Brain Research, 99 (1975) 117-123 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

I [7

Short Communications

Organ culture of the developing human cerebellum

JEAN JACQUES HAUW AND RAYMOND ESCOUROLLE Laboratoire de Neuropathologie ChaHes Foix, La SalpdtriOre, 75634 Paris (France)

(Accepted July 30th, 1975)

Although very rarely employed for the central nervous system, organotypic - in the strict meaning of the word - - methods of tissue culture 1° can bring obvious advantages for cellular identification and for the study of tissue organization in vitro. They are readily applicable to cerebellum cultures 14, especially in the h u m a n l k They allow, in this example, a striking persistent or acquired organization, very similar to what exists or should develop in situ. This appears useful to the study of isolated tissue development after deafferentation and suppression of hormonal control. We report here preliminary results concerning the human cerebellum culture in the strictly organotypic chamber of Goube de Laforest et al. s. Cerebellum of embryos and fetuses obtained from legal abortions were dissected under a binocular microscope into sagittal sections of 1 mm × 1 mm × 0.5 mm removed from the culmen cortex at the bottom of the primary fissure or from this presumed area for the youngest embryos. They were explanted onto a nutrient gelosis drop in the Goube de Laforest chamber, as already described 11,12. In comparative experiments, collagen-coated coverslips were used in Maximow double-coverslip assemblies s or in Leighton tubes is. Nutrient medium for organotypic or Leighton tubes cultures consisted of Eagle's Minimum Essential Medium, Earle base, to which 30 ~{, fetal calf serum and 500 mg/100 ml glucose (expressed in final concentrations) were added. In Maximow chambers, Allerand and Murray's medium a was used. The preliminary results reported here deal with 8 experiments each comprising 20 cultures observed during 2.5 months in vitro, with specimens ranging from 25 mm to 170 mm from crown to rump. From day 8 the cultures were regularly fixed with 10 °/,i neutralized formalin or, for electron microscopy, with 3.5-5°,', glutaraldehyde in Millonig or S6rensen buffers for 1 h and 2°.0~ osmium for 45 min. Opaque-looking organotypic cultures were discarded. Celloidin or epon-embedded explants were sectioned for light microscopy and stained with hematoxylin-eosin, Nissl and Bodian techniques or paraphenylenediamine. Uranyl acetate and lead citrate were used to stain ultra-thin sections.


119 Light microscope studies o f strictly organotypic cultures revealed the usual lens-like shape of the explants and the lack of outgrowth zone (Figs. 1 and 2). The necrosis o f the cultures was infrequent (this is probably related to the selection of clear cultures before embedding). When occurring, it could be seen as well in the center of the explant as in one of the edges. In about half of the cultures, the sections - - more frequently in the horizontal than in the vertical o n e s - - involved a favorably oriented zone allowing the unequivocal recognition of the cerebellar developing layers. The external granular layer (EGL) was most easily identified (Figs. 1 and 3). It consisted of a densely packed population of small cells with round, darkly stained nuclei measuring about 4 # m in diameter and with very scanty cytoplasm. The thickness of this layer varied from 1 to 2 rows to 4-6 rows o f cells, depending on the age of the specimen, the duration of the culture and the plane of the section. Mitotic figures were frequent (Fig. 3). Under the E G L , a less cellular layer, usually comprising some vertically or horizontally oriented spindle-shaped cells, was almost constantly seen (Fig. 3). It corresponds to the inner E G L and the developing molecular layer. The thickness o f this layer increased with the age of the embryos and with long culture durations. Beneath, slightly larger cells with lighter stained nuclei (6-8 /~m in diameter) were arranged in 3-10 rows (Fig. 3). Their cytoplasm, more apparent than those of the E G L cells, sometimes bore recognizable clear processes more or less oriented towards the latter cells. This layer, which clearly corresponds, in the older fetuses, to the prospective Purkinje cells layer (Fig. 3) might contain, in the youngest embryos, cells belonging to the deep cerebellar nuclei (Fig. 1) which are known to migrate and differentiate earlier ~7. The deeper zone consisted in a large population o f spindle-shaped or r o u n d cells with small dense nuclei and sparse cytoplasm. It obviously corresponds to the intermediate layer, the region of migrating immature cells which will develop into the inner granular layer. In a few cultures issued from very y o u n g embryos, a ventricular zone could be seen. Elsewhere, a row of pia-arachnoidal cells covered the EGL. The germinative layer and the developing meninges were separated by a conspicuous subarachnoid space (Fig. 3).

