Electron microscope study of mouse cerebellum in tissue culture

EXPERIlfENTAL

Electron

KEUROLOGY

33, 30-44 (1971)

Microscope

Study in Tissue

of

Mouse

Cerebellum

Culture

SEUNG U. KIM ’ Departwent

of Anatomy,

College

of Mcdicilae,

UGvrvsitJ*of Saskatchezua:~,

Soskatoon. Caizada Received

May

19, 1971

Explants of newborn mouse cerebellum, maintained for 14-70 days in organotypic culture, were processed for electron microscopy. The fine structural features of Purkinje cells, granule cells, and stellate cells were analyzed. The latter category included basket cells, small cortical cells, and Golgi cells. In addition, the ultrastructure of cerebellar glomeruli, myelinated fibers, neuroglia cells (both astrocytes and oligodendrocytes), and axonal growth cones were examined. On the basis of these observations, it may be concluded that the ultrastructural organization in cerebellum cultured ilz vitro has a remarkable fidelity to that of mature normal cerebellum studied in viva. Introduction

The mammalian cerebellum has been used extensively as material for tissue culture of the central nervous system. Many investigators have utilized successfully this culture system to investigate morphogenetic (4, 21, 22, 25), physiological (l$), biochemical (19, 23, 24, 33), and pathological events (3, 26, 27, 34) in the central nervous system. However, relatively little is known about the fine structural organization of this culture system. The earlier electron microscopic studies of rat cerebellum cultures ( 11, 41) placed emphasis on myelin formation in vitro and did not addsess themselves to the general fine structural organization of neurons and synaptic configurations. The establishment of normal ultrastructural morphology would enable one to examine and interpret experimentally induced changes in mammalian cerebellum cultured in Z&W. The objective of the present study was to provide such information. 1 This Multiple versity, Medical technical

work was Schlerosis College of Research assistance.

carried out in part while Society in the laboratory Physicians and Surgeons. Council of Canada. The 30

the author was a Fellow of Xational of Dr. M. R. Murray at Columbia UniThis study was supported in part by the author thanks Mr. Y. Tanaka for his

CERERELLUM

CVLTCRE

31

FIG. 1. Well-differentiated Purkinje cell with various cytoplasmic organelles including several Golgi apparatus (g), mitochondria (1111, dense bodies id), Nissl substance (ns), and subsurface cisterns (broken arrows). The perikaryal spines are indicated by black arrows. Nucleus has a large reticulated nucleolus (nu). Two asosomatic synapses (sy) are also indicated by arrow heads.

32

FIG. 2. Proximal chondria, membrane is shown here. Two

KIM

dendritic process with well-oriented microtubules, profiles of endoplasmic reticulum, and a few clusters axodendritic synapses are indicated by arrows.

slender mitoof ribosomes

CEREBELLUM

Materials

CULTt’RE

and

33

Methods

The techniques of tissue culture of newborn mouse cerebellum have been coverslips described (25). Th e cultures were explanted on fluoroplastic (35), maintained in either Maximow’s slides or in roller tubes and were fixed after 14-70 days ipz z&o in 2.5% osmium tetroxide adjusted to pH 7.4 with either veronal-acetate or phosphate buffer for 20-30 min. The preparations were then dehydrated and embedded in Epon. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with a Philips 200 electron microscope. Observations

Purfiinje Cells. The neurons most frequently encountered in cerebellum cultures were Purkinje cells and granule cells. Purkinje cells (Fig. 1) had a large round or ovoid nucleus and a reticulated nucleolus in the center. Clumping of chromatin against the nucler envelope was rare. Numerous mitochondria and free ribosomes were dispersed throughout the cytoplasm. Smooth- and rough-surfaced endoplasmic reticulum was seen in the perinuclear and peripheral cytoplasmic zones. Several Golgi bodies were located in the perinuclear zone (Fig. 1). There were also occasional dense bodies and multivesicular bodies in the cytoplasm. Some microtubules were dispersed in the cytoplasm. Few, if any, neurofilaments were seen. Subsurface cisternae were frequent, but no cisternae were closely applied to mitochondria as reported in rat Purkinje cells fixed in viva (15). Perikaryal spines of Purkinje cells have been described in 7-day-old mouse cerebellum (32). Similar, but less nunierous spines were observed in the present material. Dendritic processes contained microtubules and slender mitochondria oriented in the long axis of the dendrites. Profiles of endoplasmic reticulum with dilatations were observed also (Fig. 2). Scattered among these organelles were clusters of ribosomes and a few neurofilaments, vacuoles, and multivesicular bodies. The axons contained mitochondria, assorted vesicles, neurofilaments, microtubules, and occasional multivesicular bodies. Neurofilaments were more conspicuous in the axons than in the soma and dendrites. 3.

