Topographic Histochemstry of the Cerebellum

Topographic Histochemistry of the Cerebellum 5' -Nucleotidase, Acetylcholinesterase, Immunology of FAL

ENRICO MARANI

in cooperation with Drs. A. J. P. BOESTEN, Elisabeth Hospital, Leiderdorp, The Netherlands Dr. B. BROWN, Department of Anatomy, Emory University, Atlanta, USA Drs. A. H. EpEMA, Department of Anatomy and Embryology, Univ. Leiden, The Netherlands

With 57 Figures and 14 Tables

~

~

GUSTAV FISCHER VERLAG· STUTTGART· NEW YORK ·1986

ENRICO MARANI, Ph. D. Anatomisch-Embryologisch Laboratorium, Rijksuniversiteit Leiden, Wassenaarseweg 62, Postbus 9602, NL-2300 RC Leiden (The Netherlands)

CIP-Kurztitelaufnahme der Deutschen Bibliothek Marani, Enrico: Topographic histochemistry of the cerebellum: 5' -nucleotidase, acetylcholinesterase, immunology of FAL / Enrico Marani. In cooperation with A. J. P. Boesten ... - Stuttgart; New York: Fischer, 1986. (Progress in histochemistry and cytochemistry; Vol. 16, No.4) ISBN 3-437-11033-0 (Stuttgart) ISBN 0-89574-221-7 (New York) NE:GT

© Gustav Fischer Verlag . Stuttgart . New York· 1986 Aile Rechte vorbehalten Gesamtherstellung: Laupp & Gobel, Tiibingen 3 (Kilchberg) Printed in Germany

ISBN 3-437-11033-0 ISBN 089574-221-7 NY ISSN 0079-6336

Contents

1

2 2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.3 2.3.1 2.3.2 2.3.3 2.3.4 3

3.1 3.1.1

3.2 3.2.1 3.2.2 3.2.3 3.3

3.3.1 3.3.2 3.3.3 3.3.4 3.4 3.4.1 3.4.2 3.4.3 3.5 3.5.1 3.5.2

Abbreviations Foreword . . . Introduction . The inferior olivary complex . Introduction . . . . . . . . . Acetylcholinesterase staining in subdivisions of the inferior olivary complex . Distribution of AChE in the inferior olive of the cat . Distribution of AChE in the inferior olive of the rat . Distribution of AChE in the inferior olive of the ferret Distribution of AChE in the inferior olive of the rabbit. Discussion of the distribution of AChE in the inferior olive Enzyme histochemistry of experimentally induced inferior olivary hypertrophy in cat (A. J. P. BOESTEN and E. MARAN!) . . . . . Neuronal connections involved in olivary hypertrophy. Control series. . . . . . . . . . . . . . . . . . . Histochemistry of olivary hypertrophic neurons Discussion . . . . . . . . . . . . . . . . . . . . Histochemistry of the mature cerebellum . . . . Morphology and histology of the mammalian cerebellum AChE longitudinal pattern in the rat cerebellar granular layer Myeloarchitecture of the cerebellum . Description of the myeloarchitecture of the mouse cerebellum . Discussion of the topography of the myeloarchitecture . . Acetylcholinesterase of the monkey cerebellar fibre layer. 5' -Nucleotidase . Enzyme histochemical reaction Biochemical reaction . . . . . . Histochemical results. . . . . . Peculiar properties of mouse cerebellar 5 ' -nucleotidase. 5'-Nucleotidase isoenzymes in rodents . 5'-Nucleotidase isoenzyme in the mouse cerebellum .. 5' -Nucleotidase isoenzymes in other rodents . Quantification of the 5' -nucleotidase band pattern in the mouse cerebellum . 5'-Nucleotidase in the mouse cerebellum . Description of the 5 ' -nucleotidase pattern . Considerations on the topography of the 5' -nucleotidase pattern

VII X 1 3

3 4

6 9 10 11 11

13 15 15 16 20

24 24 34 37

38 42 43 45

47 48 50

52 55 55

60 63 64

66 72

VI . Contents

3.5.3 3.6 3.6.1 3.6.2

3.6.3 4

4.1 4.1.1

4.2 4.2.1 4.2.2 4.3 4.4 5

5.1 5.2 5.3 5.4 5.5 6 7

8

Ultrastructural localization of 5' -nucleotidase in the mouse cerebellum . Acetylcholinesterase topography in the cat cerebellum Description of the acetylcholinesterase pattern in the cat cerebellum . . Ultrastructural localization of acetylcholinesterase in the molecular layer in kittens . Description of the AChE pattern in the monkey cerebellar molecular layer Development of the cerebellum . . . . . . . . . . . . . . . . . Histology of the cerebellar development . Application of human blood monoclonals directed against FAL . . . . . . Topography of FAL monoclonal antibodies in the developing rabbit cerebellum . The longitudinal FAL pattern in the immature rabbit cerebellum. . . . . . The spinocerebellar input in the immature rabbit cerebellum . . . . . . . . Topography of FAL monoclonal antibodies in the mature mouse cerebellum . Topography of acetylcholinesterase in the developing rabbit and cat cerebellum (B. BROWN, A. H. EPEMA and E. MARANI) General discussion . . . . . . . . . . . . . . Myeloarchitecture of the cerebellum . The patterns in the inferior olivary complex . . . . The relation between the cerebellar and inferior olivary patterns Biochemical and ultrastructural considerations of cerebellar molecular layer patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . Why the longitudinal enzyme histochemical cerebellar patterns? . Appendix .. References . Subject index

79 97 97 99

106 109 109 110

114 114

115 116 117

127 127 129 130 132 133 137

144 163

Abbreviations Al - 3 AAT ACh AChE AD ANS

AP

AthChE B B (n)~

b(A)dh

C l- 3 caud ChE CN CO CR 1(11) D l- 2 DA DAB DAO dc dl dmcc E EDTA EGL EM

ER

F FAL FITC Fl(Flocc) G GABA GAD gdh

Zones projecting to fastigial nucleus Aspartate aminotransferase Acetylcholine Acetylcholinesterase Distilled water Ansiform lobule Acid phosphatase Acetylthiocholinesterase Zone projecting to Deiters' nucleus Butanol Nucleus beta of 10 Betaine aldehyde dehydrogenase Zones projecting to interposed nuclei Caudal Cholinesterase Cerebellar nucleus Cytochrome oxidase Crus 1 or II Zones projecting to lateral cerebellar nucleus Catecholamines (dopamine) Diaminobenzidine Dorsal accessory olive Dorsal cap Dorsal lamella of PO Dorsal medial cell column of 10 AChE positive edges Ethylene diamino tetraacidic acid External granular layer Electronmicroscopical Endoplasmic reticulum Fastigial nucleus Alpha fucosyl- N -acetyl-Iactosamine Fluorescein isothiocyanate Flocculus Granular layer Gamma aminobutyric acid Glutamic acid decarboxylase Glutamate dehydrogenase

VIII . Abbreviations

G6P G6Pdh H (I-X) hbdh HRP

lA

ICCL idh IGL 10 IP 1. p. iso-OMPA L ldh Lob.ant Lob.post M Ma MAO MAO mdh m.Osm. mvb MVmc MVpc N S-N NAD(tr) NADP(tr) NMN NMRI Nu P(p) PI

P2 PC PAP Parafl(occ) PAS PFLD

Glucose-6-phosphate Glucose-6-phosphate dehydrogenase Hemispherical parts of lobule(s) I-X Hydroxybutyrate dehydrogenase Horseradish peroxidase Anterior interposed nucleus Inner cortical cell layer Isocitric dehydrogenase Internal granular layer Inferior olive Posterior interposed nucleus Intraperitoneal T etra-isopropylpyrophosphoramide Lateral cerebellar nucleus Lactate dehydrogenase Anterior lobe Posterior lobe Molecular layer Marginal layer Medial accessory olive Monoamine oxidase Malate dehydrogenase Milli-osmolarity Multivesicular body Medial vestibular nucleus magnocellular part Medial vestibular nucleus parvocellular part Nuclei 5 Nucleotidase NADH tetrazolium reductase NADPH tetrazolium reductase Nicotinamidemononucleotide Naval Medical Research Institute (mouse strain) Nucleus Purkinje cell or dendrite Nuclei and debris fraction Synaptosome fraction Purkinje cell clusters or layer Peroxidase antiperoxidase Paraflocculus Periodic acid Schiff Dorsal paraflocculus I -

Abbreviations

PFLV P.Med PO PO ps-ChE r.ER ros s S2 sdh SI SSEA SV Tol.blue 4th V

vI

vlo WAG WGA-HRP 1,3-c.glyc

Ventral paraflocculus Paramedian lobule Principal olive Peroxidase Pseudo-cholinesterase Rough ER Rostral Sulcus Microsome fraction Succinic dehydrogenase Simplex lobule Stage specific embryonic antigen Superior vestibular nucleus Toluidin blue Fourth ventricle Ventral lamella of PO Ventral lateral outgrowth of PO Wistar albino glaco (rat strain) Wheat germ agglutinin-horse radish peroxidase Cellular raphes or gaps in AChE pattern 1,3-cyclic glyceromonophosphate

IX

Foreword The idea of attempting this work was conceived in the hills of Toscana. Over the excellent wine of that region, Dr. CERRO, Dr. SBARBATI and I talked about science, publish or perish, and i pollio There, I remembered the kind invitation of Prof. Dr. GRAUMANN (issued at the meeting of the Gesellschaft fur Histochemie at Gargellen, Austria) to publish my work as a monograph in the series Progress in histochemistry and cytochemistry. On the farm where I spent my holidays with my family, the fowls were allowed to roam free. One morning, I was visited in my bed by a brown hen who seemed set upon laying her eggs by my side. In my attempts to evict her from the house, I was struck by the movements of her head. Every time she took a few more steps, her head at the same time moved backwards and forwards. I was much occupied with wondering, whether chickens could also walk without shaking their heads in this particular way. Suddenly, I recollected a discussion with my colleague, NICO GERRITS, who had wanted to start a Zoo rather than being a neuroanatomist. Talking about the work of POMPEIANO on neck afferents of the cerebellum, he had told me that ducks moved alike. So I went to the ducks on the farm, with my children of course. In fact, they only did so in the water. Pigeons, in comparison, moved their heads only when walking and not when flying, while turkeys and goose did not show this behaviour. The cats, young and old, all kept their head still. I went to the horses and the dogs. They behaved as all mammals I studied and kept their heads still while walking. Neck afferents, I knew, had something to do with this phenomenon. How could the cerebellum direct this odd behaviour in the chicken, pigeon, and duck? What is the difference between these and other animals in the cerebellar function of their neck afferents? And I thought of the strong heterogeneity in brain histochemistry of mammals and birds. Encouraged by my Italian colleagues, I decided to record my diverse observations on cerebellar histochemistry. The reader finds here a strange collection of cerebella and precerebellar nuclei, since the animal world is so diverse that a unifying hypothesis on my results could only be untrue. The moral of this story is perhaps that it is better to send scientists on holidays and to supply them with wine than to set them to work on science management, as is the current trend in the Netherlands. I thank my padrone Dr. SANDRO BUZZATI and his lovely family. Chianni, Italy, Aug. 1983

1 Introduction This monograph describes the mature topographic histochemistry (FRIEDE 1966) of two enzymes in particular (5'-nucleotidase and acetylcholinesterase) in the cerebellum and of one (acetylcholinesterase) in the inferior olivary complex. An account is also given for the distribution of both monoclonal antibodies against surface antigens of the external ganular layer and of acetylcholinesterase in the immature cerebellum. 5'-Nucleotidase (E. C. 3. 1. 3. 5) is an enzyme which convertes 5'-nucleotides into nucleosides; for example, 5 ' -adenosine monophosphate into adenosine. The enzyme may therefore be involved in nucleoside incorporation in the cell nucleus or in purinergic neurotransmission. Acetylcholinesterase (E. C. 3.1. 1. 7) is an enzyme that breaks down acetic esters into acid and alcohol. Acetylcholinesterase preferentially converts the neurotransmitter acetylcholine, but breaks down other substrates as well. Often, acetylcholinesterase is erroneously thought to be exclusively involved in acetylcholine neurotransmitter conversions at the synaptic cleft (see SILVER 1967, 1974). Several monoclonal antibodies, reacting with human granulocytes, are tested for the ability to recognize their antigen in the central nervous system. These monoclonal antibodies are found to detect their antigens in the developing rabbit cerebellum. This antigen is a trisaccharide with the composition beta-galactose-N-acetylglucosamine-Lfucose (FAL). This work is derived from a research project which was started whithin the Neuroanatomy group of the State University Leiden. We undertook the project, because: (1) a longitudinal organization was postulated in the mammalian cerebellum, based on myelo-architectonic subdivisions in the white matter, the localization of olivocerebellar climbing fibres in the white matter, and the termination of certain mossy fibre systems in the cerebellar granular layer (VOOGD 1964, 1967, 1969; VAN ROSSUM 1969; VOOGD, BROERE and VAN ROSSUM 1969); (2) a rostro-caudally arranged pattern of bands, alternatively positive and negative for 5'-nucleotidase, was found in the molecular layer of the mouse cerebellum (SCOTT 1964, 1965, 1969). The purpose of this project is to describe this 5'-nucleotidase pattern in the mouse cerebellum, to determine its cytological localization, and to correlate these findings to other data on the fibre connections and the histology of the mammalian cerebellum. During the course of the project, a similar enzyme pattern was discovered for acetylcholinesterase (AChE) in the molecular layer of the cerebellar cortex of the cat (MARANI and VOOGD 1977). Moreover, a peculiar distribution of AChE was found in the inferior olive, one of the main pre-cerebellar nuclei in the cat (MARANI, VOOGD and BOEKEE 1977). This nucleus extends its axons (climbing fibres) in a longitudinal zonal pattern in the cerebellum.

2 .

Enrico Marani

The description of the AChE pattern was greatly facilitated by previous studies of the morphology of the cat cerebellum and the internal coordinate system of the division of the white matter, based on Haggqvist material, and autoradiographic experiments (for a review see VOOGD and BIGARE 1980). No adequate descriptions of the gross morphology of the mouse cerebellum or of its myeloarchitecture were available. These had to be provided (MARANI and VOOGD 1979; VOOGD, GERRITS and MARANI 1985). Light microscopy alone is insufficient to fully elucidate the structure of the molecular layer. Both 5 ' -nucleotidase in the mouse and AChE in the cat were therefore also studied with electron microsopy in the mature cerebellum 1. With respect to the biochemistry and the ultrastructural localization of 5' -nucleotidase in different organs of various species, the literature gives contradictory results. Some of the biochemical properties of cerebellar 5' -nucleotidase were reinvestigated which led to a better understanding of the enzyme histochemical method and its application in electron microscopy (MARANI 1977, 1980 a,b, 1981). The zonal distribution of efferent and afferent systems in the mammalian cerebellum (VOOGD and BIGARE 1980) are not directly comparable. Moreover, different interpretations of the zonal distribution within a single system have been given (COURVILLE et al. 1980). The histochemical band patterns serve exclusively as an independent system of reference for the study of the afferent and efferent connections of the cerebellar cortex and of its physiology and may provide a clue in the search for the functional meaning of the parasagittal alignment of these connections. The cerebellar development within the mammalian central nervous system has been extensively studied in recent years (for reviews see ALTMAN 1982; FEIRABEND 1983). However, relatively little is known about the factors which cause cerebellar neurons to establish topographically ordered connections with their target cell populations. It is also unknown, whether the cerebellar target cells are present in such a topographical ordered manner. Such an ordered topography has been put forward only for the Purkinje cells (KApPEL 1980; FEIRABEND 1983). Tritiated thymidine autoradiography has provided evidence for differences in timing of neurogenesis in sub-populations of Purkinje cells, which could account for the ultimate differences in their connections (FEIRABEND et al. 1978, 1979; MARANI and FEIRABEND 1983). Little information concerning the development of topographic pattern connections between the inferior olive, Purkinje cells, and deep cerebellar nuclear

1 The biochemistry of acetylcholinesterase has been extensively studied (see SILVER 1974, for a review of the older literature). Little experience of the ultrastructural methodology of this enyzme has been obtained in the Netherlands. On occasion of a meeting of the Anatomical Society of Great Britain and Ireland in Cambridge in 1975 (MARANI and VOOGD 1976), I had the opportunity to visit Dr. Lewis and Dr. Shute who introduced me to their method and kindly provided me with their latest version before publication (LEWIS and KNIGHT 1977).

Topographic histochemistry of the cerebellum' 3

neurons is found in the literature. The study of the developmental aspects of the cerebellar cortex has recently been stimulated by the finding that both AChE (BROWN and GRAYBIEL 1983 a, b; MARANI et al. 1983 a) and monoclonal antibodies against FAL (MARANI et al. 1983 b; MARANI and TETTEROO 1983) can be used to trace the earliest development of the cerebellar zonal arrangement. However, no adequate descriptions are available of the embryological and foetal stages of cerebellar development in mammals, as there are in the fowl (FEIRABEND 1983).

2 The inferior olivary complex 2.1 Introduction The inferior olivary complex is a nucleus within the caudal brainstem and is ultimately connected to the cerebellum by the olivocerebellar fibres, called «climbing fibers» in the cerebellum (DESCLIN and EscuBI 1974). This important pre-cerebellar nucleus delivers its axons in longitudinal zones in the cerebellar molecular layer. Each cerebellar zone can be related to circumscribed parts of the inferior olivary complex (GROENEWEGEN and VOOGD 1977, GROENEWEGEN et al. 1979). The inferior olive (10) in mammals has been divided into the dorsal accessory olive (DAO), the medial accessory olive (MAO), and the principal olive (PO) (KooY 1916; MARESCHAL 1934; BRODAL 1940). The 10 can be further subdivided on the basis of its afferent and efferent (olivo-cerebellar) fibre connections. This subdivision is illustrated particularly well for the DAO. According to BRODJ\L (1940), the caudal and ventral parts of the DAO, that receive spino-olivary fibres (BRODAL et al. 1950), project to the contralateral vermis, whereas the rostro-medial part of the DAO projects contralaterally to more lateral parts of the anterior lobe (BRODAL 1940). More recently, it was possible to show by using different techniques that the origin of projections of the DAO to the contralateral vermis is restricted to the caudal third of the nucleus (VOOGD 1969: orthograde degeneration technique; ARMSTRONG et al. 1974: antidromical stimulation technique; GROENEWEGEN and VOOGD 1977: autoradiographic technique). Because of the functional importance of the inferior olive to cerebellar function and its clear topography of efferent and afferent fibres, this structure was examined histochemically in the cat (MARANI et al. 1977), in several other mammals (in this monograph), and in the fowl and primates (MARANI, unpublished). Maps were produced of regional differences of AChE in the 10. Studies utilizing both lesions of afferent systems (BOESTEN and VOOGD 1975) or chemical destruction with 3-acetylpyridine (DESCLIN and EscuBI 1974) produced only degenerative changes in the 10; however, after hemicerebellectomy, involving deep cerebellar nuclei, a substantial increase in AChE reaction product content in cells and neuropil was measured (MARANI and BOESTEN 1979).

4 . Enrico Marani

The enlargement of neuronal perikarya after disruption of afferent and/or efferent connections is called hypertrophy. This phenomenon has been reported in the 10 in man (BEN HAMIDA 1965; VERHAART 1962), cat (VERHAART and VOOGD 1962), and dog (SINNIGE 1938), in the nucleus rotundus and ovoideus in birds (VERHAART and ZECHA, unpuplished results), in the rat subcommisural organ (M0LLGARD et al. 1978), and in the retinal ganglion cells in the goldfish (MURRAY and FORMAN 1971). At the ultrastructural level, hypertrophy in the cat inferior olive was described as characterized by an increase in diameter of the nucleolus and in the amount of rough endoplasmic reticulum, by displacement of the nucleus to the periphery of the perikaryon, and by accumulation of unknown substances in the perikaryoplasm (BOESTEN and MARANI 1979; VOOGD and BOESTEN 1976). Some of these changes are also seen in the subcommissural organ in rat (M0LLGARD et al. 1978) and in the hypertrophic goldfish retinal ganglion cells (MURRAY and FORMAN 1971). Explanations of hypertrophy are inconclusive till now. Recent publications tend to consider hypertrophic cells to be hyperactive (BOESTEN and MARANI 1979; MARANI and BOESTEN 1979) rather than being the expression of transneuronal degenerative phenomena of these cells (GAUTIER and BLACKWOOD 1961; KOEPPEN et al. 1980). Enzyme histochemistry, carried out on sections of whole goldfish heads, provides an instrument to study, whether there is a relation between retinal ganglion cell hypertrophy and hyperactivity of the regenerating optic nerve (MARANI and RUIGROK 1981; MARANI and RUIGROK 1984). Moreover, enzyme studies of regeneration of the optic nerve may contribute to the understanding of enzyme changes obtained with olivary hypertrophic cells (see part 2.3).

2.2 Acetylcholinesterase staining in subdivisions of the inferior olivary complex The distribution of AChE in the 10 has been described for the cat (see MARANI and VOOGD 1977) and the opossum (MARTINet al. 1975). The functional significance of the differential distribution of AChE in the subdivisions of the 10 is not known. Its distribution is different in various species. The positive reaction of the dorsal cap of the medial accessory olive and the negative reaction of the group beta are among the points on which all descriptions agree thus far. To facilitate and extend the comparison of AChE in the 10, maps were produced, using BRODAL'S (1940) projection method of the 10, AChE distribution was also studied in the inferior olivary complexes of rats deprived of their cells by 3-acetylpyridine (DESCLIN 1974).

Topographic histochemistry of the cerebellum' 5

Fig. 1. Four electron microscopical photographs demonstrating AChE reaction product localization around small unmyelinated fibres (A: 39.000 x), around a bouton (B: 32.000 x), within thick myelinated fibres (C: 60.000 x) and around glial protrusions (D: 51.000 x).

6 . Enrico Marani

2.2.1 Distribution of AChE in the inferior olive of the cat Enzyme activity is present in the cytoplasm and nucleus of several olivary perikarya, but it is mainly contained within the neuropil of the 10. In areas with a high or medium activity, some negative cell bodies are found against the darker background of the neuropil (MARANI 1982 b; MARANI et al. 1977). A few reactive perikarya can be encountered where the neuropil is negative. Our light microscopy cannot resolve, whether AChE is bound to axons, dendrites, or glia. The areas of the neuropil of the 10 nucleus with high or medium activity contrast strongly with the fibres of the medial lemniscus and the reticular formation surrounding the 10 which are negative for AChE. Ultrastructural studies (MARANI, unpublished, see fig. 1) show a distribution of AChE reaction product mainly around nonmyelinated fibres within the neuropil of the MAO. Besides the perikaryal localizations, glial locations are also found. Fractionation of the 10 complex (MARANI, unpublished) into Pl, P2, and 52 fractions (WHITIACKER 1964) show that the 52 fraction (microsome fraction and soluble substances) contains 62% of AChE activity, the P2 (synaptosome) fraction nearly 23%, and the P1 (nuclei and debris) 15% which can be translated in a distribution comparable to that found with ultrastructural methods. The neuropil in the cat 10 is stained in a way that its three divisions (MAO, DAO, PO) stand out from the background. However borders are slightly wider than delineated in Nissl stained series. This phenomenon and the use of young cats account for the increase in the medio-Iateral dimension in parts of the 10 in this study, when compared to those of BRODAL (1940) and others in Nissl material (MARANI et al. 1977). The distribution of this enzyme will be described from various transverse series illustrated in figs. 2, 3, 4.

Dorsal accessory olive The caudal portion of the DAO is strongly positive. The DAO becomes less reactive (fig. 3-54) approximately 700 Ilm rostrally of the caudal pole, and within about additional 100 Ilm it becomes negative. More rostrally, this negative area (fig. 3-56, 58) becomes sandwiched between two strongly positive areas and shifts laterally. At a distance of some 1600 Ilm from the caudal tip of the DAO, this negative area disappears (fig. 3-60). A new area of medium activity, situated in the middle of the DAO, appears after 100-200 Ilm (fig. 3-68). The negative area at the caudal pole of the DAO does not continue into the region of medium activity that divides the rostral part of the DAO. This discontinuity can be seen in the reconstructions of the DAO in fig. 4. The positive areas in the rostral part of the DAO overlap exactly the areas described by BOESTEN (BOESTEN 1971; BOESTEN and VOOGD 1975) and GROENEWEGEN et al. (1975) as receiving projections from the dorsal column nuclei.

Topographic histochemistry of the cerebellum . 7

Fig. 2. Micrographs df four sections through the inferior olive, schematically demonstrated in fig. 3. For abbreviations see fig. 3.

8 . Enrico Marani

Medial accessory olive The caudal two-thirds of the MAO shows a pattern of rostro-caudally directed bands of different activity (fig. 4). Caudally, its medial central part possesses a medium activity while the borders show high activity (fig. 3-44). Within another 100 Ilm the MAO can be divided into five parts. Lateral to the strongly positive dorsal cap of the principal olive, the nucleus beta appears which has a low activity, followed by a region with high activity. This area is succeeded by an area with medium activity and then an area with no activity. The most lateral margin is strongly positive. This pattern extends over the next 500 Ilm (fig. 3-46 through 50). The transition to the rostral third of the MAO is marked by an area of low or medium activity (fig. 3-56-58). The central part

,,t-

42

rI

..

54

62

via

41

.,

,~

70

56 dmcc

8734

66

PO

58

50

Fig. 3. Sections of the entire left inferior olive. The numbers of the serial brain stem sections are indicated. - dc dorsal cap, dl dorsal leaf, dmcc dorso-medial cell column, n.~ nucleus beta, vi ventral leaf, vlo ventral lateral outgrowth. Regions with high, medium, and low AChE activity are indicated respectively by black, stripes or white.

Topographic histochemistry of the cerebellum " 9

of the rostral third of the MAO has a medium or low activity, whereas its rostral, lateral, and medial margins generally exhibit higher activity. The dorso-medial cell column, located at the medial extremity of the MAO, is negative. Only its most caudal part is positive for AChE. This pattern can be recognized in the reconstruction of the MAO in fig. 4.

Principal olive The dorsal cap of the PO is strongly positive for a 900 !-lm extent (fig. 3-42 through 54). The dorsal lamella contains an area with low activity over the next 400 !-lm. The rest of the dorsal lamella has a medium (fig. 3-60 through 64) or high activity for AChE (fig. 3-66 through 70). The ventral leaf of the PO contains a zone of low activity between an area of high activity and the dorsal lamella (fig. 3-60 through 64). The rest of the ventral lamella of the PO contains medium activity at its lateral side and high activity at its medial margin. The reconstruction of the PO, as seen in fig. 4, demonstrates these aspects.

2.2.2 Distribution of AChE in the inferior olive of the rat The 10 of the rat is dis"tinguished by the extreme lateral extent of the DAO. In the projection diagrams, this lateral extension is less apparent with the projection method used in this kind of studies (BRODAL 1940). The general configuration of the inferior olive in the rat confirms to the description of BROWN et al. (1977). The borders between areas with different activities of AChE are generally distinct. The distribution of AChE in the DAO shows a pattern of alternating AChE positive and negative columns. The positive colums are narrow, narrower than the positive columns in the DAO of the cat, and the negative areas are quite extensive. The columns do not extend to the rostral pole of the DAO as they do in the cat, but are replaced by an area of medium activity that occupies the entire rostral third of the nucleus. The caudal pole of the DAO is AChE positive, as it is in the cat. AChE in the PO is distributed as a band of medium activity which occupies most of the ventral leaf, the rostral pole, and the medial part of the dorsal leaf of the PO. The lateral part of the dorsal leaf, part of the lateral bend, and the ventrolateral outgrowth which joins the dorsal leaf to the dorsal cap are negative. The main difference with the distribution of AChE in the PO of the cat concerns the apparent shift of an AChE ne~ative area from the ventral leaf in the cat to the lateral part of the dorsal leaf in the rat. The dorsal cap of both species is uniformly positive. In the MAO of the rat, a columnar distribution of AChE is present in its caudal part. The pattern is very similar to that of the cat and consists of two columns of medium activity, separated by a negative area, occupying the lateral two thirds of the caudal MAO. On their medial side, they are flanked by the group beta which is negative for AChE. The rostral half of

10 . Enrico Marani

DAO

PO

MAO

Fig. 4. Diagram summarizing the distribution of true acetylcholinesterase activity in the three. major components of the eat's inferior olive. The results represent a compilation of the data described by MARAN! et al. (1977). For abbreviations see fig. 3.

the MAO is mostly AChE positive. It lacks a central area of lower activity which is present in the other species. The dorso-medial cell column is AChE negative. When the cells of the 10 of the rat have been destroyed, the AChE pattern remains present. This could be ascertained in several rats injected with 3-acetylpyridine with survival times up to 100 days before sacrifice which were used to study the effects of the cerebellum (see 3.5). The distribution and the intensity of AChE staining in the inferior olive cannot be distinguished from those of normal control animals. Although not all olivary cells disappear, it seems likely that intrinsic connections cannot be held responsible for the presence of AChE in the neuropil of the olive (see MARAN! 1982). 2.2.3 Distribution of AChE in the inferior olive of the ferret The normal anatomy of the 10 of the ferret has not been described before. It is studied in Haggqvist stained sections. Structure and subdivision of the 10 are very similar to that of the cat. The AChE activity could be demonstrated in three brains incubated with to-3M iso-OMPA. The borders between areas with different levels of AChE reaction product usually are distinct. The MAO displays a columnar distribution of AChE in its caudal half. Two AChE positive columns are separated by an area of medium activity and the group beta is negative. The AChE positive columns extend in the rostral MAO, but are not interconnected by strands of medium activity, as they are in the cat. The dorso-medial cell column does not stain for AChE. The DAO lacks a distinct longitudinal columnar distribution of the enzyme. Its rostromedial part is AChE positive, and its rostrolateral part is negative. The caudal pole of the DAO shows a strong reaction to AChE.

Topographic histochemistry of the cerebellum' 11

The distribution of AChE in the PO is the reverse of that found for AChE in the cat. At the transition from ventral to dorsal leaf, the activity of the enzyme is high or medium, and it becomes less in the medial parts of the dorsal and ventral leaf of the PO. The dorsal cap of the ferret's 10 only is separated from the dorsal leaf of the PO by a small negative area which may correspond to the ventro-Iateral outgrowth (see MARAN! 1982). 2.2.4 Distribution of AChE in the inferior olive of the rabbit (1940) description of the 10 of the rabbit could be confirmed in normal series and was adopted in this description. The general impression of the distribution of AChE in the rabbit's 10 is that extensive areas are AChE negative (fig. 5). Positive and medium stainings, therefore, stand out clearly against an unstained background. The MAO contains a columnar distribution in its caudal part, but only one column of medium activity is present, immediately medial to the AChE negative group beta, instead of the two AChE positive columns of the caudal DAO of other species. The most rostral part of the MAO stands out by its positive reaction to AChE. The rostral DAO again is characterized by a tripartition, but in the case of the rabbit the central column is strongly AChE positive. The reverse situation is found in the cat. The lateral band region, where the dorsal and ventral leaf of the PO meet is negative for AChE, except for its most caudal portion. Areas of medium activity in the dorsal and ventral leaf shift medially in more rostral sections. Areas of high and medium activity alternate in the ventro-Iateral outgrowth (vlo) and the dorsal cap (dc). BRODAL'S

2.2.5 Discussion of the distribution of AChE in the inferior olive The distribution of AChE in the 10 is not the same in all species. The positive reaction of the dorsal cap of the MAO and the negative reaction of the group beta are among the points on which all descriptions agree. When the distributions of AChE in the 10 of rat, ferret, and rabbit are compared to published data on the cat and oppossum, some points of resemblance are immediately clear. Longitudinal columnar distributions are found in the caudal MAO and the rostral DAO of all species, the group beta is always negative, and the dorsal cap always displays positive reaction with the enzyme. The distribution af AChE in the PO is very similar for cat and rat, but in the ferret and the rabbit the distribution is almost reversed. Columnar distributions in the caudal MAO closely resemble each other, but in the rostral DAO the pattern in the rabbit is the reverse of that found in the ferret and the cat. Differences are also noted with respect to the dorso-medial cell column; it is AChE negative in cat and rat, displays medium AChE activity in the ferret, and consists of positive and negative parts in the rabbit. The distribution of AChE in the 10 cannot be fully explained on the basis of its known afferent and efferent connections, but in cat, at least, the histocherni-

12 . Enrico Marani

20

~

37

24

50

9271

de

PO

Fig. 5. Sections of the rabbit's inferior olive stained for AChE. The sections start caudally. No activity is indicated in white, medium activity striped and high activity black. At the bottom the diagrams for MAO, DAO and PO are depicted.

cal subdivisions show many points of resemblance with the subdivision on the basis of its connectivity. The similarities in the distribution of the enzyme in different subprimate species (MARAN! and VOOGD 1985) support this view.

Topographic histochemistry of the cerebellum . 13

2.3 Enzyme histochemistry of experimentally induced inferior olivary hypertrophy in cat (A. J. P. BOESTEN and E. MARANI) Experimentally induced hypertrophy of 10 cells in the cat has been the subject of studies at both light and electron microscopical level (VOOGD and BOESTEN 1975; BOESTEN 1977; BOESTEN and MARAN! 1979). It has been suggested that hypertrophic 10 cells can be considered as hyperactive cells and not as degenerative, as proposed by studies on human material (BEN HAMIDA 1968; RONDOT and BEN HAMIDA 1968; KOEPPEN et al. 1980). The proposed hyperactivity theory (BOESTEN and MARAN! 1979; BOESTEN, unpublished) of 10 cells is based on the increase in diameter of the nucleolus and increase of the cell volume, while important cell loss could not be demonstrated in the hypertrophic areas up to a survival time of 524 days (BOESTEN, unpublished). Also an accumulation of electron-dense substances has been found, with an increase in rough endoplasmic reticulum (r.E.R.) in these cells (VOOGD and BOESTEN 1975). Enzyme histochemical studies were undertaken in order to gather information on the functional state of these cells, although it is realized that severe limitations are present which are inherent to (enzyme) histochemistry with respect to the amount of enzymes and substances that can be discerned or located. Biochemistry is difficult to perform in this part of the brain, because hypertrophic areas in the 10 are small, and the borders of the hypertrophic areas are variable in different cases or experimentally induced olivary hypertrophy. Moreover, a strong individual variability is present for the 10 of the cat. Until now, the inferior olivary hypertrophy has been explained on the basis of a direct cerebello-olivary connection (BEN HAMIDA 1968). Recently, this connection was described in detail for the cat (GRAYBIEL et al. 1973; TOLBERT et al. 1976). However, this connection cannot be held exclusively responsible for olivary hypertrophy, because the hypertrophic area is smaller than the projection area of the cerebello-olivary pathway. In order to induce olivary hypertrophy, it is necessary to destroy the cerebellar nuclei which form also a relay centre in the cerebello-mesencephalic olivary circuitry or their efferents. After interruption of all cerebello-olivary connections, involving the cerebellar nuclei, olivary hypertrophy is mainly found in regions of the 10 which are related to the cerebello-mesencephalic circuitry. Olivary hypertrophy must be seen as a complex antegrade transsynaptic reaction, because the direct cerebello-olivary projections, the dentate-rubral projections, and the Darkschewitsch-olivary projections coincide with each other in the hypertrophic olivary areas, while other parts, that are subject only to retrograde degeneration after lesions of the cerebellar nuclei, show little or no degeneration or demonstrate olivary cell degeneration and loss.

OLIVO -CEREBELLAR CIRCUITS in CAT CEREBEllO·OllVAIRE CIRCUITS

dlstrlbutlon of affected cells after (henW)cetebelecton1y

0-

.-,.

"""QlIIII'a.-

l ! ) _ .... r:;] ....... ."...,.......

me

B

c

Fig. 6. A: This diagram demonstrates the neuronal connections involved in the inferior olivary complex. The inferior olive contains three main parts (MAO, DAO, PO). Cells of the inferior olive project to the cerebellar cortex as climbing fibres. The bundle of climbing fibre axons traveling across the brainstem towards the cerebellum is called the olivo-cerebellar fibre tract. Intrinsic cerebellar circuitry is needed to have signals relayed to the cerebellar nuclei (bottom corner for subdivision), the only output system of the cerebellum is towards the red nucleus and Darkschewitsch nucleus. These output axons use the cerebellar superior peduncle. However, cerebellar nuclei project also directly back to the inferior olive. A closed circuitry (the circuitry of Guillain and Mollaret) arises because both red nucleus and Darkschewitsch nucleus project back to the inferior olive by the central and medial tegmental tract respectively. Output of this circuitry is by the cerebellar nuclei axons that project on the thalamus. B: Olivary hypertrophy only arises when the cerebellar nuclei are involved in the damage. Using cerebellectomy, involving the cerebellar nuclei, the reciprocal cerebello-olivary circuits are damaged, but the mesencephalo-olivary connections (the central and medial tegmental tract) are saved. - Figs. A and B courtesly Dr. J. VOOGD. C: This figure demonstrates the areas within the inferior olive, where cells hypertrophy, degenerate and areas with unharmed cells.

Topographic histochemistry of the cerebellum . 15

2.3.1 Neuronal connections involved in olivary hypertrophy The projection areas of the Darkschewitsch nucleus and red nucleus coincide with the hypertrophic area in the cat (fig.6). In cats as in dogs (SINNIGE 1938), olivary hypertrophy can be induced by hemicerebellectomy or total cerebellectomy, involving the cerebellar nuclei. The deep cerebellar nuclei project onto the red nucleus and Darkschewitsch nucleus, and it is postulated that hyperactivity in olivary cells arises, because the cerebellar control of these olivary areas has been abolished. However, in rats, where the same connections are supposed to be present, cerebellectomy does not induce hypertrophy (unpublished results BOESTEN and VOOGo). Hemi- or total cerebellectomy in the cat results in degeneration of the cells of the lateral DAO, in hypertrophy in the rostral MAO, and in the lateral part of the ventral leaf of the PO. The rest of the olivary cells in the 10 show normal appearances (fig.6c). Table 1. Comparison of the increased enzyme activities in hypertrophic cells with the enzyme activities in non-hypertrophic cells.

Succinic dh.ase ~-hydroxy But. dh.ase NADPH tetr. red. NADH tetr. red. Cytochrome oX.ase AChE

+

o -

= = =

differentiated cells

hypertrophic cells

+/0

+ + + + + +

o o o

+/0

o

degenerating cells

increased normal absent

2.3.2 Control series Results from other studies (MARANI 1981, 1982 b), also involving control experiments in cats (C 75, H 9213, see table 1), have shown methods which are reliable for the 10. An exception was made for aspartate-amino transferase (MARTINEZ-RoDRIQUES et al. 1976), the key enzyme for glutamate-aspartate conversion, which in our hands was never reliable. Additional series with hemicerebellectomy without damage to the cerebellar nuclei have already been described (MARANI et al. 1977; 8864: 21 days, 8870: 28 days, 9194: 145 days survival). The normal enzyme distribution was studied in intact healthy cats and compared with the distribution in animals with hemicerebellectomy, not involving the deep cerebellar nuclei, and the homolateral side of the hemicerebellectomy, involving deep nuclei. The homolateral side of hemicerebellectomy, involving the deep cerebellar nuclei, shows an apparently normal distribution of enzyme activities.

16 . Enrico Marani

Fig. 7. A: Electronmicrograph demonstrating osmiophilic balls in a hypertrophic inferior olivary cell. These osmiophilic balls are present within dilatated rough endoplasmic reticulum. B: Lightmicroscopical section treated for the acid anhydride technique. Positive balls are encountered in the hypertrophic cells, indicating that accumulation of glutamate and aspartate could occur within olivary hypertrophic cells.

The increase or decrease of enzyme activity in olivary cells are reported in table 1. Some enzymes in the 10 can be located with histochemical methods to both cells and neuropil (AChE), while others are found exclusively in the neuropil (ATPase) or exclusively in cells (acid phosphatase: AP). Hypertrophic cells with their increased cell surface appear more positive with the same amount of reaction product per surface area than on contralateral normal cells. Visual assessment in these cases is not reliable, and in such cases histophotometric scans were made (see MARANI et al. 1977; MARANI and RUIGROK 1984).

2.3.3 Histochemistry of olivary hypertrophic neurons Olivary hypertrophic cells accumulate substances within dilatations of the r.E.R. (fig. 7 A). Even in uncontrasted EM sections these substances are electron dense, meaning that they are osmiophilic (VOOGD and BOESTEN 1975). The histochemistry per-

Topographic histochemistry of the cerebellum' 17

formed on these accumulated substances demonstrates that they are PAS-negative and do not react with the Millon reaction or Sudan black staining. However, the acid anhydride method colours these osmiophilic balls red, indicating that these substances may contain a local high concentration of protein bound carboxyls with amino acids, such as aspartate and/or glutamate (fig. 7B). Catecholamine fluorescence, according to DE LA TORRE et al. (1976), shows the same results in normal cats, as found by SLADEK and BOWMAN (1975) and WIKLUND et al. (1977). The rostral MAO in normal cats is devoid of catecholaminergic innervation. In hypertrophic areas, conspicuous circles of catecholaminergic fluorescence are present around olivary hypertrophic cells (fig. 8). Ultrastructural results confirm the presence of structures containing dense-core vesicles in hypertrophic areas (fig. 9). Monoamine oxidase, as studied in both normal and experimentally induced olivary hypertrophy, shows no increase in activity in the experimental group, either in neuropil or in cells. In normal inferior olivary complexes, its presence is also faint with both MAO techniques used (see Appendix 2.3). No pattern formation was anticipated for this enzyme as was found for catecholamines (WIKLUND et al. 1977). AChE has been extensively studied in normal 10 complexes (MARANI et al. 1977; MARANI 1981, 1982 b). Its pattern of distribution is shown in fig. 4. AChE, detected by adding iso-OMPA (10-SM) (see MARANI et al. 1977) to the incubation medium, is found to be increased in the rostral MAO, both in hypertrophic cells and in the

Fig. 8. Parts A and B demonstrate the presence of fluorescent varicosities around hypertrophic cells (open circles indicate the perikaryal border), while a faint line of varicosities can be followed (arrows) towards the olivary hypertrophic cells.

18 . Enrico Marani

Fig. 9. This figure shows dense-core vesicle-boutons in the neuropil around hypertrophic olivary cells. Some structures are dendritic and do contain dense-core vesicles. - D is taken from a hemicerebellectomy of 139 days, ABC of 524 days survival time (see also BOESTEN and VOOGD 1976; BOESTEN and MARAN! 1979). - Bars represent in A, B, C 0.13 nm, in D 0.23 nm.

Topographic histochemistry of the cerebellum' 19

surrounding neuropil, while the ventral leaf of the PO shows the same phenomenon (fig. 10). Esterases can be involved in the conversion of choline, therefore the last step in the break-down of choline to betaine was studied. Betaine aldehyde dehydrogenase shows no increase either in the neuropil or in the hypertrophic cells. However, beta hydroxy butyrate dehydrogenase, which diverts some of the acetyl-CoA derived from fatty acid or pyruvate oxidation shows an increase in reaction product formation in hypertrophic cells but not in the neuropil. Lytic enzymes can indicate degenerative processes within cells. Acid phosphatase is not increased in comparison to normal 10 cells in histophotometric scans. There is even no indication of changes in glucose metabolism. Enzymes of the glucose catabolic pathway and the alternative anaerobic (lactate dehydrogenase) and pentose phosphate pathways (glucose-6-phosphate dehydrogenase) were studied. These enzymes are not increased in hypertrophic cells or their surrounding neuropil. In the tricarboxylic acid cycle one enzyme is undoubtedly increased in activity. The reaction product of succinic dehydrogenase is increased in the perikaryal cytoplasm. Iso-citric dehydrogenase on the other hand does not show a consistently greater reaction product formation in different sections within the same experiment. Malate dehydrogenase is not increased in hypertrophic cells, while the neuropil contains little reaction product in both experimental and normal series. HEMICER£BELLECTOMY

C 171

MAO

Fig. 10. This figure, containing horizontal plane projections (MARANI et al. 1977), demonstrates changed AChE distribution (series C 171) within the MAO and PO of the inferior olivary complex. At the contralateral side of the hemicerebellectomy an increase in acetylcholinesterase activity can be noted. Black means high, striped medium, and white low enzyme activity.

