EXPERIMENTAL
NEUROLOGY
Tissue
50,
Culture
A Light
and
226-239
(1976)
of Human Electron SEUNG
Rcccioed
Fetal
Cerebellum:
Microscopic U.
KIM
Study
1
JTLI~ 26, 1975
Explants of cerebellar hemisphere from a human fetus of 13 weeks of gestation were cultured for up to 80 days il~ vitro and studied by light and electron microscopy. In silver-stained preparations, small multipolar neurons and ring-shaped endings suggestive of bouton terminaux were demonstrated. Electron microscopic observations demonstrated morphological development of young neurons, synpases, and astrocytes in cultures. An increase in population and structural complexity of synapses in older cultures indicated the synaptic maturation ie zlitro. The presence of immature synapses in the starting materal (13 weeks of gestation) by electron microscopy suggests that synapse formation in the human cerebellum begins on or just before 13 necks of gestation. An extensive migration of young neurons into outgron-th zone was also noted by both light and electron microscopy.
INTRODUCTION Small explants taken from avian and mammalian central nervous system (CNS) can be maintained in tissue culture for extended periods of time under controlled conditions, and demonstrate a remarkable degree of structural, metabolic, and functional developments in vitro (6, 9, 16, 18, 19, 23, 27, 28). Although there have been several tissue culture studies using human fetal brain which describe the behavior of various cell types in vitro (5, 10-12, 22, 24), a detailed account of their developmental characteristics in vitro has not been reported. The purpose of this report is to describe at the cellular and subcellular levels the morphological features of human fetal cerebellum explants maintained and matured in vitro. Electron microscopic observations of synaptic development are described in detail. 1 This study was supported by Grants and was carried out at the Department Saskatoon, Canada.
from the Medical of Anatomy, 226
Copyright 1976 by Academic Press, Inc. All rights o? reproduction in any form reserved.
Research University
Council of Canada of Saskatchewan,
FETAL
MATERIALS
227
CEREBELLUM
AND
METHODS
Explants were prepared from the cerebellar cortex of a human fetus of 13 weeks gestational age (crown to rump length of 88 mm). Two hours after the fetus was removed surgically for the purpose of terminating pregnancy, a portion of cerebellar hemisphere was dissected out from which small explants approximately 1.5 x 1.5 x 1.0 mm were prepared. Two explants were placed on each rat-tail collagen coated coverslip and fed with a drop of nutrient medium consisting of equal parts of horse serum, medium 199, Hanks’ balanced salt solution (BSS), and supplementary glucose in a final concentration of 10 mg/ml. The coverslips carrying explants were sealed in Maximow slides (3) or in roller tubes (18) and incubated at 36 C. The living cultures were observed daily with a bright field microscope using a Zeiss x40 oil immersion objective. Cultures were maintained in vitro for up to SO days (corresponds to 24 weeks of gestation in utero), and during this period, 45 of them were fixed and stained for neurofibrils by a modification of Bodian’s silver method (17). Cultures maintained for 7, 17, 25, 35, 46, 54, 65, and SO days in vitro were prepared for electron microscopy. They were washed briefly in BSS, fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) for 20 min, postfixed in 2% osmic acid in phosphate buffer for 20 min, dehydrated in graded alcohols, and embedded in Epon 812. Small blocks of 13-week fetal cerebellum, which constituted the primary explants, were similarly fixed and embedded as described above. One micrometer thick sections cut on an LKB Ultratome were stained with 0.5% toluidine blue solution and examined by conventional light microscopy. Ultrathin sections, cut from selected areas, were stained with uranyl acetate and lead citrate, and examined in Philips 200 and Siemens Elmiskop 1A electron microscope. RESULTS Primary Explant. Light microscopic examination of l-pm thick sections of 13-week fetal cerebellar cortex revealed a cytoarchitecture similar to that described by others (31) (Fig. 1). The external granular layer consisted of four to five cell layers of closely packed, small ovoid cells (68 pm) with dark nuclei and scanty cytoplasm. Beneath this layer was a thin band of the molecular layer and a sparsely populated and ill-defined Purkinje cell layer which consisted of approximately lo-15 cell layers. Purkinje cells contained small ovoid nuclei slightly larger than external granule cells and scanty cytoplasm (6-10 pm). Few of the external granule cells appeared to have migrated inward to differentiate into granule cells at this age.
SEUNG u. mar
228
FIG. 1. Cerebellar cortex, 13-week-old external granular layer (EG) consisting molecular layer (M) and underlying larger Purkinje cells. X500.
fetus; l-pm thick epon section. h’ote the of several layers of dense cells, an ill-defined Purkinje cell layer (P) containing slightly
FIG. 2. An immature Purkinje cell in cerehllar cortex of 13-week-old differeniated cytoplasm which contains mitochondria (m), profiles reticulum (er) and clusters of ribosomes. X22,000. FIG. 3. An immature synapse in cerebellar cortex of 13-week-old thickening at the junction site (arrow) seems to represent the synapase formation. X35,000.
FIG. 4. Another metrical x35,000.
membrane
of
fetus. Poorly endoplasmic
fetus. earliest
Membrane stage of
immature synapse which contains few synaptic vesicles and thickening (arrow) in cerebellar cortex of 13-meek-old
asymfetus.