Figs. I-4. Figs. 1-3 are from organotypic cultures and Fig. 4 is from an organized culture. Fig. 1. Horizontally cut section of a 21-day "in vitro' culture from a 35-ram (8 9 weeks) specimen. The nutrient gelosis (G), EGL (E.G.), marginal (M) and intermediate (1) zones can be seen. In this last layer, the lighter stained and slightly larger cells (arrows) might belong to the immature Purkinje or deep cerebellar nuclei cells. The width of the EGL is likely to be due to the tangential plane of the section. Paraffin, Bodian stain, objective 16, × 220. Fig. 2. Vertically cut section of a l-month 'in vitro" culture from the same specimen as in Fig. 1. Note the lack of any outgrowth zone and the lamination pattern barely distinguishable at this magnification. The upper surface of the explant is on the left; the nutrient gelosis, on the right. Celloidin, Nissl stain, objective 6.3, x 90. Fig. 3. Twenty days "in vitro" culture of a 130-ram specimen (17 weeks). The pia-arachnoidal cells (P.A.) and subarachnoidal space (S), EGL (E.G.), developing molecular (M) and Purkinje (P) cells can be seen. Note the mitosis (arrow). Thick section, paraphenylenediamine stain. Phase contrast, objective 40, ::~ 540. Fig. 4. One month "01 vitro" culture of a 35-mm specimen (8-9 weeks). The explant (E), neurites (N), small cells (arrow-head) and large flat cells (arrow) of the outgrowth can be seen. Collagen-coated coverslip in Leighton tube. Bodian stain, objective 16.

Figs. 5-7. Electron microscopy of tile same cuhure as in Fig. 3 (130-ram, 20 days P; l itr¢~'). Fig. 5. External granular layer. Note the subarachnoidal space (S), the basement nlembr~tne {arrox~) and granular cells (G). 330(i). Fig. 6. Pia-arachnoidal cells. Few departures fronl normal consist in cytoplasmic glycogen particle clusters and enlarged rough endoplasmic reticulum. ~ 6800. Fig. 7. Purkinje cells (P). Note the large nuclei and bulky cytoplasms containing numerous organelles. The section shows an apical dendrite (D). A synapse can be seen (arrow). ~ 7700.

121 In organized cultures grown in Maximow double-coverslip assembly or in Leighton tube, the organization of the central explant was not analyzed. As a matter of fact, it remained too thick for an accurate observation, as long as 6 weeks 'in vitro', either with phase contrast or after en bloc fixation and staining. It was surrounded by a slowly growing migrating zone where bundles of neurites and small unidentified cells spread on a meshwork of large flat cells (Fig. 4). So far, the electron microscope study relates mainly to strictly organotypic cultures. It allowed corroboration of the fair appearance of the tissue, the cellular identification already discussed and the high degree of organization. It is easy, indeed, to recognize the star- or spindle-shaped pia-arachnoidal cells, bearing slender processes and sometimes making contacts with each other. They are separated by a large extracellular space (Fig. 6). The developing subarachnoid space is lined by the basement membrane covering the EGL. This layer is composed of small cells with dense nuclei and fine, dense cytoplasmic processes (Fig. 5). In the developing molecular layer, numerous processes could be seen. Some of them may correspond, because of their direction, number, small diameter and content of 6-12 microtubules, to immature parallel fibers. Others appear to belong to migrating or differentiating cells. Typical developing Purkinje cells (Fig. 7) are recognized by their location, their large clear nuclei and their bulky cytoplasm filled with numerous organelles (ribosomes, rough endoplasmic reticulum, mitochondria and microtubules). On the contrary, the intermediate layer comprised numerous small, unidentified, round or spindle-shaped cells and many processes. Although synapses were observed 11, either in the molecular or the intermediate layer, the precise identification of the cellular processes has not yet been done. No myelin was observed. This should not be suprising on account of the immaturity of the cultivated cerebellum and of the very slow development of human nervous tisue. Human myelin can be observed 'in vitro" only after very long cultures 4. A few comments can be made on the findings reported here. The development of human cerebellum in organized cultures has been widely describedl6 ls,~'~ but few electron microscopic studies have been performed ls,~. EGL and Purkinje cells have been identified. However, no synapses were observed ~s. The 'in situ" human cerebellum development has been studied, hitherto, mostly 2v or exclusively 7 by light microscopy. The cellular organization of our cultures, examined by light microscopy, seems very similar to the one demonstrated '#l situ' at the same period of development '~v. Because there are very few electron micrograph documentations '~z,z7 it is difficult to compare out pictures in organotypic cultures with the normal 'h~ situ' counterpart. Anyway, they seem very close to the electron microscopic descriptions of developing cerebellum in mammals 6,19,23,26. Studies are in progress to compare the electron microscopic development of the human cerebellar cortex and that of the corresponding deafferented organotypic cultures. Strictly organotypic methods which prevent any cellular migration outside of the explant have been seldom used for the central nervous tissue culture. They have been applied to the study of the nervous system capillaries 12,3~ and may allow, when applied to the cerebellar cortex, a precise identification of most of the cells studied. Indeed more widely used organized cultures techniques, and especially the Bornstein

