Role of astroglial cell clones in the survival and differentiation of cerebellar embryonic neurons

247

Developmental Brain Research, 44 (1988) 247-257 Elsevier

BRD 50840

Role of astroglial cell clones in the survival and differentiation of cerebellar embryonic neurons F. Alliot 1, N. Delhaye-Bouchaud 1, M.

G e f f a r d 2 a n d B. Pessac 1

IINSERM U 178 and Centre de Biologie Cellulaire CNRS, lvry-sur-Seine (France) and 2lnstitut de Biochimie Cellulaire et Neurochimie du C. N. R.S., Bordeaux (France)

(Accepted 9 August 1988) Key words: Astroglia; Glial clone; Cerebellum; Development; Cerebellar explant

To investigate the role of astrocytes in the survival and differentiation of cerebellar neurons during development, we have used astroglial cell clones, derived from 8-day postnatal cerebellar explants and which might be the in vitro equivalents of the 3 main types of cerebeilar astrocytes, the Golgi epithelial cells and their Bergmann processes, the velate protoplasmic and the fibrous astrocytes (F. Alliot and B. Pessac, Brain Res., 306 (1984) 283-291). Nearly all single cells, dissociated from 15-day embryonic mouse cerebella and seeded at low density, adhered to layers of each of the cerebellar astroglial cell clones as well as to other glial lines or artificial substrates. However, the cerebellar embryonic neurons survived well only on monolayers of either the 'Golgi-Bergmann'-likeor the 'velate protoplasmic'-likeclones. On these layers, 60-80% of the neurons were still present after 5 days of co-culture, while only less than 5% survived on the other types of substrates. The differentiation pattern of the neurons surviving on the 'Golgi-Bergmann' and the 'velate protoplasmic" astroglial clones was studied with markers of postmitotic granule cells, the major neuronal population in adult cerebellum. The relate protoplasmic-like clone was the only one able to support the coordinate acquisition by most surviving neurons of the phenotypic characteristics of granule cells, i.e. a distinct morphology, a specific epitope binding the monocional antibody 7-8 D2 and immunoreactivity to glutamate. These data show a broad heterogeneity in the capacity of astroglial cell clones to support embryonic cerebellar neurons. In addition, they indicate that neuronal survival per se is not sufficient for the acquisition of a differentiated neuronal phenotype. INTRODUCTION A l t h o u g h the n o n - n e u r o n a l glial cells, which include astrocytes, o l i g o d e n d r o c y t e s and the microglia, o u t n u m b e r the neurons of the adult m a m m a l i a n central nervous system, their functions are not well u n d e r s t o o d tT. Previous investigations in tissue culture have shown that astroglial cells have a trophic effect on some central nervous system neurons, i.e. that either contact with or diffusible molecules from astroglial cells p e r m i t in vitro neuronal survival 3-5A1"21"22'31. H o w e v e r , these e x p e r i m e n t s have been mainly perf o r m e d on p r i m a r y cultures that comprise a mixture of the various types of astroglial cells as well as o t h e r cell types. F u r t h e r m o r e they do not address the question of a role of astrocytes in the choice of a differentiation p a t h w a y by neurons.

The cerebellar cortex is a convenient region of the central nervous system to investigate a role of astrocytes in neuronal survival and differentiation since the architectonics of the neuroglia and its relationships with the neuronal layers are well known in the adult rodent. T h e astroglial Golgi epithelial cells and their B e r g m a n n fibers are characteristic of the Purkinje and m o l e c u l a r layers, while velate protoplasmic astrocytes are localized mainly in the granular layer and the fibrous astrocytes in the white m a t t e r 23. The neuronal cells differentiate mainly in two stages. On the 15th day of embryonic development, Purkinje cells c o m p l e t e their last round of mitosis while the precursors of the o t h e r neuronal cells will go through m a n y p o p u l a t i o n doublings before differentiating 2. Thus granule cells which represent over 90% of the neurons in the adult cerebellum become postmitotic only after the first week and acquire their

Correspondence: B. Pessac, Centre de Biologie Cellulaire CNRS, 67 rue Maurice Giinsbourg, 94205 Ivry sur Seine, Cedex, France.