FETAL
CEREBELLUM
229
Electron microscopic examination of the primary explant from 13-week fetal cerebellar cortex showed that the Purkinje cells contained round electron-dense nuclei with one or more indentations and cytoplasm containing few mitochondria, poorly developed endoplasmic reticulum, and clusters of ribosomes (Fig. 2). The Golgi complex was rarely seen. Cells of the external granular layer were closely packed, had ovoid or angular nuclei with accumulation of chromatin against the nuclear envelope and had a thin rim of cytoplasm containing small condensed mitochondria, ribosomes singly or in clusters, and short elements of endoplasmic reticulum. Synaptic structures were infrequently found, and consisted of incipient and immature synapses. The earliest synapses were recognized by their membrane associated densities (Fig. 3). Synapses of a more advanced stage contained several vesicles in presynaptic terminals (Fig. 4). Such immature or primitive synaptic terminals have been described in embryonic avian or fetal mammalian central nervous system; there is general agreement that in early synaptogenesis, membrane thickening precedes the accumulation of synaptic vesicles (1, 2, 4, 8, 25, 32). The early synaptic profiles which can be determined with certainty contain membrane thickenings, few synaptic vesicles, no mitochondria and are exclusively axodendritic (4). Living Cultures. During the first 2-3 days, epithelioid cells emerged from the outer margin of the explants, and were followed by an outgrowth of nerve fibers. After 7-10 days in vitro, small, mostly unipolar or bipolar cells measuring 6-8 pm in diameter migrated from the periphery of the explants. Migration of the small cells steadily increased over the next 2-3 weeks to form clusters in the outgrowth zone. After 4-6 weeks the explants became thinner, allowing a better visualization of cell components by bright field microscopy ; cells observed in the explants were small cells measuring 610 pm in diameter (Fig. 5). Even after 2 months of ilz vitro development, I was not able to recognize large or medium sized neurons which I expected to see in the explants as mature Purkinje cells. Purkinje cells in cerebellum cultures from mouse or rat have been distinguished readily from other cells by their characteristic arrangement in arrays, large single nucleoli and vesicular nuclei surrounded by granular cytoplasm, and their size of 20-25 pm. After 4-6 weeks in vitro, several cultures developed an extensive outgrowth of astrocytes (Fig. 6). The astrocytes had cell bodies 30-40 pm in diameter, which contained peripherally located nuclei, granule-filled cytoplasm, and several long and broad processes. Silver Staining. In silver-stained cultures, the cells appeared as small multipolar neurons (6-10 pm cell body) with several fine processes which were interconnected and formed networks of nerve fibers (Fig. 7). In some areas, 20-30 of these small neurons were seen to form a cluster (Fig. 8). Some nerve fibers were observed terminating with fine ring-shaped endings
230
FIG.
periphery
SEUNG
U.
KIM
5. Numerous small cells probably immature Purkinje of an explant. Living culture 24 days irr z8ifr.o. X1000.
FIG. 6. Astrocytes are observed in the outgro\l processes. Living culture 45 days in eho. X200.
th
zone
cells
making
are
seen
mesh\,,-arks
in
the
of
which have previously been identified as synaptic boutons in cerebellum cultures (13, 14, 33). A detailed description of such ring-endings has been given in kitten cerebellum cultures (14). These boutons were usually found to be closely associated with cells (or cell nuclei), although some others were located in cell-free areas (inset, Fig. 9). The latter were probably associated with dendritic processes which failed to be stained by the silver method. In more than 40 cultures, grown for periods ranging from 7 to SO days, which correspond to 14-24 weeks gestation ix zitero, these small multipolar neurons were the only cell elements containing neurofibrils and identifiable as neurons. Our attempts to stain neurons and synaptic struc-
FETAL
CEREBELLUM
231
tures selectively by Bodian’s protargol method have not always been successful, and only a small percentage of explants exhibited silver-stained neurons. On no occasion was I able to impregnate large neurons which could, without any reservation, be identified as Purkinje cells as they were in mouse and kitten cerebellum cultures (9, 13, 15, 33). Bundles of fine nerve fibers were observed leaving the outer margin of explants and projecting into the surrounding outgrowth (Fig. 9). In the midst of these nerve fibers, ring-endings were frequently observed (Fig. 9). Electron Microscopy. In explants of 7 and 17 days in vitro, we observed small cells (6-10 pm) containing chromatin-rich, ovoid nuclei surrounded by a thin rim of cytoplasm. The cytoplasm contained a few small mitochondria, short elements of endoplasmic reticulum, and ribosomes singly or in clusters (Fig. 10). After 25 days in vvituo and onward, we observed numerous small cells (6-10 pm in diameter) which had round or ovoid nuclei with evenly distributed chromatin granules, and abundant cytoplasmic organelles such as several mitochondria, well developed granular endoplasmic reticulum, and components of Golgi complexes (Fig. 11). Frequently, these cells sent out long slender processes which contained numerous microtubules (250 K in diameter), running parallel to the long axis of the processes (Fig. 12). These small cells described above were tentatively identified as young neurons from ‘their content of organelles. such as granular endoplasmic reticulum and microtubules. Cells which could be identified as astrocytes by their content of numerous intermediate filaments (90 A in diameter) were also recognized among a large population of small neurons; in addition to the bundles of filaments they contained several mitochondria, elements of granular endoplasmic reticulum, ribosomes, and lysosomes (Fig. 13). After 7 and 17 days itt vitro, immature synapases similar to those observed in the primary explant (13-week fetal cerebellum) were seen in the cultures (Figs. 14, 15). An increased number of synapses and their structural maturation, as shown by the presence of mitochondria and increased population of synaptic vesicles, were noted progressively with the increased age of the cultures (Figs. 16, 17). Synaptic vesicles found in these synaptic terminals were mostly clear and spherical and measured 300-600 A. Dense cored vesicles and glycogen particles were found occasionally in mature synapses in older cultures (Fig. 17). All the synapses identified in the cultures were axodendritic and were asymmetric with regard to their membrane associated densities. It is not known at present between what types of neurons these synapses are formed, since there is no information available concerning early synaptogenesis in human cerebellum, in viva and in vitro. Several types of synaptic connections can be considered in cultures of human cerebellar hemisphere (in order to avoid
232
SEUNG u. KIM
FETAL
233
CEREBELLUM
including cerebellar nuclei, explants were taken from hemisphere) ; synapses between basket cells and Purkinje cells, granule cell parallel fibers and Purkinje cells, and those between Purkinje cell neurons. Since basket cell synapses are axosomatic in nature, and synapses found in cultures are always axodendritic, they cannot be basket cell synapses. Also, the size of synapses developed in cultures is rather large (the synapse in Fig. 17 measures 2.0 pm x 0.6 pm) for synapses formed by granule cell parallel fibers on Purkinje cell dendrites (approximately 0.5 pm in diameter in mouse and rat cerebellum in viva and in vitro). They are tentatively identified as synapses established between Purkinje cells. Neither myelinated axons nor oligodendrocytes were identified by electron microscopy even after 80 days in vitro development. Myelin formation was also not confirmed in living cultures by bright field microscopy. DISCUSSION Several reports have described the various cell types in cultures of human fetal CNS tissues (5, 10-12, 22, 24). Hogue ( 10, 11) described with light microscopy several neuronal types in living cultures; these observations were not confirmed by silver staining and/or electron microscopy, and certain of these “neurons” might correspond to glial or fibroblastic elements. Recently, Lapham and his associateshave reported observations on cultured explants from cerebellum and cerebrum of human fetus (5, 22, 24). They described the maturation of Purkinje cells and “granule cell” neurons by both light and electron microscopy (22). Although there is no doubt that Purkinje and granule cells develop in vitro, the criteria used by these authors for the identification of neurons appear inadequate. The absenceof asosomatic synapse and even moderately developed granular endoplasmic reticulum in their “Purkinje cell” [Fig. 3 of (22) ] and the appearance of “granule cell” nuclei with irregular morphology and accumulations of chromatin against nuclear envelope [Fig. 5 of (22) ] place their identification of neurons open to question. In addition, these authors could FIG. 7. Small multipolar neurons (n), probably and other neuronal types are seen interconnecting Bodian silver method, 35 days in vitro. X1000.
