Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium

Developmental Brain Research, 17 (1985) 17-25 Elsevier

17

BRD50121

Survival, Morphology and Adhesion Properties of Cerebellar Interneurones Cultured in Chemically Defined and Serum-Supplemented Medium ANN E. KINGSBURY, VITI'ORIO GALLO*, PETER L. WOODHAMS and ROBERT BALAZS

MRC Developmental Neurobiology Unit, Institute of Neurology, London WC1N 2NS (U. K.) (Accepted June 26th, 1984)

Key words: neuronal culture - - serum-free medium - - cell type characterization - morphological organization in vitro - - neuronal survival

Cultures obtained from early postnatal rat cerebellum, grown in either chemically defined or in serum-supplemented medium containing 25 mM K ÷, contained predominantly (>90%) small interneurones, mostly granule cells, with good and comparable viability (assessed by the retention of preloaded 51Cr). Neuronal survival was prolonged in the chemically defined medium, nerve cells living up to two weeks longer than in serum-supplemented medium, although the proportion of non-neuronal cells was not greatly increased. In the serum-supplemented medium neurones became organised into clumps connected by thick, fasciculated bundles of neurites by about one week in vitro. In comparison, in the chemically defined medium aggregation of neurones and fasciculation of neurites was markedly reduced even after 4 weeks in culture. The possible relationship between the organisation of neurones and the nature of the substratum, chemical factors in the medium as well as the surface properties of the cells is discussed. INTRODUCTION The discovery of conditions which permit the cultivation of cells in chemically defined m e d i a has o p e n e d up new opportunities for the investigation of the role of humoural factors in d e v e l o p m e n t 2. A n o t h er advantage which m a y be gained from the use of defined media is the selection for particular classes of cells in culture, as it would a p p e a r that different cell types need a specific m i c r o e n v i r o n m e n t for survival and growth2, 21. Such media, originally designed for maintaining the growth and differentiation of cell lines 4, have been used successfully, with occasional modification of the composition, for obtaining primary neural culturesS,8,9,15-17,21,24. H o w e v e r , in most studies, aimed at selecting for neuronal cells, it has been necessary to introduce certain less well defined conditions such as preculturing the cells in the presence of serum 15, or using brain extract or serum-coated substratumS,S.

Recently we have examined the e x p e r i m e n t a l conditions which select for the long-term survival of rat cerebellar interneurones in conventional serum-supplemented m e d i u m 23 (for other references on similar cultures see e.g. refs. 7 and 13). A s a basis for further studies of the factors which affect the survival and differentiation of these neurones, we have grown cultures which are similarly enriched in interneurones b u t which have not been exposed to serumS,t5 or other poorly defined supplements 5 at any stage. A similar culture of neurones from mouse cerebellum has also been r e p o r t e d recently9. Here we present a detailed comparison of the morphological organisation and the cellular composition of rat cerebellar, i n t e r n e u r o n e - e n r i c h e d cultures grown in a serum-free, chemically defined m e d i u m and in the presence of serum under the optimal conditions established in our previous studies. W e have established that even though mitotic inhibitors were not used in the serum-free cultures, non-neuronal

Present address: C.N.R. Institute of Cell Biology, Via Romagnosi, 181A, 00196 Rome, Italy. Correspondence: R. Balazs, MRC Developmental Neurobiology Unit, Institute of Neurology, 33 John's Mews, London WC1N 2NS, U.K. *

0165-3806/85/$03.30 (~) 1985 Elsevier Science Publishers B.V.

