Anatomical, physiological and biochemical studies of the cerebellum from mutant mice. II. Morphological study of cerebellar cortical neurons and circuits in the weaver mouse

94 (1975) 19-44 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Brain Research,

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A N A T O M I C A L , P H Y S I O L O G I C A L AND B I O C H E M I C A L STUDIES OF T H E C E R E B E L L U M F R O M M U T A N T MICE. II. M O R P H O L O G I C A L STUDY OF C E R E B E L L A R CORTICAL N E U R O N S AND CIRCUITS IN T H E WE&VFR MOUSE

CONSTANTINO SOTELO

Laboratoire de NeuromotThologie (U-I06 INSERM), H6pital de Port Roy.~, 75014 Paris (France) (Accepted March 10th, 1975)

SUMMARY

The vermis of the homozygous weaver mice has been examined with Golgi and electron microscopic techniques. In addition to the findings already reported by previous authors 12,29, new cytological features concerning all the cerebellar neuronal types and the synaptic reorganization of the cerebellar circuitry are described. As in other agranular cerebella, Purkinje cells do not develop spiny branchlets and have a randomly oriented dendritic tree. By contrast, their thick dendrites are studded with spines; according to their size and shape these were classified into: (a) small stubby spines which are the normal postsynaptic targets for climbing fibers; (b) tertiary-like spines, most of which are free of axonal contacts; (c) dolichoderus spines; (d) branching spines; and (e) hypertrophic spines. The last 3 types do not exist in normal cerebellum. Postsynaptic-like differentiations are frequently undercoating the smooth surface of the Purkinje dendrites. As it happens in the case of the free spines, free postsynaptic sites in the shafts of the dendrites develop an extracellular material similar to the material present in synaptic clefts. Basket and stellate cells also develop postsynapticlike differentiations undercoating the somatic and dendritic plasma membranes. These free postsynaptic sites can reach a gigantic size, being longer than 3 #m in length. The rare postmigrative granule cells which persist in w v exhibit claw-endings not only at the dendritic terminal segments, but at the proximal dendritic stems as well. Some of these granule cells, besides having fully achieved migration, undergo a degenerative process indicating that they are probably directly affected by the mutation. Concerning the cerebellar circuitry, and despite the great number of free postsynaptic sites, the large majority of the synaptic contacts keep their specificity. However, some quantitative variations have been disclosed. The surface density of climbing varicosities is increased, whereas that of mossy rosettes is decreased. Stellate and basket fibers are present and their density is also decreased. Furthermore, the pinceau formation around the initial segment of the Purkinje cell axon is missing. In addition

20 to all normal synapt iccontacts (with the exception of the 'parallel fiber omnicellular system') present in weaver, heterologous synapses have also been encountered, mainly concerning the Purkinje dendritic spines, which can be contacted by mossy rosettes, granule cell bodies and/or dendrites. Morphological signs of partial innervation of 1he free postsynaptic sites on the smooth surface of Purkinje dendrites and the perikarya and dendrites of interneurons have also been observed. These results confirm the existence of synaptic remodeling in wv cerebellum.

INTRODUCTION

The use of neurological mutations to analyze developmental processes which occur under restrained abnormal conditions has been of great value for understanding some of the cellular mechanisms taking place during neuronal migration and synapse formation (see Sidman36). One of these mutations, the weaver, gives raise to a cerebellum characterized by both a reduction in size and an almost complete depletion of granule cells 35. Although there is not a general agreement regarding the identification of the primary cellular target affected by the wv genetic abnormality27,2s,ag, it is at least known that the cause of the agranularity is the extensive cellular death observed among post-mitotic neuroblasts of the external granular layer during the first 2 weeks after birth aS. Thus, this mutation offers a natural situation where the external granular layer is damaged and this makes the wv cerebellum a more suitable model for the study of both the rearrangement of the cortical cerebellar circuitry and the morphogenesis of the Purkinje cells in the absence of parallel fibers, than the experimental lesion of the external granular layer of newborn animalsl-a,7, 9-11,14,15,21,33,34. The ultrastructural study of the adult wv cerebellum has been already the object of several publicationslZ,29, 37. However, in addition to the review of the most important features of the wv cerebellum, the aim of this paper is to present new data concerning mainly the postsynaptic differentiations in Purkinje and in the other cerebellar neurons, as well as the fate of some of these differentiations and the formation of new atypical synaptic contacts due to the observed remodeling of the cerebellar circuitry. MATERIALS A N D METHODS

The stock of mice B6 x CBA/F1 obtained from the Jackson Laboratory was raised at the Pasteur Institute. Homozygous wv/wv mice were obtained by intercrossing heterozygous - k / w v mice. Only the vermis of the wv/wv mice were studied in the present paper, since this is the most severely affected region of the cerebellum, where the agranularity is almost complete. After ether anesthesia, young adult mice (20-29 days of age) were fixed by intracardiac perfusion of 200 ml of a warm solution of 1% paraformaldehyde and 1% glutaraldehyde in 0.12 M phosphate buffer (pH 7.3). From each mouse, half of the vermis was postfixed by immersion in 2 % osmic acid solution. Blocks obtained from these vermis, after 'en bloc' staining with uranyl acetate, were embedded in Araldite. The other half of the vermis was impregnated by the rapid Golgi method (double impregnation) or by the Golgi-Rio Hortega method 31.

