larsell); lob.simpl. - lobulus simplex; M = medial accessory olive; m = medial; N.fast = fastigial nucleus; N.int = interposed nucleus; N.lat = lateral cerebellar nucleus; v.1. = ventral leaf of the principal olive; v.l.o. = ventrolateral outgrowth. Brodal (1940). (
layer of the cerebellum which may be related to the establishment of transient contacts between ingrowing afferent fibers or Purkinje cell dendrites and external granule cells. 6.3. T H E O R G A N I Z A T I O N O F T H E O L I V O C E R E B E L L A R P R O J E C T I O N The olivocerebellar projection has been studied mainly in the cat (see Brodal and Kawamura, 1980 and Voogd, 1982 for reviews). Fewer data are available for primates and sub-primates (Brodal and Brodal, 1981, 1982; Whitworth et al., 1983; Whitworth and Haines, 1986b). A more complete picture of the olivocerebellar projection in the rat is emerging (Buisseret-Delmas and Angaut, 1993; Ruigrok and Cella, 1995; Voogd, 1995). The evidence on the role of excitatory aminoacids as the transmitter in the olivocerebellar pathway was considered in Section 3.2.2. It was concluded that glutamate is the most likely transmitter of the climbing fibers. The configuration and ultrastructure of the inferior olive, the organization of the afferent connections of the olive, the olivocerebellar projection and the topographical distribution of peptidergic climbing fibers will be reviewed in the next sections.
6.3.1. Configuration and ultrastructure of the inferior olive The morphology and the subdivision of the inferior olive have been described in the comparative anatomical studies of Kooy (1917), Mar6schal (1934) and Whitworth and Haines (1986a). Brodal's (1940) subdivision of the inferior olive in the cat and his mode of representation of the olive as imagined unfolded in one plane (Fig. 157) have become generally accepted. Dorsal accessory (DAO), medial accessory (MAO) and principal (PO) subnuclei can be distinguished in most mammalian species (Figs 158 and 159). The dorsomedial subdivision of the caudal M A O was indicated as group beta by Brodal (1940). Three parallel, longitudinal cell columns, indicated from laterally to medially as a, b and c (c is the equivalent to the group beta) were distinguished in the caudal M A O of the macaque monkey (Bowman and Sladek, 1973). F o u r columns (a, b, c and beta) were delineated in the caudal M A O of the rat (Gwyn et al., 1977). This apparent discrepancy was solved by Frankfurter et al. (1977) and Ikeda et al. (1989), who showed that subnucleus b in squirrel and macaque monkeys can be subdivided into medial and lateral parts and that only the medialmost region of this subnucleus is connected with the oculomotor vermis (lobule VII) and, therefore, corresponds with subnucleus c in the rat (Hess, 1982b; Akaike, 1992). The rostral half of the M A O sometimes is indicated as the rostral extension of subnucleus a, but this does not serve a useful purpose, because its connections differ 225
Ch. I
J. Voogd, D. Jaarsma and E. Marani
substantially from the caudal half of the MAO. The dorsomedial cell column (DMCC) of the cat is located dorsomedial to the rostral half of the medial accessory olive. Bowman and Sladek (1973) identified their cell group g, which is connected to the medial tip of the ventral leaf of the PO, as the DMCC in the macaque monkey. The DMCC of the rat has been identified as a similar group, connected with the ventral leaf of the PO (Gwyn et al., 1977). However, since the DMCC is part of the MAO and forms a rostral continuation of the group beta in most species (Whitworth and Haines, 1986a),
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226
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Diagrams of the inferior olivary complex of the rhesus monkey. Dorsal accessory olive is shown in black. Medial accessory olive and attached groups (nucleus fl and dorsomedial cell column) are hatched. Principle olive, dorsal cap, and ventrolateral outgrowth are left white. Subnuclei a, b, and c of the medial accessory olive according to the nomenclature of Bowman and Sladek (1973). A series of drawings of 13 equally spaced transverse sections through the olive in the upper panel is represented from rostrally (I) to caudally (XIII). In the lower panel the inferior olive is represented as imagined unfolded (see diagram at bottom of the Figure). In the drawings of the sections showing the principal olive, borders (broken lines) are indicated between the dorsal, lateral, and ventral lamella in accordance with Bowman and Sladek (1973). The solid line at the lateral convexity of the principal olive corresponds to the vertical line in the diagram of the unfolded olive, and is used for orientation. In this diagram broken lines in the medial accessory olive refer to borders betwcen regions which are not clearly separated. In the principal olive broken lines indicate the arbitrary separations between dorsal, lateral and ventral lamella shown in the drawings of transverse sections. The dorsomedial cell column is hidden behind the medial accessory olive (dotted outlines). Brodal and Brodal (1981). Fig. 159.
227
Ch. I
J. Voogd, D. Jaarsma and E. Marani
another cell group positioned directly dorsomedial to the MAO, has been identified as the DMCC in the rat (Azizi and Woodward, 1987; Bernard, 1987; Ruigrok and Voogd, 1990). Rostrally the DMCC of both sides seem to merge. The cell group connected to rostral leaf of the PO of the rat was indicated as the dorsomedial group (DM) (Fig. 158). A dorsal and a ventral leaf usually are distinguished in the PO: in the cat the ventral leaf is continuous with the medial pole of the dorsal accessory olive. In other mammals the DAO is continuous with the dorsal leaf and the ventral leaf ends as the DMCC in macaque monkeys and at the DM in rats. The ventral leaf of the PO caudally tapers into the ventrolateral outgrowth (VLO) that continues as the dorsal cap (DC) of Kooy (1917), located dorsal to the group beta. In dorsal view the DAO of the inferior olive of the cat and the monkey is boomerangshaped, with a narrow, medially directed, caudal tail. In the rat the caudal pole of the DAO consists of a dorsal and a ventral fold, that are continuous medially. The dorsal fold is only present at caudal levels, the ventral fold extends to the rostral tip of the DAO (Azizi and Woodward, 1987) (Fig. 158). Ovoid perikarya in Nissl-stained sections of the inferior olive of the cat are dispersed in clusters comprising up to eight neurons (Sotelo et al., 1974). The dendrites of typical Golgi-impregnated olivary neurons recurve towards the cell body (Fig. 160). This type of neuron, with a compact dendritic arbor, is the main cell type of the principal olive and the rostral pole of the MAO. Other subdivisions of the inferior olive contain a mixture of compact neurons and neurons with long, unramified dendrites. The latter type of neuron was mainly found in the caudal MAO, group beta and the DC (Scheibel and Scheibel, 1955; Scheibel et al., 1956; Ruigrok et al., 1990). Dendrites of both types of neurons give rise to long and thin branching spines (Bowman and King, 1973; Sotelo et al., 1974; Gwyn et al., 1977). The ultrastructure of the inferior olive was reviewed by De Zeeuw (1990) (Fig. 161). Glomeruli are characteristic features of the olivary neuropil (Nemecek and Wolff, 1969; Bowman and King, 1973; Sotelo et al., 1974; King et al. 1975; King, 1976; Gwyn et al., 1977; Rutherford and Gwyn, 1980; Bozhilova and Ovtscharoff, 1979). Glomeruli contain a core of dendritic spines contacted by axon terminals and surrounded by a glial capsule. On average, the glomeruli contain spines derived from six different neurons. Sometimes spines of the initial axonal segment are incorporated together with the dendritic spines in the same glomerulus (De Zeeuw et al., 1990a,b). Small gap junctions, with a heptalaminar structure including a 2 nm wide cellular space, were observed between spines in and outside glomeruli (Sotelo et al., 1974, 1986). De Zeeuw et al. (1990a) excluded the presence of gap junctions between elements of the same neuron. The importance of dendrodendritic coupling is further supported by the presence of dendritic lamellar bodies that can be associated with dendrodendritic gap junctions. These organelles are ubiquitously distributed in all olivary subdivisions and their density is higher in the olive than in any other brain area (De Zeeuw et al., 1995). Another form of aggregation of dendrites of olivary neurons is the dendritic thicket (Sotelo et al., 1974; 1986, Molinari, 1987; De Zeeuw et al., 1993). Thickets are formed by several dendrites in direct apposition with each other, but without any dendrodendritic membrane specializations. The extent of the electrotonic coupling of olivary neurons was studied by injecting lucifer yellow into single cells in slices of the guinea pig brain stem. Transfer of lucifer yellow via the gap junctions between dendrites, resulted in the labelling of aggregates consisting of up to 5 cells (Bernardo and Foster, 1986). The maximal spatial extent of electrotonic coupling in the inferior olive has not been determined, but may extend 228
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Fig. 161. GABAergic terminals derived from the cerebellar nuclei innervate dendritic spines (asterisks) in the glomeruli of the principal olive and medial accessory olive in the cat. The double labelled terminals can be recognized by the 15 nm gold particles (GABA) and the reaction products of the anterogradely transported WGA-HRP (large arrows). Note in B and C that cerebellar GABAergic terminals are directly apposed to dendritic spines coupled by gap junctions (small arrows). Open arrows indicate symmetric synapses. Scale bar in A = 0.31 r in B = 0.18 r in C = 0.35 r De Zeeuw et al. (1989b).
230
The cerebellum." chemoarchitecture and anatomy
Ch.I
beyond the classical morphological borders of the neuropil of the subdivisions of the inferior olive. De Zeeuw et al. (not yet published) demonstrated that synchrony between olivary neurons that project to the left and right Crus IIa of the rat in part can be explained by direct coupling through cells in the DMCC that bridge the left and right rostral DAO. Synchronization of activity in olivary neurons derived from different subdivisions is also supported by studies from Sj61und et al. (1980) and Lang et al. (1995) who induced a hypersynchronization of the climbing fiber discharge in the projections of particular olivary subnuclei by the administration of harmalin. Three types of axon terminals were recognized in the neuropil of the olive. It was found in aldehyde-fixed material postfixed in osmium that about half of the terminals contained spherical vesicles and were associated with asymmetrical synapses (Bowman and King, 1973; Mizuno et al., 1974; Sotelo et al., 1974; Gwyn et al., 1977; Rutherford and Gwyn, 1980). A second type contains pleomorphic vesicles and is associated with symmetrical synapses. A third type of terminal contains dense core vesicles. Most of the terminals that can be double labelled with an antegrade tracer from the cerebellar nuclei and an antibody against a conjugate of GABA belong to the second type, with pleomorphic vesicles and asymmetrical synapses (Angaut and Sotelo, 1989; De Zeeuw et al., 1988, 1989a and b, 1992, 1994b, Fig. 161). At present the cerebellar and vestibular nuclei, the nucleus prepositus hypoglossi, the parasolitary nucleus and the cuneate nucleus are the only known sources for this type of GABAergic terminal, and most of these terminals disappear after lesions of these nuclei (Nelson and Mugnaini, 1985; Nelson et al., 1986; Fredette and Mugnaini, 1991; De Zeeuw et al., 1993). Less frequently, GABAergic terminals with so-called crest-synapses can be observed (De Zeeuw et al., 1994b). These terminals occur relatively often in the DC and VLO and are derived from the area of group y and the ventral dentate nucleus (Fig. 162). Two other populations of GABA-containing terminals exist. One minor population contains clear, oval vesicles and sometimes was found apposed to the perikaryon. It was infrequently labelled from the cerebellum (De Zeeuw et al., 1989b). GABA-like immunoreactivity was also found in certain terminals of the granular type, that contained a large number of dense core vesicles. The granular terminals rarely made synaptic contacts. They could not be labelled from the cerebellum and they were always found outside the glomeruli, sometimes apposed to somata. The same type of granular terminal was labelled in the inferior olive with [3H]serotonin (Wiklund et al., 1981a) or antibodies to conjugates of serotonin (King et al., 1984). It seems likely, therefore, that serotonin and GABA are co-localized in a subpopulation of these terminals. All other reported non-cerebellar afferents terminate with boutons containing spherical vesicles (nuclei at the mesodiencephalic junction: King et al., 1978; Cintas et al., 1980; De Zeeuw et al., 1988, 1989a,b; spinal cord: King et al. 1976; Mizuno et al., 1976; Gwyn et al., 1983; Molinari and Starr, 1989; Molinari, 1988). Most of these terminals were apposed to distal dendrites or spines inside and outside glomeruli, relatively few were found to contact somata. Afferents from the spinal cord and the gracile nucleus never have been found in glomeruli in close contact with gap junctions (Molinari and Starr, 1989; Molinari, 1987, 1988; Molinari et al., 1990). The preferential axo-dendritic mode of termination of dorsal column afferents may be due to the relative paucity of spines on the long, radiating dendrites of the cell type that prevails in the spinal areas of the inferior olive. Axon collaterals from olivary neurons that terminate in the same and the contralateral inferior olive have only been observed in young kittens (Ramon y Cajal, 1911) and in cases of olivary hypertrophy in the cat (Ruigrok et al., 1990; De Zeeuw et al., 1990d). 231
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J. Voogd, D. Jaarsma and E. Marani
Fig. 162. Double labelled terminals (GABA and anterogradely transported WGA-HRP) from the contralateral PrH terminate on distal and proximal portions of the neurons in the dorsal cap of the rat (A and B) and the rabbit (C). In A, a double labelled terminal (right) and a GABAergic large granular terminal (left) are apposed to dendritic spines (asterisks) coupled by a gap junction (small arrows). In B and C the terminals are apposed to somata. The large arrows indicate the WGA-HRP reaction products. The open arrows and the arrowhead indicate symmetric synapses and an asymmetric synapse, respectively. Scale bar in A - 0.24 r in B = 0.41 r in C = 0.39 r De Zeeuw et al. (1994).
Cerebellar, GABAergic and mesodiencephalic, non-GABAergic terminals contacted both spines inside glomeruli and dendritic shafts. The cerebellar (King et al., 1976; Angaut and Sotelo, 1987) and/or GAD or GABA-immunoreactive (Sotelo et al., 1986; Fredette and Mugnaini, 1991) terminals are associated with gap junctions inside the glomeruli. De Zeeuw et al. (1989a,b; 1990a and c) concluded that both GABAergic and 232
The cerebellum." chemoarchitecture and anatomy
Ch. I
non-GABAergic mesodiencephalic terminals in the PO and the rostral MAO of cat and rat innervate the same spines including those that are electrotonically coupled. However, the cerebellar GABAergic terminals, but not the mesodiencephalic terminals, showed a strong preference to be strategically located next to both dendrites coupled by a gap junction (Fig. 161). Most or all of the terminals contacting the somata of olivary neurons contained pleomorphic vesicles (Bowman and King, 1973; Gwyn et al., 1977; Rutherford and Gwyn, 1980; Sotelo et al., 1986). Moreover, large GABAergic granular terminals were observed next to the soma. The proportion of somatic GABAergic terminals, that could be labelled from the cerebellar nuclei was significantly lower than among the terminals in the entire neuropil (De Zeeuw et al., 1989b). The presence of a non-cerebellar, GABAergic innervation of the cerebellum was also suggested by Nelson and Mugnaini (1985) and Fredette and Mugnaini (1991), who showed that total cerebellectomy does not cause a total depletion of GAD-positive terminals in the olive of the rat, and by the electrophysiological studies of Andersson et al. (1988) and Weiss et al. (1990). The GABAergic innervation of the axon hillock appears to be of cerebellar origin (De Zeeuw et al., 1990b). Possible extracerebellar sources for a GABAergic innervation of the inferior olive include the raphe nuclei and the adjacent reticular formation (Bishop, 1984), the nucleus parasolitarius and the cuneate nucleus (Nelson and Mugnaini, 1989) and the intrinsic GABAergic neurons of the inferior olive. Intrinsic GABAergic neurons of the inferior olive seem to be rare. Mugnaini and Oertel (1985), Nelson and Mugnaini (1989) and Fredette et al. (1992) found only few GAD-positive cells in the inferior olive of the rat. These cells were usually small and provided with unbranched, ramifying dendrites. They were differently distributed in different species; in the cat most occur in the rostral tip of the MAO and in the dorsal fold of the DAO, in primates in the PO (Fredette et al., 1992). They were more numerous in the baboon, where they accounted for 5% of the neurons of the inferior olive (Walberg and Ottersen, 1989). Colchicine injections in rat, rabbit and cat enhanced their visibility and revealed a high density of GAD-positive neurons in the reticular formation directly dorsal to the inferior olive (Nelson and Mugnaini, 1989; Fredette et al., 1992). Large cells with dendrites penetrating in the neuropil of the olive, which resemble the cells of the reticular formation, were identified by Sotelo et al. (1974) in the cat. Similar, peri-olivary cells, containing glutaminase were illustrated by Kaneko et al. (1989) in the rat. Bishop (1984) and Bishop and King (1986) demonstrated that dendrites of intracellularly injected reticular neurons can contribute to the olivary glomeruli. 6.3.2. Afferent connections of the inferior olive
Afferent systems of the inferior olive have been reviewed by Brodal and Kawamura (1980) for the cat and by Martin et al. (1980) for the opossum. For the rat the tabulated summary of its afferent connections in the paper of Brown et al. (1977) and the review by Flumerfelt and Hryccyshyn (1985) are useful. Afferent systems of the inferior olive can be subdivided into three groups: (1) the GABAergic nucleo-olivary and vestibuloolivary projections; (2) the monoaminergic and cholinergic projections to the inferior olive; (3) the specific projections from the spinal cord, certain brain stem nuclei and the cerebral cortex will not be considered in this chapter. Their neurotransmitters are not known.
233
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6.3.2.1. The nucleo-olivary and vestibulo-olivary projections." The GABAergic afferents of the inferior olive The origin of the nucleo-olivary projection from the cerebellar nuclei (Section 5.2.) and their mode of termination (Section 6.3.1 .) has been considered before. In this section the topographical organization of the nucleo-olivary and the closely allied vestibulo-olivary projections will be reviewed. The nucleo-olivary projection is entirely GABAergic, the vestibulocerebellar projection only partially so. For the main part the nucleo-olivary and the olivocerebellar projections are reciprocally organized, in the sense that the cerebellar nuclei which are the target of a particular olivo-corticonuclear loop give rise to a nucleo-olivary projection to this particular olivary subnucleus. This reciprocity is not complete, because the olivary projections of some of the cerebellar nuclei terminate bilaterally in the olive. The olivary projection from Deiters' nucleus clearly reciprocates the olivocerebellar projection to the B zone, but for the vestibulo-olivary projections from other vestibular nuclei this reciprocity is less obvious. Nucleo-olivary fibers terminate in all parts of the inferior olive. Roughly the density of the GABAergic boutons in different subnuclei of the olive represents the density of the nucleo-olivary projection to these parts. The density of GAD immuno-reactivity is higher in the group beta of the rat, which also contains the largest reactive boutons, followed by the subnucleus c of the MAO, the DMCC, the medial and lateral poles of the DAO, the bend of the PO, the DC and the DM (Sotelo et al., 1986) (Fig. 166). Nelson and Mugnaini (1988) noticed that the dorsal fold of the rat DAO contains a higher immunoreactivity than the ventral fold and confirmed the difference in bouton-size between strongly immunoreactive and less reactive regions. Nelson et al. (1989) found the same distribution in a comparative study of GAD-immunoreactivity of the olive in rabbit, cat, rhesus monkey and man (Fig. 163). Activity is highest in the beta nucleus. The DAO contains several, differently stained regions, with high immunoreactivity in the rostromedial, and in the caudolateral parts of the nucleus. Staining in the caudal MAO is lowest in the central subnucleus b and higher in the lateral subnucleus a and the rostral portion of the MAO. Immunoreactivity of boutons in the DC (high in rabbit and monkey; low in rabbit and man) and the DMCC (high in rabbit and cat, and low in monkey) differs for different species. GAD-immunoreactive boutons disappear from the contralateral PO, the rostral MAO and the lateral half of the ventral fold of the DAO of the rat after chronic lesions of the cerebellar nuclei or the superior cerebellar peduncle in the rat. The dorsal fold of the DAO is depleted of GAD-positive boutons after lesions extending into the lateral vestibular nucleus (Fredette and Mugnaini, 1991). Additional destruction of the vestibular nuclei results in the disappearance of GAD from the group beta but not from the medial half of the ventral fold of the DAO and the caudal MAO (Nelson and Mugnaini, 1989). GAD-immunoreactive neurons in the parasolitary and cuneate nuclei could be labelled after injection of retrograde tracers in the inferior olive of the rat (Nelson and Mugnaini, 1989). These nuclei, therefore, provide additional GABAergic projections to the inferior olive. The projection of the ipsilateral parasolitary nucleus was located in the medial subnucleus c of the caudal MAO by these authors. Connections from the parasolitary nucleus (indicated as the lateral solitary nucleus) also were documented in earlier studies by Loewy and Burton (1978) and Molinari (1985) in the cat. The inhibitory connections from the cuneate nucleus in the rat are crossed and terminate in the medial DAO. A similar GABAergic cuneo-olivary pathway appears to be responsible 234
The cerebellum." chemoarchitecture and anatomy
Ch. I
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rostral Fig. 163. Schematic generalized diagram of GAD immunostaining intensities in regions of the mammalian IO based upon studies in rabbit, cat and Rhesus monkey. Intensity was visually graded on a scale of 5-1, where 5 indicates the most intense staining. The beta nucleus and medial aspect of the rostral DAO have the highest intensity in the IO (black regions). Most of the IO is stained with intermediate intensities (hatched regions). Subnucleus b of the MAO (white region) is least intensely stained. Staining in the dorsal cap and the dorsomedial cell column (dotted regions) varied by species, aMAO = subnucleus a of the medial accessory olive; beta = subnuleus beta; bMAO = subnucleus b of the medial accessory olive; DAO = dorsal accessory olive; dc = dorsal cap; dmcc = dorsomedial cell column; PO = principal olive; vlo = ventrolateral outgrowth. Nelson et al. (1989).