0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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characteristic phenotype when most astroglial cells, and in particular the velate protoplasmic astrocytes, have already differentiated j3. To obtain homogeneous populations of the different types of cerebellar astrocytes, we have established, from 8-day postnatal mouse cerebella, permanent clonal cell lines with astroglial properties. These clones could be distinguished, according to their morphology, in 3 separate types. The first one had small somata and several short processes and was comparable to the fibrous astrocytes. The second type that also had small somata but only two processes (one of which was very tong and thin) might be the in vitro counterpart of the Golgi epithelial cells and their Bergmann processes. The third type with large flat somata and no processes closely resembled the velate protoplasmic astrocytes 1. The 3 types of astroglial clones are therefore designated respectively as 'fibrous', 'Golgi Bergmann' or 'velate protoplasmic' astrocytes. They have been previously designated, respectively, as type I, type II, and type III (ref. 1) or D1A, C8S and D19 (ref. 20). The goal of the present study was to compare the survival and differentiation of 15-day embryonic cerebellar neuronal cells co-cultured on layers of these as well as other astroglial clones. The results show that the only astrocytes that permitted the survival of a high proportion (60-80%) of the embryonic neuronal cells were the 'Golgi Bergmann' and the 'velate protoplasmic' clones. In addition, the majority of the neuronal cells co-cultured on monolayers of the 'velate protoplasmic' astroglial clone acquired the granule cell-like phenotype. Taken together, these data show that astroglial cell clones differ greatly in their support of embryonic cerebellar neurons and that survival is not sufficient per se for the acquisition of a neuronal phenotype. MATERIALS AND METHODS

Materials Tissue culture (T.C.) flasks were obtained from Corning glass works, Corning NY, U.S.A. and 24well plates from Falcon, Beckton Dickinson Labware, Oxnard, CA, U.S.A. Basal medium of Eagle (BME) (cat. no. 073-1300), Hanks' solution (cat. no. 042-4065), phosphate-buffered saline (PBS) (cat. no. 042-04080 M), trypsin 2.5% (cat. no. 043-5090),

Versene (cat. no. 043-5040) were obtained from Gibco Laboratories, Grand Island, NY, U.S.A. Fetal bovine serum (FBS) was obtained from Flow Laboratories, U.K. Poly-L-a-ornithine Hbr, type lc (cat. no. P4638) was obtained from Sigma, St. Louis, MO, U.S.A. The following reagents were kindly provided: rat laminin, purified from the EngebathHolm-Swarm tumor, Marc Vigny, INSERM Ull8; purified tetanus toxin, Bernard Bizzini, Institut Pasteur Paris; mouse monoclonal antibody 7-8 D2. Michael Webb, Sandoz, London. Secondary antibodies were obtained from Kirkegaard and Perry, Gaithersburg, MD, U.S.A. or from Cappel, Coopermedical, Malvern, PA, U.S.A. Neuronal cell suspension To prepare the neuronal cell suspensions, cerebella were aseptically removed from C57BI mouse embryos between days 15 and 16 of development; after careful dissection of the meninges in Hanks' buffer, whole cerebella were cut into pieces of about 1 mm 3 and incubated in 2 ml of a 10% solution of trypsin 2.5% in Versene 1:5000 for 15 min at 37 °C. This solution was then aspirated with a Pasteur pipette and the cerebellar pieces were gently dissociatedl by pipetting in 10 ml of complete medium (BME + 10% FBS). The cell suspensions were centrifuged at 200 g for 15 min and the pellet was resuspended in 10 ml of complete medium. Cell viability, as measured by the Trypan blue exclusion test, was >85%. 4 × 103 embryonic cerebellar cells in complete medium were seeded per cm 2 in T.C. flasks or in 24-well :Falcon plates containing the preformed astrocytic monolayers. This medium was changed on days 1 and 2. Astroglial monolayers Astroglial monolayers were prepared from permanent astroglial cell clones. The cerebeUar clones have been previously described as 'fibrous', 'velate protoplasmic' or 'Golgi epithelial '1. The 'velate protoplasmic' and 'Golgi epithelial' astrocytes, labeled with an anti-GFAP antiserum, are shown in Figl 1. The F7 mes and F12 str clones were obtained after transformation, by the oncogenic virus SV40, of mouse mesencephalic and striatal cultures and are GFAP-positive 19. They were given by M. Mallat and A. Prochiantz. Suspensions of astroglial clonal cells in complete medium were seeded into Falcon plates con-