21

A

Fig. 1. Sagittal section of a Purkinje cell perikaryon stained with the Golgi Rio Hortega method. The primary dendrite is studded with spines (small anow). The perikaryon gives raise also to some thorn-like formations (large arrow). The initial segment of the axon (Ax) arises from the somatic pole opposite to the dendrite. × 2000. Fig. 2. Same material as Fig. 1. The dendritic tree of this Purkinje cell is inversed and arises from the same pole as the initial segment of the axon (Ax). The thick primary and secondary dendritic branches have a rough contour due to the numerous spines (arrows). ;. 800.

RESULTS As p r e v i o u s l y d e s c r i b e d 29 the

weaver

c e r e b e l l a r c o r t e x d o e s n o t e x h i b i t a typical

l a y e r i n g . H o w e v e r , a n d f r o m a q u a l i t a t i v e v i e w p o i n t the 5 n e u r o n a l types w h i c h c h a r a c t e r i z e the m a m m a l i a n c e r e b e l l a r c o r t e x are present. P u r k i n j e cells are the m o s t n u m e r o u s ; t h e y are n o t a l i g n e d in a single row, b u t d i s p e r s e d in 2 4 i r r e g u l a r r o w s w h i c h o c c u p y m o s t o f the c o r t i c a l a r e a with the e x c e p t i o n o f a n a r r o w superficial b a n d w h i c h is e q u i v a l e n t to the m o l e c u l a r layer. G r a n u l e cells in a strikingly rare n u m b e r are p r e s e n t at all depths. Stellate, b a s k e t a n d G o l g i cells, clearly d i f f e r e n t i a t e d in the G o l g i - i m p r e g n a t e d m a t e r i a l (Figs. 3, 4 a n d 5) are s c a t t e r e d a n d i n t e r m i x e d with the P u r k i n j e cells.

3 Fig. 3. Sagittal section of a basket cell in a Golgi double impregnated w e a v e r cerebellum. The dendritic tree (D) is less developed than in control mice. The axonal field (Ax) differs from that of controls, since it is shorter and wider than that of normal animals. This arrangement is due to the nonuniform distlibution of Purkinje cell bodies, x 500.

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Fig. 4. Stellate cell stained with tile Golgi -Rio Hortega method. WM, white matter; PS, pial surface. :~ 570. Fig. 5. Golgi cell impregnated with the rapid Golgi. The dendritic tree spreads from the deepest region of the cortical area up till the pial surface (PS). ;-: 400. Fig. 6. Rapid Golgi, double impregnation. Climbing fibers (arrow) in the superficial third of the cortical a r e a . , 800. Fig. 7. Sarne material as in Fig. 6. A mossy fiber rosette (arrow) in the superficial third of the cortical area. PS, pial surface. 800.

(A ) Morphology of neuronal elements M o s t o f the c y t o l o g i c a l features characterizing the different cell types encountered in the cerebellar cortex~, 25 are present in the wv cerebellar neurons. In this section the p e c u l i a r features o f the 3 m a i n g r o u p s o f cells t h a t is the P u r k i n j e cells, the intern e u r o n s a n d the rare relay neurons - - the g r a n u l e cells - - which persist in the a d u l t m u t a n t , will be described.

Purkinje cells T h e m a i n m o r p h o l o g i c a l characteristics o f P u r k i n j e cells in wv are similar to those described in o t h e r a g r a n u l a r cerebellae,10,1a, 2a,3a a n d can be s u m m a r i z e d as follows: their dendrites are r a n d o m l y oriented a n d often the whole dendritic tree is inversed (Fig. 2); in a d d i t i o n , a l t h o u g h these Purkinje cells do n o t develop spiny branchlets, their p r i m a r y a n d s e c o n d a r y b r a n c h e s are r a t h e r thick a n d exhibit an