122 a n d M u r r a y m e t h o d 5 or the c o l l a g e n - c o a t e d coverslip in Leighton tubes ~5, enable the r e c o g n i t i o n o f various cell types as well by light'~,5,1z, 16-18,'~0,'24,'~s,a° as by electron microscopyl8,2°,"~,"4,"5, a°. The tissue o r g a n i z a t i o n o f the explants is sometimes very c o m p l e x '~s--a0 but seldom r e m a i n s as obvious as in strictly o r g a n o t y p i c cultures: F o r example, the d e v e l o p m e n t o f a s u b a r a c h n o i d space or the presence o f a basement m e m b r a n e on the E G L has never been described in these organized cultures. A l t h o u g h limited by the i m p o s s i b i l i t y o f direct o b s e r v a t i o n o f the neurons, the o r g a n o t y p i c t e c h n i q u e might prove useful to study the m e c h a n i s m s o f d e v e l o p m e n t o f the nervous system. We are i n d e b t e d to Profs. G a u t r a y , H e r v e t a n d L a n v i n a n d Drs. Bou6, E m m a nueli, Josso a n d P a p i e r n i c k for p r o v i d i n g the specimens a n d D r . B. Berger for constructive criticism. W e t h a n k Mrs. O. Etchebehere, the Misses M. Asselineau a n d M. T o n g for accurate technical assistance a n d Miss C. Vinner for revising the English manuscript. This study was s u p p o r t e d by c o n t r a c t No. 3 A.T.P. 6.74.27 o f the l n s t i t u t N a t i o n a l de la Sant6 et de la Recherche Medicale.

1 ADLER, R., Submicroscopical study of chick embryo neural tube experimentally reopened 'in vitro', Z. Zellforsch., 86 (1968) 422-429.

2 ALLERAND,C. D., Patterns of neuronal differentiation in developing cultures of neonatal mouse cerebellum: a living and silver impregnation study, J. comp. Neurol., 142 (1971) 167-204. 3 ALLERAND,C. D., AND MURRAY,M. R., Myelin formation in vitro. Endogenous influences on cultures of newborn mouse cerebellum, Arch. Neurol. (Chic.), 19 (1968) 292-301. 4 BORNSTEIN,i . B., Personal communication, 1974. 5 BORNSTEIN,M. B., AND MURRAY,M. R., Serial observations on patterns of growth, myelin formation, maintenance and degeneration in cultures of newborn rat and kitten cerebellum, J. biophys. biochem. Cytol., 4 (1958) 449-504. 6 DEL CERRO,i . P., ANDSNIDER,R. S., Studies on the developing cerebellum, lI. The ultrastructure of the external granular layer, J. comp. NeuroL, 144 (1972) 131-164. 7 FRIEDE,R. L., Dating the development of human cerebellum, Acta neuropath. (Bed.), 23 (1973) 48-58. 8 GOUBEDE LAFOREST,P., ROmNEAUX,R., ET VOISlN,J., Perfectionnements et modalit6s d'utitisation d'une chambre ~ perfusion pour la culture d'organes 'in vitro', Ann. Inst. Pasteur, t 13 (1967) 449454. 9 KmRNAN,J. A., AND PETTIT, D. R., Organ culture of the central nervous system of the adult rat, Exp. Neurol., 32 (1971) 11 !-120. 10 HAUW, J. J., Aspects r6cents du tissu nerveux en culture, Presse mdd., 77 (1969) 939-942, 1t151116. 11 HAUW, J. J., BERGER, B., ET ESCOUROLLE,R., Presence de synapses en culture organotypique in vitro de cervelet humain, C.R. Acad. ScL (Paris), 274 (1972) 264-266. 12 HAUW, J. J., BERGER,B., AND ESCOUROLLE, R.~ Ultrastructural observations on human cerebral capillaries in organ culture, Cell Tiss. Res, in press. 13 HAUW,J. J., BOUTRY,J. M., CROSNIER-SuTTIN,N., AND ROmNEAUX,R., Morphology of cultured guinea-pig cerebellum. I. Pattern of development. Comparison of phase contrast cinematography and silver impregnations of various cell types, Cell Tiss. Res., 152 (1974) 141-t64. 14 HAUW, J. J., GOUBE DE LAFOREST,P., ANTEUNIS,A., CATHALA,F., ET ROBINEAUX, R., Culture organotypique prolong6e de tissu nerveux en chambre perfusable, C.R. Acad. Sci. (Paris), 269 (1969) 1205-1208. 15 HAUW, J. J., NOVlKOFF,A. B., NOVIKOFE,P. M., BOUTRY,J. J., AND ROBINEAUX,R., Culture of nervous tissue on collagen in Leighton tubes, Brain Research, 27 (1972) 301-309.