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Fig. 1. Immunolabelingof astroglial cerebellar clones with an anti-GFAP antiserum. Cells were fixed in cold acetone for 10 rain, incubated with a rabbit anti-GFAP antiserum diluted 1/40 (a gift of D. Dahl and A. Bignami)for 60 min and then with a fluorescein-conjugated goat IgG antirabbit immunoglobulin (CappeU). a: 'velate protoplasmic'-likecerebellar astroglial clone, b: 'Golgi Bergmann'like cerebellar astroglial clone. Bar = 25 gm.

taining uncoated glass coverslips and kept in an atmosphere of 5% CO2 in air at 37 °C. The medium was changed after 4 days. All layers were used two days later when they had reached confluency, i.e. 2.105 'Golgi-Bergmann' or 'fibrous' and 1.6 x 104 'velate protoplasmic' astrocytes per cm 2.

Poly-ornithine-laminin substrate Coverslips in 24-well culture plates were treated with 200/~1 of polyornithine solution (0.1 mg/ml in PBS) and then supplied with 200/A of laminin (10 /~g/ml in PBS) as described TM.

Immunocytochemistry After 2 or 5 days in co-culture with astroglial layers, the embryonic cerebellar neurons were labeled with tetanus toxin, a marker of neuronal cells9.24. Cultures on coverslips were washed 3 times in Hanks' buffer and incubated with tetanus toxin (20/~g/ml) in Hanks' at 4 °C for 20 min. After washing in Hanks' the cultures were fixed with formaldehyde (3% in PBS) for 30 min, washed in PBS and then incubated with a 1/100 solution of a rabbit anti tetanus toxoid antiserum, washed in PBS and incubated in a 1/20 solution of fluorescein conjugated goat antibody to rabbit IgG. For double labeling with the mouse monoclonal antibody 7-8 D2, which binds to postmitotic neurons 3°, tetanus toxin was mixed with 7-8 D2 and the fluorescent goat antibody was mixed with a rhoda-

mine-conjugated goat IgG antimouse IgG. After 6 days, co-cultures were labeled with an antiglutamate antiserum or monoclonal antibody. The glutamate (Glu) antiserum was raised from rabbits immunized with Glu conjugates. Animals were injected alternatively with Glu-glutaraldehyde (G) bovine serum albumin and Glu-glutaraldehyde-human serum albumin. After 4 injections, the anti-conjugated Glu antibody titer was increased and the antibody affinity and specificity studies were performed by competition experiments in ELISA tests. The most immunoreactive compound was Glu-G-protein (at half-displacement, apparent K d = 2.10 -8 M). The non-reduced conjugate, Glu-G-protein was 110 times less recognized than the reduced one. The other compounds: aspartate-G-protein, GABA-Gprotein, glycine-G-protein, taurine-G-protein were not at all recognized by the anti-conjugated Glu antibodies, (at half-displacement, cross-reactivity ratios superior to 1/10000). Immunocytochemical specificity studies confirmed the ELISA tests. Immunoreactivity only disappeared after pre-incubation of the Glu antiserum with Glu-G-protein (hapten concentrations at 10-5 M). No modification of the specific staining was observed after antiserum pre-incubation with either aspartate-G-protein or the other conjugates. An antiglutamate monoclonal antibody was prepared as previously described for an anti-dopamine antibody 7 (M. Geffard, in preparation).

250 For single labeling with the antiglutamate antiserum or monoclonal antibody, the co-cultures were washed 3 times in PBS, then fixed in a 1% solution of glutaraldehyde in PBS (pH 7) for 12 min in the dark. washed in PBS and incubated with a 1/1000 solution of either the antiserum or the antibody for 2 h. After washing in PBS, the cultures were incubated with a 1/20 dilution of fluorescein-conjugated IgG fraction of goat antirabbit or antimouse Ig. To investigate whether the antiglutamate antiserum and a monoclonal antibody labeled the same cells, double labeling experiments were performed, after fixation of the cocultures in 1% glutaraldehyde, as described above. Double labeling with the monoclonal antibody 7-8 D2 and the rabbit antiglutamate antiserum, was performed as follows: the cultures on coverslips were washed in Hanks', incubated with 7-8 D2 at 4 °C, washed again in Hanks', and incubated in a 1/20 dilution of rhodamine-conjugated goat IgG antimouse. After washing in PBS, the cultures were fixed in a 1% solution (pH 7) of glutaraldehyde in PBS for 12 min in the dark, washed in PBS and incubated with a 1/1000 solution of the antiglutamate antiserum for 2 h. After washing, the cultures were incubated with a 1/20 dilution of fluorescein conjugated goat IgG antirabbit immunoglobulins. For double labeling with tetanus toxin and the antiglutamate antibody, cultures were processed for tetanus toxin labeling as described above but fixed with 1% glutaraldehyde (instead of formaldehyde) and then processed for the antiglutamate labeling. All incubations were carried out for 30 min at laboratory temperature. The coverslips were mounted on slides in a mowiol containing buffered solution of glycerol and viewed with a Leitz epifluorescence microscope and appropriate filters.