23 irregular rough surface obviously due to the presence of numerous spines (Figs. 1 and 2). In agreement with these general features, the large dendritic trunks of the Purkinje cells are widespread from the white matter up to the subpial region. Only at this latter level there is an acellular band, in which the general appearance of a molecular layer is preserved (Fig. 8). The Purkinje cell perikarya has a smooth contour, although in some instances somatic immature-like spines can be present (Fig. 1). The hillock and initial segment of the axon emerge from the cell body (Fig. 1) and only in one case corresponding to a Purkinje cell situated near the pial surface, the initial segment was identified arising from a dendritic profile. A s already described 1.~,29,',~v,the electron microscopic picture which characterizes the ~vv cerebellum is the presence, at all cortical levels, of innumerable dendritic spines, similar to normal branchlet spines but devoid ofpresynaptic elements (Figs. 8 and 10). When observed at high magnification, it can be disclosed that lateral segments of the spinous unit membrane bear fuzzy cytoplasmic material, identical to a normal postsynaptic differentiation, and that these segments appear coated with granular extracellular material resembling the synaptic cleft materiap2,13 (Fig. 2). In the large majority of the cases, the pseudo-postsynaptic and cleft differentiations are facing a normal looking unit membrane belonging to a Bergmann fiber. Spines with a shorter stalk and a more rounded head, similar to those present in the primary and secondary dendritic branches of normal Purkinje cells have also been observed in this cerebellum; as in normal animals, they are synaptically contacted by climbing fibers (Fig. 29). A thorough examination of the Purkinje cell free spines discloses several aspects that have not been previously described. Thus, in addition to the typical forms, which constitute the bulk of the spine population, atypical ones have been also encountered. The atypical spines can be classified in 3 categories: (a) Dolichoderic spines: they correspond to elongated spines 21 which have a narrow neck and an elliptical head. Their free postsynaptic differentiations are usually located on the side of the head. (b) Hypertrophic spines corresponding to large stubby spines (Fig. 12) in which the implanted base can be larger than the spinous excrescence; a free postsynapticlike differentiation can undercoat most of their inner surface. (c) Branching spines. In these instances a single spinous neck divides into two 'subspines' at a short distance from its emergence. Generally, the two branches are of similar size (Fig. 1 l). In addition to the Purkinje cell free spines independently encased in the Bergmann glia, clusters composed by 2 up to 10 or more of them are present in the wv cerebellum (Fig. 14). In these clusters the segment of the spine membranes bearing the postsynaptic differentiations tend to converge, in such a way that the two opposite undercoated membranes, loosing their convexity, become straight and parallel. This direct apposition between the spines gives raise to a pseudo-attachment plate, a junction which has been never observed between spines of a normal cerebellum 1:~,37 (Fig. 13). Another very important characteristic of Purkinje cell dendrites in the wv cere-

25 bellum is the existence of postsynaptic-like dense cytoplasmic material undercoating membrane segments of variable length at their dendritic smooth surface. This postsynaptic webb-like material displays a morphology similar to those characterizing postsynaptic differentiations in Gray type 1 synapses. Such dendritic segments can face glial elements (Fig. 17), naked spines or more often axon terminals of the stellate cells (Figs. 15 and 16). Generally, these axon terminals develop neither presynaptic dense projections nor clustering of synaptic vesicles facing the dendritic postsynaptic differentiation. However, in some instances a small terminal or a protrusion arising from a terminal can deeply invaginate the Purkinje cell dendrite at the level of its postsynaptic undercoating (Fig. 16). Coated vesicles, fGrmed by a pit of the dendritic membrane, exhibiting the postsynaptic density and invaginating inside the dendrite are frequently observed (Fig. 19). Some of these pits form double walled vacuoles, containing in their inner wall a fragment of the dendritic postsynaptic density. They generally protrude into the nervous element which is facing the dendritic surface (Fig. 18). These features may indicate a process of membrane sequestration. In tgrneurons

In Golgi impregnated material the 3 types of cerebellar interneurons have been identified in the w v cerebellum (Figs. 3-5). In agreement with the results of Rakic and Sidman 29 the dendritic fields of these cells were slightly reduced when compared with those of control mice. Since the main cytological aspects of these interneurons are identical to those of control animals '~9 only their peculiar characteristics will be reported. The most important to be mentioned is the presence of numerous free postsynaptic densities undercoating segments of the perikaryal and dendritic plasma membrane (Figs. 20 and 21); their morphology and their relationships with the facing elements are identical to those described above for the Purkinje cells. The only differentiating features are quantitative, since they are more numerous and their average length is much greater than in the Purkinje cell dendrites. They are usually smaller in the perikarya (Fig. 20) than in the dendrites (Fig. 21). in this latter situation they can reach gigantic size as for instance that illustrated in Fig. 22, which overpasses 3 /~m in length. When a stellate axon terminal is directly opposed to the postsynaptic density, the synaptic vesicles can be clustered at the two ends of the density but not in a classical presynaptic position: there is no 'active' zone or synaptic complex formation since the presynaptic vesicular grid is missing (Fig. 21). However, the cleft material is clearly differentiated. Occasionally, two of these dendritic postsynaptic

Fig. 8. Low power electron micrograph of the superficial acellular band in the molecular layer. This band is mainly formed by dendritic profiles of the Purkinje cells (PD) and numerous spines encased in glial cytoplasm (GI). >: 10,000. Fig. 9. High power electron micrograph of a naked spine of a Purkinje cell dendrite. Note the postsynaptic differentiation and the extracellular cleft material (arrow) facing a glial unit-membrane. x 100,000. Fig. 10. Thick Purkinje cell dendritic profile (PD) surrounded by numerous free spines (S) with normal-looking postsynaptic differentiations lying in the glial cytoplasm. < 45,000.

mm

27 differentiations can face one to another, as some Purkinje dendritic spines do, establishing attachment plate-like junctions (Fig. 23). Granule cells