for the i n h i b i t i o n o f n e u r o n s in the D A O on s t i m u l a t i o n o f the red n u c l e u s or the c o n t r a l a t e r a l r u b r o s p i n a l t r a c t in the cat (Weiss et al., 1990). T h e t o p o g r a p h y in the c e r e b e l l a r n u c l e o - o l i v a r y p r o j e c t i o n has b e e n s t u d i e d in different m a m m a l i a n species. T h e n u c l e o - o l i v a r y fibers f r o m the i n t e r p o s e d a n d l a t e r a l nuclei r u n in a s e p a r a t e tract, v e n t r a l to the b r a c h i u m c o n j u n c t i v u m in cat ( L e g e n d r e a n d C o u r v i l l e , 1987), rat ( C h o l l e y et al., 1989) a n d r a b b i t (Tan et al., 1995b). F a s t i g i o - o l i v a r y 235
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J. Voogd, D. Jaarsma and E. Marani
and vestibulo-olivary fibers take other routes (Nelson and Mugnaini, 1989; Ruigrok and Voogd, 1990). It is generally assumed that the anterior- and posterior interposed nuclei project to the rostral DAO and MAO respectively and the lateral, dentate nucleus to the PO (cat: Tolbert et al. 1976b; Buisseret-Delmas and Batini, 1977; Dietrichs and Walberg, 1981, 1985, 1986; Courville et al., 1983a; monkey: Kalil, 1979; Asanuma et al., 1983; Gonzalo-Ruiz and Leichnetz, 1990; opossum: Martin et al., 1976, 1980; rat: Brown et al., 1977; Angaut and Cicirata, 1982; Haroian, 1982; Swenson and Castro, 1983 a and b; Nelson and Mugnaini, 1989; Ruigrok and Voogd, 1990). Points of discussion were, and still are the presence and the extent of the fastigio-olivary projection, the nucleo-olivary connections of subdivisions of the dentate nucleus and of certain cerebellar subnuclei in rat (dorsolateral protuberance of the fastigial nucleus, dorsolateral hump), the presence of ipsilateral nucleo-olivary projections and the relations of the cerebellar nucleo-olivary projection with the GABAergic and non-GABAergic vestibulo-olivary connections. A projection of the medial cerebellar nucleus to the caudal MAO was denied by Brown et al. (1977) and Haroian (1982) for the rat, but was found by Achenbach and Goodman (1968), Angaut and Cicerata (1982) and Swenson and Castro (1983a and b). In the cat negative findings were published by Graybiel et al. (1973), Tolbert et al., (1976b) and Courville et al. (1983a), but the connection was found to be present by
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236
The cerebellum." chemoarchitecture and anatomy
Ch. I
Sugimoto et al. (1980) and Dietrichs and Walberg (1985). In the monkey negative results were obtained in the anterograde tracer studies from the fastigial nucleus by Batton et al. (1977), Kalil (1979) and Asanuma et al. (1983). Ikeda et al. (1989), however, reported projections from the caudal part of the fastigial nucleus to medial portions of the caudal MAO in the macaque monkey. Ruigrok and Voogd (1990) in a recent study, using anterograde transport of Phasaeolus vulgaris lectin injected in the cerebellar nuclei of the rat, established projections from rostral regions of the fastigial nucleus to a lateral zone in the caudal MAO and of the caudal fastigial nucleus to the group beta (Fig. 164). The projection of the dorsolateral hump is located in a circumscribed area in the medial subnucleus c, that coincides with the tectal recipient zone of Akaike et al. (1992) that projects to the vermis of lobule VII and the lateral extension of the A zone in the hemisphere of the lobules VI and VII. The central portion of the caudal MAO (subnucleus b) lacks a nucleo-olivary projection. No terminations were found in the DC, which has been reported to receive fastigial projections by Angaut and Cicerata (1982), Swenson and Castro (1983a and b) in the rat and Dietrichs and Walberg (1985) in the cat. The results of experiments on nucleo-olivary projections from different parts of the dentate nucleus to the PO are conflicting. According to Beitz (1976) and Tolbert et al. (1976b) in the cat and Kalil (1979) in the monkey the dorso-ventral and medio-lateral relations are maintained in the nucleo-olivary projection of the dentate nucleus. Dietrichs and Walberg (1985) denied this and proposed a complicated rostro-caudal relationship between both structures. According to Angaut and Cicirata (1982) the medial dentate of the rat, including the dorsolateral hump, projects to the ventral leaf, and the lateral dentate to the dorsal leaf of the PO. Swenson and Castro (1983a and b) described a projection of the rostral dentate of the rat to the dorsal leaf and of the caudal dentate to the ventral leaf of the PO. Chan-Palay (1977) claimed that the projection in the monkey is reversed: caudal dentate projecting to the ventral PO. Some of her experiments, however, show an exclusive projection of the caudal dentate to the ventral leaf of the PO. Ruigrok and Voogd (1990) confirmed Angaut and Cicerata's (1982) topology for the rat (Fig. 164). They located the nucleo-olivary projection of the dorsolateral hump in the dorsomedial group (DM), the enlarged medial portion of the ventral leaf of the PO. The DMCC that was often confused with the DM of the rat, did not receive a nucleo-olivary projection. The nucleo-olivary projection is not completely crossed. Some fibers from the dorsolateral hump, IP and the ventromedial lateral nucleus recross at the level of the inferior olive, and terminate in the ipsilateral DM, rostral MAO and ventral leaf of the PO respectively. Ipsilateral labelling was sparse or absent in other parts of the inferior olive. Projections from the vestibular nuclei terminate in subnuclei that do not receive a nucleo-olivary projection, such as the DMCC, the group beta and the central region (subnucleus b) of the caudal MAO in cat (Fig. 165) (Saint-Cyr and Courville, 1979; Gerrits et al., 1985a), opossum (Martin et al., 1980) and rat (Nelson and Mugnaini, 1989). These projections are mainly crossed, and, in the rat at least, entirely or partially GABAergic. An ipsilateral projection from the superior vestibular nucleus to a lateral zone in the caudal MAO and the bend region of the PO in the cat was reported by Gerrits et al. (1985a). The uncrossed, GABAergic projection from the parasolitary nucleus to subnucleus c (Nelson and Mugnaini, 1989), and the bilateral projections from the nucleus prepositus hypoglossi to DC and VLO (Gerrits et al., 1985a; McCrea and Baker, 1985; De Zeeuw et al., 1993) should be included in the vestibulo-olivary projection. Terminations from the nucleus prepositus hypoglossi were located both in subnuclei dominated by descend237
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 165. The vestibulo-olivary projection in the cat. Localization of terminal fields with antegrade axonal transport of [3H]leucine. A. Contralateral projection from the superior vestibular nucleus. B. Ipsilateral projection from the medial vestibular nucleus. C. Bilateral projections from the nucleus prepositus hypoglossi. MAO = medial accessory olive; PO = principal olive. Gerrits et al. (1985a).
ing visual afferent connections (DC and VLO) and in the caudal MAO, where they overlap with vestibular afferents from the medial and the spinal vestibular nuclei. The prepositus hypoglossi nuclear projection to the DC in rat and rabbit is bilateral and deals mainly with the caudal DC. It contains GABAergic (De Zeeuw et al., 1993) and cholinergic (Barmack et al., 1991) components. Recently it was shown that these two neurotransmitters are co-localized in the same terminals (De Zeeuw et al, unpublished). The GABAergic projections from the nucleus prepositus hypoglossi to the VLO and 238
The cerebellum." chemoarchitecture and anatomy
Ch. I
subnucleus c are exclusively contralateral and much sparser. The VLO and the rostral DC receive their major GABAergic input from the ventral dentate nucleus and the adjacent group y in the rabbit (De Zeeuw et al., 1994b). 6.3.2.2. Monoaminergic and cholinergic projections A plexus of varicose catecholaminergic fibers has been mapped in the inferior olive of cat, monkey and in several rodents with histofluorescence methods (Fuxe, 1965a and b; Hoffman and Sladek, 1973; Sladek and Bowman, 1975; Sladek and Hoffman, 1980). The distribution of these varicose fibers is heterogeneous and displays marked species variations. The distribution of serotoninergic fibers is also heterogeneous and speciesdependent, but it differs from the distribution of the catecholamines (Wiklund et al., 1977, 1981a,b; Sladek and Hoffman, 1980; Takeuchi and Sano, 1983; Bishop and Ho, 1984; King et al., 1984; Par6 et al., 1987; Compoint and Buisseret-Delmas, 1988). Immunocytochemical methods, using antibodies against conjugates of serotonin, were applied in the investigations reported in the last five papers. The catecholaminergic innervation of the inferior olive of the rat is strongest for the dorsal leaf of the PO and the rostral MAO (Fig. 166) (Sladek and Bowman, 1975). For serotonin the most intensely innervated subnucleus of the inferior olive of the rat is the lateral portion of the rostral lamella of the DAO (Fuxe, 1965a,b; Takeuchi and Sano, 1983; Bishop and Ho, 1984; Par6 et al., 1987; Compoint and Buisseret-Delmas, 1988), the dorsal leaf of the DAO is spared (Fig. 166) (Takeuchi and Sano, 1983). For the MAO and the PO the descriptions differ. Takeuchi and Sano (1983) found a somewhat higher innervation of the rostral MAO. Bishop and Ho (1984) described a dense innervation by varicosities of the subnuclei a and b of the caudal MAO, moderate numbers of serotonin-like immunoreactive fibers in subnucleus c and the caudal PO and few elements in group beta and the DC (Fig. 166). Par6 et al. (1987) confirmed this distribution for the caudal MAO. Compoint and Buisseret-Delmas (1988) stressed the presence of an immunoreactive plexus around the caudal MAO, with few fibers penetrating the center of the nucleus. Both catecholaminergic and serotoninergic fibers predominate in the lateral part of the caudal MAO of the cat. At more rostral levels only the medial MAO and the DMCC are densely innervated by serotoninergic fibers. The catecholaminergic and serotoninergic innervation of the DAO are complementary, with a serotoninergic plexus occupying the lateral and caudal DAO with the exception of its most lateral border region (Wiklund et al., 1977; Takeuchi and Sano, 1983). A central column of the caudal MAO contains a dense plexus of serotoninergic fibers in monkey (Takeuchi and Sano, 1983) (Fig. 167) and opossum (King et al., 1984). Other parts of the MAO of the opossum, including subnucleus c and the rostral pole of the MAO are less densely innervated. Serotoninergic fibers also predominate in the lateral DAO of the monkey (Takeuchi and Sano, 1983) but a serotoninergic plexus is present in the entire DAO of the opossum (King et al., 1984). The distribution of serotoninergic fibers in the PO differs for the different species, with an innervation of the ventral leaf of the cat (Sladek and Bowman, 1975), both the ventral and the dorsal leaf in the monkey (Takeuchi and Sano, 1983) (Fig. 168), and the dorsal leaf of the PO in the opossum (King et al., 1984). Serotonin was localized in axons and their terminals at the ultrastructural level by Wiklund et al. (198 l a) in the DAO of the rat with high resolution autoradiography of [3H]serotonin and by King et al. (1984) and Compoint and Buisseret-Delmas (1988) with 239
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Fig. 166. The innervation of the inferior olive of the rat by catecholaminergic, serotoninergic, substance P, and gamma-aminobutyric acid decarboxylase (GAD)-immunoreactive fibers and the distribution of acetylcholinesterase (ACHE). a = subnucleus a of the medial accessory olive; b = subnucleus b of the medial accessory olive; beta = subnucleus beta; D A O = dorsal accessory olive; d c = dorsal cap; dl = dorsal eaf principal olive; D M = dorsomedial subnucleus; dmcc = dorsomedial cell column; MAO = medial accessory olive; PO = principal olive; vl = ventral leaf principal olive; vlo = ventrolateral outgrowth; XII = hypoglossal nerve.
preembedding immunocytochemistry in the same species. The label was located in thin axons and varicosities, containing large dense core vesicles and small, clear vesicles or tubulo-vesicular elements. Few terminals engage in synaptic contacts (Wiklund et al.: 5%; King et al.: 2%), mainly with dendritic shafts, and never with the spines located in the glomeruli. Configurations suggesting axo-somatic contacts were found in the subnucleus b of the caudal MAO in the opossum (King et al., 1984). Similar terminals in the rostral MAO of the cat, containing large dark core vesicles, displayed GABA-like immunoreactivity (De Zeeuw et al., 1989b). The possibility that certain, non-cerebellar GABAergic projections are derived from the raphe nuclei or the adjoining medial reticular formation (Bishop 1984), and that they are co-localized with serotonin still needs to be explored. According to Compoint and Buisseret-Delmas (1988) the serotoninergic innervation of the inferior olive of the rat, at least in part, is derived from cell groups surrounding it. The nucleus raphe obscurus and the reticular formation dorsal to the olive innervate the MAO, the reticular formation and cell groups lateral to the DAO innervate the latter subnucleus. Co-localization of serotonin and substance P, that display a similar distribution in the olive of the rat
DAO
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Fig. 167. Schematic representation of the distribution of serotonin fibers in the inferior olivary complex of the monkey (a, rostral; i, caudal). Abbreviations: see Fig. 168. Takeuchi and Sano (1983).
241
Ch. I
J. Voogd, D. Jaarsma and E. Marani ~
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Fig. 168. Schematic representation of the distribution of serotonin fibers in the inferior olivary complex of the cat (a, rostral; i, caudal). B = group beta; DA--DMC = dorsomedial cell column; DAO = dorsal accessory olive; DC = dorsal cap; MAO = medial accessory olive; PO = principal olive; POD - dorsal lamella of the principal olive; POV = ventral lamella of the principal olive. Takeuchi and Sano (1983).
(Bishop and Ho, 1984; Par6 et al., 1987) (Fig. 166), is another possibility (H6kfelt et al., 1978; Pelletier et al., 1981; Par6 et al., 1987). A differential distribution of AChE has been described in the neuropil of the inferior olive of cat (Marani et al., 1977) (Fig. 169), ferret, rabbit (Marani, 1986,), rat (Fig. 166), and the opossum (Martin et al., 1975). These distributions are fairly consistent as to the columnar distribution of the enzyme in the caudal MAO and the rostral DAO, the absence of AChE in the group beta and the presence of AChE in the DC. There are some points of resemblance with the distribution of serotonin in the DAO and the caudal MAO of the cat, but a causal relationship between the presence of AChE and the distribution of certain afferent or efferent (peptidergic) systems of the olive has not been established. A dense plexus of ChAT-immunoreactive fibers pervading the entire inferior olive of the cat, was reported by Kimura et al. (1981). Receptor binding using labelled ~bungarotoxin for nicotinic receptors (Hunt and Schmidt, 1978) and [3H]propyl-benzilyl choline for muscarinic receptors (Rotter et al., 1979a) was stronger in the PO and the MAO, than in the DAO of the rat. Wamsley et al. (1981) and Swanson et al. (1987) reported on the presence of muscarinic and nicotinic receptors in the olive, but did not distinguish between the subdivisions of the inferior olive. 6.3.3. The connections between the inferior olive and the cerebellum
When the olivocerebellar projection was studied in the cat with antegrade axonal transport of tritiated aminoacids it appeared that the labelled climbing fibers were arranged 242
Ch.I
The cerebellum." chemoarchitecture and anatomy
42
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Fig. 169. Diagram of the distribution of AChE in the inferior olive of the cat. A. Transverse sections, numbered from caudally to rostrally. B. Diagrams of the unfolded inferior olive. Black: heavy staining; stippled: medium staining; white: low staining. Abbreviations: DAO = dorsal accessory olive; dc = dorsal cap; dmcc = dorsomedial cell column; MAO = medial accessory olive; nfl = subnucleus beta; PO = principal olive; vlo = ventrolateral outgrowth. Marani et al. (1977).
in longitudinal strips (Courville, 1975) and that this zonal pattern closely resembled the zonal organization in the corticonuclear projection and the associated compartmental subdivision of the feline cerebellum (Voogd, 1969; Groenewegen and Voogd, 1977; 243
Ch.I
J. Voogd, D. Jaarsma and E. Marani
Groenewegen et al., 1979; Voogd and Bigar6, 1980). Electrophysiological studies of the olivocerebellar projection (Armstrong et al., 1973, 1974) and of spino-olivocerebellar climbing fiber paths (SOCPs) (Oscarsson, 1969, 1973, 1980) generally supported the zonal distribution of the climbing fibers. To express the similarity with the anatomical olivocerebellar climbing fiber zones Oscarsson and Sj61und (1977a) adopted a modification of Voogd's (1969) nomenclature to designate the zonal projections of the S O C P s . 4 Brodal and Kawamura (1980), who reviewed their extensive studies with retrograde labelling of the olivocerebellar pathway, similarly used Voogd's paradigm in the interpretation of their data. Information on the olivocerebellar projection in primates is less complete, but the available data suggest that its overall organization is similar to the cat. The principles underlying Voogd's nomenclature also have been applied to the olivocerebellar projection in the rat (Buisseret-Delmas, 1988a,b; Buisseret-Delmas and Angaut, 1993; Voogd, 1995) but in rodents other schemes have been proposed (Azizi and Woodward, 1987; Apps, 1990). The olivocerebellar projection to the anterior lobe, the hemisphere of the posterior lobe, the caudal vermis and the flocculus and the collateral projections to the cerebellar and vestibular nuclei will be reviewed. Some reports on the olivocerebellar projection in non-mammalian vertebrates are available. In the chicken the inferior olive consists of a dorsal and ventral laminae. The homologies of these laminae with the mammalian olive are complicated (see Furber, 1983; Arends and Voogd, 1989). The projection to the cerebellum is crossed and arranged in longitudinal zones (Freedman et al., 1977). A detailed topographical map of the relations between the olive and the cerebellum of the pigeon was published by Arends and Voogd (1989). Different lines of evidence on the myelo-architecture and the mossy and climbing fiber connections, therefore, support the longitudinal subdivision of the avian cerebellum. The inferior olive of the turtle is difficult to identify with histological staining methods. Ktinzle and Wiklund (1982) identified it on the basis of specific retrograde transport of D-[3H]aspartate. The olivocerebellar projection in the turtle also is organized in bands. Both in reptiles and birds the climbing fibers and the smooth parts of the Purkinje dendrites on which they terminate are limited to the deep parts of the molecular layer (compare the extent of the spiny branchlets in Purkinje cells of fish, birds and mammals in Fig. 12). 6.3.3.1. The olivocerebellar projection to the anterior lobe
The situation in the cat, where most data are available, will be considered first. The olivocerebellar projection to the anterior lobe in primates and the rat will be reviewed at the end of this section. Three broad zones, e.g. the vermis, the pars intermedia and the lateral zone or hemisphere proper, can be distinguished in the anterior lobe of the cat, in accordance with the classical description of Brodal (1940). The anterior vermis receives its climbing fibers from the caudal portions of the MAO and the DAO, the pars intermedia from the rostral MAO and DAO and the hemisphere from the PO. Voogd (1969), Armstrong et al. (1974) and Groenewegen and Voogd (1977) in the cat distinguished between a medial A zone, receiving fibers from the caudal half of the MAO and a lateral B zone which receives its climbing fibers from the caudal DAO (Fig. 119).
Zones defined by electrophysiological criteria usually are designated by lower case characters (i.e. the a, x, cl-3, d 1-2 zones; see Fig. 175).
4
244
The cerebellum." chemoarchitecture and anatomy
Ch. I
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Fig. 170. Laterality and somatotopical organization of the projection zones of the ventral funiculus spinoolivocerebellar climbing fiber path (VF-SOCP) in the cerebellum of the cat. A. Rostral aspect. B. Caudal aspect of the cerebellum. The position of the caudal border of the inferior colliculus (INF.COLL.) is indicated. Oscarsson and Sj61und (1977b).
Equivalent SOCP's terminating in the a and b zone of the anterior vermis, that could be activated from the isolated ventral and dorsal funiculus of the cord, were discovered by Oscarsson c.s. (see Oscarsson, 1969; 1973). The ventral funiculus (VF-) SOCP carries information from skin, joints and high and low threshold muscle afferents. The VFSOCP to the a zone is an ipsilateral pathway from the hindlimb. The VF-SOCP to the b zone is bilateral, the projection from the forelimbs was found to be located medially in the b (b~) zone, the hindlimbs in the more lateral b2 zone (Oscarsson and Sj61und, 1974, 1977a,b,c) (Figs 170 and 171). The a zone is also present as a wide, paramedian zone in lobule VIII; the b zone is restricted to the anterior vermis (Figs 170 and 183). The dorsal funiculus (DF-)SOCP synapses in the dorsal column nuclei and converges with the VF-SOCP at the level of the inferior olive. The DF-SOCP is activated from high threshold muscle afferents and from both tactile and nociceptive cutaneous afferents 245
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 171. Termination zones in the anterior lobe of the dorsal (A, DF-SOCPs), ventral (B, VF-SOCPs), dorsolateral (C, DLF-SOCPs) and lateral (D, LF-SOCP) spino-olivo-cerebellar paths. The diagrams represent those parts of lobules IV and V in the left half of the anterior lobe which are accessible at the cerebellar surface. Borders between zones a-d2 are indicated with lines. Areas activated from hindlimb and forelimb nerves through direct and indirect paths from the dorsal funiculus nuclei (DFN) or spinal cord to the inferior olive (IO) are indicated (see key). The DF-SOCP projection (from forelimb nerves) and the LF-SOCP projection to the c2 zone, and the VF-SOCP projection to the b zone are bilateral, whereas all other projections are ipsilateral. B-D based on Larson et al. 1969a, 1969b and Oscarsson and Sj61und 1977a, 1977b. Ekerot and Larson (1979a)
(Ekerot and Larson, 1979a; Ekerot et al., 1991a,b). Ipsilateral DF-SOCP's to the a zone and to the lateral b 2 zone, carry hindlimb information (Ekerot and Larson, 1979a; Ekerot and Larson, 1977, 1979a,b, 1982; see also Andersson and Erikson, 1981) de246
The cerebellum." chemoarchitecture and anatomy
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CAMPBELL & ARMSTRONG'85 Fig. 172. Origin from the border region of the rostral and caudal medial accessory olive of the olivocerebellar projection to the electrophysiologically identified x zone in the cat. a = a zone; b = b zone; fl = subnucleus beta; cl-c3 = cl-c3 zones; coll = inferior colliculus; d~, d2 = dl and d 2 zones; dmcc = dorsomedial cell column; M A O = medial accessory olive; ml = mldline; pvg = lateral border of vermis; x = x zone; IV, V = lobules IV and V. R e d r a w n from Campbell and A r m s t r o n g (1985).
scribed a third, 'x' zone in the anterior lobe of the cat, located between the a and b zones (Fig. 171). The x zone is not present in all lobules of the anterior lobe, but is restricted to the lobules IV and V. It receives an ipsilateral projection through the DF-SOCP from the forelimb, through climbing fibers that branch between the x zone and zone in the pars intermedia (Fig. 175). The VF-SOCP does not terminate in the x zone. The climbing fibers to the x zone take their origin from the junction of the caudal and rostral halves of the MAO (Fig. 172) (Campbell and Armstrong, 1985; Voogd, 1989). Olivocerebellar fibers terminating in the A, X and B zones and the Purkinje cell fibers from these zones occupy the corresponding white matter compartments (Figs 119 and 120) (Voogd, 1969; Groenewegen and Voogd, 1977; Voogd and Bigar6, 1980; Voogd, 1989). The projections of the Purkinje cells of the VF-SOCP-innervated x and b zones were verified by Andersson and Oscarsson (1978a) and Trott and Armstrong (1987a,b) and were found to correspond to the anatomical corticonuclear projection zones X and B. The border between the B zone and the most medial C1 zone of the pars intermedia may be difficult to assess. This border was not recognized with retrograde labelling by Brodal and Walberg (1977a), who considered the B and C1 zone as a single area innervated by the caudal and lateral DAO. However, the presence of separate B and C1 compartments in the white matter and the characteristic electro-physiological properties of the b and c~ zones of Oscarsson validate their distinction in cat cerebellum. The problem in defining the border between the caudal portion of the DAO, projecting to the B zone and the more rostral regions, projecting to the C1 and C3 zones was discussed by Brodal and Kawamura (1980) and Voogd (1989). There is agreement on the differential origin of the two projections, but a precise border has never been established. Collateral projections to the fastigial and Deiters' nucleus take their origin from the 247
Ch. I
J. Voogd, D. Jaarsma and E. Marani
NOCICEPTIVECLIMBINGFIBER INPUT P
DAO DAO
A
1"I
MAO
~ULILr~TIVE NON-NOCICEPTIVE NOT STUDIED
Fig. 173. A summary diagram of nociceptive and non-nociceptive cutaneous climbing fiber input to lobules IV and V of the cerebellum of the cat. Forked arrows show branching of olivary axons to innervate pairs of zones (Ekerot and Larson, 1982). PF, primary fissure; DAO, dorsal accessory olive; MAO, medial accessory olive. Garwicz (1992).