Moreover, 90% of the viable cells in the initial embryonic suspension could be seen as small (<10/~m) birefringent cells attached to the large flat 'velate protoplasmic' astrocytes. Neurons represent at least 80% of the total population of embryonic cerebellar cells ~5. After 24 and 48 h, neuronal cells that had adhered to the astroglial layers were identified by their binding of tetanus toxin (the clonal astroglial cells are not labeled by tetanus toxina). Indeed, 87 and 78% of the embryonic cells seeded, respectively, on the 'Golgi-Bergmann' and the 'velate protoplasmic" astrocytes were tetanus toxin-positive, displayed a neuronal morphology and had already extended long processes. The percentage of tetanus toxin-positive cells after 5 days of co-culture on each astroglial clone is shown in Table I. The vast majority of neuronal cells had survived on monolayers of "Golgi-Bergmann" and 'velate protoplasmic' clones and had developed a characteristic network of processes (see below). In contrast, while neuronal cells had adhered to the 'fibrous' astrocytes as well as to the other astroglial

TABLE I Survival and differentiation of neuronal cellsfrom 15-day mouse embryonic cerebella Cerebellar cells were seeded at a density of 4.103 cells per cm: on monolayers of the astroglial clones as well as on polyornithine laminin. After 5 days neuronal cells were double labeled with tetanus toxin and the monoclonal antibody 7-8 D2 as described in Materials and Methods. Results are expressed in thousands of seeded cells per 25 cm2 with S.E.M. and as the percentage of neurons labeled by 7-8 D2. Substrates

RESULTS The vast majority o f cerebellar embryonic neurons survive very well on layers o f the 'Golgi Bergmann' and 'relate protoplasmic' astroglial clones Cerebella from 15-day embryonic mice were dissociated into single cells and seeded at a density of 4 x 103 cells/cm 2 on preformed layers of each of the 3 astroglial clones. After 3 h, fewer than 10% of the seeded cells remained free in the medium of the 3 co-cultures. Thus the embryonic cells adhered rapidly and with the same efficiency to the 3 astroglial layers.

Number Number of of seeded neurons (in cells (in thousands) thousands)

% Of neurons labeled by 7-8 D2

100

78.5±6.8

20±2.4

100 1O0

58.3±5.5 <5

67.7±2.5 N.D.

lOO 100

<1 <3

N.D. N.D.

100

<1

N.D.

Cerebellar astroglial clones

Golgi Bergmann Velate protoplasmic Fibrous Astroglial clones derived from mesencephalon (F7) striatum (FI2) Polyornithine laminin

251 clones, fewer than 5% were present after 5 days and they did not appear in good health, These data show that permanent astroglial cell lines derived from 8 day postnatal cerebella differed Strikingly in their capacity to promote neuronal survival. Therefore, it appeared of interest to investigate the effect on the survival of neuronal cells from embryonic cerebellum of other astroglial lines. Clones F7 and F12, respectively, derived from embryonic mesencephalic and striatal cultures transformed by SV-40, are GFAPpositive and have a protoplasmic-like morphology19. However, as shown in Table I, neuronal survival was very poor on these astroglial clones: after 5 days of culture no more than 3% of the neurons survived on the F7 and F12 clones. The rate of survival of the 15-day embryonic neuronal cells was also measured on artificial substrates, i.e. poly-ornithine or poly-ornithine-lamininIs, in conditions similar to those used for co-cultures on astrocytes. Although 90% of the cells attached readily to these substrates, their survival was very poor. Indeed after two days at least 95% of the embryonic cerebellar cells took up Trypan blue and therefore can be considered as dead.