represent immature Purkinje each other by fine nerve
FIG. 8. A group of small neurons are shown making Note a ring-ending terminating near the neuronal method ; 46 days in vitro. X1000.
close soma
contacts (arrow).
cells, fihers.
with each other. Bodian silver
FIG. 9. Fine nerve fibers assembled in bundles are seen projected into outgrowth zone. Two ring-endings are also indicated (arrows). Bodian silver method; 46 days 0 terminates on the soma of a small neuron ilz vitro. X1.500. Inset: A ring-endin, (arrow). Bodian Silver method; 46 days C vitro. X1500.
FETAL
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not demonstrate the formation of synapses in culture, which I did in the present study. It should be noted that on no occasion was I able to demonstrate large neurons which could be identified as mature Purkinje cells in cultures stained with silver method, although smaller multipolar neurons were frequently observed. Electron microscopic examination of cultures also failed to reveal large mature neurons suggestive of Purkinje cells, although such mature neurons were found electron microscopically in mouse cerebellar cultures without difficulty (16). It appears that small neurons, identified as such by means of the silver method and electron microscopy in the present study, are in fact a mixed population of immature Purkinje cells, granule cells, and possibly basket and small stellate cell neurons. It is known that young neurons maintained in culture continue to mature and increase in size; in cultures of chick neural tube, neuroblasts increase 150-250 times in size within 3 weeks in vitro (20). In this regard, it is intriguing to note that even after 80 days development in vitro, the immature Purkinje cells did not increase in size. A possible explanation for this difference is that the gestation period in man is 300 days and that of the chick is 22 days, with the result that the morphological development of the neuronal elements proceeds much more slowly in human than in chick cultures. The development of synpatic complexes in the CNS has been studied by numerous investigators, and the question whether membrane thickening at a contact region represents an initial stage in synaptic formation has been discussed extensively. Many authors believed that membrane thickenings appear prior to synaptic vesicles in developing synapses (1, 2, 4, 8, 25, 32). At variance with these authors was a conclusion forwarded by Oppenheim and Foelix that immature synapses contain both membrane thickening and synaptic vesicles from a very early stage (29). It is likely that most, but not all, of the avesicular complexes with membrane thickenings found in developing CNS in V&JO and in vitro represent an initial stage of synaptogenesis. These complexes are seen in the- starting material (13-week gestation) and in early cultures, but not found in older mature cultures. If these FIG. 10. A migrating young neuron in outgrowth zone. Several mitochondria (m), granular endoplasmic reticulum (er), and a Golgi complex (g) are indicated; 17
days
in z&o.
FIG.
X13,000.
11. Relatively
well
differentiated
Purkinje
cell,
the cytoplasm
of which
contains
granular endoplasmic reticulum (er) and elements of Golgi complexes (g) ; 54 days in vitro. FIG. tubules
x28,000. 12. A neuron (t) ; 54 days
with a long dendrite irt z&a. X28,000.
which
contains
numerous
parallel
micro-
236
SEUNG
U.
KIM
FETAL
237
CEREBELLUM
complexes are junctions of attachment nature, they should also be found in mature cultures. To my knowledge, this study is the first to describe synapse formation in tissue culture of human brain. Although there is no neurophysiological evidence of functional activity of the synapses observed in these cerebellar cultures, complex synaptically-mediated bioelectric activity has previously been recorded in a similar organotypic culture of a &week human fetal spinal cord after weeks of maturation in vitro (7). In cultures of human fetal spinal cord and brain stem, HGsli et al. (12) have also reported the recording of resting membrane potentials from “neurons.” Even after repeated and careful observations by light and electron microscopy, no myelin was found in cultures grown for up to 80 days. The total period of growth of cells in these cultures (period grown ipl z&o combined with that in z&o) was close to the 24-weeks’ gestation period for human fetus. This absence of myelin formation in my cultures is not surprising, since myelination is one of the last steps of brain development. Myelinated axons in vizo are first detected in human fetal cerebellum of 6 months (28 weeks) gestation as determined by myelin staining (21). It is not until after birth that myelination occurs in the cerebellar hemisphere (21). These results, therefore, suggest that further cellular development is necessary to achieve myelination in cultures of human fetal cerebellar cortex. There is, however, another piece of evidence reported previously that may dispute the above explanation. Peterson et al. (30) have observed myelinated axons in a 75-day-old culture of 6-week human fetal spinal cord. The total period of growth in this explant is equal to 17 weeks of gestation for human fetus. However, it is known that myelinogenesis in human spinal cord precedes that in cerebellum, therefore, it seems &hat in human cerebellar cultures a far longer period of ilz &fro development is required to demonstrate myelin formation. FIG. 13. An astrocyte observedin outgrowth. The cytoplasmcontainsnumerous gliofilaments(f), mitochondria(m), and lysosomesfly) ; 6.5days insvitro. X13,000. FIG. 14. Symmetricalmembrane thickening at the junction (arrow) representsan early developingsynapse ; 7 days i*t Gtro. X35,000. FIG. 15. An axodendritic synapse(arrow) displays several synaptic vesicles,a mitochondria,and a slightly widened synaptic cleft which contains dense material; 17 days
ilt oifro.
X35,000.
FIG. 16. The increased the structural maturation
amount of synaptic vesicles in presynaptic of the synapse; 46 days in dro. X3.5,000.
ending
indicates
FIG. 17. A large presynaptic ending makes three synaptic contacts with dendritic processes and containsnumeroussynaptic vesicles (arrows). Densecored vesicles (dv) and glycogengranules(g) are also indicated.65 days in vitro. X28,000,
238
SEUNG
U.