20 . Enrico Marani

The glutamate pathway can be assessed using glutamate dehydrogenase. Reaction product from this enzyme is not raised in the neuropil or hypertrophic cells (fig. 11). In the respiration chain, three enzymes are found to give enhanced reaction product formation, as compared to controls. NADPH tetrazolium reductase, NADH tetrazolium reductase, and cytochrome oxidase are all increased in hypertrophic cells. At the longest survival time, 5'-nucleotidase activity, as determined according to SCOTT (1965), is decreased in activity in the hypertrophic perikarya, but keeps its high activity in the neuropil. These enzyme histochemical results are summarized in a diagram (fig. 11). 2.3.4 Discussion Palatal myoclonus is a recognized disorder in clinical neurology and is known to be connected with olivary hypertrophy (FRENKEN 1977). This paragraph is therefore of clinical importance; at the same time it contributes to the understanding of an exceptional neuronal reaction: hypertrophy. Other light microscopical examples of neuronal hypertrophy after axonal lesions are known (see Chapter 2.1). Enzyme histochemistry has been performed only on retinal ganglion cells in the goldfish (see MARANI and RUIGROK 1981, 1984) and in humans (FRENKEN 1977). Hypertrophy has been studied in retinal ganglion cells in the goldfish. After cutting the optic nerve, degeneration of the optic nerve occurs within a few days, involving all endings in the target organs (MARANI and RUIGROK 1981). The retinal ganglion cells react by an increase of axonal flow and an increase in protein metabolism (GRAFSTEIN and MURRAY 1969; MURRAY and FORMAN 1971). Depending on the temperature the goldfish retinal ganglion cells establish new connections with the tectum and other target nuclei within two to three weeks after cutting (SPRINGER and AGRANOFF 1977). This regeneration phenomenon in the goldfish optic nerve has been described. For example, it is known that the duration of the hypertrophy of the retinal ganglion cells is longer than the period of time necessary for reestablishing the contacts with the tectum. On the other hand, the start of the increase in cell volume cannot easily be related to the onset of regeneration in the optic nerve (see MARANI and RUIGROK 1984). After the administration of serotonin neurotoxins the loss of the 5'-hydroxytryptamine terminals leads to hypertrophy in the cells of the subcommissural organ of the rat. This hypertrophy is characterized on the ultrastructural level by distended r.E.R. cisternae filled with pale substances or electron-dense granules (M0LLGARD et al. 1978). Retinal ganglion hypertrophy is also marked by distended r.E.R. cisternae filled with pale substances. Moreover, comparable ultrastructural features have been reported for olivary hypertrophic cells, though with mainly osmiophilic balls in the cisternae of the r.E.R. (BOESTEN and VOOGD 1976; BOESTEN and MARANI 1979). Hyperactivity, as supposed in the olivary hypertrophy hypothesis (BOESTEN and MARANI 1979; MARANI

Topographic histochemistry of the cerebellum· 21

and BOESTEN 1979), has at least been proved for the secretory activity of the subcommissural organ in the rat (M0LLGARD et al. 1978). The results published in this paragraph are based on earlier ultrastructural studies performed by Dr. BOESTEN. However, neither ultrastructural studies nor connection studies can indicate the type of neurotransmitter involved in olivo-cerebellar projections. Aspartate (ITo 1978; WIKLUND et al. 1982) and glutamate (MAO et al. 1974) have been postulated as neurotransmitters of the climbing fibres, and, indeed, the dendritic area of the Purkinje cells, on which climbing fibres make synaptic contacts, is glutamate sensitive (CHUYO et al. 1975). It must be noted that aspartate is easily converted to glutamate and vice versa, a reaction catalyzed by the enzyme aspartate-amino transferase. In our opinion, the electron-dense osmiophilic granula must be an accumulation of proteins rich in protein-bound carboxyls, since the acid anhydride colouration was positive. Amino acids, like glutamate or aspartate in these proteins, can account for this positive colouration reaction and have an.affinity for OS04 (PEARSE 1972, 1980) which in its turn would be responsible for the black appearance of these granules. Moreover, our histochemical studies rule out the possibility that the accumulated substance in the distended r.E.R. is glycogen, because of the ultrastructural feature and the PAS results. Proteins with a high tyrosin content are excluded, too. The conversion enzymes studied from the glutamate breakdown pathway, glutamate dehydrogenase and succinic-semialdehyde dehydrogenase are not increased. Therefore, the production of glutamate (or aspartate) is believed to be normal. There is also no change in activity of the enzymes of the side pathways to the oxidative breakdown of glucose. LDH and G-6PD are not increased or diminished. The storage is increased. In our opinion this is due to a blockade of neurotransmitter release which in its turn is due to the destruction of the climbing fibre and its target by hemicerebellectomy. Within the hypertrophic cells, several biochemical systems show an increase in enzyme reaction product formation as measured by their key enzymes (see HARDONK and KOUDSTAAL 1976). Succinic dehydrogenase is markedly increased, but appears not to be coupled to an increase of other enzymes in the Krebs-cycle, because serious doubt exists concerning the isocitric dehydrogenase results. Succinic dehydrogenase increase alone is also found in human olivary hypertrophic cells (FRENKEN 1977). The terminal respiration cycle shows a distinct increase in enzyme reaction products. These results point towards an increased metabolic activity within the olivary hypertrophic cells and argue strongly against a degenerative process in these cells. Especially, since acid phosphatase is not increased, autophagosomes are not found ultrastructurally, and no increase in glial phagosomes was observed. The increase in AChE in the neuropil coincides with an increase of AChE reaction product formation within the hypertrophic cells themselves. The distribution of AChE in the olive of the cat (see 2.2) remains unaffected by longstanding ablations of the

22 . Enrico Marani

cerebellar cortex and lesions of the dorsal column nuclei which are known to project in a columnar fashion to portions of the 10 with a similar columnar distribution of AChE (MARAN!, VOOGD and BOEKEE 1977). A contribution of intrinsic neurons of the 10 to the AChE pattern can be excluded in rats by destroying the 10 with 3-acetylpyridine (MARAN! 1982 b). Ablation of the cerebellum together with the central cerebellar nuclei in cats is therefore the only intervention which has so far been shown to cause a permanent change in the AChE pattern of the 10 complex.

neuropil

hypertrophic cell

D
LU'uut[

atp.ase gepdh

..... gdh

OIO.~U

c_

ItA. .

SueC'NfC II

ALDl.M't'OI

lute...,,'.

0I410 ilClTAfI

)>-_ _----:...-_---J

..-.n... - - - - -......

bdh

...

.~

Fig. 11. This summary diagram demonstrates main glutamate pathways in neuronal and/or inferior olivary cells (MAO et al. 1974; SCHMIDT et al. 1977; ITo 1978). Enzymes tested and scheduled in this diagram are stippled, when enzyme reaction products are equal, or black at increased enzyme reaction products. The increase in catecholaminergic fluorescence outside the cell is indicated with DA. The acetylcholinesterase is augmented both in and outside the cell. The hatched arrows indicate the enzyme systems, which are not increased by glutamate conversion.

Topographic histochemistry of the cerebellum' 23

The enhanced AChE activity is present exactly and reproducibly in those parts of the 10, the rostral MAO, and part of the PO which also show hypertrophic cell changes after cerebellectomy. It is not present in the other atrophic parts of the 10 which nevertheless retain their original distribution of AChE activity. It has been attempted to explain the particular localization of neuronal hypertrophy in the 10 of the cat on basis of fibre connections. Crossed nucleo-olivary fibres issuing from the central cerebellar nuclei have been described in the cat (GRAYBIEL et al. 1973; TOLBERT et al. 1976). Destruction of the central nuclei, however, equally affects the nucleo-olivary fibres from the dentate nucleus to the PO, from the anterior interposed nucleus to the rostral DAO, and from the posterior interposed nucleus to the rostral MAO, whereas only part of the PO and the rostral MAO are involved in 10 hypertrophy. Two indirect pathways from the central nuclei to the 10 focus on the rostral MAO and the dorsal leaf of the PO respectively, namely from the posterior interposed nucleus through the contralateral Darkschewitsch nucleus and from the dentate through the contralateral red nucleus (fig. 6; BUSCH 1961; VOOCD 1964). Although the terminations of these tracts roughly coincide whith the regions of the 10 showing hypertrophy, this still does not explain the phenomenon as such. Protracted hyperactivity ofaxotomized nerve cells should involve the establishment of new connections. These connections could assume the configuration of connections with other hypertrophic cells through dendro-dendritic synapses which were noticed by BOESTEN (unpublished results) in hypertrophic 10 or electrotonic junctions (SOTELO et al. 1974). The localization of increased AChE activity should therefore be investigated in its relation to the reorganization of the neuropil of the hypertrophic 10. Sprouting of hypertrophic cells and the rebuilding of their axonal connections would increase the need for choline to produce phospholipids (VAN DER WAARD 1974). This effect is supported by the increase in beta hydroxy butyrate dehydrogenase, a key enzyme in the fatty acid metabolism (HARDONK and KOUDSTAAL 1976). Further investigation seems desirable; particularly, as the mechanism of AChE increase remains unclear. AChE increase could not be metabolically related to choline breakdown, since betaine aldehyde dehydrogenase is not increased. It is likely, according to the ultrastructural studies, that the hypertrophic 10 cells form new contacts. Rebuilding axonlike structures needs choline in order to produce phospholipids, characterized by the increase in beta hydroxybutyrate dehydrogenase, while their ingrowth into the neuropil of the rostral MAO could induce the increase of AChE around these endmgs. It is proper to speculate here, why these cells become hypertrophic. From our study, it seems clear that these cells, as well as the environmental neuropil, are reoccupied by catecholaminergic structures. Whether the occupancy of the hypertrophic area by catecholaminergic structures is the cause of the sprouting of the 10 cells or the result of the sprouting of the 10 cells, is uncertain. It could be supposed that the cells which receive mesencephalic input are preferentially reoccupied by catecholaminergic end-

24 . Enrico Marani

ings. Moreover, dendrite-like structures also contain dense-core vesicles, which makes it extremely difficult to discern changes in the neuropil on the ultrastructural level. Although a large number of enzymes were studied, only a few demonstrate an increase in enzyme reaction product formation (Appendix 2.3, table 13). These few are mainly key enzymes related to the oxidative energy metabolism of these hypertrophic cells. The demonstrated increase in beta hydroxybutyrate dehydrogenase can be related to extra fatty acid turnover or to pyruvate oxidation. Increased activity of beta hydroxybutyrate dehydrogenase has seldom been reported for the brain and has so far been mainly related to ketone body utilization as a partial replacement for glucose (SOKOLOFF 1973). In our opinion the results presented in this chapter favour strongly the hyperactive hypothesis for hypertrophic olivary cells and make it unlikely that the histochemical changes are an expression of the degeneration of these cells (see BEN HAMIDA 1965; GAUTIER and BLACKWOOD 1961; KOEPPEN et al. 1980 for arguments in favor of degeneration). Whether a long-lasting state of hypertrophy will result in the degeneration of these cells, is not known. It has been found that with the longest survival time 5' -nucleotidase activity shifts towards a decrease in reaction product formation within hypertrophic cells.

3 Histochemistry of the mature cerebellum 3.1 Morphology and histology of the mammalian cerebellum The cerebellum constitutes the roof of the rostral metencephalic portion of the fourth ventricle. It consists of a three layered cortex and a central white matter, containing the central cerebellar nuclei. Three cerebellar peduncles connect the cerebellum with the brainstem. Afferent connections from the spinal cord, the medulla oblongata, and the pons, enter the cerebellum laterally of the efferent connections of the cerebellum. Afferents from the pontine nuclei constitute the laterally located middle cerebellar peduncle. The inferior cerebellar peduncle contains fibres from spinal and bulbar origin in its lateral portion (restiform body) and cerebellar efferents in its medial part (juxtarestiform body). The superior cerebellar peduncle, that is the main efferent tract of the cerebellum, occupies a medial position. A great many transverse fissures divide the cerebellar surface in lobes, lobules and folia. The deep primary fissure separates the anterior from the posterior lobe. Two longitudinal sulci of varying depth demarcate the median vermis and hemispheres from the two cerebellar hemispheres. A distinction of vermis and hemispheres is not always possible in the anterior lobe, but it is a constant feature of the posterior lobe (for a review see JANSEN and BRODAL 1958).

Topographic histochemistry of the cerebellum . 25

The configuration of the lobules of the cerebellum of the cat was described by LARSELL (1936, 1952, 1953), LARSELL and Dow (1935), VOOGD (1964), and recently by BIGARE (1980). The general mammalian pattern, as described in detail for the mouse

(MARANI

and

VOOGD

1979), can also be recognized in the cat. The anterior lobe is divided into five lobules I-V and

displays a faint indication of its subdivision in vermis and hemispheres. Lobule VI is located caudally of the primary fissure. It constitutes the vermis of the simple lobule which has the same structure as the anterior lobe. The posterior lobe, caudal of the simplex lobule, is divided by the deep paramedian sulcus in the caudal vermis (lobules VII-X) and the hemispheres (ansiform and paramedian lobules, dorsal- and ventral paraflocculus and flocculus, numbered HVII to HX). The folia of the caudal vermis form a loop in the region of lobules VII and VIII. The cortex of the caudal vermis and the hemisphere is completely interrupted in the depth of the paramedian sulcus. This conexless area extends laterally far into the intercrural sulcus, into the centre of the folial loop of the ansiform lobule, and in between the dorsal and ventral paraflocculus. The cortex is always continuous between the successive lobules of the folial chains of vermis and hemispheres (see also MARAN I and VOOGD 1979; VOOGD 1975; VOOGD, GERRITS and MARANI 1985 for a detailed description). The continuity of the cortex within the folial chains finds its expression in the continuity of the individual longitudinal cortical zones of vermis and hemisphere. The concept of the division of the cerebellar cortex in longitudinal zones is based on the fibre connections of the cat. According to VOOGD (1964, 1967, 1969) and VOOGD and BIGARE (1980), the afferent and efferent connections of the Purkinje cells of the cortex are arranged in longitudinal strips. The first indication for this arrangement is the localization of the Purkinje cell fibres from each longitudinal zone on their way to a central cerebellar nucleus in a specific compartment of the central white matter (VOOGD 1964, 1969). BIGARE (1980) verified this idea with retrograde transport of horse radish peroxidase injected into the central cerebellar and vestibular nuclei of the cat. She was able to delineate a number of longitudinal Purkinje cell zones each projecting to a single nucleus (fig. 12). At least seven zones can be distinguished. Zones A and B belong to the vermis. They are uninterrupted from the anterior into the posterior lobe and project to the fastigial nucleus and Deiters' vestibular nucleus, respectively. The zones Cl, C2, and C3, which project to different parts of the interposed nucleus, continue from the anterior lobe along the folial chain of the hemisphere. Cl and C3 stop at the junction of the paramedian lobule with the dorsal paraflocculus. C2 continues to the flocculus and possesses an offshoot in the caudal vermis. The most lateral zones (Dl and D2) connect with the dentate nucleus and also extend over the entire length of the hemisphere. This hypothesis of the subdivision of the cerebellar cortex is called the multi-zonal, mono-nuclear hypothesis (BIGARE 1980). The zonal pattern can be recognized from the arrangement of climbing fibre terminals from certain parts of the inferior olive on longitudinal strips of Purkinje cells (GROENEWEGEN and VOOGD 1977; GROENEWEGEN et al. 1979). GROENEWEGEN concluded that climbing fibres from a particular part of the

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VI

ANTERIOR

Fig. 12. Concluding scheme of the cortico-nuclear and cortico-vestibular projections of the cat cerebellum. At the top left the cerebellar and vestibular nuclei are indicated. In reconstructed views of the cerebellum the different projection zones of the cortex are indicated. Within the schemes of the vestibular and cerebellar nuclei the same symbols are used. - Courtesey F. BIGARE.

Topographic histochemistry of the cerebellum' 27

inferior olive terminate both on Purkinje cells and on the central cerebellar nucleus which receives the axons of these Purkinje cells. The organization of the olivocerebellar projection in the cat is summarized in fig. 13. The number of zones, their extent and their distribution are probably essentially similar in the corticonuclear and olivocerebellar projection (VOOGD and BIGARE 1980; VOOGD et al. 1981).

A

B

......--ROSTRAAL

c

flocculus

CAUDAAL--+

Fig. 13. Olivo-cerebellar projection in the cat (GROENEWEGEN et al. 1979). Corresponding cortex zones and inferior olivary parts are indicated with the same symbols. A: Transversal section through the inferior olive (DAO dorsal accessory olive, MAO medial accessory olive, PO principal olive). B: Concluding diagram of the olivo-cerebellar projection.

Recent studies on the vestibular input into the cerebellum and the cortico-vestibular subdivisions demonstrate that AI, a subdivision of zone A, projects to the magnocellular part of the medial vestibular nucleus, while A2 is connected to the fastigial nucleus. EKEROT and LARSON (1982) discovered an extra zone X, which seems to be related to the dorsolateral protuberance of the fastigial nucleus. This band is exclusively located in the anterior lobe. Together with the B zone, these zones constitute the control system for the motoneurons in the anterior horn of neck and deep muscles of the back (fig. 14). All other zones (Cl till D2) are related via their cerebellar

28 . Enrico Marani

nuclei and motoneurons in the anterior horn of the spinal cord with arm and leg (fore- and hindpaw) musculature. The zones C1 through D2 are thus related to the guidance of proximal located musculature within the extremities, while the zones A to B are related to more centrally placed muscle groups (fig. 14). Cytologically the cerebellar cortex is subdivided into an inner layer containing the granule cells and an outer molecular layer. In between, the large perikarya of the Purkinje cells constitute a monolayer. The dendrites of the Purkinje cells branch in the molecular layer. The dendritic tree of the Purkinje cell is confined to a single plane oriented perpendicularly to the transverse fissures. The myelinated axons of the Purkinje cells enter the cerebellar white matter and terminate in the central cerebellar and vestibular nuclei. During their course through the granular layer, the Purkinje cell axons give off myelinated collaterals which can be traced back to the Purkinje cell layer, where they constitute an infra- and supraganglionic plexus and terminate mainly on the Purkinje cell somata and primary dendrites of neighbouring Purkinje cells (PALAY and CHAN-PALAY 1974). In the direction of the transverse fissures, the spread is (far) greater, including even neighbouring lobules, according to some (FREZIK 1963). In the adult, the transverse spread of the Purkinje cell axon collaterals is presumably less than in the neonate. This is perhaps one of the examples of the remodelling which occurs during the histogenesis of the cerebellar cortex (PALAY and CHAN-PALAY 1974). The axons of the Purkinje cells constitute the only efferent system of the cerebellar cortex. The afferent systems of the cortex can be subdivided into climbing fibres which terminate in the molecular layer on dendrites of the Purkinje cells and mossy fibres which terminate on granule cells which, in their turn, give rise to axons of parallel fibres which form synapses with the Purkinje cell. More recently, other types of afferents were discovered by using fluorescent histochemical methods for monoamines arising from the locus coeruleus and the raphe nuclei. Climbing fibres are thought to originate exclusively from the inferior olive in the medulla oblongata. Olivocerebellar fibres branch in the cerebellar white matter. Each Purkinje <;ell is provided with a single branch of the climbing fibre (SZENTAGOTHAI and RAJKOVITS 1959). Both the branching of the olivocerebellar fibres (AMSTRONG et al. 1973) and the terminal arborization of the climbing fibre are oriented perpendicularly to the transverse fissures. During the histogenesis of the cerebellar cortex, there exists a close relationship between the outgrowth of the Purkinje cell dendritic tree and the climbing fibre terminals (RAMON Y CAJAL 1972; VAN GEHUCHTEN 1891; RETZIUS 1892 a,b; LARRAMENDI 1969). Originally, the climbing fibre terminals surround the immature Purkinje cell body and the stem of its dendritic tree like a cape. Innervation of a single Purkinje cell by multiple climbing fibres is quite common at this stage. The synaptic region of the climbing fibre in the adult is shifted from the cell body to the initial smooth portion of its dendrites and multiple innervation is no longer observed (LARRAMENDI 1969). Climbing fibres form synapses on short, stubby spines present on «smooth» dendrites, but do not reach the characteristic terminal «spiny» branchlets of the Purkinje cell dendritic tree with their long thin-necked spines (PALAY and CHANPALAY 1974). For a long time, the origin of the climbing fibres has been a matter. of dispute.

Topographic histochemistry of the cerebellum . 29

DESCLIN (DESCLIN 1974; DESCL"iN ~~d ESCUBI 1974) was able to show the complete degeneration of the climbing fibres after chemical destruction of the 10 in the rat with 3' -acetylpyridine. With this experiment, the exclusive origin of the climbing fibres from the 10 seems to have been settled. Mossy fibres originate from different groups of cells in the spinal cord, the medulla oblongata, and the pons. They terminate with multiple voluminous terminals, called «mossy fibre rosettes», on the cells of the granular layer. The rosette with its surrounding of granule cell dendrites and the terminals of some inhibitory interneurons (Golgi cells) constitute a complex synapse or cerebellar glomerulus. The terminal branching of mossy fibres mainly occurs on a plane perpendicular to the transverse fissures, but, unlike the climbing fibre, this branching is much more diffuse and its parasagittal orientation is less precise. Granule cells are among the smallest nerve cells in the central nervous system. The perikaryon consists of a nucleus with a small rim of cytoplasm. The axon ascends towards the molecular layer, where it divides in a T-shaped manner into parallel fibres which are situated parallel to the transverse fissures and perpendicularly to the Purkinje cell dendritic trees. Parallel fibres form synapses with the dendrites of Purkinje cells on their way. These synapses are found on the spines of the spiny branchlets which constitute the terminal portions of the Purkinje cell dendritic tree. Parallel fibres are unmyelinated, although some of these fibres in the deepest part of the molecular layer may have a myelin sheath. Their exact length has never been settled. Parallel fibres in the deep portion of the molecular layer, which supposedly originate from the deepest portion of the granular layer, are shorter than the more superficially located fibres (MUGNAINI 1976). The maximal length of parallel fibres, which has been established with experimental methods, is 3.0 mm (MUGNAINI 1976; SCHILD 1980). Parallel fibres form synapses in the molecular layer, also on the dendrites of a number of interneurons of the cerebellar cortex, apart from the dendrites of the Purkinje cells. These interneurons are the stellate cells located in the upper part of the molecular layer, the basket cells with their perikaryon located in the inner part of this layer, and the Golgi cells which are located among or below the Purkinje cell layer. The axons of stellate and basket cells terminate on Purkinje cells. The dendrites of stellate and basket cells and the axonal arborization of the basket cells with their characteristic axonal baskets around the cell body and the initial axonal segment of the Purkinje cell are oriented perpendicularly to the transverse cerebellar fissures. The dendrites and the dense axonal plexus of the Golgi cells do not display such specific orientation. The axon terminates with multiple boutons on the periphery of the glomeruli in the granular layer.

Extensive electrophysiological investigations of the cerebellar cortex, summarized by ECCLES, ITo and SZENTAGOTHAI in their book «The cerebellum as a neuronal machine» (1967) show the interneurons of the cerebellar cortex to be inhibitory. Mossy, climbing, and parallel fibres which belong to the main cerebellar circuit are all provided with excitatory synapses. The ultimate effect of the cortical machinery is expressed in the activity of the Purkinje cells which was found to be inhibitory on the cells of the central cerebellar or vestibular nuclei (ECCLES, ITO and SZENTAGOTHAI 1967, see also ITO 1984). Concomitant investigations of the ultrastructure of the synapses in the cerebellar cortex demonstrate a consistent difference between excitatory

30 . Enrico Marani

and inhibitory synapses under certain conditions of fixation and processing of the material (UCHIZONO 1965). Synaptic vesicles in the mossy fibre rosettes and the synapses of the parallel and climbing fibres on Purkinje cell dendrites are spherical, whereas basket cell axons and Golgi cell boutons contain flattened vesicles. The central nuclei of the cerebellum are located in the central white matter in the roof of the fourth ventricle at the base of the white matter of different lobes and lobules. According to WEIDENREICH (1899) and OGAWA (1936), four cerebellar deep nuclei can be distinguished. In between the medial cerebellar nucleus or fastigial nucleus and the most lateral nucleus or the dentate nucleus, the interposed nucleus is located. This nucleus is subdivided into a caudo-medial posterior interposed nucleus and a rostro-lateral anterior interposed nucleus. The Deiters' nucleus (nucleus vestibularis lateralis), although located within the vestibular area, receives Purkinje cell axons and is therefore considered as a true cerebellar nucleus. Afferents of the central nuclei, apart from the Purkinje cell fibres, consist of the collaterals of the mossy fibres and climbing fibres which terminate in the cortex. Similarities in the type of terminal boutons in the cortex and the central nuclei make it possible to discern the terminals of these mossy and climbing fibre collaterals in normal material of the central nuclei (CHAN PALAY 1977). In addition, other types of boutons related to interneurons of the central nuclei have been observed by the same author. The significance of the dual innervation of the cerebellar cortex by climbing and mossy fibres is not well understood. Both provide paths from the periphery and the cerebral cortex to the cerebellum. According to the «learning hypothesis» of MARR (1969), climbing fibres change the efficacy of the synapses of the parallel fibres with the Purkinje cell dendrites on the basis of previous experience. The ultimate effect of the cerebellar cortex is exerted through inhibition of the central cerebellar and vestibular nuclei on motor centres in the cortex, the brain stem, and the spinal cord (fig. 14). This effect in mainly indirect. Cerebellar glia is interstitial tissue which can be subdivided into ependyma, the lining cells of the ventricle, and the neuroglia proper. The neuroglia is subdivided in macroglia (ectodermal of origin) and microglia (mesodermal- or from mixed origin). Cerebellar macroglia mainly consists of the protoplasmic type of astrocytes. Oligodendrocytes form the myelin sheet around axons in the cerebellum. The astrocytes in the cerebellum can be subdivided into Golgi-epithelial cells, because they are present within the molecular layer. Their protrusions into the molecular layer are known as the Bergmann fibres. The arborization of the Golgi-epithelial cell is mainly situated in the parasagittal plane. Each cell gives rise to several protrusions which bear irregular thickenings over their trajectory to the pial surface. These glial cells are related to the Purkinje cell dendritic tree. The Bergmann glial protrusions end on the pial surface but also on capillaries with terminal knobs (PALAY and CHAN PALAY 1974). The neurotransmitters involved in signal processing of the cerebellar cortex are only partially known. Detection of small amounts of suspected transmitter substances is now possible with gas chromatography and radioimmunoassays. Unfortunately, only few putative neurotransmitters can be visualized directly. An accurate localization of these substances at the light and electron microsopical level is recently made possible with immunohistochemical methods (STEINBUSCH 1982). Often, the presence of their synthetizing or catabolizing enzymes is used to indicate their presence. Additional

Topographic histochemistry of the cerebellum' 31

Fig. 14. This imaginal unfolded plane of the brain demonstrates the main cerebello-cortical and cerebello-spinal pathways. The corticofugal and rubro-spinal tracts are indicated too.

information about the presence and the localization of transmitter substances was derived from genetic malformations, missing one or more cell types in their cerebellar cortex. Neuropharmacological techniques include the local application of suspected neurotransmitters (<
32 . Enrico Marani

and SCHMIDT 1978). For most mossy fibre systems, the neurotransmitters are not yet known. Several afferent systems containing aminergic transmitters have been identified with the FALCK-HILLARP method (FALCK et al. 1962). Fibres from the locus coeruleus contain noradrenaline (BLOOM et al. 1971, 1974; HOFFER et al. 1971, 1978; SIGGINS et al. 1971), while projections of the raphe nuclei to the cerebellum consist of serotoninergic fibres (SHINNAR et al. 1975; KAWASAKI and SATO 1980). It has also been claimed that some mossy fibres contain serotonin (CHAN PALAY 1977). SOTELO (1981), however, found no immunoreactivity for the synthetizing enzymes of serotonin in mossy fibre terminals in the granular layer. The morphological characteristics of fibres containing noradrenalin or serotonin have not yet been definitely settled. Some of the amino acid neurotransmitters, such as aspartate or glutamate, are always excitatory; others such as GABA and glycine are always inhibitory. All of these amino acids, including taurine, are probably present as neurotransmitters in the cerebellar cortex. Recently, selective uptake and retrograde transport of aspartate by climbing fibres (WIKLUND et al. 1982) has strengthened the case for aspartate as the neurotransmitter in the olivocerebellar system, although glutamate has not yet been ruled out definitely. The inhibitory neurotransmitter GABA has been shown to be the neurotransmitter of the Purkinje cell (ITo 1972). GABA is produced from glutamate by glutamic acid decarboxylase (GAD). The presence of GAD has been demonstrated with immunohistochemical methods in cerebellar neurons (Purkinje, basket, and stellate cells) which are supposed to use GABA as a neurotransmitter (see OERTEL et al. 1981). GABA is broken down into succinic semialdehyde. The end product of the conversion of succinic semialdehyde, gamma hydroxybutyric acid, is known to be highly concentrated in the cerebellum (ROTH and GIARMAN 1966). The basket cells in the molecular layer presumably produce GABA at transmission (ITo 1978). GAD is localized in these cells and their terminals (OERTEL et al. 1981). The stellate cells can be subdivided into cells of the upper and lower portion of the molecular layer (PALAY and CHAN PALAY 1974). The cells in the upper part are considered to use taurine as a neurotransmitter, those in the lower portion react with antibodies against GAD and, therefore, are GABA-ergic (ITO 1978; WIKLUND et al. 1982). The results of OERTEL et al. (1981) demonstrate, however, with a new antiserum for GAD that the upper stellate cells are also GAD immunoreactive. The Golgi cells, which constitute an inhibitory feed back loop between the parallel fibres in the molecular layer and their cells of origin in the granular layer, are supposed to be glycinergic (WIKLUND et al. 1982). Arguments supporting GABA as the neurotransmitter of the Golgi cells have been put forward (OERTEL et al. 1981). The granule cell axon, the parallel fibre, uses an excitatory amino acid as the neurotransmitter in its synapses in the molecular layer. Both glutamate and aspartate have been named as possible candidates (WIKLUND et al. 1982).

Topographic histochemistry of the cerebellum· 33

lansofcrmos S I

I~

f po5lotQ

!t,;lI'ft""--or'

par3lotculus floc",lus

A ••

II

' .........

B

ACM ..... _

.. _

c

Fig. 15. Comparison of the distribution of acetylcholinesterase in the rat, the spino-cerebellar and the vestibulo-cerebellar endings in the mouse and rabbit (A). In (B) a map of the longitudinal bands for AChE in the rat granular layer is demonstrated in lobule IX and VIII. In the diagram of the unfolded cerebellum of the mouse (C) the longitudinal spino-cerebellar mossy fibre bands are indicated.

Purines such as ATP and AMP, and, in addition, adenosine have been proposed as neurotransmitters or neuromodulators of the so-called purinergic neurons (see STONE 1981, for a review). Effects of purines on the fire frequency of Purkinje cells have been described by KOSTOPOLOUS et al. (1975), although, originally they were denied (BLOOM et al. 1971).

34 . Enrico Marani

Recently, adenosine receptors have been described in high concentrations within the molecular layer of the cerebellar cortex (GOODMAN et al. 1983), while it was proposed that these adenosine receptors are located presynaptically in several brain areas (PHILLIS and Wu 1981). Adenosine application selectively blocks presynaptically parallel fibre mediated synaptic activity, but does not block climbing fibre mediated synaptic activity. Adenosine may be contained within the same vesicles as the primary transmitter and may be released as a co-transmitter (PHILLIS and Wu 1981; SILINSKY 1975; MARANI 1982 a, b). 3.1.1 AChE longitudinal pattern in the rat cerebellar granular layer Within the cerebellar granular layer of the rat and, less clearly, in that of the mouse, AChE activity is present with a peculiar topography. Previously it was categorically stated that AChE is exclusively located in the nodule, uvula, and flocculus (ARVY 1966), but this was questioned by SHUTE and LEWIS (1964) and ODUTOLA (1970). ODUTOLA described the correct topography of AChE in the adult rat cerebellum. AChE is also found to be present in vermallobules VI and VII and vermallobules I and II. Most AChE studies look at the distribution in sagittal series, missing the additional details of the medio-lateral extent which can only be noticed in transverse series. Within the caudal as well as the rostral vermis, a longitudinal pattern can be noticed for the AChE positivity within the rat and mouse cerebellar granular layer. This pattern is described below. Because of its sagittal topography, the AChE distribution has been related to the input of the nucleus reticularis tegmenti pontis. This nucleus is considered to be an intermediate in the audio-visual cerebellar circuitry (ODUTOLA 1970). Recent studies in our laboratory on the vestibulo-oculo-cerebellar circuit, also concerning the nucleus reticularis tegmenti pontis (GERRITS, EPEMA and VOOGD 1984), result in an autoradiographic distribution of 3H-leucine which is not concordant with the AChE distribution in the granular layer (see also WALBERG 1982). The nucleus reticularis tegmenti pontis does not project to lobule X, and ponto-cerebellar input in general is mainly directed to the hemispheric parts of the cerebellum (VOOGD 1969; GERRITS, EPEMA and VOOGD 1984). None of the cerebellar mossy fibre input systems (VOOGD 1969) so far described coincide with the distribution of AChE positive mossy fibre endings. After lesions of the cerebellar peduncles (SHUTE and LEWIS 1964), AChE piles up in cells and axons. AChE positive fibres have been found in each of the peduncles. Since specific systems use a certain peduncle and none of the mossy fibre input systems totally overlaps with the AChE positive mossy fibre distributions, heterogeneity of the mossy fibres must be accepted (see also SILVER 1974). There are, however, cerebellar mossy fibre systems which have an equivalent longitudinal distribution: the spinocerebellar and the vestibular systems. Hemichor-

Topographic histochemistry of the cerebellum' 35

Fig. 16. The distribution of secondary mossy fibres after WGA-HRP injections into the vestibular nuclei. Antegrade HRP positive mossy fibre endings coincide with HRP labelled Purkinje cells. dotomies in the mouse gave the same topography of degenerated mossy fibres as that of AChE. The spino-cerebellar input, however, extends further than the AChE pattern (see fig. 15 A) and differs in its localization in lobule VIII and the copula pyramidis. AChE is contributed to mossy fibres, a fact which is beyond doubt (CSILLIK, Jo and KASA 1963; ODUTOLA 1970). An early report (SHIMIZU and ISHII 1966) already demonstrated its ultrastructural localization around mossy fibres. This localization is an extracellular one; therefore, a contribution of the surrounding structural elements cannot be excluded. In fact, parts of granular cell dendrites have been found to be AChE positive, an observation which is confirmed in our ultrastructural studies in cat and rat (MARANI, unpublished). The other strictly mossy fibre input, which has recently be studied again is the vestibulo-cerebellar secondary mossy fibre system. Antegradely HRP-Iabelled mossy fibres show a homologous distribution after several injections of WGA-HRP in the vestibular nuclei (fig. 16; EPEMA, GULDEMOND and VOOGD 1985). The distribution of

36 . Enrico Marani

the mossy fibre strips is concordant with the numbers of AChE positive zones in lobules I-II, III, IV-V, and IX-X. Both the vestibular mossy fibres and the AChE positive mossy fibres are present in lobule VI-VII. The vestibulo-cerebellar mossy fibres (are believed to) reach the cerebellum not only via the corpus restiforme. The corpus juxtarestiforme is also involved, while part of these fibres are directed rostrally and turn back near the brachium conjunctivum to reach the dorsal part of the corpus juxtarestiforme. The pedunculus flocculi is AChE positive and embraces the brachium pontis. Within the granular layer, the AChE reactivity is confined light microscopically to mossy fibres. The density of AChE-positive fibres is different in various lobules (fig. 15), as has been described by OnuToLA (1970). In transverse sections, concentrations of AChE mossy fibres are noticed. Their appearance, arrangement and quantity are similar in successive sections. In reconstructions of lobule X and IX, they are found to form longitudinal strips (fig. 15 B, C). On each side of the broad midline strips of lobules X and IX, three distinct zones are present. Sometimes the first zone at each side of the midsagittal one has a shallow subdivision. Laterally, where the cortexless area in the uvula commences a fourth, but mostly indistinct zone, AChE-positive concentration can be distinguished on each side of the midline. Lobule VIII contains three positive zones on each side of a smaller positive midline zone. Sometimes, a weak concentration of positive mossy fibres can be noticed in the copula pyramidis. At the transition of lobule VIII to VII, strongly positive fibres cross the midline and can be followed backwards into rostral part of the brachium pontis. These strongly positive fibres give off AChE-positive mossy fibres diffusely to the simplex, ansiform, and paramedian lobules. The granular layer of lobule VI and part of the dorsal granular layer of VII contains positive glomeruli of which the zonal pattern is indistinct. The anterior lobe vermis contains positive mossy fibres. The midline zone and its bordering strips form a continuum in lobules I to III and can only be separated into distinct subzones in lobule III to V. Three positive strips can be discerned on each side of the midline. All lobules contain AChE-positive fibre clusters below the AChEpositive mossy fibre zones. In the paraflocculus and flocculus, no zonal pattern for AChE positive mossy fibres can be discerned. The flocculus and only the ventral paraflocculus are strongly AChE positive. The fibres entering the flocculus (pedunculus flocculi) are strongly positive for AChE, as is the stalk of the paraflocculus. The presence of AChE mossy fibre terminals in the rat granular layer and the supposed localization of this enzyme in the archicerebellum have led to great controversy both in anatomical and in physiological studies (see SILVER 1974). The vestibular nuclei were originally held responsible for the AChE pattern (CSILLIK et al. 1963; SHUTE and LEWIS 1964; KASA and SILVER 1969). Although AChE positive mossy fibres are also found outside the archicerebellum (OnuToLA 1970), the statement that

Topographic histochemistry of the cerebellum' 37

the vestibular input is restricted to the archicerebellum is a false one (fig. 15). The overlap of AChE positive mossy fibres and the input of secondary mossy fibres, as detected with modern anatomical techniques, is striking. Both spinocerebellar and pontine mossy fibres have quite a different topography. The positive fibres of the brachium pontis which are directed to the most lateral parts of the hemispheric sublobules, where an additional very weak concentration of AChE terminals is present, are not consistent with a strictly vestibular input. 3.2 Myeloarchitecture of the cerebellum The variations in fibre calibre of the cerebellar medullary ray contrast with the uniform histology of its cortex. Within the cerebellar white matter, subdivisions are present in which thick or thin fibres predominate. Corticonuclear thick fibres in the white matter of the cerebellum of the cat, ferret, and fowl are arranged in parasagittal sheets which alternate with regions where thin afferent fibres predominate. In transverse sections through the individual folia, a bundle of thick, myelinated fibres and the area of thinner fibres, bordering it on its lateral side (the «raphe», VOOGO 1964), are called a compartment. Compartments of successive folia fuse in the central white matter of the cerebellum and align with their appropriate deep cerebellar nuclei (for the cat and ferret: VOOGO 1969; VOOGO and BIGARE 1980; for the fowl: FEIRABENO et al. 1976). The distribution pattern of the corticonuclear fibres has led to the hypothesis that Purkinje cells, projecting to a particular cerebellar or vestibular nucleus, are located in discrete, longitudinal zones. This phenomenon has been confirmed by axonal transport and antegrade techniques (Van ROSSUM 1969; HAINES 1975 a,b; VOOGO and BIGARE 1980), as well as by enzyme techniques (see 3.5 and 3.6). Afferent thin fibres are not only present in raphes in the lateral part of the compartments, but also occur as scattered fibres among the thicker cell fibres. Some of the thin fibres represent olivocerebellar fibres. These are climbing fibres (see 2 and 3.1) and demonstrate the same distribution in parasagittal sheets as the corticonuclear fibres. Mossy fibres constitute the other basic afferent system in the white matter of the folia. Although a parasagittal sheet-like distribution has been described for several mossy fibre systems (VAN ROSSUM 1969; VOOGO, BROERE and VAN ROSSUM 1969; RUSSCHEN, GROENEWEGEN and VOOGO 1976; VIELVOYE 1977; GERRITS and VOOGO 1979; NAS, GERRITS and VOOGO 1981), the topographical situation is less clear. The description of the parasagittal organization of the cerebellar cortical afferent and efferent fibres has elucidated the organization of the input and output to the cerebellar cortex, and thus leads to a greater understanding of this part of the central nervous system. This longitudinal organization of the cortex and the typical relation of each zone to one cerebellar or vestibular nucleus are described in the cerebellar multizonalmononuclear hypothesis (BIGARE 1980).

38 . Enrico Marani

Three principles form the basis for the analysis of compartments in the cerebellar white matter. The first is that each compartment is related to a central cerebellar nucleus. The second concerns the fibre pattern, i. e. the calibre of the fibres within each compartment. The third is the principle of continuity of the compartments, as determined by the successive parallel folia and the orientation of the cortex in the vermis and hemispheres. Previous description of the gross anatomy of successive parallel folia of the mammalian cerebellum have assumed that the vermis and hemispheres constitute three parallel and independent folial chains (BOLK 1906; VOOGD 1975; MARANI and VOOGD 1979). In the posterior lobe, that folial chain of the hemisphere forms two laterally directed loops, the ansiform lobule and the paraflocculus. The cortex is said to be always uninterrupted from one folium to the next within a folial chain. However, in between the limbs of a loop (i. e. between the crura of the ansiform lobule and the dorsal and ventral paraflocculus) and between two successive loops (i. e. in between crus II and the dorsal paraflocculus), the cortex is generally interrupted. These relations have been described in detail for the mouse cerebellum (MARANI and VOOGD 1979) and in the rat (VOOGD, GERRITS and MARANI 1985; see also MARANI and VOOGD 1979). The position of the underlying white matter of the compartments, containing the efferents and afferents of the cortex, is also determined by the direction of the folial chain (VOOGD and BIGARE 1980). Some of the cortical zones have been found to divide and thus to extend into both vermis and the hemisphere. If a cortical zone branches (e. g. C2), the corresponding compartment straddles the (paramedian) sulcus and bridges the area devoid of cortex which may be present in the floor of this sulcus.

3.2.1 Description of the myeloarchitecture of the mouse cerebellum

Myeloarchitecture of the anterior lobe Symmetrically disposed compartments can be distinguished in all folia of the anterior lobe. However, it is only possible to trace the continuity of a few compartments throughout the entire lobe, due to the addition of new compartments in more caudal anterior lobe lobules. Four compartments can be recognized (fig. 17-213 to 221) in the white matter lobule I-II. The medial two compartments (numbers 1 and 2 in the figures) are of equal width, but markedly wider than the lateral compartments 3 and 4. In the caudal part of this lobule, the most lateral compartment is flat and it spreads out into the white matter dorsal to the superior cerebellar peduncle (fig. 17-166). Five compartments can be recognized bilaterally in rostral sections through the white matter of lobule III. The medial compartment is further subdivided in 1a and Ib by an indistinct raphe of scattered small fibres. More caudally (fig. 17-166), the compartments la, 1b, and 2a fuse with the compartments 1 and 2 of lobules I-II. Compartment 2b of lobule III can only be distinguished in the dorsal part of this lobule, more ventrally it disappears into the raphe bordering the lateral side of compartment 2. The border between compartments 3 and 4 blends within the white matter dorsally to the central nuclei. This region contains bundles of fibres which arch medially from

Topographic histochemistry of the cerebellum' 39

the restiform body and the ventral spinocerebellar tract. Purkinje cell fibres from the most lateral portion of the lobule cannot be traced to their destination. In transverse sections, the white matter of the lobules IV-V forms an almost complete semicircle that contains numerous compartments on both sides of the midline (fig. 17-172). The most medial compartments subdivide again into compartments la and lb. More caudally (fig. 17178), these compartments and the next compartment 2a fuse with their counterparts in lobule III. It is unclear, whether compartment 2b of lobule IV-Vis the continuation of compartment 2b that disappeares in the ventral part of lobule III. Compartment 3 is located at the point, where the white matter of the lobules IV-V curves ventrally (fig. 17-172). Two narrow compartments (4 and 5) occupy the lateral, sloping part of the white matter. In more caudal sections, this part of the white matter is connected to the white matter dorsal to the central nuclei. Whether compartments 3 and 4 of lobules IV-V fuse with the third compartment of lobule III, could not be established. The corpus of the fibres in the more lateral portions of the medullary ray is obscured by the presence of bundles of semicircular fibres, passing dorsally to the central nuclei.

A

184

Fig. 17. Sections through the cerebellum of the mouse. Raphes bordering compartments in the white matter are indicated in black in both the Haggqvist- (left) and the 1 f.lm sections (right side). (A) are sections in the posterior lobe, while (B) contains more rostral sections.

40 . Enrico Marani

Fig. 18 depicts the borders of the compartments projected on the anterior and dorsal surface of the lobules of the anterior lobe. These graphic reconstructions summarize the subdivision of the white matter of the anterior lobe. However, these orthogonal projections do not imply that these borders correspond exactly to the position of the comparable longitudinal cortical zone, due to the projection method used. There are compartments of the anterior lobe that cannot be traced into the central nuclei. This is caused by the lateral position of the fastigial nucleus, relative to the medial two or three compartments, and the presence of the heavy layer of semicircular fibres arching over the central nuclei. Fibres from compartments 1,2 and 3 can be followed into the fastigial nucleus, but it is not clear, whether all of them terminate here of whether some contribute to the fibres passing to Deiters' nucleus too. A characteristic fibre pattern could be established for most compartments in the anterior lobe, which is repeated in each successive lobule. The pattern of fibre calibres is essentially the same as described in the cat (VOOGD and BIGARE 1980).

Myeloarchitecture of the posterior lobe The rostral margin of the simple lobule (lobule VI) overhangs the anterior lobe (MARANI and VOOGD 1979). The caudal-medial aspect of the simple lobule borders lobule VII and is indicated by a shallow sulcus (fig. 17). Laterally, the simple lobule continues into two hemispherical folia. An area devoid of cortex separates this lobule from the medial tapering part of the ansiform lobule. The lobules VIII, IX, and X are well demarcated. Lobule VIII (the pyramis) continues laterally in the copula pyramides which forms the ventral part of the paramedian lobule. The cortex of lobules IX and X does not continue into the hemisphere. Parasagittal compartments are present in the white matter of the posterior lobe vermis and in the paramedian lobule. The raphes are much wider and the areas containing the bundles of thicker Purkinje cell fibres are narrower in the vermis of the lobules VI and VII than in the anterior lobe. Moreover, dense bundles of thin, pontocerebellar fibres occupy the lateral white matter of the simple lobule and enter the vermis of the lobules VI and VII caudally. Most of these fibres cross in the white matter of lobule VII (fig. 17-196, 202) and obscure the parasagittal arrangement of the corticonuclear fibres in the transition region of VI-HVI and VII-HVII. A wide raphe which contains bundles of thicker fibres, separates compartments 1 and 2 of the simple lobule. Compartment 1 is rather wide, 2 and 3 are narrower. In the rostral sections, the lateral, sloping part of the white matter contains compartments 4 and 5 (fig. 17-184). The parasagittal division of lobule VI is more prominent in the ventral white matter adjoining the primary fissure. The dorsal part of the white matter of lobule VI contains decussating pontocerebellar fibres which increase caudally. Caudal to the primary fissure compartments 1, 2,and 3 fuse with the corresponding divisions of lobules VIII and IX. These compartments can be traced rostrally into the fastigial nucleus. The position of the compartments 1 and 2 is approximatively the same as the corresponding compartments of the anterior lobe. These relations are not as distinct in the more lateral subdivisions. Compartments 1,2, and 3 continue rostrally into lobule VII, but their borders become less distinct. Most of the compartments in the lobules VIII and IX can be traced into their corresponding central nuclei. Therefore, it seems justified to apply VOOGD'S nomenclature (1969) for this part of the cerebellum. Three medial compartments (A-I, A-2, and A-3) can be distinguished in both lobules. In lobule VIII, they are narrower than in lobule IX. The raphes are connected to the separating ones from compartments 1-3 of the simple lobule (fig. 17-196). More laterally, the fibre spectrum of a narrow bundle of large Purkinje cell fibres permits identification of compartment C-l that is present in lobule VIII at the transition of vermis and hemisphere. Dorsally this

Topographic histochemistry of the cerebellum . 41

compartment widens and becomes distinctly subdivided within the medial white matter of the paramedian lobule. The C-1 compartment does not continue into lobule IX. This is apparent at the junction of lobules VIII and IX ventrally. A more laterally situated area of thinner corticofugal fibres (C-2) is present in the paramedian lobule, the copula, and the extreme lateral part of lobule IX. Even more laterally larger Purkinje cell fibres occupy the C-3 compartment in the copula and the more dorsal folia of the paramedian lobule. An additional raphe indicates tq.e presence of D-1 and D-2 compartments in the most lateral part of these folia. The A-I and A-2 compartments cQntinue into the medial and central parts of the fastigial nucleus at the base of the caudal folia. Fibres from A-3 reach the ventral part of the dorsolateral protuberance of the fastigial nucleus. Fibres from the medial part of the hemisphere, medial to the C-1 zone and the lateral vermis can be seen in semi-thin sections to converge on the dorsolateral protuberance. It is not clear, whether these fibres should be included in A-3, therefore, this region has been indicated by a question mark in fig. 18. The C-2 compartment with its characteristic homogeneous pattern of thin fibres, can be traced into the posterior interposed nucleus. It is located between the larger fibres of C-1 and C-3 which surround the dorsomedial crest and the dorsolateral hump region of the anterior interposed nucleus.

DORSALASP.~E~C~T

t

~-r-~

CAUDAL ASPECT

c d

H99B3

A H991l3

B

Fig. 18. A: Borders of the compartments in the white matter of the cerebellum as projected on the dorsal aspect of the lobules I-II, III and IV-V of the posterior lobe. B: Borders of the compartments in the white matter of the cerebellum as projected on the rostral aspects of the lobules I-II, III and IV-V of the anterior lobe, the simple lobule and the caudal aspects of the posterior lobe.