A majority of cerebellar embryonic neurons cultured on 'relate protoplasmic' monolayers differentiate into granule-like cells As reported in the preceding paragraph, 15-day embryonic cerebellar neuronal cells seeded at low density survive best on monolayers of the 'GolgiBergmann' and 'velate protoplasmic' astroglial clones. Therefore, the next step of this investigation was to determine whether these astroglial cells had any effect on the differentiation program of the 15day embryonic cerebellar neuronal cells. Since the granule cells represent around 95% of the neuronal population in the adult cerebellum, we investigated the expression of 3 distinct markers that are expressed by postmitotic granule cells: their morphological pattern, in particular that of the neuronal processes, the appearance of an epitope found only in postmitotic granule cells in vivo3° and immunoreactivity to glutamate 27, the probable neurotransmitter of granule cells 16'28,29. We first investigated the morphological differentiation pattern of the embryonic cerebellar neuronal cells. After one day in co-culture on the 'velate proto-

plasmic' layers, most tetanus toxin-positive cells had a small (<10/~m) round or oval soma which usually gave rise to 2 or 3 processes (Fig. 2a). After two days, the short processes produced digitiform branches that tended to grow parallel to each other and from one of which a long thin process often originated (Fig. 2c). After 5 days, the neuronal cells were no longer dispersed but aggregated to each other (Fig. 2e). The axon-like processes were very long, up to 0.7 mm (Fig. 2h). They were often parallel to each other, whether they arose from a bifurcation or from adjacent cells and they could be straight or curved. Some of the processes had many varicosities (Fig. 2e). These neuronal cells were reminiscent of postnatal granule cells in vivo23. Furthermore their morphological appearance was strikingly comparable to that of 8 day postnatal granule cells co-cultured on the 'relate protoplasmic' astroglial layers (Fig, 2g) or to neurons dissociated from 8-day mice cerebella and cultured for 11 days 1°. The morphological pattern of embryonic cerebellar neuronal cells co-cultured on the 'Golgi-Bergmann' astroglial layers was quite different from that of the same neuronal cells plated on the 'velate protoplasmic' astrocytes. After one day most neuronal cells extended many processes of various lengths (Fig. 2b). 24 h later these processes were already interdigitating (Fig. 2d). After 5 days, the smooth thin processes extended in all directions and formed an intricate network (Fig. 2f). These data indicated that the majority of embryonic neuronal cells acquired a granule cell-like morphology only when they were co-cultured on the 'velate protoplasmic' astrocytes. In order to characterize the neurons which differentiate on the astrocytic monolayers, specific markers were necessary. To this end, we used the recently described monoclonal antibody 7-8 D2 which binds to the internal granular and molecular layers of postnatal rat cerebella. In particular, expression of the 78 D2 antigen appears in the cerebellum to be highly specific to postmitotic cerebellar granule neurons 3°. In preliminary experiments we have verified that 7-8 D2 labels tetanus toxin-positive cells in primary cultures from 8 day postnatal mouse cerebella but that neither tetanus toxin nor 7-8 D2 label the 'Golgi Bergmann' and 'velate protoplasmic' cell clones. The embryonic cerebellar cells co-cultured on the astro-

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Fig. 2. Morphological pattern of cerebellar neuronal cells co-cultured on layers of the 'velate-protoplasmic' and 'Golgi-Bergmann' astrogliaJ clones. Single cell suspensions from cerebella of 15-day embryos (a-f,h) or g-day mice (g) were plated on layers of the 'relateprotoplasmic' (a,c,e,g,h) or 'Golgi-Bergmann' layers (b,d,f) and cultured for one (a,b), two (c,d) or 5 (e-g) days. Neuronal cells were visualized with the indirect immunofluorescence technique by the tetanus toxin binding as described in Materials and Methods. h: montage showing an axon-like process 0.7 mm tong. Bar = 25/~m except in h (20/~m).

cytic layers were double labeled with tetanus toxin and the monoclonal antibody 7-8 D2. All cells labeled with 7-8 D2 were tetanus toxin-positive. After two days of co-culture on the 'velate protoplasmic' astroglial layers, 23% of the neuronal cells were already labeled by 7-8 D2. After 5 days, 67.7% of the tetanus toxin-positive cells bound 7-8 D2 (Table I and Fig. 3a,b). The cell bodies were intensely labeled while only a few processes and varicosities could be visualized (Fig. 3b). The vast majority of cells labeled with 7-8 D2 had a granule cell-like morphology. Conversely, most, if not all, tetanus toxin-positive cells that displayed the granule cell morphology shown in Fig. 2e and 3a were 7-8 D2-positive. In contrast, on the 'Golgi-Bergmann' astroglial layers, after 2 or 5 days of co-culture, respectively, 5 or 20% of the embryonic cerebellar neurons were faintly labeled by 7-8 D2. Glutamate appears to be the neurotransmitter liberated from the granule cells in cerebellum 16'28'29. To investigate the presence of this neurotransmitter in 15-day embryonic mouse cerebellar neuronal cells co-cultured on the 'velate protoplasmic' and 'GolgiBergmann' astroglial clones, we used an antiserum and a monoclonal antibody for glutamate (M. Gel-