Axosomatic

contains

several

FIG.

bag

synapse (arrow) on a Purkinje mitochon:lria, a dense body, and

perikaryon. The numerous vesicles.

presynaptic

FIG. 4. Asodendritic synapse contains several mitochondria (m). a multivesicular body (mv), and numerous vesicles which accumulate at the synaptic contact. A granular dense-core vesicle (gv) is also shown here. Fibrillar substance in the synaptic cleft is indicated by an arrow. FIG. 5. Cerebellar giomerulus with five synaptic contacts is shown here. ous mitochondria and synaptic vesicles in the mossy fiber ending (mf). synaptic vesicles are of the clear-core type.

Note numerMost of the

34

KIM

CEREBELLUM

35

CULTURE

Two types of junctions, axosomatic (Figs. 1. 3) and axodendritic (Figs. 2, 4) synapses, were frequently observed. Their fine structural details corresponded to those described in rat spinal cord cultures (5). However, in the present study, it was observed that some of the asodendritic synapses had an enlarged synaptic cleft. Such synaptic clefts were filled with dense substance of a bridging fibrillar structure (Fig. 4). The asodendritic synapses occurred on the dendritic trunk (Fig. 2), branches and spines. In agreement with the report of Gray (14), the dendritic spines of the Purkinje cell contained no spine apparatus. Most of the synapses observed in the present study could be classified as type 1 (13) or asymmetric synapses (S). The presence of an axoasonic synapse was not confirmed in our study. Crrcbellav Glomcruli. Cerebellar glomeruli are multiple synaptic contacts between granule cell dendrites and the terminal of an incoming mossy fiber. The presence of such glomeruli in mouse cerebellum culture has been reported previously (2.5). Presynaptic terminals of mossy fibers contained several mitochrondria, numerous synaptic vesicles (300-600 A), a few granular dense-core or spiked vesicles (or both) and large vesiclelike profiles of endoplasmic reticulum (Fig. 5). Granule cell dendrites which made synaptic contacts with a mossy fiber ending contained mitochondria, microtubules, numerous membrane profiles of endoplasmic reticulum, and an occasional multivesicular body (Fig. 5). Each cerebellar glomerulus contained from two to seven synaptic contacts. Such contacts were distinguished by distinct membrane thickenings between the granule cell dendrites and the preterminal mossy fiber ending. The occurrence of glycogen in mossy fiber endings has been reported in neonatal mouse cerebellum fixed is vii-lo (29, 31 J. However, no such structures were observed in the present study. GrunuZe Cells. Inasmuch as observations on the ultrastructure of granule cells (Fig. 6) in the present study agreed with previous ones (25). details are omitted here. Stellate CL&. In accordance with Herndon (16), the term “stellate cells” is applied in a broad sense and includes Golgi neurons as well as basket cells and small cortical cells. The small stellate cells (i.e., basket and small cortical cells) had a round or oval nucleus with dispersed chromatin and one or two dense nucleoli. The nuclear envelope had one or cells. Axosomatic and axodendritic synapses on Purkinje cells occur but

FIG. 6. Three granule the center is ensheathed

FIG. myelin

cells with by myelin.

thin rim of cytoplasm A mossy fiber ending

7. Several well-myelinated axons sheaths in the center are loosely

are shown here. The one in (mf) can also be seen.

are shown in this electron applied around the axons.

micrograph

T\vo

36

FIG.

KIM

8. Small

stellate

cell

(SC)