olivocerebellar fibers to the A and B zones respectively (Groenewegen and Voogd, 1977). Retrograde labelling after injections of the fastigial nucleus of the cat was found in cells of the caudal MAO, the group beta and the dorsal cap (Hoddevik et al., 1976; Courville et al., 1977; Ruggiero et al., 1977). The projection of the lateral part of the caudal MAO to the lateral fastigial nucleus (Dietrichs and Walberg, 1985) may include the collateral projection of the olivocerebellar fibers to the x zone, that was never studied separately. Somatotopical patterns in the anterior vermis are arranged as mediolaterally disposed subzones or microzones. Information on the somatotopy of the A zone is scarce. According to Oscarsson and Sj61und (1977a,c) and Ekerot and Larson (1979a,b) the a zone is dominated by the hindlimb. The DF-SOCP projection to the x zone is dominated by the forelimb but lacks a somatotopical organization (Ekerot and Larson, 1977, 1979b, 1982). The exclusive forelimb connections of the x zone are in accordance with the cervical and cuneate projections to the border region of the caudal and rostral halves of the MAO (Boesten and Voogd, 1975; Gerrits et al. 1984a) that contains the olivary neurons projecting to the x zone (Campbell and Armstrong, 1985). Climbing fibers to the x zone receive tactile input and can be strongly activated by noxious pinch of the 248
The cerebellum." chemoarchitecture and anatomy I
Ch. I
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skin (Fig. 175). The receptive fields for tactile and noxious stimulation coincide (Garwicz et al., 1992 in Garwicz, 1992). Climbing fibers to the b zone are responsive to cutaneous and deep stimuli but do not receive a nociceptive input (Figs 170, 171 and 175). Successively more rostral body segments (tail, hindlimb, thorax, forelimb, head) project to successively more medial microzones in the b zone (Anderson and Oscarsson, 1978b; Andersson and Eriksson, 1981). The climbing fibers innervating a microzone share the same receptive field. In the b zone each microzone has been shown to project to a separate set of neurons of Deiters' nucleus (Andersson and Oscarsson, 1978a). The microzones and the olivocorticonuclear micro-complexes to which they belong can be considered as the basic functional units of the cerebellum. Contrary to the cortical zones to which they belong, the microzones do not possess anatomical borders because they are physiological artefacts isolated from a somatotopical continuum. Climbing fiber projections from the contralateral sensorimotor cortex to the lateral part of the anterior vermis were described by Provini et al. (1967, 1968). They noticed a strong correspondance in the localization of the cortical and peripheral projections to this region. Andersson and Eriksson (1981) and Andersson and Nyquist (1983) concluded that the projections of the posterior sigmoid gyrus to the a, x and b zones were closely matched to those of the VF-SOCP with respect to somatotopical organization and laterality. The projections to the a and b zones were bilateral and those to the x zone were crossed. Andersson and Nyquist (1983) identified short latency projections from separate forelimb and hindlimb areas in the posterior sigmoid gyrus to zones in the pars intermedia (Fig. 174). The short latency projections to the b zone took their origin from a single area, intermediate between and overlapping with the hindlimb and forelimb areas. Responses to stimulation of the more medial portion of this area, adjoining the localization of the hindlimb were located in the lateral b 2 zone, responses from more laterally situated sites, located next to the forelimb area, were located in the 249
Ch. I
J. Voogd, D. Jaarsma and E. Marani
medial b 1 z o n e . The x zone only received a short latency projection from the forelimb area. The subdivision of the pars intermedia into three zones, with the C1 and C3 zones receiving climbing fibers from the rostral two thirds of the dorsal accessory olive with collateral projections to the anterior interposed nucleus and the C2 zone from the rostral portion of the MAO with collateral projections to the posterior interposed nucleus (Fig. 119) (Groenewegen et al., 1979), agrees with the organization of the corticonuclear projection from these zones. C2 does not reach as far ventrally as the C1 and C3 zones and is absent from the lobules I and III, where the C~ and C3 zones become contiguous (compare Fig. 170). This arrangement agrees with other, anterograde and retrograde studies of the anterior lobe in the cat (Armstrong et al., 1974; Brodal and Walberg, 1977a; Kawamura and Hashikawa 1979; Gibson et al., 1987). It is generally assumed that the three anatomical C zones are identical to their electrophysiological namesakes identified as the terminations of the different SOCP's. The rostral segments of the c~ and c3 zones receive a converging short latency input from the ipsilateral hind-limb through the VF- and the DF-SOCP (Oscarsson and Sj61und, 1974; Ekerot and Larson, 1979a). Ipsilateral forelimb components of the DF-SOCP and of a pathway ascending in the dorsolateral funiculus (DLF-SOCP) (Larson and Oscarsson and Larson et al., 1969) converge upon the rostral segments of these zones. A third zone, receiving forelimb input through the DF-SOCP, was identified by Ekerot and Larson (1979a,b) in the extreme lateral part of the anterior lobe and indicated as the d 2 zone. The Cl, c3 and d2 zones receive branches from a common set of climbing fibers (Ekerot and Larson, 1977, 1982). One group innervates the medial halves of the Cl and the c3 zone, another set branches between lateral c3 and the d 3 zone, a third system of climbing fibers, gives off branches to the x zone and the lateral c~ (or cx) (Campbell and Armstrong, 1985) zone (Figs 171 and 173). The cl, c3 and d 2 z o n e s belong to a collection of zones, that also includes the x zone, receiving short latency input through the DF-SOCP. Moreover, these zones are distinguished from the intercallated b, c2 and d 1 zones, because they are strongly activated by nociceptive stimuli (Ekerot et al., 1991a,b; Garwicz et al., 1992, in Garwicz, 1992) (Fig. 175). The innervation of these zones from the inferior olive is not uniform. The medial Cl, the c3 and the d 2 z o n e are supplied by the rostral DAO, but the x and cx zones from the MAO (Fig. 172) (Campbell and Armstrong, 1985; Apps et al., 1991; Trott and Apps, 1991). As a consequence the anatomical
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Fig. 177. Reconstructions of transverse sections through the anterior lobe from two experiments showing the
AChE banding pattern in the molecular layer, the responsive properties of the climbing fibers, and the locations of marking lesions. A. Transverse section through sublobules Vc to Vf showing a midline and bilateral lateral bands of AChE-positive activity. The area between the midline AChE band and the lateral border of the lateral band corresponds to zone A, as defined by Marani and Voogd (1977). All the units recorded within zone A were unresponsive to mechanical stimulation, except for one response that was located on the lateral edge of the lateral zone. Lateral to zone A, all the climbing fiber responses encountered represented areas of the extremities. B. Another experiment (similar to A except that the plane of the electrode penetrations and the histological section involved sublobules Vb to lobule III) where unresponsive units were encountered within zone A but lateral to the lateral AChE band; all of the climbing fiber responses were elicited by tactile stimulation. Robertson and Logan (1986).
and physiological nomenclatures for these zones no longer correspond. In an anatomical sense the cx (or lateral c~ zone), that receives climbing fibers from a caudomedial region of the rostral MAO, should be considered as part of the C2 zone. However, the physiological properties of the cx zone are indistinguishable from the (medial) c~ zone, which is innervated by the rostral DAO and represents the entire anatomical C1 (Fig. 173). A similar discrepancy may exist for the d 2 z o n e which, as the equivalent of the anatomical D 2 zone, supposedly receives climbing fibers from the dorsal leaf of the PO, but shares a climbing fiber projection with the c3 zone, that is innervated from the DAO. The intercallated c2 and d~ zones receive long latency projections through the DFSOCP, bilateral to the c2 zone, ipsilateral to d]. In addition a pathway in the lateral funiculus of the cord (LF-SOCP) (Larson et al., 1969b) provides a bilateral long-latency input to the c2 zone and the DLF-SOCP innervates the d] zone. Climbing fibers inner252
The cerebellum." chemoarchitecture and anatomy
Ch. I
vating the c3 and dl zones cannot be activated by nociceptive stimuli (Garwicz et al., 1992 in Garwicz, 1992) (Fig. 175). The multisynaptic pathways from the dorsal column to the inferior olive for these long-latency projections to c2 and dl, have not been identified. They may include connections from the dorsal column nuclei to the nuclei of the mesodiencephalic junction. The DF-SOCP projection to the c3 and presumably to the medial cl and d2 zones, is somatotopically organized. The rostro-caudal, hindlimb-forelimb organization of the pars intermedia has been known since Adrian (1943) and Snider and Stowell (1944). A corresponding rough topography in the projection of the lateral and rostromedial DAO to rostral and caudal segments of the C1 and C3 zones in the anterior lobe has been recognized with anatomical tracing methods (Fig. 119) (Brodal and Walberg, 1977a; Groenewegen et al., 1979; Gibson et al. 1987). The studies of Ekerot and Larson (1979b) and, especially, the mapping of nociceptive receptive fields of climbing fibers projecting to the anterior lobe by Ekerot et al. (1991a,b) showed a remarkable degree of somatotopical organization in the c3 zone (Fig. 176). The representation of the ipsilateral body half was double, with a mirror image of the sequence of receptive fields found in the medial and lateral c3 zones. A quite different, patchy and non-zonal type of somatotopical localization was described by Robertson (see Robertson, 1987 for a review), using natural stimulation in anaesthetized intact cats to record climbing fiber responses in the Purkinje cells. They found a relatively unresponsive area corresponding to the A zone, which was defined on the basis of AChE histochemistry (Fig. 177) (Robertson and Logan, 1986). A patch-like organization of the body representation exists throughout the anterior lobe hemisphere. Patches differ in size, isolated representations can be encountered within or adjacent to a patch involving a different area and adjacent patches seldom have representations of neighbouring skin areas. The somatotopical arrangement with forepaw in caudal and hindpaw in rostral parts of the anterior lobe is roughly maintained. The patchy representations in these areas resemble the 'fractured somatotopy' in mossy fiber systems (see Section 6.4.2.) and lack a definite sagittal zonal disposition. Branching of climbing fibers supplying the same sagittal zone, or a set of functionally similar zones, was first described by Armstrong et al. (1973). Systematic branching between the x and lateral c~, medial c~ and medial c3 and lateral c3 and d2 was described by Ekerot and Larson (1977, 1979a) and is illustrated in Figs 171,173 and 175. Branching between equivalent regions of the anterior lobe and the paramedian lobule has been anatomically substantiated by Brodal et al. (1980) and Rosina and Provini (1983) for the projections of the rostral DAO and MAO and for the PO of the cat, and by Eisenman and Goracci (1983), Payne et al. (1985); Wharton and Payne (1985) and Hrycyshyn et al. (1989) for the rat. Apps et al. (1991), who studied the branching between the x and cx zones of the cat cerebellum with double labelling methods, found only few doublelabelled neurons projecting to both zones in the border region of the rostral and caudal halves of the MAO. Branching to the medial c~, c3 and d2 zones was never studied with anatomical methods. Neurons supplying climbing fibers to the medial Cl zone are located in more lateral and caudal regions of the rostral DAO than those innervating the c3 zone (Trott and Apps, 1991). Neurons with branching axons should be located in the regions where the projections to c l and c3 overlap, and in the border region between the DAO and the ventral leaf of the PO, that innervates the D2 zone. It can be concluded that the anatomical and electrophysiological subdivisions of the pars intermedia of the anterior lobe of the cerebellum of the cat are not completely concordant (Fig. 173). Short-latency DF-SOCP innervated zones with cutaneous no253
Ch. I
J. Voogd, D. Jaarsma and E. Marani
ciceptive input alternate with zones lacking this input. This pattern extends into the vermis and the lateral zone. The short latency DF-SOCP zones are innervated from the MAO, rostral DAO and the PO. The lateral zone or hemisphere proper of the anterior lobe receives a projection from the principle olive. The distinction of this projection by Brodal (1940) was based on its absence in the cat, and its presence in the anterior lobe of the cerebellum of the rabbit, that extends further laterally. Projections of the PO to the extreme lateral part of the anterior lobe of the cat have been documented by Armstrong et al. (1974), Brodal and Walberg (1977a), Groenewegen et al. (1979) and Kawamura and Hashikawa (1979). Brodal and Kawamura (1980) discussed this projection and tentatively concluded that both the ventral and dorsal lamella of the PO projected to the anterior lobe, the dorsal lamella to the medial D1 zone and the ventral lamella to the lateral D 2 z o n e . It cannot be decided whether the dl and d 2 z o n e s identified in the electrophysiological studies of Ekerot and Larson (1979a, 1982) correspond to either D~ or the D2 zone. Some pertinent observations on the projection of the somatosensory cortex to the anterior lobe were made by Andersson and Nyquist (1983) (Fig. 174) in the cat. The c~, c2 and c3 zones receive short latency, somatotopically organized projections from the posterior sigmoid gyrus. The somatotopical organization of the c] and c3 zones is similar to that observed after peripheral stimulation, for the c2 zone no somatotopical organization was observed for the peripheral projections through the VF- and the DLF-SOCP. The corticocerebellar projection to the c2 zone is bilateral and the connections with the c~ and c3 zones are crossed, which is in accordance with the peripheral input to their zones. An exclusive projection to c2 was found to be present from the second somatosensory area. Short latency projections from the anterior sigmoid gyrus only terminated
dao
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\ C2 C~BA D2 Fig. 178. Semidiagrammatic illustration showing the zonal arrangement in lobules IV and V of Saimiri based on projections from specific subnuclei of IO to each zone. A = A zone; B - B zone; Cl_3 - C].3 zones; Dr, 2 = DI,2 zones; caudmao = caudal medial accessory olive; daom3 = medial/lateral part of the dorsal accessory olive; dlpo = dorsal leaf of the principal olive; rostmao - rostral medial accessory olive; vlpo - ventral leaf of the principal olive. Whitworth and Haines (1986b).
254
The cerebellum. chemoarchitecture and anatomy
Ch. I
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Fig. 179. Diagram of lamellar and zonal distribution of olivary afferents and efferents in the rat. The two lamellae (folds) of the dorsal accessory olive (DAO, 1 and 2) and the horizontal lamella of the medial accessory olive (MAC, 3) appear to receive afferents mainly from the spinal cord and dorsal column nuclei while projecting to anterior vermis and parts of intermediate cerebellum. The medial MAC (vertical lamella, 4) receives afferents from the vestibular and visual areas and projects to the posterior vermis as well as the flocculus. The rostral lamella of MAC and both lamellae of the principal olive (PC) receive projections from higher centers and send fibers to the lateral hemispheres. In the lower part of the figure, three drawings of the inferior olive demonstrate the lamellae corresponding to their sagittal zones of projection in the cerebellum. Azizi and Woodward (1987).
bilaterally in the dl zone. This zone could also be activated from parietal cortical areas. The d 2 z o n e was not investigated, it seems likely that its cortical afferentation is similar to c~ and c3, i.e. a crossed, somato-topically organized input from the posterior sigmoid gyrus. 255
Ch. I
J. Voogd, D. Jaarsma and E. Marani
The organization of the olivocerebellar projection to the anterior vermis in primates appears to be similar to the cat (Brodal and Brodal, 1981; Whitworth and Haines, 1986b) (Fig. 178). Olivocerebellar projections to the A, X and B zones were identified in a preliminary report on Macaca fascicularis by Voogd et al. (1987a,b, 1990). The projection to the A zone resembles the situation in the cat with respect to the presence of A1 and A2 subzones. Olivocerebellar projections to the X zone take their origin from intermediate levels of the MAO, that also project to the C2 zone. A collateral projection to Deiters' nucleus detaches from olivocerebellar fibers to the B zone. Projections from the DAO and the dorsal and ventral leaf to the hemisphere of the anterior lobe were described by Brodal and Brodal (1981) in macaque monkeys and C1, C 2 and C 3 and D zones were identified by Whitworth and Haines (1986) in the olivocerebellar projection to the anterior lobe in Saimiri sciureus. Studies of the olivocerebellar projection in the rat included the anterior lobe (ChanPalay et al., 1977; Furber and Watson, 1983; Campbell and Armstrong, 1983; Azizi and Woodward, 1987; Buisseret-Delmas, 1988a,b; Buisseret-Delmas and Angaut, 1989b, 1993; Buisseret-Delmas et al., 1993). Olivocerebellar projection zones in the cerebellum of the mouse were studied by Beyerl et al. (1982). Of these studies the paper of Azizi and Woodward (1987) is of interest because it introduced a new scheme for the identification of the zones (Fig. 179). Azizi and Woodward (1987) subdivided the MAO in a horizontal lamella (i.e. the subnuclei a and b of the caudal MAO), a vertical lamella (including the subnucleus c, the group beta and the dorsal cap) and a rostral lamella (corresponding to the rostral MAO). They distinguished a dorsal fold of the caudal DAO, which is joined laterally to the rest of the DAO, that was indicated as the ventral fold. The different lamellae and folds were found to project in a systematic manner to sagittal zones (Fig. 179). In the anterior lobe they distinguished projections from the horizontal lamella of the MAO to a medial vermal zone (corresponding to the A zone) and from the dorsal fold of the DAO to a lateral vermal zone (corresponding to B). The ventral fold projects to a single zone in the pars intermedia (corresponding to C~) and the rostral lamella of the MAO to a zone in the lateral cerebellum (corresponding to C2). The dorsal lamella of the PO projects to a medial zone in the hemisphere (D~) and the ventral lamella of the PO to the most lateral zone of the hemisphere (D2). With respect to the situation in the cat, therefore, the projections of the dorsal and ventral leaf of the PO to the D~ and D2 zones of the anterior lobe of the rat cerebellum are reversed. The projection of the DAO to the C3 zone was not identified. Buisseret-Delmas (1988a and b), Buisseret-Delmas and Angaut (1989b, 1993) and Buisseret (1993) using the same retrograde transport methods as Azizi and Woodward (1987), identified projections to the A, X, B and C1-C3 zones from the same subnuclei of the inferior olive as in the cat. They confirmed the projection of the dorsal fold of the caudal DAO to the B zone and traced projections to the C1 and C3 zones from the rostrolateral and rostromedial DAO respectively (Fig. 141). The main differences between their scheme lobe and the situation in the cat are the the presence in the anterior lobe of the rat cerebellum of an additional Do zone and the reversal in the projection of the dorsal and the ventral leaf of the PO to the D~ and D2 zones. The olivocerebellar projection to the pars intermedia is similar in rat and cat. The differences in the connections of the C~_3zones of rat cerebellum concern the corticonuclear projection of the zones of the pars intermedia (see Section 6.1.4.). The X zone was indentified in the lobules IV-VI of the cerebellum of the rat by Buisseret-Delmas et al. (1993) as a zone located lateral to the A zone, projecting to the junctional region of the fastigial and interposed nuclei (their 'interstitial cell groups'). 256
The cerebellum." chemoarchitecture and anatomy
Ch. I
rostral
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1ram Fig. 180. Schematic illustration of the result of D-[3H]aspartate injection into lobules IV and V of the cerebellum of the rat. In the sketches of the cerebellar sections, retrogradely labelled axons and axon collaterals are indicated by lines and dots. Filled dots in the olives indicate the location of labelled cells. Retrograde labelling in cells of the inferior olive is also illustrated in more detail in the diagrams on the right. BP = brachium pontis; D A O = dorsal acessory olive; D N = Deiters' nucleus; F N - fastigial nucleus; LL = lateral lemniscus; LLV = ventral nucleus of the lateral lemniscusw; M A O - medial accessory olive; OI = inferior olive; OS = superior olive; PN - pontine nuclei; PO - principal olive; RB - restiform body; I-X lobules I-X. Wiklund et al. (1984).
It receives a projection from the subnuclei a, b and c of the caudal MAO. The origin of this projection in the rat appears to be more extensive than in the case of the X zone of the cat (compare Fig. 172) (Campbell and Armstrong, 1985). They also identified a CX zone in the lobules V and VI, medial to the C1, on the basis of its projection to the interstitial cell groups. It received its olivocerebellar projection from the group c at middle and rostral levels of the MAO. Buisseret-Delmas and Angaut (1989b, 1993) defined three D-zones in the lateral part of the anterior lobe on the basis of their corticonuclear projection to the dorsolateral hump (Do), the dorsal magnocellular part of the lateral cerebellar nucleus (D1) and the ventral parvicellular part of this nucleus (D2). They receive their olivocerebellar projections, respectively, from the DM group and the medial half of the ventral leaf of the PO (Do), the dorsal leaf of the PO (D,) and the lateral half of the ventral leaf of the PO (D2) (see Fig. 141). Several aspects of the olivocerebellar projection have been studied mainly in the rat. 257
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 181. A comparison of the zonal distribution of Zebrin-immunoreactive Purkinje cells (left diagram) and the olivocerebellar projection (right diagram) in the rat. Diagram of the olivo-cerebellar projection is based on data from Furber and Watson (1983), Eisenman (1984), Azizi and Woodward (1987), Buisseret-Delmas and Angaut (1989b, 1993), Sugita et al. (1989), Apps (1990), Akaike (1992) and Ruigrok and Voogd (1992). Subnuclei of the inferior olive (bottom diagram) and their projection zones are indicated with the same symbols. With the exception of the olivocerebellar projection to the lateral extension of the A zone (A~) of Buisseret-Delmas (1988a) (Figs 141 and 144), the correspondance between the Zebrin zones and the olivocerebellar projection has not been verified experimentally. 1-7 = Zebrin antigenic zones P1 - P7; a = subnucleus a of the MAO; A = zone A; A~ = lateral extension of the A zone of Buisseret-Delmas (1988a); b = subnucleus b of the MAO; B = zone B; 13 = group beta; c = subnucleus c of the MAO; C1-3 = zones C1-C3; CrI = crus I of the ansiform lobule; CrlI = crus II of the ansiform lobule; D = zone D; Do = zone Do of Buisseret-Delmas and Angaut (1989b); D A O = dorsal accessory olive; dc = dorsal cap; dl = dorsal leaf of the PO; D M = dorsomedial subnucleus of the ventral leaf of the PO; dmcc = dorsomedial cell column; F L O = flocculus; M A O = medial accessory olive; P F L = paraflocculus; P M D = paramedian lobule; PO = pricipal olivary nucleus; SI = simple lobule; vl = ventral leaf of the PO; vlo = ventrolateral outgrowth; X = zone X; I-X = lobules I-X.