KIM
Although the earliest synapse formation in fetal human brain has been reported in cerebral cortex of an U-week-old fetus (2S), a comparable study has not been made in cerebellum. Electron microscopic observations of the present study have shown that synapses found in the cerebellar cortex of a 13-week-old fetus are structurally immature (Figs. 3,4), suggesting that synapse formation in the human fetal cerebellum probably begins on or just before the 13-week gestation. In two separate reviews, Murray (27) and Lumsclen (23) stated that the migration of neurons from explants is a rare phenomenon in CNS cultures. In the present study, an extensive migration of young neurons into the outgrowth zone from the explants was consistently observed. Similar observations were made by Markesbery and Lapham (24), and Choi and Lapham (5) in cultures of human fetal cerebrum and cerebellum. At present it is not clear whether this neuronal migration is regulated by an inherent capability of young neurons to migrate to the final destination or a passive movement of young neurons influenced by migration of glial cells and fibroblasts into the outgrowth zone. REFERENCES J. 1971. Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat. Brake Res. 30 : 311-322. BODIAN, D. 1966. Development of fine structure of spinal cord in monkey fetuses. I. The motor neuron neuropil at the time of onset of reflex activity. Bltll. JO/W Hopkins Ho&al, 119 : 129-149. BORKSTEIN, 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. Biopl~ys. Biochc~z. Cytol. 4: 499-504. BUEGE, M. B., R. P. BUNGE, and E. R. PETERSON. 1967. The onset of synapse formation in spinal cord cultures as studied by electron microscopy. nrnitz Rrs. 6: 728-743. CHOI, B. H., and I-. W. LAPAIIAX 1974. Autoradiographic studies of migrating neurons and astrocytes of human fetal cerebral cortex in vitro. Exp. Mol. Pathol. 21: 204-217. CRAIN, S. M. 1966. Development of “organotypic” bioelectric activities in central nervous tissue during maturation in culture. Inf. Rev. Neurobiol. 9: l-43. GRAIN, S. M., and E. R. PETERSON. 1964. Complex bioelectric activity in organized tissue cultures of spinal cord (human, rat and chick). J. Cell. Corrrp. Pltssinl. 64: l-13. GLEES, P., and B. L. SHEPPARD. 1964. Electron microscopical studies of the synapse in the developing chick spinal cord. 2. Zellforsch. 62 : 356-362. HILD, W. 1966. Cell types and neuronal connections in cultures of mammalian central nervous tissue. 2. Zellforsch. 69: 155-188. HOGUE, M. J. 1947. Human fetal brain cells in tissue culture: Their identification and motility. /. Exp. Zool. 106 : 85-108. HOCUE, M. J. 1950. Brain cells from human fetuses and infants cultured in e8itr.o after death of the individuals. Axat. Rec. 108: 457-475.
1. ALTMAN, 2.
3.
4.
5.
6. 7.
8. 9.
10. 11.
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12. H~SLI, L., E. HOSLI, and P. F. Aaroa~s. 1973. Light and electroyhysiological studies of cultured human central nervous tissue. Eztr. Ncrtrol. 9: 121-130. 13. KIM, S. U. 1963. Neurons in tissue culture. Arch. Histol. Jap. 23: 401-429. 14. KIM, S. U. 1965. Neurons in tissue culture. Observations on terminal boutons in cultures of mammalian central nervous tissue. Arch. Histol. lap. 25: 371381. 15. KIM, S. U. 1970. Observations on cerebellar granule cells in tissue culture. A silver and electron microscopic study. 2. Zellforsch. 107 : 454-465 16. KIM, S. U. 1971. Electron microscope study of mouse cerebellum in tissue culture. Exp.
Ncurol.
33 : 30-44.
17. KIM, S. U. 1971. Neuronal (Basel)
29:
types in long-term
cultures of avian retina. E.z-perioda
264-265.
18. KIM, S. U., T. H. OH, and D. D. JOHNSON. 1972. Developmental changes of actylcholinesterase and pseudocholinesterase in organotypic cultures of spinal cord. Exp. Neurol. 35: 274-281. 19. KIM, S. U., and G. TUNNICLIFF. 1974. Morphological and biochemical development of chick cerebrum cultured in vitro. Exp. Neurol. 43: 515-526. 20. KIM, S. U., and E. L. WENGER. 1972. De novo formation of synapse in cultures of chick neural tube. Nature (New Biol.) 236: 152-153. 21. LANGWORTHY, 0. R. 1932. Development of behavior patterns and myelinization of tracts in the nervous system. Arch. Neurol. Psychiat. (Chicago) 28: 1365-1382. 22. LAPHAM, L. W., and W. R. MARKESBERY. 1971. Human fetal cerebellar cortex: Organization and maturation of cells in vitro. Science 173 : 829-832. 23. LUMSDEN, C. E. 1968. Nervous tissue in culture, pp. 68-41. In “The Structure and Function of Nervous Tissue.” Vol. 1, G. H. Bourne [Ed.]. Academic Press, New York. 24. MARKESBERY, W. R., and L. W. LAPHAM. 1974. A correlated light and electron microscopic study of the early phase of growth in vitro of human fetal cerebellar and cerebral cortex. J. Neatropathol. Exp. Neurol. 33: 113-127. 25. MELLER, K., and R. HAUPT. 1967. Die Feinstruktur der Neuro-, Glio- und Ependymoblasten von Hiihnerembryonen in der Gewebe kultur. 2. Zellforsch. 76 : 260-277. 26. MOLLIVER, M. E., I. KOSTOVIC, and H. VAN DER Loos. 1973. The development of synapses in cerebral cortex of human fetus. Brain. Res. 50 : 403-407. 27. MURRAY, M. R. 1965. Nervous tissue in vitro, pp. 373455. In “Cells and Tissues in Culture.” Vol. 2, E. N. Willmer [Ed.]. Academic Press, New York. 28. NELSON, P. G. 1975. Nerve and muscle cells in culture. Physiol. Rev. 55: 161. 29. OPPENHEIM, R. W., and R. F. FOELIX. 1972. Synaptogenesis in the chick embryo spinal cord. Nature (New Biol.) 235: 126128. 30. PETERSON, E. R., S. M. CRAIN, and M. R. MURRAY. 1965. Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human). 2. Zellforsclt. 66: 130-154. 31. RAKIC, P., and R. L. SIDMAN. 1970. Histogenesis of cortical layers in human cerebellum, particularly the lamina dissecans. J. Camp. Neural. 139: 473-500. 32. WECHSLER, W. 1966. Elektronmikroskopischer Beitrage zur Nervenzelldifferenzienung und Histogenese der grauen Substanz des Riickenmarks von Hiihnerembryonen. 2. Zellforsch. 74: 401-422. 33. WOLF, M. K. 1964. Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum. J. Cell. Biol. 22: 259-279.