The amount of postmigratory granule cells in the vermis of the young adult weaver can vary from one mouse to another, but in all cases they are rare, and their

number never overpasses 1 ~o of the cellular population of the ccrebellar cortex. Those in the superficial half have most of their dendrites oriented towards the central white axis as observed for arrested granule cells tT. Those in the deeper cortical level have fewer and longer irradiating dendrites (Fig. 24) than in control animals. Furthermore, the dendritic claw-like formations are extremely frequent in the intermediary segments of the main granule cell dendrites (Figs. 24 and 25), contrary to what happens in control animals, where these formations are almost exclusively placed at the terminal segments of the main dendritic branches and at the endings of the few short collateral branches a°. The light microscopic observation of thick plastic sections stained with toluidine blue discloses the occasional presence of degenerating granule cells in the deep half of the cortex. The ultrastructural analysis of this vermal region has allowed the identification of some necrotic grains near-by other normal granule cells in the proximity of Purkinje cell perikarya (Fig. 26), and even at deelzer levels, intermixed there with the myelinated fibers of the white matter. The possible reasons for the death of some postmigratory granule in the wv cerebellum have been discussed in a previous paper 39. In addition to the organelles present in normal granule cells, the corresponding weaw, r neurons can contain clusters of rounded synaptic-like vesicles in their perikarya and in their dendrites. Most of these clusters are in the peripheral cytoplasm, near the somatic and/or dendritic membrane, where they contact Purkinje cell dendritic spines. The morphology of these somatospinal or dendrospinal contacts corresponds to that of Gray type 1 synapses (Figs. 27 and 28). (B)

N e u r o n a l circuits

With the exception of parallel fiber synapses, which are practically absent in the weaver cerebellum all the other kinds of cerebellar synapses have been identified in this cerebellum 29. In addition, new synaptic contacts, due to the reorganization of the remaining presynaptic elements and postsynaptic targets, have been encountered. In

Fig. 11. Cross-section of a Purkinje cell dendrite. The hypolemmal cistern system is well developed. The arrow points to a branching spine arising from the dendrite, x 25,000. Fig. 12. The upper right corner of the micrograph is occupied by a portion ofa Purkinje cell dendritic profile (PD). The rest of the picture is occupied by dendritic spines encased in glial cytoplasm. Note the hypertrophic spine (HS) undercoated by a long postsynaptic differentiation. The arrow points to the origin of a branching spine. × 27,500. Fig. 13. High magnification of an attachment plate-like junction formed by two free postsynaptic sites of two apposing Purkinje dendritic spines. ;< 75,000. Fig. 14. Low power electron micrograph ofa Purkinje cell dendrite (PD) encircling a cluster of numerous free Purkinje dendritic spines. × 8400.

29 the following section the m o r p h o l o g y of the ' n o r m a l circuitry' and of the 'synaptic remodeling' will be described.

'Normal' circuittT A x o n terminals belonging to the 2 afferent systems - - climbing and mossy fibers - - (Figs 6 and 7) and to the interneurons - - stellate, basket and Golgi cells - establish synaptic connections with their usual postsynaptic targets. Most of the ultrastructural features of these synapses in weaver are identical to those of control animals allowing a very easy recognition of the cerebellar circuitry. However, there are important differences, specially quantitative between the ' n o r m a l ' circuitry of the mutants and that of the control animals. Climbing fiber input. Climbing fiber varicosities which establish synapses 'en passant' are a b u n d a n t in the wv cortical region. They are widespread all over the cortex from the white matter up to the subpial surface. A l t h o u g h the absolute numbering of climbing fiber varicosities is impossible to establish without using elaborate quantitative methods, an approximative estimation can be obtained comparing the relative density of climbing fibers per surface area in the mutant mice with that of control. F r o m this simple quantification it can be concluded that the climbing fibers may be at least twice more dense in the weaver. Even in the youngest studied mice (20 days old) the large majority of the climbing fiber varicosities have already established synaptic contact with spines arising from Purkinje dendrites; only in few instances, these spines have a somatic origin (Fig. 30). These observations indicate that despite the absence of parallel fibers, the process of maturation of the climbing fiber synapses is not delayed; thus, the Purkinje cell bodies are relieved from their spiny processes at the proper time. Most of the postsynaptic targets of the climbing fibers are small spines, which exhibit the characteristics of those arising from the large dendritic branches of the normal Purkinje cells (Fig. 29) 19. However, larger spines similar to those belonging to spiny branchlets can also be innervated by the climbing fibers. In addition, the formation of climbing fibers collaterals also takes place in weaver. These collaterals mainly innervate the soma and the dendrites of the interneurons (,Fig. 31). MossyJiber input. C o n t r a r y to what happens with the climbing fiber varicosities,

Fig. 15. The arrows point to the boundaries of a long postsynaptic differentiation undercoating the smooth surface of a Purkinje cell dendritic profile. A neuronal process, probably belonging to a stellate cell axon, is facing this postsynaptic differentiation. Note the absence of presynaptic vesicular grid in the axonal profile (SA). × 63,000. Fig. 16. Purkinje cell dendritic profile. An axon terminal (AT) deeply invaginate the smooth surface of the dendrite. At this level there is a postsynaptic differentiation (arrows). A long and narrow spine emerge from the dendrite (arrow head). × 27,500. Fig. 17. Purkinje cell dendritic profile. A small area of its smooth surface is undercoated by a postsynaptic differentiation (arrow). This area is facing glial cytoplasm (GI). × 27,500. Fig. 18. A portion of the Purkinje cell dendritic postsynaptic differentiation (arrow) invaginates into the neuronal profile directly apposed, forming a double walled coated vesicle, morphological sign of membrane sequestration, x 60,000. Fig. 19. Similar situation as in Fig. 18. In this instance, lhe coated vesicle (arrow) is only formed by the dendritic plasma membrane. × 65,000.

|

31

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W:

~--i,3

Fig. 24. Postmigrative granule cell impregnated with the Golgi-Rio Hortega method. This neuron has only two dendritic processes. They are longer than in control animals. The arrow points to the claw endings located in the stem of the dendrites, x 1200. Fig. 25. Same material as in Fig. 24. The arrows point to the claw endings not located at the dendritic terminals, but emerging from the proximal dendritic stems. :< 1200. Fig. 26. Electron micrograph taken from the deeper half of the cortical area. Two postmigrative granule cells are near-by a Purkinje cell body (PCB). One of the granule cells (G 1) exhibits a normal appearance, whereas the other (G~) is an advance stage of dark degeneration. :: 12,000.