The zonal distribution of the olivocerebellar projection was first demonstrated by ChanPalay et al. (1977), using autoradiography of antegrade axonal transport of 35S-methion258
The cerebellum." chemoarchitecture and anatomy
Ch. I
ine. She advocated a bilateral projection of the olive, but this observation has never been confirmed. The labelling of climbing fibers in the molecular layer in her experiments always was discontinuous. The unlabelled spaces between the labelled climbing fiber strips were assumed to receive climbing fibers of extra-olivary origin. This argument subsequently was disproved by Campbell and Armstrong (1983), who showed that labelling of climbing fibers from the inferior olive in the molecular layer was continuous. The investigations of Wiklund et al. (1984), using selective retrograde transport of [3H]D-aspartate were done in rats. The injection sites in the cerebellar cortex were relatively large and defied a detailed analysis of the material in terms of zones. Their experiments clearly confirmed the presence of branching olivocerebellar fibers between the anterior lobe and lobule VIII of the caudal vermis and the paramedian lobule. Moreover, their experiments clearly showed the presence of collateral projections to the cerebellar nuclei and to Deiters' nucleus (Fig. 180). The zonal disposition of Zebrin-immunoreactive and non-immunoreactive Purkinje cells has not been compared in any detail to the olivocerebellar projection. According to Gravel et al. (1987) some of the borders between Zebrin-positive and negative zones coincide with borders of certain climbing fiber strips, but these strips were not further identified. Judging from the reported identity of some of the Zebrin-positive and -negative zones with certain corticonuclear and cortico-vestibular projection zones it seems likely, that the correspondence between the zonal organization in the olivocerebellar projection and the Zebrin pattern will be close (Fig. 181). 6.3.3.2. Olivocerebellar projection to the hemisphere of the posterior lobe According to Groenewegen et al. (1979) the olivocerebellar projection zones in the cat continue uninterruptedly from the anterior lobe across the primary fissure into the simple lobule, where they diverge laterally to enter the ansiform lobule (Fig. 119). The projection of the caudal MAO to the B zone cannot be traced beyond the border of the simple lobule (lobule VI) and lobule VII. The projections from the rostral DAO to the C~ and C3 zones in the ansiform lobule are narrow or interrupted, but reappear in the caudal folia of the crus II (the ansula). C3 is only present in the dorsal folial rosette of the paramedian lobule, C1 continues into the most ventral folia of this lobule. The projection of the rostral MAO to the C2 zone can be traced as an uninterrupted zone, through the medial ansiform lobule, and the centrolateral paramedian lobule into the paraflocculus, where it occupies a ventral position in the dorsal paraflocculus and a dorsal position in the ventral paraflocculus. The final segment of the C: zone innervates the caudo-medial portion of the flocculus (Gerrits and Voogd, 1982). The PO is connected with the D~ and the D2 zones of the entire hemisphere and sends collaterals to the lateral cerebellar nucleus. The antegrade [3-H]leucine tracing experiments of Groenewegen et al. (1979) did not allow for an analysis of the possible differential origin of the climbing fibers terminating in the D1 and D2 zones. The olivocerebellar connections of the simple and ansiform lobules were studied in detail by Rosina and Provini (1982; see Kotchabhakdi et al. 1978, for an earlier report). Their conclusions are in accordance with the scheme of Groenewegen et al. (1979), specifying the presence of a C~ zone in the central axis of the folial rosette, and the attenuation of the C3 zone in the lateral bend of the lobule. They found a projection of the ventral leaf of the PO to the medial D1 zone and of the dorsal leaf to the lateral D 2 zone of the ansiform lobule (Fig. 182). The olivocerebellar projection to the crus II and the paramedian lobule of the cat was 259
Ch. I
J. Voogd, D. Jaarsma and E. Marani
A
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Fig. 182. The olivocerebellar projection to the lobulus simplex and the ansiform lobule of the cat. A. Diagrammatic surface view of the cerebellar injected areas in the four different groups of folia of cerebellum. Thin lines indicate H R P injected areas in the medial crus I-simple lobule; dotted lines in the lateral crus I; thick lines in the lateral crus II; dashed lines in the intermedio-medial crus II. Arrows indicate the subdivisions of the ansiform lobule adopted in this study: (a) intercrural sulcus and border between crus I and lateral crus II; (b) border between lateral and intermediate crus II; (c) intracrural sulcus 2 and border between intermediate and medial crus II. B. Summarizing diagram of the olivary areas retrogradely labelled by H R P injections into the four different groups of folia, drawn on the schema of the IO imagined unfolded. The olivary areas corresponding to the different groups of cerebellar injected folia are marked by thin, dotted, thick and dashed lines as indicated in A. Four different hatchings mark the four olivary subdivisions (MAO, vl, dl and lateral bend, DAO) which project to the four different longitudinal strips (C2, D1, D2 and C1-C3) of the ansiform lobule, as reconstructed in C. C. Surface view of the localization of the longitudinal zones as reconstructed by these reported results (D1 and D2 zones) and by previous studies (C2 and C1-C3 zones). Abbreviations: CrI, II = crus I, II of the ansiform lobule; C1-3 = C1-3 zones; D1,2 = D1,2 zones; D A O = dorsal accessory olive; dl = dorsal leaf of the PO; 1 = lateral; L.pm = paramedian lobule; L.sim = simple lobule; m = medial; M A O = medial accessory olive; Pfl.d = dorsal para-flocculus, PO = principal olive; vl = ventral leaf of PO. Rosina and Provini (1982).
the subject of series of reports, that reflect the changing views of the main actors in the course of time (Brodal and Courville, 1973; Courville et al., 1973; Brodal et al., 1975; Brodal and Walberg, 1977b; Walberg and Brodal, 1979). Brodal and Kawamura (1980), reviewing this work, concluded that C~, C2 and D zones are present in the paramedian lobule. They were unable to identify C3 in the dorsal lobules and stated that the dorsal and ventral leaf of the PO project to narrow D1 and D 2 z o n e s respectively. A reverse origin of the projection of the ventral and the dorsal leaf of the PO to the D~ and D 2 was advocated by Groenewegen et al. (1979) and substantiated by Rosina and Provini (1982) for the ansiform lobule. Electrophysiological studies of the configuration of the termination of climbing fibers in the paramedian lobule are scarce. Trott and Apps 260
The cerebellum." chemoarchitecture and anatomy
d
c3
Ch. I
C2 ci
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| Fig. 183. Activation of climbing fibers projecting to the caudal paramedian lobule (PM) in (B) by stimulation of collaterals terminating in the c~, c2, c3 and d zones of the anterior lobe indicated in (A). Redwawn from Oscarsson and Sjolund (1977b).
(1993) identified c~, c 2 and c3 zones in the rostral paramedian lobule on the basis of their electrophysiologically defined climbing fiber input and their projections to the cerebellar nuclei. They concluded that the presence of the c3 zone was variable. Oscarsson and Sj61und (1977b) delimited an ipsilateral hindlimb VF-SOCP projection to cl and c3 zones in the pars copularis of the paramedian lobule: the zones fuse into a wide area occupying almost the entire ventralmost paramedian folium. Stimulation of the c1_3and the d zones in the anterior lobe activated climbing fibers in the corresponding zones of the paramedian lobule (Fig. 183). Jeneskog (1974) recorded climbing fiber evoked potentials in the c~ and d zones of the anterior lobe and the paramedian lobule, after stimulation in and around the red nucleus. Rubral and spino-olivary pathways converge upon these zones: the DLF-SOCP terminates in the d zone, and the hindlimb and forelimb components of the DF-SOCP in the ventral and dorsal portions of the Cl zone of the paramedian lobule (Jeneskog, 198 l a). Tentatively they identified a C3 zone in the dorsolateral paramedian lobule and located the C2 zone in the central region of this lobule after mesencephalic stimulation. The total picture of the olivocerebellar projection to the paramedian lobule is less complete, but still very similar to the anterior lobe, with fusion of the C~ and C3 zones in its ventralmost part. The discontinuity of the C3 zone in the dorsolateral and ventrolateral parts of the lobule allows the C2 zone to leave the paramedian lobule to enter the paraflocculus. 'The paraflocculus is probably the most enigmatic cerebellar lobule from a functional point of view' (Brodal and Kawamura, 1980), but the pattern of its climbing fiber 261
Ch. I
J. Voogd, D. Jaarsma and E. Marani
afferents is simple and straightforward, with concentric projections of the rostral MAO to the C2 zone along its medial border and of the PO to the D zones along its periphery (Fig. 119) (see Brodal and Kawamura, 1980, for a discussion of the results of retrograde tracing studies from the paraflocculus). The main problem in the zonal structure of the paraflocculus is posed by the presence of a distinct subdivision of the D compartment and clear evidence for the projection of D~ and D 2 z o n e s to different parts of the dentate nucleus and the lack of evidence on a corresponding differentiation in the olivocerebellar projection from the PO. An educated quess can be made on the basis of the generally acknowledged nucleo-olivary projections of the rostromedial dentate to the dorsal leaf and the caudolateral dentate to the ventral leaf of the PO. Because the D~ zone projects caudolaterally and the D 2 z o n e of the paraflocculus rostromedially in the dentate, it seems likely that the D~ zone is innervated by the ventral leaf and the D 2 z o n e by the dorsal leaf of the PO. The olivocerebellar connections of the hemisphere of primates have been studied in the classical studies of Holmes and Stewart (1908), that were reviewed and updated by Jansen and Brodal (1958) (see also Voogd et al., 1990). Information on monkeys is limited to the retrograde transport experiments of Brodal and Brodal (1981, 1982) with injections of HRP in the simple, ansiform and paramedian lobules, in macaques, that suggest that C~, C2, C3 and D zones are present in these lobules. The diagram of Buisseret-Delmas and Angaut (1993) of the organization of the olivocerebellar projection in the rat (Fig. 141), appears very similar to the situation in the cat, but closer inspection learns that there are several important differences. One difference concerns the presence in the hemisphere of the posterior lobe of an additional zone, which has never been observed in the cerebellum of either cat or primates, located in the medial region of the hemisphere of the simple lobule, the ansiform lobule and the rostral folium of the paramedian lobule. This zone projects to the dorsolateral protuberance of the fastigial nucleus. It is absent from the anterior lobe and the copula pyramidis (see Section 6.1.4.). The olivocerebellar projection from the medial subnucleus c to this zone in the hemisphere (the lateral extension of the A zone of Buisseret-Delmas (1988a)) was recognized with retrograde transport from small WGA-HRP injections in this area (Fig. 142). She considered it to belong to and and to be continuous with the vermal A zone. The projection of the caudal MAO to the hemisphere was not noticed by Campbell and Armstrong (1983) and was not accounted for in Azizi and Woodward's (1987)
Fig. 184. The tecto-recipient zones in the vermis (lobule VII) and the hemisphere (paramedian lobule, Crus II and simple lobule) of rat cerebellum. Note their absence from anterior lobe, Crus I and copula pyramidis. Compare Fig. 142. ANT = anterior lobe; COP = copula pyramidis; CrI(II) = crus I(II) of ansiform lobule; PMD = paramedian lobule; SI = simple lobule. IV-IX = lobules IV-IX. Redrawn from Akaike (1992).
262
The cerebellum." chemoarchitecture and anatomy
Ch. I
scheme of the olivocerebellar projection in the rat. Akaike (1986bc, 1987, 1989, 1992) identified a similar zone with stimulation of the ipsilateral superior colliculus. It receives climbing fibers from the rostral subnucleus c (the tectorecipient zone of the MAO), that branch between medial VIb and the crus II (Fig. 184). It differs from the lateral extension of the A zone of Buisseret-Delmas (1988a) because it is absent from the crus I and from the rostral folia of the paramedian lobule. According to Akaike (1986c) it is separated from a similar zone in the vermis of lobule VII that also receives a climbing fiber input from the tectum, by a narrow non-tectal zone which straddles the paramedian sulcus. The tectal zone of the hemisphere is bordered on its lateral side by a zone receiving climbing fiber input from the ipsilateral wishker area (Akaike, 1989). The olivary neurons projecting to the tectal zone of lobule VII are also located in the tectorecipient area of subnucleus c, medial to the neurons projecting to the tectal zone in the hemisphere (Akaike, 1986a). The two populations are completely separated and do not collateralize between vermis and hemisphere (Akaike, 1986a,b,c, 1992). The position of the lateral extension of zone A in the hemisphere of the simple lobule differs from Buisseret-Delmas' (1988b) diagram, because it is located lateral and not medial to the B zone (see Section 6.1.4.). This peculiar position was confirmed in an experiment with an injection of the antegrade tracer Phaseolus vulgaris lectin in the rostral subnucleus c of the rat (Fig. 144) (Voogd, unpublished), with climbing fiber labelling in this zone and a collateral projection to the dorsolateral protuberance of the fastigial nucleus. To make the picture complete, it should be remembered (Section 6.3.2.1.) that the rostral subnucleus c of the caudal MAO is the target of the nucleoolivary projection of the dorsolateral protuberance (Fig. 164) (Ruigrok and Voogd, 1990). Counterstaining with the Zebrin I antibody showed that the termination of the climbing fibers coincides with the area of the Zebrin immunoreactive zones P4b and 5a extending from the simple lobule into the paramedian lobule. C1, C2, C3 and D zones receiving climbing fibers from the rostral DAO and MAO and the PO presumably are present in more lateral parts of the hemisphere of the simple lobule. The crus I of the ansiform lobule seems to lack a tectal response zone (Akaike, 1986b, 1987, 1992), and B, C~ and C3 zones are not represented in the ansiform lobule (Furber and Watson, 1983; Buisseret-Delmas, 1988a,b). The lateral extension of the A zone (tectal response zone of Akaike) and the C1, C2 and C3 zones reappear in the crus II and the dorsal folia of the paramedian lobule (Furber and Watson, 1983; Buisseret-Delmas, 1988a,b). The lateral extension of the A zone (Buisseret-Delmas, 1988a) and the C3 zone are absent from the copula pyramidis, that contains representations of the C1, C2 and D zones (Fig. 185) (Eisenman, 1981; Azizi and Woodward, 1987; Apps, 1990). Multiple D zones with projections from the PO are present in the entire hemisphere. According to Azizi and Woodward (1987) and Buisseret-Delmas and Angaut (1989b) the medial D~ zone (zone 6 of Azizi and Woodward, 1987) and the lateral D2 zone (zone 7 of Azizi and Woodward), receive their climbing fibers from the dorsal and the lateral half of the ventral leaf of the PO respectively (Figs 141 and 179). The dorsolateral hump sends a nucleo-olivary projection to the dorsomedial subnucleus (DM) of the olive, that is attached to the ventral leaf of the PO and has often been confused with the DMCC (Azizi and Woodward, 1987; Ruigrok and Voogd, 1990). Olivocerebellar projections from this subnucleus to the hemisphere of the simple lobule, the crus II, the paramedian lobule and the copula, but not to the crus I, have been noticed by Furber and Watson (1983) and Buisseret-Delmas and Angaut (1989b, 1993: their Do zone) (Fig. 141). The projection of the DM to the Do zone, therefore, is limited to the same lobules as the projection of the DAO to the C~ and C3 zones. Little is known about the olivocerebellar 263
Ch. I
J. Voogd, D. Jaarsma and E. Marani
B8 a
9b c
drn
9
drn 7.
caudal Fig. 185. The olivocerebellar projection to the pyramis and the uvula (lobules 8 and 9) of the rat cerebellum. A,B. Olivocerebellar projection zones of the lobules 8 and 9. C. Origin of these projections, indicated in diagrams of transverse sections through the inferior olive, a = subnucleus a of the medial accessory olive; b = subnucleus b of the medial accessory olive; beta = group beta; c = subnucleus c of the medial accessory olive; d - dorsal accessory olive; dm = dorsomedial subnucleus; rn - medial accessory olive; pr = principal olive; 8 and 0 = lobules VIII and IX of Larsell. Relabelled and reproduced from Eisenman (1984).
projection to the paraflocculus of the rat. According to Furber and Watson (1983) and Azizi and Woodward (1987) and Buisseret-Delmas and Angaut (1993) it receives climbing fibers from the rostral MAO and the PO. There is a good correspondence between the organization of the olivocerebellar projection in the rat and the cat. The main exceptions are the presence of the lateral extension of the A zone (or tectal response zone) and the Do zone in the hemisphere of 264
The cerebellum." chemoarchitecture and anatomy
Ch. I
the rat cerebellar hemisphere, that seem to be absent in the cat. The interruption of C1 and C3 zones in the crus I and the presence of a C3 zone in the crus II and the paramedian lobule, and its absence in the copula are in accordance with the findings in the cat. Projections of the dorsal and the ventral leaf of the PO to the D1 and D2 zone respectively, are still controversial in the cat, but seem to be well established for the rat. With the exception of the olivocerebellar projection to the lateral extension of the A zone, which coincided with the area of the P4b/P5a Zebrin immunoreactive Purkinje cell zones (Fig. 144), no direct comparisons of the olivocerebellar projection with the Zebrin pattern are available. However, in view of the similarity of the corticonuclear projection zones to the zonal arrangement in the olivocerebellar projection (see Section 6.1.4.), it seems likely that the P5b+ zone corresponds to C: and that the Zebrin-negative zones that border upon P5 correspond to the C~ and C3 zones (Fig. 181). 6.3.3.3. The olivocerebellar projection to the caudal vermis and the flocculus
In all species the zonal arrangement of the olivocerebellar projection to the vermis of the simple lobule (lobule VI) is very similar to the anterior lobe. Caudal MAO and DAO projections to A and B zones were traced to lobule VI in the cat (Groenewegen and Voogd) (1977) (Fig. 119) and the rat (Buisseret-Delmas and Angaut, 1993) (Fig. 141). The olivocerebellar projection to the rest of the caudal vermis differs substantially from that to the anterior lobe, with characteristic patterns for each of the lobules VII-X. Collateral projections to the fastigial nucleus have been observed with injections of antegrade tracers in the MAO and its subnuclei and labelling of (sub)zones in the caudal vermis (Groenewegen and Voogd, 1977), but these collaterals have not been systematically studied. Brodal and Kawamura (1980) and Sugita et al. (1989) noticed that the cells of the inferior olive projecting to the A zone of the lobules VI-VIII in cat and rat respectively, were arranged along the periphery of the caudal MAO, surrounding a central region projecting to the A zone of the anterior lobe (Fig. 186). The differences between their diagrams concern the origin of the projection to lobule VI, and the involvement of the group beta, which provides a sparse projection to lobule VII of the cat, (Hoddevik et al., 1976) but projects heavily to the lobules VI and VIII of the rat (Sugita et al., 1989; Apps, 1990). The A zone of lobule VI of the cat receives a single projection from a cell column in the medial caudal MAO, located lateral to the cells projecting to lobule VII (Fig. 186). The situation in the rat is more complicated with two labelled foci in the lobules Via and b after injections of WGA-HRP. One is located in the group beta, the other is a column situated along the lateral margin of the caudal MAO and extending along the border with the rostral half of this nucleus. The localization of the latter column is very similar to the cells projecting to the X zone of the anterior lobe in the cat (Campbell and Armstrong, 1985), and may correspond to the olivary neurons projecting to the X zone in the rat (Buisseret-Delmas et al., 1993). Retrograde labelling in the subnucleus c appears with injections of the caudal lobule VIC of the rat (Sugita et al., 1989). The existence of multiple sites in the caudal MAO that project to lobule VI of the rat is in accordance with the subdivision of this lobule in multiple corticonuclear projection zones and Zebrin-positive and -negative Purkinje cell strips (see Sections 5.6.1.3. and 6.1.4.). Laterally the A zone is bordered by the projection of the caudal DAO (cat: Hoddevik et al., 1976) or the dorsal fold of the DAO (rat: Buisseret-Delmas, 1988a; Azizi and Woodward, 1987) to the B zone. The B zone ends at the border of lobule VI and lobule VII in the cat, and at the VIa/VIb border in the rat. 265
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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According to Hoddevik et al. (1976) the main projection to lobule VII in the cat takes its origin from the rostromedial portion of the caudal MAO. This region receives afferents from the contralateral superior colliculus (Weber et al., 1978; Saint Cyr and Courville, 1982) and projects in a topical manner to the lobules VI and VII, with the rostral superior colliculus being located in the medial part of these lobules and the caudal colliculus more laterally (Kyuhou and Matsuzaki, 199 l a,b). An equivalent projection to lobule VII (the vermal visual area) was traced in macaque monkeys from the medial, Z-shaped portion of subnucleus b of the caudal MAO (Frankfurter et al., 1977; Brodal and Brodal, 1981; Yamada and Noda, 1987; Ikeda et al., 1989). The olivocerebellar projection to lobule VII of the rat differs from the adjoining lobules VI and VIII in being derived from a single cell column in the medial MAO (Sugita et al., 1989). This projection to lobule VII of the rat of the tectal response zone of the subnucleus c of the caudal MAO has been studied by Hess (1982b) and in a series of publications of Akaike (see Akaike, 1992 for a review). The neurons projecting to 266
The cerebellum." chemoarchitecture and anatomy
Ch. I
lobule VII are located in the caudal half of the tectorecipient zone of the caudal MAO, caudal to and intermingled with cells projecting to the lateral extension of the A zone in the medial hemisphere (see also the previous Section 6.3.3.2.). There is no climbing fiber branching between lobule VII and this zone in the hemisphere. The climbing fiber responses on stimulation of the superior colliculus occupy the medial two thirds of lobule VII (Akaike, 1985, 1986b,c, 1992). A similar, medial localization of the climbing fiber evoked potentials on tectal stimulation was reported in the earlier study of Jeneskog (1983) in the cat. The nature and the precise origin of the climbing fiber afferents to the lateral, non-tectal zone of lobule VII are not known. The area may be related to the X-zone or to a climbing fiber zone in more caudal lobules receiving afferents from the group beta. In the rat this non-tectal zone bridges the paramedian sulcus and consists of an 1 mm wide strip of cortex between the vermal and hemispheral zones supplied by the neurons of the tectorecipient area of subnucleus c of the caudal MAO. The relatively simple schemes of the olivocerebellar projection to lobule VIII based on studies in the cat by Hoddevik et al. (1976), Groenewegen and Voogd (1977) and Brodal and Kawamura (1980) now have evolved into the more complicated patterns defined by Eisenman (1981, 1984) and Apps (1990) for the rat. These patterns are of special interest because they bear a strong resemblance to the distribution of Zebrinpositive and -negative zones in lobule VIII of the rat (Hawkes and Leclerc, 1987) (see also Section 6.1.3.) and to the compartmentalization of this lobule in AChE-stained material of macaque monkeys (Hess and Voogd, 1986). Eisenman (1981) found two strips of climbing fibers in lobule VIII innervated by subnucleus a of the caudal MAO that were separated by a projection from the group beta. Climbing fibers terminating at the junction of lobule VIII with the copula pyramidis originated from the middle portion of the lateral DAO. Apps' (1990) observations were rather similar. The two strips receiving projections from subnucleus a constitute isolated regions, because projections from subnucleus a (and b: Sugita et al., 1989; Apps, 1990) are absent from lobule VII and scarce in IX. The group beta projection to lobule VIII, shifts medially in lobule IX, where it occupies a position next to the midline (Fig. 185) (Eisenman, 1984). This situation is reminiscent of the presence of distinct Zebrin-negative P 1 - and P 2 - strips, separated by P2+ in lobule VIII of the rat that disappear or become much narrower in the adjacent lobules VII and IX. The presence of an X zone in lobule VIII of the rat was postulated by Buisseret-Delmas et al. (1993). The olivocerebellar projection to lobule IX has been most extensively studied in cat and rabbit, data on rat and primates are less complete. Groenewegen and Voogd (1977) and Groenewegen et al. (1979) distinguished three zones in lobule IX of the cat: a medial zone, receiving a projection from the caudal MAO and/or the group beta, an intermediate zone receiving a projection from the DMCC and a lateral zone corresponding to C2, with climbing fibers from the rostral MAO. Similar patterns in the olivocerebellar projection to the uvula were reported by Brodal (1976) and Brodal and Kawamura (1980). Sato and Barmack (1985) in the rabbit and Kanda et al. (1989) in cat, distinguished projections of the group beta to two medial zones from the projection of the caudal MAO to a slightly more lateral area, and located a narrow strip receiving a projection from the ventral leaf of the PO between the lateral DMCC and rostral MAO (C2) zones (Fig. 187). Presumably, this pattern is shared by the rat (Eisenman, 1984; Bernard, 1987; Apps, 1990). Buisseret-Delmas et al. (1993) considered the projections from the DMCC and subnucleus c of the MAO to lobule IX of the rat to belong to the X and CX zones. Correlations of the olivocerebellar projection with the Zebrin pattern of lobule IX have not yet been verified. Retrograde labelling from lobule IX in the same 267
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subnuclei of the inferior olive was observed in the opossum (Linauts and Martin, 1978), in primates (Brodal and Brodal, 1981, 1982) and in subprimates (Whitworth et al., 1983). Zonal projections of the inferior olive to the nodulus (lobule X) and the flocculus have been substantiated in different species. Purkinje cells of these lobules are uniformly Zebrin-positive in rat, mouse, opossum, squirrel monkey (see Section 6.1.3.) and rabbit (Jaarsma unpublished observations). A compartmental subdivision of the white matter of the nodulus and the flocculus was demonstrated with AChE-histochemistry in the rabbit and the monkey (Section 6.1.5.) and correlated with the olivocerebellar projection to these lobules (Voogd et al., 1987a,b; Tan et al., 1995a,b). The pattern in the olivocerebellar projection to lobule X is quite distinct from lobule 268
The cerebellum." chemoarchitecture and anatomy
Ch. I
IX. The zones provided with climbing fibers from the group beta, the DMCC and the rostral MAO continue for some distance over the cortex of the nodule, but they are replaced by climbing fibers from the dorsal cap (DC) and the ventrolateral outgrowth (VLO). A large proportion of the climbing fibers from the latter two subnuclei are branches from climbing fibers which also terminate in the flocculus (Takeda et al., 1989a,b; Maekawa et al., 1989). Projections from rostral MAO, DMCC, group beta and DC, but not from the VLO were traced in the rabbit with HRP injections of lobule X and ventral IX by Alley et al. (1975). Zonally organized projections to lobule X from DC and VLO were demonstrated with anterograde axonal transport by Groenewegen and Voogd (1977) and Kawamura and Hashikawa (1979) in the cat. Retrograde labelling in these subnuclei after injections of retrograde tracers in lobule X was confirmed by Whitworth et al. (1983) for Galago and by Walberg et al. (1987) for the cat. Katayama and Nisimaru (1988) based their description of the olivocerebellar projection to lobule X on retrograde axonal transport experiments in the rabbit (Fig. 150). The projection from the group beta occupies the same medial position as in lobule IX. The lateral zones that are innervated from the DMCC and the rostral MAO, only extend over the dorsal surface of lobule X. The intermediate region contains a central zone, receiving a projection from the VLO, flanked by two strips innervated from the DC. The results of the anterograde and retrograde tracing experiments of Balaban and Henry (1988), also in the rabbit, were rather similar. They found the VLO zone to extend on the dorsal surface of lobule X and the ventral lobule IX and the two DC zones to be restricted to its ventral surface. These observations on the extent of the DC and VLO innervated zones are in complete accordance with the complex spike recordings in nodule Purkinje cells of the rabbit of Kano et al. (1990). The projections from the DC to lobule X and the flocculus were traced with parvalbumin-immunohistochemistry in rat pups by Wassef et al. (1992a,b). She showed that the cells of the DC (but not the cells of the VLO) transiently express immunoreactivity for antibodies against parvalbumin from birth till the 15th postnatal day. During this period two bundles of parvalbumin-immunoreactive climbing fibers were present in lobule X, corresponding to the medial and lateral DC innervated zones described in the rabbit. Olivocerebellar projections from the DC and the rostral one third of the MAO were first traced to the flocculus by Alley et al. (1975) with retrograde transport of HRP in the rabbit. The origin of the olivocerebellar projection to the flocculus was subsequently confirmed, using the same method, by Hoddevik and Brodal (1977), Yamamoto (1979) and Tan et al. (1995b) in the rabbit, Gerrits and Voogd (1982), Gould (1980) and Sato et al. (1983a) in the cat, Brodal and Brodal (1982) and Langer et al. (1985a) in macaque monkeys, Whitworth et al. (1983) in Galago and Blanks et al. (1983) and Bernard (1987) in the rat. The zonal organization of the olivocerebellar projection to the flocculus is similar in all species that were studied. Paired climbing fiber zones innervated by the caudal DC (the zones 2 and 4 of the rabbit flocculus) interleave with two zones innervated by the rostral DC and the VLO (zones 1 and 3) (Fig. 188). A fifth, lateral C2 zone is innervated by the rostral MAO. In the rabbit these zones correspond with the 5 corticonuclear and corticovestibular projection zones and the climbing fibers innervating a zone occupy the same white matter compartment as the axons of the Purkinje cells of this zone (Tan et al. 1995a,b,c) (Figs 147, 149 and 188) (see Section 6.1.5.). This arrangement is of special interest because it has been implicated in the spatial organization of eye movement control. Briefly the caudal DC of the rabbit transmits information about contralateral 269
Ch. I
J. Voogd, D. Jaarsma and E. Marani
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Fig. 188. Diagram of the origin of the climbing fiber afferents and the projections of the Purkinje cells of the five Purkinje cell zones of the flocculus of the rabbit. The zones and the corresponding white matter compartments containing their Purkinje cell axons and climbing fiber afferents are indicated with the same symbols. 1-4 = compartments and zones 1-4; DC = dorsal cap; fl-4 = folia 1-4 of the rabbit flocculus; fm = medial folium of the flocculus; fp = folium p of the ventral paraflocculus; Ip = posterior interposed nucleus; M A O = medial accessory olive; MV = medial vestibular nucleus; SV = superior vestibular nucleus; VLO = ventrolateral outgrowth; Y = group y. From Tan et al. (1995b,c).