42 . Enrico Marani

The white matter of lobule X and the flocculus is narrow. Only rostrally, in the nodule, three A compartments can be distinguished in 1 f.lm sections. It is inconclusive, whether C-Z is present in the lateral portion of lobule X.

3.2.2 Discussion of the topography of the myeloarchitecture Some reservations have been made as to the value of myeloarchitectonic studies (COURVILLE et al. 1974, 1980). Parasagittal zones could not be distinguished through successive lobules ofthe posterior lobe. In our analysis of the white matter of the cerebellum of the mouse we encounter similar problems. Compartments can be traced through the anterior lobe and the posterior lobe vermis. However, in the cerebellar hemisphere positive identification in Haggqvist-stained series is also possible in the copula pyramidis and the adjoining folia of the paramedian lobule. Compartments are present in the white matter of the ansiform and simple lobules, but their continuity cannot be established conclusively (fig. 17). However, 1 mf.l sections and Haggqvist series demonstrate similar fibre components within white matter of ansiform lobule and partly the simple lobule, more distinct in 1 f.lm series. In the caudal part of the posterior lobe, the number of the compartments, their fibre pattern, and their relation to the central nuclei are identical to the situation in the cat, as outlined by VOOGD and BIGARE (1980). The medial compartment (A-3) seems to be related to the dorsolateral protuberance of the fastigial nucleus. The intermediate compartment C-Z continues with the posterior interposed nucleus. Compartments C-1 and C-3 terminate in the medial and lateral parts of the anterior interposed nucleus, which have been described in the rat as the dorsomedial crest and the dorsomedial hump (GOODMAN et al. 1963). The relationship between the most lateral D compartments with the lateral nucleus cannot be established with certainty. The number of compartments in the anterior lobe increases in the more dorsal folia. There are four compartments in lobule I-II, five in III, and seven or eight in the combined lobules IV-V for each hemi-lobule. Part of this increase is due to the subdivisions of compartment 1 and the insertion of compartment zb in lobules III-V. Moreover additional compartments appear in the lateral sloping part of lobule IV-V, which are not present in more ventral lobules. The compartmentalization of the mouse in the anterior lobe closely resembles the pattern observed in cat and ferret. In carnivores, the medial (A) compartment in the dorsal part of the anterior lobe is subdivided into A-1 and A-Z and an X compartment is intercalated between A and B (EKEROT and LARSON 1979; VOOGO 1982). The C-2 compartment of pars intermedia does not continue as far as the junction of the lobules II and III. In the anterior lobe of the mouse, the «B» compartment, therefore, could be represented by compartment 3 (large dots in fig. 18). If this is correct, then four compartments are present in the medial vermis of the mouse (la, 1b, Za, and Zb) instead of the three compartments (A-1, A-Z, and X), as found in carnivores. The continuity of compartments between the anterior and posterior lobes of the mouse cerebellum is difficult to prove. Purkinje cells of the B-zone in carnivores, which project to the Deiters' nucleus, demarcate the lateral border of the vermis of the anterior lobe and the simple lobule (VOOGD 1964; BIGARE 1980). The paramedian sulcus almost completely interrupts the cortex between the vermis and cerebellar hemispheres caudally to the simple lobule. In the mouse the B zone cannot be identified in the posterior lobe, and a paramedian sulcus is only present laterally to the uvula and nodulus.

Topographic histochemistry of the cerebellum' 43

3.2.3 Acetylcholinesterase of the monkey cerebellar fibre layer The use of the Haggqvist method for studying the myeloarchitecture is limited in neurosciences, due to its laborious and time consuming technique. AChE has been used in pathway tracing procedures, mainly in conjunction with lesion techniques or anterogradely and retrogradely transported labels (BUTCHER 1983). Most AChE topographic studies in the brain are restricted to the localization of presumed cholinergic cell groups (RAMON-MoLINAR 1972; SNELL 1961). Cholinesterase positive fibre systems have been traced topographically (KRNJEVIC and SILVER 1965) only with limited success, because AChE is very low in the white matter, with some variance in different fibre systems (FRIEDE 1966). Slight differences within the cerebellar fibre layer for AChE were already noticed in cat and rat fibre layer (midline raphe), but sharp contrast was firstly noticed in monkeys (Saimiri sciureus and Macaca rhesus). It was found again as very slightly positive strips in the fibre layer of orang (courtesy Dr. Brown and Dr. J. Tigges, Dept. of Anatomy, Emory University and Primate Center, Atlanta, USA, and Dr. Hess, MIT, Cambridge, USA). These strongly contrasted AChE positive and negative fibre areas alternate. A comparison with the results of 3H-Ieucine injections in the inferior olive of Macaca shows that borders of AChE positive and negative areas in the fibre layer are concomittant with boundaries for the compartments, as delineated with autoradiographic climbing fibre localizations (VOOGD, SEDO and GERRITS 1982, 1983). Direct evidence that AChE borders coincide with boundaries of compartments is derived from the relations of these positive and negative strips with the cerebellar nuclei (fig. 19). In the squirrel monkey (Saimiri), AChE positive fibre bundles sometimes form not only borders between compartments but label a part of the compartment (B). Other compartments seem to be totally loaded by AChE positive fibres (C2). This interchangeability of partially labelled compartments and simple borders of compartments that are positive for AChE makes it possible to follow these compartments over the whole extent of the cerebellum. Within the anterior lobe vermis of the squirrel monkey, compartment A can be subdivided in Al and A2 by a small raphe which is AChE positive. The B c~mpartment comprises a medial broad fibre area positive for AChE (perhaps the zone X?) and, lateral to it, a negative part of this compartment. The Cl compartment is delineated by a small positive raphe from Band can be easily discerned from C2 which is totally AChE positive. C3, Dl and D2 can be followed into hemispherical parts of the anterior lobe. A subdivision can be made within C3 by a small positive area. Dl and D2 are bordered by positive raphes. The A compartment can be followed into the fastigial nucleus. The medial positive area of compartment B lies to the lateral side of the fastigial nucleus. This nucleus can be subdivided on account of its AChE content into a dorsal AChE neuropil positive part and a ventral AChE neuropil negative part. The fastigial neurons are positive for AChE. The raphe B/Cl can be followed into the fibre area between' fastigial and

B

Fig. 19. From Saimiri sciureus (B) and Macaca rhesus (A) is given a drawing of the myeloarchitecture and a reconstruction of the anterior lobe. C: Section through the white matter of the cerebellum, demonstrating the AChE positive strips in and around the cerebellar nuclei. (The Saimiri material was a gift from Dr. HESS; Macaca rhesus sections were sent by Dr.BROWN and Dr. TIGGES).

Topographic histochemistry of the cerebellum· 45

.interposed posterior nucleus. C2 can be clearly related to the interposed posterior nucleus. The D compartments are clearly visible in the anterior lobe, but are difficult to relate to the lateral cerebellar nucleus. Within the posterior lobe, in both vermis and hemispheres, these compartments can be related to the cerebellar nuclei. The AChE positive strips within the posterior lobe vermis are easier to follow than those in the hemispheres. However, within paraflocculus and flocculus white matter, clear distinctions are reached with this technique. Four compartments are discerned, one of which can be related to the C2 compartment. Macaca rhesus demonstrates even better this AChE pattern within the cerebellar fibre layer. In the anterior lobe, the A compartment can be subdivided in Al and A2, and these compartments can be followed into the fastigial nucleus. The B compartment is directed to the white matter between the interposed and fastigial nucleus. The medial part of this compartment is AChE positive. Its lateral part is AChE negative. The border between Cl and"B is small but AChE positive. The C2 compartment is AChE positive over its whole extent. The C3 area is distinguishable from the D compartments by a small AChE positive raphe. Within the posterior lobe, the compartments are distinct over the whole extent of the fibre layer. An extensive description will be published on the exact course of the compartments in the cerebellum of Macaca rhesus (VOOGD, SEDO and HESS, in prep.). The results of both monkeys demonstrate that AChE can be used as a marker for the localization of fibre compartments in the cerebellar white matter. Such a distinction is not encountered within the fibre layer of rodents or ungulates. Only some AChE positivity can be traced in the white matter of these species. The comparison of both AChE patterns in Saimiri sciureus and Macaca rhesus leads to the conclusion that they are alike. The sharp distinctions within the Band C2 area, due to the high content of AChE reaction product, seem to be correlated with the smaller calibre ofaxons (axons within raphes, X, and C2 belong to the smaller ones, VOOGD 1983; VOOGD and BIGARE 1980). Ultrastructural and tracing research is needed to couple the AChE positivity to a certain type of axon.

3.3 5'-Nucleotidase 5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, E.C. 3.1.3.5) is an enzyme that converts 5'-nucleotides into nucleosides (fig. 20). The original definition of this enzyme by the discoverer was «Phosphatase specifique pour les esters 5'-monophosphoriques des ribosides puriques» (REIS 1934, see also BODANSKY and SCHWARTZ 1968). The comparison of 5'-nucleotidases from many different species and different organs has led to the view that the 5'-nucleotidases are a somewhat heterogeneous group of enzymes with many differences; yet they have certain properties in common (DRUMMOND and YAMAMOTO 1971).

46 . Enrico Marani ribose-I-phosphate

ADENINE 5'phosphoribosyl-l - pyrophospha I e

Pi

•\

p.xine nucleoside phosphorylase

h

INOSINE

ADENOSINE

ribose-lphosphate PP i

5'-Pho$pho ribosyl-ll¥ophosph:Jte

fumarate

adenylosuccinate lyase

GOP. P,

ADENYLOSUCCINATE

~

PP,

IMP

adenybsuccirote synthetase

Fig. 20. Possible pathways of adenosine metabolism. The inclusion of a pathway in this figure does not necessarily signify that it is important in animal tissues. - Courtesy Dr. J. S. R. ARCH, from ARCH and NEWSHOLM (1978). .

Most 5' -nucleotidases act on a variety of nucleoside monophosphates, but their substrate specificity is less rigid than has been assumed before. Since REIS' (1934) discovery of the enzyme, 5' -nucleotidases have been found in insect imaginal discs that also convert ATP (SPREY 1970). Conversely, ATP-ases have been described that break down AMP (SCHLAEFFER et al. 1969). Moreover, it seems likely from biochemical studies that 5' -nucleotidases are capable of converting substrates of acid or alkaline phosphatases (BELFIELD and GOLDBERG 1970; BODANSKY and SCHWARTZ 1968).

The limited substrate specificity of 5'-nucleotidase has important consequences for the histochemical demonstration of 5' -nucleotidase and for its differentiation from acid and alkaline phosphatases. Magnesium ions have been used as an activator for 5'nucleotidase in most histochemical studies concerning this enzyme. The role of magnesium ions as a universal activator for 5'-nucleotidase should be questioned, however, because EDTA, a substance that removes magnesium ions from the enzyme or its environment, has proved to have an highly variable effect on 5' -nucleotidases from different sources (DRUMMOND and YAMAMOTO 1971). The influence of ions on enzyme activity is of practical importance in the histochemistry of 5' -nucleotidase and also of theoretical interest, because 5'-nucleotidase in brain tissue is subjected to great fluxes in the concentration of ions.

Topographic histochemistry of the cerebellum' 47

3.3.1 Enzyme histochemical reaction The enzyme histochemical reaction is complicated because lead ions (capture agent) have an inhibitory effect on 5' -nucleotidase activity ( MARANI 1982 b). 5'-Nucleotidase is affected by the fixatives which are commonly used in enzyme histochemistry. It is inhibited by glutaraldehyde, and this inhibition of 5'-nucleotidase in lymphocytes is pH dependent (UUSITALO and KARNOVSKY 1977 a, b). Most other fixatives are known to have a destructive effect on 5'-nucleotidase in the cerebellum of the mouse (SCOTT 1965). In general, the arguments for its localization are derived from biochemical studies in purified fractions ofaxolemma (DE VRIES 1976) or plasma membranes (EVANS and GARD 1973; RIORDAN and SLAVIK 1974) as well as in fat globules and isolated milk fat globule membranes (PATTON and TRAMS 1971) or isolated intact or disrupted cells (DE PIERRE and KARNOVSKY 1974 a, b, c). Histochemical studies have demonstrated the localization of 5'-nucleotidase to be on the outer surface of the plasma membrane, for example in UUSITALO and KARNOVSKY'S (1977) study of the localization of 5'-nucleotidase reaction product in lymphocytes. Although most authors regard 5'-nucleotidase as a membrane marker (EMMELHOF et al. 1964; SONG, KApPERS and BODANSKY 1969; SONG and RAy 1970; BOSMANN and PIKE 1970; DE VRIES 1976), they always mention that 5'-nucleotidase may also be demonstrated at other sites. Other important localizations are the membranes of the endoplasmic reticulum (WIDNELL and UNKELESS 1968; WIDNELL 1972; MAGNUSSON, HEYDEN and SVENNSON 1974) and in the nuclear fraction (ISRAEL and FRANCHONMASTOUR 1970). The main concentrations of 5' -nucleotidase within the brain of rat and mouse (SCOTT 1965, 1967) are found in the cerebellum and the striatum. High activity is also found in the olfactory bulb, in the mitral cell layer, and deeper internal plexiform and granular layers. The amygdaloid complex contains strong 5' -nucleotidase activity, as do the septal nuclei. 5'-Nucleotidase in the hippocampus is restricted to certain layers (entorhinal area, parasubiculum and presubiculum, the ammons horn, the outer molecular layer and the innermost zone of the area dentata). The cerebral cortex contains some areas that are positive for 5'-nucleotidase. In the cerebellum, 5'nucleotidase occurs in the molecular layer in a peculiar pattern of rostro-caudally directed bands. Within the white matter and the cerebellar nuclei, a weak form of 5'nucleotidase activity is present, too (SCOTT 1964, 1965). Early studies on 5'-nucleotidase have been concerned with the effect of coma on 5'-nucleotidase localization (CHESSICK 1954), the distribution of phosphomonoesterases in the particulate fractions of the dog cerebrum (WAKED and KERR 1955), and the histochemical demonstration of 5' -nucleotidase after 18-22 hours fixation with formol calcium or neutral phosphate buffered formalin (BARRON and BOSHES 1961). Biochemical studies inquired the effects of paraffin embedding and freezing of speci-

48 . Enrico Marani

men (PRArr 1953), and the effects of capture and activator agents (PRArr 1954) were studied. Histochemical studies of 5' -nucleotidase were conducted by FELGENHAUER (1963), looking at the effects of different lead concentrations on brain tissue and the presence of unspecific phosphomonoesterases. The description of the distribution of 5' -nucleotidase in the cerebellum of the rat (TEWARI and BOURNE 1963) localizes 5' -nucleotidase activity in the white matter and granular layer. The glomeruli are positive and the granule cells negative in the granular layer. The molecular layer is positive, and the Purkinje cell layer is strongly positive for intracellular reaction product. However, the rostro-caudally directed bands (Scorr 1963, 1964, 1965, 1967, 1969) are missing in the rat molecular layer in this study. Possible actions of the 5' -nucleotidase activity range from a hormonal function to a neurotransmitter or modulator function (for reviews see ARCH and NEWSHOLM 1978; Fox and KELLY 1978; STONE 1981). The KREUTZBERG group postulates a neuromodulator function of 5' -nucleotidase which regulates the availability of adenosine (KREUTZBERG and BARRON 1978; KREUTZBERG et al. 1978; SCHUBERT et al. 1979; SCHUBERT and KREUTZBERG 1978; SCHUBERT and MITZDORF 1979; ROSE and SCHUBERT 1977). They implicate glia in the production of adenosine. In certain parts of the brain, ATP is released at synpatic transmission (BURNSTOCK et al. 1970). ATPase and 5'nucleotidase which are attached to outer membranes of glia cells, convert ATP into adenosine in two steps. Although the involvement of adenosine in synaptic transmission is still under discussion (BURNSTOCK et al. 1970), a glial localization was indeed demonstrated by this group for 5' -nucleotidase. This function of 5' -nucleotidase in the production of adenosine as a neuromodulator involves not only adrenergic systems (HEDQVIST and FREDHOLM 1976), but also cholinergic synapses (VIZI 1977). Several isoenzymes of 5 ' -nucleotidase have been described for various tissues. «The five types of 5' -nucleotidase localization may reflect functional stages or contain certain informations about the actual transmitter in the synapses» (BERNSTEIN et al. 1978 a, b). A functional implication of the postulated isoenzymes in the mouse cerebellum is also described by Scorr (1965).

3.3.2 Biochemical reaction 5'-Nucleotidase converts 5'-nucleotides to 5 ' -nucleosides, liberating a phosphate molecule (fig. 20). This reaction normally occurs at pH 7.2. The amounts of phosphate or of nucleosides produced by this reaction can be determined. Phosphate is determined by most authors, using the FISKE and SUBBAROW (1952) technique. We never found this method reliable, because it is dependent on its reagent concentrations. When AMP is given as a substrate, adenosine is produced by the action of 5' -nucleotidase. As an alternative method, the amount of adenosine produced can be determined by converting it into inosine by adding the enzyme adenosine deaminase and by measuring the amount of NH) produced by this reaction.

Topographic histochemistry of the cerebellum' 49

Within homogenates of the cerebellum, other enzymes are present that can convert AMP. The substrate retention method is employed in order to prevent the production of adenosine by other enzymes. Beta-glycerophosphate (VAN DER SUK 1975) or even glucose-6-phosphate (DE PIERRE and KARNOVSKY 1974 a,b) is added to the AMP containing medium in biochemical determinations and is preferentially broken down by disturbing enzymes (PERSIJN and V.D.SUK 1969, 1970). This phenomenon is called the competitive substrate retention. Five years after the start of this study radioactive methods for the determination of 5'-nucleotidase were developed. Moreover, radioactive substrates became available after 1975 for routine determinations. These autoradiographic methods are not used in the biochemical investigations referred to in this monograph. A biochemical approach in considering the fixatives and their effects on 5'-nucleotidase activity, as used in the electronmicroscopical part of this study, therefore, is difficult, because both glutaraldehyde and formalin destruct the mediator enzyme adenosine deaminase. Qualitative comparison in our histochemical series confirms the semiquantitative results of SCOTT (1965) for formalin. Formalin itself inhibits 5'nucleotidase activity, although, after a 1 hour fixation with 4% formalin or paraformaldehyde,S' -nucleotidase activity can still be demonstrated with histochemical methods, providing prolonged incubations are used. Glutaraldehyde rapidly destroys 5' -nucleotidase activity. A 1% glutaraldehyde solution completely abolishes 5' -nucleotidase activity in cryostate sections after only 15 min (see 3.5.3.1, and fig. 37). The effect of the capture agent (lead ions) in SCOTT'S (1965) histochemical reaction for 5' -nucleotidase was determined in homogenates of the mouse cerebellum according to PERSIJN and VAN DER SLIK (1969). The lead ion effects can be determined, because the effects on adenosine deaminase can be evaluated from the test blanco. The Pb2+ effect on mouse cerebellar 5 ' -nucleotidase activity is measured in the presence and absence of magnesium ions in the «PERSIJN and VAN DER SLIK" Na-veronal buffer and in the Na-succinate buffer in the presence of magnesium. The absence of magnesium ions in the Na-succinate buffer influences the determinations using control solutions; therefore, Na-succinate-lead effects are only given in the presence of magnesium ions. In all cases (fig. 21), lead concentrations in the millimolar range result in a severe inhibition of 5'-nucleotidase activity. It is this concentration that is used in 5'nucleotidase enzyme histochemistry. However, high protein concentrations reduce such an inhibitory effect (PERSIJN et al. 1961). Acid phosphatase activity at pH 5.0 is inhibited by fluoride ions. Non-specific activity at pH 7.2 can be considered as a residual activity of acid phosphatase, alkaline phosphatase activity, or both. High concentrations up to O.lM NaF inhibit 65% of the non-specific phosphatase activity at pH 7.2. However, a total inhibition with NaF of unspecific phosphatase activity cannot be achieved. The effect of NaF on 5' -nucleotidase activity is an inhibitory one. High concentrations of NaF (O.1M or O.OlM) abolish nearly all 5' -nucleotidase activity in homogenates of the mouse cerebellum. Levamisole (l-tetramisole) as an inhibitor of cerebellar alkaline phosphatase should

so . Enrico Marani

100

50

10

• 3

4

5

6

_log(Pb +)

Fig. 21. Inhibitory effect of Pb2+ on 5' -nucleotidase activity. Pb 2+ effects were determined in 0.03 M Na-succinate pH 7.5 with Mi+ and in 0.033 M Na-veronal pH 7.5, with and without Mi+. 5 ' -Nucleotidase activity is expressed as percentage of the mouse cerebellar blanco homogenate (50 III of a 2.5% w/v suspension, centrifugated for 30 ' , 5000 rpm, 2650 g). S.D. is indicated, n = 6.

unmask the pure 5 ' -nucleotidase actlVlty (BORGERS 1973). Levamisole added to homogenates of mouse cerebella, shows an inhibitory effect on alkaline phosphatase at pH 9 (MARAN! 1981). A problem arises since it is observed from these biochemical studies that addition of levamisole to homogenates of cerebella increases the AMPase activity at pH 8-10 (see MARAN! 1981). 3.3.3 Histochemical results After his discovery of the longitudinal pattern of 5 ' -nucleotidase in the molecular layer of the mouse (SCOTT 1963, 1964), SCOTT published an extensive study on the histochemical method for demonstrating 5'-nucleotidase (SCOTT 1965). When we started to use SCOTT'S incubation method (MARAN! and BOEKEE 1973) we found it to be extremely reliable, as long as the appropriate sodium succinate buffer was used. Several samples of Na-succinate (BDH), obtained in the years 1971-1974, inhibited 5 ' -nucleotidase activity, but in all other cases this inhibition is absent. In certain experiments, the nucleotide or the lead acetate was changed. Incubations with AMP, UMP, IMP, and GMP or with lead nitrate produce the same pattern, confirming SCOTT'S results (1965). An influence of anaesthetics, including barbiturates

Topographic histochemistry of the cerebellum . 51

on the activity or distribution of 5' -nucleotidase, cannot be detected. Lowering the lead concentration at a constant pH of 7.2 results in artifactial deposits of reaction product on the cell nuclei. At higher concentrations, inhibition of the 5' -nucleotidase activity occurs and the band pattern in the molecular layer becomes less distinct. The 5'-nucleotidase reaction product is localized in rostro-caudally directed bands which are exclusively present in the molecular layer. The pattern arises after 25 min of incubation, but for reconstruction purposes (see MARAN! 1981) the incubation time is prolonged to 90 min. Incubation for 5' -nucleotidase results in lead deposits in the Purkinje cell layer and in spots in the granular layer which are difficult to trace to smaller somata near Purkinje cell clusters, like Bergmann glia. Basket cell bodies certainly contain lead precipitates. Stellate perikarya are negative in strongly positive bands of the mouse molecular layer, but are slightly positive in negative areas. The fibre layer is equally positive. The choroid plexus and pia mater are strongly positive. The 5'-nucleotidase band pattern in the molecular layer is unaffected by fluoride ions, even at high concentrations such as O.lM NaF. When fluoride ions are added to the incubation medium, lead deposits at the Purkinje cell layer cannot longer be observed, and the positivity of the granular and fibre layers is strongly reduced. The stellate cells in the molecular layer now stand out as negative spaces in strongly positive 5'-nucleotidase bands. Acid phosphatase, determined according to BARKA and ANDERSON (1962) in the mouse, is found in perikarya of most nerve cells of the cerebellar cortex and deep nuclei. A comparison with Nissl counter-stained sections reveales that the acid phosphatase positive cells in the mouse granular layer are Golgi cells. Sodium fluoride abolishes all acid phosphatase activity in the cerebellum of the mouse at both concentrations used (0.0001 M and 0.01 M). The choroid plexus, however, stays slightly positive. Non-specific phosphatase, as determined with the LAKE (1965) method at pH 7.2, is found within perikarya of Purkinje cells and stellate cells. Endothelial cells are clearly positive. Both basket cells and Bergmann's glia are positive for this enzyme, although the non-specific phosphatase reaction product in Bergmann glia is difficult to distinguish from the granular deposits in the Purkinje cells. The pia mater and choroid plexus are strongly positive. A slight activity is present in the fibre layer. Fluoride ions also inhibit the localization of non-specifc phosphatase reaction product at pH 7.2. Sections of the mouse cerebellum, processed for alkaline phosphatase with betaglycerophosphate as substrate, show heavy deposits of lead sulfide in the mouse cerebellar white matter. This activity of alkaline phosphatase is not inhibited by the addition of levamisole (0.005 M) to the incubation medium. In sections for 5' -nucleotidase, both the 5' -nucleotidase longitudinal band pattern in the molecular layer and the reaction product in the fibre layer are still seen after addition of levamisole. These histochemical results could indicate that the biochemical increase of AMPase activity in homogenates of mouse cerebellum is due to an increased break-down of AMP by

52 . Enrico Marani

alkaline phosphatase. The importance of 5' -nucleotidase pure localization in axons in the fibre layer is argued by the inconclusive results given in literature (TEWARI and BOURNE 1963). It can be concluded from the histochemical results that: (1) Lead ions concentration for the histochemical reaction seems to be correct, as proposed by SCOTT (1965), despite the biochemical results. (2) Fluoride ions are usefull as an inhibitor of non-specific phosphatase activity at pH 7.2, despite the biochemical results. (3) Levamisole is questionable as a histochemical inhibitor for mouse cerebellar phosphatase. 3.3.4 Peculiar properties of mouse cerebellar 5'-nucleotidase A series of publications have studied the properties of the 5 ' -nucleotidase longitudinal pattern in the mouse cerebellum, the K+ and Na+ activation (MARANI 1980b), the ATP and neurotransmitter effects on this enzyme (MARANI 1982 b), the presence of circadian rhythmicity for 5'-nucleotidase (MARANI 1980 a), and its subcellular distribution with differential centrifugation techniques (MARANI 1977). The effect of monovalent and bivalent ions on 5' -nucleotidase activity have repeatedly been observed (MARANI 1982 b). Generalizations concerning the effect of ions on 5'-nucleotidase are not forthcoming from the literature. In particular, we are not aware of any study that proves a K+ and or Na+ influence on brain 5'-nucleotidase as is commonly accepted for K+, Na+ activated ATPase (SKOU 1957). Cerebellar 5' nucleotidase is subjected to important changes in K+ and Na+ concentrations during synaptic transmission (ABOOD 1972). Moreover, adenosine, 5' -nucleotidase's reaction product, has been nominated as an «intercellular communication molecule which is produced on nerve cell activation in addition to the principal transmitter, which may modulate synaptic function» (SCHUBERT and KREUTZBERG 1978), while adenosine interrelations with purinergic and glutamate transmission are described in earlier studies (MARANI and VOOGD 1977; PULL and McILWAIN 1972; SCHMIDT et al. 1976, 1977). Adenosine receptors have been demonstrated on neurones in various brain areas, including the cerebellum (BRUNS, DALY and SNYDER 1983; WOJCIK and NEFF 1983a, b). Increase of K+ and Na+ permeability has been proved to be necessary for the effects of several cerebellar neurotransmitters. Any dependence of 5 ' -nucleotidase activity on the concentrations of these cations, therefore, is of obvious importance in assessing enzymatic function. The possible influence of Mg2+ (MARANI 1980 b) or other bivalent ions, such as calcium (MARANI 1982 b) on cerebellar 5'-nucleotidase is excluded in biochemical experiments by the addition of EDTA. Both K+ and Na+ each separately enhance 5 ' -nucleotidase activity in our experiments, without the addition of magnesium ions. The combined administration of these

Topographic histochemistry of the cerebellum' 53

ions in physiological concentrations in the absence of magnesium ions consistently produces higher levels of enzyme activity than results from the addition of K+ or Na+ alone (MARANl 1980 b). These data suggest that, although the micro-environmental concentration of K+ and Na+ can change, the 5 ' -nucleotidase is not affected by these changes and maintains its high activity, although the enzyme is sodium and potassium dependent. Since adenosine may be considered a «local hormone» which exerts its action within the region in which it is produced, the level of adenosine in the brain must be well regulated. This would be possible through the regulation of 5 ' -nucleotidase activity (ARCH and NEWSHOLM 1981). The intraneuronal levels of adenosine, however, are very low (STONE 1981). Intracellularly produced adenosine, therefore, must be rapidly converted or excreted into the extracellular space. An extracellular membrane bound 5'-nucleotidase might exert its action on extracellular AMP which is derived from either extracellular ATP or cyclic AMP. ATP is indeed lost to the extracellular space from excitable tissues during depolarization (ABOOD et al. 1966). More specifically, ATP is produced at certain nerve endings, alone or in combination with certain neurotransmitters. The «purinergic neuron» hypothesis even regards ATP as a neurotransmitter. Sheep cerebellar 5 ' -nucleotidase has been shown to be inhibited by ATP (IPATA 1966, 1967, 1968 a, b). Still it was considered to be of interest to investigate the influence of various substances involved in the excretion of ATP on cerebellar 5 ' nucleotidase and to correlate these findings with the purinergic hypothesis (MARAN! 1982 b). The actions of a number of cerebellar neurotransmitters (acetylcholine, noradrenaline, GABA, and glutamate) and of ATP on cerebellar 5 ' -nucleotidase are to be reported. The effect of calcium ions is also included, because it is known to be liberated together with ATP from macromolecular complexes in the plasma membrane during depolarization at synaptic transmission (ABOOD et al. 1966), and because of its wellknown functions in the storage and release of neurotransmitters in nerve endings. The addition of acetylcholine to the supernatant in different concentrations does not affect the measured 5'-nucleotidase activity. Even high concentrations of acetylcholine do not influence the amount of adenosine produced by the activity of 5 ' nucleotidase. By contrast, noradrenaline influences 5 ' -nucleotidase activity at high concentrations. An inhibition of 20% is measured at a concentration of 0.001 M noradrenaline. Lower concentrations of noradrenaline do not affect 5 ' -nucleotidase activity. GABA and L-glutamate show no effect on mouse cerebellar 5'-nucleotidase activity. The administration of ATP to cerebellar homogenates of mice causes severe inhibition of 5'-nucleotidase activity. The inhibitory effect at high concentrations reaches nearly 90% of the mouse cerebellar 5' -nucleotidase activity. These results are comparable to the effects on purified sheep cerebellar 5' -nucleotidase, as described by IpATA (1966, 1967, 1968a, b). Effects of low concentrations of ATP on 5' -nuc-

S4 . Enrico Marani

leotidase activity in homogenates are difficult to measure, because cerebellar homogenates contain ATP-ases that convert the added ATP. The action of ATP at concentrations between 10-3 and 10-4 M is variable. Neither GTP, ADP, or IDP altered mouse cerebellar 5'-nucleotidase activity (see also IpATA 1966,1967, 1968a,b). Ouabain effects on ATP-ase activity and 5'nucleotidase activity cannot be determined in mouse cerebellar homogenates, since adenosine deaminase is inhibited by ouabain.

Calcium ions alone do not affect 5' -nucleotidase activity, either at high (10-2 M or 10-3 M) nor low (10- 7 till 10-9 M) concentrations. At physiological concentrations of 10-3 M and 10-4 M (ABOOD 1966; DODGE and RAMINOFF 1967; STOCKLE and TEN BRUGGENCATE 1978), calcium has not been found to affect mouse cerebellar 5' -nucleotidase activity. It also does not affect 5' -nucleotidase activity in the presence of noradrenalin and glutamate. Cerebellar 5' -nucleotidase, thus, is not influenced by cerebellar neurotransmitters or calcium ions. It is inhibited by ATP alone and not by GTP. These facts do not support, and are difficult to reconcile with an extraneuronal degradation of ATP which is secreted by nerve endings. ATP is secreted alone or in combination with neurotransmitters and is degraded by ATP-ase and 5' -nucleotidase located on the glial surface, as proposed by KREUTZBERG (KREUTZBERG et al. 1978 a,b). A possible mechanism that explains the inhibition of 5 ' -nucleotidase by ATP is the action in the homogenates of an ATP-ase which produces enough phosphate ions to inhibit 5' -nucleotidase activity at higher concentrations (10-2 till 10-4 M). High concentrations of phosphate ions do inhibit liver 5 ' -nucleotidase acitivity (HARDONK, personal communication). However, most ATP-ases also convert other nucleotide triphosphates, like GTP, while GTP itself does not inhibit 5' -nucleotidase. No answer has been given concerning the constancy of the 5 ' -nucleotidase pattern in time. So far, we have not considered the possibility of the rhythmical changeability in the pattern or the presence of an on-off mechanism. It is known that the cerebellum contributes to the phasic suppression of motor activity during REM-sleep. Moreover, the impulse frequencies of afferent cerebellar systems do show a circadian rhythmicity. Such a circadian rhythmicity has also been postulated for cerebellar neurotransmitters (HOFFER et al. 1978). Circadian studies on 5 ' -nucleotidase and nonspecific acitivity fail to show a circadian rhythmicity for 5' -nucleotidase, but confirm the presence of this phenomenon for nonspecific phosphatase. 5 ' -Nucleotidase histochemistry performed at several time intervals within 24 hours confirm the topography of the pattern to be constant (MARANI 1980 a). The differential centrifugation results (MARANI 1977; PILCHER and JONES 1970) suggest a 5' -nucleotidase localization in a specific type of synapse in the molecular layer of the mouse cerebellum. Although 5' -nucleotidase in this localization is particularly soluble and there are minor differences between the results of MARANI (1977) and PILCHER and JONES (1970), it seems clear that a localization of 5' -nucleotidase on

Topographic histochemistry of the cerebellum' 55

synaptic structures is supported by results obtained with differential centrifugation techniques (fig. 22). The peculiar properties of 5'-nucleotidase within the cerebellum of the mouse, particularly its inhibition by ATP, its sodium and potassium dependence, and the synaptosomal localization, show that 5' -nucleotidase is related to neuronal structures rather than to glial structures, while its function in a purinergic system using ATP seems doubtful.

%

100 0 ACTIVITY

50

1.2

0.86

0.4M.

Fig. 22. Linear sucrose gradients from crude P2 fractions. Each point in the figure is the mean of three experiments. Open circles represent the 5' -nucleotidase activity, asterisks represent the monoamine oxidase activity, and crosses indicate the protein determinations: Filled squares indicate the lactate dehydrogenase activities. All points are represented as percentages of the fractions with the highest content of enzyme activity or protein. The recoveries are: protein 104 %, 5' -nucleotidase 95 %, monoamine oxidase 92 %, and lactate dehydrogenase 75 %.

, 3.4 5'-Nucleotidase isoenzymes in rodents 3.4.1 5'-Nucleotidase isoenzyme in the mouse cerebellum 5' -Nucleotidase in brain tissue has been studied biochemically over the last few years (IpATA 1967, 1968a,b; HARDONK and DE BOER 1968; BOSMANN and PIKE 1970; SURAN 1974 a, bj ARCH and NEWSHOLME 1978; BERSTEIN and LUPPA 1978 a, b). SCOTT

56 . Enrico Marani

(1965) found histochemically, at high magnification, product of a specific mouse brain 5'-nucleotidase, reacting with AMP, CMP, and TMP (pH optimum 7.2) inside the perikarya of Purkinje cells, whereas the reaction product was located outside the perikaryon, when the substrates UMP, GMP, IMP, and NMN were used. The results of SCOTT'S experiments, in which various cations were added to the incubation medium, furnish additional proof for the existence of different isoenzymes (see table 2). Table 2. Species variation in 5'-nucleotidase activity as influenced by pH and cation concentration. HARDONK (Mouse) M~+

Mn2+ Ni2+ Zn2+ Co2+ Cu2+ FeH pH

7.5

IpATA (Sheep) 5mM

BOSMANN (Rat) 10 mM

SCOTT (Mouse) 10 mM

0 0

+ +

+ + + -~:.

7.3

0 0 0 6.8 ± 0.2

7.2

o=

no influence, + = activation, - = inhibition ':. MARAN! and BOEKEE 1973

HARDONK and DE BOER (1968) have investigated rat and mouse 5'-nucleotidase by means of agar electrophoresis. In several organs of the mouse, various isoenzymes were found. In homogenates of mouse whole brain, however, only one form of the 5'-nucleotidase enzyme (pH optimum 7.5) was found to be present. This paragraph determines, whether several forms of the enzyme 5'-nucleotidase contribute to the formation of the longitudinal pattern in the molecular layer of the mouse cerebellum or whether this pattern is due to quantitative differences in enzyme product. This information is a necessary prerequisite for the quantification of the 5'-nucleotidase band pattern with the method of SCOTT (1969) and for the ultrastructural localization of the 5'-nucleotidase (BERSTEIN and LUPPA 1978; KREUTZBERG, BARRON and SCHUBERT 1978 b). The reason for the discrepancy between some of our findings and those reported in the literature may be found in the extraction procedure and the advantages of disc electrophoresis (BREWER 1970) over agar electrophoresis, which is normally used in older studies. Therefore the activities of 5' -nucleotidase, acid, neutral, and alkaline phosphatase are also determined in cerebellar homogenates extracted with butanol in our initial experiments, following the procedure of HARDONK and DE BOER (1968). In later experiments butanol was excluded.

Topographic histochemistry of the cerebellum' 57 PH

ACTIVITY

CURVES

••t. 0.11

0.10

0.09 0.08 0.07

0." o.o~

0.04

0.01

0.11

0,02 0,01

10

Fig. 23. Acid phosphatase-pH activity curves using the method of ANDERSCH and SZCYPINSKI (1947) illustrating the effect of NiH (5'10- 3 M). The cerebellar homogenates were prepared in electrophoretic buffer.

In table 3, the specific activities of the butanol, electrophoretic buffer, and Triton X 100 extracts are given as percentages of the enzyme activity in the homogenates of the same cerebellum. These results indicate that enzyme activities of homogenates treated with butanol are consistently lower than in comparable homogenate extracts, and since butanol may also exert an influence on the isoenzyme pattern (KABARA and KONVICH 1972), disc electrophoresis of homogenates treated with butanol was repeated with aqueous extracts in electrophoretic buffer and with extracts of Triton X100. No important differences are noticed.

58 . Enrico Marani

Table 3. This table represents the influence of Triton X100, electrophoretic buffer and butanol on each of the five assays conducted in this investigation. In each case the numerical values indicate the activity found in the supernatants and the value determined in the total homogenate. Each value is followed by the standard error. Activity is expressed in lUll/min. The interpretation of the information presented in this table is contained in the text. BUTANOL Homogenate Supernatant Acid phosphatase 36.13 ± 0.86 0.86 ±0.46 5'-Nucleotidase 231.00 ±14.1 24.90 ±3.2 Alk. Phosphatase 39.26 ± 4.2 32.13 ±3.1 Protein mg/ml 8.36 ± 0.03 2.16 ±0.06 Non spec. phosphatase 14.56 ± 0.46 2.56 ±0.92

ELECTROPHORETIC BUFFER TRITON Xl00 (0.1%) Homogenate Supernatant Homogenate Supernatant 40.35 ±0.82 153.16 ±4.32 22.11 ±0.64 6.98 ±0.09 16.20± 0.69

14.30 ±1.08 103.50 ±2.73 12.24 ±0.46 3.92 ±0.04 5.22 ±0.20

42.13 ±2.91 322.00 ±7.25 43.80 ±3.4 8.30 ±0.05 15.13 ±0.23

30.43 ± 1.20 280.00 ±16.00 28.33 ± 3.88 7.12± 0.11 11.70 ± 0.24

Already IpATA (1966) indicates that 5' -nucleotidase coincides with non-specific phosphatases. HARDONK and DE BOER (1968) using beta-glycerophosphate, however, found hardly any acid phosphatase activity in butanol extracts of the mouse brain at pH 7.2. In order to substantiate our findings of acid phosphatase activity with beta-glycerophosphate in butanol and aqueous extracts at pH 5.0, the incubations of butanol extracts with para-nitrophenylphosphate as a substrate were repeated. The same densitograms result after incubation of the gels. Subsequently, a pH activity curve for nonspecific phosphatase was prepared, using para-nitrophenylphosphate as a substrate (fig. 23, 24). Although the pH optimum lies at pH 5.0, we find an activity of acid and alkaline phosphatase of 30-40% of the maximum between pH 6.8 and 7.4. We can conclude, therefore, that a residual acitivty of acid phosphatase is present at the pH at which the reaction for 5' -nucleotidase is measured. In order to demonstrate which part of the densitogram of 5' -nucleotidase is really due to this enzyme, acid phosphatase must be selectively inhibited. Inhibition is obtained with NiH and 1,3-c. glyceromonophosphate2. According to SCOTT (1965), NiH in a concentration between 10-3 M and 10-4 M does not affect 5' -nucleotidase activity. It has been found that NiH added to the incubation medium for acid phosphatase in a concentration of 5·1O-3M strongly inhibits the activity of butanol-treated homogenates. This can be shown for both the pH activity curve (fig. 23) and the densitometric scans of acid phosphatase. In histological preparations, the NiH concentration causes a slight reduction of the 5 ' -nucleotidase activity, however, localization of the reaction product in sections remains unchanged. The densitometric scans of butanol extracts with NiH added to the incubation medium for acid phosphatase show a 50-100% inhibition in 80% of the mouse cerebella, when 5·10-3M NiH is added (MARANI 1981, 1982 b). It is not possible to obtain selective inhibition of acid phosphatase in 5'-nucleotidase incubations by increasing the concentration of NiH, because at higher concentrations the 5' -nucleotidase itself would be strongly inhibited (Scorr 1965). The great 2 For preparation and composition of 1,3-c. glyceromonophosphate see

MARAN!

(1982 b).

Topographic histochemistry of the cerebellum' 59

variability in inhibition obtained in different mice may be due to the variation inherent in the butanol extraction referred to previou"sly.In some mice, the acid phosphatase is completely suppressed. •

AMP. ADA.p-nPP

*AMP. ADA.1,3·cGMP 0

90 / I /I

.

I

/

.---6 / .'" " / I

I

I

I

/ I

I

I

I"

/"';~ "."

20 3

4

- -~,\.

*

/

OAMP.ADA

,

I

I

/ 1 .--e/

/ /

\ •

1

01

/

,

..,..". ...

/

.p.nPP

0--- ~

I

I

* \

\' \

\

~,.....

I

6

7

*"

*

"

__

......

" ~""

" o-+~

,,* '"

5

\

\ " 0, .....

'" ·~l

,,, ~ '.

\

1\

"'.///

8

./

9

./

. . /.

//

0,

'\ \\

\~\ \ \

10

~

11

\

0

12

pH

Fig. 24, Comparison of cerebellar analysis for AMP-ase activity and para-nitrophenylphosphatase activity in electrophoretic buffer. Details of the assay procedure(s) are: AMP in concentrations of 2.5 mM, and 10 mM, 1,3-c. glyceromonophosphate (PERSIJN, VAN DER SLIK and TIMMER 1969; PERSIJN and VAN DER 5LIK 1970); in the method of ANDERSCH and SZCYPINSKI (1947) 5.5 mM para-nitrophenylphosphate was added. Adenosine deaminase was administered in a total activity of 500 mIU. Activity is expressed in lUll. The results indicate almost complete suppression of acid phosphatase activity in the presence of 1,3-cyclic glyceromonophosphate.

Since Scon (1965) found different localizations for the reaction products of isoenzymes of 5' -nucleotidase reacting with two different groups of nucleotides, densitograms, using a nucleotide from each group (UMP and AMP), have been compared. Identical densitometer patterns with AMP as with UMP have been found. Also, after incubation with AMP+ UMP+ NiH (5'1O-3M), only one peak has been found in some of the densitograms. This may signify that only one isoenzyme of 5'-nucleotidase is present in the mouse cerebellum and reacts with both AMP and UMP. It has also been looked for substances other than NiH that would selectively inhibit acid phosphatase without affecting 5'-nucleotidase activity. This was necessary, because NiH is variable in its inhibition of acid phosphatase, and it interferes with the ultrastructural demonstration of 5 ' -nucleotidase activity. Such an effect is obtained with 1,3-c. glyceromonophosphate. When the beta-glycerophosphate from the incubation medium for acid phosphatase is replaced by 1,3-c. glyceromonophosphate, a total inhibition is consistently observed in the densitograms, also after a single preincubation

60 . Enrico Marani

Table 4. Comparison of cerebellar analysis for AMPase activity with para-nitrophenylphosphate and 1,3-cyclic glyceromonophosphate. pH

Substrates AMP + p-nitroAMP + 1,3-c. glycerophenylphosphate monophosphate

7 8

11 22 50 77 83 77

9

57

80

50

61

3 4

5 6

10 11

12

11 20 24

35

50 77

50

66

61

61

Details of the assay procedures (PERSIJN and VAN DER 5LIK 1970) are: AMP 2.5 mM, 1,3-c. glyceromonphosphate 10 mM, 5.5 mM para-nitrophenylphosphate. Adenosine deaminase was administered in a total activity of 500 I. U. Activity is expressed as percentage of the total AMPase activity at pH 7.0. Each measurement was peformed in six different homogenates.

of the gels during 5 min. The influence of 1,3-c. glyceromonophosphate on the activity of 5'-nucleotidase is measured according to the method of PERSIJN and VAN DER SLIK (1970) and amounts to 90% of the control value after 2 hours. A comparison of the method of PERSIJN and VAN DER SLIK (1970) and the use of 1,3-c. glyceromonophosphate is found in table 4, indicating that the 1,3-c. glyceromonophosphate suppresses a higher amount of acid phosphatase activity than the substrate retention method does. After preincubation of the gels with 1,3-c. glyceromonophosphate and incubation with either AMP + 1,3-c. glyceromonophosphate or AMP + UMP + 1,3-c. glyceromonophosphate with or without Triton X100, only one band is consistently observed in the densitograms (fig. 25). The same results are obtained with the same quantities of butanol-treated homogenates. Therefore, it is concluded that 5' -nucleotidase in the cerebellum of the mouse is present in the form of only one isoenzyme. 3.4.2 5'-Nucleotidase isoenzymes in other rodents Previously SCOTT (1965) showed that the band pattern of 5' -nucleotidase is not universally present in the cerebella of all mammals, nor can it be found in all rodents. In this paragraph, the isoenzyme properties of 5'-nucleotidase occurring in rodents having a band-like and a uniform distribution of this enzyme in the molecular layer is compared.

Topographic histochemistry of the cerebellum' 61

C

8

A

250 fli (+8)

100 pi (+8)

A'

500 fli (-8)

250 pi (-8)

50 pi (-8)

8'

500 }AI

(+8)

C'

Fig. 25. Densitograms of gels incubated for 5 ' -nucleotidase with AMP, UMP, and 1,3-c. glyceromonophosphate. A: homogenate made with Triton X100, B/C: homogenate made with electrophoretic buffer, (-B) without butanol, (+ B) with butanol. In all experiments only one form of enzyme was found. - Scan rate 2, chart speed 0.5, calibration 1.0; ordinate: absorption, absciss: mm.