contains

various

cytoplasmic

organelles

such

as

CEREBELLUM

CULTURE

37

more invaginations (Fig. 8). In the thin rim or perinuclear cytoplasm (0.1-3 p) mitochondria, rough- and smooth-surfaced endoplasmic reticulum, free ribosomes and an occasional dense or multivesicular body (or both) occurred (Fig. 8). The Golgi complex was usually located in the more peripheral cytoplasm. The fine structural difference between small stellate cells and granule cells was that axosomatic synapses were frequent on stellate cell perikarya, but rare on granule cells. The rare occurrence of synaptic contact between mossy fibers and granule cell perikarya has previously been reported (25). On occasion axosomatic synapses on the perikaryal spine of a stellate cell were observed (Fig. S) . Because the fine structure of the large stellate cells (Golgi neurons) resembled that of Purkinje cells in most respects, it was not possible to differentiate between the two neurons (Fig. 8). A similar opinion was expressed by Herndon in adult rat cerebellum fixed in viva ( 16). MyAn Shcatlzs. The general fine structure of myelin sheaths observed in cerebellum cultures (Fig. 7) has been described (11, 41). In the present study apparent direct connection between a myelin sheath and a participating neuroglia cell was rarely seen. Infrequently, two axons were observed together within a single myelin sheath, the lamination of which was apparently a normal occurrence. Nodes (Kanvier) displayed typical digital arrangements of myelin lamellae in the paranodal regions (Fig. 9). There was a difference in the number of myelin lamellae present at the proximal and distal parts of a node of Ranvier (Fig. 9). This observation tempts one to assume that myelin sheaths in the central nervous system may be formed segmentally by neuroglia cells in a mechanism similar to that believed to be esecuted hy neurilemma cells in peripheral myelinogenesis. Figure 9 also demonstrates that there was a constriction of the axoplasm at the node. This constriction resulted in an apparent accumulation of various axoplasmic organelles on both sides of the node (mitochondria, membrane profiles of endoplasmic reticulum, dense bodies, and multivesicular bodies). Two peculiar structures have been observed in the present study. The first was a partial or complete myelination of some neuronal somas of both the Purkinje and granule cell populations (Fig. 6). Such structures were described previously in vitro (25, 28). The second peculiar structure consisted of collapsed portions of myelin mitochondria(m), Golgi complex (g), endoplasmic reticulum,and has severalaxesomaticsynapses(arrow heads) on its soma.Perikaryal spine with two synaptic contactsis indicatedby arrows. Large stellatecell (Golgi cell-gc) and/or Purkinje cell is alsoshownhere.

38

KIM

CEREBELLUM

39

CrLTURE

sheaths. Such portions contained neither axon nor lumen (Fig. 10). The opposing myelin lamellae were closely associated. Such collapsed myelin sheaths were usually closely applied to the plasma membrane of a cell soma (Fig. 10) or to a myelinated axon. Similar structures have been described in detail by Rosenbluth ,in zri’z,o in toad cerebellum (40). Axonal Gvozuth Cones. Axonal growth cones were reported in vizjo in newborn cat cerebellum (7), in newborn mouse cerebellum (32)) and in fetal monkey spinal cord (2). Such structures were infrequently seen in the present study. When observed, the growth cones consisted of a large (2-4 p in diameter) bag which contained several mitochondria and numerous large (500-1200 Nezcroglia Cells.

A)

vesicular

structures

(Fig.

11).

The astrocytes had an ovoid nucleus with evenly distributed chromatin granules (Fig. 12) and one or two nucleoli. The nuclear envelope usually had one or more indentations. The cytoplasm was less compact than that of the oligodendrocytes and granule cells. The cytoplasm contained several mitochrondria, dense bodies, multivesicular bodies, free ribosomes, scattered membranous or tubular profiles of endoplasmic reticulum, and one or two small Golgi bodies (Fig. 12). Glioiilaments were a characteristic feature of astrocytes, and were gathered into bundles in the cell bocly and in the processes. The oligodendrocytes had a round or oval nucleus, similar in size to that of the granule cells. A slight accumulation of chromatin against the nuclear envelope was observed (Fig. 13). The chromatin of oligodendrocyte nuclei was denser than that of astrocytes and similar to that of granule cells. The cytoplasm contained several mitochondria, one or two small Golgi bodies, rough- and smooth-surfaced endoplasmic reticulum, and numerous free ribosomes. Dense and multivesicular bodies were usually seen in the cytoplasm (Fig. 13). Over-all density of the cytoplasm was very high when compared with that of the astrocytes. A connection of an oligodendrocytic process with a myelin sheath was not observed. Discussion

Most of the fine structural characteristics of mammalian cerebellum fixed in z,k~ (10. 12, 14-16, 30, 32, 37, 43) can be observed also in of myelin lamellae of node is shown here, The causes an accumulation of various organelles suchas mitochondria(m), multivesicularbodies(mv), densebody (d), and tubular profiles of endoplasmic reticulum (er) FIG.