fard, in preparation). Preliminary experiments had shown that, at the concentration used in this study, the antiglutamate antiserum or antibody did not label the astroglial clones, while the somata of some neuronal ceils were clearly immunoreactive, but only when the medium had been changed only once, 24 h after the beginning of the culture. However, in these conditions, the rate

TABLE II Glutamate immunoreactivity in neuronal cells from 15-day mouse embryonic cerebella after 6 days in co-culture on the "Golgi Bergmann' or the 'velate protoplasmic' astroglial monolayers

Cerebellar cells were seeded at a density of 2.104 per cm 2 on the astroglial monolayers. The co-cultures were changed once, 24 h after the beginning of the culture. After 6 days, the neurons were doubly labeled with tetanus toxin and the antiglutamate monoclonal antibody. Astroglial layer

Percentage of neurons

Percentage of glutamateimmunoreactive neurons

Goigi Bergmann Velate protoplasmic

33.6 + 4.9 29.2 + 3.8

11.1 + 2.7 27.7 + 3.1

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Fig. 3. Double immunolabeled neuronal cells from 15-day embryonic mice cerebella co-cultured on a monolayer of 'velate-protoplas~ mic' astrocytes, a,b: the same microscopic field showing cells labeled by tetanus toxin and the monoclonal antibody 7-8 D2, respectivcly, after 5 days of co-culture, as described in Materials and Methods. Note that some neuronal cells labeled by tetanus toxin are not immunoreactive with 7-8 D2. c,d: the same field labeled by the antiglutamate antiserum and 7-8 D2, respectively, after 6 days of co-culture, as described in Materials and Methods. Bar = 251tm.

of neuronal survival was very low. In contrast when embryonic cerebellar cells were seeded at a density of 2 × 104 (instead of 4 × 103) per cm 2, 3 0 - 3 5 % of the neurons were still present after 6 days of co-culture on monolayers of the astroglial clones even when the medium had been changed only once. Further studies with the antiglutamate antiserum or antibody were therefore carried out at the higher density of embryonic cerebellar cells. These experiments showed that after 6 days, 28% of the embryonic neurons were immunoreactive for glutamate when cocultured on the 'velate protoplasmic' astrocytes and only 11% on the 'Golgi Bergmann' layers (Table II). Furthermore, double labeling experiments have shown that the antiserum and the antibody labeled the same cells (data not shown). When the embryon-

ic neurons co-cultured on the 'velate protoplasmic' astrocytes were double labeled with the antiglutamate serum and 7-8 D2 (Fig. 3c,d), about 55% of the 7-8 D2-positive neurons were immunoreactive for glutamate and conversely at least 80% of the neurons immunoreactive for glutamate were 7-8 D2-positive. DISCUSSION This study shows that astroglial cell clones, derived from 8-day PN mouse cerebella differ greatly in their capacity to support 15-day embryonic cerebellar neurons. Furthermore, the clone that may be the in vitro equivalent of the 'velate protoplasmic' astrocytes of the granular layer in the adult cerebellum triggers the coordinate expression of granule cell markers.