If 1,3-c. glyceromophosphate is added to the incubation media, acid phosphatase activity is strongly reduced, and the activity of 5' -nucleotidase at the pH range 7-8 is high enough to be demonstrated by disc electrophoresis. Histochemical localization of the enzyme 5'-nucleotidase and the occurrence of iso-enzymes is checked in four rodents: Mus musculus (NMRI), Rattus norvegicus (WAG), Clethrionomys glarolus and Microtus arvalis. In accordance with SCOTT (1965, 1969), a longitudinal pattern in the distribution of 5' -nucleotidase in the molecular layer is found to be present in Mus musculus and Rattus norvegicus. In the rat the pattern is less distinct. Negative areas as found in the mouse, are absent, and strongly positive areas alternate with less intense ones. In Clethrionomys glarolus the distribution is similar to that in the mouse. The positive bands are less reactive than in the mouse, when the same incubation time is chosen. In Microtus arvalis the distribution of 5'-nucleotidase in the molecular layer is homogenous. No bands are found and the layer is uniformly positive. Agar electrophoresis is performed to check, whether enzyme movement of 5'-nucleotidase and nonspecific phosphatase occurs in both directions. In all four species considered, movement occurs in the same direction. Our results for rat and mouse in agar electrophoresis confirm the

62 . Enrico Marani findings of HARDONK and DE BOER (1968). In disc electrophoresis of a series of homogenates of the cerebella of all four species of rodents, only one form of the enzyme 5 ' -nucleotidase can be found when 1,3-c. glyceromonophosphate is added to a medium containing AMP and UMP (MARAN! 1981, 1982). The Nj2+ in its effect on 5' -nucleotidase activity was measured. The same concentration as in the mouse (S'IO-3M) was chosen to block unspecific phosphatase activity.

Up to now, the distribution of 5 ' -nucleotidase in the molecular layer has been described in ten mammals including man (see table 5). From the suborder Myomorpha, two species of the family Cricetidae (Clethrionomys glarolus and Microtus arvalis) and two species of the family Muridae (Rattus rattus and Mus musculus) have been studied. From this small sample of rodents, it can be ascertained that the distribution of 5' -nucleotidase in the molecular layer differs between members of the same family. In the family Cricetidae, the negative areas are present in Clethrionomy, whereas in Microtus the molecular layer is uniformly positive. Mus from the family Muridae has a distinct pattern of negative and positive bands. In the molecular layer of Rattus the negative areas are less distinct. Irrespective of the type of distrubtion in the molecular layer, only one isoenzyme of 5' -nucleotidase is present in all species. It is impossible, therefore, that one isoenzyme in the molecular layer forms the band pattern, whereas others are uniformly distributed or fill up the less positive areas between bands. It would be interesting to know, whether primates and carnivores, which also lack a 5' nucleotidase band-like distribution, have only one form of the enzyme 5'-nucleotidase, too. MANOCHA and SHANTA (1970) postulate a connection between the cells, with an inhibitory function in the cerebellum and the localization of the enzyme 5' -nucleotidase. In chapters 3.3.1, and 3.5.3 it has been shown that the 5'-nucleotidase activity in Golgi cells, stellate cells, and Purkinje cells is due to residual inherent acid phosphatase activity and not to the enzyme 5'-nucleotidase. Based on our results, we cannot agree Table 5. The distribution of 5 ' -nucleotidase in the molecular layer of ten mammals including man.

Sorex araneus Man Rhesus monkey Mouse Rat Microtus arvalis Hamster Clethrionomys glarolus Lemmus lemmus Cat

Pattern

Order

Author

+

Insectivora Primates Primates Rodentia Rodentia Rodentia Rodentia Rodentia Rodentia Carnivora

MARAN! (unpublished) SCOTT (1967) SCOTT (1967) SCOTT (1967) SCOTT (1967) MARAN! (1982 b) SCOTT (1967) MARAN! (1982b) MARAN! (1982b) SCOTT (1967) MARAN! (1982 b)

+ + +

Topographic histochemistry of the cerebellum' 63

with the postulation of MANOCHA and SHANTA (1970). Only ultrastructural localization studies can yield more information concerning the function of the 5'-nucleotidase in the cerebellum.

3.4.3 Quantification of the 5' -nucleotidase band pattern in the mouse cerebellum The amount of PbS produced by the 5' -nucleotidase reaction product can be used for the quantification of the 5'-nucleotidase pattern (SCOTT 1969). To give an idea of the relative absorbance values corresponding to the arbitrarily chosen levels of 5'-

300,um

200,um

930~m

•••••••••••••••••• • • • • • • • • • • • • • •••••••••••••••••• • • • • • • • • • • • • •••••••••••••••••• •• •• •• •• •• •• •• •• •• •• •• • •••••••••••••••••• , ", , , 7' 5 61 62 63 +

•

0 •

.' 600 500 •. .' 700 . ' 800 ··1000 Fig. 26. Relative absorbance values compared in several bands in the anterior lobe in a section treated with 1,3-c. glyceromonophosphate. The bands are numbered according to 3.5.1.

64 . Enrico Marani

nucleotidase activity in the mouse cerebellar layer, histophotometric scans of several areas containing various positive strips are produced. A Zeiss scanning microscope coupled to a PDP-12 computer was used (MARANI, VOOGD and BOEKEE 1977). Measurements were performed in serial sections incubated together for a short incubation time (30 min) or over-incubated (1,5 hours). The relative absorbance values for 5' -nucleotidase activity in scans show that positive strips alternate with areas distinctly lower in reaction product (fig. 26). In the short incubation time series the absorbance of negative areas in between positive 5' -nucleotidase strips is equal to the absorbance values of areas in the brain stem negative for 5' -nucleotidase. Central vermal bands always contain higher absorbance values than more lateral positive strips. Several adjacent bands in one section contain different relative absorbance values. In those areas, where bands are difficult to discern (lobule V, the bands 6,1; 6,2; 6,3; 7), the initial description (MARANI 1981, 1982b) is fully confirmed by the histophotometric scans (see fig. 26). Since one isoenzyme was demonstrated in the molecular layer of the mouse cerebellum for 5' -nucleotidase, these relative absorbance values can directly be related to the relative differences in 5'-nucleotidase enzyme content per surface unit. It can be concluded from these data that the amount of 5 ' -nucleotidase present per square surface unit in various bands differs. The negative areas between 5 ' -nucleotidase positive bands do contain only reaction product in over-incubated series. It is therefore concluded that the negative areas in between positive bands do not contain this enzyme. Perhaps research can identify individual 5 ' -nucleotidase bands by their relative amount of reaction product within one series.

3.5 5 ' -Nucleotidase pattern is the mouse cerebellum Scon (1964, 1965, 1967) described a 5' -nucleotidase pattern of positive and negative parasagittal bands in the molecular layer of the cortex of the mouse cerebellum. He reported in some detail on the course of bands in the vermis, but his descripton of the localization of the enzyme in bands in the hemisphere was incomplete. The bands were supposed to be continuous over the whole rostro-caudal extent of the cerebellum from the nodule (lobule X) to the lingula (lobule I) (Scon 1967). Since then more evidence has been accumulated for a parasagittal, modular organization of the cerbellar cortex. Parasagittal mature distribution patterns have been described for the enzyme acetylcholinesterase in the molecular layer of the vermis in the cat (MARANI and VOOGD 1977; BROWN and GRAYBIEL 1983a,b, see part 3.4) and for climbing fibre terminals on Purkinje cell dendrites in the cerebellum of several species (COURVILLE 1975; GROENEWEGEN and VOOGD 1977; GROENEWEGEN, VOOGD and FREEDMAN 1979, cat; CHAN PALAY et al. 1977, rat), including mice (BEYERL et al. 1982). Parasagittal parcellations have

Topographic histochemistry of the cerebellum' 65

been observed in the distribution of several incoming systems (see chapters 3.1 and 3.1.1), including the spinocerebellar tracts in mice. The pattern of alternating 5'-nucleotidase positive and negative bands once more demonstrates the histochemical heterogeneity of the Purkinje cells, exemplified in CHAN PALAY'S studies of the cerebellar localization of motilin, GABA, and taurine (CHAN PALAY et al. 1981, 1982). It is deemed of interest, therefore, to reinvestigate and complete SCOTT'S description of the 5' -nucleotidase pattern in the cerebellum of the mouse. The distribution of 5' -nucleotidase in the molecular layer of the cerebellar cortex of mouse and rat bears a certain resemblance to the distribution of the climbing fibre terminals of the olivocerebellar system (see chapter 3.1). The gaps between the climbing fibres strips, which have been observed in all antegrade tracer studies of the olivocerebellar projection, are the result of the incompleteness of injections of the 10. Others maintain that these gaps are real and correspond to Purkinje cells without olivocerebellar climbing fibres (COURVILLE 1975; CHAN PALAY et al. 1977). In rats containing a 5' -nucleotidase pattern too (see 3.4.2), it is possible to injure the inferior olive selectively by 3'-acetylpyridine (HICKS 1955). The 10 of mice is found to be resistant against 3'-acetylpyridine poisoning (DESCLINS, personal communication). A modified method, avoiding 3'-acetylpyridine damage to other medullary nuclei, which is responsible for the high mortality rate in earlier experiments, was introduced by LLINAS et al. (1975) in their study of the role of the 10 in motor learning. In our study of the effects of 3'-acetylpyridine on 5'-nucleotidase (MARANI 1982b), the results confirm the observations of DESCLINS (1974) on subtotal destruction of the 10 in rats treated with high dosage. The combination of niacinamide and harmaline (LLINAS 1975) give the best results. Animals rarely die, and destruction of the 10 is almost complete. 3' -Acetylpyridine treated rats, which show manifest cerebellar ataxia and loss of the 10, display a normal topographic distribution of 5'-nucleotidase activity in the

Table 6. Biochemical determination of protein, 5'-nucleotidase- and acid phosphatase activity in rats injected i. p. with 75 mglkg 3-acetylpyridine, 15 mglkg harmaline and 300 mglkg niacinamide. Protein giL" Not treated rats 3-acetylpyridine treated rats':":'

5'-Nucleotidase"

Acid phosphatase

2.73 0.19

359 lUlL 14

34 lUlL 3

SD

2.78 0.15

338 lUlL 19

35 lUlL 1

SD

':. Determinations according to ,:." Survival times 56 days

MARAN!

(1982 b)

mean mean

66 . Enrico Marani

molecular layer. Our biochemical results, moreover, fail to demonstrate differences in the activity of 5' -nucleotidase and acid phosphatase between 3 ' -acetylpyridine treated rats and normal rats (table 6, MARANI 1982 b). 5'-Nucleotidase, therefore, is not bound to the climbing fibres. It is of interest in this respect to note that after the longest survival times of 100 days, which are characterized by the outgrowth of new Purkinje cell dendritic spines and parallel fibre contacts (SOTELO et al. 1974) the 5' -nucleotidase pattern remains unchanged, but stands out more clearly. 3.5.1 Description of the 5'-nucleotidase pattern In this description, the 5' -nucleotidase positive bands of the anterior lobe and the simple lobule are indicated by single digits (1-7), those of the posterior lobe with double digits (11-17). When a band splits and later rejoins, it is considered as a single unit, and the original number is retained, with a letter assigned to indicate the split portions of the band (i. e. 4 splits in 4a and 4b). Branching of a band in separate bands over several sublobules is indicated by adding a digit to the original number (i. e. band 6 gives rise to bands 6.1 and 6.2).

The distribution of 5'-nucleotidase in the molecular layer is essentially similar an all cases examined. Reaction product is located in strips, which extend from the Purkinje cell layer to the pial surface. The intensity of the staining differs for different strips and for different lobules. The distribution of the reaction product is uniform, and binding to specific structures of the molecular layer cannot be recognized. The borders of the 5'-nucleotidase strip, which is situated at the midline, are distinct. The lateral borders of the more laterally located strips generally are sharper defined than their medial borders. Sections incubated without inhibitors for unspecific phosphatase display reaction product in the perikarya of the Purkinje cells, in the nerve cells of the molecular layer, and in basket cell fibres. The areas between the positive bands are not completely negative for the reaction product of the enzyme 5' -nucleotidase, but they show much less activity in series incubated one and two hours. In the diagrams and the drawings of the reconstructions, these areas are indicated as white areas between the positive bands, as they are in shorter incubations. When 1,3-c. glyceromonophosphate (see part 3.4.1) or F- are added to the incubation medium, these localizations are no longer seen, but the 5'-nucleotidase pattern in the molecular layer has not changed (MARAN I 1981, 1982 b; see also 3.4.1).

The 5' -nucleotidase pattern in the anterior lobe and the simple lobule 5' -Nucleotidase in the anterior lobe and the simple lobule is distributed in a symmetrical pattern, consisting of 15 positive bands, separated by negative areas. Some of the bands are subdivided over part of their trajectory. Band 1 is located at the midline, whereas bands 2-7 are located at either side. The bands 2 and 3 are present in the lingula (lobules I and II). They continue over the anterior lobe into the simple lobule. Bands 4,

Topographic histochemistry of the cerebellum . 67

5, and 6 are added more laterally in the central lobule (lobule III, fig. 28A), and band 7 appears in the culmen (lobules IV and V, fig. 28B) at the most lateral, 5' -nucleotidase positive tip of this lobule. The spacing between the bands becomes wider in the dorsal

Fig. 27. Transverse sections through the anterior lobe of the mouse cerebellum. Incubations were performed in the presence of 1,3-c. glyceromonophosphate, except for E.

68 . Enrico Marani

parts of the anterior lobe and the simple lobule (fig. 31). The staining in the bands 1 through 4 is of equal intensity. The contrast with the negative bands and the intensity of the staining of the bands 5-7 in the lateral parts of the lobules is generally lower, and these bands are more difficult to distinguish (fig. 27 and 28C; see also 3.4.3).

12

A

B

c

Fig. 28. A: View at the rostral side of lobule I and II, III. The precentral fissure is distended to enable having a view nearly over the whole rostral part of lingula and lobulus centralis. The bands are indicated by numbers. B: Caudal view at the lobules. The fibres and the granular layer are not reconstructed. Clearly visible is that the bands 1, 2, and 3 have a continuation from lingula to lobulus centralis. The bands 2 and 3 have some connections. C: Reconstruction of the culmen. The caudal view of the lobule shows the pattern unaffected by the division of this lobule.

Topographic histochemistry of the cerebellum' 69

In the lingula, the bands 2 and 3 sometimes split and reunite (fig. 28A, left side). Band 4 is subdivided into 4a and 4b. 4a sometimes fuses with band 3 at the transition of the lobules III and IV and in the culmen (fig. 28A and B). The lateral part of the culmen contains two positive areas, located between the bands 6 and 7. They do not constitute bands and do not extend into the lateral part of the culmen. On the caudal and rostral surface of the culmen, these areas mix with band 6 (fig. 28B). They are indicated as 6.2 and 6.3 to distinguish them from band 6 which more caudally is indicated as band 6.1. The band pattern of the simple lobule (lobule VI) is very similar to that of the anterior lobe (fig. 31). Bands 1-3 are present in its medial, vermal portion (the declive). Band 4 is located at the junction of the medial, horizontal, and lateral sloping of the parts of the lobule. It is subdivided into bands 4a and 4b. Band 5 is present as a single strip of 5'nucleotidase activity. Five additional positive bands are present in the hemisphere of the simple lobule. The medial three bands are continuations of the bands 6.1, 6.2, and 6.3 of the anterior lobe. The lateral two bands are subdivisions of band 7.

Fig. 29. Horizontal section through the mouse cerebellum. The bands are numbered according to the text. The incubation was performed without inhibitors and for 2 hrs at

3rc.

70 . Enrico Marani

The 5' -nucleotidase pattern in the posterior lobe The topography of the 5'-nucleotidase band pattern in the posterior lobe is more complicated than in the anterior lobe and the simple lobule. The following description of this pattern is based on our previous account of the morphology of the posterior lobe (MARANI and VOOGD 1979). One of the main features of the posterior lobe is the discontinuity of its cortex at the transition of the vermis and the hemisphere, laterally to vermallobule VII in the centre of the intercrural sulcus of the ansiform lobule, and laterally to the uvula (lobule IX) and the nodule (lobule X). No such discontinuities are present in the cortex of the anterior lobe and the simple lobule and between the pyramis (lobule VIII) and the copula pyramidis. The 5' -nucleotidase positive bands in the lobules X-VII are generally wider than in the anterior lobe and the simple lobule. The width of the negative areas is compressed. The intensity of the staining is much stronger in the bands 11 and 13 than in the intervening band 12 and its subdivisions (fig. 29). The cortex, which covers the ventral side of the nodule, contains the positive band 11 at the midline, flanked by two areas which show a strong and uniform reaction for 5' -nucleotidase (figs. 33 and 34). At the dorsal side of the nodule, where its cortex is continuous with the uvula, the positive areas resolve into the bands 12 and 13 (fig. 30). Band 12 is the widest of all the bands of the posterior lobe, but its reaction for 5'nucleotidase is the lowest. Over part of its trajectory, through the lobules X, IX, and VIII, band 12 can be subdivided into 12 a and 12b (fig. 29). The bands 13 and 15 are located in the lateral part of the uvula. Band 16.1 is difficult to discern and usually is present at the lateral margin of the ventral uvular cortex only (figs. 29, 32B, 34 and 35). Band 15 marks the lateral border of the dorsal cortex of the uvula. Band 14 splits off from band 14 at the transition of the cortex of the uvula and the pyramis. The midline band 11 decreases in width, but can be followed across the lobules VIII and VII, where it seems to end at the caudal surface of the lobule (figs.32A and 35). The bands 12-14

1 2

3 a

3

1 2

0

5

12

5

~II

b

IV-V

t1~I-1I

Fig. 30. Diagram of the supposed connection between the bands from lingula (I), lobulus centralis (II-III), and culmen (IV-V). Uncertain connections are indicated by a question-mark.

Topographic histochemistry of the cerebellum' 71

Fig. 31. A transverse section through the posterior lobe of the mouse cerebellum. Incubation was performed in absence of unspecific phosphatase inhibitors.

72 . Enrico Marani

are continuous over the pyramis into lobule VII, where they constitute a complicated pattern of interweaving bands, called the pars mixta. At the rostral side of lobule VII, this complex area gives rise to a new midline band which continues into band 1 of the simple lobule and the anterior lobe. Three parasagittal bands, which similarly emerge from this area, continue into the bands 2, 3, and 4 of the simple lobule (figs.32A and 35). Band 15 crosses lobule VIII at the border of the pyramis and the copula pyramidis and bifurcates in lobule VII. Its two branches 15a and 15b become located medially and laterally to the cortexless area in the intercrural sulcus at the border of vermallobule VII and the ansiform lobule (fig. 32). Band 15 is not engaged in the fusion of the bands 12-14. The split portions of band 15 reunite in the simple lobule and are continuous with band 5 of the anterior lobe. Bands 5 and 15, therefore, are located at the transition of vermis and hemispheres. The bands 6.1, 6.2, 6.3, 7.1, and 7.2 of the simple lobule continue as the bands 16 and 17 of the ansiform and paramedian lobules (figs. 36). At the copula pyramidis the bands 16.1 and 16.2 fuse and disengage to continue as separate bands on the the uvula and copula pyramidis. In this way, the cortexless area in the paramedian sulcus lateral to the uvula becomes located in between band 16.1 in the lateral cortex of the uvula and 16.2 in the medial part of the copula pyramidis (figs. 32B and 35). The bands 17.1 and 17.2 are difficult to trace in the copula, and no topographical relation between these bands and the 5' -nucleotidase positive areas in the paraflocculus and the flocculus can be established. 3.5.2 Considerations on the topography of the 5'-nucleotidase pattern The 5' -nucleotidase pattern in the molecular layer of the cerebellum of the mouse is summarized in figs.32A and B. Most of the bands are concentrated in the central portions of the lobules. The laterally located bands are more difficult to follow, because the contrast in the staining is often low, and the borders of the 5' -nucleotidase positive areas become less distinct, when they are not sectioned perpendicularly. The centrally located bands 1-4, which arise from the bands of the lingula and the uniformly 5'nucleotidase positive areas of the nodule, diverge in the dorsal part of the cerebellum. Some of the bands subdivide, whereas others are added more laterally. The horizontally hatched bands 2-4 of the anterior lobe and the simple lobule are not continuous with the bands 12-14 of the posterior vermis through their reallocation in lobule VII (see MARAN! 1982, the pars mixta; fig. 36). The number of 5'-nucleotidase positive bands in the anterior lobe increases from the lingula through the central lobule into the culmen by the addition of band 3 and the subdivision of band 4 in 4a and 4b. In lobule VII, the midline band 1 of the anterior lobe and the simple lobule divides and continues into the paired medial branches of band 12. The midline band 11 of the caudal vermis, therefore, is not continuous with its coun-

Fig. 32. A: View on the anterior lobe and simplex lobule from rostral. Bands are indicated on a different way. The midsagittal band is indicated in black, while the vertical hatching indicates the first longitudinal zone that is passing lateral of the sulcus intercruralis (MARANl and VOOGD t 979). Longitudinal bands present in between the midsaggital and the longitudinal zone passing lateral of the intercrural sulcus are horizontal hatched. Zones lying lateral of the vertical hatched zone are all indicated by tiger skin structures. This reconstruction was made-to indicate the relation of the bands of the anterior lobe with those present in the posterior lobe. B: Caudolateral view on the reconstructed 5' -nucleotidase pattern from series H9992.

74 . Enrico Marani

terpart in the anterior lobe. Such a partial intermingling is also present for the bands 12, 13, and 14 (see fig. 35). Stepwise fusion of the bands 12, 13, and 14 in the caudal vermis leads to the disappearance of the bands in the nodule which shows a uniform, high positivity for 5' -nucleotidase. Band 5/15 (vertical hatching) indicates the border between vermis and hemisphere. It occupies a lateral position in the caudal vermis, shifts to the border of the pyramis and the copula pyramidis, and divides into branches which pass medially and laterally to the intercrural sulcus of the ansiform lobule. It is continuous with band 5 of the anterior lobe and the simple lobule which ultimately may fuse with band 2 of the lingula. Band 16.1 (tigerskinned) occupies the extreme lateral part of the uvula and part of the copula pyramidis and, therefore, straddles the paramedian sulcus. For most of its trajectory it is located in the hemisphere, where it gives rise to the bands 16.1, 16.2, and 16.3 and continues in band 6 of the anterior lobe. The most laterally located bands are 17.1 and 17.2. They participate in the formation of the 5'-nucleotidase pattern in the molecular layer of the paraflocculus, but this cannot be analysed in more detail in the available material. The 5'-nucleotidase band pattern in the molecular layer is largely independent from the transverse lobular pattern of the cerebellum, although certain differences between successive lobules exist. Sometimes, the 5' -nucleotidase positive bands seem to cross sulci. This happens with band 16 between the uvula and the hemisphere. The bands cross the primary fissure between the anterior and posterior lobes without any apparent change. With respect to the number and the appearance of the 5' -nucleotidase positive bands, the simple lobule (lobule VI and HVI) and the anterior lobe clearly belong together. Changes in the band pattern occur at the caudal border of VI and in lobule VII with its complicated order of interlacing bands and at the prepyramidal and secondary fissures which separate the lobules VII, VIII, and IX. The laterally located bands pass without interruption from the hemisphere of the simple lobule into the ansiform and paramedian lobules. A description of the myeloarchitecture of the mouse cerebellum is given in chapter 3.2.1. On many points, the arrangement of parasagittal compartments in the cerebellar white matter resembles the pattern of 5' -nucleotidase positive and negative bands in the molecular layer. Differences are present, of course, because the two patterns are located in different layers with their own constituents, and also because the circumferences of the inner fibre layer and outer molecular layer differ. Both patterns consist of two juxtaposed tissue elements. In the case of the myeloarchitectonic pattern, each compartment is built up by two elements: a medial area of thick and thin fibres and a lateral area of thin fibres. The 5'-nucleotidase pattern consists of a positive band always bordered by a negative one. The lateral border of a compartment at the thin fibre area is sharp, and the medial border, where the thin fibres merge with thick fibres, is indistinct. The lateral borders of the 5'-nucleotidase posi-

Topographic histochemistry of the cerebellum . 75

Fig. 33. View on the caudal aspect of the reconstructed 5 ' -nucleotidase pattern. At the bottom of this figure a view ventral on the cerebellum is demonstrated.

tive bands are also sharply defined, and the medial borders gradually merge into the negative zone. The midline of the cerebellum is not indicated in the structure of the cortex, but is clearly marked by a higher activity of 5' -nucleotidase (bands 1 and 11) and by an accumulation of small fibres in the cerebellar white matter with only few thick fibres. The midline accumulation of thin fibres is wide and tapers towards the anterior lobe. The features of the midline bands 11 and 1 are identical. On both sides of the midline,

76 . Enrico Marani

the number of negative and positive bands for 5'-nucleotidase generally equals the number of compartments in the cerebellar white matter. The 5'-nucleotidase pattern and the myeloarchitectonic compartments both diverge in the dorsal part of the cerebellum. 5'-Nucleotidase zones come together in the ventral part of the anterior lobe. This feature is less clear for the subdivisions of the white matter, because the fibre contents of the compartments increases in the ventral part of the cerebellum, and their borders tend to slope laterally. The number of compartments in the lobules I-III is the same as the number of 5'nucleotidase zones, and the split of compartment 2 in 2a and 2b corresponds to the division of band 4 in 4a and 4b. The division of the white matter is much less clear in the caudal part of lobule VI and VII, where the 5'-nucleotidase bands become rearranged in the pars mixta. The compartments AI, A2, and A3 of lobules IX and X correspond with the bands 12, 13, and 14. The C2 compartment, which straddles the

Fig. 34. View from rostral into the inside of lobules X, IX, VIII, and the copula pyramidis. The midsagittal band is black. Band 13 which belongs to the horizontal hatched bands is here hatched vertically to demonstrate its relation to band 15 in the copula pyramidis. Due to the angel of which this inside is viewed, band 15 is invisible at the lateral edges of lobule IX a and b.

Topographic histochemistry of the cerebellum . 77

paramedian sulcus between the caudal vermis and the copula pyramidis,corresponds in position with band 16.1. The three C compartments in the copula coincide with the bands 16.1, 16.2, 16.3 and the D compartments with the bands 17.1 and 17.2. The

4

6

8019

7

A

Fig. 35. A: Horizontal sections throught the culmen, declive and tuber-folium (lobules IV-V, VI, VIII). The cutting direction is indicated at the right top of this figure. Successive sections are plotted for their 5'-nucleotidase positive areas (stippled band), while the deeper situation (sections 8-10A) is shown for caudal lobule VII. At the right bottom a scheme is indicated, demonstrating the transition of posterior lobe bands (II-Db) into anterior lobe bands (1-3) in the «pars mixta». B: Shows the fusion of the bands 12 to form band 1 at the transition of lobule VII to lobule VI.

78

Enrico Marani

medial border of the hemispheres in the posterior lobe is indicated over most of its extent by band 15. The myeloarchitecture of the corresponding region is not quite clear, but the region gives rise to corticonuclear fibres terminating in the fastigial nucleus (see 3.2.1) and as such should be included with the compartments A1-A3 in the cerebellar vermis. The continuation of band 15, i. e. band 5 of the anterior lobe, corresponds to compartment 3. As a consequence, the border between vermis and hemisphere in the anterior lobe would be located between the compartments 3 and 4, and laterally to 5' -nucleotidase band 5. The correspondence of the longitudinal 5' -nucleotidase pattern and the myeloarchitecture of the cerebellum is still purely topographical. A correlation of the localization in the cerebellar white matter of the afferent and efferent connections of the molecular layer with the 5' -nucleotidase band pattern is not yet possible. Attempts to correlate the localization of olivocerebellar fibres in the cerebellar white matter and their climbing fibre afferents in the molecular layer with the 5'-nucleotidase band pattern fail, because it is found that the 5' -nucleotidase in the molecular layer is not bound to climbing fibres (MARANI 1982 a, b). A relation between certain Purkinje cells and the 5'-nucleotidase pattern is established by electronmicroscopy (MARANI 1982a,b; see also part 3.5.3). As a consequence, two kinds of Purkinje cells should be distinguished, namely those located within and those located outside 5' -nucleotidase positive bands. If the hypothesis that a 5' -nucleotidase negative and a 5'-nucleotidase positive band correspond to a compartment is correct, this would mean that the fibres from 5'-nucleotidase negative Purkinje cells are located within the medial, large fibred portion of the compartment and axons of 5'-nucleotidase positive cells within its lateral thin fibred portion. Small calibre Purkinje cell fibres are indeed found in the thin fibred part of the compartment (FEIRABEND and CHOUFOUR 1985). Thin fib red Purkinje cell axons express AChE enzymatic activity in contrast to thick fib red Purkinje cells (see 3.2.3) within the fibre layer of monkeys. The assumption of a subdivision of the cortical zones A to D in two subunits, determined by a different enzymatic content for 5' -nucleotidase, brings about the question of the purinergic transmission. After administration of AMP microiontophoretically (KOSTPOPOULUS et al. 1975) to Purkinje cells, the effect on the spontaneous firing capacity is different after the administration of adenosine. These kinds of experiments suggest that the alternating zones of high and low 5' -nucleotidase activity create gradients within one longitudinal zone of the spontaneous firing capacity of the corresponding Purkinje cells. The histochemical heterogeneity hypothesis (MARAN! and VOOGD 1977) for acetylcholinesterase seems to hold for the 5'nucleotidase pattern, too. This histochemical heterogeneity seems to explain functional differences of Purkinje cells, as has been stressed by CHAN PALAY et al. (1981, 1982), too.

Topographic histochemistry of the cerebellum' 79

3.5.3 Ultrastructural localization of 5' -nucleotidase in the molecular layer of the mouse cerebellum3 3.5.3.1 Preliminary remarks and experiments Terminology

This part deals with three different enzymes apart from 5' -nucleotidase. They will be defined on the basis of histochemical studies on the cerebellum: (1) 5'-nucleotidase. The enzyme that converts 5/-nucleotides into their nucleosides at an optium pH of 7.0-7.6, as defined by SCOTT (1965, 1967). (2) Acid phosphatases. Enzymes that convert most phosphomonoesters (at least beta-glycerophosphate) at an optimum pH of 4.5-5.5 (MARAN! 1981; see 3.3.2). (3) Nonspecific phosphatases. Enzymes that convert phosphomonoesters (at least beta-glycerophosphate) at pH 7.0-7.6. They can be considered as rest activities of acid or alkaline phosphatases, see 3.4. (4) Alkaline phosphatases. Enzymes that convert phosphomonoesters (at least betaglycerophosphate) at an optimum pH of 8.5-9.5 (MARAN! and KURK, unpublished results; see 3.3.2 and 3.4). The last three phosphatases are also capable of breaking down 5' -nucleotides. The discrimination between 5'-nucleotidase and other phosphomonoesterases (by histochemical methods) depends on the use of other substrates, different pH's, and inhibitors. I. anterior

I. simplex

I. ansiformis

I. paramedianus

copula pyramidis

parallocculus s. parafloccularis

flocculus

s. paramedianus

Fig. 36. Summary diagram (MARAN! and VOOGD 1978) indicating the various 5 ' -nucleotidase bands that can be distinguished in the mouse. 3

A shortened version appeared in: Neurotransmitter Interaction and Compartmentation (ed. H. F.), pp. 557-572. Plenum Pub!. Co., New York 1982.

BRADFORD,

o

o

.

.

Fig. :)f. 1 ne ngures 1\-lJ show Y -nucleotidase reaction pro uct localization in cryostate sections of rat uvula (lobule IX) after 0' (A), 5' (B), 10' (C), 30' (D) fixation with 1% glutaraldehyde in A.D. Incubation time was 1.5 hours in Scott's medium. E and F demonstrate the effect of paraformaldehyde alone for 15 min (E), and (F) the appearance of overall reaction product localization due to 5'-nucleotidase solubility (method A, 1 min incubation, according to SCOTT 1965).

Topographic histochemistry of the cerebellum' 81

Fixation The inhibitory effect of a number of fixatives on the activity of phosphomonoesterases is well-known (for cerebellar 5 ' -nucleotidase see SCOTT 1967; for other phosphomonoesterases see HOPWOOD 1972). Among these fixatives, glutaraldehyde is commonly used for its ultrastructural preservation property (SABATINI, MILLER and BARNETT 1964). In general, glutaraldehyde suppresses enzymatic activity more than other fixatives, e. g. formaldehyde (BREDEROO et al. 1968; HOPWOOD 1972; PEARSE 1972). 5' -Nucleotidase actitivty, as determined with SCOTT'S medium (1967) in 16 !-lm cryostate sections, almost disappears after fixation for 10 to 15 min in unbuffered 1% glutaraldehyde (fig. 37A-D), while after 1 hour's fixation in unbuffered 1% paraformaldehyde its activity is still present, although greatly diminished (see also SCOTT 1967). In electron microscopy, however, formaldehyde alone cannot be used because of the inadequate morphological preservation (for example see the unsatisfactory preservation of the molecular layer, fig. 37E). The use of unfixed tissue blocks (ESSNER, N OVIKOFF and MASEK 1958) is not considered in this study. Therefore, despite its strong inhibitor effect, glutaraldehyde must be utilized in order to obtain sufficient preservation of elementary ultrastructure of the molecular layer, and to be able to compare it to descriptions of the normal ultrastructure of the cerebellar cortex (PALAY and CHAN-PALAY 1974).

Solubility All biochemical studies (BERNSTEIN and LUPPA 1978 a; BOSMAN and PIKE 1970; HARDONK and DE BOER 1968; IPATA 1966, 1967, 1968a, b; ISRAEL and FRANCHONMASTOUR 1970; MARANI 1977), with one exception (PILCHER and JONES 1970), show that brain 5'-nucleotidase is partially soluble. Solubility can be enhanced by using detergents. In histochemical studies, the solubility of 5 ' -nucleotidase is not apparent. In the preceding chapter 3.4.1 (see also MARANI 1981) the solubility of mouse cerebellar 5' -nucleotidase isoenzymes is studied (table 7).

Table 7. Total 5'-nucleotidase activities with their SD measured according to PERSIJN and VAN DER SLIK (1969) in lUll of supernatants (2650 g) after treatment of the whole homogenate (2,5% w/v) for identical times with different solvents. Butanol (n-butanol)

Distilled water

Electrophoretic buffer (28,8 g glycine, 6.0 g Tris/L)

Triton

29.4 ± 3.2

47.1 ± 6.5

103.5 ± 2.7

280 ± 16.0

X

100 (0.1%)

82 . Enrico Marani

Perfusion with buffers, or even saline, will probably dissolve some of the enzyme(s). The effects of cacodylate or phosphate buffers used in electron microscopy lie in between those of the Tris-glycine buffer and AD effects documented in our previous study (MARANI 1981). Even after very short fixation time, the reaction product is usually scattered all over the cerebellar molecular layer. This product is thought to be the result of soluble 5' -nucleotidase (fig. 37) However, in our studies, perfusion with saline followed by fixatives does improve 5' -nucleotidase localization.

Artifacts The site of enzyme activity can be misleading, if based upon the localization of 5' nucleotidase reaction product, because of nonspecific lead deposits and enzyme product captured by lead ions at some distance from the enzymatic reaction (DAEMS et al. 1972). A generally recognized artifact (fig. 38), produced by lead salt methods, consists of the deposition of intranuclear lead ions (GOMORI 1952; MOSES, ROSENTHAL, BEAVER and SCHUFFMANN 1966; ROSENTHAL et al. 1966; ROSENTHAL et al. 1969, a, b; TANDLER 1956). Although easily detected, lead does produce errors in brain 5'-nucleotidase determinations (BERNSTEIN, WEISS and LUPPA 1978b; SURAN 1974a, b). Electron micrographs of experiments containing these artifacts are not considered for the localization of 5 ' -nucleotidase. After glutaraldehyde fixation, a prolonged incubation time (over 1 hour) is required for 5' -nucleotidase. Biochemical studies, using gel electrophoresis for cerebellar enzymes, indicate that gels with separate bands and high enzyme activity produce lead phosphate outside the gels within three minutes. In addition, lead phosphates adhere at protein fronts and at the brownish myelin front in gels (fig. 38E; see also STACH, OCKENFELS and PILGRIM 1977, for the same phenomenon studied in agar isoenzyme). The onset of this process is easily recognized by the appearance of a white cloudiness in the incubation medium. Cerebellar sections after prolonged incubation behave in the same way: therefore, incubation media have to be regularly renewed, and reaction product localization at edges of cerebellar vibratome sections (mostly found at intercellular location, fig. 38B) are discarded. The Scott incubation medium for 5 ' -nucleotidase (SCOTT 1967) produces fewer penetration artifacts than we obtained in our acetylcholinesterase studies (MARANI 1981). Moreover, spontaneous or Pb2+ -catalyzed non-enzymatic hydrolization of AMP is low for this substrate (ROSENTHAL et al. 1966).

Activation Enzyme histochemical incubations usually require Mg2+ for activation of 5' -nucleotidase. An exception to this rule seems to be mouse cerebellar 5 ' -nucleotidase, for which Mg2+ is not important as an activator, since mouse cerebellar 5 ' -nucleotidase is K+, N a+-sensitive (MARANI 1980 b). Therefore, incubation media for mouse cerebellar

Topographic histochemistry of the cerebellum' 83

5' -nucleotidase must contain high concentrations of sodium or potassium ions. The incubation medium of SCOTf (1967) meets this condition. Most divalent cations like NiH or CaH , can increase brain 5' -nucleotidase activity (MARANI and BOEKEE 1973). Mercury or PV+, on the contrary, inhibit 5'-nucleotidase activity (DE PIERRE and KARNOVSKY 1974 a, b, c; SURAN 1974a, b). Despite the use of lead salt methods in 5' -nucleotidase histochemistry, a detailed study concerning Pb H effects on brain 5'-nucleotidase is not available (see also fig. 21). Nonspecific phosphatase activity Nonspecific cerebellar phosphatase activity at pH 7.2 in mice is an acid phosphatase rest activity retaining 30-40% of its optimum activity (see 3.4.1). To distinguish between nonspecific phosphatase and 5' -nucleotidase at this pH, inhibitors for acid phosphatase can be used, e.g. P-, NiH or 1.3-c. glyceromonophosphate (see 3.4). A shift towards alkaline pH range for the 5' -nucleotidase determination is also possible because mouse cerebellar 5' -nucleotidase has a second optimum between pH 10-11 (see 3.4), demonstrating the same histological band pattern (MARAN! and KURK, unpublished results). However, levamisole, a biochemical inhibitor for alkaline phosphatase (BORGERS 1973), is unreliable as a histochemical inhibitor on cerebellar cryostate sections (see 3.4 and MARANI 1981, 1982 b). Cryostate sections incubated for the demonstration of nonspecific phosphatase activity, using beta-glycerophosphate as a substrate and sections incubated for 5' -nucleotidase to which 1.3-c. glyceromonophosphate is added, are therefore made at pH 7.2. Nonspecific phosphatase activity is present in: Purkinje cell somata, stellate cell somata, basket cell somata, and blood vessels (figs. 38 C and D; see also MARANI 1982 b). Nevertheless, it is unclear, whether Bergmann's glial cell bodies belong in this category, as these cell bodies are in close proximity to the positive Purkinje cell bodies. Bergmann's glial cell somata do contain acid phosphatase activity at pH 5.0. Enzyme-substrate protection By coupling the enzyme with substrate before or during fixation, some enzymes are protected from fixative denaturation (PAPADIMITRIOU and VAN DUYN 1970a, b). However, addition of AMP to the saline perfusion solution or the fixative produces severe morphological alterations of the molecular layer (MARANI, unpublished results). 3.5.3.2 Description of the ultrastructural 5' -nucleotidase localization Biochemical effect of EM substances on 5' -nucleotidase Tests for possible inhibitory effects of substances used in electron microscopy (table 8), except fixatives, indicate that none of the chemicals used produces total 5' -nucleotidase inhibition.

84 . Enrico Marani

5 NUCLEOTIDASE

E

Topographic histochemistry of the cerebellum . 85 Table 8. Electron microscopic chemicals':' tested for their influence on cerebellar 5 ' -nucleotidase activity (in lUll) according to PERSIJN and VAN DER SUK (1969). Mean Blanco':":' Na chloride (150 mM) Na cacodylate (160 mM) Na cacodylate (16 mM) Na succinate (70 mM) Sucrose (160 mM) Ca chloride (1 mMy':":' Pb acetate (2 mM)

47.1

106.7 186.9 95.4 191.0 36.9 52.0 27.3

± S. E. M. 2.9

4.8

6.8 6.1 15.0 1.5

4.0 1.6

':. Routine chemicals were used, not all are grade analar. ,:.,:. All determinations were without Mg2+ from 6 homogenates, 2.5% w/v, in A. D.; adenosine deaminase activity was not altered by these chemicals. ".,:.,:. Lower Ca++ concentrations do not affect 5' -nucleotidase activity.

Nonspecific localizations of lead ions Incubations, omitting the capture agent lead ions in the SCOTT incubation medium (1965), do not produce localized reaction product. Perfusion fixation with the SCOTT medium, omitting the substrate AMP, show nonspecific lead ion deposits at the surface membranes of endothelial cells (fig. 38). Acid phosphatase studies with the lead betaglycerophosphate technique at pH 5.0 show imperfect reaction product formation even in lysosomal structures (BREDEROO et al. 1968). This is the case for nonspecific phosphatase activity, too. These lead ion responses are to be rejected as being indicative of 5 ' -nucleotidase activity.

5' -Nucleotidase localization in the cerebellar molecular layer A characteristic feature of 5'-nucleotidase is its arrangement in longitudinal zones of alternating positive bands (see 3.5). The lateral borders of positive bands are sharply

Fig. 38. Nonspecific lead precipitate in a stellate cell nucleus in an uncontrasted section (A). B: demonstrates nonspecific lead precipitate due to penetration artifact at the edges of a vibratome section. Inset shows a detail of this intercellular localization, which is only found after prolonged fixation and is due to the enhancement of unspecific phosphatase activity by prolonged fixation. The contrasted photographs on the right side show nonspecific lead precipitate locations at the endothelial inner surface (C). Detail of another blood vessel is demonstrated in (D) after normal incubation. AMPase at the external endothelial membrane is present (arrows), but cannot be due to 5' -nucleotidase activity. E: shows the effects on gels of short incubation times (1 min) with the Scott incubation (overall AMPase activity) as compared to long incubation times (30 min). Protein lead adherence is indicated by (a), while (b) is the lead phosphate adherence to the myelin front. The true AMPase activity lies in between (a) and (b).

Fig. 39. The top figures (A) show vibratome sections (200 lim, series E 614) treated with sulfide after incubation for 5' -nucleotidase. Three parallel positive bands in the anterior lobe of the mouse cerebellum are seen. B: shows the sharp contrast even in E.M. photographs of the transition of 5'-nucleotidase positive to negative areas in an oblique section through the molecular layer. The interrupted vertical lines indicate the positive zone (right) and the nonreactive area (left).

Topographic histochemistry of the cerebellum . 87

delineated from negative areas, while their medial borders sometimes show a gradual transition into the negative area bordering it. The identification of these sharp lateral borders in ultrathin sections proves that the lead precipitates in these areas represent 5'-nucleotidase reaction product. Figure 39 shows one such border in the vermal part of lobule IV-V in an ultrastructural photograph of the mouse cerebellar molecular layer. 5'-Nucleotidase reaction product is found within the dendritic tree of Purkinje cells in sections treated with and without a nonspecific phosphatase inhibitor. Reaction product for 5'-nucleotidase cannot be demonstrated in the cell somata of Purkinje cells. It was absent from rough endoplasmic reticulum with all types of incubations used for 5'-nucleotidase demonstration. In the primary dendrites, 5'-nucleotidase reaction product is situated in structures comparable to the smooth endoplasmic reticulum (fig. 40), while just beneath the cell membrans they are found within the subsurface cisternae. Reaction product is always within the lumen of the cisternae (fig. 41). No connections of the 5'-nucleotidase positive subsurface cisternae with the intradendritic smooth endoplasmic reticulum have been found, while our studies do not provide any findings suggestive of the origin of the 5'-nucleotidase producing system within Purkinje cell dendrites. It must be pointed out, however, that ribosome-like structures occur between the packed cisternae. The fine branches of the Purkinje cell dendritic tree are replete with spines. Dendritic spines are small protrusions onto which the axon of the granule cell (parallel fibre) synapses with Purkinje cells. These spines contain a spine apparatus which is 5'-nucleotidase positive (MARAN! 1977, fig. 42). The spine apparatus has the same ultrastructural appearance as the subsurface cisternae. The 5'-nucleotidase reaction product position appears to be identical in the subsurface cisternae and spine apparatus (compare figs. 41 and 42). Parallel fibres are small calibre unmyelinated axons which make synaptic contact with spines, stellate cells, basket cells, and Golgi cells by producing boutons en passage on these structures. The parallel fibre contains reaction product, which accumulates in tubular structures (i. e. microtubules). This electron density is absent from control series. 5'-Nucleotidase reaction product is not only found in the axon, but also in the parallel fibre boutons en passage (figs.42 and 43). In these boutons en passage, 5'nucleotidase reaction product is localized in membrane bound spaces, closely adjacent to mitochondria. Sometimes, the mitochondria are engulfed by cisternae containing the reaction product (fig. 42). Cisternae .containing reaction product have irregular contours that resemble buds (fig. 44). The appearance of these buds strongly suggests the formation of the synaptic vesicles from these buds in the parallel fibre bouton. Other cerebellar strucutures and 5' -nucleotidase

Stellate cells cannot be regarded as containing 5'-nucleotidase activity (see 3.5.3.1, for nonspecific phosphatase). Cells treated with inhibitors for unspecific phosphatase

Fig. 40. Localization of 5/-nucleotidase reaction product in the smooth endoplasmic reticulum of Purkinje cell dendritic trees are demonstrated. A: shows a Purkinje cell dendrite with 5'nucleotidase reaction product located exclusively in the smooth endoplasmatic reticulum (s-ER), indicated by arrows, Band C: show two cisternae of the s-ER that are positive for 5/ -nucleotidase. D: taken at the base of the main Purkinje cell dendrite tree. E: reveals a circular cisterna of the s-ER filled with 5'-nucleotidase reaction product. Note that in E ribosomes are present near this circular cisterna (method B, see Appendix [3.5.1 D.

Fig. 41. This figure shows four E.M. photographs of Purkinje cell dendritic subsurface cisternae (asterisks), in which 5'-nucleotidase reaction product is localized. MVB: multivesicular body.