Fig. 20. Electron micrograph of a stellate cell body. Two postsynaptic differentiations (arrows) of different sizes are facing neuronal elements, probably of dendritic nature. S, free spine, x 19,000. Fig. 21. A dendritic profile belonging to a stellate neuron (SD) is facing a stellate axon terminal (SA). At the axonal region opposing the dendritic postsynaptic differentiation there are neither presynaptic dense projections nor clusters of synaptic vesicles. The extracellular space at this level is, however, larger and occupied by a dense material similar to the synaptic cleft material, x 44,000. Fig. 22. Dendritic profile belonging to an interneuron. Two long postsynaptic differentiations undercoat the plasma membrane of the dendrite. The postsynaptic differentiation located at the lower region of the micrograph (arrows) reaches a gigantic size. x 40,000. Fig. 23. Electron micrograph of two dendritic profiles belonging to interneurons. The postsynaptic differentiations of the dendrites are facing one to another forming a pseudo-attachment plate (arrows). • 48,000.

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Fig. 27. Isolated granule cell in the central region of the cortical area. The arrow points to a somato. spinal synapse between the granule cell and a Purkirlje dendritic spine S, free spines. >~ 23,000. Fig. 28. Cerebellar glomerulus in an early 'claw-stage'. The mossy rosette (MR) is surrounded by 6 granule cell dendritic profiles. One of them contains synaptic vesicles clustered against the membrane facing a Purkinje dendritic spine and forming a dendrospinal synapse (arrow). 23,000.

the density of mossy rosettes is reduced in the w v cerebellum. M o s s y fiber terminals are m o r e frequent in the deeper h a l f of the cortical area, but they are also present in the superficial half, even in a subpial position. Since granule cells are a l m o s t absent, the typical g l o m e r u l a r a r r a n g e m e n t between the m o s s y rosette a n d the dendritic digits o f granule cells is an exceptional finding. However, in regions occupied by small clusters o f 3 - 4 granule cells, simple g l o m e r u l a r f o r m a t i o n s can exist (Fig. 28). They l o o k like the early 'claw stage' glomeruli described by L a r r a m e n d i is in mice 10-14 days old. M o r e c o m m o n l y observed are the peculiar g l o m e r u l a r f o r m a t i o n s , a l r e a d y r e p o r t e d by R a k i c a n d S i d m a n 29 in w e a v e r . They consist o f a central mossy rosette c o m p l e t e l y s u r r o u n d e d by G o l g i cell dendrites; in some instances, they are s u r r o u n d e d by a single

Fig. 29. Electron micrograph of the main dendritic stem of a Purkinje cell. A climbing varicosity (CF), is in synaptic contact with 5 small stubby spines. The opposite site of the dendritic surface is occupied by 2 steltate axon terminals (SA). :< 18,000. Fig. 30. Climbing varicosities (CF) in synaptic contact with somatic thorns (T) belonging to a Purkinje cell (PCB). 22-day-old w v / w v . < 15,000. Fig. 31. Electron micrograph of a climbing varicosity (CF) in synaptic contact with a stellate cell body (arrow) and 3 small spines (S). ;< 25,000.

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35 enwrapping dendritic profile (Fig. 32). The identification of such profiles as belonging to a Golgi cell dendrite is favored by the following features: (1) the large diameter of the dendrite; (2) the presence of multiple 'active' zones at the synaptic interface between the mossy terminal and the dendrite, and (3) the rugose surface of the dendrite, which sends a series of small protrusions or ridges into the mossy rosette. This last feature is characteristic of the synapse 'en marron 'z5 between a mossy terminal and a Golgi cell. Small synaptic boutons, containing a pleomorphic population of synaptic vesicles and identified as belonging to axons of the Golgi cells, are frequently located at the peripheral region of the pseudo-glomerular arrangement. These axon terminals are in synaptic contact with the surface of the Golgi cell dendrites. Basket-stellate input. In Golgi-impregnated material the basket cell axon forms pericellular nets which cover 8-10 Purkinje cells disposed in various cellular rows (Fig. 3). The electron microscopic analysis of environment of the Purkinje perikarya discloses the presence of long pericellular basket profiles, as well as small boutons containing flattened vesicles which probably represent the endings of the recurrent collaterals from Purkinje cell axons. The large majority of the Purkinje cell somatic surface is covered by neuroglial processes, most of which belong to Golgi epithelial cells. The axonal profiles of the pericellular baskets give off boutons 'en passant' as in a normal cerebellum. A peculiarity of these profiles, illustrated in Fig. 33, is that they often branch. From the two divided branches, one keeps its position around the Purkinje cell body, whereas the other bifurcates in a divergent orientation to reach a pericellular position of a different Purkinje perikaryon. As described for the experimentally induced agranular cerebellum 21, in weaver, neither the typical basket formation around the axon hillock (Fig. 34), nor the pinceau formation around the initial segment (Fig. 35) are observed. Instead, the hillock and the intial segment of the Purkinje cell axon are surrounded by a thin discontinuous layer of glia. The places uncovered by glia are occupied by axon terminals of the basket fibers. Occasionally, the basket terminals establish synaptic contacts with the surface of the initial segment of the Purkinje cell axon (Fig. 35). From a qualitative viewpoint, basket fibers are present both as pericellular nets and surrounding the initial segment of the Purkinje axon; however, they appear much less numerous than in control animals, and they do not form the thick axon-glial plexus which characterizes the basket pinceau. Some ascending collaterals of the basket fibers and more abundant smaller boutons belonging to stellate axons are in synaptic contact with Purkinje cell dendrites (Fig. 29) and with the perikarya or dendrites of the interneurons. The described relationship between climbing fibers and ascending collaterals of the basket fibers 25 are partially preserved in weaver.