NEUROLOGY
Tissue
50,
Culture
A Light
and
226-239
(1976)
of Human Electron SEUNG
Rcccioed
Fetal
Cerebellum:
Microscopic U.
KIM
Study
1
JTLI~ 26, 1975
Explants of cerebellar hemisphere from a human fetus of 13 weeks of gestation were cultured for up to 80 days il~ vitro and studied by light and electron microscopy. In silver-stained preparations, small multipolar neurons and ring-shaped endings suggestive of bouton terminaux were demonstrated. Electron microscopic observations demonstrated morphological development of young neurons, synpases, and astrocytes in cultures. An increase in population and structural complexity of synapses in older cultures indicated the synaptic maturation ie zlitro. The presence of immature synapses in the starting materal (13 weeks of gestation) by electron microscopy suggests that synapse formation in the human cerebellum begins on or just before 13 necks of gestation. An extensive migration of young neurons into outgron-th zone was also noted by both light and electron microscopy.
INTRODUCTION Small explants taken from avian and mammalian central nervous system (CNS) can be maintained in tissue culture for extended periods of time under controlled conditions, and demonstrate a remarkable degree of structural, metabolic, and functional developments in vitro (6, 9, 16, 18, 19, 23, 27, 28). Although there have been several tissue culture studies using human fetal brain which describe the behavior of various cell types in vitro (5, 10-12, 22, 24), a detailed account of their developmental characteristics in vitro has not been reported. The purpose of this report is to describe at the cellular and subcellular levels the morphological features of human fetal cerebellum explants maintained and matured in vitro. Electron microscopic observations of synaptic development are described in detail. 1 This study was supported by Grants and was carried out at the Department Saskatoon, Canada.
from the Medical of Anatomy, 226
Copyright 1976 by Academic Press, Inc. All rights o? reproduction in any form reserved.
Research University
Council of Canada of Saskatchewan,
FETAL
MATERIALS
227
CEREBELLUM
AND
METHODS
Explants were prepared from the cerebellar cortex of a human fetus of 13 weeks gestational age (crown to rump length of 88 mm). Two hours after the fetus was removed surgically for the purpose of terminating pregnancy, a portion of cerebellar hemisphere was dissected out from which small explants approximately 1.5 x 1.5 x 1.0 mm were prepared. Two explants were placed on each rat-tail collagen coated coverslip and fed with a drop of nutrient medium consisting of equal parts of horse serum, medium 199, Hanks’ balanced salt solution (BSS), and supplementary glucose in a final concentration of 10 mg/ml. The coverslips carrying explants were sealed in Maximow slides (3) or in roller tubes (18) and incubated at 36 C. The living cultures were observed daily with a bright field microscope using a Zeiss x40 oil immersion objective. Cultures were maintained in vitro for up to SO days (corresponds to 24 weeks of gestation in utero), and during this period, 45 of them were fixed and stained for neurofibrils by a modification of Bodian’s silver method (17). Cultures maintained for 7, 17, 25, 35, 46, 54, 65, and SO days in vitro were prepared for electron microscopy. They were washed briefly in BSS, fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) for 20 min, postfixed in 2% osmic acid in phosphate buffer for 20 min, dehydrated in graded alcohols, and embedded in Epon 812. Small blocks of 13-week fetal cerebellum, which constituted the primary explants, were similarly fixed and embedded as described above. One micrometer thick sections cut on an LKB Ultratome were stained with 0.5% toluidine blue solution and examined by conventional light microscopy. Ultrathin sections, cut from selected areas, were stained with uranyl acetate and lead citrate, and examined in Philips 200 and Siemens Elmiskop 1A electron microscope. RESULTS Primary Explant. Light microscopic examination of l-pm thick sections of 13-week fetal cerebellar cortex revealed a cytoarchitecture similar to that described by others (31) (Fig. 1). The external granular layer consisted of four to five cell layers of closely packed, small ovoid cells (68 pm) with dark nuclei and scanty cytoplasm. Beneath this layer was a thin band of the molecular layer and a sparsely populated and ill-defined Purkinje cell layer which consisted of approximately lo-15 cell layers. Purkinje cells contained small ovoid nuclei slightly larger than external granule cells and scanty cytoplasm (6-10 pm). Few of the external granule cells appeared to have migrated inward to differentiate into granule cells at this age.
SEUNG u. mar
228
FIG. 1. Cerebellar cortex, 13-week-old external granular layer (EG) consisting molecular layer (M) and underlying larger Purkinje cells. X500.
fetus; l-pm thick epon section. h’ote the of several layers of dense cells, an ill-defined Purkinje cell layer (P) containing slightly
FIG. 2. An immature Purkinje cell in cerehllar cortex of 13-week-old differeniated cytoplasm which contains mitochondria (m), profiles reticulum (er) and clusters of ribosomes. X22,000. FIG. 3. An immature synapse in cerebellar cortex of 13-week-old thickening at the junction site (arrow) seems to represent the synapase formation. X35,000.
FIG. 4. Another metrical x35,000.
membrane
of
fetus. Poorly endoplasmic
fetus. earliest
Membrane stage of
immature synapse which contains few synaptic vesicles and thickening (arrow) in cerebellar cortex of 13-meek-old
asymfetus.