movements of the surround around a vertical axis. Cells in the rostral DC and the VLO are excited by movements around an oblique horizontal axis that is oriented roughly parallel to the axis of the ipsilateral anterior semicircular canal (see Van Der Steen et al., 1994 for a review). Groenewegen and Voogd (1977) first described the projection of the caudal DC to a central zone in the cat flocculus flanked by two zones innervated by the rostral DC/VLO (zones 2 and 4). This pattern was extended by Gerrits and Voogd (1982) in tracing studies in the cat. The 7 floccular zones distinguished by these authors correspond to the 5 zones of the rabbit flocculus, because two of them are double (Fig. 149). Moreover, Gerrits and Voogd (1982) noticed that the zonal pattern in the olivo270
The cerebellum." chemoarchitecture and anatomy
Ch. I
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caudal Fig. 189. Diagram of the projection from the inferior olive to the flocculus and the ventral paraflocculus in the rat. The medial accessory olive (MAO) and the principal (PO) are drawn as diagrams of the unfolded inferior olive; the cortex of the flocculus and the ventral paraflocculus are unfolded. Different climbing fiber zones are indicated with different symbols. The climbing fiber zones of the rat flocculus corresponds to those in the rabbit (Fig. 188). F D corresponds to zone 1 of the rabbit; FE to zone 2; F D ' to zone 3 and F E ' to zone 4. F D and F D ' continue as zone D of the paraflocculus. C2 - C2 zone; caud -- caudal; d c = dorsal cap; FD, F D ' = F D ( ' ) zone (projections of vlo and PO); FE, F E ' = FE ( ' ) zone (projections of dc); F L O d and v = dorsal and ventral surface of the flocculus; M A O = medial accessory olive; PO = principal olive; rost = rostral; vlo - ventrolateral outgrowth; V P F L = ventral paraflocculus. Ruigrok et al. (1992)
cerebellar projection extends beyond the posterolateral fissure in the ventral paraflocculus (the 'ME': the medial extension of the ventral paraflocculus) (Fig. 149). In the anterograde axonal tracing studies of Tan et al. (1995b) in the rabbit the olivocerebellar projection was matched with the white matter compartmentalization in adjacent AChE-stained sections and, consequently, with the corticovestibular and corticonuclear projection of the correspooding zones (Fig. 188) (see Section 6.1.5.). The medial and central compartments 4 and 2 contained fibers from the caudal DC and the more laterally located compartments 3 and 1 conducted fibers from the rostral DC and the VLO to corresponding zones in the molecular layer. The fifth, most lateral compartment contained fibers of the rostral MAO to the Cz zone. The rostral DC and VLO 271
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Fig. 190. Distribution of C R F and CGRP-immunoreactive climbing fibers in P7 mouse cerebellum (A-C) and in neurons of the inferior olive (D-F). CRF-immunoreactive climbing fibers and neurons are indicated by dots, CGRP-immunoreactive climbing fibers and neurons by open circles, a, b and c = subnuclei a, b and c of the medial accessory olive; beta + group beta C1, C2 = Crus I and II; D A O - dorsal accessory olive; dc = dorsal cap; E G L . M L = external granular layer and molecular layer; F -- flocculus; I G L = internal granular layer; PF = paraflocculus; M A O = medial accessory olive; PO = principal olive; PL - Purkinje cell layer. Redrawn from Yamano and Tohyama (1993). (
projections extended, across the posterolateral fissure in folium P of the ventral paraflocculus, where the compartments 1 and 3 fuse around the tip of compartment 2, containing the olivocerebellar fibers of the caudal DC. The olivocerebellar projection to the flocculus of primates was not yet studied in detail. Zone 2 of the flocculus of macaques receives a projection from the caudal dorsal cap and, therefore, corresponds to zone 2 of the rabbit flocculus (Fig. 149). Recently Ruigrok et al. (1992) analysed the olivocerebellar projection to the flocculus and the adjacent paraflocculus in the rat with anterograde axonal tracing with the lectin Phaseolus vulgaris and retrograde transport of WGA-HRP (Fig. 189). Their results confirmed and extended the observations in other species, discussed in the previous paragraphs (Fig. 149). Two pairs of interdigitating zones, innervated by the caudal DC and the rostral DC/VLO, and a lateral C2 zone could be distinguished in the rat. The distal segments of these zones crossed the posterolateral fissure and were found to receive climbing fibers from more rostromedial levels of the DC (the FE/FE' zones, corresponding to zones 2 and 4 of the rabbit) and the ventral leaf of the PO (the FD'/FD zones, corresponding to zones 1 and 3). The rostral shift in the origin of climbing fibers directed to more distal (rostral) segments of a set of continuous, olivocerebellar projection zones is in accordance with Yamamoto's (1979) observations on the projection of intermediate levels of the DC and of the PO to the folium P of the ventral paraflocculus of the rabbit, and with Gerrits and Voogd's (1982) distinction of a lateral and rostral shift in the projection of successively more rostral parts of the DC and the VLO/PO to flocculus and ventral paraflocculus of the cat. Collateral projections of the olivocerebellar pathways from the DC and the VLO to the medial and superior vestibular nuclei were described by Balaban (1984), but have been denied by Groenewegen and Voogd (1977), Gerrits and Voogd (1982) and Ruigrok et al. (1991). Ruigrok et al. (1992) observed a strong projection to the parvocellular lateral cerebellar nucleus concomitantly with the climbing fiber labelling in the FD'/D zones. The anatomical and electrophysiological studies of Takeda and Maekawa (1989a and b), Maekawa et al. (1989), Kusonoki et al. (1990) and Kano et al. (1990, 1991) in the rabbit have shown that 36% of the climbing fibers innervating the flocculus and 64% of the climbing fibers in the nodule that could be driven by optokinetic stimuli, could be activated by collaterals terminating in the complementary lobule of the vestibulocerebellum. Retrograde labelling from flocculus and nodule with different fluorescent tracers resulted in double labelling of 9-27% of the DC neurons and 12-48% of the cells of the VLO. There was no distinct spatial segregation of the cells projecting to the two lobules.
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Fig. 191. Schematic line drawings of the unfolded opossum cerebellum modified after Larsell and Jansen (1972). The broken lines indicate the boundaries of the corticonuclear zones A-D after Klinkhachorn et al. (1984a). The distribution of the three types of enkephalinergic axons is indicated by the frequency and size of the symbols: the beaded axons by asterisks (C), the mossy fibers by dots (A), and the climbing fibers by triangles (B). I-X indicate vermal lobules; CR I, II, crura I and II, F, flocculus; LS, lobulus simplex; PFL, paraflocculus; PML, paramedian lobule. D. Distribution of enkephalinergic axons in a horizontal section through the cerebellar nuclei. D, dentate nucleus, F, fastigial nucleus; IPA, anterior interposed nucleus; IPP, posterior interposed nucleus. From King et al. (1987). (
6.3.4. The distribution of peptides and calcium binding proteins in climbing fibers and cells of the inferior olive A transient immunoreactivity in subpopulations of climbing fibers during the maturation of the cerebellum was described for somatostatin and CGRP. Somatostatin-immunoreactive climbing fibers are located in midsagittal and parasagittal bands in the vermis of lobules VI-VIII, some of which remain present in the flocculus and paraflocculus of adult rats (Vincent et al., 1985; Villar et al., 1989) (see Section 3.1.3., Fig. 20). Neuronal labelling in the inferior olive was limited to the MAO. Transient immunoreactivity for CGRP in immature climbing fibers, surrounding the somata of Purkinje cells, and in cells of the inferior olive was noticed in the early postnatal period in rats and mice (Kubota et al., 1987, 1988; Morara et al., 1989, 1992; Rosina et al., 1992; Chedotal and Sotelo, 1992; Yamano and Tohyama, 1993, 1994). These immunoreactive climbing fibers define longitudinal bands, first next to the midline, later in paravermal regions, the hemisphere and in the flocculus. Chedotal and Sotelo (1992) used the expression of CGRP immunoreactivity during prenatal development in the rat to trace olivocerebellar fibers to the cerebellar anlage in the fetal rat at El7. They identified the transient CGRP-immunoreactive climbing fibers in early postnatal stages bands as the A, B and C3 zones. Yamano and Tohyama (1994) noticed in early postnatal mice, that the bands of CGRP-immunoreactive climbing fibers alternate with CRF-immunoreactive climbing fibers (Fig. 190A-C). In postnatal mice CGRP disappears from the climbing fibers and the number of climbing fibers expressing CRF increases. Immunoreactivity for CGRP and CRF was never observed in the same neurons of the inferior olive. Both CGRP and CRF containing neurons occur in the group fl, subnucleus c of the MAO, the dorsal fold of the DAO, and in the dorsal cap (Fig. 190D-F), but the two cell populations occupy slightly different regions of these subnuclei. Their results on the localization of alternating bands of CRF and CGRP-immunoreactive climbing fibers in lobule X resemble the identification of parvalbumin-immunoreactive climbing fibers in two dorsal cap-innervated stripes in this lobule of the immature rat cerebellum by Wassef et al. (1992a,b) (see also Section 6.3.2.1.). The results of Yamano and Tohyama (1994), however, are difficult to interpret in terms of olivocerebellar projection, because their definition of the olivary subnuclei in the mouse is not precise enough. Enkephalin (ENK)-like immunoreactivity in subpopulations of climbing fibers and cells of the inferior olive has only been found in the opossum (King et al., 1986a, 1987) but not in other adult species. A more wide-spread distribution of corticotrophin releasing factor (CRF)-like immunoreactivity in certain climbing fibers has been observed in the cerebellum of the opossum (Cummings et al., 1989) and several other species including rat and mouse (Cummings et al., 1983; Van den Dungen et al., 1987), rabbit (Errico 275
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and Barmack, 1993), cat and sheep (Kitahama et al., 1988; Cummings et al., 1988, 1989), and Saimiri sciurus and Macaca fascicularis (Foote and Cha, 1988; Cha and Foote, 1988). The distribution of CRF, ENK and CCK in the cerebellum and the precerebellar nuclei, the species-differences and the co-localization of these peptides, recently was reviewed by King et al. (1992). Climbing fibers containing ENK are located in patches, that constitute discontinuous 276
The cerebellum." chemoarchitecture and anatomy
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midline and parasagittal bands in the vermis of opossum cerebellum (Figs 191B and 192) (King et al., 1986a, 1987). The position of the parasagittal band in the anterior lobe approximately corresponds to the border of the A and B zones, as determined by Klinkhachorn et al. (1984a and b) from the corticonuclear projection in this species. This description also would fit a localization in the X zone, which has not yet been described in the opossum. The presence of immunoreactive somata in the lateral cell group a of the caudal MAO (Walker et al., 1988; King et al., 1989) is in accordance with such an enkephalinergic climbing fiber projection to the X-zone. ENK-immunoreactive climbing or mossy fibers have not been observed in the cerebellum of rat, cat or primates. However, opiate receptor binding is present over the molecular and to a lesser extent over the granular layer of rat cerebellum (Zajac and Meunier, 1980; Robson et al., 1984). A detailed description of the localization of CRF in climbing fibers is available for the opossum (Cummings et al., 1989). CRF is present in all lobules but the numbers of reactive climbing fibers and of CRF-like immunoreactivity in individual fibers show local differences. CRF-immunoreactive climbing fibers are concentrated in midline and parasagittal bands in the vermis (Fig. 193), the density of the CRF climbing fiber innervation is high for the vermis of the lobules VI-VII and lobule X and the flocculus. Concentrations of less reactive climbing fibers occur in the hemisphere. CRF-containing climbing fibers are concentrated in the flocculus and the adjacent part of the paraflocculus. The density of CRF-immunoreactive climbing fibers and the density of CRF receptors, determined by radioligand binding of [125I]-Tyr-ovine CRF, show a close correspondence for this region (Cummings et al., 1989). The distribution of CRF in neurons of the inferior olive of the opossum was determined with immunohistochemistry and in-situ hybridization (Cummings et al., 1989). All subdivisions of the olive contain CRF-positive neurons, but the number of labelled neurons and their staining intensity varies within and among these subnuclei. Staining levels were consistently lower in subnucleus a of the caudal MAO, when compared to the subnuclei b and c (which includes the neurons of the less immunoreactive group beta). Cells in the dorsal cap and the rostral MAO stained intensely with both methods. Rostral DAO, especially its lateral part, contains more labelled cells than medial and caudal regions of this subnucleus. Cell bodies in the PO reveal less CRF immunoreactivity, but the caudal PO contains strongly immunoreactive neurons. In the opossum CRF-like and ENK-like immunoreactivity co-exist in climbing fibers in midsagittal and parasagittal bands at the border of the lobules VII and VIII, from here they shift to a position in the base of the paramedian lobule, laterally in lobule X and ventrally in the flocculus. CRF is more widely distributed in the climbing fibers and single-labelled and double-labelled climbing fibers co-exist in these foci. CRF-like immunoreactive neurons are double-labelled for ENK in the subnuclei a and c of the caudal MAO and in the dorsal cap (Cummings and King, 1990). This pattern corresponds with the known projections of the periphery of the caudal MAO (subnuclei a, c and group beta) to the caudal vermis and of the dorsal cap to a lateral zone in lobule X and to the flocculus and the adjacent paraflocculus (see Section 6.3.3.3.). A few CCK-labelled climbing fibers are located in the same region of the caudal vermis of the opossum as the ENK/CRF (double) labelled fibers. They may originate from CCKpositive neurons in subnucleus c, where most cells are also irnmunoreactive for CRF (King and Bishop, 1990). The widespread distribution of CRF-containing climbing fibers in the cat resembles the situation in the opossum (Cummings et al., 1988; Cummings, 1989). Some of the CRF in climbing fibers is concentrated in bands. Bands are prominent in midline and 277
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Fig. 193. Camera lucida drawings of 60/lm transverse sections (A,B) through the opossum cerebellum that illustrate the distribution of corticotrophin releasing factor immunoreactive (CRF-IR) climbing and mossy fibers. Stripes in the molecular layer represent climbing fibers; the dots represent mossy fibers. The density and intensity of each symbol represent the relative number of fibers and staining intensity for respective fibers within each region. Roman numerals I-X indicate cerebellar lobules. PML, paramedian lobule~ LS, lobus simplex; CRII, crus II; PF[, para-flocculus; F, flocculus; DN, deep nuclei; x7. C. Camera lucida drawing of a 60 pm sagittal section of vermal lobule X. M, molecular layer; P, Purkinje cell layer; G, granule cell layer; pc, Purkinje cell; mf, mossy fibers; CF, climbing fibers. Note greater density of mossy fibers at the ventral medial aspects of folium. Bar in A,B = 1 ram, in C = 70 pm. Cummings et al. (1989). <.-
paramedian regions of the anterior lobe, in the intermediate part of the anterior lobe and the simple lobule, in the caudal Crus II, the paramedian lobule and the caudal vermis (Fig. 194). The distribution of CRF-containing climbing fibers in the flocculus and the adjacent ventral paraflocculus is heterogeneous, CRF-like immunoreactivity is less prominent in the rest of the paraflocculus. When we compared the immunoreactivity to the distribution of AChE in the anterior lobe, the paramedian band of CRF-containing climbing fibers appears to coincide with the lateral border of the paramedian AChE-positive band in the molecular layer, i.e. with the X-zone, and/or the lateral A-zone. The same position was found for the paramedian band of CRF-containing climbing fibers in the opossum. The two prominent bands of CRF-containing climbing fibers in the intermediate zone probably are located within the C1 and the C2 zones, the C3 zone seems to be less immunoreactive. CRF-containing cell bodies in the inferior olive of the cat occur in all subdivisions, with a columnar distribution in medial and lateral parts of the caudal MAO, CRF-positive cells in the groups beta, the dorsal cap and the ventromedial cell column, and a predominant localization in caudal and lateral D A O (Kitahama et al., 1988; Cummings, 1989). All cells of the inferior olive of the monkey express some degree of CRF-immunoreactivity (Cha and Foote, 1988), activity is highest in the caudal and central MAO. CRFcontaining climbing fibers in the cerebellar cortex are present in most of the cortex. A high and uniform distribution is present in lobule IX, dorsal VIII and in the ventral paramedian lobule (the authors identified this lobule as belonging to the paraflocculus) and as midline paramedian and mid-hemispheral bands in the A and C2 zones of the anterior lobe (Figs 195 and 196). The localization in the C2 zone would be in accordance with the situation in the cat. The zonal distribution in the ventral anterior lobe is more complicated. CRF-receptor binding, using [~2sI]-ovine C R F was higher over the molecular layer (Millan et al., 1986). In the human brain immunoreactivity was present in the great majority of the neurons of all subdivisions of the inferior olive and in climbing fibers in the molecular layer of the anterior vermis (Powers el al., 1987). Originally immunoreactivity with an antibody against C R F could not be detected in rat cerebellum (Swanson et al., 1982). C R F immunoreactive fibers in the molecular layer were observed by Olschowka et al. (1982), Cummings et al. (1983), Merchenthaler (1984), Sakanaka et al. (1987) and Van den Dungen et al. (1987, 1988). Palkovits et al. (1987) identified these fibers as climbing fibers with light and EM-immunohistochemical methods. They found CRF-immunoreactive neurons in all subdivisions of the inferior olive with a predominance in the PO. C R F m R N A was transcribed by the cells of the inferior olive and depleted at'ter a lesion of the contralateral olivocerebellar tract. C R F receptor binding, using [I~25]-ovine CRF, was highest over the granular layer of rat cerebellum (DeSouza et al., 1985). It is not possible to correlate the distribution of immunoreactive cells in the adult 279
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inferior olive with the olivocerebellar projection more precisely without double labelling experiments of olivocerebellar fibers with CRF-immunocytochemistry. It seems likely, however, that CRF-immunoreactivity is not confined to certain subsystems and that gradients in the different subdivisions of the inferior olive are responsible for the banded 281
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J. Voogd, D. Jaarsma and E. Marani
Fig. 196. Dark-field photomicrograph of a sagittal section through the vermis of the cerebellum of the squirrel monkey. Note that some areas of the molecular layer (M) contain dense CRF-immuno-reactive axons while others do not. However, intensely immuno-reactive axons are evident in the Purkinje cell layer (P) even in those regions in which such axons are not evident in the molecular layer. Occasional labelled axons are evident in the granular layer (G) and in the white matter (W). Scale bar = 100/lm. Cha and Foote (1988).