Fig. 32. Electron micrograph of an atypical glomerular formation between a mossy rosette (MR) and an encircling dendrite of a Golgi cell (GD). Note the small protrusions that the Golgi dendrite sends into the mossy terminal. At the periphery, Golgi axon terminals (GA) establish synaptic contacts on the dendritic surface. The arrow points to a minute 'gap' junction between 2 dendritic profiles. :< 16,500.

37

'Synaptic remodeling' In the normal cerebellum, the axons of the granule cells establish synaptic contacts with all the other cortical neurons. In weaver, this 'parallel fiber-omnicellular system' is practically absent. However, most of the postsynaptic targets which in normal conditions are innervated by parallel fibers develop, in weaver, postsynapticlike differentiations. Thus, the weaver cerebellum is characterized as other agranular cerebella by the presence of large amounts of free postsynaptic sites, which reproduce the morphological picture of a partially deafferented central region (see review in Gentschev and Sotelo6). Probably, due to the existence of free postsynaptic sites 'calling' for innervation, there is a true reorganization of the cerebellar circuits. This synaptic remodeling mainly affects the Purkinje cells at different levels (the dendritic spines, and the long postsynaptic densities developed under the plasma membranes of the dendritic shafts) and the perikarya or the dendrites of the interneurons.

Heterologous innervation of tke dendritic spines of the Purkin]e cells The most numerous free postsynaptic sites encountered in the wv cerebellum are by far the dendritic spines of the Purkinje cells. Thus, these elements are the most frequently used for the formation of heterologous synapses. Various kinds of axon terminals can establish new synapses with the free Purkinje spines. (a) Mossyfiber input on Purkinje cells. These afferents only find a very small proportion of their normal postsynaptic targets, i.e., Golgi cell dendrites and rare granule cell dendrites. However, they are able to differentiate into rosettes, which can be observed in Golgi-stained material (Fig. 7) and in electron micrographs (Fig. 32). In some instances, these rosettes can be in synaptic contact exclusively with the dendritic spines of the Purkinje cells (Fig. 3 from Sotelo3V). More often, a portion of the rosette membrane synapses on Golgi cell dendrites or on granule cell dendrites and the other portion on the Purkinje spines (Fig. 36). These heterologous synapses have also been described in other agranular cerebellaZ, 21. (b) Heterologous climbing fiber input on Purkinje cells. Climbing fiber varicosities can innervate some of the large dendritic spines resembling those normally contacted by parallel fibers. The reality of such heterologous synapses is demonstrated not only by the size and shape of some of the spines, but also by the presence of some of them partially innervated b~, the climbing varicosity, as illustrated in Fig. 37. In this last instance, there is a partial apposition of the axon terminal to the free postsynaptic side. The spinous postsynaptic density is shared by the invading terminal, which

Fig. 33. Electron micrograph of a basket profile (BT). This axon terminal is forming the pericellular basket of one Purkinje cell body (PCB1). At a given point, it bifurcates (arrow) given a collateral which enters in the pericellular basket formation of another Purkinje cell body (PCB2). ~ 24,000. Fig. 34. Purkinje cell axon hillock (PCH). The hillock is not surrounded by basket fibers, but by glial profiles. A basket fiber (BT) is directly apposed to the hillock. × 14,000. Fig. 35. Initial segment of a Purkinje cell axon (PCIS). Some basket fiber profiles (BT) are in the neighborhood of the initial segment. One of them establishes a synaptic contact (arrow) with the initial segment. The scarceness of basket fibers contrasts with the abundance of such profiles in the pinceau formation. Gl, glial cytoplasm. × 30,000.

t~ O~

39 develops a new 'active' zone with half of the spinous postsynaptic differentiation. (c) Granule cell somatic and/or dendritic input on Purkinje cells. This newly formed type of somatospinal or dendrospinal synapses is the less frequent. As has been reported above in the description of the granule cells and the mossy rosettes, the rare remaining granule cells do not only give raise to parallel fibers with abundant varicosities, but also have synaptic-like vesicles scattered on their perikarya and dendrites. Generally, when a Purkinje dendritic spine is directly apposed to these perikarya or dendrites, the synaptic-like vesicles are clustered in the region of the granule cell membrane facing it. The morphology of these junctions closely resembles that of a Gray type 1 synapse (Figs. 27 and 28).