90 . Enrico Marani

(F- and 1.3-c. glyceromonophosphate) are negative for 5'-nucleotidase. Howe~er, some structures synapting on stellate cells or on stellate protrusions do show a5'nucleotidase positive reaction. These structures have the characteristics of parallel fibre boutons and contain 5' -nucleotidase reaction product in their cisternae engulfing mitochondria (fig. 45). The baskets around Purkinje cell somatas are never found to be positive for 5'nucleotidase reaction product. In our experiments basket cells themselves never contain 5'-nucleotidase activity. In contrast, in sections treated for light microscopic purposes with chloroform:ether (1:1), the baskets around Purkinje cells definitely become positive for localized 5'-nucleotidase reaction product. In our material the climbing fibre axon and the climbing fibre boutons were never negative. Profiles of Bergmann's glia within the molecular layer are not found to be 5' -nucleotidase positive. 3.5.3.3 Discussion of the ultrastructural 5' -nucleotidase localization It should be clear from section 3.5.3.1 that the lead products identified with our electron microscopic cytochemical methods can only represent a small fraction of the activity of the enzyme 5' -nucleotidase present in vivo. Inhibition by glutaraldehyde and lead ions (see 3.5.3.1) diminish 5'-nucleotidase activity, and the resolution of the method is diminished by the solubility of 5'-nucleotidase (see 3.4.1 and 3.5.3.1). Nevertheless. since only one isoenzyme is present in the cerebellum of the mouse (see 3.4.1), all remaining activity must belong to the same type of 5' -nucleotidase. Still, the question remains, whether these results are representative of the overall 5'-nucleotidase localization. It is obvious that negative results do not exclude the presence of 5'-nucleotidase. The positive results, however, consistently show localization in Purkinje cell dendrites and parallel fibres. The results of our study demonstrate an intracellular localization of 5' -nucleotidase in cisternae of both pre- and postsynaptic elements of the parallel fibre-Purkinje cell synapses. Localization for 5 ' -nucleotidase is only deemed unacceptable, when these localizations coincide with nonspecific phosphatase activity. Arguments supporting the 5'-nucleotidase localizations are: (a) Results from differential centrifugation studies show that 5'-nucleotidase is present in the synaptosomal fractions under biochemically optimal conditions (MARAN! 1977; BALABAN et al. 1984). (b) Destruction of climbing fibres with 3-acetylpyridine leaves the homologous 5'nucleotidase pattern in rats unaltered and does not diminish the 5'-nucleotidase activity biochemically (see table 6; BALABAN et al. 1984, 1985), thereby excluding a primary localization for 5'-nucleotidase in climbing fibres (MARAN! 1982b). (c) Light microscopic results are consistent with distribution in Purkinje cell dendrites (Scon 1965, 1967; see 3.5)

Fig. 42. The left electron micrographs demonstrate Purkinje cell dendritic spines, with or without synapting parallel fibres. 5' -Nucleotidase ~eaction product is confined to the cisternal spaces of the spine apparatus. The two micrographs at the right reveal Purkinje cell dendritic spines positive for 5' -nucleotidase, synapting on 5 ' -nucleotidase positive parallel fibre boutons (examples taken from method A and B).

n . Enrico Marani (d) Transection of the parallel fibres in the molecular layer of mice does not result in the disappearance of the 5'-nucleotidase banding pattern (MARANI 1982 b). This indicates that other structures (i. e. Purkinje cells) must be involved in its generation. Our findings differ from those obtained by BERNSTEIN and LUPPA (1978 a), BERNSTEIN et al. (1978 b), KREUTZBERG and BARRON (1978 a), KREUTZBERG et al. (1978 b), SCHUBERT, KOMP and KREUTZBERG (1979), and SCHUBERT and KREUTZBERG (1978). BERNSTEIN et al. (1978 b) observe an intracellular localization of 5' -nucleotidase in boutons of the CA3 hippocampus region in the rat, as well as axolemmal and nuclear locations. In our opinion, insufficient arguments are given for the presence of 5'nucleotidase in the nucleus, particularly because BERNSTEIN and LUPPA (1978 a) and BERSTEIN et al. (1978 b) show the presence of an intranuclear beta glycerophosphatase activity which could be identical to the rest activity of acid phosphatase known to be present in the mouse cerebellum. In their isoelectrofocusing study they demonstrate the lack of nonspecific phosphatase activity at pH 7.2 with NiH at a concentration of 10- 3 M which totally inhibits any 5'-nucleotidase activity. Our study on mice cerebellar isoenzymes (see 3.4) indicates that 5·1O- 2M NiH inhibits nonspecific phosphatase and not 5'-nucleotidase activity (see also SCOTT 1965). Moreover, nonspecific phosphatase activity is not only present in mouse cerebellum, but also in mouse and rat hippocampus (MARANI, unpublished). However, caution should be taken, because different actions of NiH on 5' -nucleotidase activity have been described for different tissues (SCHWARTZ and BODANSKY 1964). As to the presence of nonspecific phosphatase activity at pH 7.2, the different localizations that are achieved for intraperikaryonal 5' -nucleotidase activity are, in our opinion, due to the presence of nonspecific or residual activity of phosphatases. In the electron microscopic cytochemical studies of the rat central nervous system of Kreutzberg's group (e. g. KREUTZBERG and BARRON 1978 a; KREUTZBERG et al. 1978 b), 5'-nucleotidase reaction product is found to be exclusively present in glial cells. When criteria derived from our mouse cerebellar study are applied to their results, with extensive long glutaraldehyde and paraformaldehyde fixations, they may be considered to provide nonspecific phosphatase activity at pH 7.2. This would be in good agreement with a glial localization. In these studies, inhibitors for 5' -nucleotidase are not used, and the presence of nonspecific phosphatase is not considered. When inactivation of 5 ' -nucleotidase by fixation was measured by them (KREUTZBERG et al. 1978 b), 65% inhibition was found. However, the inhibitory lead effects are not found (for the opposite results see SURAN 1974; table 8, and fig.21). Still, the remaining 35% could theoretically be contributed to other enzymes, as has been demonstrated in 3.4. It is found that 30%-40% nonspecific phosphatase activity remains in the mouse cerebellum at pH 7.2. In general, the inherent rest activity of acid phosphatase improves after prolonged paraformaldehyde fixation (BREDERoo and DAEMS 1970) at pH 7.2 and can easily be mistaken for 5'-nucleotidase activity after prolonged fixation. The studies of SURAN

"Topographic histochemistry of the cerebellum' 93

Fig. 43. A: demonstrates the overall localization of 5 ' -nucleotidase in the molecular layer neuropil. B, C, D, E: show parallel fibre boutons synapting on Purkinje cell dendritic spines. These boutons are 5 ' -nucleotidase positive within the cisternal space surrounding mitochondria. B: also reveals that parallel fibres contain 5 ' -nucleotidase activity (examples taken from method A and B), while the bouton is localized near a stellate perikaryon.

94 . Enrico Marani

Topographic histochemistry of the cerebellum· 95

(1974a, b) on 5 ' -nucleotidase and acid phosphatase activity in the mouse spinal cord were partially repeated (MARANI, VAN DEN VOORT and WILLIK, unpublished results). Trigeminal nerve root lesions produce the related somatotopy (RUSTIONI et al. 1971), but 5'-nucleotidase activity remains unchanged, while acid phosphatase activity disappears. The indication that two different types of enzymes are present in the substantia gelatinosa Rolandi is also expressed by the difference in rostral-caudal extension of both enzymes. At the time SURAN'S papers appeared (1974a, b), the same description appeared for the precise area and the identical enzyme in the rat (COIMBRA et al. 1974). The localizations described for acid phosphatase by both authors are contradictory. But the prolonged fixation time (over 24 hours with 3% glutaraldehyde), after which both 5' -nucleotidase and acid phosphatase activity were determined at the ultrastructural level, are indeed, mitigating SURAN'S results. Their differing results concerning 5 ' nucleotidase and acid phosphatase are due to a number of factors: (1) variation in the concentrations of fixatives, (2) variation in fixation times, (3) improvement of nonspecific phosphatase at pH 7.2 after prolonged fixation.

So it might be accepted that too long a fixation is the responsible factor for bad 5'nucleotidase localization at the ultrastructural level. One of the main results obtained from this study is evidence for the existence of two biochemically distinct populations of Purkinje cells in the cerebellum of the mouse which differ in respect to the presence of 5' -nucleotidase in their dendrites. This difference may be related to the presence of two classes of Purkinje cells in the rat: those which react to the administration of adenosine and those which do not respond (KosTOPOULOS et al. 1975). The somata of the Purkinje cells are 5' -nucleotidase negative but possess nonspecific phosphatase activity at pH 7.2. This nonspecific phosphatase activity is absent from the rat Purkinje cell body. In mice, this appearance is only obtained, when an inhibitor for acid phosphatase is added to the incubation medium. The presence of 5 ' -nucleotidase, both in the subsurface cisternae and in the spine apparatus of dendritic thorns, supports the notion of the continuity of these parts of the endoplasmic reticulum (PALAY and CHAN PALAY 1974). Because 5 ' -nucleotidase is thought to be a glycoprotein, the specific binding of Concanavalin A in subsurface cisternae of rat Purkinje cell dendrites (WOOD et al. 1974) closely agrees with our results (see also the FAL results, Chapter 4.3). The site of 5 ' -nucleotidase synthesis is not known. The absence of the enzyme from other cell organelles and the presence of ribosome-like structures between the cisternae suggest a local synthesis in this part of the endoplasmic reticulum. Fig. 44. These four electron micrographs show 5' -nucleotidase reaction product within parallel fibre boutons. The fine cisternae, located near mitochondria, seem to be undergoing exocytosis. A, B, and C demonstrate bulges that contain reaction product, while D demonstrates that these cisternae are more complex than would be expected from A, B, and C. (Examples taken from methods A and B).

96 . Enrico Marani

Fig. 45. These figures demonstrate 5 ' -nucleotidase positive parallel fibre boutons, synapting on stellate cell soma. (Examples taken from methods A and B).

Topographic histochemistry of the cerebellum· 97

It must be concluded from our observations that parallel fibres are 5' -nucleotidase positive only over part of their length. The length of parallel fibre in mice certainly exceeds the width of the 5 ' -nucleotidase positive bands which varies between 0.1 and 0.4 mm. 5' -Nucleotidase reaction product appears to be present in the parallel fibre boutons in structures which look like synaptic vesicles. This would bring adenosine in direct access to neurotransmitters at the parallel fibre-Purkinje cell synapses. Adenosine could be considered being a modulator or co-neurotransmitter. Recent studies (see 5.5) support the location of 5'-nucleotidase in Purkinje cell dendrites and parallel fibres.

3.6 Acetylcholinesterase topography in the cat cerebellum RAMON-MOLINAR (1972) demonstrated the distribution of acetylthiocholinesterase (AthChE) in the brainstem of cats four months old. His illustrations indicate that this activity is present in a band-like distribution in the molecular layer. However, the presence of AChE in the molecular layer of the cerebellum is disputed. AChE can only be demonstrated during development from the third day till the twentieth day postnatum with histochemical techniques (see Chapter 4.4). Conversely, biochemical determinations in cats show activity in the granular layer to be nearly as strong as in the molecular layer (AUSTIN and PHILLIS 1965; GOLDBERG and MCCAMAN 1967; MARANI, unpublished). In this part of the monograph the AChE distribution in both cat and monkey cerebellum is discussed. . 3.6.1 Description of the acetylcholinesterase pattern in the cat cerebellum 4 The molecular layer of most lobules of the anterior and posterior lobe vermis contains symmetrically disposed AChE positive areas alternating with narrow negative zones (fig. 46). The somata of the Purkinje cells are negative, and in transverse sections AChE activity is present in narrow striations, reaching from the somata of the Purkinje cells to the pial surface. The neuropil of the granular layer is strongly AChE positive, but the granular cells are negative. No activity is found in our cryostate sections of the white matter. HESS (personal communication) demonstrated that AChE positive strips are present within the white matter of several species after strong fixation. The subdivision of the cat white matter is now being studied and compared to Haggqvist stained senes. The margins of the midline positive area within the molecular layer are ill-defined. It 4 An extensive description of the AChE band pattern appeared in J.Anat. (Lond.) 124, 335-345 (1977). In this monograph, only a summary of these results is given. For an extensive discussion concerning AChE in the molecular layer of several species, see SILVER (1967, 1974) and MARANI and VOOGD (1977) or MARANI (1981, 1982).

98 . E nnco . Ma ram.

.

<.
~

Topographic histochemistry of the cerebellum· 99

consists of some closely packed vertically arranged striations in the anterior lobe. Lateral to these negative bands, symmetrically disposed positive bands are present both in the anterior and posterior lobe vermis (fig. 46). The transition between positive and negative bands is sharp. Often, this lateral border is located opposite a constriction of the white matter positive for AChE in fixated series that corresponds to the lateral border of compartments. The bands deviate progressively laterally from lobule I to lobule V in the anterior lobe. Consequently, both negative and positive bands become wider in the dorsal part of the anterior lobe. The area containing the midline positive band at its medial side and the first positive band at its lateral side, including this positive strip in the anterior lobe, is identified as zone A (MARANI and VOOGD 1977; BROWN and GRAYBIEL 1983b; VOOGD, HESS and MARANI, in press). The next negative zone with its positive band is then zone B, although the lateral positive border can only be distinguished in lobule III and sometimes lobule IV (see also VOOGD, HESS and MARANI, in press). In the posterior lobe, the bands have the same appearance as in the anterior lobe. The contrast between positive and negative bands is less obvious, but both in negative and positive areas striations are present. The Purkinje cell bodies are negative for AChE. The longitudinal bands are restricted to certain lobules of the posterior lobe vermis. In lobule VI, the midline and two lateral bands continue from the anterior lobe. In lobule VII, the molecular layer is uniformly positive for AChE, and no negative bands are present. In lobule VIII, the midline and lateral positive bands are wide, and the negative areas are narrow (fig. 46). In lobule IX, the midline band is occupied by a positive midline band, and on both sides, two wide, symmetrically disposed negative bands are found (fig. 46). In lobule X and the hemispheres, these bands are difficult to discern.

3.6.2 Ultrastructural localization of acetylcholinesterase in the molecular layer in kittens AChE in the cerebellar molecular layer has a strong affinity to acetylthiocholine over butyrylthiocholine, which is also accentuated by the use of pseudocholinesterase inhibitors (MARAN I and VOOGD 1977; MARANI 1981). Therefore, this acetylthiocholinesterase activity is considered to be «true AChE activitY'>' Biochemical determinations, however, (MARANI, unpublished; GOLDBERG and MCCAMAN 1967, cat; AUSTIN and Fig. 46. A: Sections from animal 8734, indicating the band pattern in the posterior lobe vermis. Incubation conducted according to KARNoVSKy-RooTS method. No inhibitors were added. When the reaction for AChE was equivocal, the loci in the molecular layer were not stippled. Therefore these sections represent the minimal extent of AChE-bands. B: Reconstruction of the posterior lobe vermis of animal 8734. The mid-sagittal plane is indicated by a dotted line. In lobules X to VIII, five positive bands are present alternating with six negative zones. Lobule VII is devoid of alternating areas with and without AthChE acitivity, except for the most caudal lobules, where three positive and four negative zones are present.

100 . Enrico Marani

PHILLIS 1965, cat), have shown that AChE activity is still present in the molecular layer of mature animals, although it can no longer be demonstrated there with cryostate histochemical methods in rabbit and cat. However, after fixation, the pattern can be found again in mature cats (BROWN and GRAYBIEL 1983 b). AChE in the granular layer has been localized in rat and mouse around granule cells. In the rabbit, a comparable distribution of AChE is present. However, a distinct localization is demonstrated in the molecular layer. Cells identified as a group of granular cells, surrounding an AChE positive Golgi cell, are present at different places within the molecular layer (SPACEK et al. 1973). Such cell clusters are absent in rat, mouse, and cat. The distribution of AChE within the granular layer of mouse, rat, and rabbit is in a distinct zonal distribution which is topographically comparable with the longitudinal 5'-nucleotidase pattern in mouse and rat (see 3.5). The cat granular layer also contains a longitudinal distribution which involves the granular cells and mossy fibre endings. This pattern also contains borders that are identical with AChE borders iIi the molecular layer (MARANI and VOOGD 1977). Just around birth the developing cerebellum of rabbit demonstrates a zonal arrangement for AChE, which starts to be present in an area containing the Purkinje cells (see 3.4). Although several other different distributions in mammals (SILVER 1974), and birds (FRIEDE 1966) are described, some of which are studied for this monograph, too (sheep, chicken), these studies are never worked out in a topographical sense (see MARANI 1981) and are therefore not further considered. AChE in the molecular layer of teleost fishes remains present in maturity and has been localized with electron microscopical enzyme histochemistry at climbing fibres (CONTESTABILE et al. 1978; VILLANI et al. 1978), although it is uncertain, whether climbing fibres exist in fish. In this paper, the true AChE, which is responsible for the pattern of longitudinal bands in the kitten's molecular layer, is found to be situated at parallel fibres and Purkinje cell dendrites, using the LEWIS and KNIGHT (1977) technique for ultrastructural localization of AChE.

Incubation methods The combination of the three techniques used for ultrastructural determination of AChE activity leads to a preference for the LEWIS and KNIGHT (1977) method over techniques using Hatchett's brown precipitates, such as the KARNOVSKy-RoOTS (1964) and the HANKER et al. (1973) techniques, because the penetration of the incubation medium is better and the reaction product is present throughout the tissue and is combined with a good preservation of the ultrastructural morphology. The Karnovsky-Roots as well as Hanker method suffers from penetration artifacts. Moreover, these techniques spoil the ultrastructure of the molecular layer of the kitten. To get improved results with the Lewis and Knight method, a low pH has to be used to prevent aspecific reaction product formation over nuclei and to promote penetration and precipitation.The disadvantages of the low pH which is far distant from the pH optimum

Topographic histochemistry of the cerebellum· 101

(pH 8.0) or AChE are the scanty formation and the spotty distribution of the reaction product.

Localization of the reaction product The Lewis and Knight method suffers from two main types of artifacts: (1) The appearance of reaction product intracisternally in mitochondria and holes which appear during ultrathin sectioning at places where high concentrations of reaction product is present. (2) Sometimes the washing procedure at the end of the incubation induces displacement of the precipitate over other structures, but this aspecific localization can be easily recognized. Light microscopy of cryostate sections processed with the Karnovsky-Roots technique shows that the perikarya and dendrites of the Purkinje cells in newborn and 25-day old kittens are positive for AChE. The Purkinje cells in older ·animals become negative for AChE, and the reaction product in transverse sections through the molecular layer obtains its typical striated appearance. When whole cat cerebella are incubated with the Lewis and Knight procedure, the AChE band pattern becomes visible at the cerebellar surface (fig. 47), because cholinesterase activity is absent from the pial membrane.

Fig. 47. Whole cat cerebellum incubated with the Lewis and Knight (1977) method, demonstrating the AChE band pattern. View from rostral on the anterior lobe.

102 . Enrico Marani

The Lewis and Knight method does not produce reaction product in the cerebellum of 2-3 days old kittens with the ultrastructural preservation techniques and the incubation times employed in this study. No explanation can be offered for this absence of reaction product formation in the Purkinje cells which are AChE positive in the light microscopy. All three techniques show reaction product formation in the AChE positive bands to be located in and around Purkinje cell dendrites in the lumen of the same subsurface cisternae (fig. 48). The subsurface cisternae have connections with the spine apparatus of Purkinje cell dendritic spines, and AChE reaction product is also found in the cisternal lumina of the spine apparatus. Reaction product is also present extracellularly, just at the outside of the Purkinje cell dendritic membrane. No interconnections of AChE reaction product in or outside the dendritic membrane are encountered during this study, and no indications for exo- or endocytosis of AChE, like AChE positive coated vesicles, are found. Within the parallel fibres, intracellular deposits of reaction product sometimes are present in relation to the microtubules (fig. 49). However, one is inclined to consider this location as an artifact, because false reaction product formation is known to occur on microtubuli, and structures normally negative for AChE and occurring within negative bands, such as the parallel fibres, sometimes contain positive microtubuli. Around parallel fibres, strong reaction product formation is found (fig. 49). The reaction product is located intercellularly and can surround several parallel fibres. At the synaptic contacts of parallel fibre boutons and Purkinje cell dendritic spines, AChE is never found in the intrasynaptic cleft (fig. 49). From light microscopy, it is clear that the basket cell protrusion structures are also found to be positive in the electronmicroscopy study (see MARAN! 1982 a, b). The location is an extracellular one, just at the basket cell membrane. No arguments are found in either light or electronmicroscopy holding that basket cell somata are alone positive for AChE. Within the lower part of the molecular layer, cells are encountered which are positive in their granular endoplasmatic reticulum. It is difficult to identify them unequivocally as stellate or as basket cells, because the fine structural characteristics deteriorate during incubation. The series shows positive endothelial cells in capillaries of the molecular layer. The ultrastructural distribution of AChE can be determined most accurately by a combination of the three techniques for this enzyme (MARAN! 1981). It has been established in previous studies that the band pattern in the kitten's molecular layer contains «true AChE». Although pseudocholinesterase activity seems to be absent here, high concentrations of the pseudocholinesterase inhibitor iso-OMPA were used in this investigation. AChE activity in the cerebellum of the cat is relatively insensitive to fixation. Incubation of 14-16 I-tm thick cryostate sections for 1 hour in 2.5% glutaraldehyde in 0.16 M cacodylate buffer at pH 7.2 or in 4% paraformaldehyde in the same buffer shows no decrease of enzyme activity compared to the untreated part of the same section (MA-

Topographic histochemistry of the cerebellum' 103

Fig. 48. Intracisternal AChE reaction product localization in Purkinje cell dendrite subsurface cisternae. Top: HANKER et al. (1973) incubation. Bottom: LEWIS and KNIGHT (1977) incubation.

104 . Enrico Marani

RANI 1981). Perfusion fixation, therefore, is compatible with enzyme histochemistry of AChE in the cat cerebellum. Although the precipitation of the reaction product is much lower with the LEWIS and KNIGHT (1977) techniques, compared to the KARNOVSKy-ROOTS (1964) and HANKER et al. (1973) methods, it allows a more accurate localization, due to a better preservation of the ultrastructure. In this case, the sensitivity to small quantities of enzyme «must be sacrificed to precision in localization» (GEREBTZOFF 1959; MAZZA et al. 1973). The criticism of KLINAR and BRZIN (1977 a, b) that the formation of the precipitate in the Lewis and Knight technique may be delayed has no bearing on our experiments, because the concentration of is sufficiently high. No precipitation of CUl2 ever takes place in our solutions. AChE reaction product formation within mitochondria (only observed in the Lewis and Knight incubation method) is considered an artifact (P. LEWIS, personal communication). The possibility exists that it represents a rest activity of pseudocholinesterase, because «pseudocholinesterase inhibitors do inhibit all other butyrylthiocholine enzyme locations, except reaction product in the mitochondria» (MAZZA et al. 1973). The cytochemically demonstrated distribution of AChE within various brain structures (rat striatum: KLINAR and BRZIN 1977 a, b; rat hippocampus: LEWIS and SHUTE 196; rat hypothalamus: CARSON et al. 1978; cerebellar cortex of fishes: CONTESTABILE et al. 1977; VILLANI et al. 1977) shows constant features: an intracellular localization of reaction product in the granular endoplasmatic reticulum of somata and their dendrites, and an extracellular localization around small-calibre nonmyelinated fibres (see also 2.2.1 and fig. 1). The localization of AChE in the cerebellar molecular layer differs from other regions of the brain by its presence in the subsurface cisterns and at the outside of the membrane of the Purkinje cell dendrites. Reaction product in the granular endoplasmatic reticulum is only detected in stellate or basket cell somata. The localization in Purkinje cell dendrites contributes to the discussion whether Purkinje cells in several species do contain this enzyme (SILVER 1974; CONTESTABILE et al. 1977; VILLANI et al. 1977; see also 3.6.1), because contradictory explanations on the cholinergic properties of Purkinje cells in cerebellum also arise from the absence or presence of AChE in cerebellar enzyme histochemistry. AChE in the molecular layer is present in and around structures which themselves are not supposed to be cholinergic or cholinoceptive. Purkinje cells and basket cells (see 3.1) use GABA as a neurotransmitter and receive parallel fibres which are now believed to contain an amino acid neurotransmitter (see WIKLUND 1982). Although direct effects of injected AChE on non-cholinergic or cholinoceptive cells have been demonstrated in the substantia nigra (GREENFIELD 1981), the functional significance of the transient presence of AChE in the molecular layer remains enigmatic. The presence of cholinesterase activity in capillaries in the outer third of the molecular layer may have a bearing on the hypothesis of KREUTZBERG (1969, 1973) that AChE secreted by neurons in the intercellular space is taken up by the vascular endothelium.

r

Topographic histochemistry of the cerebellum· 105

Fig. 49. AChE reaction product is localized around parallel fibres. Microtubulus reacts positive (arrow), but this is considered an artifact.

106 . Enrico Marani

Biochemical data do not confirm the disappearence of AChE from the molecular layer in cats at an age of 5-6 months in unfixated sections (AUSTIN and PHILLIS 1965; GOLDBERG and MCCAMAN 1967; MARAN!, unpublished). The presence of equal amounts of AChE activity in the granular and molecular layers of mature animals may be caused by impurities introduced by the manual dissection of the layers. On the other hand, it remains possible that the preference for acetylthiocholine in the histochemical procedure is abolished at maturity, because membrane structures protect or change the enzyme configuration. At this stage, a clear answer to these questions and the effect of fixation cannot be given. Results presented in this part support the conclusions on the location of AChE in the molecular layer from an earlier light microscopical study (MARAN! and VOOGD 1977) and supply information on the location of AChE around parallel fibres. The absence of AChE in light and electron microscopical studies within the Purkinje cell somata and its presence in and around Purkinje cell dendrites and parallel fibres, taken together with its overall distribution in positive and negative bands and its ultimate disappearance explains much of the confusion which prevails around the occurrence of this enzyme in the molecular layer. The localization in the molecular layer of AChE in the cat and of 5'-nucleotidase in the mouse have many features in common. Both are distributed in a pattern of longitudinal zones. Both are found inside the Purkinje cell dendritic tree and at the parallel fibres. However, AChE is found extracellularly, whereas 5' -nucleotidase is found intracellularly in the parallel fibres. Despite this difference, it can be concluded that both enzymes are localized at the transition of the parallel fibre and the Purkinje cell.

3.6.3 Description of the AChE pattern in the monkey cerebellar molecular layerS The AChE distribution was studied in several rhesus monkeys (HESS, SEDO and VOOGD, in prep.), and graphic reconstructions were made (fig. 50). The molecular layer of all lobules of the anterior lobe vermis contains symmetrically disposed AChE positive strips alternating with wide negative areas (fig. 50). The transverse sections demonstrate that the AChE activity is present in narrow striations of 1 or 2 Purkinje cells wide. They reach from the somata of the Purkinje cells to the pial surface (fig. 50). The somata themselves are not clearly positive for AChE. The neuropil of the granular layer is strongly positive, but the granular cells are negative. Within the white matter, positivity is found in these strongly fixated cerebella (see 3.2.3). The narrow strips of AChE positivity are well defined and sharply delineated. No subdivisions can be discerned within these small positive strips in the molecular layer. 5 In December 1984 JosE 5EDO died. This part of the study could only be written due to his work on the monkey cerebellum.

Topographic histochemistry of the cerebellum' 107

Fig. 50. A representative part of a transverse section through the anterior lobe of Saimiri (lower part). Small positive AChE strips can be recognized in the molecular layer (arrows). The upper part shows a reconstruction of the AChE positive bands in the anterior lobe. (This material was a gift from Dr. HESS.)

108 . Enrico Marani

The transition between positive and negative areas is always sharp on both sides of the AChE positive strips. These positive strips can be followed throughout successive lobules of the anterior lobe vermis and, therefore, can constitute longitudinal zones. In part 3.2.3 it is pointed out that the borders of compartments in the rhesus monkey can also be recognized with AChE positivity (see als SEDO, HESS and VOOGD 1984). Within transverse sections the AChE positivity of the raphes can be followed through the granular layer into the molecular layer and compared to climbing fibre projections in the molecular layer after 3H-Ieucine injections into the inferior olivary region. Thus, borders of marked compartments (see 3.2.3) can directly be transformed to AChE positive zones in the molecular layer and be confirmed in other autoradiographic climbing fibre series (SEDO, HESS and VOOGD 1984). The anterior lobe vermis of lobule I to III contains midsagittally a positive band in the molecular layer. On every side of the midsagittal band, zones can be found in the molecular layer determining the A-B, the B-C1 and the C1-C2 border. In lobules III to V an extra positive band seems to be present between A and B, presumably the X zone, although until now no experimental evidence is present for such a supposition. This X zone seems to be continuous with the AChE positive fibre strip in between the compartments A and B (fig. 19). In the posterior lobe vermis, the bands have the same appearance as in "the anterior lobe. The midsagittal band reaches to the pial surface. However, more laterally situated bands do not arrive at the upper part of the molecular layer. Within the molecular layer, four positive bands can be discerned on each side of the midsagittal one, separating five negative areas. The second lateral positive strip is indistinct. The negative area between the third and the fourth positive band is small. The Purkinje cell bodies are negative for AChE. In the depth of the fissures between lobule VIII and IX, these bands are clear. In lobule VIII, the positive strips form the borders between A1-A2 and presumably A2-A3 or Cl. The more lateral areas are difficult to denominate. The lobules VI, VII, and X contain also positive bands. The midsagittal zone can be followed into all lobules of the posterior lobe. The more laterally placed bands are difficult to discern and are less clear to follow into lobules. Within the hemispheres of the monkey, positive strips are present. However, these strips cannot be followed through one lobule to another and, therefore, constitute no longitudinal bands. On the basis of our light microscopic histochemical results, the AChE activity in the molecular layer of the rhesus monkey has no known structural basis in the molecular layer. Based on our ultrastructural results in the cat and the light microscopic appearance of these strips, a Purkinje cell dendritic localization is suggested. Whether parallel fibres are also involved is questionable. However, in cat and guinea pig (KASA, Joo and CSILLIK 1965) cholinesterase activity is bound to parallel fibres too (see 3.6.2). The number of bands and also the absence of bands in the hemispherical parts

Topographic histochemistry of the cerebellum . 109

correspond in cat and monkey. The thickness of the positive zones clearly differs. Within the hemispheres in the cat, some negative strips can be discerned. Clear positive strips are present in the hemispheres of the monkey. In both species these strips are difficult to follow through one lobule. After lesions of the inferior olivary complex in cats, positive areas stand out more precisely in the lateral vermis, but also in the hemispheres (BROWN 1985; see chapter 5).

4 Development of the cerebellum 4.1 Histology of the cerebellar development In any given neuroanatomical or electrophysiological experiment, only parts of the cerebellar modules (see 3.1) can be demonstrated. Histochemical methods, on the other hand, show the cortical longitudinal strips along the whole rostro-caudal extent of the cerebellum (see 3.5 and 3.6). The adult pattern of connectivity of the longitudinal zones has been worked out, but a coherent picture of the developmental parameters, needed to produce such a complexly interrelating topographical pattern, is missing. Questions such as: - is there an orderly relationship between sequences of neurogenesis in the specific subdivisions of a module and their ultimate connectivity? - is cell death a factor in the formation of these topographic patterns? - is there a simple ancestral pattern present which is responsible for the mature pattern? - is the topographic pattern of the connections a consequence of the timing of arrival ofaxons? cannot be answered at present. Our lack of knowledge of these issues is probably due to the relative ignorance of matters such as chemoaffinity and chemoheterogeneity, mechanical guidance of glial structures, and the differences in the topography of cell mitosis (FEIRABEND et a1. 1978) in studies of the mammalian cerebellum. In the past the morphological studies of cerebellar ontogenesis were focussed on the development of the fissures and lobules. Most investigators arrived at the assumption of a principally transverse division of the mammalian and bird cerebellum (for a review see JANSEN and LARSELL 1970). Such a subdivision can be accounted for in the anterior lobe, but in the adult cerebellum fissures within the posterior lobe are seldom continuous (BOLK 1906; VOOGD 1975; MARANI and VOOGD 1979). The independence of vermis and hemispheres in the posterior lobe was stressed by BOLK (1906; see also VOOGD 1975). The arguments for independent longitudinal growth centers, developing into the folial chains of vermis and hemispheres, which were postulated by BOLK (1906), were not replicated in t;he literature. Therefore, BOLK'S ideas are seldom recognized in modern literature (see VOOGD 1964, 1975).

110

Enrico Marani

The ontogenesis of the cerebellum takes place in the rhombencephalon within the rhombic lip (HIS 1890). Several discussions can be found in literature, concerning: - the presence of a rhombic lip and its subdivisions (HOCHSTETIER 1929), - the origin of the cerebellum from two bilateral growth centers in the rhombencephalon (STREETER 1903; ARIENS !\.APPERS 1921), - the presence of segmental and longitudinal neurogenetic cell columns (RUDEBERG 1961) within the cerebellar anlage. In more recent publications using 3H-thymidine (ALTMAN 1972; FEIRABEND and VOOGD 1979; FEIRABEND et al. 1978; FUJITA 1966; MIALE and SIDMAN 1961), the neuroblast layers were found to be divided into inner and outer layers, separated by a marginal layer. The inner neuroblast (inner mantle) layer gives rise to the Purkinje cells and perhaps to Golgi cells. The outer cerebellar mantle layer produces the external granular layer and the deep cerebellar nuclei (FEIRABEND 1983).

4.1.1 Application of human blood monoclonals directed against FAL (Fucosyl-N -acetyl-Iactosamine) Monoclonal antibodies, raised against human monocytes or granulocytes, are capable of recognizing antigens in various parts of the central nervous system of mammals or on neuroblastoma cells (DALCHAU et al. 1980; HOGG et al. 1981; KEMSHEAD et al. 1981; MARANI and TETTEROO 1983 a; MARANI et al. 1983 b), while monoclonal antibodies against human T-Iymphocytes have been shown to specifically label cerebellar Purkinje cells of many species (GARSON et al. 1982). Series of monoclonal antibodies, raised against blood cells, are now available in most departments of haematology in different countries. This makes it possible to use these monoclonal antibodies for research in neuroscience (GARSON et al. 1982; MARANI and TETIEROO 1983 a; MARANI et al. 1983 b). This chapter summarizes our results, with the intention that it will invite other investigators to use these monoclonals. Therefore, the antibodies are listed by name with their place of origin, their synonyms, and with an indication where they react under our test conditions (tables 9 and 10). None of the monoclonal antibodies reacted with the human cerebellum or brain stem. A similar lack of reactivity was observed in the domestic chicken, guinea pig, and common marmoset. The monoclonal antibodies that did react in the rat, rabbit and rhesus monkey are summarized in table 11. In the rhesus monkey, antibodies B37.4 and VIMD5 reacted with Purkinje cells and a certain type of cell in the granular layer (Golgi cells ?). The cells of the cerebellar nuclei reacted with these antibodies, as did some unidentified structures in the brain stem. In the rat, monoclonal antibody B 4.3 was found to react with cells in the neural tube. Nonspecific reactions were found with the ectoderm of young embryos using the PAP technique. In rats, 3-4 weeks postnatally, the antibodies UJ308, VIMD5, and M11 N1 reacted in the cerebellar molecular layer. B2.12 antibody was bound in the ependy-

Topographic histochemistry of the cerebellum . 111 Table 9. Nineteen monoclonal antibodies (McAb), their isotope and specificity, the molecular weight of the glycoprotein carrier molecules (antigen molecular weight), the chromosomal localization of the gene(s) involved in the expression of the antigens recognized and the type of antigen or the protein determined of the monoclonal antibody. McAb

Synonym

Ig class

Antigen

Specificity (human tissues)

Mol Weight* Human chr.

Gift from

B4.3 BI3.9 B2.12 MI/NI UJ308 VIMD5 FMCIO FMC11 FMCI2 FMC13 55.7 L13.1 54.7 L5.11 L12.2 53.12 B44.1 B48.4 B37.4

CLB gr/2 CLB gr/I CLB Mrg/2

IgM IgG IgM IgM IgM IgM IgG IgM IgM IgG IgM IgM IgM OgG

FAL

gran.lneurobl. gran. gran.lmono/T cells gran.lneurobl. gran.lneurobl. gran.lneurobl. gran.lneurobl. gran. gran. gran. gran.lT cells gran. mon.lgran. proliferating cells gran.lmelano gran.lT cells

ISO/IDS 92K 120/170 ISO/IDS ISO/IDS 150/105 ISO/IDS 65 K ISO/IDS 150/105 20 K

P. LANDSDORP P. LANDSDORP P. LANDSDORP J. KEMSHEAD J. KEMSHEAD W. KNAPP H.ZoLA H.ZOLA H.ZoLA H.ZoLA G. ROVERA G. ROVERA G. ROVERA G. ROVERA G. ROVERA G. ROVERA B. PERUSSIA B. PERUSSIA B. PERUSSIA

Sugar FAL Sugar Sugar Sugar Glycolipid Sugar Transferin recep.

IgM IgM IgM IgM

ISO 87 110 29

K K K K

K

II

K K K K K

11 11 II II

K K 11 3

mono

Sugar

gran.lmono gran.

11

':. Molecular weight of antigens detected by B4.3, Ml/Nl, Uj308, VIMDS, FMC10 are determined by TEITEROO and VISSER, antigens detected by 54.7.13 determined by ROVERA. FAL: Gal-(~ 1.4)-GlcNAc - -

I

a (1-3)

I

L-Fuc

Table 10. Species, which are screened for granulocyte monoclonal antibodies. 0 screened, • reactive for one or more monoclonal antibodies. CNS mature fetal Birds Fowl Rodentia Rat Cavia Rabbit Monkeys Macaca rho Marmoset Man

o

•o o

•o o

Head mature fetal

Other organs mature fetal

o

•o •

o

•

• •

112 . Enrico Marani

Fig. 51. A: demonstrates FAL positivity in the submandibular gland of the rabbit. In (B) reactivity for B2.12 is shown in the ependyma of the third ventricle of the rat. C: overall view of a part of a lobule of the cerebellum. D: Shows a detail; note the absence of FITC fluorescence in the stellate cells and Purkinje cell somata (22 days old rat).

Topographic histochemistry of the cerebellum' 113

rna of the third ventricle of the rat. Moreover B4.3, B37.4, VIMD5, and MlINl reacted in the mature rat hippocampus with the neuropil around pyramidal cells. The results in the rabbit central nervous system showed that from day 20 after conception (a. c.) till day 5-10 after birth (a. b.) monoclonal antibodies B 4.3, VIMD5, MlIN1, and B37.4 reacted in the external granular layer of the cerebellum (see also MARAN! and TETTERoo 1983 a). B 4.3 was also found to react in several brain stem areas and in the innervated areas in the developing internal ear. Among the extraneuronal tissues screened for these monoclonal antibodies, the salivatory glands and their ducts reacted positively for B4.3, 54.7, and MlINl (fig. 51). 50me reactivity for B4.3 was also found at the base of rabbit whiskers. By now, the availability and characterization of human granulocyte monoclonal antibodies and the positive results obtained with human neuroblastoma cell lines would be sufficiently well established so that it seems justified to use them for screening in the central nervous system. Most of the monoclonal antibodies which gave positive results in our study (B4.3, MlIN1, UJ308, VIMDS, B37.4, 54.7) recognize coupled monosaccharides. For two of these antibodies the antigen (see table 9) has been identified as the trisaccharide 3-fucosyl-N-acetyl-lactosamine (FAL) (TETTERoo et al. 1984). The sequence of monosaccharides, rather than the position of a single residue, determines the specificity (CLICK and 5ANTER 1982; RAUVALA 1983). Monoclonal antibodies, raised against neuroblastoma-, granulocyte-, monocyte-, and T lymphocyte cell membranes, recognize these particular sequences of monosaccharides (DALCHAU et al. 1980; GARSON et al. 1982; HOGG et al. 1981; KEMSHEAD et al. 1981; KEMSHEAD, personal comTable 11. Areas in the eNS of rat, rabbit and Macaca rhesus, which react for granulocyte monoclonal antibodies (McAb). Some other tissues are reported for the rabbit in the table too.a. c.: after conception; a. b.: after birth.

Rat

Rabbit

Rhesus Monkey

Age

McAb

Tissue

12 days a. c. 22 day a. b. mature mature

B4.3 Uj308, VIMD5, Ml/Nl B4.3, B37.3, VIMD5, Ml/Nl B2.12

neural tube cerebellum, molecular layer hippocampus ependyma III ventricle

20 days a. c. till 5-10 days a. b. 26 days a. c. 26 days a. c.

B4.3, VIMD5, Ml/Nl B37.4 B4.3, S4.7, Ml/Nl B4.3 B4.3

external granular layer

mature

B37.4, VIMD5

mature

B37,4

submand.gland stomach (?) hair follicle cerebellum, Purkinje cell layer and granular layer brain stem

114 . Enrico Marani

munication). These sequences of monosaccharides may well play an important role in the differentiation of the central nervous system, too (GARSON et al. 1982; KEMSHEAD et al. 1981; MARAN! and TETTERoo 1983 a; MARAN! et al. 1983 b; MONMOI and YOKOTA 1981 ). The carbohydrate composition of certain glycoproteins on the cell surface changes during differentiation and is the normal expression of the genome in response to internal or external stimuli during differentiation. Moreover, the carbohydrate composition of a glycoprotein, its position, and its anomeric linkage to other monosaccharides gives the cell biological specificity (CLICK and SANTER 1982; RAUVALA 1983).

4.2 Topography of FAL monoclonal antibodies in the developing rabbit cerebellum The monoclonal antibodies against FAL have been shown to specifically label neuronal structures (see 4.1). This chapter will report the results with B4.3 monoclonal antibody in the immature rabbit cerebellum. The B4.3 monoclonal antibody reacts with 105.000-150.000 dalton proteins with the sugar determinant fucosyl-N-acetyllactosamine. The B4.3 monoclonal antibody is detected in the immature rabbit cerebellum in a longitudinally oriented band pattern of alternating strips that are positive and negative for this antibody (MARAN! and TETTERoo 1983 a). Moreover, its ultrastructurallocalization has been studied in cell suspensions of the immature rabbit cerebellum (MARAN! et al. 1983). 1!-tm sections of the cell suspensions show cells with and without peroxidase reaction product at their periphery. In contrasted and uncontrasted sections these FAL-positive cells (PAP technique, see MARAN! et al. 1983 b), can be recognized as granular cells. The ultrastructural study demonstrates exclusive localization of the reaction product on plasma membranes of external granular cells (MARAN! et al. 1983 b). No cytoplasmic localization can be demonstrated in either damaged or undamaged cells. However, positive cells show non-specific DAB localizations in the mitochondria of damaged cells. This is due to the reduction of DAB by the enzyme systems present in the cells which are still unfixated at the time of the antibody treatment. The reaction product is located immediately above the cell membrane surface, indicating a glycocalyx localization. Extensive rinsing procedures with buffer, fixative, or alcohol remove the reaction product at the cell membrane and cause non-specific localization in the embedding medium of the cell suspensions (MARAN! et al. 1983 b). 4.2.1 The longitudinal FAL pattern in the immature rabbit cerebellum Between day 18-19 (a. c.) and day 15-16 (a.b.), FAL positivity is present in a longitudinal band pattern. The FAL positivity extends from the external granular layer

Topographic histochemistry of the cerebellum· 115

into the upper part of the Purkinje cell layer before birth. Around birth strong positivity is noticed in the upper part of the Purkinje cell layer. However, after birth the FAL positivity retracts up to higher parts of the external granular layer. The granule cell raphes are negative for FAL, although the positivity of the external granular layer above the raphes protrudes within the top of them. After day 16 a.b. the antibody reaction diminishes in the rabbit cerebellum. The antigen FAL, detected by the monoclonal antibody B4.3, as studied in several series from day 20 a.c. till day 26 a.c., is organized in a longitudinal pattern. The pattern in this period of cerebellar development restricts itself clearly to the posterior lobe, and only few short strips are present in the anterior lobe. The distinction between posterior and anterior lobe is possible, because the primary fissure is already present. In the posterior lobe the paramedian sulcus can be recognized as a deep sulcus still containing an external granular layer. The midsagittal area of the vermis contains a wide strip of FAL positivity, which runs from the most caudal end up to the primary fissure. This positive strip does not extend over the primary fissure into the anterior lobe vermis. Within each paramedian sulcus a longitudinal zone of FAL positivity is present that only runs towards the primary fissure but stops halfway into the paramedian sulcus. Whether this is the area of the intercrural sulcus, is unclear. In the hemispherical part of the posterior lobe, one FAL positive band is present at each side of the paramedian sulcus. These zones traverse somewhat the primary fissure and are present only in the caudal aspects of the anterior lobe. In the rostral aspect of the anterior lobe, two distinct, but short FAL positive strips can be recognized at the most lateral margin of the anterior lobe. All FAL positive zones are separated from each other by a negative area of the external granular layer. The midline band contains no negative strip in the midsagittal line. None of the bands is continuous over the whole rabbit cerebellum in these stages. The positivity seems not to continue into the primary fissure, indicating that most strips are restricted either to the posterior or anterior lobe. In older stages, until after birth, the FAL positivity remains present in a longitudinal pattern. The anterior lobe contains one broad midline positive band, separated by negative strips. These negative strips are bordered by a positive band, which is present in the hemispherical part of the anterior lobe. The patterns within the posterior lobe, paraflocculus, and flocculus are too intricate to be described here in short.

4.2.2 The spinocerebellar input in the immature rabbit cerebellum The spinocerebellar systems are present early in the development of the rabbit cerebellum. After injections of WGA-HRP into the spinal cord of rabbit foetuses, ranging in age from day 22 a.c. to one week after birth, spinocerebellar fibres can be traced into the rabbit cerebellum. At day 22 a.c. the fibres already cross over into the cerebellar commissure and are present just below the ?urkinje cell clusters. Later in the development the incoming fibre systems arrange them-

116 . Enrico Marani

selves in a longitudinal pattern in the internal granular layer (LAKKE and VAN KEULEN, in prep). However a direct relation of the FAL positivity pattern and the early incoming spinocerebellar fibre system is unclear, although the spinocerebellar systems end upon the internal granular. cells, which are derived from the external granular layer. Other systems, that can exist early in the development of the cerebellum, are the tecto-cerebellar, trigemino-cerebellar, and presumably the climbing fibres.

4.3 Topography of FAL monoclonal antibodies .in the mature mouse cerebellum The mouse cerebellum contains several glycoproteins. In fact, the enzyme 5'-nucleotidase is considered to be a glycoprotein (see 3.4 and 3.5). Glycoproteins containing glucose and mannose can be detected with concanavalin A in the subsurface cisternae of rat Purkinje cell dendrites (WOOD et al. 1974). Carbohydrate antigen systems have stage specific expression in the early embryos (GOOI et al. 1981). Antibodies against FAL detect one of these stage specific embryonic antigens. Expression of this FAL antigen has been shown for a certain developmental stage (see 4.2). However, FAL monoclonal antibodies also detect the antigen in mature organs (see 4.1.1). Within the mature mouse cerebellum, FAL positivity can be demonstrated in the molecular layer in a longitudinal pattern. After perfusion fixation, FAL positivity is restricted to the Purkinje cell layer and molecular layer. FAL antigens are determined around the Purkinje cells in the baskets and in fibre systems in the granular layer. The Purkinje cell somata are negative. The FAL positivity constitutes a longitudinal pattern of positive and less positive bands. These bands can be followed throughout several lobules of the posterior and anterior lobe. The topography of the FAL pattern coincides with the topography of the 5'nucleotidase pattern (see 3.5). The FAL bands stand out distinctly, and the borders are sharply delineated, while in the positive areas striations can be noticed. Fucosyl-N-acetyl-Iactosamine is also known as X-hapten, Lacto-N-fucopentose III, or S.S.E.A-1. It is present in sphingolipids (URDAL et al. 1983) and glycoproteins (URDAL et al. 1983; TETTERoo et al. 1984, and in press). Recently, other monoclonal antibodies showing a longitudinal band pattern in the rat molecular layer have been found. The antigen of these patterns is unknown. Since the topography of 5'-nucleotidase and FAL in the mouse molecular layer is identical and 5'-nucleotidase is a glycoprotein, one is inclined to consider FAL to be a part of this enzyme. However, other glycoproteins of non-enzymatic origin could also be responsible for the FAL pattern. Ultrastructural and biochemical research is needed to solve this problem.