9. Typical

constriction

digital

arrangement

of axoplasm at the node

FIG. 10. An overgrowth here. The myelin structure

of myelin sheath with its collapsed is closely applied to a cell soma.

FIG. 11. A structure which seems mitochondria and numerous vesicular

to hc an axonal structures.

grolvth

middle cone

(g)

portion contains

is shown several

40

KIM

FIG. 12. Astrocyte with watery cytoplasm which mitochondria (m), dense body (d), and bundles of The surface of the culture is noted as sf.

contains gliofilaments

Golgi (f)

apparatus is shown

(g), here.

CEREBELLUM

11

CULTURE

mouse cerebellum cultured in efitro. Among the neuronal types identified at the ultrastructural level in the present study were Purkinje cells, granule cells, and stellate cells. The latter category included basket cells, small cortical cells, and Golgi cells. Although stellate cells are not yet differentiated at the age at which the present cerebellum explants were taken (36, 38: 39, 4.5), such cells subsequently became differentiated under the culture conditions used. Purkinje cells and neurons of subcortical nuclei might be confused, but since the population of Purkinje cells is overwhelming in cultures (21 j, large neurons observed in the present study were regarded as Purkinje their origin from basket cells, collaterals of Purkinje axons, or granule cell parallel fibers is uncertain. The climbing and mossy fibers have been described as afferents of extraneous origin (10, 20, 38). It is reasonable, therefore, to assume that such fibers are severed from their perikarya during the process of explantation and degenerate in the subseuqne phases of the culture. Such a condition would explain our inability to demonstrate any structure which might indicate the survival of climbing fibers in culture. On the other hand, it is clear that mossy fibers survive in the culture conditions, as evidenced by the presence of cerebellar glomeruli composed of mossy fiber endings and granule cell dendrites. The possible explanation for this finding is that there is a chance of explanting part of the vestibular nucleus with the cerebellum due to the close anatomical relation of the two CNS regions. However, all the cerebellum cultures fixed and examined by electron microscope yielded the occurrence of mossy fiber endings. Since it is unlikely that all the cerebellum cultures contain segments of vestibular nucleus, there is probably an alternative source of mossy fibers. I suggest that the mossy fibers are actually axon terminals of neurons intrinsic to cerebellum such as the recurrent collaterals of the Purkinje axons or axons of cerebellar nuclei neurons, although there have been no observations reported to support this hypothesis. The factors which enabled me to identify granule cells i>z vitro were their size and fine structural details. Such characteristics correspond well to those observed in z&o. They are the most numerous cell type seen in culture (42, 44). Although the occurrence of glycogen granules in synaptic endings has been reported in mossy fiber terminals of neonatal mouse cerebellum (29, ~--

~__

FIG. 13. Oligodendrocyte with oval nucleus is shown here. Cytoplasmic organelles, such as Golgi apparatus (g) , mitochondria (m), and dense bodies (d) are indicated, respectively. Note the numerous free ribosomes scattered throughout the cytoplasm. Observe higher density of the nucleus and cytoplasm compared with that of the astrocytes.