FETAL
CEREBELLUM
229
Electron microscopic examination of the primary explant from 13-week fetal cerebellar cortex showed that the Purkinje cells contained round electron-dense nuclei with one or more indentations and cytoplasm containing few mitochondria, poorly developed endoplasmic reticulum, and clusters of ribosomes (Fig. 2). The Golgi complex was rarely seen. Cells of the external granular layer were closely packed, had ovoid or angular nuclei with accumulation of chromatin against the nuclear envelope and had a thin rim of cytoplasm containing small condensed mitochondria, ribosomes singly or in clusters, and short elements of endoplasmic reticulum. Synaptic structures were infrequently found, and consisted of incipient and immature synapses. The earliest synapses were recognized by their membrane associated densities (Fig. 3). Synapses of a more advanced stage contained several vesicles in presynaptic terminals (Fig. 4). Such immature or primitive synaptic terminals have been described in embryonic avian or fetal mammalian central nervous system; there is general agreement that in early synaptogenesis, membrane thickening precedes the accumulation of synaptic vesicles (1, 2, 4, 8, 25, 32). The early synaptic profiles which can be determined with certainty contain membrane thickenings, few synaptic vesicles, no mitochondria and are exclusively axodendritic (4). Living Cultures. During the first 2-3 days, epithelioid cells emerged from the outer margin of the explants, and were followed by an outgrowth of nerve fibers. After 7-10 days in vitro, small, mostly unipolar or bipolar cells measuring 6-8 pm in diameter migrated from the periphery of the explants. Migration of the small cells steadily increased over the next 2-3 weeks to form clusters in the outgrowth zone. After 4-6 weeks the explants became thinner, allowing a better visualization of cell components by bright field microscopy ; cells observed in the explants were small cells measuring 610 pm in diameter (Fig. 5). Even after 2 months of ilz vitro development, I was not able to recognize large or medium sized neurons which I expected to see in the explants as mature Purkinje cells. Purkinje cells in cerebellum cultures from mouse or rat have been distinguished readily from other cells by their characteristic arrangement in arrays, large single nucleoli and vesicular nuclei surrounded by granular cytoplasm, and their size of 20-25 pm. After 4-6 weeks in vitro, several cultures developed an extensive outgrowth of astrocytes (Fig. 6). The astrocytes had cell bodies 30-40 pm in diameter, which contained peripherally located nuclei, granule-filled cytoplasm, and several long and broad processes. Silver Staining. In silver-stained cultures, the cells appeared as small multipolar neurons (6-10 pm cell body) with several fine processes which were interconnected and formed networks of nerve fibers (Fig. 7). In some areas, 20-30 of these small neurons were seen to form a cluster (Fig. 8). Some nerve fibers were observed terminating with fine ring-shaped endings
230
FIG.
periphery
SEUNG
U.
KIM
5. Numerous small cells probably immature Purkinje of an explant. Living culture 24 days irr z8ifr.o. X1000.
FIG. 6. Astrocytes are observed in the outgro\l processes. Living culture 45 days in eho. X200.
th
zone
cells
making
are
seen
mesh\,,-arks
in
the
of
which have previously been identified as synaptic boutons in cerebellum cultures (13, 14, 33). A detailed description of such ring-endings has been given in kitten cerebellum cultures (14). These boutons were usually found to be closely associated with cells (or cell nuclei), although some others were located in cell-free areas (inset, Fig. 9). The latter were probably associated with dendritic processes which failed to be stained by the silver method. In more than 40 cultures, grown for periods ranging from 7 to SO days, which correspond to 14-24 weeks gestation ix zitero, these small multipolar neurons were the only cell elements containing neurofibrils and identifiable as neurons. Our attempts to stain neurons and synaptic struc-
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tures selectively by Bodian’s protargol method have not always been successful, and only a small percentage of explants exhibited silver-stained neurons. On no occasion was I able to impregnate large neurons which could, without any reservation, be identified as Purkinje cells as they were in mouse and kitten cerebellum cultures (9, 13, 15, 33). Bundles of fine nerve fibers were observed leaving the outer margin of explants and projecting into the surrounding outgrowth (Fig. 9). In the midst of these nerve fibers, ring-endings were frequently observed (Fig. 9). Electron Microscopy. In explants of 7 and 17 days in vitro, we observed small cells (6-10 pm) containing chromatin-rich, ovoid nuclei surrounded by a thin rim of cytoplasm. The cytoplasm contained a few small mitochondria, short elements of endoplasmic reticulum, and ribosomes singly or in clusters (Fig. 10). After 25 days in vvituo and onward, we observed numerous small cells (6-10 pm in diameter) which had round or ovoid nuclei with evenly distributed chromatin granules, and abundant cytoplasmic organelles such as several mitochondria, well developed granular endoplasmic reticulum, and components of Golgi complexes (Fig. 11). Frequently, these cells sent out long slender processes which contained numerous microtubules (250 K in diameter), running parallel to the long axis of the processes (Fig. 12). These small cells described above were tentatively identified as young neurons from ‘their content of organelles. such as granular endoplasmic reticulum and microtubules. Cells which could be identified as astrocytes by their content of numerous intermediate filaments (90 A in diameter) were also recognized among a large population of small neurons; in addition to the bundles of filaments they contained several mitochondria, elements of granular endoplasmic reticulum, ribosomes, and lysosomes (Fig. 13). After 7 and 17 days itt vitro, immature synapases similar to those observed in the primary explant (13-week fetal cerebellum) were seen in the cultures (Figs. 14, 15). An increased number of synapses and their structural maturation, as shown by the presence of mitochondria and increased population of synaptic vesicles, were noted progressively with the increased age of the cultures (Figs. 16, 17). Synaptic vesicles found in these synaptic terminals were mostly clear and spherical and measured 300-600 A. Dense cored vesicles and glycogen particles were found occasionally in mature synapses in older cultures (Fig. 17). All the synapses identified in the cultures were axodendritic and were asymmetric with regard to their membrane associated densities. It is not known at present between what types of neurons these synapses are formed, since there is no information available concerning early synaptogenesis in human cerebellum, in viva and in vitro. Several types of synaptic connections can be considered in cultures of human cerebellar hemisphere (in order to avoid
232
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including cerebellar nuclei, explants were taken from hemisphere) ; synapses between basket cells and Purkinje cells, granule cell parallel fibers and Purkinje cells, and those between Purkinje cell neurons. Since basket cell synapses are axosomatic in nature, and synapses found in cultures are always axodendritic, they cannot be basket cell synapses. Also, the size of synapses developed in cultures is rather large (the synapse in Fig. 17 measures 2.0 pm x 0.6 pm) for synapses formed by granule cell parallel fibers on Purkinje cell dendrites (approximately 0.5 pm in diameter in mouse and rat cerebellum in viva and in vitro). They are tentatively identified as synapses established between Purkinje cells. Neither myelinated axons nor oligodendrocytes were identified by electron microscopy even after 80 days in vitro development. Myelin formation was also not confirmed in living cultures by bright field microscopy. DISCUSSION Several reports have described the various cell types in cultures of human fetal CNS tissues (5, 10-12, 22, 24). Hogue ( 10, 11) described with light microscopy several neuronal types in living cultures; these observations were not confirmed by silver staining and/or electron microscopy, and certain of these “neurons” might correspond to glial or fibroblastic elements. Recently, Lapham and his associateshave reported observations on cultured explants from cerebellum and cerebrum of human fetus (5, 22, 24). They described the maturation of Purkinje cells and “granule cell” neurons by both light and electron microscopy (22). Although there is no doubt that Purkinje and granule cells develop in vitro, the criteria used by these authors for the identification of neurons appear inadequate. The absenceof asosomatic synapse and even moderately developed granular endoplasmic reticulum in their “Purkinje cell” [Fig. 3 of (22) ] and the appearance of “granule cell” nuclei with irregular morphology and accumulations of chromatin against nuclear envelope [Fig. 5 of (22) ] place their identification of neurons open to question. In addition, these authors could FIG. 7. Small multipolar neurons (n), probably and other neuronal types are seen interconnecting Bodian silver method, 35 days in vitro. X1000.