Topographic histochemistry of the cerebellum' 117

4.4 Topography of acetylcholinesterase in the developing rabbit and cat cerebellum (B. BROWN, A. EpEMA, and E. MARANI, Emory University, Atlanta and Leiden University) During cerebellar development, a temporary arrangement of Purkinje cells in a number of parasagittally oriented clusters (Purkinje cell clusters of theiif§t ord~r) can be found (VAN VALKENBURG 1913; WINKLER 1926; HAYASHI 1924;]AKOB 1928; KApPEL, 1980; KORNELIUSSEN 1968a, b; FEIRABEND et al. 1976; FEIRABEND and VOOGD 1977, 1979; MAAT 1978). These clusters are situated below the cell poor marginal layer within the inner neuroblast layer. In early stages of development a limited inward migration of small «granule cell raphes» out of the external germinal layer subdivides these Purkinje cell clusters of the first order into smaller second order Purkinje cell clusters. In later stages of the development a massive inward migration of external germinal layer cells accompanies the spreading of the second order Purkinje cell clusters to form a uniformly distributed monolayer (FEIRABEND 1983). Based on experimental data, the fetal longitudinal subdivision of the Purkinje cell neuroblasts and the mature cerebellar subdivision are expected to be related. Therefore, the development of the longitudinal zones was studied with histochemical methods, because the mature histochemical patterns were found to originate from fetal histochemical patterns (BROWN and GRAYBIEL 1983 a; MARANI et al. 1983a, b; MARANI and TETTEROO 1983 a). The aim of this part is to give an overall view of the changes in the AChE pattern which occur during the development of the rabbit and cat cerebellum. The description of these changes in the AChE staining pattern in the developing cerebellum is a time consuming job (VOOGD et al. 1986). Therefore, a brief description of the occurring histological changes will be given first (for prelimenary descriptions see BROWN 1985; MARANI et al. 1983 a; MARANI and TETTEROO 1983 a), as the basis for a later, more detailed description of the topographical relations of AChE staining to several Purkinje cell clusters and the deep cerebellar nuclei. This will necessarily be a general description of a number of different stages, without claiming to cover all the details of the subsequent changes in the zonal localization of the AChE reaction product. Nissl, hematoxylin, and additional hematoxylin-eosin stained series demonstrate characteristic developmental stages for rabbit and cat cerebellum which are similar to those described by FEIRABEND (1983) for chicken. In rabbit cerebella 18-20 days after conception (a.c.), first order Purkinje cell clusters are evident. The main subdivisions of the Purkinje cell clusters in rabbits are similar to the subdivisions proposed in monkeys (KAPPEL 1980). These first order Purkinje cell clusters are then subdivided into smaller, more numerous second order Purkinje cell clusters at the time of appearance of the granule cell raphes. In mammals a total of 8 to 9 second order Purkinje cell clusters are postulated by FEIRABEND (1983),based on a comparison of his results with the description of KORNELIUSSEN (1968a, b) which was also confirmed by KAPPEL (1980). During the formation of the Purkinje cell monolayer, the raphes disappear and are replaced by a massive overall inward migration of the cells of the external granular layer (fig. 52). After this time, no further subdivision of Purkinje cell groups is possible, based on strictly cytoarchitectural criteria.

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/l\RO M

MASSI VE I

/l\RO MIGRATION

Q.USTER1P-K; I HE PURKI NJE CELl LAYER

Fig. 52. Diagram showing the successive histogenetic events in the primitive rabbit cerebellar cortex. For explanation see text. (Courtesey of Dr. FEIRABEND, adapted for the rabbit).

The reconstruction of several series yields convincing evidence that AChE is localized in bands of alternating positive and less positive areas, forming a longitudinal pattern, both in the rabbit and cat cerebellum (BROWN and GRAYBIEL 1983; MARANI and FEIRABEND 1983). This longitudinal organization can be demonstrated both preand postnatally. The determination of true AChE or pseudo-cholinesterase, as present in the rabbit cerebellum, was carried out only on sections of newborn or mature rabbits. The combination of substrates and inhibitors (see MARANI, VOOGD and BOEKEE 1977) shows true AChE to be involved in the rabbit immature cerebellum. The two different types of incubation reactions (LEWIS and KNIGHT 1975; KARNOVSKY and ROOTS 1964) most frequently used in this study produce the same pattern of AChE staining. In several series the two reactions were performed on adjacent sections. Due to the lower pH used in the LEWIS and KNIGHT incubation (MARAN I 1982 b), a longer incubation time is needed for this type of reaction

Description of cellular and cluster localization of AChE in the developing rabbit Acetylcholinesterase activity arises between day 17-20 a.c. In unfixated counterstained sections AChE reaction product is found on day 20 a.c. restricted to Purkinje cell clusters. Slight AChE activity in longitudinally oriented positive and negative alternating areas is seen around the Purkinje cells. Several patches containing AChE (in total four at each side of the midline) are present. In the midline two AChE positive patches fuse ventrally. At day 23 a fifth patch of AChE positivity is noted just above the brachium pontis. The AChE reaction product extends to the deep cerebellar nuclei which can be recognized at day 20 a.c. up to day 24 a.c. These strips of AChE

Topographic histochemistry of the cerebellum' 119

positivity pass mainly medially to the Purkinje cell clusters towards the deep cerebellar nuclear area. In the midline, however, these fan-like strips do not fuse, as the AChE positivity in the Purkinje cell clusters just above them does. The developing marginal layer at this age is negative for AChE, as the emergent external granular layer lacks AChE reaction product. In contrast, the deep cerebellar nuclei do contain some reaction product within their cells. At day 24 a.c., a distinct marginal layer is present and contains patches of AChE activity. These patches form longitudinal strips and correspond with reaction product localization in the Purkinje cell clusters, where AChE is found around Purkinje cells. The strips of AChE positivity, extending into the deep nuclei are faint at this age (fig. 53). The correspondence of AChE activity, both in marginal layer and Purkinje cell clusters, is present up to day 26 a.c. The AChE pattern constitutes a clear longitudinal distribution within the vermal area of the marginal layer. At this age, the Purkinje cells start to spread out, while the formation of granule cell raphes can be noticed. AChE activity is now confined to both Purkinje cell layer and marginal layer. From day 26 a.c. on, the evolution of the AChE pattern is different in time sequence within different parts of the cerebellum, with the anterior lobe leading the other regions in terms of the timing of evolution of the AChE staining pattern. Despite the differences in timing, all regions of the cerebellar cortex ultimately appear to undergo the same cytological and topographical differentiations. However, the final mature expression of remaining AChE activity is different in the various parts of the rabbit cerebellum.

The AChE pattern in the rabbit anterior lobe At day 28 a.c., the AChE activity is present at the Purkinje cell layer, around the Purkinje cells, and in Purkinje cell dendrites, thus producing a positive marginal layer. The longitudinal band pattern is still present, and areas strongly positive, weakly positive, and negative for AChE activity can be discerned from now on. Just after birth (0-1 day a.b.), AChE activity is present both in somata and dendrites of Purkinje cells. AChE positivity is now found in the internal granular layer, too. Five days -after birth, the AChE reactivity disappears from the somata. AChE activity within the internal granular layer remains high until 6 weeks a.b., with its peak at day 7 a.b. At maturity, parts of the granular layer of the rabbit cerebellum contain high AChE activity, as described for the rat in chapter 3.1.1. Within the rabbit molecular layer, some clumps of cells remain positive for AChE. These are Golgi cells surrounded by some granular cells (SPACEK et al. 1973). Using the Gomori technique, the area of the whole molecular layer above the Purkinje cells is slightly positive.

The AChE pattern in the rabbit posterior lobe vermis The AChE positivity is present in the marginal layer at day 28 a.c. The AChE positivity around Purkinje cell somata is evident at day 0-1 a.b. in Purkinje cell den-

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tIg. 53. A - E: AChE positivity in Purkinje cell clusters of 21 day old fetal rabbit cerebellum. F: Banding in a 1 day (a.b.) old rabbit cerebellum. G: Reconstruction of the longitudinal pattern of acetylcholinesterase in the developing rabbit (1 day) cerebellum.

Topographic histochemistry of the cerebellum' 121

drites and somata until day 2-3 a.b. The granular layer starts to be positive at day 0-1 a.b. and exhibits a strong AChE activity until one week after birth. At maturity, strong AChE activity in the internal granular layer is present, as described for the mature rat in chapter 3.1.1. The AChE activity is confined to mossy fibre endings, while the longitudinal pattern described for the rat is also present for the rabbit.

The AChE pattern in the rabbit hemispheres Within the hemispheral parts of the rabbit cerebellum at 28 days a. c., AChE activity is still found in Purkinje cell clusters of the second order around the cell perikarya. The marginal layer is strongly positive in some parts. Strongly and weakly positive as well as negative areas of AChE staining can be noted. The alternation of strongly and weakly AChE positive regions in the marginal layer constitutes a longitudinal pattern, while the negative areas belong to cortexless areas (MARANI and VOOGD 1979). This longitudinal pattern is evident in the marginal layer up to 2-3 days after birth. From then on, the same sequence of displacement of AChE positivity into the granular layer begins, ending with a nearly AChE negative internal granular layer in the hemispheres one week after birth. Paraflocculus and flocculus are even later in this development, and there the developmental sequence ends at 6 weeks after birth. The mature pattern for AChE in flocculus and paraflocculus is identical to the AChE localization described in the rat granular layer (chapter 3.1.1).

Distribution of AChE in the developing cat cerebellum Both developing rabbit and cat cerebellum show a distribution of AChE in longitudinally oriented bands which appear to be forerunners of the adult zonal pattern. However, the developmental changes in the AChE staining pattern differ somewhat for the two species. This probably reflects differences in the length of gestation for the two species (approximately 30 days for the rabbit, and 65 days for the cat), as well as differences in the developmental end point that is reached; that is, the mature AChE staining pattern. In mature rabbit the molecular layer is negative for AChE, as is most of the granule cell layer. There exists patchy staining of the posterior lobe vermis, apparently corresponding to the localization of mossy fibre endings, which displays a longitudinal pattern. In mature cat, on the other hand, most of the molecular layer is stained positively for AChE, although the staining is not uniform. The presence of areas which are positive, less positive, and negative for AChE creates a pattern of longitudinally oriented strips in the molecular layer (BROWN and GRAYBIEL 1983 b; MARANI and VOOGD 1977). Combined tract-tracing and AChE-histochemical studies have yielded evidence that the boundaries of these AChE positive areas mark the boundaries of VOOGD'S (1964, 1969; VOOGD and BIGARE 1980) A and B corticonuclear and olivocerebellar zones (BIGARE and VOOGD 1977; BROWN and GRAYBIEL 1983b; MA-

122 . Enrico Marani

RANI and VOOGD 1977). The granule cell layer of the mature cat, however, is more or less uniformly positive for AChE. In developing cat cerebellum, AChE activity is first evident at around 24 days a.c. At this time Purkinje cells are still undergoing neurogenesis in the ventricular proliferative zone (BROWN 1985). Patchy AChE activity is present superficially in the cerebellar primordium but not in the ventricular zone. Thus, it would appear that, as Purkinje cells complete their final cell division and migrate toward their ultimate positions in the cerebellar cortex, they migrate toward areas which are already differentiated in terms of their AChE activity. During the next two weeks, the localization of AChE positive areas is largely superficial, with respect to the localization of the primitive Purkinje cell clusters (fig. 54). However, around 37-38 days a.c. the localization of AChE staining appears to shift relative to the developing Purkinje cell clusters, so that some ~reas of AChE positivity now lie deeper than the Purkinje cells. In cell-stained sections, it can be seen that the developing Purkinje cells are already segregated into discontinuous plates or clusters which form longitudinally oriented strips. Superficial and deep with respect to the developing Purkinje cell plates lie AChE positive and negative areas, which also form longitudinally oriented strips. At this stage, the laminar distribution of AChE positive areas and developing Purkinje cell clusters is almost entirely complementary. Most importantly, however, edges or discontinuities in the AChE staining pattern correspond to the location of gaps between the developing Purkinje cell clusters. The deep cerebellar nuclei, which are realtively well-developed compared to the cortex, also contain areas of AChE positivity (fig. 54). At 46-47 days a.c., the laminar complementarity of the Purkinje cell clusters and AChE staining is still fairly striking, but there is some overlapping of AChE positive areas and areas containing Purkinje cells, particularly near the midline around the fissura prima. In cell-stained sections it can be observed that the gaps between Purkinje cell clusters are starting to disappear. However, longitudinal zones are still distinctly evident in sections stained for demonstration of AChE. At this stage (and continuing up to the week before birth), the midline of the anterior lobe is marked by two AChE positive strips, one on either side of the actual midline. The development of the caudoventral and lateral portions of the cerebellar cortex seems to lag behind that of the vermis of the anterior lobe; so while the Purkinje cell clusters are merging in the anterior lobe vermis, they remain more distinct in the hemispheres, the lateral parts of the anterior lobe, and the posterior lobe vermis. The caudal and hemispheral parts of the cerebellar cortex also lag behind in terms of development of AChE positivity, with these regions staining less intensely for AChE than parts of the anterior lobe (fig. 54). By 50 days a.c., there is almost no indication of zonal boundaries in medial parts of cerebellar cortex in the cell-stained sections. Purkinje cells in the medial parts of the anterior lobe have spread out and almost form a monolayer. In other parts of the developing cortex, however, they remain in multi-layered clusters. In some areas of

Topographic histochemistry of the cerebellum' 123

Ie

Fig. 54. Adjacent sections of the developing cat cerebellum, stained with cresyl violet (left side) and for demonstration of AChE (right side). A: Parasagittal sections through the cerebellar primordium of a fetus, 33 days a.c. B: Frontal sections through the cerebellum of a fetus, 38 days a.c. C: Frontal sections through the cerebellum of a fetus, 47 days a.c. - For abbreviations see fig. 55.

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cerebellar cortex, cellular «raphes» are evident. These appear to correspond to the granule cell raphes described by FElRABEND (1983). In adjacent sections stained for AChE it can be seen that the location of these raphes lines up rather consistently with edges of AChE positive zones or gaps in the AChE staining pattern. The AChE staining pattern continues to provide a distinct indication of zonal subdivision of the cerebellar cortex. The laminar complementarity between areas of AChE positivity and the Purkinje cell layer has been lost along the midline, and in these areas AChE positivity is evident around Purkinje cell somata. The entire cerebellum, except for the ventral flocculus, now shows some evidence of AChE positivity, although the vermal areas are more intensely stained than the lateral parts of the cerebellum (fig. 55). At 56-59 days a.c. the Purkinje cells have spread out into a monolayer over a larger part of the cerebellar cortex. Cellular raphes are prominent in medial regions of cerebellar cortex, and in cell-stained sections these provide the main remaining indication of a zonal subdivision of the medial parts of the cortex. Some Purkinje cell clusters are still evident in the developing hemispheres. In sections stained for AChE, a very intense staining of Purkinje cell bodies and dendrites is now evident in parts of the vermis and paramedian lobule. In the vermis the AChE staining is fairly uniform across the mediolateral extent of the cortex. However, gaps in the AChE staining continue to provide some indication of a zonal subdivision of the cerebellar cortex. Comparison of cell-stained sections with adjacent sections processed for AChE, reveals that the gaps in the AChE staining pattern line up rather consistently with locations of cellular raphes. All areas of cerebellar cortex, including the flocculus, now contain AChE positivity. The two midline bands fuse to form a single band at about this time (fig. 55). During the first postnatal week, cell-stained sections give very little indication of any zonal organization of the cerebellar cortex, except for faint remnants of cellular raphes in parts of the cortex. Rather, the cerebellar cortex appears to be more or less histologically uniform throughout, as in the adult. The external granular layer is still present superficially over all the parts of the cerebellar cortex. The entire cerebellar cortex now exhibits strong AChE positivity. In most regions, Purkinje cell bodies and dendrites are darkly stained. The overall intensity of AChE staining makes it difficult to discern any zonal organization, and minor gaps in AChE staining constitute the clearest indicator of cortical zonal organization. By the end of the second postnatal week, cell-stained sections show essentially an adult pattern, except that the external granular layer is still present. Sections stained for AChE show that this is a time of transition toward the adult pattern of AChE staining. The (internal) granular layer, which has been more or less negative up to now, begins to show AChE positivity in the ventral parts of the vermis and the flocculus. In these same areas, Purkinje cell bodies are now negative for AChE, while the dendrites remain positive. The increase in positivity of the (internal) granule cell layer generally proceeds from ventral parts of the cerebellum to dorsal parts and from medial to lateral,

Topographic histochemistry of the cerebellum' 125

Fig. 55. Adjacent sections of fetal cat cerebellum and kitten cerebellum stained with cresyl violet (left side) and for demonstration of AChE (right side and bottom). A: Frontal sections through the cerebellum of a fetus, 50 days a.c. B: Frontal sections through the cerebellum of a fetus, 57 days a.c. C: Frontal sections through the cerebellum of a kitten, 13 days a.b. EGL external granular layer, Ma marginal layer, ICCL inner cortical cell layer, 4V fourth ventricle, ros rostral, caud caudal, G gaps between Purkinje cell clusters, Fl flocculus, PC Purkinje cell clusters or Purkinje cell layer, CN cerebellar nucleus, E AChE positive edges, « cellular raphes, / / gaps in AChE staining pattern, IGL internal granular layer.

126 . Enrico Marani

although a maturational gradient does not appear to be involved. The loss of AChE positivity in Purkinje cell bodies proceeds in a similar pattern (fig. 55). The mature pattern of AChE staining in cat cerebellum is achieved near the end of the first postnatal month (BROWN and GRAYBIEL 1983a). This is described briefly above and, in more detail, by MARANI and VOOGD (1977).

Considerations The first description of cerebellar heterogeneity of AChE in the molecular layer was given by MARANI and VOOGD (1977) for the cat. Electron microscopic techniques have shown AChE in the molecular layer of the cat cerebellum to be localized in the Purkinje cell dendritic tree and in the intercellular space between parallel fibres (see 3.6.2). In this monography, additional AChE longitudinal patterns are described for the monkey in the molecular layer (3.6.3) and for the rat in the granular layer (3.1.1). The first description of an AChE pattern in the developing cerebellum was given by MARANI and FEIRABEND (1983) for rabbit and chicken and by BROWN and GRAYBIEL (1983 a). Recent findings on chemical heterogeneity in the developing cerebellar cortex have been reported by EPEMA, MARANI and TETTEROO (1983). Two main criteria are still discussed concerning the chemical heterogeneity in the cerebellum: - the usefulness of AChE as a marker for the chemical and neuroanatomical heterogeneity of the cerebellum is widely not accepted, - endogenous chemical labels of migrating structures appear t~ have less credibility as markers than experimentally administered exogenous labels, such as 3H-thymidine. The usefulness of AChE as a marker is widely not accepted for embryological and fetal developmental studies. This is due to the fact that the function of AChE is badly understood even in mature nervous system (see SILVER 1974; CHUBB, HODGSON and WHITE 1980). In this chapt-er AChE in early development is demonstrated to be present in a longitudinally banded distribution which appears to be a forerunner of the adult zonal pattern of organization. The publication of WASSEF and SOTELO (1984) demonstrates a chemical subdivision of Purkinje cell clusters in the rat developing cerebellum, using antibodies to c.GMP proteinkinase, which appears to be similar to the AChE topography of Purkinje cell clusters. The c.GMP proteinkinase topography also concerns an endogenous label. This enzyme is known to respond to c.GMP in that it starts specific functions in the cell. These endogenous chemical labels are among the earliest expressions of longitudinal pattern formation in the cerebellum. Whether the chemical heterogeneity is needed for climbing or mossy fibre system ingrowth is unknown, although evidence is emerging now that early mossy fibre ingrowth in chicken, just at the time of Purkinje cell cluster formation, is longitudinally oriented (LAKKE and FEIRABEND 1983, 1984). One of the main issues in cerebellar topography concerns the alignment of various afferent and efferent systems in the mature or developing cerebellar cortex (VOOGD and

Topographic histochemistry of the cerebellum . 127

BIGARE 1980; GERRITS and VOOGD 1982; GERRITS et a1. 1984; KApPEL 1980). The comparison of boundaries of longitudinal zones, visible with AChE staining and staining for FAL immunoreactivity (see Chapters 3.6 and 4.2), indicates that, at least during certain stages of development, zonal boundaries in the marginal layer and the external granular layer are shifted with respect to one another. The comparison of the AChE borders with the location of granule cell raphes in rabbit and chick also demonstrates a non-alignment of both systems. The main conclusion from these results is that different principles may govern the development of the longitudinal topographical organization, producing non-alignment of these zones in the different cerebellar layers. This possibility has been underestimated in literature. This chapter describes the zonal distribution of AChE in the developing cerebellum of the rabbit and cat. In both species the localization of the enzyme within the layers of the cerebellar cortex may change during development. In the rabbit, AChE positivity is displaced into the granule cell layer toward the end of gestation and is then extinguished in the early postnatal period over most of the granule cell layer, except in the vestibulo-cerebellum which remains AChE positive. In the cat, both the granule cell layer and the molecular layer remain positive, although some areas of low AChE positivity can be identified in the molecular layer which allow delineation of a persisting longitudinally oriented AChE band pattern in the adult cat. The changes in AChE localization during development and the loss of AChE positivity in large parts of the granule cell layer of the rabbit and rat cerebellum shortly after birth (see 4.4) suggest that transient AChE positivity is related to developmental occurrences. Recent findings, reported by LAKKE et a1. (in press), suggest the interesting possibility that the transient AChE positivity is closely related to the ingrowth of early arriving mossy fibre systems (which also display a longitudinal organization) and to subsequent shifts in the localization of these fibre systems from the marginal layer to the (internal) granular layer (LAKKE and FElRABEND 1983, 1984; see also ALTMAN and BAYER 1978a, b).

5 General discussion 5.1 Myeloarchitecture of the cerebellum From the description of its myeloarchitecture we see that the longitudinally organized pattern in the mouse cerebellum is identical to that found in the ferret and monkey (fig. 56). Such a subdivision of the cerebellar white matter is also described in the cat (VOOGD 1964, 1967, 1969) and the chicken (FEIRABEND 1983). More subzones could be identified in the mouse cerebellum (MARANI 1982 b) than in the other mammals. However, an additional subzone is now recognized in the rat and cat, too

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v IV

III

II

c

MYELOARCHITECT Ie COMPART E TS I THE A TERIOR LOBE

Fig. 56. Reconstructions of the myeloarchitectural patterns in Macaca (A), ferret (B), and mouse (C).

Topographic histochemistry of the cerebellum· 129

(VOOGD, GERRITS and MARANI 1985; VOOGD 1983). These results show that the multizonal-mononuclear hypothesis of VOOGD is valid for rodents (see also EPEMA and MARANI for the rabbit myeloarchitecture, in prep.). Support for the inherent coordinate system in the white matter of mammalian cerebella came from AChE studies. AChE in the white matter seems to display the same pattern, at least in the case of monkeys. Fro~ unpublished results we now know that AChE mirrors the myeloarchitecture in the cat, ferret, rabbit, and rat. Besides the autoradiographic results of 3H-Ieucine injections into the inferior olive of the cat (GROENEWEGEN and VOOGD 1977), rabbit (EPEMA, VOOGD and LAKKE, in prep.), and monkey (VOOGD, SEDO and GERRITS 1983), injections with HRP or WGA-HRP make the same architectonic subdivisions in the cerebellar white matter visible. Hence, the value of the Haggqvist method as one of the oldest histochemical myelin colourations has again been proved for neuroanatomical studies (see also VERHAART 1964).

5.2 The patterns in the inferior olivary complex There is serious evidence for the conjecture that the AChE subdivisions of the inferior olivary complex coincide with the borders of the afferent and efferent fibre systems of this nucleus (MARAN I 1981). As to the relation of the AChE pattern in the cerebellum and in the inferior olive of the cat, BRODAL and KAWAMURA (1980) quoted MARANI et al. (1977): that the correlation of the distribution of AChE with sites of the origin of efferents and with sites of termination of afferent fibres «does not yet allow a correlation of AChE and function for this cerebellar relay nucleus». In the meantime, however, autoradiographic studies (MARANI, unpublished) and HRP injections (BROWN and GRAYBIEL 1983a, b) yield some interesting results pointing to a different picture. The histochemical subdivision of the cat's inferior olive does in many ways resemble the subdivision of the inferior olive on the basis of its connectivity. Not only the similarities but also the dissimilarities in the distribution of the enzymes in different species support these findings. Longitudinal columnar distributions are found in the caudal MAO and in the rostral DAO of all species. The group beta is always negative, while the dorsal cap generally gives a positive reaction for the enzyme. The distribution of AChE in the PO of the cat is very similar to that found in the rat, whereas the distribution in the ferret is almost the reverse of that in the rabbit. Differences in the distribution are also noticed in the dorsomedial cell column. Recent studies (GERRITS and VOOGD 1982; CAMPBELL and ARMSTRONG, in press) support these subdivisions of the the inferior olive in the cat. Many anterograde transport and degeneration studies of afferent cerebellar systems have been made in the cat. Unfortunately, this is not the case for many other species, where only relatively few such studies have been performed. To facilitate and extend the comparison of AChE types in the inferior olive of

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several species, maps were produced using BRODAL'S (1940) projection method. These descriptions can be used in studies concerning afferent or efferent projections into the inferior olivary complex in these species. The constant inherent AChE borders can easily be compared with injection or projection areas in one and the same section or in successive sections. Recent publications on spino-olivary pathways (see ARMSTRONG et al. 1982) support the idea that the AChE subdivisions mirror the afferent projection borders in the cat (MARANI 1981), although neuroanatomists tend to underestimate the importance of the AChE borders found in the inferior olivary complex. The differences in projection methods, used to plot diagrammatically degeneration, HRP, or autoradiographic results in most neuroanatomical studies, can be considered to be responsible for the differences between AChE borders and afferent or efferent projection borders. For example, the spino-olivary distribution of lumbar and cervicothoracic levels in the cat seems to correspond exactly to less positive areas in the rostral DAO and the negative column in the caudal MAO respectively (AMSTRONG et al. 1982). Also, the border between the dorsal cap and the ventro-Iateral outgrowth, as described by GERRITS and VOOGD (1982), coincides exactly with the AChE borders in this area. However, these similarities are not easily discernible in the given diagrams.

5.3 The relation between the cerebellar and inferior olivary patterns The presented results are more directly related than can be concluded from the purely histochemical descriptions given in this monograph. Recent research efforts have established several connections between the seemingly less related topics discussed in various chapters. The autoradiographic results after injection of 3H-Ieucine into the inferior olive (VOOGD, HESS and MARANI 1986, in press) were compared with the localization of AChE zones within the cat's cerebellar molecular layer and the Haggqvist stain in the same series. Autoradiography and acetylcholinesterase incubation were performed on the same sections. Successive sections were used for Haggqvist staining. These results show a direct alignment of the borders of the AChE zones and of the borders of the climbing fibre zones. In fact BROWN and GRAYBIEL (1983 b) showed by HRP injections into the deep cerebellar nuclei that Purkinje cell zones correspond remarkably well to AChE zones. Therefore, the AChE zonal pattern can even be used in fixated cat cerebella as an internal reference system for the localization of climbing fibre zones and Purkinje cell zones. The AChE longitudinal pattern seems to be the direct chemical expression of the modules postulated by VOOGD (1964, 1967, 1969; VOOGD and BIGARE 1981 ). The modules can only exist if certain groups of olivary neurons project in a bandlike distribution onto the cat cerebellum. As already pointed out in the chapter on AChE in

Topographic histochemistry of the cerebellum' 131

the inferior olivary complex, more refined neuroanatomical studies relate the inferior olivary AChE borders with borders of cell groups projecting to certain zones. Evidence that the AChE pattern in the cerebellum is also linked to the inferior olive is now accumulating. AChE is attributed to Purkinje cells and parallel fibres, intracellular and extracellular respectively. However, long-term lesions of the inferior olivary nucleus (BROWN 1985) lead to the increase of activity of longitudinal AChE bands over a period of six weeks. It is known that lesioning of climbing fibres also causes the disappearance of climbing fibres bands within 48 hours (DESCLINS 1974). The long-lasting absence of climbing fibres in certain longitudinal zones apparently causes the increase of the AChE activity bound to Purkinje cell dendrites and parallel fibres. It may, therefore, be concluded that the A ChE pattern in the cat cerebellar molecular layer is maintained and perhaps induced by climbing fibres. The results with the 5'-nucleotidase pattern show the same situation. Administration of 3'-acetylpyridine, which causes total destruction of both inferior olivary complexes, does not change the topography of the band pattern in the rat molecular layer, even after 100 days (MARAN! 1982 b). On the contrary, it stands out better through wider negative zones. Biochemical determination of 5'-nucleotidase in 3' -acetylpyridine administrated rats, compared with normal rats, both treated with harmaline, indicates far more S'-nucleotidase activity in the cerebellum of normal rats (BALABAN etal. 1984). Our own biochemical results show no decrease of S'-nucleotidase 56 days after the administration of 3' -acetylpyridine (MARAN! 1982). Injections of 35 5_ methionine into the inferior olivary complex of rats show a pattern of longitudinal zones of climbing fibres identical to the 5'-nucleotidase pattern in rat and mouse. These longitudinal radioactive labelled zones are subdivided by zones containing no labelling (CHAN PALAY et al. 1977). Recent results of AMSTRONG et al. (1982), after radioactive methionine administration in the rat inferior olive, found that the whole molecular layer is filled with labeled climbing fibres. It should be mentioned here that preferential uptake of 35S-adenosyl methionine by those olivary cells, that produce the longitudinal 5'-nucleotidase band pattern in the rat molecular layer, may take place. The breakdown of adenosyl methionine produces adenosine which is an important substance for the production of AMP. Although it is unclear why olivary cells would preferentially take up 35S-adenosyl methionine, it still could serve as an explanation of the contradictory results obtained by CHAN PALAY'S and AMSTRONG'S group.

S'-Nucleotidase is absent from the mouse and rat inferior olive but present in the cat's inferior olive. Olivary hypertrophy is a phenomenon which till now is only found in the cat, dog, and human olivary complex. 5'-Nucleotidase in these hypertrophic cells demonstrates only a change after very long survival times of nearly over one year. Perhaps the 3' -acetylpyridine results obtained within the molecular layer of the rat would also benefit from time periods of more than 100 days. Arguments exist which suggest that hypertrophic cells sprout within the hypertrophic area (BOESTEN, unpublished). The sole change within the neuropil recognized

132 . Enrico Marani

in hypertrophic areas is an increase in the catecholaminergic fluorescence (a phenomenon which appears with the onset of hypertrophy) and an increase in AChE content. In view of the AChE inducing capacity of climbing fibres within the molecular layer, an analogous process could be occurring in the hypertrophic inferior olive. This could be explained by the sprouting of climbing fibres, inducing an increase in AChE activity in its neuropil. It must be noted that long-lasting ablation of the hemicerebellum with its deep cerebellar nuclei does not induce hypertrophy in the rat's inferior olive. The prevailing belief among neuroanatomists that the wiring of the brain is identical in all mammals causes difficulties in understanding the absence of hypertrophy in the rat. Olivary hypertrophy in the cat is explained by the presence of a rubro-cerebellar projection, because it is considered as an antegrade, transsynaptic reaction. Olivary hypertrophy mainly resides within the rostral MAO which is partially the target of both the direct cerebello-olivary projection and an indirect cerebellomesencephalo-olivary projection. The study of the cerebello-olivary connections has produced conflicting data, especially in the case of the rat. Fastigio-olivary projections have been described in the rat by ACHENBACH and GOODMANN (1968) and ANGAUT and CIClRATA (1982), but were not found by BROWN et al. (1977). The study of HAROIAN (1982) also failed to demonstrate the fastigial projection. The studies confirming a fastigio-olivary connection show the fastigial nucleus and also the interposed posterior nucleus projecting into the caudal MAO in the rat. In the cat the hypertrophic rostral MAO only contains projections from the interposed posterior nucleus (BRODAL and KAWAMURA 1980). ,These results could be considered as arguments in favour of a different cerebello-olivary wiring fin the rat and would thus lead to an explanation of the absence of hypertrophy in the rat inferior :'olivary complex.

5.4 Biochemical and ultrastructural consideration on cerebellar molecular layer patterns The ultrastructural localization of 5'-nucleotidase is badly described in the literature, due to a careless application of enzyme histochemistry. Serious damage to the 5'nucleotidase localizations led to the assumption that exclusively glial cells are concerned (see 3.5.3 for ref.). Recently, however, even the KREUTZBERG group accepted the presence of 5' -nucleotidase in or at neurons (KREUTZBERG, Proceedings 25. Symposion, Gesellschaft fur Histochemie, 1983). It was already known that, for example, damage of the hypoglossal nerve decreases the AChE content in its neurons and increases glial 5'-nucleotidase activity (DAVIDOFF et al. 1973; MARAN!, unpublished). Therefore, glial localizations are present in the brain, which is generally accepted. Advocating an exclusive 5'-nucleotidase activity in or on glial cells contradicts the results of most studies concerning 5 ' -nucleotidase in the brain over the last twenty years. They all show a differential localization of 5 ' -nucleotidase. The fact that only one isoenzyme for 5' -nucleotidase is present in the mouse cerebellum makes quantification possible which, in its turn, supports the fact that the

Topographic histochemistry of the cerebellum' 133

amount of enzyme present in several bands is different. This must mean that a differential effect of adenosine can be present in the enzyme positive longitudinal bands of the mouse cerebellum. The biochemical results demonstrate the peculiar properties of 5 ' -nucleotidase. To our knowledge, this is the first type of 5 ' -nucleotidase, having a K+ and Na+ activation, that is severely inhibited by high concentrations of ATP, while Mg2+ ions are not needed as an activator. Most neurotransmitters tested have no effect on 5' -nucleotidase, with the exception of high concentrations of norepinephrine (MARANI 1982 b). A localization of 5' -nucleotidase within the subsurface cisternae and spine apparatus of the Purkinje cells and in the cisternae and transmitter vesicles of the parallel fibre bouton seems odd. However, this localization is repeated for AChE, except that the AChE localization is not in but around the parallel fibre bouton. Recent studies in the rat (KOCSIS et al. 1984), also having a 5 ' -nucleotidase pattern, show that adenosine is involved in the parallel fibre - Purkinje cell synapse. In addition, all the ultrastructural and biochemical results gathered on 5 ' -nucleotidase in the mouse cerebellum also suggest this ultrastructural localization of 5' -nucleotidase.

5.5 Why the longitudinal enzyme histochemical cerebellar patterns? In his «Cerebellum and Neuronal Control» ITo (1984) describes how complex, highly ordered neuronal circuitry, like the cerebellar cortex, can constitute a «memory» device. This idea has been incorporated into learning network models of the cerebellum (MARR 1969; ALBUS 1971). Experimental evidence supports the plasticity assumption of the MARR-ALBUS model of the cerebellum. The MARR assumption states that a parallel fibre - Purkinje cell synapse augments its transmission efficacy towards a fixed maximum value, when a parallel fibre is active at about the same time as the climbing fibre input to the same Purkinje cell. A certain coincidence of incoming parallel fibre and climbing fibre signals is needed. Purkinje cells can be made highly responsive to sets of parallel fibre inputs mediated via the mossy fibres, as long as a simultaneous climbing fibre input is present. Modifications of this system can be induced by the release of a change factor (e. g. adenosine), which modifies the active synapses that are controlled by the climbing fibre action and by simultaneous pre- and postsynaptic depolarization (ITo 1984). Another situation can be understood according to a simple perceptron model (ITo 1976). After inputting a set of spike patterns into a simple perceptron programmed with a specific class to which the spikes should belong, every pattern in the set will be correctly recognized. According to ITO (1984): «the possible algorithms for adjusting the weights in a simple perceptron are:

134 . Enrico Marani

1- if a pattern is correctly classified, an increase occurs in all weights coming from active association cells, 2- if a pattern is incorrectly classified, a decrease occurs of all the weigths coming from active association cells.» The climbing fibre pattern is presumably imprinted on the mossy fibre pattern and, therefore, the capacity to store information concerning the relative firing rates of climbing fibre patterns would be present in a cerebellar perceptron. ALBUS (1971) put the climbing fibres in the position of «teachers» which change the weigths according to the algorithms (ITO 1984). ALBUS (1971) assumed that the cerebellum functions according to possibility 2. Thus, after a wrong signal from the Purkinje cells, cell synapses coming from active parallel fibres will be weakened. Indeed it was found (ITo 1984) that during conjunctive stimulation of climbing fibre and parallel fibre - Purkinje cell synapse the latter undergoes a long-term depression. Climbing fibre activation often causes Purkinje cells to pause. If the Purkinje cell has to learn to pause, parallel fibre excitation must be decreased (ITo 1984). Molecular mechanisms are supposed to underlie the long-term depression influencing the subsynaptic chemosensitivity of Purkinje cells which have yet to be investigated. The studies in this monograph show that enzymatic patterns are present within the molecular layer of the cat, monkey and mouse cerebellum. These patterns are related to the Purkinje cell - parallel fibre synapse and, therefore, could be involved in the learning-plasticity capacities of the cerebellum. Arguments supporting this hypothesis are: - Harmaline experiments in mice with intact and destroyed inferior olives; harmaline induces a rhythmic tremor in mammals. Synchronous rhythmic discharge of inferior olivary neurons is brought via the climbing fibres to the Purkinje cell dendrites. Harmaline increases the cerebellar 5' -nucleotidase activity in normal mice in the P2 fraction (the synaptosome fraction). This increase is absent in mice whose inferior olive has been destroyed (BALABAN et al. 1984). - Adenosine selectively blocks parallel fibre mediated synaptic potentials, due to the presence of an adenosine presynaptic receptor on the parallel fibre bouton (KOCSIS et al. 1984). It follows from our studies on the ultrastructural localization of 5' -nucleotidase that adenosine is produced in the parallel fibre - Purkinje cell synapse and in Purkinje cell dendrites. It was already stated by KOSTOPOULOS et al. (1975) that microiontophoretic application of AMP and ATP has a depressing effect on nearly all rat Purkinje cells tested. However, application of adenosine has a depressing effect only in 80% of the Purkinje cells tested. In the rat the negative zones for 5'-nucleotidase are small, when compared to the ones in the mouse cerebellum. In the mouse cerebellum, 5'-nucleotidase is clearly present in alternating positive and negative longitudinal zones. From a comparison of our myeloarchitectonic results with the topography of the 5'-

Topographic histochemistry of the cerebellum' 135

nucleotidase pattern, it can be concluded that each zone of VOOGD is subdivided into a 5' -nucleotidase positive and negative zone (MARANI and VOOGD 1981; VOOGD and MARANI 1979). The same holds for the AChE pattern in the cat (BROWN and GRAYBIEL 1983; MARANI, unpublished) and monkey (SEDO, HESS and VOOGD 1984). Climbing fibre action within a zone will have different effects in a 5'-nucleotidase positive and a 5'-nucleotidase negative zone. Conjunctive stimulation within a 5'nucleotidase positive zone causes for the parallel fibre - Purkinje cell synapse (fig. 57):

climbing fiber

parallel fi ber

PURKINJE cell granule cell

III

climbing fiber

B b mossy fibers

II

from: spinal cord external cuneate nu. reticular nuclei pontine nuclei

INFERIOR OLIVE efferent pathways of the central nuclei

climbing fiber

A

c

Fig. 57. A: Explains the adenosine effects on the Purkinje cell - parallel fibre synaps. Input systems like climbing fibres and mossy fibres activate the cerebellar nuclei (a and b) while cerebellar output is also relayed back to the inferior olive (c). B: Parallel fibre bouton activation results in the release of the cotransmitter adenosine (1) in 5'-nucleotidase positive bands. The presynaptic adenosine receptors block the parallel fibre transmission (2,3). C: Effect of the climbing fibres in 5 ' -nucleotidase positive bands. It is assumed that the climbing fibre will release by its transmitter action (1) adenosine from the Purkinje cell dendrite (2) or from the spine apparatus (2), that will block the parallel fibres by the presynaptic receptors (3).

"136 . Enrico Marani

(a) activation of the parallel fibre bouton, (b)release of adenosine contained within synaptic vesicles, (c) activation of the inhibitory presynaptic adenosine receptors on the parallel fibre bouton, (d) weakening of the parallel fibre transmission. Non-conjunctive stimulation causes the same effect for parallel fibre transmission (fig. 57). For climbing fibre excitation of the Purkinje cell dendrites, it is assumed that climbing fibre action results in adenosine release in the extracellular space via the subsurface cisternae and the spine apparatus, thus also resulting in a weakening of the parallel fibre transmission. It is also accepted by ITo (1984) as a possible mechanism that climbing fibre action on Purkinje cell dendrites results in a Purkinje cell dendrite mediated action on the parallel fibre synapse. No adenosine effect is present in a 5'nucleotidase negative zone. Such a device would not make sense, unless a set of input patterns could be relayed by the branching climbing fibres to a positive strip and to a negative strip in another zone or vice versa, for example, olivary neurons projecting both into C1 and C3 (OSCARSSON 1969, 1973, 1980). Each set of input patterns would then be discriminated in both 5'-nucleotidase positive and negative zones. Perhaps the programmed specific classes I and 0 of ALBUS (1971) to which the spikes belong, would be created in such a way. Or perhaps these strange longitudinal patterns have something to do with the proposed recorder in the expansion recorder perceptron (ALBus1971). There are arguments supporting the plasticity assumption by MARR (1969), ALBUS (1971), and FUJITA (1982) and the associated involvement of adenosine: - Intracerebroventricular administration of theophylline, an adenosine receptor antagonist, decreases harmaline tremor, indicating that purinergic mechanisms participate in cerebellar-inferior olivary harmaline tremor (BALABAN et al. 1984). - What happens to the heterosynaptic interaction between Purkinje cell and parallel fibre, when the instruction of the climbing fibre disappears? A reasonable answer would be that the learning perceptron ceases to function and, therefore, maintainance of the longitudinal patterns would not be expected. Indeed, partial destruction of the inferior olive in cats, leads to the disappearance of climbing fibres in several AChE bands and the increase of AChE activity in these bands after a survival time of 6 weeks. 3'-Acetylpyridine administration in rats does not affect the topography of the pattern" but causes it to stand out more clearly, perhaps due to an increase in the width of its negative bands and a corresponding increase in the positive bands. Therefore it has to be accepted as an inductive principle that the climbing fibres maintain the level of the longitudinal enzymatic patterns.

Topographic histochemistry of the cerebellum' 137

6 Appendix Several parts of this monograph contain data not yet published. In this appendix, material and methods of several chapters are gathered. The numbers in front of the parts refer to the chapters involved. [2.3] Lesions, material and methods for experimentally induced inferior olivary hypertrophy For the study of olivary hypertrophy we used experimental material available from previous electron microscopic (VOOGD and BOESTEN 1975) and light microscopic studies (VERHAART and VOOGD 1962). Fresh material was obtained by using healthy adult cats (table 12). The surgical procedure was performed as described by BOESTEN and VOOGD (1975), except that the occipital bone was removed further cranially. Hemi-cerebellectomies or total cerebellectomies were carried out, half or the whole cerebellum being removed with due care to destroy the cerebellar nuclei. Postoperatively the cats were treated with antibiotics (tetracyclinum). After the chosen appropriate survival times (table 12) the cats were anaesthetized (Evipan-Na, 1 ml/kg) again, and their heads were fixed in the stereotaxic apparatus, where the skull and the first vertebra was opened using a dorsal approach. The brain stem and residual cerebellum were removed within 7 min, using cuts in the spinal cord and rostral to the cerebellum (MARANI 1981 ; MARANI, VOOGD and BOEKEE 1977). The cerebellar remnant was separated from the brain stem and processed with routine stains to determine the extent of the lesion (Toluidin blue or KluverBarrera; VOOGD and FEIRABEND 1981). The brain stem was frozen for enzyme histochemistry (MARANI 1978). In the inferior olive region all sections were collected, while in the adjacent areas in cranial or caudal direction 1 of 10 sections was collected. A summary of the lesions and survival times of the various histological and histochemical treatments carried out in this study can be found in tables 12 and 13. All methods using NAD as coenzyme were applied as described by PEARSE (1968, 1970). However, 5' -nucleotidase reaction was carried out according to SCOTT (1965; see MARANI 1981), ATP-ase according to GUTH and ALBERS (1974), MAO according to both WILLIAMS et al. (1975), and PEARSE (1968, 1970), and aspartate amino transferase according to MARTINEZ-RoDRIQUES et al. (1976). Membrane techniques (MEYER 1980) were used for soluble enzymes. Catecholamine fluorescence was performed according to DE LA TORRE et al. (1976).

Table 12. Summary of the hemi- and total cerebellectomies and control series used in this studies. E C C C C

251 235 171 88 131

hemicerebellectomy hemicerebellectomy hemicerebellectomy hemicerebellectomy total cerebellectomy

E.M. Hist. Hist. Hist. Hist.

139 150 153 235 379

days days days days days

H 9213

hemicerebellectomy not involving cerebellar nuclei

Hist.

4 days

Hist.

o days

Control series

C

75

138 . Enrico Marani Table 13. The (enzyme)histochemical reactions performed on hypertrophic and control animals (e7S and H9213).

Kliiverffol. blue

E251

Cl71

C88

C131

C235

C75

x

x

x x x x

x x

x

x x x x

Millon PAS Sudan black Acid anhydrite Catachol-fluorescence SD

x

x x x

G-6-Pdh H-B-A-dh

x

L-dh NADPH-tr NADH-tr G-dh ATPase 5'-N AP AChE CO

x x x x x x x x

AAT M-dh

x

BA-dh iso C-dh

x

x x x x x x x x x x x x x x

x x x x x x x x x x x x x x

x x x x x x x x x x x x x x x x

ps-ChE G-6-Pase MAO

x

x x x x x x x x x x x x

x

PO

H9213

x x x x

x x x

x

S.D.: succinic dehydrogenase, G-6-Pdh: glucose-6-phosphate dehydrogenase, HBAdh: ~ hydroxy butyric acid dehydrogenase, L-dh: lactate dehydrogenase, NADP(H)-tr: NADP(H) tetrazolium reductase, Gdh: glutamate dehydrogenase, 5'-N: 5-nucleotidase, AP: acid phosphate, AChE: acetylcholinesterase, CO: cytochrome oxidase, AAT: aspartate amino transferase, Mdh: malate dehydrogenase, BA-dh: betaine aldehyde dehydrogenase, iso-C-dh: iso citric dehydrogenase, PO: peroxidase, ps-ChE: pseudo cholinesterase, G-6-Pase: glucose-6-phosphatase, MAO: mono amine oxidase, Succ. SA. dh: succinic semi aldehyde dehydrogenase.