Heterologous innervation of the long postsynaptic densities on Purkinje cell dendritic shafts and on interneurons As stated before, the thick postsynaptic densities which undercoat areas of the plasma membrane of most of the neuronal elements present in the wv cerebellum are generally facing axon terminals and less often glial profiles or other postsynaptic densities. In addition they can be innervated by axon terminals belonging to stellate or basket cells. The morphological criteria used to identify this innervation are: (1) the presence of presynaptic dense projections and clusters of synaptic vesicles on the axonal membranes facing the postsynaptic densities (Fig. 38); (2) the partial occupation of the free postsynaptic sites by axon terminals (Fig. 39). This last criterion corresponds to the one already described for the heterologous synapses between climbing varicosities and branchlet-like spines. The heterologous synapses between stellate and/ or basket axon terminals and Purkinje dendrites or interneurons can be easily differentiated from their homologous or normal ones. In the latter case, the postsynaptic differentiation is generally mueh more reduced in size and the synaptic complex exhibits the features of a Gray type 2 synapse. DISCUSSION

Even if the primary cellular target of the wv genetic locus is not clearly established 2v,28,39, the main disturbance affecting this cerebellum is the almost total absence of granule cells due to a failure on their migration 3e. Rakic and Sidman e9 have already emphasized the advantages, over the experimentally induced agranular

Fig. 36. Electron micrograph of a mossy rosette (MR). The region of the rosette located at the left of the picture establishes normal synaptic contacts with granule cell dendrites (D). The region at the right is synapsing on dendritic spines (S) of Purkinje cells. × 28,000. Fig. 37. Climbing varicosity (CF) in synaptic contact with two large tertiary-like spines ($1, SD. The postsynaptic differentiation of one of the spines (SD is only partially occupied by the presynaptic terminal. The other part (arrow) is facing a glia[ process. × 36,000. Fig. 38. Long postsynaptic differentiation (arrow heads) under the smooth surface of a Purkinje dendrite (PD). The basket fiber collateral (BT) facing this postsynaptic differentiation has developed presynaptic dense projections and clusters of vesicles (arrows), morphological sign of innervation. 34,000. Fig. 39. Postsynaptic differentiation under the plasma membrane of a stellate cell. A stellate axon terminal (SA) partially innervates this postsynaptic differentiation (arrow). × 36,000.

40 cerebella, of using this mutant tbr the study of the reorganization of the cerebellar elements in the absence of granule cells. In any case, the dendritic tree in w e a v e r exhibits a similar morphology to the one observed in other agranular cerebella ~,t°.~l .:~4. This similarity provides a morphological proof that the size, shape and orientation of the Purkinje cell dendrites depend oa the local milieu. That is to say, the normal pattern of the dendritic tree of the Purkinje cells is at least partially, if not more, induced by its interaction with parallel fibers ~. A surprising finding emerging from the cytological study of the wv cerebellum is that the neurons (which in normal conditions are innervated by parallel fibers) develop postsynaptic-like differentiations many of which exhibit the same features than their normal counterparts, as is the case for the majority of the free dendritic spines of the Purkinje cellsl'~,'~9,a7. Other postsynaptic sites have an abnormal appearance, mainly characterized by their overgrowth (branching and hypertrophic spines, gigantic postsynaptic densities under the plasma membrane of the interneurons) or by their unusual location (postsynaptic densities on the dendritic shafts of the Purkinje cells). The most obvious explanation for the presence of such free postsynaptic differentiations is that they are formed by an autonomous process, which has to be independent from interactions with parallel fibers. Concerning the free dendritic spines of the Purkinje cells, however, a different interpretation has been suggested by Hfimori 8. This author postulates an indirect or heterotopic induction of Purkinje spines by 'afferent elements (climbing fibers) contacting other postsynaptic sites of the same neuron'. According to this postulate, climbing fibers would be necessary during development for the induction of Purkinje dendritic spines, and also for their maintenance throughout the whole life of the animal. This assumption would represent a morphological basis, correlating the theory advanced by Marr 2a for 'learning in the cerebellum'. Indeed Marr predicted that 'the synapses from parallel fibers to Purkinje cells are facilitated by the conjunction ofpresynaptic and climbing fiber (or postsynaptic) activity'. The arguments used by Hfimori s to reinforce his speculation are mainly: (1) pyramidal cells kept in a dissociate cell culture do not develop dendritic spines (afferent fibers are necessary for the growth of spines in pyramidal neurons, thus also in Purkinje cells); (2) after chronic isolation of the cerebellar cortex, the majority of the Purkinje dendritic spines disappear. Since the undercutting of the cerebellar white matter produces a degeneration of climbing and mossy fibers, it follows that the presence of climbing fibers are necessary for the maintenance of the spines. However, many facts are against these elegant interpretations. Concerning the development of Purkinje dendritic spines, there are some indications, mainly arising from other tissue culture experiments, in favor of an autonomous spine formation. The most interesting results in this respect are those obtained from cultures of newborn rat cerebellum, cultivated in presence of the antimitotic MAM (methyl-azoxymethanolacetate) 4,'6. Under these circumstances, Purkinje cells are developed in an almost extreme situation of synaptic isolation, since the external granular layer and the two afferent systems degenerate. Even in these instances, Purkinje cells are able to develop somatic and dendritic spines.