255 Astrocytes and neuronal survival The rate of adhesion of embryonic neuronal cells to the various astroglial layers used in this study was similar, i.e. that at least 90% had adhered within 3 h. However, the capacity of the different astrocytes to promote the survival of the cerebellar embryonic neurons varied greatly and appeared independent of their morphology. Indeed, the two kinds of astroglial layers that promoted the best neuronal survival were the 'Golgi-Bergmann' and the 'velate protoplasmic' clones that display a very different morphology but on which 60-80% of the neurons counted after one day were still present after 5 days of co-culture. In contrast, few embryonic neurons survived on the 'fibrous' cerebeUar clone or on the F7 and F12 clones, although these have a protoplasmic-like morphologY. The simplest explanation for the excellent neuronal survival on the 'Golgi Bergmann' and the 'velate protoplasmic' monolayers is that these astroglial clones might produce neurotrophic factors. However, preliminary experiments indicate that the trophic effect of the astroglial clones cannot be mimicked by that of their conditioned medium but appears to require contact with the neuronal cells. In this context, it has been reported that factors associated with the astrocyte surface, but not conditioned media were primarily responsible for the vigorous neurite outgrowth seen on astrocytes 12. In contrast, the disappearance of the embryonic neurons co-cultured on the 'fibrous' astrocytes appears mediated through a soluble factor(s) (data not shown). It might be tempting to extrapolate these in vitro data to the in vivo situation. The 'Golgi Bergmann cells', which have been shown to guide the migration of granule cells25, might also play a role in their survival as well as the velate protoplasmic astrocytes which compartmentalize the granule cells in the granular layer. Conversely the fibrous astrocytes located in the white matter might be implicated in the process of neuronal death 8. Most embryonic neuronal cells co-cultured on the velate protoplasmic astroglial clone differentiate into granule cells Although we have only observed very rare mitotic figures in the embryonic neurons during the first two

days of co-culture, the total number of neuronal cells did not increase during this period. This indicates that most 15-day embryonic cerebellar neuronal cells, which go through many cell cycles in the course of in vivo development, become postmitotic in cocultures with the 'velate protoplasmic' astroglial clone derived from 8-day postnatal cerebella. The fact that the neuronal cells stop dividing in vitro much earlier than during normal development does not appear to prevent the expression of their differentiation program into postmitotic granule cells. Indeed the majority of the tetanus toxin-positive neuronal cells that are present on the 'velate protoplasmic' monolayers acquire differentiation markers of postmitotic granule cells. The small size of the somata (<10/am in diameter) and the number, length and branching pattern of processes are reminiscent of granule cells in vivo23. It has been shown recently that laminin and heparan sulfate proteoglycan (HSPG) have distinct effects on the outgrowth of central nervous system neurons; while laminin induces the emergence and branching of newly formed neurites, HSPG only promotes neurite elongation 14. The 'Golgi Bergmann' and 'velate protoplasmic' clones synthesize both laminin and HSPG; however only HSPG, but little if any laminin, can be seen on the surface of 'velate protoplasmic' astrocytes (data not shown). This may account for the fact that the pattern of neurite outgrowth on these astrocytes is comparable to that of other central nervous system neurons on an HSPG substrate TM. Embryonic cerebellar neurons co-cultured on the 'velate protoplasmic' layers acquired the epitope 7-8 D2 which appears to be specific in the cerebellum for neurons originating from the external granular layer. The number of neuronal cells expressing this epitope increased markedly during the co-culture. Furthermore this epitope and the intensity of the labeling were only present at the surface of neurons whose morphological pattern was similar to that of granule cells and conversely almost all granule-like cells were 7-8 D2-positive. About 55% of the granule celMike, 7-8 D2-positive cells, were clearly immunoreactive for glutamate. Preliminary experiments had shown that the immunoreactivity appeared only when the medium was changed once, the day after the co-cultures were initiated. It thus appears that repeated medium

256 changes may prevent the accumulation of glutamate in the neuronal granule-like cells. It should be stressed however that accumulation of immunoreactive glutamate took place only in neuronal cells, since in these conditions neither the 'velate protoplasmic' nor the 'Goigi B e r g m a n n ' astroglial cells showed immunoreactivity to glutamate. A simple hypothesis that could account for the selective acquisition of the granule cell p h e n o t y p e by embryonic cerebellar neuronal cells co-cultured on monolayers of the 'velate protoplasmic' astroglial clone would be that the progenitors of the granule cells preferentially adhere to and are therefore selected by the 'velate protoplasmic' clonal astrocytes. This is not the case however since the cerebellar neuronal cells adhere to and survive as well, if not better, on the 'Golgi B e r g m a n n ' astroglial cells. The n u m b e r of cerebellar e m b r y o n i c neuronal cells present on the ' G o l g i - B e r g m a n n ' layers is almost identical between co-culture days 2 and 5. Indeed, 78% of the seeded cells are tetanus toxin-positive and extend a complex array of processes after 5 days of co-culture. In addition, 20% of these neuronal cells bind the M a b 7-8 D2 which recognizes postmitotic granule cells. Thus, it appears that the granule cell precursors do adhere to the 'Golgi-Bergm a n n ' astrocytes but that only a small p r o p o r t i o n initiates a partial differentiation p r o g r a m along the

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