Topographic histochemistry of the cerebellum· 139

[3.4} Material and methods for isoenzyme studies Mice aged 6 months to 1 year (USA Naval Medical Research Institute, NMRI strain), were anaesthetized with chloroform. Only females were used, because HARDONK and]ONKERS (1972) reported on sex differences in the liver concerning the the localization of 5/ -nucleotidase. The cerebellum was dissected within 5 min, homogenized, and suspended in 2 ml elecrophoretic buffer at +4°C. Concentrated electrophoretic buffer (28.8 g glycine, 6.0 g Tris, 1000 ml distilled water) was diluted 1:10 with distilled water. The final pH was 7.7. (1) One group of homogenized cerebella, each suspended in 2 ml electrophoretic buffer at +4°C was immediately centrifugated for 30 min at +4°C and 2650 g. (2) To another group 1 ml prim. butanol was added. This homogenate was stirred for 30 min at +4°C and subsequently centrifugated during 30 min at +4°C, 3200 g (HARDONK and DE BOER 1968) or at 2650 g. (3) A third group of cerebella was homogenized in a 0.05 M veronal buffer, pH 7.2, to which 0.1 % Triton X100 was added. This homogenate was stirred for 30 min at +4°C and centrifugated at 2650 g. A small amount of sucrose was added to the aqueous phase before use in the disc electrophoresis. All solutions for the preparation of polyacrylamide gels were stored in the refrigerator. The glass tubes were treated with Desicoat (Beckmann & Co., USA) and acetone before use. A glass tube of 26 em was connected to a glass tube of 9 em by a plastic hose. The glass tubes were filled with polyacrylamide gel solution within 5 em from the top, and the remaining volume was filled with distilled water. Polymerization was started with UV light. The glass tubes were filled up to 1 em from the top of the long tube with a 3.5% solution of polyacrylamide (pH 6.8; FELGENHAUER 1971) and placed in the electrophoretic apparatus. 50 111 of the homogenized mouse cerebellum with sucrose and some bromophenol blue were added to each glass tube. Electrophoresis was stopped, when the bromophenol blue reached the middle of the 9 em glass tube. In order to determine whether the enzymes we looked for had indeed arrived at the short distal segment of the glass tube, the 9 em tubes were checked for 5/-nucleotidase or acid phosphatase activity, after the bromophenol blue had been displaced 20, 24, 28, and 32 em. The aqueous phase of the homogenized mouse cerebellum, moreover, formed a three-layered brownish front that could be followed in the disc electrophoresis. In this connection it should be remembered that, according to HARDONK and DE BOER (1968), 5/-nucleotidase in the brain does not move in the opposite direction. We confirmed this result for homogenates not treated with butanol using the agar electrophoretic method of WIEME (1965). Electrophoresis was performed for 3 to 4 hours at 600 V and 6 rnA per tube. The polyacrylamide gels were removed from the 9 em tube by water pressure. Incubations were performed at +37°C for 1 min in 5/nucleotidase media of Scon (1965), using AMP and/or UMP as 5/-nucleotide and the acid phosphatase medium of LAKE (1965). Lead nitrate was used to capture the phosphate ions. In a final series of experiments, inhibitors for acid phosphatase were added to the incubation medium. 10 mM 1,3-c. glyceromonophosphate 6 (MARANI 1982 b) was added to the medium for 5/ -nucleotidase with AMP and UMP. The concentration of NiH (nickel nitrate, CAMPBELL 1962) when added to the incubation media, was always 5 mM. Preincubation was performed 1 min with 5 mM NiH or 10 mM 1,3-c. glyceromonophosphate, also for 1 min after the electrophoresis and 10 min for the determination of the pH curve. 6 The composition of 1,3- cyclic glyceromonophosphate was analyzed by The Netherlands Organization for Applied Scientific Research T. N. O. (Organisch Centrum Instituut T. N. O. Element Analyse, Utrecht), and N. M. R.-scans were performed by Dr.]. Lugtenburg, Faculty of Chemistry.

140 . Enrico Marani

Acid phosphatase assays were determined with para-nitrophenylphosphate (ANDERscH and SZCYPINSKY 1947). 5' -Nucleotidase was defined according to PERSIJN et al. (1969, 1970). Homogenates of mouse cerebellum were prepared as described previously (MARANI 1982) and extracted with butanol, following the procedure of HARDONK and DE BOER (1968). In other experiments, aqueous extracts and extraction with Triton X 100 were used. Proteins were determined with the method of LOWRY et al. (1951). All incubations were stopped by washing with distilled water. Densitometric readings of the polyacrylamide gels were made with a Gilford 2400 at 500 nm. To determine pH activity curves, 50 mM para-nitrophenylphosphate of homogenized cerebellum were added to 1 ml 0.05M veronal-acetate buffer (pH range 4.0 - 10.0), containing 5.5 mM para-nitrophenylphosphate. . Incubation time was 30 min at +3rc. After incubation, 10 ml 0.02 N NaOH containing 0.01 M NaF was added to the incubation medium. All densitometer readings were made at 405 nm with a Carl Zeiss equipment. [3.5.1] Material and methods for 5/ -nucleotidase topography

The mice used in this description of the 5' -nucleotidase pattern were of the NMRI (USA) strain. The mice were anaesthetized with chloroform. Production of series (n=200) occurred according to MARANI (1978). Of the cryostate sections every tenth section was mounted. Most serial sections had not been prefixed but were postfixed with Baker's fixative. The histochemical reaction according to SCOTT (1965) was used with AMP as nucleotide in the incubation medium at +37°C. Other nucleotides were also used, but the description is based on series with AMP as substrate. 1,3-c. Glyceromonophosphate (10- 3M) and NaF (10- 1M to 10- 6M) were used as inhibitors for unspecific phosphatase (see 3.3.2). l,3-c. glyceromonophosphate was added in some series which were also preincubated with this inhibitor for 10 min in the SCOTT buffer (1965) at the same concentration. Levamisole was used in some series as inhibitor for alkaline phosphatase (see MARANI 1981, and 3.3.2). Generally, the incubation time was one hour, except for some series in which it was two hours. Styropor reconstructions (TINKLENBERG 1979, 1980; VOOGD and FEIRABEND 1981) were made from the separate lobules of the anterior lobe (H8553), the entire anterior lobe (H9992), and the caudal vermis (C 150). For the study of the posterior lobe additional serial sections were prepared of the mouse cerebellum (H9997, H9998, C1, C25, C37, C38, C47, C57) and reconstructed with the technique of reference tissue (MARANI 1978). Histophotometric scans were made as described in 3.4.3). [3.5.3] Procedures for 5/ -nucleotidase ultrastructural studies Experimental procedures

Mature female and male mice were anaesthetized with chloroform or ether. These anaesthetics did not influence 5' -nucleotidase activity. Sex differences were not present at the light microscopic level. Experiments were initiated late in the morning, thereby taking into account the circadian rhythms of nonspecific phosphatase activity reported for rats (MARANI 1980 a). A description of the series and the experiments performed for electron microscopy is provided in table 14. The thorax was opened via the abdominal cavity, and an intracardial injection (0.1 ml) of equal volume parts of heparin® (5000 UI/ml) and 1% NaN0 2 was given. The left ventricle was opened, the perfusion needle inserted and clamped with a forceps. After opening the right atrium, aortic perfusion was performed at 10-15 mllmin. Fixation was preceded by saline perfusion. The perfusate contained 0.9% NaCl, 0.01 % CaCl2 , 0.016 M cacody-

Topographic histochemistry of the cerebellum' 141 Table 14. Summation of the used series for E.M. studies on mouse cerebellar 5'-nucleotidase. Experiment

Perf. Fix I

Perf. Fix II

H8243/45 H8521/22 H8320/32 H8523/24 H8525/26 H8527/30 C95/96 C97/99,132/36 Cl04 Cl05/106 H8953/56 C138/139 C140/141 C607 C615/622

+ + + + + + + + + + + + +(AMP) + +

+ + + + + + + + + + + +(24h) +(AMP) +

Perf. Inc.

Pb2+

+

Scott Inc.

1,3.cG SCOll +1,3.cG

bGlyc.

bGlyc +NaF

Parallel sectIons

+ + + +

+ + +

+ +(P) +

+ +(P)

+ pos + pos

+(Pre) +(Na+,K+)

+(time) + + + +

+

+

+ + + + +

pos neg pos pos pos

Abbreviations AMP: AMP added to fixatives, Glyc.: beta-glycerophosphatase incubation, 1,3.cC: AMPase incubation according to SCOTT (1965) with ± 10mM 1,3.c-glyceromonophosphate added, P: perfusion, Pre: preincubation, Perf. Fix I: paraformaldehyde solution perfused, Perf. Fix II: glutaraldehyde solution perfused, Perf. Inc.: Scott's incubation medium perfused, Pb2+: Scott's medium omitting AMP perfused, pos.: 5'-nucleotidase band pattern present, neg.: 5'-nucleotidase band pattern absent, time: different incubation times were checked, +: method or possibility carried out.

late buffer pH 7.4, 300 mOsm, and was injected at room temperature for 2-2.5 min. The first fixative (4% paraformaldehyde, freshly prepared in 0.16 M cacodylate buffer, with 0.01% CaClz, pH 7.4, 1850 mOsm, 0 to +4°C) was allowed to flow for 2-3 min. This first fixation was immediately followed by a 2 min (method A) or 5 min (method B) perfusion fixation at room temperature with a 2.5% glutaraldehyde solution (in 0.16 M cacodylate buffer with 0.01 % CaCl z, pH 7.4, containing 5.4 gil sucrose, 600 mOsm). Note that phosphate buffers are omitted, as in the solutions utilized by RINVIK and GROFOVA (1970). For normal ultrastructural studies, the second perfusion fixation was increased to 15 min, followed by one hour of immersion fixation in the same fixative, before vibratome sections were made (200 Jlm) and osmification was performed. Enzyme studies were carried out in two ways. Perfusion fixation (method B) was followed by a 5 min perfusion with the incubation medium. The brain was then removed from the skull, vibratome chopped (200 Jlm), and incubated. Alternatively, after perfusion fixation (method A), the cerebellum was quickly removed from the skull, sectioned on the vibratome in 200 Jlm sections, and then incubated. With the latter method, the first sections for incubation are obtained within 10 min after the start of the perfusion fixation. Sections obtained about 45 min after perfusion fixation no longer reveal a band pattern of enzyme activity and were subsequently discarded. All incubation media used had to be clear. Clouded media were discarded. The pH was checked before and after incubation. No pH changes greater than 0.2 units were noticed. The incubation media used are: (1) Scon's (1967) medium for 5' -nucleotidase to which sometimes freshly prepared 1,3.-c. glyceromonophosphate (10 mM) was added; (2) LAKE'S medium (MARANI 1977) for the determination of acid phosphatase (for the advantage of the LAKE method (MARANI 1977) over the. GOMORI (1952) method see BREDEROO et a1. (1968». Nonspecific phosphatase was determined by changing the pH of the buffer solution of the LAKE method (MARANI 1977) to pH 7.2. For light microscopic purposes, acid phosphatase was

142 . Enrico Marani

also determined by the method of BARKA and ANDERSON (1962). Media were regularly renewed during incubation periods. After incubation for 1-1.5 hours, the sections were washed for 15 min in cacodylate buffer of the same composition as the fixatives and postfixed in 2.5% glutaraldehyde fixative for 30 min. Osmification was performed at room temperature with OS04 (1 %) in 0.16 M cacodylate buffer pH 7.4. Dehydration in a graded series of ethanol was followed by 20 min in propyleneoxide and by one hour in Epon-propyleneoxide (1 :1). Sections were stored overnight under vacuum in Epon 812. Polymerization occurred 48 hours after embedding in freshly prepared Epon kept at +60°C. Ultrathin sections were made on a LBK III or a Reichert OMU III microtome. Sections were contrasted and stained with lead hydroxyde and/or uranyl. Adjacent uncontrasted sections were always examined. The electron microscopes used were a Siemens Elmiskop or a Philips EM 200, both at an accelerating voltage of 80 kV or 60 kV for uncontrasted sections. For electron microscopy, every third vibratome section was incubated and stained with 0.5% (NH 4 )zS to visualize the band pattern (fig.40A) and to decide which regions of the two remaining sections were to be examined. The band pattern was indicated on enlarged negative prints of these stained sections. Most sections were taken from vermal parts of both the anterior and posterior lobes. Biochemical effects on 5/ -nucleotidase All substances used in the electron microscopical procedure were checked for their biochemical effects on 5/ -nucleotidase activity in homogenates (2.5% w/v) determined according to PERSIJN and VAN DER SUK (1969). For a comparison see MARANI (1977, 1980). [3.6.2J Material and methods for ultrastructural AChE studies Newborn kittens and older cats of up to 5.4 months of age (see MARANI and VOOGD 1977) were used in this study. Animals were anaesthetized with Nembutal® i. p. (60 mg/kg body weight) or chloroform (newborn ones). The thorax was then opened via the abdominal cavity, and an intracardiac injection (0.25 ml) of equal quantities of heparin (5000 IU/ml) and 1% NaNO z was given. The left ventricle was opened and the perfusion needle inserted and clamped (with a forceps). After opening the right atrium, aortic perfusion was carried out with a flow of 80 mil min, except for the newborn ones (30-35 mllmin). The perfusion solutions were the same as in a previous study on the ultrastructural Sf-nucleotidase localization (MARANI 1977; MARANI 1982 a). The perfusion with saline, formalin, and glutaraldehyde fixations, all lasted 5-8 min. Incubations After perfusion fixation the brains were removed, the cerebellum was vibratome chopped (50-100 Jlm thick sections), and incubated at room temperature according to KARNOVSKy-RoOTS (1964), HANKER et al. (1973), and LEWIS and KNIGHT (1977). Before incubation the sections were rinsed for half an hour in an incubation solution from which the substrate, i. e. acetylthiocholine-iodide, was omitted. Incubations lasted for two hours. The pseudocholinesterase inhibitor iso-OMPA was. added to the preincubation and to incubation solutions in a concentration of 10- 4M (MARANI and VOOGD 1977). Incubations for the LEWIS and KNIGHT technique were carried out at a low pH (5.8-6.0). Variations were introduced for the duration of postrinsing the sections in the LEWIS and KNIGHT procedure (see MARANI 1981). Sections were counterstained with sulfide in the LEWISKNIGHT technique after intervals of 1 and 1.5 hours to check the presence of the band pattern in the molecular layer. In two experiments, whole anterior lobes were incubated and counterstained

Topographic histochemistry of the cerebellum' 143

(VOOGD et al. 1981). Incubated sections were postfixed for half an hour in the second fixative and cut for vermal and lobular subdivisions. Most material studied ultrastructurally was taken from lobules III, IV, V, and VI (for the topography of the bands in these lobules see fig. 9.1 in MARAN! and VOOGD 1977). After cutting of the sections, the remaining parts were embedded in the routine way for electronmicroscopy and were oriented. After ultrathin sectioning on a LKB III ?r Reichert OMU III, uncoloured sections were always compared to one stained with Pb or U IOns. The electron microscopes used were a Siemens Elmiskop and a Philips EM 200, both at an accelerating voltage of 80 Kv or 60 Kv for uncontrasted sections. [4.1.1J Methods for FAL immunology

Several monoclonal antibodies were screened for their capacity to detect specific structures in the brain of various species in the Neuroanatomy Division of the Department of Anatomy and Embryology, University of Leiden, in cooperation with the Central Laboratories of the Blood Transfusion Services (Immunohaematology Department). The monoclonal antibodies were donated (table 9) by several researchers. ELISA tests for sugar antigens were performed in cooperation with Chembiomed Ltd., Alberta, Canada. The antigenic structures recognized by several monoclonal antibodies have been catalogued by TETIEROO and coworkers (TETIEROO et al. 1984, and in press). The species used in this study are listed in table 10. Monkey brains were available from the rhesus monkey Macaca rhesus, two whole brains) and the common marmoset (Callithrix jacchus, six cerebella with brain stem). Human brains (no pathology) came from two female patients, 60 and 80 years old. The human brains were obtained 8 hrs p. m. via the Department of Neuropathology, University of Leiden. The screening procedure was the same as described for the monoclonal antibody B 4.3 (MARAN! and TETIEROO 1983 a). The other monoclonal antibodies were used in the same concentration (1 :1000), and FITC-goat-antimouse Ig antiserum was diluted 1:50. All sections were initially processed for the FITC technique. If a positive reaction was observed, the PAP technique (MARAN! and TETIEROO 1983 a) was used additionally on adjacent sections. The monoclonal antibodies that were screened are summarized in table 9, together with Ig class, specificity, and molecular weight of the protein carrier(s) of the antigenic determinant. For several antibodies, the antigen was known or could be deduced from (recent) research performed at the CLB (TETIERoo, personal communication). [4.4J Material and methods for developmental AChE studies

For the studies on cat, a total of 70 fetuses or young kittens were used. All cerebellar tissues were fixed by immersion (typical for animals younger than 40 days a. c.) or perfusion (typical for animals older than 40 days a. c.) with aldehyde solutions. Kittens were anaesthetized with Nembutal® (sodium pentobarbital, 40 mg/kg, administered intraperitoneally) prior to transcardial perfusion with fixative. Various fixatives were used: 4% paraformaldehyde, or 3.5% glutaraldehyde with 2% sucrose in O.lM phosphate buffer, or 10% formaldehyde with 0.9% sodium chloride. Neither the particular aldehyde used nor the fixation procedure (immersion vs. perfusion) seemed to make any difference in the AChE staining pattern. Cerebella were kept refrigerated in O.lM phosphate buffer with 30% sucrose until they were ready for cutting. All tissues were sliced on a cryostat at 3D-50 !J.m thickness. Alternate tissue sections were processed for visualization of AChE, using Graybiel's (GRAYBIEL and RAGSDALE 1980) modification of GENESER-jENSEN and BLACKSTAD's (1971) procedure, with 5.10- 4 M ethopropazine in the incubation medium to suppress pseudo-cholinesterase activity. Adjacent sections were usually stained for visualization of histological detail, using cresyl violet or haematoxylin.

144 . Enrico Marani

Moreover, a hundred young and mature rabbits of the Dutch belted breed were used. The mature animals were anaesthetized with Hypnorm® (fluanosyl 10 mg/ml, fentanyl 0.2 mg/ml). The fetuses and newborns were perfused (n=30) or decapitated (n=70). Perfusion occurred with 1.25% glutaraldehyde and 2% paraformaldehyde in O.1M cacodylate buffer pH 7.2 to which 0.007% CaCh was added. In case of decapitation, all brains were processed according to MARANI (1978). One out of six sections was treated for acetylcholinesterase histochemistry (see MARANI 1981, 1982). Pseudo-cholinesterase activity was suppressed with 10- 3M iso-OMPA in preincubation and incubation fluids (see MARANI and VOOGD 1977; MARANI, VOOGD and BOEKEE 1977). Three different acetylcholinesterase procedures were used: LEWIS and KNIGHT (1977), KARNOVSKY and ROOTS (1964), and the reaction described by GOMORI (1952), with or without silver intensification. Some of the incubated and adjacent sections were stained with haemotoxylin for 30 sec and rinsed in tap water or treated for monoclonal antibody localization (MARANI and TETTEROO 1983) or other enzyme incubations. Postfixation was always performed on cryostat sections with Baker's formalin.

7 References ABOOD, L. G.: Excitation and conduction in the neuron. - In: Basic Neurochemistry (ALBERS, R. W. et aI., eds). - Little Brown, Boston 1972. -: Interrelationships between phosphates and calcium in bioelectric phenomena. - Int. Rev. Neurobiol. 9, 223-261 (1966). ACHENBACH, K. E., GOODMAN, D. c.: Cerebellar projections to pons, medulla and spinal cord in the albino rat. - Brain Behav. Evol. 1, 43-57 (1968). ALBERS, R. W., SIEGEL, G. W., KATZMAN, R., AGRANOFF, B. W.: Basic Neurochemistry. Little-Brown, Boston 1972. ALBUS, J. S.: A theory of cerebellar function. - Math Bisoci. 10, 25-61 (1971). ALTMAN, J.: Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. - J. compo Neural. 145, 339-364 (1972). -: Morphological development of the rat cerebellum and some of its mechanisms. - In: The Cerebellum - New Vistas (ed. PALAY, S. L., CHAN-PALAY, V.). - Exp. Brain Res. 86, 8-49 (1982). ALTMAN, J., BAYER, S. A.: Prenatal development of the cerebellar system in the rat. I. Cytogenesis and histogenesis of the deep nuclei and the cortex of the cerebellum. - J. compo Neurol. 179, 23-48 (1978 a). -: Prenatal development of the cerebellar system of the rat. II. Cytogenesis and histogenesis of the inferior olive, pontine gray and the precerebellar nuclei. - J. compo Neurol. 179, 49-76 (1978 b). ANDERSCH, M., SZCYPINSKI, A. J.: Use of p-nitrophenylphosphate as the substrate in determination of serum acid phosphatase. - Amer. J. clin. Path. 17, 571-574 (1947). ANGAUT, P., BRODAL, A.: The projection of the <
Topographic histochemistry of the cerebellum' 145

ARIENS KApPERS, C. U.: Die vergleichende Anatomie des Nervensystems der Wirbeltiere und des Menschen, Bd.2, 625-768. - De Erven Bohn, Haarlem 1921. ARMSTRONG, D. M., CAMPBELL, N. c., EDGLEY, S. A., SCHILD, R. F., TROTT, J. R.: Investigations of the olivocerebellar and spino-olivary pathways. - In: The Cerebellum-New Vistas (eds. PALAY S. L., CHAN-PALAY, V.). - Exp. Brain Res. 6 (1982). AMSTRONG, D. M., HARVEY, R. J., SCHILD, R. F.: The spatial organization of climbing fiber branching in the cat cerebellum. - Exp. Brain Res. 18, 40-58 (1973). -: Topographical localization in the olivo-cerbellar projection: an electrophysiological study in the cat. - J. compo Neurol. 154, 287-302 (1974). ARMSTRONG, D. M., SCHILD, R. F.: An investigation of the cerebellar corticonuclear projections in the rat using an autoradiographic tracing method. I. Projections from the vermis. - Brain Res. 141, 1-19 (1978a). -: An investigation of the cerebellar corticonuclear projections in the rat using an autoradiographic tracing method. II. Projections from the hemisphere. - Brain Res. 141, 235-249 (1978b). ARVY, L.: Cerebellar enzymology. - Int. Rev. Cytol. 20, 277-359 (1966). AUSTIN, L., PHILLIS, J. W.: The distribution of cerebellar cholinesterases in several species. - J. Neurochem. 12, 709-717 (1965). BALABAN, C. D., WURPEL, J. N. D., SEVERS, W. B.: A specific harmaline-evoked increase in cerebellar 5 ' -nucleotidase activity. - Neurosci. Lett. 50, 111-116 (1984). -: Intracerebroventricular theophylline depresses harmaline tremOr in rats. - FASEB Abstr. (1985). BARCHAS, J. D., AKIL, H., ELLIOT, G. R., HOLMAN, R. B., WATSON, S. J.: Behavioral neurochemistry: neuroregulators and behavioral states. - Science 200, 964-973 (1978). BARKA, T., ANDERSON, P. J.: Histochemical methods for acid phosphathase using hexazonium pararosanilin as coupler. - J. Histochem. Cytochem. 10, 741-753 (1962). BARRON, K. D., BOSHES, R.: Histochemical demonstration of 5 ' -nucleotidase in central nervous system: effects of manganous ion and pH. - J. Histochem. Cytochem. 9, 455-457 (1961). BELFIELD, A., GOLDBERG, D. M.: Comparison of sodium beta-glycerophosphate and disodium phenylphosphate as inhibitors of alkaline phosphatase in determination of 5'-nucleotidase activity of human serum. - Clin. Biochem. 3, 105-110 (1970). BEN HAMIDA, M. E. M.: Contribution a l'etude anatomique du couple olivo-dentele. - Thesis, Paris 1965. BERNSTEIN, H. G., LUPPA, H.: Short communications: Isoelectric focusing studies of the neutral 5 ' -nucleotidase from Wistar rat hippocampus. Evidence for the absence of isoenzymes. Histochemistry 56, 341-343 (1978a). BERNSTEIN, H. G., WEISS, L., LUPPA, H.: Cytochemical investigations on the localization of 5' -nucleotidase in the rat hippocampus with special reference to synaptic regions. - Histochemistry 55,261-267 (1978b). BEYERL, B. D., BORGES, L. F., SWEARING, B., SIDMAN, R. L.: Parasagittal organization of the olivo-cerebellar projection in the mouse. - J. compo Neurol. 209, 339-346 (1982). BIGARE, F.: De efferente verbindingen van de cerebellaire schors van de kat. - Thesis, Leiden University, Maes copy, Brussels, Belgium (1980). BIGARE, F., VOOGD, J.: Cerebello-vestibular projections in the cat. - Acta morpho neerl.-scand. 15, 323-325 (1977). BLOOM, F. E., HOFFER, B. J., SIGGINS, G. R.: Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. I. Localization of the fibers and their synapses. - Brain Res. 25,501-521 (1971). BLOOM, F. E., SIGGINS, G. R., HOFFER, B. J. : Interpreting the failures to confirm the depression of cerebellar Purkinje cells by cyclic AMP. - Science 185, 627-628 (1974).

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BODANSKY, 0., SCHWARTZ, M. J.: 5'-Nucleotidase. - Adv. clin. Chern. 11,277-328 (1968). BOESTEN, A. J. P.: Systemes afferents et ultrastructure de la parolive externe chez Ie chat. - Bull. Ass. Anat. 152, 459-464 (1971). -: Neuronal hypertrophy of the inferior olive in the cat: an experimental light and electron microscopical study. - Proc. 11th World Congr. Neurol. Excerpta Medica (1977). BOESTEN, A. J. P., MARANI, E.: A light- and electron microscopic study of olivary hypertrophy in the cat. - Neurosci. Lett. 53, 158 (1979). BOESTEN, A. J. P., VOOGD, J.: Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat. - J. compo Neurol. 161,215-239 (1975). -: Transneuronal reactions with chronic hypertrophy of granular endoplasmic reticulum, in neurons of the inferior olivary nucleus of the cat. - Ultramicroscopy 2, 113-140 (1976). BOLK, L.: Cerebellum der Saugetiere. - F. Bohn, Jena 1906. BORGERS, M.: The cytochemical application of new potent inhibitors of alkaline phosphatse. - J. Histochem. Cytochem. 21, 812-824 (1973). BOSMANN, H. B., PIKE, G. Z.: Membrane marker enzymes: isolation, purification and properties of 5' -nucleotidase from rat cerebellum. - Biochem. biophys. Acta (Arnst.) 227, 402-413 (1970). BRAITENBERG, V., ATWOOD, R. P.: Morphological observations on the cerebellar cortex. - J. compo Neurol. 109, 1-34 (1958). BREDEROO, P., DAEMS, W. TH.: Submicroscopic cytology of guinea pig exudates. I. The heterogeneity of the granules of neutrophilic granulocytes. - In: FAVARD, P., ed., Microscopie electronique 1970, Resume, 7e Congr. into Grenoble (1970). BREDEROO, P., DAEMS, W. TH., VAN DUYN, P., VAN DER PLOEG, M.: Quantitative investigations on the effect of aldehyde fixation on acid phosphatase activity. - Electron Microscopy, Pre-congress Abstracts 4th European Regional Conference Rome, vol.2, 79-80 (BOCCIARELLI, D. S. ed.). - Tipografia Poliglotta Vaticana, Roma 1968. BREWER, G. J.: An introduction to isoenzyme techniques. - Acad. Press, New York-London 1970. . BRODAL, A.: Experimentelle Untersuchung iiber die olivo-cerebellare Lokalisation. - Z. ges. Neurol. Psychiat. 169, 1-153 (1940). -: The cerebellum of the rabbit. A topographical atlas of the folia as revealed in transverse sections. - J. compo Neurol. 72, 63-82 (1940). BRODAL, A., KAWAMURA, K.: Olivocerebellar projection: A review. - Adv. Anat. Embryol. Cell. BioI. 64, 1-137 (1980). BRODAL. A., WALBERG, F., BLACKSTADT, TH.: Termination of spinal afferents to inferior olive in cat. - J. Neurophysiol. 13, 431-454 (1950). BROWN, B. L.: Changes in acetylcholinesterase staining in the molecular layer of the cat cerebellar cortex following climbing fiber destruction. - Anat. Rec. 211, 27 A (1985). -: Non-isochronous generation of Purkinje cells destined for single cerebellar cortical zones: A tritiated thymidine autoradiographic study in the cat. - Proc. Amer. Neurosci. Ass. (1985). BROWN, B. L., GRAYBIEL, A. M.: Development of the acetylcholinesterase band pattern in cat cerebellum. - Neurosci. (Abstr.) 9, 1091 (1983a). -: Zonal organization in the cerebellar vermis of the cat. - Anat. Rec. 205, 25-26a (1983b). BROWN, J. T., CHAN-PALAY, V., PALAY, S. L.: A study of afferent input to the inferior olivary complex in the rat by retrograde axonal transport of horseradish peroxidase. - J. compo Neurol. 176, 1-22 (1977). BRUNS, P., DALY, S., SNYDER, S.: Adenosine receptor-binding structure activity analysis generates extremely potent xanthine antagonists. - Proc. nat. Acad. Sci. BioI. (Wash.) 80, 2077-2080 (1983).

Topographic histochemistry of the cerebellum' 147

BUELL, M. V., LOWRY, O. H., ROBERTS, N. R., CHANG, M. W., KUPPHAHN, J. I.: The quantitative histochemistry of the brain. V. Enzymes of glucose metabolism. - J. bioI. Chern. 232, 979-993 (1958). BURNSTOCK, G., CAMPPBELL, G., SATCHELL, D. G., SMYTHE, A.: Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut. - Brit. J. Pharmacol. 40, 668-688 (1970). BUSCH, H. F. M.: An anatomical analysis of the white matter in the brain stem of the cat. Thesis Leiden. - Van Gorcum Co., Assen (The Netherlands) 1961. BUTCHER, L. L.: Acetylcholinesterase histochemistry. - In: Handbook of Chemical Neuroanatomy, Vol. 1, 1-49 (BJORKLUND, A. B., HOKFELT, T.). - Elsevier, Amsterdam 1983. CAMPBELL, D. M.: Determination of 5' -nucleotidase in blood serum. - Biochemical J. 82, 43p (1962). CAMPBELL, D. M., ARMSTRONG, D. M.: The olivocerebellar projection in the rat: an autoradiographic study. - Brain Research 275, 215-233 (1983). CARSON, K. A., NEMEROFF, C. B., RONE, M. S., NICHOLSON, G. F., KIZER, J. S., HANKER, J. S.: Experimental studies on the ultrastructural localization of acetylcholinesterase in the mediobasal hypothalamus of the rat. - J. compo Neurol. 182, 201-219 (1978). CHAN-PALAY, V.: Cerebellar dentate nucleus. - Springer Verlag, Heidelberg-New York 1977. CHAN-PALAY, V., NILAVER, G., PALAY, S. L., BEINFELD, M. c., ZIMMERMAN, E. A., Wu, J. Y., O'ONOHUE, T. L.: Chemical heterogeneity in cerebellar Purkinje cells: existence and coexistence of glutamic acid decarboxylase-like and motiline-like immunoreactivities. - Proc. nat. Acad. Sci. (Wash.) 78, 7787-7791 (1981). CHAN-PALAY, V., PALAY, S. L., BROWN, J. T., VAN hALlE, c.: Sagittal organisation of olivocerebellar and reticulocerebellar projections: Autoradiographic studies with 35S_ methionine. - Exp. Brain Res. 30, 561-576 (1977). CHAN-PALAY, V., PALAY, S. L., Wu, J. V.: Sagittal cerebellar microbands of taurine neurons. Immunocytochemical demonstration by using antibodies against the taurine synthetizing enzyme cysteine sulfinic acid decarboxylase. - Proc. nat. Acad. Sci. (Wash.) 79, 4221-4225 (1982). CHESSICK, R. D.: The effect of coma on the enzymes of the brain: histochemical and biochemical study. - J. Histochem. Cytochem. 2, 122-126 (1954). CHUBB, J. W., HODGSON, A. J., WHITE, G. H.: Acetylcholinesterase hydrolyzes substance P.Neurosci. 5, 2065-2072 (1980). CHUJO, T., YAMADA, Y., YAMAMOTO, c.: Sensitivity of Purkinje cell dendrites to glutamic acid. - Exp. Brain Res. 23, 293-300 (1975). CLICK, M. c., SANTER, U. V.: Cell function and differentiation, Part A, 371-383. - Alan R. Liss New York 1982. COIMBRA, A., SODRE-BoRGES, B. P., MAGALHAES, M. M.: The substantia gelatinosa Rolandi of the rat. Fine structure, cytochemistry (acid phosphatase) and changes after dorsal root section. - J. Neurocytol. 3, 199-217 (1974). CONTESTABILE, A., VILLANI, L., CIANI, F.: Ultrastructural analysis on acetylcholinesterase localization in the cerebellar cortex of teleosts. - Anal. Embryol. 152, 15-27 (1977). COOPER, J. R., BLOOM, F. E., ROTH, R. H.: The biochemical basis of neuropharmacology.Oxford Press, Oxford 1974. COURVILLE, J. : Distribution of olivocerebellar fibres demonstrated by a radioautographic tracing method. - Brain Res. 95, 253-264 (1975). COURVILLE, J., FARACO-CANTIN, F.: Topography of the olivocerebellar projection: An experimental study in the cat with an autoradiographic tracing method. - In: The Inferior Olivary Nucleus (ed. COURVILLE, J.. et al.), 235-277. - Raven Press, New York 1980.

148 . Enrico Marani COURVILLE, J., FARACO-CANTIN, F., DIAKIW, N.: A functional important feature of the distribution of the olivo-cerebellar climbing fibers. - Canad. J. Phys. Pharm. 52, 1212-1217 (1974). CSILLIK, B., Joo, F., KASA, P.: Cholinesterase activity of archicerebellar mossy fibre apparatuses. - J. Histochem. Cytochem. 11, 113-114 (1963). DAEMs, W. TH., CORNELISSE, C. J., POELMANN, R. E.: Interpretation of the results of metal salt methods for electromicroscopy. - Proc. 5th European Congr. Electr. Micr. 1972 (COSSLETI, V. E., ed.) 264-269. - The Institute of Physics, London 1972. DALCHAU, R., KIRKLEY, J. FABRE, J. W.: Monoclonal antibody to human brain granulocyte. Tlymphocyte antigen probably homologous to the W 3/13 antigen of the rat. - Europ. J. Immunol. 10, 745-749 (1980). DAs G. D., DAs, K. G., BRASKO, J., ALLEMANGO, J.: Neural transplants, volumetric analysis of their growth and histopathologic changes. - Neurosci. Lett. 41, 73-79 (1983). DAVIDOFF, M.: Ober die Glia im Hypoglossuskern der Ratte nach Axotomie. - Z. Zellforsch. 141,427-442 (1973). DE PIERRE, J. W., KARNOVSKY, M. L.: Ectoenzymes of guinea-pig polymorphonuclear leukocyte, I. Evidence for an ecto-adenosine monophosphatase, ecto-adenosine triphosphatase and ecto-nitrophenyl-phosphatase. - J. bioI. Chern. 249, 7111 (1974a). -: Ectoenzymes of granulocytes: Sf-nucleotidase. - Science 184, 1096-1098 (1974b). -: Ectoenzymes of guinea-pig polymorphonuclear leukocyte. II. Properties and suitability as markers for plasmamembrane. - J. bioI. Chern. 249, 7121 (1974c). DESCLIN, J. c.: Histological evidence supporting the inferior olive as the major source of cerbellar climbing fibers in the rat. - Brain Res. 77, 349-364 (1974). DESCLIN, J. c., ESCUBI, J. : Effects of 3' -acetylpyridine on the central nervous system of the rat, as demonstrated by silver methods. - Brain Res. 77, 349-364 (1974). DE VRIES, G. H.: Isolation ofaxolemma-enriched fractions from bovine central nervous system. - Neurosci. Lett. 3, 117-122 (1976). DODGE, J. A., RAMINOFF, R.: Cooperative action of calcium ions in transmitter release at the neuromuscular junction. - J. Physiol. 193, 419-426 (1967). DRUMMOND, YAMAMOTO, M.: Nucleotide phosphomonoesterases. - In: The Enzymes IV (Ed. D. BOYER), 337-352. - Academic Press, New York-London 1971. EBELS, E. J.: De rijping van het cerebellum. - Thesis University Groningen (1969). ECCLES, J. c., ITo, M., SZENTAGOTHAI, J.: The cerebellum as a neuronal machine. - Springer Verlag, Heidelberg-New York 1967. EKEROT, C. F., LARSON, B.: The dorsal spino-olivocerebellar system in the cat. I. Functional organization and termination in the anterior lobe. - Exp. Brain Res. 36, 201-217 (1979). -: Branching of olivary axons to innervate pairs of sagittal zones in the cerebellar anterior lobe of the cat. - Exp. Brain Res. 48,185-198 (1982). ELLMAN, G. L., COURTNEY, K. D., ANDRES, V. JR., FEATHERSTONE, R. M.: A new rapid colorimetric determination of acetylcholinesterase activity. - Biochem. Pharmacol. 7, 88-95 (1961). EMMELHOF, P., Bos, C. S., BENNEDETII, E. J., RUMcKE, P. H.: Studies on plasma membranes. I. Chemical composition and enzyme content of plasma membranes isolated from rat liver. Biochem. biophys. Acta (Arnst.) 90, 126-145 (1964). EpEMA, A. H., GULDEMOND, J., VOOGD, J.: Reciprocal connections between the caudal vermis and the vestibular nuclei in the rabbit. - Neurosci. Lett. 57, 273-278 (1985). EpEMA, A. H., MARANI, E., TETIEROO, P. A. T.: Longitudinal bandpattern in the developing rabbit cerebellum: immuno- and enzyme histochemistry. - Abstr. 4th Int. Congr. Developm. Neurosci. Salt Lake City (1983).

Topographic histochemistry of the cerebellum' 149

ESSNER, E., NOVIKOFF, A. B., MASEK, B.: Adenosine triphosphatase and 5'-nucleotidase activities in the plasma membrane of liver cells as revealed by electron microscopy. - J. biophys. biochem. Cytol. 4, 711-716 (1958). EVANS, W. H., GARD, J. W.: Properties of 5'-nucleotidase purified from mouse liver plasma membranes. - Biochem. J. 133, 189-199 (1973). FALCK, B., HILLARP, N. A., THIEME, G., THORP, A.: Fluorescence of catecholamines and related compounds condensed with formaldehyde. - J. Histochem. Cytochem. 10, 348-354 (1962). FEIRABEND, H. K. P.: Anatomy and development of longitudinal patterns in the architecture of the cerebellum of the white leghorn (Gallus demesticus). - Thesis Leiden (The Netherlands) 1983. FEIRABEND, H. K. P., CHOUFOUR, H.: Morphometric analysis of Purkinje cell fibers in the cerebellum of the white leghorn. - Acta morpho neerl.-scand. 23, 72 (1985). FEIRABEND, H. K. P., VAN LUXEMBURG, E. A., VOOGD, J.: Developmental aspects oflongitudinal cerebellar organization as studied with 3H-thymidine in the white leghorn. - Neurosci. Lett. 5 1, 144 (1978). FEIRABEND, H. K. P., VIELVOYE, G. J., FREEDMAN, S. L., VOOGD, J.: Longitudinal organization of afferent and efferent connections of the cerebellar cortex of the white leghorn (Gallus domesticus). - In: Afferent and intrinsic organization of laminated structures in the brain (0. CREUTZFELD, ed.). - Exp. Brain Res. 5.1, 72-78 (1976). FEIRABEND, H. K. P., VOOGD, J.: Development of longitudinal banding in the cerebellar cortex. - Acta anat. (Basel) 99, 265 (1977). -; The development of longitudinal subdivisions in the cerebellum of the white leghorn (Gallus domesticus). - Acta morpho neerl.-scand. 17, 238-239 (1979). FELGENHAUER, K.: Information on the specific and non-specific phosphatases of the nervous system. - Histochemie 3,222-232 (1963). FISKE, C. H., SUBBAROW, Y.: The colorimetric determination of phosphorus. - J. bioI. Chern. 66, 375-400 (1952). FONNUM, F.: Application of microchemical analysis and subcellular fractionation techniques to the study of neurotransmitters in discrete areas of mammalian brain. - Adv. Biochem. Psychopharmacol. 6, 75-88 (1972). Fox, C. A., HILLMAN, D. E., SIEGESMUND, K. A., DUTTA, C. R.: The primate cerebellar cortex. - In: Fox, C. A., SNIDER, R. S., eds., The cerebellum. - Progr. Brain Res. 25, 174-225. Elsevier Pub!. Co., Amsterdam 1967. Fox, J. H., KELLY, W. N.: The role of adenosine and 2'-deoxyadenosine in mammalian cells.Ann. Rev. Biochem. 47, 655-686 (1978). FRENKEN, C. W. G. M.: De niet-epileptische myoclonieen. - Thesis Nijmegen; P. Smits, Tilburg (The Netherlands) 1977. FREZIK, J.: Associative connections established by Purkinje cell axon collaterals between different parts of the cerebellar cortex. - Acta morpho!. Acad. Sci.hung. XII, Fl, 9-14 (1963). FRIEDE, R. L.: Topographic brain chemistry. - Academic Press, New York 1966. FUJITA, M.: Adaptive filter model of the cerebellum. - BioI. Cybern. 45, 195-206 (1982). FUJITA, S.: 3H-thymidine autoradiographic studies on the cell proliferation and differentiation in the external and internal granular layer of the mouse cerebellum. - J. compo Neurol. 128, 191-208 (1966). GARSON, J. A., BEVERLY, P. C. L., COAKHAM, H. B., HARPER, E. J.: Monoclonal antibodies against human T-lymphocytes label Purkinje neurones of many species. - Nature (Lond.) 298, 375-377 (1982). GAUTIER, J. c., BLACKWOOD, W.: Enlargement of the inferior olivary nucleus in association with axons of the central tegmental tract or dentate nucleus. - Brain 84, 341-361 (1961).

150 . Enrico Marani

GENESER-JENSEN, F. A., BACKSTAD, T.: Distribution of acetylcholinesterase in hippocampal region of guinea pig. I. - Z. Zeli. Mike. 114, 460-468 (1971). GEREBTZOFF, M. A.: Cholinesterase, a histochemical contribution to the solution of some functional problems, vol. 3. - Pergamon Press, Oxford 1959. GERRITS, N. M., EPEMA, A. H., VOOGD, J.: The mossy fiber projection of the nucleus reticularis tegmenti pontis to the flocculus and adjacent ventral paraflocculus in the cat. - Neuroscience 12, 627-644 (1984). GERRITS, N. M., VOOGD, J.: Cerebellar mossy fiber systems in cat. - Acta morphol. neer!.scand. 17, 236-237 (1979). -: The climbing fiber projection to the flocculus and adjacent paraflocculus in the cat. - Neuroscience 7, 2971-2991 (1982). GOLDBERG, A. M., MCCAMAN, R. E.: A quantitative microchemical study of choline acetyltransferase and acetylcholinesterase in the cerebellum of several species. - Life Sci. 6, 1493-1500 (1967). . GOMORI, G.: Histochemical demonstration of sites of cholin esterase activity. - Proc. Soc. expo BioI. Med. (N. Y.) 68, 354-358 (1948). -: Microscopic histochemistry: Principles and practice. - Chicago Univ. Press, Chicago 1952. GOODMAN, D. c., HALLET, R. E., WELCH, R. B.: Patterns of localization in the cerebellar corticonuclear projections of the albino rat. - J. compo Neurol. 121, 51-63 (1963). GOODMAN, R. R., KUHAR, M. J., HESTER, L., SNIJDER, S. H.: Adenosine receptors, autoradiographic evidence for their localization on axon terminals of excitatory neurons. - Science 220, 967-969 (1983). Go 01, H. C., FEIZI, T., KAPADIA, A., KNOWLEST, B. B., SOLTER, D., EVANS, M. J.: Stage specific embryonic antigen involves alpha 1-3 fucosylated type 2 blood group chains. Nature (Lond.) 292, 156-158 (1981). GOULD, B. B., RAKIC, P.: The total number, time of origin and kinetics of proliferation of neurons comprising the deep cerebellar nuclei in the rhesus monkey. - Exp. Brain Res. 44, 195-206 (1981). GRAFSTEIN, B., MURRAY, M.: Transport of protein in goldfish optic nerve during regeneration.Exp. Neurol. 25, 494-508 (1969). GRAYBIEL, A. M.: A stereometric pattern of distribution of acetylcholinesterase in the deep layers of the superior colliculus. - Nature (Lond.) 272, 5653, 539-541 (1978). GRAYBIEL, A. M., NAUTA, H. J. W., LASEK, R. J., NAUTA, W.'J. H.: A cerebello-olivary pathway in the cat: an experimental study using autoradiographic tracing techniques. - Brain Res. 58, 205r-211 (1973). GRAYBIEL, A. M., RAGSDALE, C. W.: Clumping of acetylcholinesterase activity in the developing striatum of the human fetus and young infant., - Proc. nat. Acad. Sci. (Wash.) 77, 1214-1218 (1980). GREENFIELD, S. A., STEIN, J. F., HODGSON, A. J., CHUBB, I. W.: Depression of nigral pars compacta cell discharge by exogeneous acetylcholinesterase. - Neuroscience 6, 2287-2296 (1981 ). GROENWEGEN, H. J., BOESTEN, A. J. P., VOOGD, J.: The dorsal column nuclear projections to the nucleus ventralis posterior lateralis thalami and the inferior olive in the cat. An autoradiographic study. - J. compo Neurol. 162, 505-518 (1975). GROENEWEGEN, H. J., VOOGD, J.: Olivocerebellar climbing fiber projections in the cat. An autoradiographic and degeneration study. - Acta morphol. neerl.-scand. 14, 247-249 (1976). -: The parasaggital zonation within the olivocerebellar projection in the vermis of the cat cerebellum. - J. compo Neurol. 174, 417-488 (1977). GROENEWEGEN, H. J., VOOGD, J., FREEDMAN, S. L.: The parasagittal zonation within the

Topographic histochemistry of the cerebellum' 151

olivocerebellar projection. II. Climbing fiber distribution in the intermediate and hemispheric parts of cat cerebellum. -]. compo Neurol. 183, 551-602 (1979). GUTH, L., ALBERS, R. W.: Histochemical demonstration of (Na+-K+-)activated adenosine triphosphatase. - ]. Histochem. Cytochem. 22, 320-326 (1974). HAGGQVIST, G.: Analyse der Faserverteilung in einem Riickenmarkquerschnitt (Th3). - Z. mikr.-anat. Forsch. 39, 1-34 (1936). HAINES, D. E.: Cerebellar corticovestibular fibers of the posterior lobe in a prosimian primate the lesser bushbaby (Calago senegalensis). - ]. compo Neurol. 160, 363-398 (1975a). -: Cerebellar cortical efferents of the posterior lobe vermis in a prosimian primate (Calago) and the tree shrew (Tupaia). -]. compo Neurol. 163, 21-40 (1975b). HANKER, ]. S., THORNBURG, L. P., YATES, P. E., MOORE, H. G.: The demonstration of cholinesterase by the formation of osmium black at the sites of Hatchett's brown. - Histochemie 37, 223-242 (1973). HARDONK, M. J., DE BOER, H. G. A.: 5 ' -Nucleotidase. III. Determinations of 5'-nucleotidase isoenzymes in tissue of rat and mouse. - Histochemie 12, 29-41 (1968). HARDONK, M. ]., ]ONKERS, A.: 5 ' -Nucleotidase. IV. Sex differences in the localization of rat liver 5/ -nucleotidase activity. - Histochemie 32, 363-368 (1972). HARDONK, M. ]., KOUDSTAAL, ].: Enzyme histochemistry as a link between biochemistry and morphology. - Progr. Histochem. Cytochem. 8/2, 1-68 (1976). HAROIAN, A. ].: Cerebello-olivary projections in the rat: An autoradiographic study. - Brain Res. 235, 125-130 (1982). HAYASHI, M.: Einig~ wicht'ige Tatsachen aus der ontogenetischen Entwicklung des menschlichen Kleinhirns. - Dtsch. Z. Nervenheilk. 81, 74-82 (1924). HEDQVIST, P., FREDHOLM, B. P.: Effects of adenosine on adrenergic neurotransmission: prejunctional inhibition and postjunctional enhancement. - Naunyn-Schmiedeberg's Arch. expo Path. Pharmak. 293, 217-223 (1976). HICKS, S. P.: Pathologic effects of antimetabolites. Acute lesions in hypothalamus peripheral ganglia and adrenal medulla caused by 3-acetylpyridine. - Amer.]. Path. 31, 189-199 (1955). HIS, W.: Die Entwicklung des menschlichen Rautenhirns vom Ende des ersten bis zum Beginn des dritten Monats. - Abhandl. K.S. Gesellsch. Wissensch. XXIX, 4-71 (1880). HOCHSTETTER, F.: Beitrage zur Entwicklungsgeschichte des menschlichen Gehirns. II. Die Entwicklung des Mittel- und Rautenhirns. - Franz Deuticke, Wien-Leipzig 1929. HOFFER, B. J., FREEDMAN, R., WOODWARD, D. ]., MOISES, H.: A functional role for adrenergic input to the cerebellar cortex: interaction of norephinephrine with mossy- and climbing fiber excitation and GABA mediated inhibition. - In: Interactions between putative neurotransmitters in the brain, Chap. 13 (GARATTINI, S. et aI., ed.) - Raven Press, New York 1978. HOFFER, B.]., SIGGINS, G. R., BLOOM, F. E.: Studies on norepinephrine containing afferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis. - Brain Res. 25, 523-534 (1971). HOGG, N., SCHLUSSERANKO, M., COHEN, J., REISER, J.: Monoclonal antibodies with specificity for monocytes and neurons. - Cell. 24,875-884 (1981). HOPWOOD, D.: Theoretical and practical aspects of glutaraldehyde fixation. - Histochem.]. 4, 267-303 (1972). HUNT, S., SCHMIDT, ].: Some observations on the binding patterns of a bungarotoxin in the central nervous system of the rat. - Brain Res. 157, 213-232 (1978). IPATA, P. L.: Resolution of 5/ -nucleotidase from non-specific phosphatase from sheep brain and its inhibition by nucleoside triphosphates. - Nature (Lond.) 214, 618 (1966). -: Studies on the inhibition by nucleoside triphosphates of sheep brain 5' -nucleotidase. Biochem. Biophys. Res. Comm. 27, 337-343 (1967).