represent immature Purkinje each other by fine nerve
FIG. 8. A group of small neurons are shown making Note a ring-ending terminating near the neuronal method ; 46 days in vitro. X1000.
close soma
contacts (arrow).
cells, fihers.
with each other. Bodian silver
FIG. 9. Fine nerve fibers assembled in bundles are seen projected into outgrowth zone. Two ring-endings are also indicated (arrows). Bodian silver method; 46 days 0 terminates on the soma of a small neuron ilz vitro. X1.500. Inset: A ring-endin, (arrow). Bodian Silver method; 46 days C vitro. X1500.
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not demonstrate the formation of synapses in culture, which I did in the present study. It should be noted that on no occasion was I able to demonstrate large neurons which could be identified as mature Purkinje cells in cultures stained with silver method, although smaller multipolar neurons were frequently observed. Electron microscopic examination of cultures also failed to reveal large mature neurons suggestive of Purkinje cells, although such mature neurons were found electron microscopically in mouse cerebellar cultures without difficulty (16). It appears that small neurons, identified as such by means of the silver method and electron microscopy in the present study, are in fact a mixed population of immature Purkinje cells, granule cells, and possibly basket and small stellate cell neurons. It is known that young neurons maintained in culture continue to mature and increase in size; in cultures of chick neural tube, neuroblasts increase 150-250 times in size within 3 weeks in vitro (20). In this regard, it is intriguing to note that even after 80 days development in vitro, the immature Purkinje cells did not increase in size. A possible explanation for this difference is that the gestation period in man is 300 days and that of the chick is 22 days, with the result that the morphological development of the neuronal elements proceeds much more slowly in human than in chick cultures. The development of synpatic complexes in the CNS has been studied by numerous investigators, and the question whether membrane thickening at a contact region represents an initial stage in synaptic formation has been discussed extensively. Many authors believed that membrane thickenings appear prior to synaptic vesicles in developing synapses (1, 2, 4, 8, 25, 32). At variance with these authors was a conclusion forwarded by Oppenheim and Foelix that immature synapses contain both membrane thickening and synaptic vesicles from a very early stage (29). It is likely that most, but not all, of the avesicular complexes with membrane thickenings found in developing CNS in V&JO and in vitro represent an initial stage of synaptogenesis. These complexes are seen in the- starting material (13-week gestation) and in early cultures, but not found in older mature cultures. If these FIG. 10. A migrating young neuron in outgrowth zone. Several mitochondria (m), granular endoplasmic reticulum (er), and a Golgi complex (g) are indicated; 17
days
in z&o.
FIG.
X13,000.
11. Relatively
well
differentiated
Purkinje
cell,
the cytoplasm
of which
contains
granular endoplasmic reticulum (er) and elements of Golgi complexes (g) ; 54 days in vitro. FIG. tubules
x28,000. 12. A neuron (t) ; 54 days
with a long dendrite irt z&a. X28,000.
which
contains
numerous
parallel
micro-
236
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KIM
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complexes are junctions of attachment nature, they should also be found in mature cultures. To my knowledge, this study is the first to describe synapse formation in tissue culture of human brain. Although there is no neurophysiological evidence of functional activity of the synapses observed in these cerebellar cultures, complex synaptically-mediated bioelectric activity has previously been recorded in a similar organotypic culture of a &week human fetal spinal cord after weeks of maturation in vitro (7). In cultures of human fetal spinal cord and brain stem, HGsli et al. (12) have also reported the recording of resting membrane potentials from “neurons.” Even after repeated and careful observations by light and electron microscopy, no myelin was found in cultures grown for up to 80 days. The total period of growth of cells in these cultures (period grown ipl z&o combined with that in z&o) was close to the 24-weeks’ gestation period for human fetus. This absence of myelin formation in my cultures is not surprising, since myelination is one of the last steps of brain development. Myelinated axons in vizo are first detected in human fetal cerebellum of 6 months (28 weeks) gestation as determined by myelin staining (21). It is not until after birth that myelination occurs in the cerebellar hemisphere (21). These results, therefore, suggest that further cellular development is necessary to achieve myelination in cultures of human fetal cerebellar cortex. There is, however, another piece of evidence reported previously that may dispute the above explanation. Peterson et al. (30) have observed myelinated axons in a 75-day-old culture of 6-week human fetal spinal cord. The total period of growth in this explant is equal to 17 weeks of gestation for human fetus. However, it is known that myelinogenesis in human spinal cord precedes that in cerebellum, therefore, it seems &hat in human cerebellar cultures a far longer period of ilz &fro development is required to demonstrate myelin formation. FIG. 13. An astrocyte observedin outgrowth. The cytoplasmcontainsnumerous gliofilaments(f), mitochondria(m), and lysosomesfly) ; 6.5days insvitro. X13,000. FIG. 14. Symmetricalmembrane thickening at the junction (arrow) representsan early developingsynapse ; 7 days i*t Gtro. X35,000. FIG. 15. An axodendritic synapse(arrow) displays several synaptic vesicles,a mitochondria,and a slightly widened synaptic cleft which contains dense material; 17 days
ilt oifro.
X35,000.