123 16 HOGUE, M. J., Human foetal brain cells in tissue culture: their identification and motility, J. exp. Zool., 106 (1947) 85-107. 17 HOsH, L., HOSLI, E., AND ANDRES, P. F., Light microscopic and electrophysiological studies of cultured h u m a n central nervous tissue, Eu,vp. Neurol., 9 (1973) 121-130. 18 LAPHAM, L. W., Human fetal cerebellar cortex: organization and maturation of cells ht vitro, Science, 173 (1971) 829-832. 19 LARRAMENDI, L. M. H., Analysis of synaptogenesis in the cerebellum of the mouse. In R. LHNAS (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass. Educ. Res. Found., Chicago, Ill., 1969, pp. 803-843. 20 LUMSDEN,C. E., Nervous tissue in culture. In G. H. BOURNE (Ed.), The Structure and Function of Nervous Tissue, Vol. 1, Academic Press, New York, 1968, pp. 67-140. 21 LYSER, K. M., Differentiation of glial cells and glia limitans in organ cultures of chick spinal cord, hi Vitro, 8 (1972) 77 84. 22 MARKESBER¥,W. R., AND LAPHAM, L. W., A correlated light and electron microscopic study of the early phase of growth in vitro of human fetal cerebellar and cerebral cortex, J. Neuropath. exp. Neurol., 33 (1974) 113-127. 23 MELLER, K., AND GLESS, P., The development of the mouse cerebellum. A Golgi and electron microscopical study. In R. LL1NAS (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. reed. Ass. Educ. Res. Found., Chicago, Ill., 1969, pp. 783 801. 24 MURRAY, M. R., Nervous tissues 'in vitro'. In E. N. WtLLMER (Ed.), Cells and Tissues in Culture, Vol. 1I, Academic Press, New York, 1965, pp. 373-455. 25 RAINE, C. S., Ultrastructural applications of cultured nervous system tissue to neuropathology. In H. M. ZIMMERMAN(Ed.), Progress in Neuropathology, Vol. H, Grune and Stratton, New York, 1973, pp. 27-68. 26 RAK1C, P., Kinetics of proliferation and latency between final cell division and onset of differentiation of cerebellar stellate and basket neurons, J. comp. Neurol., 147 (1973) 523-546. 27 RAK~C, P., AYD SIDMAY, R. L., Histogenesis of cortical layers in human cerebellum, particularly the lamina dissecans, J. comp. Neurol., 139 (1970) 473-500. 28 SELL, F. J., Neuronal groups and fiber patterns in cerebellar tissue cultures, Brain Research, 42 (1972) 33-51. 29 SOBKOWICZ, H. M., BLEIER, R., BEREMAN,B., AND MONZAIN, R., Axonal growth and organization of the mammillary nuclei of the newborn mouse in culture, J. Neuroo'tol., 3 (1974) 431 447. 30 WOLF, M. K., AYD DUBOIS-DALCQ, M., Anatomy of cultured mouse cerebellum. 1. Golgi and electron microscopic demonstrations of granule cells, their afferent and efferent synapses, J. comp. Neurol., 140 (1970) 261 280. 3l WOLFF, J. R., RAJAN, K. l., AND NOACK, W., The fate and fine structure of fragments of blood vessels in CNS tissue cultures, Cell Tiss. Res., 156 (1974) 89-102.

Organ culture of the developing human cerebellum.

Brain Research, 99 (1975) 117-123 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands I [7 Short Communications Organ...
4MB Sizes 0 Downloads 0 Views