distribution in the cortex. Interestingly, the a m o u n t of C R F - l i k e i m m u n o r e a c t i v i t y in cells of the inferior olive increases in pontine cats ( K i t a h a m a et al., 1988). C R F messenger R N A m e a s u r e d with a [35S]-labelled oligonucleotide probe increased in neurons of the caudal dorsal cap of rabbits after long-lasting optokinetic stimulation of the contralateral eye ( B a r m a c k and Young, 1990) (Fig. 197). By itself C R F has little or no effect on n e u r o n a l activity, but it potentiates the excitatory effects of g l u t a m a t e and aspartate, the putative n e u r o t r a n s m i t t e r s of the climbing fibers on the Purkinje cells (Bishop, 1990; Bishop and K e r r 1992). K i t a h a m a et al. (1988) also raised the question whether the C R F in precerebellar nuclei is under endocrinological control, but they were unable to observe effects of a d r e n a l e c t o m y or hypophysectomy. Some calcium-binding proteins occur in subpopulations of climbing fibers. Transient labelling with antibodies against p a r v a l b u m i n was observed in neurons of the dorsal cap
Fig. 197. Optokinetically induced increase in corticotrophin releasing factor (CRF) mRNA in caudal dorsal cap of the inferior olive of the rabbit revealed by darkfield photomicrograph of an emulsion-coated brain-stem section. The rabbit received 37 hr of binocular optokinetic stimulation in the posterior to anterior direction with respect to the left eye, causing a 360% increase in levels of CRF mRNA in the right dorsal cap. A,B. Bright-field and dark-field views of the same tissue section are shown. The finer spatial resolution of the emulsion demonstrates clustering of silver grains over individual olivary neurons. Scale bar = 200 r (Barmack and Young, 1990) 282
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and in climbing fibers in the two DC-innervated zones in lobule X and in a single zone in the rat flocculus by Wassef et al. (1992a,b) in rat pups. Calretinin-immunoreactive climbing fibers were observed in the cerebellum of the chicken (Rogers, 1987) and the rat (Floris et al., 1994). The precise topography of these climbing fibers has not yet been reported. 9 ~.-.
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6.4. MOSSY FIBER SYSTEMS
6.4.1. Concentric and discontinuous, lobular arrangements of mossy fiber systems Mossy fibers take their origin from many different sites in the brain stem and the spinal cord. The topographical organization of mossy fiber systems differs considerably from the climbing fiber projection. Mossy fiber systems enter the cerebellum through the restiform body and the middle cerebellar peduncle, lateral to and along the brachium conjunctivum. The main orientation of the stem fibers of the mossy fibers is transverse. These stem fibers are known as semicircular fibers, which constitute a layer situated rostral and dorsal to the cerebellar nuclei and peripheral to the olivocerebellar fibers, which are immediately apposed to the surface of the cerebellar nuclei and their efferent tracts (Fig. 198). Medially the layer of semicircular fibers continues into the cerebellar commissure, that consists of a central part, containing the decussation of the uncinate tract and the more peripherally located decussation of the mossy fibers (Voogd, 1964). Most mossy fiber systems are partially crossed. Secondary vestibulocerebellar and ventral spinocerebellar fibers, that enter the cerebellum in a ventral and medial position, occupy a ventral and rostral position in the cerebellar commissure; pontocerebellar fibers that enter the cerebellum laterally and caudally, occupy a dorsal and caudal position in the commissure. These fibers are located in the dorsal white matter of the anterior lobe, in the medullary ray of the lobules VI and VII and in the base of lobule IX. On their way towards the commissure the pontocerebellar fibers come to the meningeal surface, in the bottom of the intercrural fissure, where cortex is absent in the center of the ansiform lobule and in the interparafloccular sulcus in the center of the parafloccular loop, where the cortex is also interrupted. Mossy fiber systems within the cerebellum display a concentric arrangement: vestibulocerebellar fibers are located most centrally, and terminate in the cortex in the bottom of the fissures; spino-, cuneo-, and reticulocerebellar fibers extend more peripherally and
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284
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pontocerebellar fibers cover the apex of the lobules. An electrophysiological corrolary of this concentric arrangement is the observation of superficial and deep terminations of exteroceptive and proprioceptive components of the cuneocerebellar tract respectively (Ekerot and Larson, 1972). As a consequence mossy fiber projections are discontinuous in the direction across the folium: being absent at the base or apex of the folium, and reappearing in the next (Jasmin and Courville, 1987b; Tolbert et al., 1993). Another consequence is that the distribution of mossy fibers is mainly transverse and lobular, with the restriction that these systems generally populate several adjacent lobules and that the borders between different mossy fiber systems do not necessarily correspond to the bottom of the fissures. The presence of a concentric arrangement in their distribution does not preclude a large degree of overlap between different mossy fiber systems. The question of the presence of overlap in their terminations is complicated by the termination of some mossy fiber systems in alternating longitudinal strips. The origin and distribution of primary and secondary vestibulocerebellar mossy fibers has been studied repeatedly. Vestibular root fibers enter the cerebellum from the superior vestibular nucleus, as part of the ascending branch of the vestibular root (Mannen et al., 1982; Sato et al., 1989). The development of the primary vestibulocerebellar projections was studied in rat embryos, where the root fibers can be distinguished by their parvalbumin-immunoreactivity (Morris et al., 1988). The literature on the projection of the vestibular nerve was reviewed in the study of Gerrits et al. (1989) on the primary vestibulocerebellar projection in the rabbit. The evidence on the origin and distribution of secondary vestibulocerebellar projections was reviewed for the cat by Brodal (1974), Kotchaphakdi and Walberg (1978), Batini et al. (1978, lobules VI and VII), Matsushita and Okado (1981, lobules I and II), Sato et al. (1983b, flocculus), Magras and Voogd (1985) and Blanks (1990, flocculus), for the monkey by Brodal and Brodal (1985), Langer et al. (1985a, ftocculus) and Frankfurter et al. (1977, lobule VII), for the rat by Rubertone et al. (1995) and for the rabbit by Thunnissen et al. (1989), Epema et al. (1990) and Tan and Gerrits (1992). The general conclusions on the distribution of the vestibulocerebellar projection are depicted in Figs 199 and 200, taken from the papers on the rabbit from the last three authors. Both primary and secondary vestibulocerebellar fibers terminate in lobule X and ventral lobule IX, in the lobule I and II and in the cortex in the depth of the vermal fissures. This projection is mostly ipsilateral for the fibers of the vestibular root and bilateral for the secondary vestibulocerebellar projection. The secondary vestibulo-cerebellar projection to the hemisphere is restricted to the flocculus and the adjacent cortex of the ventral paraflocculus (Fig. 203). A primary vestibulocerebellar projection to the flocculus is absent in the rabbit (Gerrits et al., 1989). Vestibulocerebellar mossy fibers take their origin from neurons in all vestibular nuclei, with the exception of the Deiters' nucleus and a sparse projection from the magnocellular medial vestibular nucleus (Figs 200 and 201). The distribution of neurons projecting to either flocculus or caudal vermis or to both is rather similar and is bilaterally symmetrical. Most neurons were found in the medial, superior and descending vestibular nuclei in this order. Neurons projecting to lobules IX and X, to the flocculus and to both parts of the cerebellum occur in a ratio of 12:4:1 (Epema et al., 1990). A statistical preference was found for the superior vestibular nucleus for a projection to the contralateral flocculus (Tan and Gerrits, 1992). Widespread projections of the nucleus prepositus hypoglossi and neighbouring perihypoglossal nuclei terminate bilaterally with an ipsilateral predominance in the vermis, the flocculus and the paraflocculus and in the cerebellar nuclei (see McCrea and 285
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Fig. 199. Primary and secondary vestibulocerebellar mossy fiber projections in the rabbit, determined with antegrade axonal transport of [3H]leucine and WGA-HRP. Upper panels: sagittal sections; lower panels: transverse sections through the caudal vermis. K196: ipsilateral distribution of fibers of the vestibular nerve. Gerrits et al., (1989); C2098: bilateral distribution of fibers from the medial vestibular nucleus (MV); K82: bilateral distributions of fibers from the superior vestibular nucleus (SV; Thunnissen et al., 1989). Dense termination in the sagittal sections is indicated with heavy hatching, scattered labelled mossy fiber rosettes with light hatching and dots. Note similarity in the distribution of primary and secondary vestibulocerebellar projections. (
Baker, 1985 and Roste, 1989 for reviews). Barmack et al. (1992b) noticed a particularly strong projection of the nucleus prepositus hypoglossi to the ventral paraflocculus (folium p) in the rabbit (Figs 84 and 201). Some cerebellum-projecting neurons of the nucleus prepositus hypoglossi and the caudal parts of the vestibular nuclear complex contain acetylcholine and/or C R F (see Section 3.10.1. and 6.4.5.). Anatomical classifications of the spinocerebellar tracts are based on their level and nuclei of origin, their decussation within the spinal cord, their position in the lateral funiculus, their entrance route into the cerebellum and their lobular and zonal distribution. These different criteria are not necessarily correlated. Oscarsson (1973) in his comprehensive review of the functional organization of spinocerebellar paths, distinguished the classical dorsal and ventral spinocerebellar tracts and added a third tract, the rostral spinocerebellar tract (Oscarsson and Uddenberg 1964), that originates from the cervical cord. Moreover, Oscarsson (1973) included the cuneocerebellar tract as one of the direct spinocerebellar pathways. The dorsal spinocerebellar and cuneocerebellar tracts are ipsilaterally ascending and terminating pathways that transmit information about external events from the lower and upper extremity respectively. Both contain proprioceptive and exteroceptive components. The ventral spinocerebellar and rostral spinocerebellar tracts are hindlimb and forelimb tracts, that convey information to the cerebellum about interneurons mediating flexor reflex afferents and related motor centers in the spinal cord. These tracts terminate bilaterally in the cerebellum. A fifth direct spinocerebellar pathway arises from the central cervical nucleus (Wiksten, 1979b). Although Oscarsson's criteria were functional rather than anatomical and the great flow of new information on the origin and termination of these tracts had not yet started, his classification is still useful. The dorsal spinocerebellar tract takes its origin from Clarke's column and from a group of neurons in Rexed's (1954) dorsal laminae IV-V! (see Yaginuma and Matsushita, 1987; Matsushita and Hosoya, 1979; Matsushita et al., 1979; and Grant and Xu, 1988, and Xu and Grant, 1994, for complete references on rat and cat). It terminates, mainly ipsilaterally, in nine strips in the vermis, the pars intermedia and the extreme lateral part of the lobules III-V of the anterior lobe (Fig. 206C), bilaterally in lobule VIII of the caudal vermis and in parts of the paramedian lobule. The cuneocerebellar projection takes its origin from the external cuneate nucleus and the internal cuneate and gracile nuclei (Gordon and Seed, 1961). The exteroceptive and proprioceptive components of the cuneocerebellar tract synapse in the internal- and external cuneate nuclei respectively (Cooke et al., 1971). The differential termination of exteroceptive and proprioceptive mossy fibers in the apical and basal part of the folia is known from an electrophysiological analysis of the two components of this tract (Ekerot and Larson, 1972). These proximo-distal differences are reflected to some degree in the antegrade tracer studies with injections of the external and internal cuneate nucleus. The projection of the internal cuneate, moreover, is predominantly uncrossed 287
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Fig. 200. Origin of secondary vestibulocerebellar mossy fibers in the rabbit. A-C. Transverse sections through the vestibular nuclei. D. Dorsal view of a reconstruction of the vestibular nuclei. E-G. Transverse sections with retrogradely labelled cells, from injections of fast blue in the lobules IX and X (dots), of nuclear yellow in the flocculus (open circles) and from both injection sites (astarisks). H. Distribution of cells retrogradely labelled from the flocculus. I. Distribution of cells retrogradely labelled from nodulus and uvula, bc = brachium conjunctivum; CO = cochlear nulei; cr = restiform body; d = dorsal part of group y; dfb = direct fastgiobulbar tract; DV - descending vestibular nucleus; gVII = genu of facial nerve; IN = iinterstitial vestibular nucleus; LV = lateral vestibular nucleus; MV = medial vestibular nucleus; MVc = caudal part of medial vestibular nucleus; MVmc = magnocellular medial vestibular nuleus; MVpc = parvocellular medial vestibular nucleus; nV = spinal root of trigeminal nerve; NVpar = parabrachial cerebellar nucleus; PH = nucleus prepositus hypoglossi; S = nucleus of the solitary tract; SV = superior vestibular nucleus; tu = uncinate tract; v = ventral part of group Y; VI = nucleus of the 6th nerve; X = group X of the vestibular nuclei; Y = group y. Bar = 0,6 ram. Epema et al. (1990). (
and directed to the hemisphere (Fig. 202) (Gerrits et al., 1985a; Jasmin and Courville, 1987a,b). The cuneocerebellar tract is distributed to the anterior lobe and the lobules VI, VIII and the paramedian lobule of the posterior lobe (Grant, 1962). The distribution of cuneocerebellar fibers in the hemisphere is complementary to the distribution of the dorsal spinocerebellar tract; in the vermis both projections overlap. This overlap may be related to the predominantly mediolateral organization of somatotopical projections to the anterior vermis and the existence of rostro-caudal somatotopical gradients in the hemisphere in spino-olivo-cerebellar climbing fiber paths (Oscarsson, 1973). Somatotopy in the dorsal spinocerebellar tract was discussed by Xu and Grant (1988) and for the cuneocerebellar projection by Jasmin and Courville (1987b) and Hummelsheim et al. (1985). The ventral spinocerebellar tract is a composite pathway that contains crossed components from lower sacrococcygeal segments, from lumbar spinal border cells and from different cell groups in the lumbar intermediate zone (Matsushita and Hosoya, 1979; Matsushita et al., 1979; Grant et al., 1982; Matsushita and Ikeda, 1980; Matsushita and Yaginuma, 1989 and Yaginuma and Matsushita, 1989). The lower lumbar and sacrococcygeal component terminates preferentially in the apical part of the rostralmost lobules I and II of the anterior lobe. Spinal border cells project to a more extensive area, including the lobules I-V (Fig. 206D). The terminations of the spinal border cells are mainly ipsilateral to their origin, i.e. the fibers recross in the cerebellar commissure. They are distributed to the apical part of the lobules II-V of the anterior lobe (Yaginuma and Matsushita, 1986, Xu and Grant, 1990). The rostral spinocerebellar tract takes its origin from cell groups in Rexed's (1954) laminae VI, VII and VIII of the intermediate zone and a cell group in lamina V of the dorsal horn. The fibers from lamina VIII cross within the cord, the others ascend in the ipsilateral lateral funiculus (Petras, 1977; Petras and Cummings, 1977; Snyder et al., 1978; Matsushita et al., 1978, 1979; Wiksten and Grant, 1980, 1986). The rostral spinocerebellar tract terminates more dorsally than the dorsal and ventral spinocerebellar tract, in the lobules IV, V of the anterior lobe and in the lobules VI, VIII and the paramedian lobule. Their distribution is bilateral, but mainly ipsilateral. Spinocerebellar fibers from lower cervical segments terminate mainly in the vermis of the simple lobule (Matsushita et al., 1985; Matsushita and Ikeda, 1987). The central cervical-cerebellar projection is crossed, with a bilateral distribution in the lobules of the anterior lobe, the bottom of the primary fissure and lobule VIII (Fig. 206A) (Wiksten, 1979a,b; see Matsushita and Tanami, 1987 for a survey of the literature). The termination of the central cervical fibers overlaps with the secondary vestibu289
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locerebellar projection (Matsushita and Wang, 1987) (Fig. 206B) rather than with other spinocerebellar tracts. An important collateral projection to the cerebellar nuclei takes its origin from the central cervical nucleus (Matsushita and Yaginuma, 1995). The central cervical nucleus itself is a site for convergence of vestibulospinal and propriospinal input from neck muscles (Hirai et al., 1978, 1984; Hirai, 1987). Systematic and complete studies with antegradely transported axonal markers on the 290
The cerebellum." chemoarchitecture and anatomy
Ch. I
distribution of mossy fibers from the paramedian and lateral reticular nuclei are few. A rough idea of their lobular distribution can be gained from the reviews and the papers on retrograde axonal transport of Somana and Walberg (1978), Dietrichs and Walberg (1979b), Gould (1980), Qvist (1989a) and Ruigrok and Cella (1995). The trigeminocerebellar projection recently was studied and reviewed by Ikeda and Matsushita (1992). The basal pontine nuclei, the nucleus reticularis tegmenti pontis and the adjacent paramedian pontine reticular formation are connected with the cerebellum through the middle cerebellar peduncle. Both the nucleus reticularis tegmenti pontis and the paramedian pontine reticular formation send their fibers through the midline raphe into the pes pontis (fibrae rectae), where they deflect laterally to occupy the deep stratum of the middle cerebellar peduncle. The paramedian pontine reticular formation of the cat projects bilaterally to lobule VII and the caudal part of lobule VI and to the ansiform lobule, i.e. to the visual areas of the cerebellum involved in control of saccades (Gerrits and Voogd, 1986; Yamada and Noda, 1987). Fibers of the reticular nucleus of the pons distribute bilaterally, with ipsilateral predominance, to all lobules of the cerebellum, with the exception of the lobules I and X and the dorsal paraflocculus (Kawamura and Hashikawa, 1981; Gerrits and Voogd, 1986). This projection includes the flocculus and the adjacent part of the ventral paraflocculus (Fig. 203) (Gerrits and Voogd, 1989) and collateral projections to the cerebellar nuclei (see Section 5.6.). The projection through the middle cerebellar peduncle of rostral and caudal parts of the pes pontis to the cerebellum is reversed (Von Bechterew, 1885; Spitzer and Karplus, 1907; Voogd, 1964; Voogd et al., 1990). Fibers originating in the ventral and superficial layers of the pes pontis travel in superficial layers of the middle cerebellar peduncle and terminate preferentially in caudal and ventrolateral parts of the cerebellum. Von Bechterew (1885) was able to distinguish this pathway (his 'cerebral', i.e. rostral system) because it acquires its myelin rather late. One of its main constituent is the corticopontocerebellar projection from the visual cortex to the caudal vermis and the paraflocculus. Fibers from caudal and central portions of the pes pontis occupy deeper layers of the peduncle and distribute to more rostral parts of the cerebellum. This pathway corresponds to the early myelinating 'spinal' (i.e. caudal) component of the middle cerebellar peduncle of Von Bechterew (1885). It conveys the cortico-pontocerebellar projection to the anterior lobe. Attempts to analyse the pontocerebellar projection in more detail have shown that the organization of this pathway is extremely complicated. Injections of retrograde tracers in single lobules usually result in bilateral labelling of multiple cell columns or shells. The afferent projections from the cerebral cortex and other sources showed the same degree of dispersion. Our knowledge of informationflow in different cortico-pontocerebellar subsystems, therefore, remains incomplete. The cortical and non-cortical afferents and the efferent connections of the pons were studied and reviewed by Brodal and Jansen (1946), Mower et al. (1979), Rosina and Provini (1980, 1984), R Brodal (1968a,b, 1971, 1972, 1978a,b, 1982, 1987), Gerrits and Voogd (1986, 1987, 1989), Keizer et al. (1984), Ugolini and Kuypers (1986), Gerrits and Voogd (1989), Aas (1989), Bjaalie et al. (1991), R Brodal and Bjaalie (1987, 1992) and Nikundiwe et al. (1994) for the cat; Eisenman (1980), Eisenman and Noback (1980), Azizi et al. (1981, 1985), Anderson and Flumerfelt (1984), Angaut et al. (1985), Kosinski et al. (1986), Leggen et al. (1989), Wells et al. (1989), Mihailoff et al. (1989), Mihailoff (1993), Voogd (1995) and Ruigrok and Cella (1995) for the rat; Mihailoff et al. (1980) for the opossum; R Brodal (1980, 1982), Langer et al. (1985a), Schmahmann
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Fig. 202. Cuneocerebellar projection to ipsilateral cerebellum in the cat. Left side: diagrams from antegrade axonal transport of [3H]leucine in transverse sections. Borders of compartments in adjacent Hfiggqvist-stained sections are indicated on the right. Abbreviations: A(1-3) - A(1-3)compartment; 'A' = concentration of mossy fibers in A compartment; B = B compartment; bp = brachium pontis; 'C1 + C 2 ' = concentration of mossy fibers in C1 and C2 compartments; C1-3 = C1-3 compartment; 'C3' - concentration of mossy fibers in C3 compartment; cr = restiform body; D = Dcompartment; ' D ' = concentration of mossy fibers in D compartment; F = fastigial nucleus; F L - flocculus; HVI - hemisphere of lobule VI (simple lobule); IA = anterior interposed nucleus; L = dentate nucleus; PAR = paramedian lobule; P F L D = dorsal paraflocculus; PFLV = ventral paraflocculus; X = X compartment; 'X/B' --- concentration of mossy fibers in border region of X and B compartments. Gerrits et al. (1985b). (
and Pandya (1989, 1993), Fries (1990), Glickstein et al. (1985, 1994), Stein and Glickstein (1992) and Yhielert and Thier (1993) for the monkey. 6.4.2. Zonal arrangement in the termination of mossy fibers: Correlations with cytochemical maps
During their course as semicircular fibers mossy fibers branch extensively. Some branches are distributed to the anterior and the posterior lobe. This type of branching, which was studied by Heckroth and Eisenman (1988), may explain the mirrored somatotopy which is present in the two lobes. The branches in the medullary rays are of a small calibre. In the granular layer the mossy fibers branch preferentially in a direction across the long axis of the folium (Scheibel, 1977), i.e. with the same orientation as the climbing fibers (Fig. 6). The termination of entire mossy fiber systems in the granular layer often consist of a number of parallel longitudinal arrays of mossy fiber rosettes (van Rossum, 1969). These strips are not as sharply delimited as the terminal zones of the climbing fibers in the molecular layer. Discrete strips often are visible only in the periphery of the projection field; in the center the strips coalesce into a single field. Corresponding concentrations of stem fibers are present in the cerebellar white matter. The termination of mossy fibers in longitudinal strips was first observed and illustrated for the termination of the spinocerebellar fibers in the rabbit (Voogd, 1967; Van Rossum, 1969). It was also reported for the spinocerebellar projections in Tupaia glis and the ferret (Voogd, 1969), the cat (Voogd, 1969 and the series of papers of Matsushita c.s., cited above), Trichosuris vulpecula (Watson et al., 1976), the Virginia opossum (Hazzlet et al., 1971) and the rat (Gravel and Hawkes, 1990; Tolbert et al., 1993). A zonal pattern is also characteristic for the termination of the cuneocerebellar tract in the cat (Voogd, 1969; Gerrits et al., 1985b; Jasmin and Courville, 1987a and b) and rat (Ji and Hawkes, 1994), projection of the lateral reticular nucleus both in cat (Ktinzle, 1975; Russchen, 1976) and rat (Chan-Palay et al., 1977) and in the secondary vestibulocerebellar projections to lobule IX and the anterior lobe (Epema et al., 1985; Matsushita and Wang, 1987) (Fig. 206B). Zonation has not been observed in the distribution of the primary and secondary vestibulocerebellar root fibers to lobule X and the flocculus and was only observed in some parts of the vermis for the projections from the basal pontine nuclei and the reticular nucleus of the pons in the cat and the tree shrew (Voogd, 1969; Gerrits and Voogd, 1986; Kawamura and Hashikawa, 1981). The zonal distribution of spinocerebellar and pontocerebellar fibers in Tupaia glis (Voogd, 1969) is illustrated in Figs 204 and 205, made from silver impregnated sections with large lesions of the pes pontis and low cervical cordotomies. The zonal pattern is striking in the tree shrew and the zonation in the spinocerebellar projection is generally similar to that in other species. A precise comparison of the spinocerebellar and ponto293
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Fig. 203. Mossy fiber projections to the flocculus and the adjacent paraflocculus in the cat. Based on antegrade tracing experiments with tritiated leucine. Notice the lack of basal pontine and reticulopontine projections to the flocculus and their presence in the medial extension (ME) and the caudal lobules (PFLVc) of the ventral paraflocculus in the upper three diagrams. Vestibulo-cerebellar fibers in lower two diagrams terminate both in the flocculus and the ME. A, AP - stereotactic planes; DV = descending vestibular nucleus; FL = flocculus; LV = lateral vestibular nucleus; ME = medial extension of the ventral paraflocculus; MV = medial vestibular nucleus; N P - nuclei pontis = NRTP = nucleus reticularis tegmenti pontis; P F L D - - dorsal paraflocculus; PFLV(c) = (caudal folium of the) ventral paraflocculus; SV - superior vestibular nucleus. Gerrits and Voogd (1989). (