FIG. 16. The increased the structural maturation
amount of synaptic vesicles in presynaptic of the synapse; 46 days in dro. X3.5,000.
ending
indicates
FIG. 17. A large presynaptic ending makes three synaptic contacts with dendritic processes and containsnumeroussynaptic vesicles (arrows). Densecored vesicles (dv) and glycogengranules(g) are also indicated.65 days in vitro. X28,000,
238
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KIM
Although the earliest synapse formation in fetal human brain has been reported in cerebral cortex of an U-week-old fetus (2S), a comparable study has not been made in cerebellum. Electron microscopic observations of the present study have shown that synapses found in the cerebellar cortex of a 13-week-old fetus are structurally immature (Figs. 3,4), suggesting that synapse formation in the human fetal cerebellum probably begins on or just before the 13-week gestation. In two separate reviews, Murray (27) and Lumsclen (23) stated that the migration of neurons from explants is a rare phenomenon in CNS cultures. In the present study, an extensive migration of young neurons into the outgrowth zone from the explants was consistently observed. Similar observations were made by Markesbery and Lapham (24), and Choi and Lapham (5) in cultures of human fetal cerebrum and cerebellum. At present it is not clear whether this neuronal migration is regulated by an inherent capability of young neurons to migrate to the final destination or a passive movement of young neurons influenced by migration of glial cells and fibroblasts into the outgrowth zone. REFERENCES J. 1971. Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat. Brake Res. 30 : 311-322. BODIAN, D. 1966. Development of fine structure of spinal cord in monkey fetuses. I. The motor neuron neuropil at the time of onset of reflex activity. Bltll. JO/W Hopkins Ho&al, 119 : 129-149. BORKSTEIN, 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. Biopl~ys. Biochc~z. Cytol. 4: 499-504. BUEGE, M. B., R. P. BUNGE, and E. R. PETERSON. 1967. The onset of synapse formation in spinal cord cultures as studied by electron microscopy. nrnitz Rrs. 6: 728-743. CHOI, B. H., and I-. W. LAPAIIAX 1974. Autoradiographic studies of migrating neurons and astrocytes of human fetal cerebral cortex in vitro. Exp. Mol. Pathol. 21: 204-217. CRAIN, S. M. 1966. Development of “organotypic” bioelectric activities in central nervous tissue during maturation in culture. Inf. Rev. Neurobiol. 9: l-43. GRAIN, S. M., and E. R. PETERSON. 1964. Complex bioelectric activity in organized tissue cultures of spinal cord (human, rat and chick). J. Cell. Corrrp. Pltssinl. 64: l-13. GLEES, P., and B. L. SHEPPARD. 1964. Electron microscopical studies of the synapse in the developing chick spinal cord. 2. Zellforsch. 62 : 356-362. HILD, W. 1966. Cell types and neuronal connections in cultures of mammalian central nervous tissue. 2. Zellforsch. 69: 155-188. HOGUE, M. J. 1947. Human fetal brain cells in tissue culture: Their identification and motility. /. Exp. Zool. 106 : 85-108. HOCUE, M. J. 1950. Brain cells from human fetuses and infants cultured in e8itr.o after death of the individuals. Axat. Rec. 108: 457-475.
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12. H~SLI, L., E. HOSLI, and P. F. Aaroa~s. 1973. Light and electroyhysiological studies of cultured human central nervous tissue. Eztr. Ncrtrol. 9: 121-130. 13. KIM, S. U. 1963. Neurons in tissue culture. Arch. Histol. Jap. 23: 401-429. 14. KIM, S. U. 1965. Neurons in tissue culture. Observations on terminal boutons in cultures of mammalian central nervous tissue. Arch. Histol. lap. 25: 371381. 15. KIM, S. U. 1970. Observations on cerebellar granule cells in tissue culture. A silver and electron microscopic study. 2. Zellforsch. 107 : 454-465 16. KIM, S. U. 1971. Electron microscope study of mouse cerebellum in tissue culture. Exp.
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17. KIM, S. U. 1971. Neuronal (Basel)
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18. KIM, S. U., T. H. OH, and D. D. JOHNSON. 1972. Developmental changes of actylcholinesterase and pseudocholinesterase in organotypic cultures of spinal cord. Exp. Neurol. 35: 274-281. 19. KIM, S. U., and G. TUNNICLIFF. 1974. Morphological and biochemical development of chick cerebrum cultured in vitro. Exp. Neurol. 43: 515-526. 20. KIM, S. U., and E. L. WENGER. 1972. De novo formation of synapse in cultures of chick neural tube. Nature (New Biol.) 236: 152-153. 21. LANGWORTHY, 0. R. 1932. Development of behavior patterns and myelinization of tracts in the nervous system. Arch. Neurol. Psychiat. (Chicago) 28: 1365-1382. 22. LAPHAM, L. W., and W. R. MARKESBERY. 1971. Human fetal cerebellar cortex: Organization and maturation of cells in vitro. Science 173 : 829-832. 23. LUMSDEN, C. E. 1968. Nervous tissue in culture, pp. 68-41. In “The Structure and Function of Nervous Tissue.” Vol. 1, G. H. Bourne [Ed.]. Academic Press, New York. 24. MARKESBERY, W. R., and L. W. LAPHAM. 1974. A correlated light and electron microscopic study of the early phase of growth in vitro of human fetal cerebellar and cerebral cortex. J. Neatropathol. Exp. Neurol. 33: 113-127. 25. MELLER, K., and R. HAUPT. 1967. Die Feinstruktur der Neuro-, Glio- und Ependymoblasten von Hiihnerembryonen in der Gewebe kultur. 2. Zellforsch. 76 : 260-277. 26. MOLLIVER, M. E., I. KOSTOVIC, and H. VAN DER Loos. 1973. The development of synapses in cerebral cortex of human fetus. Brain. Res. 50 : 403-407. 27. MURRAY, M. R. 1965. Nervous tissue in vitro, pp. 373455. In “Cells and Tissues in Culture.” Vol. 2, E. N. Willmer [Ed.]. Academic Press, New York. 28. NELSON, P. G. 1975. Nerve and muscle cells in culture. Physiol. Rev. 55: 161. 29. OPPENHEIM, R. W., and R. F. FOELIX. 1972. Synaptogenesis in the chick embryo spinal cord. Nature (New Biol.) 235: 126128. 30. PETERSON, E. R., S. M. CRAIN, and M. R. MURRAY. 1965. Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human). 2. Zellforsclt. 66: 130-154. 31. RAKIC, P., and R. L. SIDMAN. 1970. Histogenesis of cortical layers in human cerebellum, particularly the lamina dissecans. J. Camp. Neural. 139: 473-500. 32. WECHSLER, W. 1966. Elektronmikroskopischer Beitrage zur Nervenzelldifferenzienung und Histogenese der grauen Substanz des Riickenmarks von Hiihnerembryonen. 2. Zellforsch. 74: 401-422. 33. WOLF, M. K. 1964. Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum. J. Cell. Biol. 22: 259-279.