KIM

31) and in synapses of fetal rat spinal cord maintained in vitro (5)) I did not observe any glycogen granules in synaptic configurations. I previously suggested that synaptic connections could be newly formed in cultures of central nervous tissue (‘21). I observed ring-shaped terminal boutons in silver impregnated cultures of kitten cerebellum. Subsequent published communications (1, 17, 22) have also demonstrated similar synaptic configurations in cultures of central nervous tissue. Fine structural accounts of these synaptic connections have been shown in cultured materials (5, 25, 34a). Further evidences of the operation of synaptic connection in cultures were advanced by the means of neurophysiological methods (9). Furthermore, the present paper suggests that not only axosomatic and axodendritic synapses are present on the Purkinje and stellate cells but that complex synaptic configuration of cerebellar glomeruli also are demonstrable in mouse cerebellum cultures. To my knowledge, a general account of the fine structural features of mammalian cerebellum in vitro has never before been described in detail. The earlier electron microscope studies (11, 25, 41, 42) covered limited aspects of the fine structural features of rat and mouse cerebellum cdtures. These studies were fragmentary and the details of cellular organization within the cultures were not studied. On the basis of the observations in the present study, it may be concluded that the fine structural features of cerebellum cultures have remarkable fidelity to those of mature normal cerebellum studied in viva. References 1. ALLERAND, C. D. 1969. Regeneration of synapses irz zitvo. Erp. Nezwol. 25: 482493. 2. BODIAN, D. 1968. Development of fine structure of spinal cord in monkey fetuses. II. Prereflex period to period of long intersegmental reflexes. J. Co?rtp. Nezbrol. 133 : 113-166. 3. BORNSTEIN, M. B. 1965. Tissue culture studies of demyelination. A+z+z. N.Y. Acad. Sci. 122 : 28@-286. 4. BORNSTEIN, M. B., and M. R. MURRAY. 1958. Serial observations on patterns of growth, myelin formation maintenance and degeneration in cultures of newborn rat and kitten cerebellum. J. Biophys. Biochem. Cytol. 5 : 499-504. 5. BUNGE, R. P., M. B. BUNGE, and E. R. PETERSON. 1%5. An electron microscore study of cultured rat spinal cord. J. Cell Biol. 24 : 163-191. 6. CALLAS, G., and W. HILD. 1964. Electron microscopic observations of synaptic endings in cultures of mammalian central nervous tissue. Z. Zellfovsch. 63: 686-691. 7. CERRO, M. P. DEL, and R. S. SNIDER. 1968. Studies on the developing cerebellum. Ultrastructure of the growth cones. J. Comp. Newol. 133: 341-362. 8. COLONNIER, M. 1968. Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscopic study. Brai Res. 9: 268-287.

CEREBELLUM

CULTURE

33

9.

GRAIN, S. M. 1966. Development of ‘organotype’ bioelectric activities in centrai nervous tissue during maturation in culture. Iltt. Rczf. X~~wol~ioI. 9: 143. 10. ECCLES. J. C., M. ITO, and J. SZENTAGOTHAI. 1967. “The Cerebellum as a Neuronal Machine.” Springer, Berlin. 11. FIELD, E. J., D. HUGHES, and C. S. RAINE. 1968. Electron microscopic observations on the development of myelin in cultures of neonatal rat cerebellum, J. Nezwol. sci. 8 : 4940. 12. Fox, C. A., D. E. HILLMAN, K. -4. SIEGESMUND, and C. R. DUTTA. 1967. The primate cerebellar cortex: A Golgi and electron microscopic study. Progr. Brain Res. 25 : 174225. 13. GRAY, E. G. 1958. Xxo-somatic and axo-dendritic synapses of the cerebral cortex: An electron microscopic study. J. ,41taf. 93 : 420-433. 14. GRAY, E. G. 1961. The granule cells, mossy synapses and Purkinje spine synapses of the cerebellum : light and electron microscope observations. J. hat 95 : 345356. 15. HERNDOX, R. M. 1963. The fine structure of the Purkinje cell. J. Cell Biol. 18: 167-180. 16. HERXDON, R. M. 1964. The fine structure of the rat cerebellum. II. The stellate neurons, granule cells and glia. J. Cell Biol. 23 : 277-293. 17. HILL), W. 1966. Cell types and neuronal connections in cultures of mammalian central nervous tissue. 2. Zellfovsch. 69 : 155-188. 18. HILD, W., and I. TAS\I;I. 1962. Morphological and physiological properties of neurons and glial cells in tissue culture. J. Neurofihy~ioE. 25: 277-304. 19. HOSEIN, F. C. G., and C. D. ALLERAXD. 1968. Metabolism of specifically labelled glucose by explants of newborn mouse cerebellum. J. Neztrochcm. 15: 427-432. 20. JASSEP~, J., and -4. BRODAL. 1954. “.4spects of Cerebellar Anatomy.” J. Grndt Tanum Forlag, Oslo. 21. KIM, S. U. 1963. Neurons in the tissue culture. Arch. Histol. Jap. 23: 401-429. 22. KM, S. U. 1965. Neurons in the tissue culture. Observations on terminal boutons in cultures of mammalian central nervous tissue. Arcll. Histol. Jab. 25: 371-381. 23. KIM, S. TJ. 1966. Histochemical demonstration of oxidative enzymes associated with carbohydrate metabolism in cerebellar neurons cultured in vitro. Avck. Histol. lap. 2'? : 465471. 24. K~ar, S. U. 1970. Cytochemical demonstration of “marker” enzymes in nerve cells cultured in vitro. Expevicnfia 26 : 292-293. 25. I-211, S. U. 1970. Observations on cerebellar granule cells in tissue culture. A silver and electron microscopic study. 2. Ze/lforsch. 107 : 454465. 26. KIM, S. U. 1971. Neurotoxic effects of alkyl mercury compound on myelinating cultures of mouse cerebellum. E.rp. Nrftro2. (In press). 27. KIX, S. U., hf. R. MURRAY, LV. LIT. TOURTELLOTTE, and J. A. PARKER. 1970. Demonstration in tissue culture of myelinotoxicity in cerebrospinal fluid and brain extracts from multiple sclerosis patients. J. ~Vmropathol. E.rj~. Xcwol. 29 : 420-431. 28. KIWI, S. U., and Y. TASAKA. 1971. Myelinated neuronal soma in organized cultures of mouse central nervous tissue. E-t-p. Xcllrol. 30: 190-193. 29.