cerebellar projections is not possible, because the experiments were done in different animals. It is not known whether they interdigitate or overlap. More detailed observations on the zonation in the spinocerebellar and trigeminocerebellar projection were reported by Matsushita c.s. for different spinocerebellar pathways in the cat. They plotted the mossy fiber rosettes on reconstructions of the surface of the individual folia. Data on the distribution of mossy fibers on the dorsal (caudal) surface of lobule IV are assembled in Fig. 206. Spinocerebellar fibers from the central cervical nucleus (Fig. 206A) (Matsushita and Tanami, 1987) and the medial vestibular nucleus (Fig. 206B) (Matsushita and Wang, 1987) terminate in three, presumably overlapping zones 1-3 in the bottom of the lobule. Zone 3 is stated to be located at the border of the zones A and B. This interpretation is supported by our map of the distribution of AChE in rostral lobule IV (Fig. 206G). Spinocerebellar fibers from the cervical enlargement are located in the same three zones, in more apical parts of the lobule (Matsushita et al., 1985; Matsushita and Ikeda, 1987). Spinocerebellar fibers from the thoracic cord distribute to the midline and to three parasagittal zones, numbered 2-4. Zone 4 is situated in the medial B zone, a number of patches are present in the lower portion of the hemisphere (Fig. 206C) (Yaginuma and Matsushita, 1987). Spinal border cells project to more apical parts of the hemisphere, with the most medial zone 1 being located at the border of the B and C1 zone (Fig. 206D) (Yaginuma and Matsushita, 1986; Matsushita and Yaginuma, 1989). Fibers from the lower lumbar cord distributed widely over vermis and hemisphere in a distinct zonal pattern (Yaginuma and Matsushita, 1989). The termination of lower lumbar, sacral and coccygeal fibers in the apical parts of the lobules is mostly restricted to a single band around two mm. from the midline, i.e. overlapping the B zone (Matsushita, 1988). Similar plots from the cuneocerebellar (Fig. 206E) and the basal pontocerebellar projection (Fig. 206F) were reproduced from Gerrits (1985). Cuneocerebellar fibers terminate in bands in the vermis and the hemisphere, the gap that separates them presumably corresponds to the B zone. Pontocerebellar fibers are mainly restricted to the apical hemisphere, i.e. the C and D zones, and are scarce in the vermis. The mossy fibers in lobule IV, therefore, belong to different pathways, that terminate in transversely oriented projection fields with a different baso-apical distribution. Vestibulo-, spino- and cuneocerebellar fields can be subdivided into rostro-caudally oriented concentrations of mossy fiber rosettes. The different fields and their concentrations of terminals partially overlap. Correlations between the terminations of mossy fibers and cytochemical maps are rare. Gerrits et al. (1985b) mapped the localization of cuneocerebellar fibers with respect to the borders of white matter compartments in adjacent, H~iggqvist-stained sections (Fig. 202). The projections of low thoracic-lumbar cord to the cerebellum of the rat were compared to the localizations of Zebrin l-immunoreactive Purkinje cells in surface maps of all relevant cerebellar lobules by Gravel and Hawkes (1990). These authors noticed 295
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a detailed correspondence between the zonal distribution of spinocerebellar fibers in the rat with that reported for other species. The P1 + Zebrin band in the anterior lobe usually overlaps with a spinocerebellar cluster, a second cluster is located under P 1-, few terminals underlie P2+ and a third concentration of mossy fiber rosettes coincides with 296
The cerebellum." chemoarchitecture and anatomy
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P 2 - (i.e. the B zone) extending into P3+ (Fig. 207). Matsushita et al. (1991), who mapped fibers from the cervical cord in Zebrin-I stained sections of rat cerebellum found less correspondence of the concentrations of rosettes with the borders of the immunoreactive Purkinje cell zones. Ji and Hawkes (1994) showed that cuneocerebellar mossy fiber terminals are located between the concentrations of lumbar spinocerebellar mossy fiber rosettes in P1 +, P1- and P2- of lobules II and III of the rat cerebellum (Fig. 207). A close correspondence between multiple patches of mossy fibers with vibrissal receptive fields and the Zebrin-negative P 1-, P2- and P3- zones of lobule IX of the rat cerebellum, was observed by Chockkan and Hawkes (1994). An organization of the spinocerebellar projection in medio-laterally oriented bands, located mainly near the junctions of the lobules I-II, II-III and III-IV (i.e. in the bottom 297
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Fig. 207. Lobules II/III of the rat anterior lobe vermis: a schematic representation of the relationships between Zebrin+/- bands of Purkinje cells and the mossy fiber projections from the external cuneate nuclei, and lumbar, thoracic and cervical levels of the spinal cord. There is one Zebrin + PC compartment at the midline (P1 +) and two others (P2 + and P3 +) positioned laterally. P1 + and P2 + are separated by P 1-, P2 + and P3 + by P2-. More laterally there is also a weak P4+ compartment (not illustrated). Terminals of the cuneocerebellar tract are clustered under P1 + (the Cul cluster), and within P 1 - (Cu2) and P 2 - (Cu3). Only the medial edge of Cu3 clearly corresponds to a Zebrin+/- boundary. The lumbar and thoracic spinocerebellar terminals (Gravel and Hawkes, 1990) have very similar distributions (the L1/T12, L2/T2 and L3/T3 fields) and are complementary to the cuneocerebellar projection. Statistical analysis suggests that the lumbar L2 terminal field is narrower than the thoracic T2 and so is illustrated here as fading towards its medial edge. The empty space aligned with Cul and splitting L1 and T1 at the midline is questionable: neither a split within L1/T1 nor the presence of Cul is unambiguous. The lateral edge of Cu3 abuts the medial edge of L3/T3. More laterally, L3/T3 are drawn as aligned with the lateral edge of P3 +: this is speculative as the P3+ band cannot confidently be identified in double-labelled sections. The MF terminals from the cervical spinal cord are shown as spread uniformly across the vermis. Ji and Hawkes (1994).
of the fissures), separated by terminal-free areas and the presence of anterior-posteriorly aligned regions within these bands with high and lower densities of the terminals, was shown in computer reconstructions of the unfolded cerebellar cortex of the rat by Tolbert et al. (1993). It was implied by these authors that the anterior-posteriorly oriented zonation in the spinocerebellar projection was less distinct than had been suggested by previous authors. We would agree that some of the published diagrams of the zonation in the spinocerebellar projection exaggarate the sharpness of this projection. In reality the localization is more diffuse, especially in the bottom of the fissures. Parasagittal focussing in mossy fiber systems is most pronounced in the white matter, in the granular layer the mossy fibers disperse.
6.4.3. The somatotopical organization in mossy fiber pathways The somatotopical organization and the convergence in somatosensory mossy fiber paths has been investigated in great detail in the micromapping studies in the rat and other species using natural stimulation of Welker and his collaborators (see Welker, 1987 299
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of the fractured somatotopy of the mossy fiber projections in the cerebellum of the rat. Patches with similar receptive fields are indicated with abbreviations for the stimulation sites on the head and the extremities. Redrawn from Welker (1987). Cr = crown; El - eyelids; Fbp = furry buccal pad; F L = forelimb and hand; G = gingiva; H L = hindlimb; I, I] = crus I and II; Li = lower incisor; L1 -- lower lip; Lob.ant. = anterior lobe; lob.sim = lobulus simplex; N = nose; Nk = neck; P = pinna; P F L = paraflocculus; P M L = paramedian lobule; PY = pyramis; Rh = rhinarium; Ui = upper incisor; U1 = upper lip; UV = uvula.
300
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for a review). They observed a mosaic of small (0.1-0.3 m m 2) columnar modules or patches in the granular layer that receive short latency somatosensory projections from receptive fields located within a specific body structure (Fig. 208). Receptive fields from specific body structures often are represented in multiple patches on different folia and this distribution is not necessarily longitudinal. Topographical continuities in the periphery are not maintained in the projection to the patches in the granular layer. The arrangement of adjacent patches is asomatotopic and the mossy fiber sources of adjacent patches are disjunctive. This type of fractured somatotopy is found in parts of the hemisphere (Crus I and II, paramedian lobule) and vermis (lobules VIII and IX) in rat, opossum and cat; it is folia-specific and highly reproducible. Branching was found of axon collaterals from single cells in the spinal trigeminal nucleus between pairs of patches with the same receptive fields located on the same or on a different folium, or between patches located in vermis and hemisphere (Woolston et al., 1981). Projections from the somatosensory cortex and the superior colliculus, moreover, were found to conform to the patchy mosaic of projections of the periphery to the granular layer of the rat cerebellar hemisphere (Bower et al., 1981; Kassel, 1980). Welker's observations raise interesting questions about the sites of convergence of mossy fiber pathways mediating peripheral, cortical and tectal information, about the significance of a detailed somatotopical localization (fractured, or longitudinal) in the first link of a mossy fiber-parallel fiber pathway, which would get blurred or even lost in the transvere projections of the parallel fibers and, finally, about the relationship between fractured somatotopy and longitudinal zonation in corticonuclear and climbing fiber pathways and even in the mossy fiber systems themselves. Convergence in mossy fiber pathways may occur at precerebellar level. Convergence of tecto- and corticocerebellar pathways takes place in the pontine nuclei; a direct tectocerebellar pathway does not exist. It is not clear how much of the convergence between peripheral and cortical pathways takes place at a precerebellar level. For certain climbing fiber pathways from the cerebral cortex and the spinal cord it has been shown that this convergence occurs in the sensory relay nuclei (Andersson, 1984). The terminations of certain spinocerebellar tracts certainly overlap with the pontocerebellar projection in the apex and in the hemispheral portions of the lobules (Figs 205 and 206) and the same probably holds for the trigeminocerebellar projection (Jasmin and Courville, 1987a). Systematic convergence of peripheral and cortico-pontine input on the same glomeruli or on the same group of granule cells, therefore, is possible, but has never been studied at the necessary level of precision with anatomical methods. Electrophysiological investigations of somatotopic localization in the granular layer, be it longitudinal zonal (Ekerot and Larson, 1980) or fractured (Bower and Woolston, 1983) both adduced evidence that the same somatotopical pattern is transmitted to the overlying Purkinje cells. These authors found no evidence for lateral spread along the parallel fibers. Llinas (1982) (see also Welker, 1987) explained the preferential connection of granule cells with the Purkinje cells overlying them by the greater greater number of parallel-Purkinje cell synapses on the ascending part of the parallel fiber, but this leaves the function of the long sidebranches of the parallel fiber unexplained. The presence of multiple representations of the same body part in the granular layer is in accordance with the description of mossy fibers as mainly transversely oriented, bilaterally distributed semicircular fibers, which give off collaterals during their course. Multiple representations of the same body part are also present as microzones in different longitudinally oriented climbing fiber systems. Cerebral and peripheral input has been found to converge on to these microzones (Andersson and Eriksson, 1981; 301
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Andersson and Nyqvist, 1983) (see Section 6.3.3.1., Fig. 176). Since zonal borders were never determined in Welker's studies, it is not known whether the mossy fiber patches and the climbing fiber-microzones correspond. A crude, somatotopically arranged convergence of certain climbing and mossy fiber paths from the peripheral nerves and the somatomotor cortex to the cerebellum has been noticed by several authors (Provini et al., 1967, 1968; Ekerot and Larson, 1973, 1980). A topographical correspondence also has been noticed for mossy and climbing fibers containing the same peptides (see next Section 6.3.4.). The convergence of mossy and climbing fibers, that share the same somatotopical and neurochemical properties, on to the same regions of the cerebellar cortex, is remarkable because they reach their targets by quite different routes. This convergence may be explained by the presence of a common developmental factor that determines the distribution of these afferents over the cortex (see Section 6.2.). 6.4.4. Collateral projections of mossy fiber systems to the cerebellar nuclei. The nuclear projection of the red nucleus
Projections from mossy fiber systems to the cerebellar nuclei (see also Section 5.6.) were traced from the spinal cord, the lateral reticular nucleus and the nucleus reticularis tegmenti pontis and adjacent parts of the pontine nuclei. Projections from the vestibular nuclei are still disputed. No collateral projections to the nuclei were traced from the cuneocerebellar system. A special position is taken by the projection from the red nucleus to the interposed nucleus. Evidence for a collateral origin from mossy fibers terminating in the cerebellar cortex was provided by Qvist (1989a and b) with retrograde double-labelling from the cortex and the cerebellar nuclei and by Shinoda et al. (1992) with intraaxonal labelling for the collateral projections from the lateral reticular nucleus, the reticulo-tegmental nucleus and the pontine nuclei of the cat. Spinal projections to the cerebellar nuclei have been reported by several authors in cat (Szentagothai in Eccles et al. 1967; Voogd, 1969; Matsushita and Ikeda, 1970; Ikeda and Matsushita, 1973; Robertson et al., 1983), from the cervical enlargement and the central cervical nucleus in the rat (Matsushita and Yaginuma, 1990, 1995) and Tupaia (Fig.204). They terminate bilaterally in both interposed nuclei and in the fastigial nucleus. The projection from the central cervical nucleus in the rat terminates mainly contralaterally in the rostral fastigial nucleus and mainly in central portions of the anterior and posterior interposed nuclei, excluding the caudal fastigial nucleus, the dorsolateral protuberance, the dorsolateral hump, the lateral cerebellar nucleus and the nucleus of Deiters. The collateral projections from the lateral reticular nucleus terminate in the same regions of the cerebellar nuclei as the direct spinocerebellar projections. According to Matsushita and Ikeda (1976) they are absent from the lateral cerebellar nucleus in the cat, but according to Dietrichs (1983b) certain parts of the lateral nucleus receive lateral reticular afferents. A weak projection of the lateral reticular nucleus to the lateral vestibular nucleus that was described by Dietrichs and Walberg (1979a) in the cat, recently was confirmed in the rat (Ruigrok et al., 1995). Collateral projections from the pontine nuclei were mostly traced from the nucleus reticularis tegmenti pontis. Smaller contributions from the dorsolateral and medial pontine nuclei were found by Gerrits and Voogd (1987) in the cat and Mihailoff (1993) in the rat. Their termination is mostly in the lateral part of the posterior interposed nucleus and in the lateral cerebellar nucleus. The caudal pole of the fastigial nucleus receives a projection in cat and Tupaia (Fig.204). It appears as though the collateral 302
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projections of spinocerebellar and reticulotegmental pontine mossy fibers are found in parts of the nuclei that receive a projection of the Iobules that are strongly innervated by these fibers. This topic was illustrated and discussed by Gerrits and Voogd (1987) for the connections of the nucleus reticularis tegmenti pontis with the cerebellar nuclei and the paraflocculus of the cat. The complementarity in the projections of the spinal cord and the pontine nuclei (including the nucleus reticularis tegmenti pontis) in Tupaia is illustrated in Fig. 204. A connection from the red nucleus to the cerebellar nuclei of the cat was described with the retrograde cell degeneration method by Brodal and Gogstad (1954). This connection was confirmed with retrograde tracers in the rat (Huisman et al., 1983; Naus et al., 1985). Huisman et al. (1983) using fluorescent double-labelling techniques, showed that the rubrocerebellar fibers arise as collaterals from the rubrospinal tract. Rubrocerebellar fibers were found to terminate in the anterior interposed nucleus with axonal degeneration methods in the cat (Courville, 1968; Courville and Brodal, 1966). Walberg and Dietrichs (1986) suggested that the rubronuclear projection is either small or absent. Rubrocerebellar fibers were found to terminate in intermediate parts of the cerebellar cortex (Dietrichs and Walberg, 1983). Retrograde degeneration or labelling of rubral cells in previous studies was explained by the interruption of fibers of passage by large lesions or injections of the cerebellar nuclei. 6.4.5. The chemoarchitecture of mossy fibers It is generally assumed that the distribution of mossy fiber rosettes over the cerebellar cortex is uniform and that all mossy fibers use glutamate as a neurotransmitter (Raymond et al., 1984; Beitz et al., 1986; Clements et al., 1986, 1987; Kaneko et al., 1987, 1989). Intense immunoreactivity, using antibodies against phosphate-activated glutaminase or conjugates of glutamate was present in many neurons of the precerebellar nuclei of the rat, including the basal and reticular tegmental pontine nuclei, the vestibular ganglion, the medial and superior vestibular nuclei and the groups x, y and f, the nucleus prepositus hypoglossi, the external cuneate nucleus, the lateral reticular nucleus and the paramedian reticular formation (Beitz et al., 1986; Kaneko et al., 1989). There is strong evidence, however, for more heterogeneity because certain mossy fibers have been shown to be cholinergic and others to contain several neuroactive peptides. Serotonin has been demonstrated in certain mossy fibers with high resolution autoradiography after topical or ventricular infusion of [3H]serotonin in rodents (Chan-Palay, 1975; Beaudet and Sotelo, 1981). Authors using immunocytochemical methods for serotonin in rat, opossum and cat, however, concluded that varicose serotoninergic fibers distribute throughout the cerebellar cortex, but that few if any terminate as mossy fibers (Bishop and Ho, 1985; Bishop et al., 1985; Takeuchi et al., 1982). The presence of ChAT-immunoreactive mossy fibers in the lobules X and IX of the caudal vermis and the flocculus in several mammalian species was discussed in Section 3.10.1. ChAT-positive mossy fiber rosettes were most numerous in the caudal vermis of the rat, the rosettes were large in X and smaller in ventral lobule IX and the lobules I-III of the anterior lobe (Fig. 83). The ChAT-positive mossy fiber innervation of the flocculus was restricted to the ventral folium and the ventral half of the dorsal folium of this lobule. It was less dense than the innervation of lobule X, but a particularly dense plexus of thin, beaded fibers, which may detach from the mossy fibers, was present in this lobule. ChAT-immunoreactive mossy fibers innervating the lobules I, II, IX and X of the rabbit, were most numerous in the banks of the precentral and posterolateral 303
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fissures. The flocculus receives a sparse innervation, but a heavy concentration of ChAT-positive mossy fibers is present in the ventral paraflocculus of the rabbit. ChAT activity measurements (Fig. 84) revealed that the highest ChAT activity of any cerebellar region in rat, rabbit, cat or monkey ever measured was found for folium 2 of the ventral paraflocculus of the rabbit (Barmack et al., 1992a). The origin of these cholinergic mossy fibers was traced from the caudal medial vestibular nucleus and the nucleus prepositus hypoglossi, with double-labelling of retrogradely transported HRP from injections in the caudal vermis and the flocculus and ChAT immunohistochemistry, both in rat and rabbit (Fig. 201) (Barmack et al., 1992b). Single and double-labelled ChAT-immunoreactive neurons were primarily found in the caudal part of the medial vestibular nucleus, the nucleus prepositus hypoglossi and the vestibular efferent nucleus. They were absent from the superior nucleus. Single HRP labelled cells were present in all vestibular nuclei with the exception of the lateral vestibular nucleus. The cholinergic projection to the ventral paraflocculus of the rabbitis exclusively derived from the nucleus prepositus hypoglossi. In other brain stem nuclei only a few neurons in the lateral reticular nucleus were double labelled for HRP and ChAT following injections in the lobules X and IX in rat and rabbit. Several peptides (enkephalin-ENK, CCK, CRF, calcitonin gene-related peptide, CGRP) have been localized in mossy fibers. When they are present these mossy fibers occur ubiquitously, but they display a preferential localization in certain lobules and, for ENK-CCK and CRF, at least, in midline and parasagittal bands in vermis and paravermis. These concentrations of mossy fiber rosettes usually are aligned with the climbing fiber bands containing the same peptide (Figs 191-195). ENK-, CRF- and CCK-like immunoreactivity in mossy fibers has been reported in the opossum (King et al., 1986a,b, 1987; Cummings and King, 1990; Cummings et al., 1989; King and Bishop, 1990). With respect to their distribution the three peptides differ. Enkephalin containing mossy fibers are limited to the vermis and the the flocculonodular lobe. CRF-containing fibers are more numerous; the vermis of lobule VI and the flocculonodular lobe are densely CRF-innervated. Sagittal and parasagittal concentrations of mossy fibers containing CRF-like immunoreactivity are particularly evident in the anterior lobe and lobules VII-IX. Scattered mossy fibers are present in the hemispheres. CRF and ENK-like immuno-reactivity co-exists in some mossy fibers (and climbing fibers) in the midsagittal and parasagittal bands in the caudal vermis and in the flocculus (Cummings and King, 1990). Numerous CCK-containing mossy fibers are present in all lobules of the opossum cerebellum, with indications of a zonal distribution in the vermis of lobule III and in parts of the caudal vermis (Fig. 193). Enkephalinergic mossy fibers have also been reported in the rat, where they are universally distributed, with a preference for the vermis (Schulman et al., 1981). CRF also occurs in mossy fibers in cat and sheep (Cummings et al., 1988; Cummings, 1989), rat (Van den Dungen et al., 1988) and rabbit (Errico and Barmack, 1993). The distribution of CRF-immunoreactive mossy fibers in the cerebellum of the cat is very similar to the opossum, with concentrations of mossy fibers underlying the stained bands of immunoreactive climbing fibers in vermis and pars intermedia, and heavy labelling in the flocculonodular lobe (Cummings, 1989) (Fig. 194). The localization of CGRP-immunoreactive mossy fibers over the cerebellum of the cat differs substantially from that of the other peptides. They are present in the paraflocculus, the paramedian and ansiform lobules and in the pars intermedia of the simple lobule and the anterior lobe. In the anterior vermis they are located in the apices of the lobules. 304
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In the posterior vermis they are concentrated in the lobules VII and VIII and in the deep part of the granular layer of dorsal lobule IX. They are absent from ventral IX, X and the flocculus. Some CRF-positive mossy fibers can be labelled in monkeys (Saimiri sciureus and Macacafasciculara, Foote and Cha, 1988) but they seem to be less frequent than in other mammalian species. Some of these CRF-immunoreactive rosettes may originate as collaterals from the climbing fibers in these species. Neurons in several precerebellar nuclei which are known to give rise to mossy fibers are immunoreactive for one or more of these peptides. CRF-like immunoreactivity was located in the medial and descending vestibular nuclei (including groups f and x), the nucleus prepositus hypoglossi, the lateral reticular nucleus and other parts of the medullary reticular formation and the solitary tract nucleus in rat (Olschowka et al., 1982; Sakanaka et al., 1987; Van den Dungen et al., 1987, 1988), opossum (Cummings et al., 1989; Cummings and King, 1990) and cat (Cummings, 1989). These authors also found such cells in the external cuneate nucleus of the cat (but not in the opossum) and in the spinal trigeminal nucleus, the locus coeruleus and the raphe nuclei of both species. The profusion of CRF-immunoreactive neurons that could be retrogradely double-labelled from injections in the caudal vermis of the rabbit cerebellum was stressed by Errico and Barmack (1993). Some of these nuclei also contain CCK-like immunoreactive neurons in the opossum and ENK-like immunoreactive cells both in rat and opossum (i.e. the nucleus prepositus hypoglossi, the medial vestibular nucleus, the lateral reticular nucleus, parts of the medullary reticular formation and certain raphe nuclei (Finley et al., 1981; Williams and Dockray, 1983; King et al. 1987; Walker et al., 1988; Cummings and King, 1990). ENK- and CCK immunoreactive neurons could be double-labelled with injections of HRP in the cerebellum of the opossum (Walker et al., 1988; King and Bishop, 1990). CCK/HRP double-labelled neurons also were present in the medial vestibular and prepositus hypoglossi nucleus. Co-existence of these peptides in single neurons of these nuclei has also been demonstrated for ENK with CRF (Cummings and King, 1990) CGRP-containing neurons were double-labelled with HRP from injections in the cerebellum of the cat in the lateral reticular nucleus, the external cuneate nucleus, the descending vestibular nucleus and in the lateral and ventral divisions of the basilar pons (Bishop, 1992). The origin of a major contingent of CGRP-immunoreactive mossy fibers from the pontine nuclei may explain their preferential distribution to the cerebellar hemisphere.