41 In relation to the role played by the climbing fibers in the maintenance of Purkinje dendritic spines, work in progress (Sotelo, Hillman, Llin~.s and Zamora, in preparation) designed to study the chronic effect on adult Purkinje cells of total climbing fiber deafferentation shows, in different species and using different experimental approaches, that Purkinje cell tertiary spines can survive in absence of climbing fibers. The results obtained by H/tmori s on the chronic isolated cerebellar slabs are very probably due to retrograde changes following the Purkinje cell axotomy and/or vascular changes secondary to the surgical isolation. In agreement with Mugnaini's hypothesis 24 that the postsynaptic partner is determinant for synapse formation, the results reported in this paper prove that ],~ the w v cerebellum not only Purkinje dendritic spines are present but also the postsynaptic differentiations in other cerebellar neurons, which in normal conditions are contacted by parallel fibers. Therefore, if we assume that neurons of the cerebellar cortex have an autonomous development of their different receptive surfaces, several hypotheses can be formulated to explain the presence of normal and abnormal postsynaptic sites: (a) cerebellar neurons are programmed for the synthesis of a specific amount of receptor proteins. The surface area of the dendritic trees belonging to the different cerebellar neurons being smaller in w e a v e r than in control animals, the postsynaptic differentiations, morphological counterpart of receptive areas, are larger and in some instances have an unusual location; (b) in normal cerebellum the establishment of functional synapses between parallel fibers and their postsynaptic partners may serve as a feedback mechanism to regulate the autonomous synthesis of the specific receptor protein. In w e a v e r , since this normal synaptic function is not established, the cerebellar neurons are devoid of their regulatory synaptic input, and would pursue the synthesis of the receptor protein, thus the overgrowth of the postsynaptic differentiations. Another interesting problem to discuss concerns the reorganisation of the cerebellar circuitry. The increased density of climbing fiber varicosities, already described by Hamori 7 in an agranular cerebellum, can be correlated with recent electrophysiological studies on irradiated rats cerebella 4"~(Crepel and Delhaye-Bouchaud, in preparation) which tend to prove that in this material each single Purkinje cell is innervated by more than one climbing fiber. However, this apparent increase in the density of climbing fiber varicosities is not an absolute morphological argument to prove such multiple innervation. The presence of basket and stellate cells in the w e a v e r ~9 makes this cerebellum more similar to the ferret cerebellum degranulated after virus infection 21 than to that of rats irradiated at birth z. In fact, Llinhs e t a l . 21 have demonstrated not only the morphological presence of stellate and basket cells in their agranular cerebellum, but the presistence of their normal inhibitory action upon Purkinje cells as well. However, according to the results reported here, there are important quantitative differences between the basket fiber investment of Purkinje cells in w e a v e r and in control animals. In the former, the pericellular basket is incomplete and the pinceau is missing. These differences cannot be associated with either a failure of a delay in the translocation of climbing fiber varicosities from the somatic thorns to the dendrites, as may be the

42 case for another mutation ( s t a g g e r e r ) affecting the cerebellum of the mouse aS. In the young adult w e a v e r the perikarya of Purkinje cells are smooth and almost totally devoid of climbing fiber synapses. A tentative interpretation to explain the basket fiber deficit can be the following: the main synaptic input to the basket cells is constituted by the parallel fibersZ0; since these fibers have never existed in weaver, it is possible that the development of the dendritic and axonic fields of the basket fibers is less conspicuous than in normal cerebellum. This hypothesis assumes that the normal growth of the dendritic and axonic fields of a given neuron must be directly related to the number of the synaptic inputs that it receives. The presence of free postsynaptic differentiations in deafferented neurons has been associated with spontaneous neuronal hyperactivity16. One of the possible mechanisms involved in such hyperactivity can be the facility of access of the extracellular materials (ions, t r a n s m i t t e r s . . . ) to the receptor site 41. However, and in spite of the presence of innumerable free spines in the Purkinje cells of the agranular cerebella their spontaneous hyperactivity has not been reported in the studies devoted to the electrophysiology of these cerebella (refs. 21 and 42 and Crepel and Delhaye-Bouchaud, in preparation). In spite of the great number of free postsynaptic sites encountered in the wv cerebellum, the large majority of the synaptic contacts have kept their specificity, and they can be classified as homologous synapses. However, heterologous synapse formation, already described in other agranular cerebella 2,21, is also a common process in weaver. According to the present results, heterologous synapses are more diversified in w e a v e r than in the other agranutar cerebella. In this respect, the neuron of the former react in a similar way than other central neurons partially deafferented by experimental lesions6,e2,40,4t: they can be innervated by heterologous synapses, attempting in this way to the reorganization of the cerebellar circuitry. ACKNOWLEDGEMENTS The author wishes to thank Drs. J. P. Changeux, F. Crepel, J. Mallet and J. Mariani for useful discussions. Mice were raised at the Institut Pasteur by M, Huchet and J. L. Guenet. D. Le Cren is also acknowledged for photographic assistance. This work was partially supported by the Institut National de la Sant6 et de la Recherche M6dicale (A.T.P. 6-74-27 No. 20).

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