A. 1966. Occurrence of glycogen in developing cerebellar mossy fiber : An electron microscopic study. drcl~. Histol. Jap. 27 : 451464. KORNGUTII, S. E.. J. IV. ANDERSOX, and G. SCOTT. 1967. Obervations on the ultrastructure of the developin g cerebellum of the RIacaca mulatta. J. Cuwp. Nellrol. 130 : l-21. KONI~HI,

endings

30.

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44 31.

L. M. H. 1965. Maturation of the glomerular synapses of the cerebellum. An electron microscopic study. Anat. Rec. 151: 376. 32. LARRAMENDI, L. M. H. 1969. Analysis of synaptogenesis in the cerebellum of the mouse, pp. 803-843. In “Neurobiology of Cerebellar Evolution and Development.” R. Llinas (ed.) , Med. Ass. Educ. Reser. Found., Chicago. 33. LEHRER, C. M., M. B. BORNSTEIN, C. WEISS, M. FURMAN, and C. LICHTMAN. 1970. Enzymes of carbohydrate metabolism in the rat cerebellum developing in situ and in vitro. Exp. Neurol. 27 : 41&425. 34. LUMSDEN, C. E. 1965. The clinical neuropathology of multiple sclerosis, pp. 243393. In “Multiple Sclerosis, A reappraisal.” D. McAlpine, C. E. Lumsden, and E. D. Acheson (eds.), Williams & Wilkins, Baltimore. 34a. LUMSDEN, C. E. 1968. Nervous tissue in culture, pp. 68-141. 11%“The Structure and Function of Nervous Tissue.” Vol. 1. G. H. Bourne (ed), Academic Press, New York. 35. MASUROVSKY, E. B., and R. P. BUNGE. 1965. Fluoroplastic coverslips for longterm nerve tissue culture. Stain Technol. 43 : 161-165. 36. MIALE, I. L., and R. L. SIDMAN. 1961. An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp. Ncwol. 4 : 277-296. 37. O’LEARY, J. L., J. PETTY, J. M. SMITH, M. O’LEARY, and J. INUKAI. 1968. Cerebellar cortex of rat and other animals. A structural and ultrastructural study. J. Cow@. Newol. 134 : 401432. 38. RAMON Y CAJAL, S. 1909-1911. “Histologie du systeme nerveux de l’homme et des vertebres.” L. Azoulay (trans.) Instituto Ramon y Cajal, Madrid. (Reprint, 1952). 39. RAMON Y CAJAL, S. 1960. “Studies on vertebrate neurogenesis.” L. Guth [trans.], Thomas, Springfield, Ill. 40. ROSENBLUTH, J. 1966. Redundant myelin sheaths and other ultrastructural features of the toad cerebellum. J. Cell Biol. 28 : 73-93. 41. Ross, L. L., M. B. BORNSTEIN, and G. M. LEHER. 196.2. Electron microscopic observations of rat and mouse cerebellum in tissue culture. J. Cell Biol. 14: 19-30. 42. SEIL, F. J., and R. M. HERNDON. 1970. Cerebellar granule cells in vitro. A light and electron microscope study. J. Cell Biol. 45 : 212-220. 43. SMITH, K. R., JR. 1963. The cerebellar cortex of the rabbit. An electron microscopic study. J. COP@. Neural. 121: 4591183. 44. SUYEOKA, O., and M. OKAMOTO. 1966. Granule cells of the mouse cerebellum cultured in vitro : Their identification, degeneration and perikaryal myelin. Arch. Histol. Jab. 27 : 117-130. 45. UZMAN, L. L. 1960. The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. J. COTJI~.Neural. 114 : 137-159. LARRAMENDI,