7. P O S T S C R I P T
7.1. BIOCHEMICAL CORRELATES OF CELL TYPES AND FIBER SYSTEMS The chemical neuroanatomy of the cerebellum offers many examples of cell types, grisea and their connections, that can be recognized on the basis of the expression of particular biochemical markers. However, its significance extends far beyond mere recognition of what was already known. In fact, previous unrecognized or neglected cell types, such as the unipolar brush cell (Section 3.6.2.) and the glycinergic interneuron of the cerebellar nuclei (Section 5.3.), and new connections like the multilayer plexus of monoaminergic and cholinergic afferents (Sections 3.8., 3.9. and 3.10.), have been uncovered by virtue of their biochemical properties. Existing cell populations could be split. In 305
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particular, the Purkinje cells can be subdivided into multiple, biochemically distinct subpopulations, distinguished by their contents of certain neuropeptides (Sections 3.1.2. and 3.1.3.) protein kinase C subtypes (PKC~: Section 3.1.5.), receptors (GABAB receptors, muscarinic receptors, and possibly substance P and vasopressin receptors: Sections 3.7.2., 3.10.2. and 6.1.3.) or other biochemical markers (Section 3.1.8.). Several of these biochemically distinct subpopulations are located in longitudinal zones and/or are restricted to certain lobules of the cerebellum. Purkinje cells proved to be particularly rich in markers related to the inositol phosphate-second messenger pathways and the control of intracellular CaZ+-concentration (Section 3.1.4.). Some markers such as cyclic GMPdependent protein kinase, L7 and the InsP3-recptor protein have proven useful to outline the development, the somatodendritic and axonal extent and the ultrastructure of the Purkinje cells (Sections 3.1.4., 3.1.8., 6.2.). Specific markers for granule cells are less numerous. Granule cells, however, uniquely express specific types of glutamate and GABA receptor sububits (see Sections 3.3. and 3.7.). Granule cells are heterogeneous in their expression of calretinin and a zonal distribution has been reported for nitric oxide synthase (Section 3.4.) in granule cells. Among the GABAergic interneurons of the cerebellar cortex, the Golgi cells and the Lugaro cells are biochemically distinct from the basket and stellate cells, in that basket and stellate cells both react with antibodies against calmodulin (Section 3.6.1.), and contain nitric oxide synthase (Section 3.4.). Golgi cells and Lugaro cells both have been identified with type-specific antibodies (Section 3.6.2.). Sub-populations of Golgi cells were distinguished on the basis of their enkephalin- and somatostatin-like immunoreactivities, the co-localization of GABA with glycine-like immunoreactivity (Section 3.6.2.) and the presence of the metabotropic glutamate receptor subunits mGluR5 and mGluR2/3 in (different?) populations of Golgi cells (Section 3.3.2.). Biochemical markers may be helpful in future studies to characterize subtypes of Golgi cells. One factor that hinders such a correlation is our insufficient knowledge of their axonal trajectories of the Golgi cells. Clues for a morphological taxonomy of the Golgi cells are embodied in the finding of a mainly transverse orientation of Golgi cell axons in the direction of the long axis of the folium by De Zeeuw et al. (1994c) and the discovery of the candelabra cell in the cerebellar cortex as a new type of (Golgi?) cell with a transversely oriented axon, distributed to the molecular layer by Lain6 and Axelrad (1994) (Section 2). The distinction of the small, GABAergic neurons of the cerebellar nuclei that give rise to the nucleo-olivary projections, from the large, non-GABAergic relay cells (Mugnaini and Oertel, 1981) (Section 5.2.) certainly represents one of the most consistent correlations between a type of neuron with a specific projection, and its neurotransmitter. However, the dichotomy of the neurons of the cerebellar nuclei into GABAergic and non-GABAergic neurons was complicated by the discovery of small interneurons that co-localize GABA and glycine, by reports on the presence of GABAergic nucleocortical projections (Section 5.3.) and the occurrence of both excitatory aminoacid neurotransmitters as well as acetylcholine, glycine and cholecystokinin in large neurons of the cerebellar nuclei (Section 5.4.). No specific markers are available for mossy and climbing fibers. However, subsets of mossy and climbing fibers can be distinguished by their immunoreactivity towards antibodies against selected neuropeptides (Sections 6.3.4. and 6.4.4.). A subset of secondary vestibulo-cerebellar mossy fibers, taking their origin from the medial and spinal vestibular nuclei and the nucleus prepositus hypoglossi, reacts with antibodies against choline-acetyltransferase (Sections 3.10.1. and 6.4.1 .). 306
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7.2. NEUROTRANSMITTERS AND THEIR RECEPTORS One of the goals of chemical neuroanatomy is to uncover the chemical nature of signalling pathways. As in other parts of the brain the amino acids glutamate and GABA are likely to be the predominant neurotransmitters in the excitatory (mossy, parallel and climbing fiber) and inhibitory (intrinsic cortical and corticonuclear) pathways of the cerebellum respectively (Sections 3.2.1 .-3.2.3.). The characteristics of the excitatory and inhibitory responses of glutamate and GABA in part depend on the type of receptor expressed post-synaptically. Molecular biology has unraveled a great variety of ionotropic and metabotropic glutamate and GABA receptor types, and recent studies have shown that each cell type is provided with a characteristic set of glutamate and GABA receptor types (Tables 2 and 3). In particular granule cells express a large array of glutamate and GABA A receptor subunits. The functional significance of this is puzzling in view of the single type of excitotory mossy fiber and inhibitory Golgi cell input to the granule cells. Studies with transgenic mice may be useful in unravelling the specific roles of particular subunits in the cerebellar circuitry. Diffusely projecting extracerebellar afferents that contain monoamines (noradrenalin, serotonin, histamine and, possibly, dopamine, Sections 3.8. and 5.7.) and acetylcholine (Sections 3.10.1. and 3.10.2.) and their receptors have been identified. They terminate in a multilayer plexus in the cortex and in the cerebellar nuclei. It is generally assumed that these pathways play a modulatory role in the cerebellar circuitry, but their cellular targets are still largely unknown. Their functional importance was highlighted in papers on the cellular physiology of serotonin (e.g. Bishop and Kerr, 1992) and in the studies of the modulation of compensatory eye movements by adrenergic or cholinergic agonists and antagonists by Pompeiano and Collewijn and co-workers (Pompeiano et al., 1991; Tan and Collewijn, 1991, 1992a,b; Tan et al., 1991,1992,1993a,b; Van Neerven et al., 1991; Collewijn et al., 1992). 7.3. LOBULES AND ZONES The stock in trade of the classical neuroanatomist includes the subdivision of the cerebellum in the vestibulocerebellum and the largely somesthetic corpus cerebelli, the modular organization of the cerebellum and the subdivision of the cerebellar nuclei and the zonal and lobular patterns in the termination of mossy and climbing fibers. The distinction of the vestibulocerebellum as a specific subdivision of the cerebellum received support from studies of the transient biochemical properties of its primary vestibular mossy fibers (Morris et al., 1988) (Section 6.4.1.) and its climbing fiber afferents from the dorsal cap (Wassef et al., 1992a,b) (Sections 6.1.5. and 6.2.) during early stages of their development. Early observations on the high context of the vestibulocerebellum of acetylcholinesterase (ACHE) and cholinacetyltransferase (CHAT) were confirmed and extended by the preferential termination of ChAT-immunoreactive mossy fibers in the vestibulocerebellum of different mammalian species (Barmack et al., 1992a,b) (Sections 3.10.1. and 6.4.1.). Calretinin-immunoreactivity differentiates the adult vestibulocerebellum from other lobules by the strong staining of the unipolar brush cells that prevail in the vestibulo-cerebellum, and the relatively low immunoreactivity in granule cells and parallel fibers (Section 6.1.5.). The distribution of calretinin-immunoreactivity also emphasized the extension of the vestibulocerebellum, beyond the posterolateral fissure, into the ventral uvula and the paraflocculus. The compartmental organization of the white matter of the cerebellum as an expres307
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sion of the distribution of the Purkinje cells in longitudinal zones, the precise correspondence in the parasagittal arrangement of the olivocerebellar and the cortico-nuclear projections and the near perfect correlation of the anatomical zones with their electrophysiological counterparts have been known for several decades (Voogd, 1964, 1969; Oscarsson, 1969; Armstrong et al., 1973; Groenewegen and Voogd, 1977; Groenewegen et al., 1979; Voogd and Bigar6, 1980). It was concluded that the output of the cerebellum is organized as a series of modules that incorporate one or more Purkinje cell zones, their cerebellar or vestibular target nuclei, their climbing afferents and the reciprocally organized nucleo-olivary connections. Still, the discovery by Hawkes et al. (1985) (Sections 3.1.8. and 6.1.3.) in the rat, of a longitudinal zonal pattern in the distribution of Zebrinpositive and -negative Purkinje cells, with its perspective of a biochemical diversity of the modules, came as a surprise. As a pattern the zonal distribution of Zebrin was not new; it was preceded by the discovery of the 5'-nucleotidase patttern in the molecular layer of the mouse cerebellum by Scott (1964) (the identity of the two patterns was shown by Eisenman and Hawkes 1989) (see Section 6.1.3.) and of the AChE-positive and -negative zones in the molecular layer of the cerebellum of the cat (Marani and Voogd, 1977) (Sections 3.10.3. and 6.1.1 .). The great impact of the discovery of Zebrin on studies of the cerebellum was due to the unique localization of the Zebrin epitopes in the Purkinje cells, the distinctness of, and the facility in demonstrating the Zebrin pattern in different species with antibodies against Zebrin I and, later, against Zebrin II (Dor6 et al., 1990). The Zebrin pattern is positively or negatively correlated with the distribution of other Purkinje cell markers, discussed by Hawkes (1992) and Leclerc et al. (1992), and in Sections 3.1.8. and 6.1.3. of this chapter. Notably, the distributions of PKC~ (Chen and Hillman, 1993a) (Section 6.1.3.) and of nerve growth factor receptor protein (Section 3.1.10.) in the Purkinje cells conform to the Zebrin pattern. The distribution of certain substances in the Bergmann glia appears to be linked to the Zebrin pattern: this may be true for 5'-nucleotidase (Section 3.5.) and has been established for 3-fucosyl-acetyllactosamine (Bartsch and Mai, 1991) (Section 3.11.) that occurs preferentially in the Bergmann glia of the Zebrin-negative zones. Does the Zebrin pattern result from the interdigitation of two sets of Purkinje cells that differ in their biochemical properties and in their afferent and efferent connections? The truth, probably, is less simple. Purkinje cells of the A and B zones of rat cerebellum, that project to the lateral vestibular nucleus, are uniformly Zebrin-negative and are delimited by Zebrin-positive bands and satellite bands (Fig. 143). Other zones, that can be defined by their corticonuclear and olivocerebellar connections, such as the lateral extension of the A zone of Buisseret-Delmas (1988a), include both Zebrin-positive and Zebrin-negative regions (Fig. 144). Morever, uniformly Zebrin-positive lobules, like lobule VII, the nodulus, the flocculus and the paraflocculus, contain a complex zonal substructure (Sections 6.1.4., 6.1.5. and 6.3.3.3.). The recent maps of the corticonuclear and olivocerebellar connections of the cerebellum of the rat (Buisseret-Delmas and Angaut, 1993) are fairly accurate and based on the same principles as applied in the cat by Groenewegen and Voogd (1977), Groenewegen et al. (1979) and Voogd and Bigar6 (1980). The Figures 145 and 181 that compare the zonal organization of these connections with the Zebrin pattern are still largely hypothetical. There is no doubt, however, that the two patterns are correlated, but there is no simple 1:1 relationship. The borders between corticonuclear and olivocerebellar projection zones often are located within, rather than in between, the Zebrinpositive and -negative zones. The observation that the cerebellar midline divides the 308
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Zebrin-positive P l+ zone in independent left and right subzones (Hawkes and Leclerc, 1987) and the subzonation resulting from the partial overlap of different markers, discussed by Hawkes (1992) and Leclerc et al. (1992) should be taken into account for a more detailed comparison of the two patterns. Do cerebellar modules differ in other respects than in the biochemical properties of their Purkinje cells? True; they differ in the efferent connections of their target nuclei, but their internal organization seems to be very similar. Differences have been reported for the organization of the nucleocortical projections from the different nuclei (Section 5.3.) and it has been claimed that all nucleocortical projections to the C1 and C2 zones of the pars intermedia of the cat take their origin from the posterior interposed nucleus, i.e. from the target nucleus of C2 zone (Trott and Armstrong, 1990). The nucleo-olivary projections from the posterior interposed nucleus to the rostral medial accessory olive is dense (Nelson et al. 1989), and the amount of electrotonic coupling in this olivary subnucleus is the highest in the olive (Llinas and Yarom, 1981). Moreover, a recurrent excitatory loop connects the posterior interposed nucleus, via the Darkschewitsch nucleus and the medial tegmental tract, with the rostral medial acccessory olive (Ruigrok and Voogd, 1995). The C2-nucleus interpositus posterior module, therefore, may be an example of a special type of module, characterized by well-regulated mass action, tight interconnections and a relative lack of internal specialization. The C2 zone, moreover, lacks a somatotopic organization (Section 6.3.3.1.). The C2 module differs in most of these respects from the C1/C3-nucleus interpositus anterior module. Nucleocortical projections from this nucleus are rare or absent (Trott and Armstrong, 1990), the nucleoolivary projection from the anterior interposed nucleus to the dorsal accessory olive (Nelson et al., 1989; Ruigrok and Voogd, 1990) and the degree of electrotonic coupling of the neurons of the subdivision of the inferior olive are rather weak. Moreover, it lacks a recurrent nucleo-mesencephalo-olivary pathway. Both the olivo-cerebellar pathway from the dorsal accessory olive to the C~ and C3 (and d2) zones and the cortico-nuclear projection of these zones to the anterior interposed nucleus display a detailed somatotopical (microzonal) organization (Section 6.3.3.1.). The search for differences in the internal organization of the modules and their biochemical and electrophysiological correlates, therefore, should continue. 7.4. THE ROLE OF BIOCHEMICALLY DEFINED SYSTEMS IN CEREBELLAR MOTOR CONTROL Important contributions of the chemical anatomy of the cerebellum concern our understanding of its role in motor control. The perpendicular arrangement of the parallel fibers and the Purkinje cell zones and microzones is one of the fundamental structural properties of the cerebellum. This arrangement optimalizes the chance that any given mossy fiber input of the cerebellum may interact, through the parallel fibers, with the Purkinje cells or the climbing fibers of a particular zone or microzone. This 'suggests that a mossy fiber input to a restricted part of (a) zone can influence a number of microzones via the parallel fibres. If the strength of the parallel fiber-Purkinje cell transmission is modified separately in each microzone, specific combinations of microzones may be selected for each mossy fiber input. This modification would be performed by the climbing fibers system, which has been shown to exert both short-term (Ebner and Bloedel, 1984) and long-term (Ito et al., 1982; Ekerot and Kano, 1985, 1989) effects on the parallel fiber-Purkinje cell transmission. (...) The propagation of activity along the parallel fibers from (one) zone into the (next) zone would result in output units 309
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involving parts of both zones. Probably spread of activity across the borders of the sagittal zones is a general feature. This would allow far more complex synergies as, for example, combinations of distal and postural muscles' (Garwicz and Andersson, 1992, p. 621). The processes responsible for the selection of the appropriate output for a particular mossy fiber-parallel fiber input may function at the level of the synapses of the mossy fibers with granule cells, unipolar brush cells, or Golgi cells and at the level of the parallel fiber-Purkinje cell synapse. Several systems may be involved in this selection process. Nitric oxide, that is synthetized by subsets of granule cells (Schilling et al., 1994; Hawkes and Turner, 1994) and in stellate and basket cells (Bredt et al., 1990) (Section 3.4.) has been shown to be involved in the production of long-term depression (LTD) of transmission in synapses of the parallel fibers with the Purkinje cell dendritic spines (see Section 3.1.6.), but may also function at the level of the granular layer. Parallel fiber-Purkinje cell transmission also can be blocked by adenosine, acting on the A1adenosine receptors on the parallel fibers (Section 3.5.). The production of adenosine and the levels of the enzyme 5'-nucleotidase in the molecular layer that degrades adenosine monophosphate to adenosine, both are under the influence of the climbing fibers, and, in certain species, 5'-nucleotidase is distributed in bands. An important piece of information on the selection process concerns the heterogeneous distribution at the synapses between the parallel fibers and the Purkinje cell dendritic spines of the ionotropic glutamate receptors, that are blocked during LTD, and of the metabotropic glutamate receptors that elicit this reaction. Somogyi and co-workers have shown that immunoreactivity for the mGluR1 unit of the metabotropic receptor was never associated with the postsynaptic density of the synapse, but was localized at perisynaptic and extrasynaptic sites. This localization is in marked contrast with the ionotropic receptor subunits that are primarily located at the postsynaptic membrane (see Sections 3.3.1. and 3.3.2.). Induction of LTD could be inhibited by in-vitro immunoinactivation of the mGluR1 subunit (Shigemoto et al., 1994) and LTD could not be elicited in transgenic mice that lack this subunit (Aiba et al., 1994). A major drawback of many studies of the chemical neuroanatomy is that they were conducted in only one species, the rat. There is extensive evidence for species differences in the distribution of the synthetizing enzyme of acetylcholine (CHAT), muscarinic cholinergic receptors and acetylcholinesterase (see Section 3.10.), and there is reason to assume that a similar interspecies variability exists for other transmitter systems. The expression of Zebrin by certain subpopulations of Purkinje cells, and the zonal patterns in the distribution of 5'-nucleotidase, only occur in certain species. It is a fortunate coincidence for the experimental neuroscientist that the Zebrin zonal pattern is expressed in rats, but in other species like the cat or macaque monkeys all Purkinje cells are Zebrin-immunoreactive. Many species-differences in the chemical neuroanatomy of the cerebellum may be due to the selectivity of the antibodies employed in the immunocytochemical techniques, but other differences may be real and may reflect true variations in structure or in the transmission and second messenger systems of the cerebellum.
8. ACKNOWLEDGEMENTS The authors wish to express their gratitude to the many scientists and publishers who gave permission to reproduce illustrations from their publication and made the original 310
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prints available to us. Dr. Greet Schalekamp, Dr. Tom Ruigrok and Dr. Doug Hess allowed the use of some of their unpublished material. The secretarial assistance of Edith Klink, the photography of Eddie Dalm and the artwork of Karin Voogd are gratefully acknowledged. Figures 1 and 4 were redrawn from Ramon y Cajal (1911) by Philip Wilson FMAA, AIMI.
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