152 . Enrico Marani -: Sheep brain 5' -nucleotidase. Some enzymic properties and allosteric inhibition by nucleoside triphosphates. - Biochem. 7, 5407-515 (1968 a). -: Inhibition of sheep brain 5 ' -nucleotidase by nucleoside triphosphatases. - Progr. Brain Res. 29, 527-534 (1968 b). ISRAEL, M., FRANCHON-MASTOUR, P.: Fractionnement du cortex cerebral de rat, distribution subcellulaire de la 5 ' -nucleotidase et du cholinesterases. - Arch. Anat. micro Morph. expo 59, 382-392 (1970). ITo, M.: Neuronal design of the cerebellar motor control system. - Brain Res. 40, 81-84 (1972). -: Cerebellar learning control of vestibulo-ocular mechanisms. - In: Mechanisms in Transmission of Signals for Conscious Behaviour (DESIRAJU, T., ed.), 1-22. - Elsevier, Amsterdam 1976. -: Recent advancement in the cerebellar physiology and pathology. - Neurology 21, 59-84 (1978). -: The cerebellum and neuronal control. - Raven Press, New York 1984. JACOB, A.: Das Kleinhirn. - In: v. Mollendorff's Handbuch der Mikroskopischen Anatomie des Menschen, IV/1:Nervensystem, 674-906. - Springer Verlag, Berlin 1928. JANSEN, J., BRODAL, A.: Das Kleinhirn. - In: v. Mollendorff's Handbuch der Mikroskopischen Anatomie des Menschen, IV18. - Springer Verlag, Berlin 1958. JANSEN, J., LARSELL, 0.: The comparative anatomy and histology of the cerebellum from monotremes through apes. - Univ. Minnesota Press, Minneapolis 1970. KABARA, J. J., KONVICH, D.: The extraction of brain isoenzymes with solvents of varying polarity. - P.S.E.B.M. 139, 132Cr1333 (1972). KAN, K. S. K., LIPEN, C. H. A. 0., ENG, L. F.: Immunohistochemical localization of cholinacetyltransferase in rabbit spinal cord and cerebellum. - Brain Res. 146, 221-230 (1978). KApPEL, R. M.: The development of the cerebellum of Macaca mulatta. - Thesis, Leiden (The Netherlands) 1980. KARNOVSKY, M. J., ROOTS, L.: A «direct-coloring» thiocholine method for cholinesterases. - J. Histochem. Cytochem. 12, 219-221 (1964). KASA, P., Joo, F., CSILLIK, B.: Histochemical localization of acetylcholinesterase in the cat cerebellar cortex. - J. Neurochem. 12, 31-35 (1965). KASA, P., SILVER, A.: The correlation between cholinacetyltransferase and acetylcholinesterase activity in different areas of the cerebellum of the rat and guinea pig. - J. Neurochem. 16, 389-396 (1969). KAWASAKI, T., SATO, Y.: Afferent projections from the dorsal nucleus of the raphe to the flocculus in cats. - Brain Res. 197, 49Cr502 (1980). KEMSHEAD, J. T., BICKNELL, D., GREAVES, M. F.: A monoclonal antibody detecting an antigen shared by neural and granulocytic cells. - Pediatr. Res. 15, 1282-1286 (1981). KLINAR, B., BRZIN, M.: A comparison between the one step and the two step copper thiocholine procedure for the cytochemical localization of cholinesterases. - Histochemie 50, 313-318 (1977 a). KLINAR, B., BRZIN, M.: Cytochemical localization of acetylcholinesterase in the rat striatum. Acta histochem. ena) 58, 223-231 (1977 b). KOCSIS, D. J., ENG, D. L., BHISITKUL, R. B.: Adenosine selectively blocks parallel fiber mediated synaptic potentials in rat cerebellar cortex. - P. N. A. S. 81, 6531-6534 (1984). KOEPPEN, A. H., BARRON, K. D., DENTlNGER, M. P.: Olivary hypertrophy. Histochemical demonstration of hydrolytic enzymes. - Neurology 30, 471-480 (1980). Kooy, F. H.: The inferior olive in vertebrates. - De Erven Bohn, Haarlem (The Netherlands) 1916. KORNELIUSSEN, H. K.: On the ontogenetic development of the cerebellum (nuclei, fissures and

a

Topographic histochemistry of the cerebellum' 153 cortex) of the rat, with special reference to regional variations in corticogenesis. - ]. Hirnforsch. 10, 379-412 (1968 a). -: On the morphology and subdivision of the cerebellar nuclei of the rat. - ]. Hirnforsch. 10, 109-122 (1968b). KOSTOPOULOS, G. K., LIMACHER, ]. ]., PHILLIS, ]. W.: Action of various adenine derivatives on 'cerebellar Purkinje cells. - Brain Res. 88, 162-165 (1975). KREUTZBERG, G. W.: Neuronal dynamics and axonal flow. IV. Blockage of intra-axonal enzyme transport by colchicine. - P. N. A. S. 62, 722-728 (1969). KREUTZBERG, G. W., BARRON, K. D.: Sf-Nucleotidase of microglial cells in the facial nucleus during axonal reaction. -]. Neurocytol. 7, 601-610 (1978 a). KREUTZBERG, G. W., BARRON, K. D., SCHUBERT, P.: Cytochemical localization of Sf-nucleotidase in glial plasma membranes. - Brain Research 158, 247-257 (1978 b). KREUTZBERG, G. W., SCHUBERT, P., TOTH, L., RIESKE, E.: Intradendritic transport to postsynaptic sites. - Brain Res. 62, 399-404 (1973). KRNJEVIC, K., SILVER, A.: A histochemical study of cholinergic fibres in the cerebral cortex. -]. Anat. (Lond.) 99, 711-759 (1965). LAKE, B. D.: The histochemistry of phosphatases: the use of lead acetate instead of lead nitrate.]. roy. micro Soc. 85,73-75 (1965). LAKKE, E. A.]. F., FEIRABEND, H. K. P.: Development of the spinocerebellar tracts in the white leghorn. - Proc. 4th Int. Congr. Develop. Neurosci. 44 (1983). -: Development of sagittal organization of the spinocerbellar tracts in the white Leghorn. Neurosci. Lett. S 18, 293 (1984). LAKKE, E. A. ]. F., GULDEMOND, ]. M., VOOGD, ].: The ontogeny of the spinocerebellar projection. A study using WGA-HRP as a tracer. - Acta histochem. Gena), in press. LARRAMENDI, L. M. H.: Analysis of synaptogenesis in the cerebellum of the mouse. - In: Neurobiology of cerebellar evolution and development (ed. R. LLINAS.), p. 803-843. A. M. A., Chicago 1969. LARSELL, 0.: Cerebellum and corpus pontobulbare of the bat (Myotis). - ]. compo Neurol. 64, 275-302 (1936). -: The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat. -]. compo Neurol. 97, 281-356 (1952). -: The cerebellum of the cat and the monkey. -]. compo Neurol. 99, 135-200 (1953). LARSELL, 0., Dow, R. S.: The development of the cerebellum in bat (Corynorhinus sp.) and certain other mammals. -]. compo Neurol. 62, 443-468 (1935). LARSELL, 0., ]ANSEN, ].: The comparative anatomy and histology of the cerebellum from monotremes through apes. - University of Minnesota Press, Minneapolis 1970. LEWIS, P. R. and KNIGHT, D.P.: Staining methods for sectioned material. - In: Practical in electron microscopy (GLAUERT, A. M., ed.), 137-223. - North-Holland, Amsterdam 1977. LEWIS, P. R., SHUTE, C. C. D.: The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. -]. Cell Sci. 1, 381-390 (1966). LLINAS, R., WALTON, K., HILLMAN, D. E., SOTELO, c.: Inferior olive: its role in motor learning. - Science 190, 1230-1231 (1975). LONG, ]. P.: Structure activity relationships of the reversible anti-cholinesterase agents. Handb. expo Pharmakologie, Bd. 15 (ed. G. B. KOELLE), 374-427. - Springer Verlag, Berlin 1963. LOWRY, O. H., ROSENBROUGH, N. ]., FARR, A. L., RANDALL, R. ].: Protein measurement with the folin phenol reagent. -]. bioI. Chern. 193, 265-275 (1951). MAAT, G. ]. R.: Embryologie van het cerebellum. I. Het menselijk cerebellum. - In: Boerhaave cursus «Het cerebellum en zijn functiestoornissen». - Leiden 1978.

154 . Enrico Marani -: The onset of the formation of the cerebellar nuclei in man. - Neurosci. Lett. 55, 441 (1980). MAGNUSSON, B. c., HEYDEN, G., SVENNSON, S. E.: Histochemical observations on 5'-nucleotidase activity in squamous cell carcinomas of the rat. - Histochemistry 42, 1-8 (1974). MANOCHA, S. L., SHANTA, T. R.: Macaca Mulatta, Enzyme histochemistry of the nervous system. Chap. X: Cerebellum. - Acad. Press, New York 1970. MAo, C. c., GUIDOTTI, A., COSTA, E.: The regulation of c. GMP in rat cerebellum. Possible involvement in putative amino acid neurotransmitters. - Brain Res. 79, 510-514 (1974). MARANI, E.: Subcellular distribution of 5' -nucleotidase activity in the mouse cerebellum. - J. expo Neurol. 57,1042-1048 (1977). -: A method for orienting cryostate sections for three dimensional reconstructions. - Stain Technol. 53, 265-268 (1978). -: 5' -Nucleotidase in der Molekularschicht des Rattenkleinhirns (rhythmische Veranderungen der unspezifischen Phosphatasen). - Acta histochem. Gena), Suppl. XXI, 237-242 (1980a). -: K+- and Na+ activation of cerebellar 5'-nucleotidase. - J.exp. Neurol. 67, 412-422 (1980b). -; Enzyme Histochemistry. - In: Methods in Neurobiology, Vol. 1, 481-581 (ed. R. LAHUE). Plenum Press, New York 1981. -; The ultrastructural localization of 5' -nucleotidase in the molecular layer of the mouse cerebellum. - In: Neurotransmitter interaction and compartmentation (H. F. BRADFORD, ed.), 558-572. - Plenum Press, New York 1982 a. -: Topographic enzyme histochemistry of the mammalian cerebellum. 5' -Nucleotidase and acetylcholinesterase. - Thesis, Leiden 1982 b. MARANI, E., BOEKEE, A.: Aspects histoenzymologiques de la localisation de I'adenylcyclase de la 3-c', 5' -nucleotide phosphodiesterase, de la 5' -nucleotidase et de I'alpha-glucanphosphorylase dans Ie cervelet de la souris. - Bull. Assoc. Anat. (Nancy) 57, 555-564 (1973). MARANI, E., BOESTEN, A. J. P.: Histochemistry of experimental hypertrophy of the inferior olive in the cat. - J. Anat. (Lond.)129, 203-205 (1979). MARANI, E., FEIRABEND, H. K. P.: AChE distribution in the developing cerebellum of the rabbit. An enzyme histochemical and immunological study. - J. Anat. (Lond.) 137,429-430 (1983). MARANI, E., FEIRABEND, H. K. P., EPEMA, A. H., TETTERoo, P. A. T.: A longitudinal band pattern for acetylcholinesterase and the monoclonal antibody B4,3 in the developing rabbit and fowl cerebellum. - Acta morpho neerl.-scand. 21, 303-304 (1983a). MARANI, E., RUIGROK, T. J. H.: Enzyme histochemical changes in some optic projection areas of the goldfish after optic nerve lesions. - Neurosci. Lett. 23, 223-238 (1981). -: Enzyme histochemical changes in retinal ganglion cells and the optic nerve after goldfish optic nerve lesion. A regenerative and hypertrophic phenomena study. - Acta morpho neerl.-scand. 22,97-114 (1984). MARANI, E., TETTERoo, P. A. T.: A longitudinal band-pattern for the monoclonal human granulocyte antibody B4,3 in the cerebellar external granular layer of the immature rabbit. Histochemistry 78, 157-161 (1983). MARANI, E., TETTERoo, P. A. T. , VAN DER VEEKEN, J. : The ultrastructural localization of the monoclonal human granulocyte antibody B4,3 in cell suspensions of the immature rabbit cerebellum. - Cell BioI. into Rep. 7, 763-769 (1983b). MARANI, E., VOOGD, J.: Some aspects of the localization of the enzyme 5 ' -nucleotidase in the molecular layer of the cerebellum of the mouse. - Acta morpho neerl.-scand. 11, 353-354 (1973). -; An acetylcholinesterase band pattern in the molecular layer of the cat cerebellum. - J. Anat. (Lond.) 122, 710-712 (1976). -: An acetylcholinesterase band pattern in the molecular layer of the cat cerebellum. - J. Anat. (Lond.) 124,335-345 (1977).

Topographic histochemistry of the cerebellum' 155

-: The ultrastructural localization of 5 ' -nucleotidase reaction products in the molecular layer of the mouse cerebellum. - Neurosci. Lett. 51, 151 (1978). -: The morphology of the mouse cerebellum. - Acta morpho neerl.-scand. 17, 33-52 (1979). -: Comparison of the myeloarchitecture of the fibre layer and the 5 ' -nucleotidase pattern in the molecular layer of the mouse cerebellum. - J. Anat. (Lond.) 133, 136-137 (1981). -: Acetylcholinesterase staining in subdivisions of the inferior olive in several species. - Anat. Rec. 211, 122A (1985). MARANI, E., VOOGD, J., BOEKEE, A.: Acetylcholinesterase staining in subdivisions of the eat's inferior olive. - J. compo Neurol. 174, 209-226 (1977). MARtsCHAL, P.: L'olive bulbaire. Anatomie, ontogenese, phylogenese, physiologie et physiopathologie. - Doin, Paris 1934. MARTIN, G. F., DOM, R., KING, J. S., ROBARDS, M., WATSON, C. R. R.: The inferior olivary nucleus of the opossum, its organization and connections. - J. compo Neurol. 160, 507-534 (1975). MARTINEZ-RoDRIQUEZ, R., GARCIA-SEGURA, L. M., TOLEDANO, A., MARTINEZ-MuRILLO, R.: Localization of aspartate aminotransferase and glutamic dehydrogenase in the Edinger-Westphal and oculomotor nuclei of Lacerta lepida. - Neurosci. Lett. 6, 65-68 (1977). MARR, D.: A theory of cerebellar cortex. - J. Physiol. 202, 437-470 (1969). MAZZA, J. P., HANKER, J. S., DIXON, A. P. : Ultrastructural localization of cholinesterase activity in the trigeminal ganglion of the rat. - J. Anat. (Lond.) 115, 65-78 (1973). MIALE, I. L., SIDMAN, R. L.: An autoradiographic analysis of histogenesis in the mouse cerebellum. - Exp. Neurol. 4, 277-296 (1961). MCGEER, E. G., KIMURA, H., MCGEER, P. L., PENG, J. H.: Cholinergic systems in the CNS.In: G. F. BRADFORD (ed.), Neurotransmitter Interaction and Compartmentation, pp.253-290. - Plenum Press, New York 1982. MEIJER, A. E. F. H.: Semipermeable membrane techniques in quantitative enzyme histochemistry. - In: Trends in Enzyme Histochemistry and Cytochemistry. Ciba Found. Symp. 73, 103-119 (1980). M0LLGARD, K., LUNDBERG, J. J., WIKLUND, L., LACHENMAYER, L.: Morphologic consequences of serotonin neurotoxin administration: neuron target cell interaction in the rat subcommissural organ. - Ann. NY. Acad. Sci. 305,262-288 (1978). MOSES, H. L., ROSENTHAL, A. S., BEAVER, D. L., SCHUFFMANN, S. S.: Lead ion and phosphatase histochemistry. II. Effect of adenosine triphosphatase hydrolysis by lead ion on the histochemicallocalization of adenosine triphosphatase activity. - J. Histochem. Cytochem. 14, 702-710 (1966). MONMOI, T., YOKOTA, Y.: Alterations of glycolipids of human leukemia cell line HL-60 during differentiation. - J. nat. Cancer Inst. 70, 229-236 (1983). MUGNAINI, E.: Organization of cerebellar cortex. - Exp. Brain Res. Suppl. I, 8-19 (1976). MURRAYA, M., FORMAN, D. S.: Fine structural changes in goldfish retinal ganglion cells during axonal regeneration. - Brain Res. 32,287-298 (1971). NAIDOO, D.: The activity of 5 ' -nucleotidase determined histochemically in the devloping rat brain. - J. Histochem. Cytochem. 10,421-436 (1962). NAS, W. S. c., GERRITS, N. M., VOOGD, J.: The erebeliar projection of the external cuneate nucleus in the cat. - Acta morpho neerl.-scand. 19, 95-96 (1981). ODUTOLA, A. B.: The topographical localization of acetylcholinesterase in the adult rat cerebellum: a reappraisal. - Histochemie 23,98-106 (1970). OERTEL, W. H., SCHMECHEL, D. E., MUGNAINI, E., TAPPAZ, M. L., KOPIN, I. J.: Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neurosci. 12, 2715-2735 (1981).

156 . Enrico Marani

OGAWA, T.: Beitrage zur vergleichenden Anatomie des Zentralnervensystems der Wassersaugetiere. Dber die Kleinhirnrinde der Pinnipedien und Cetaceen. - Arb. Anai. Inst. Serndai 17,63-136 (1936). OSCARSSON, 0.: The sagittal organization of the cerebellar anterior lobe as revealed by the projection patterns of the climbing fiber system. - In: Neurobiology of cerebellar evolution and development (ed. LLINAS, R.), 525-537. - A. M. A., Chicago 1969. -: Functional organization of spino-cerebellar paths. - In: Handbook of Sensory Physiology, Vol. II (ed. A. IGGo), Chap. 11. - Springer Verlag, Berlin 1973. -: Functional organization of olivary projection to the cerebellar anterior lobe. - In: Inferior Olivary Nucleus: Anatomy and Physiology (ed. COURVILLE, J.), 279-289. - Raven Press, 1980. OZEKI, M., SATO, M.: Potentiation of excitatory junctional potentials and glutamate induced responses in crayfish muscle by 5' -ribonucleotides. - Compo Biochem. Physiol. 3, 203-218 (1970). PALAY, S. L., CHAN-PALAY, V.: Cerebellar cortex. - Springer Verlag, Heidelberg-New York 1974. PAPADIMITRIOU, ]. M., VAN DUYN, P.: Effect of fixation and substrate protection on the isoenzymes of aspartate aminotransferase studied in a quantitative cytochemical model system. - ]. Cell BioI. 47, 71-83 (1970a). -: The ultrastructural localization of the isoenzymes of aspartate aminotransferase in murine tissues. - J. Cell. BioI. 47, 84-98 (1970b). PASQUINI, J. M., SOTO, E. F.: Extraction of proteolipids from nervous tissue with n-butanolwater. - Life Sci. 11, part II, 433-443d (1972). PATTON, S., TRAMS, E. G.: The presence of plasma membrane enzymes on the surface of bovine milk fat globules. - FEBS Lett. 14, 230-232 (1971). PEARSE, A. G. E.,: Histochemistry: Theoretical and Applied, Vol. 1, 4th ed. - Churchill/Livingstone, Edinburgh-London 1980. -: Histochemistry: Theoretical and Applied, VoU, 3rd ed., Churchill/ Livingstone, Edinburgh-London 1972. PERSIJN, ]. P., DAEMS, W. J., MAN, J. c., MEIJER, A. E.: The demonstration of adenosine triphosphatase activity with the electron microscope. - Histochemie 2, 372-382 (1961). PERSIJN, J. P. , VAN DER SLIK, W. A.: A new method for the determination of serum 5'nucleotidase activity in the presence of beta-glycerophosphate. - Clin. Biochem. 2, 335-337 (1969). PERSIJN, J. P. , VAN DER SLIK, W. A., TIMMER, C. J.: On the determination of serum 5'nucleotidase activity in the presence of beta-glycerophosphate. - Clin. Biochem. 2, 335-337 (1970). PHILLIS, J. W., Wu. P. H.: The role of adenosine and its nucleotides in central synaptic transmission. A review. - Progr. Neurobiol. 16, 187-239 (1981). PILCHER, C. W. T., JONES, D. G.: The distribution of 5' -nucleotidase in subcellularfractions of the mouse cerebellum. - Brain Res., 24, 143-147 (1970). PRATT, O. E.: The proportion of phosphatase activity demonstrable in brain by histological techniques. - Biochem. J. 55, 140-145 (1953). -: Some factors affecting rat brain phosphatase activity in fresh tissue suspensions and in histochemical methods. - Biochim. biophys. Acta (Arnst.) 14, 380-389 (1954). PULL, J., McILWAIN, H.: Metabolism of [14C] adenine and derivatives by cerebral tissues, superfused and electrically stimulated. - Biochem. J. 126, 965-973 (1972). RAMON Y CAJAL, S.: Histologie du systeme nerveux de l'homme et des vertebres, Vol. 1,2. Maloine, Paris 1909-1911. Reprint Concejo Superior de Invest. Cientif. Madrid 1972.

Topographic histochemistry of the cerebellum' 157

RAMON MOLINAR, E.: Acetylcholinesterase distribution in the brain stem of the cat. - Ergebn. Anat. 46, 1-52 (1972). RAUVALA, H.: Cell surface carbohydrates and cell adhesion. - Trends neurochem. Sci. 8,323-326 (1983). REIS, J.: La nucleotide et sa relation avec les desanimations des nucleotides dans Ie coeur et dans Ie muscle. - Bull. Soc. Chim. BioI. 16, 385 (1934). RETzIUs, G.: Die nervosen Elemente der Kleinhirnrinde. - Biologische Untersuchungen, Neue Folge Bd. III, 17-24. - Samson/Wallis, Stockholm 1892a. -: Kleinere Mitteilungen von den Gebieten der Nervenhistologie. I. Ober die Golgischen Zellen und Kletterfasern Ramon y Cajal's der Kleinhirnrinde. - Biologische Untersuchungen, Neue Folge Bd. IV, 57-59. - Samson/Wallis, Stockholm 1892b. RINVIK, E., GROFOVA, J.: Observations on the fine structure of the substantia nigra in the cat.Exp. Brain Res. 11, 229-248 (1970). RIODAN, J. R., SLAVIK, M.: Interactions of lectins with membrane glycoproteins. Effects of concanavalin A on 5 ' -nucleotidase. - Biochem. biophys. Acta (Arnst.) 373,356-360 (1974). RILEY, H. A.: A comparative study of the Arbor vitae and the folial pattern of the mammalian cerebellum. - Arch. Neurol. Psychiat. 20, 895-1034 (1928). RONDOT, P., BEN HAMIDA: Myoclonies du voile et myoclonies squelettiques. Etude clinique et anatomique. - Revue neurol. 119, 159 (1968). ROSE, G., SCHUBERT, P.: Release and transfer of eH) adenosine derivatives in the cholinergic septal system. - Brain Res. 121, 252-257 (1977). ROSENTHAL, A. S., MOSES, H. L., BEAVER, D. L., SCHUFFMANN, S. S.: Lead ion and phosphatase histochemistry. I. Non-enzymatic hydrolysis of nucleoside phosphatases by lead ions. - J. Histochem. Cytochem. 14, 698-701 (1966). ROSENTHAL, A. S., MOSES, H. L., GANOTE, CH. E., TICE, L.: The participation of nucleotide in the formation of phosphatase reaction product: a chemical and electron microscopic autoradiographic study. - J. Histochem. Cytochem. 17, 839-847 (1969b). ROSENTHAL, A. S., MOSES, H. L., TICE, L., GANOTE, CH. E.: Lead ion and phosphatase histochemistry. III. The effects of lead and adenosine triphosphate concentration on the incorporation of phosphate into fixed tissue. - J. Histochem. Cytochem. 17, 608-612 (1969a). ROSSlER, J.: Choline acetyltransferase: A review with special reference to its cellular and subcellular localization. - Int. Rev. Neurobiol. 29, 283-337 (1977). ROSSUM, J. VAN: Corticonuclear and corticovestibular projections of the cerebellum Thesis. Van Gorcum Co., Assen (The Netherlands) 1969. ROTH, R. H., GIARMAN, N. J.: Gamma-butyrolaceton and gamma-hydroxybutyric acid. I. Distribution and metabolism. - Biochem. Pharmacol. 15, 1333-1381 (1966). RUDEBERG, S. I.: Morphogenetic studies on the cerebellar nuclei and their homologization in different vertebrates including man. - Thesis Lund (Sweden) 1961. RUSSCHEN F. T., GROENEWEGEN, H. J., VOOGD, J.: Reticulocerebellar connections in the cat. An autoradiographic study. - Acta morpho neerl.-scand. 14, 245-246 (1976). RUSTIONI, A., SANYAL, S., KUYPERS, H. G. J. M.: A histochemical study of the distribution of the trigeminal divisions in the substantia gelatinosa of the rat. - Brain Res. 32,45-32 (1971). SABATINI, D. P., MILLER, F., BARNETT, R. J.: Aldehyde fixation for morphological and enzyme histochemical studies with the electron microscope. - J. Histochem. Cytochem. 12, 57-71 (1964). SCHILD, R. F.: Length of the parallel fibers in the rat cerebellar cortex. - J. Physiol. (Lond.) 303, 25P (1980). SCHLAEFFER, W. W., LA VALLE, M. c., TORACK, R. M.: Cytochemical demonstration of nucleoside phosphatase activity in myelinated nerve fibers of the rat. - Histochemie 18, 281-292 (1969).

158 . Enrico Marani SCHMIDT, M. J., RYAN, J. J., MOLLOY, B. B.: Effects of kainic acid, a cyclic analogue of glutamic acid, on cyclic nucleotide accumulation in slices of rat cerebellum. - Brain Res. 112, 113-126 (1976). SCHMIDT, M. J., THORNBERRY, J. F., MOLLOY, B. B.: Effects of kainic acid and other glutamate analogues on cyclic nucleotide accumulation in slices of rat cerebellum. - Brain Res. 121, 182-189 (1977). SCHUBERT, P.,. KOMP. W., KREUTZBERG, C. W.: Correlation of 5'-nucleotidase activity and selective transneuronal transfer of adenosine in the hippocampus. - Brain Res. 168, 419--424 (1979). SCHUBERT, P., KREUTZBERG, C. W.: Transport, release and electrophysiological effects of adenosine. - Neurosci. Lett., 5 1, 183 (1978). SCHUBERT, P., MITZDORF, V.: Analysis and quantitative evaluation of the depressive effect of adenosine on evoked potentials in hippocampal slices. - Brain Res. 172, 186-190 (1979). SCHWARTZ, M. K., BODANSKY, 0.: Properties of activity of 5 ' -nucleotidase in human serum and application in diagnosis. - Amer. J. Path. 42, 572-580 (1964). SCOTT, T. G.: A unique pattern of localization within the cerebellum. - Nature (Lond.) 200, 793 (1963). -: A unique pattern of localization within the cerebellum of the mouse. - J. compo Neurol. 122, 1-7 (1964). -: The specificity of 5 ' -nucleotidase in the brain of the mouse. - J. Histochem. Cytochem. 13, 675-667 (1965). -: The distribution of 5' -nucleotidase in the brain of the mouse. - J. compo Neurol. 129, 97-113 (1967). -: The quantative assay of 5 ' -nucleotidase in the brain by histophotometry. - Histochem. J. 1, 215-229 (1969). SEDO, J. R., HESS, D. T., VOOGD, J.: Longitudinal organization in the primate cerebellum. Neurosci. Lett. 518, 384 (1984). SHIMIZU, N., ISHII, S.: Electron microscopic observations on nucleolar extrusions in nerve cells of the rat hypothalamus. - Z. Zellforsch. 67, 367-372 (1966). SHUTE, C. C. D., LEWIS, P. R.: The cholinergic innervation of the cerebellum. - J. Anat. (Lond.) 98,466 (1964). SIGGINS, G. R., HOFFER, B. J., BLOOM, F. E.: Studies on norepinephrine containing afferents to Purkinje cells of rat cerebellum. III. Evidence for mediation of norepinephrine effects by cyclic 3' ,5' -adenosine monophosphate. - Brain Res. 25, 535-553 (1971). SILINSKY, E. M.: On the association between transmitter secretion and the release of adenine nucleotides from mammalian motor nerve terminals. - J. Physiol. (Lond.) 247, 145-162 (1975). SILVER, A.: Cholinesterase of the central nervous system with special references to the cerebellum. - Int. Rev. Neurobiol. 10, 57-108 (1967). -: The biology of cholinesterases. - In: North Holland Research Monographs «Frontiers in Biology», Vol 36 (NEUBERGER, A., TATUM, E. L., ed.). - North Holland/American Elsevier, Amsterdam 1974. SINNIGE, J. L. M.: Anatomisch onderzoek over de verbindingen van de kleine hersenen bij de hondo - Noord Hollandse Vitg. N.V. Mij Amsterdam (1938). SKOU, J. c.: The influence of some cations on an adenosine triphosphatase from peripheral nerves.- Biochim. biophys. Acta (Arnst.) 23, 394--401 (1957). SLADEK, J. R., BOWMAN, P.: The distribution of catecholamines within the inferior olivary complex of the cat and rhesus monkey. - J. compo Neurol. 163, 203-215 (1975). SNELL, E. E.: Metallo enzymes in the study of active enzyme sites. - Fed. Proc. 20, 2, 81-83 (1961).

Topographic histochemistry of the cerebellum' 159

SOKOLOFF, L.: Metabolism of ketone bodies by the brain. - Anm. Rev. Med. 24, 271-280 (1973). SONG, C. S., KAPPERS, A., BODANSKY, 0.: 5' -Nucleotidase of plasma membranes of the rat liver. Studies on the subcellular distribution. - Ann. New York Acad. Sci. 166, 565-573 (1969). SONG, C. S., RAy, T. K.: A modified method for the isolation of plasma membranes from rat liver. - Biochem. biophys. Acta (Arnst.) 196, 1-19 (1970). SOTELO, c.: Cerebellar neuroglia. Morphological and histochemical aspects. - In: The cerebellum. - Progr. Brain Res. 25, 226-250 (1967). SOTELO, c., LUNAS, R., BAKER, R.: Structural study of the inferior olivary nucleus of the cat. Morphological correlates of electrotonic coupling. - J. Neurophysiol. 37, 541-559 (1974). SPACEK, j., PARIZEK, J., LIEBERMAN, A. R.: Golgi cells, granule cells and synaptic glomeruli in the molecular layer of the rabbit cerebellar cortex. - J. Neurocytol. 2, 407-428 (1973). SPREY, T. E.: Localization of 5 ' -nucleotidase and its possible significance in some of the imaginal disks of Calliphora erythrocephala. - Netherl. J. Zool. 20, 419-432 (1970). SPRINGER, A. D., AGRANOFF, B. W.: Effect of temperature on the rates of goldfish optic nerve regeneration: an autoradiographic and behavioural study. - Brain Res. 128, 405-415 (1977). STACH, E., OCKENFELS, H., PILGRIM, c.: Electrophoretic separation and properties of acid phosphatase isoenzyme from rat brain. - Acta histochem. Gena) 60, 121-131 (1977). STEINBUSCH, H. W. M.: Serotoninergic neurons in the central nervous system of the rat. Thesis,Nijmegen (The Netherlands) 1982. STOCKLE, H., TEN BRUGGENCATE, G.: Climbing fiber mediated rhythmic modulations of potassium and calcium in cat cerebellar cortex. - Exp. Neurol. 61, 226-230 (1978). STONE, T. W.: Physiological tools for adenosine and adenosine 5 ' -triphosphate in the nervous system. - Neuroscience 6,523-555 (1981). STREETER, G. L.: Anatomy of the floor of the fourth ventricle. - Amer. J. Anat. 11 (1903). STROUD, B. B.: The mammalian cerebellum. - J. compo Neurol. 5, 71-118 (1895). SURAN, A. A.: 5 ' -Nucleotidase and acid phosphatase of spinal cord. Comparative histochemistry and specificity in mouse and cat spinal cords. Cytological localization in mouse substantia gelatinosa. - J. Histochem. Cytochem. 22, 802-811 (1974a). -: 5 ' -Nucleotidase and acid phosphatase of spinal cord. Quantitative histochemistry in cat and mouse spinal cord and in mouse brain. - J. Histochem. Cytochem. 22, 812-818 (1974b). SVENDSEN, P. j.: On the procedure of preparative istachophoresis. - Science Tools 20, 1-3 (1973). SZENTAGOTHAI, j., RA]KOVITS, D.: Uber den Drsprung der Kletterfasern des Kleinhirns. - Z. Anat. Entwickl.-Gesch. 121, 130-141 (1959). TANDLER, C. j.: An acid soluble component of the nucleolus: The cytochemical specificity of the lead acetate reaction. - J. Histochem. Cytochem. 4, 331-340 (1956). TETTEROO, P. A. T., LANSDORP, P. M., GEURTS VAN KESSEL, A. H. M., HAGEMEI]ER, A., KR VON DEM BORNE, A. E. G.: Serological and biochemical characterization of human myeloid associated antigens and the expression in human-mouse cell hybrides. - In: BERNHARD, A., BOUMSELL, L., eds., First Int. Workshop on Human Leucocytes Differentiation Antigens (1984). TETTEROO, P. A. T., VISSER, F. j., Bos, M. J. E., GEURTS VAN KESSEL, A. H. M., TROMP, J. F., KR VON DEM BORNE, A. E. G.: Serological, biochemical and cytogenetic studies with the granulocyte monoclonal antibodies of the 'M Protecol'. - Second Int. Workshop on Human Leucocytes Differentiation Antigens (in press). TEWARI, H. B., BOURNE, G. H.: Histochemical studies on the distribution of alkaline and acid phosphatase and 5'-nucleotidase in the cerebellum of the rat. - J. Anat. (Lond.) 97, 65-72 (1963).

160 . Enrico Marani

TINKLENBERG, ].: Graphical reconstruction, microdissection with a pencil. - Audiov. Media Med.2, 102-106 (1979). -: Graphical reconstruction and stereoscopy. - Audiov. Media Med. 3, 68-71 (1980). TOLBERT, D. L., BANTU, H., BLOEDEL, ]. R.: Anatomical and physiological evidence for a cerebellar nucleo-cortical projection in the cat. - Neurosciene 1,205-217 (1976). TORRE, J. C. DE LA, SURGEON, J. W.: A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method. - Histochemistry 49, 81-93 (1976). TSUJI, S.: On the chemical basis of thiocholine methods for demonstration of acetylcholinesterase activities. - Histochemistry, 42, 99-110 (1974). UCHIZONO, K.: Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat. - Nature (Lond.) 207, 642-543 (1965). URDAL, D. L., BRENTHAL, T. A., BERNSTEI, J. D., HAKOMORI, S. I.: Granulocyte reactive monoclonal antibody 1G10 identifies the GAL-beta-1-4 (Fuc-alpha-1-3) GIcNAc (X determinant) expressed in HL-60 cells on both glycolipid and glycoprotein molecules. - Blood 62, 1022-1026 (1983). UUSITALO, R. J., KARNOVSKY, M. J.: Surface localization of 5' -nucleotidase on the mouse lymphocyte. - J. Histochem. Cytochem. 25, 97-103 (1977a). -: Surface localization of 5' -nucleotidase on the mouse lymphocyte. - J. Histochem. Cytochem. 25, 87-96 (1977b). VAN DER SUK, W.: Determination and diagnostic value of 5' -nucleotidase. - Thesis Leiden University. - Krips Repro, Meppel1975. VAN DER WAARD, M. : Choline en choline derivaten in ratte hersenen. In vitro onderzoek over het metabolisme. - Thesis Leiden University. - Pasmans, Den Haag 1974. VAN GEHUCHTEN, A.: La structure des centres nerveux. La moelle epiniere et Ie cervelet. Cellule 7, 83-122 (1891). VAN ROSSUM, J.: Corticonuclear and corticovestibular projections of the cerebellum. - Thesis. Van Gorcum Co., Assen (The Netherlands) 1969. VAN VALKENBURG, C. T.: Bijdrage tot de kennis eener localisatie in de menselijke kleine hersenen. - Ned. Tijdschr. Geneesk. 57, 6-24, (1913). VERHAART, W. J. c.: The red nucleus, the cerebellum and the inferior olive in man. - Acta neurol. scand. 38/1, 67-68 (1962). -: A stereotactic atlas of the brainstem of the cat. - Van Gorcum Co., Assen (The Netherlands) 1964. VERHAART, W. J. c., VOOGD, J.:Hypertrophy of the inferior olives in the cat. - J. Neuropath. expo Neurol. 21, 92-103 (1962). VIELVOYE, G. J.: Spinocerebellar tracts in the white leghorn (Gallus domesticus). - Thesis, Leiden (The Netherlands) 1977. VILLANI, L., CIANI, F., CONTESTABILE, A.: Electron microscope histochemistry of acetylcholinesterase in the optic tectum of teleosts. - J. Hirnforsch. 20, 539-551 (1979). VIZI, E. S.: Presynaptic modulation of neurochemical transmissions. - Vth Int. Neurobiol. Sympos. Magdeburg (GDR) 1977. VOOGD, J.: The cerebellum of the cat. - Thesis, Leiden University. - Van Gorcum Co., Assen (The Netherlands) 1964. -: Comparative aspects of the structure and fibre connections of the mammalian cerebellum. In: Progress in Brain Res. 25, The cerebellum. (eds. Fox, C. A., SNIDER, R. S.), pp. 99-175.Elsevier, Amsterdam 1967. -: The importance of fiber connections in the comparative anatomy of the mammalian cerebellum. - In: LUNAS, R., ed., Inst. for Biomed. Res. Symp. Chicago, 493-514 (1969).

Topographic histochemistry of the cerebellum' 161 -: BOLK'S subdivision of the mammalian cerebellum. Growth centres and functional zones. Acta morpho neerl.-scand. 13, 35-54 (1975). -: The olivocerebellar projection in the cat. - In: The cerebellum, New Vistas (eds. PALAY, S. L., CHAN-PALAY, V.). - Exp. Brain Res. 56, 134-161 (1982). -: Anatomical evidence for a cortical X zone in the cerebellum of the cat. - Abstr. 13th Annual Meet. Soc. Neurosci., 1091 (1983). VOOGD, J., BIGARE, F.: Topographical distribution of olivary and cortico-nuclear fibres in the cerebellum: A review. - In: The inferior olivary nucleus (COURVILLE, J., ed.), 207-234 (1980). VOOGD, J., BIGARE, F., GERRITS, N. M., MARANI, E.: Structure and fiber connections of the cerebellum. - In: Glial and Neuronal Cell Biology, 259-268. - Liss Inc., New York 1981. VOOGD, J., BOESTEN, A. J. P.: A light and electron microscope study of inferior olivary hypertrophy. - 16th Dutch Fed. Meet. 159-163 (1975). -: A light and electron microscopical study of inferior olivary hypertrophy in the cat. - J. Anat. (Lond.) 122, 712-713 (1976). VOOGD, J. BROERE, G., VAN ROSSUM, J.: The medio-lateral distribution of the spinocerebellar projection in the anterior lobe and the simplex lobule in the cat and a comparison with some other afferent systems. - Psychiat. Neurol. Neurochir. 72, 137-151 (1969). VOOGD, J. FEIRABEND, H. K. P.: Classical methods in neuroanatomy. - In: Chap. 5, Methods in Neurobiology, 301-364 (LAHUE, R., ed.). - Plenum Press, New York 1981. VOOGD, J., GERRITS, N. M., MARANI, E.: The cerebellum of the rat. - In: The Rat Nervous System, Vol. 2 (ed. PRAXINOS, G.). - Academic Press, New York 1985. VOOGD, J., HESS, D. T., MARANI, E.: The parasagittal zonation of the cerebellar cortex in cat and monkey. Topography, distribution of acetylcholinesterase and development (KING, J. S., COURVILLE, J., eds.). - Liss Inc., New York 1986 (in press). VOOGD, J., MARANI, E.: A comparison of the 5' -nucleotidase bandpattern and the myeloarchitecture in the cerebellum of the mouse. - Proc. 20th Dutch Fed. Meet., 447 (1979). VOOGD, J. SEDO, J., GERRITS, N. M.: Olivocerebellar projections in the rhesus monkey: an autoradiographic study. - Neurosci. Lett. 5 10, 509 (1982). -: The olivocerebellar projection in the rhesus monkey: an autoradiographic study. - Acta morpho neerl.-scand. 21, 324-325 (1983). WAKED, N., KERR, S. E.: The distribution of phosphomonoesterases and pyrophosphatases in the particulate fractions of dog cerebellum. - J. Histochem. Cytochem. 3, 75-84 (1955). WALBERG, F.: The vestibulo- and reticulocerebellar projections in the cat as studied with horseradish peroxidase as a retrograde tracer. - In: The Cerebellum, New Vistas (eds. PALAYS, S. L., CHAN-PALAY, V.). - Exp. Brain Res. 56, 477--500 (1982). WASSEF, M., SOTELO, c.: Asynchrony in the expression of guanosine 3',5'-phosphate dependent protein kinase by clusters of Purkinje cells during the perinatal development of rat cerebellum. - Neuroscience 13, 1217-1241 (1984). WEIDENREICH, F.: Zur Anatomie der zentralen Kleinhirnkerne der Siiuger. - Z. Morph. Anthrop. 1, 259-312 (1899). WERMAN, R.: Criteria for identification of a central nervous system transmitter. - Compo Biochem. Physiol. 18, 745-766 (1966). WHITTAKER, V. P., MICHAELSON, J. A., KIRKLAND, R. J. H.: The separation of synaptic vesicles from nerve-ending particles (,synaptosomes'). - Biochem. J. 90, 293-303 (1964). WIDNELL, C. c.: Cytochemical localization of 5' -nucleotidase in subcellular fractions isolated from the rat liver. I. The origin of 5'-nucleotidase in microsomes. - J. Cell BioI. 52, 542-558 (1972). WIDNELL, C. c., UNKELESS, J. c.: Partial purification of a lipoprotein with 5'-nucleotidase activity from rat liver cells. - Proc. nat. Acad. Sci. (Wash.) 61, 1050-1052 (1968).

162 . Enrico Marani WIEME, R. J.: Agar gel electrophoresis. - Elsevier Publ. Co., Amsterdam 1965. WIKLUND, L., BJ0RKLUND, A., SJ0LUND, B.: The indolaminergic innervation of the inferior olive. 1. Convergence with the direct spinal afferents in the areas projecting to the cerebellar anterior lobe. - Brain Res. 131, 1-21 (1977). WIKLUND, L., TOGGENBURGER, G., CUENOD, M.: Aspartate as transmitter candidate of cerebellar climbing fibers. - Science 216, 78-80 (1982). WILLIAMS, J. E., GASCOIGNE, J. E., WILLIAMS, E. D.: A tetrazolium technique for the histochemical demonstration of multiple forms of rat brain monoamine oxidase. - Histochem. J. 7, 585-597 (1975). WINKLER, c.: Het cerebellum en zijn verbindingswegen. - Handboek der Neurologie III, 109-373 (1926). WOJCIKW, J., NEFF, W. H.: Adenosine A1 receptors are associated with cerebellar granule cells. - J. Neurochem. 41, 759-763 (1983 a). -: Differential location of adenosine A1 receptors and A2 receptors in the striatum. - Neurosci. Lett. 41,55-69 (1983 b). WOOD, J. G., McLAUGHT, B. J., BARBER, R. P.: The visualization of Concanavalin A binding sites in Purkinje cell somata and dendrites of rat cerebellum. - J. Cell BioI. 63, 541-549 (1974).