18 contamination was similarly low under both conditions but that nerve cells were able to survive longer in serum-free medium (see also ref. 9). However, in contrast to the recent report 9 of mouse cerebellar interneurones in serum-free culture in a slightly different medium, our most striking finding was that the organisation of neurones in chemically defined medium was markedly different from that seen in the reference, serum-supplemented cultures. The question of whether the pronounced differences in the morphological organisation of these cells seen in chemically defined and serum-supplemented medium may be related to alterations in plasma membrane properties has been addressed in the accompanying paper 10. MATERIALS AND METHODS Dissociated cells from pooled cerebella of 8-dayold Porton rats were obtained by the methods described previously l,ls,2s. Cells were suspended in the appropriate medium and seeded (2.5 x 106 cells/ dish) onto 35 mm Falcon dishes coated with poly-Llysine (PLL), 5 ktg/ml, Sigma, (mol.wt. 70-150 x 103) and were incubated at 37 °C in a humidified atmosphere, 5% CO2-95% air. The chemically defined medium (CDM) was 3 parts Dulbecco's Modified Eagle's Medium (DMEM; Gibco) to one part Ham's F12 (Flow Labs.), containing the N2 supplement of Bottenstein and Sato 4, 5 ktg/ml insulin, 100/tg/ml transferrin, 20 nM progesterone, 100 nM putrescine, 30 nM selenium plus 2 mM glutamine and 50 units/ml penicillin-50 #g/ml streptomycin. In serum-free medium the KCI concentration was 4.75 mM and gentamicin was not used as it appeared to be toxic under serumfree conditions. The serum-supplemented medium (SSM) was Basal Eagle's Medium (BME; Gibco) supplemented with 10% fetal calf serum (FCS; Imperial Labs.), KC1 (25 mM final concentration), glutamine (2 raM) and gentamicin (100 #g/ml) (Flow Labs.). Cytosine arabinofuranoside (araC, I0/~M) was added to serum-containing cultures after 16 h in vitro but not to serum-free cultures.

Immunocytochemistry Cells were labelled with the appropriate marker,

using the indirect sandwich technique 22 directly on the culture dishes. Unless otherwise stated, cells were fixed with cold acid alcohol (5% acetic acid in 95% ethanol at -10 °C for 15 min). The following fluorescein-labelled conjugates were used to visualize cell markers (supplier and dilution given in parentheses); sheep anti-horse immunoglobulins (Wellcome; diluted 1:20); sheep anti-rabbit immunoglobulins (Wellcome; 1:20); goat anti-rabbit immunoglobulins (Miles Yeda; 1:50); sheep anti-human immunoglobulins (Wellcome; 1:20); goat anti-mouse immunoglobulins (Miles Yeda; 1:50). Nerve cells were identified by the binding of tetanus toxin. Cultures were washed with D M E M and incubated with 10 ktg/ml tetanus toxin (the generous gift of Dr. R. O. Thompson, Wellcome Research Labs.) for 40 rain at room temperature, washed with DMEM and then incubated with horse anti-tetanustoxoid serum, (Wellcome Reagents) 1:20 for 30 rain. Following visualization with anti-horse serum conjugated to fluorescein, cultures were fixed with acid alcohol. in some experiments neurons were identified using brain plasma membrane antiserum (anti BPM-serum) 14. Cells were washed in D M E M and incubated for 40 min with anti-BPM serum, 1:20 at room temperature. Cells were fixed with acid alcohol after labelling and visualization. Astrocytes were identified by their content of glial fibrillary acidic protein (GFA). The cultures were washed in D M E M and fixed with acid alcohol, as described above and incubated for 1 h with anti-GFA serum (1:50) prepared in our laboratory as described previously27. 28. Vimentin-containing cells were identified after fixation with rabbit anti-vimentin serum (the generous gift of Dr. S. Fedoroff)diluted 1:20. Endothelial cells were labelled with human Chagasic serum 26 after acid alcohol fixation; fibroblasts were labelled with monoclonal anti-Thy-1 tissue culture supernatant (mouse, MRC 0 X-7; the generous gift of Dr. A. F. Williams) and after labelling fixed with 4% paraformaldehyde at room temperature for 15 min. Oligodendrocytes were labelled with a monoclonal galactocerebroside antibody (a-Gal C19), the generous gift of Dr. C. Linnington. All preparations were mounted in 50% glycerol in PBS and photographed using a Leitz Ortholux II mi-

19

Fig. 1. Phase contrast and epifluorescence micrographs of cerebellar neuronal cultures in serum-free medium (a, b, e and 10 and serum-supplemented medium (c, d, g and h) at 7 DIV, stained with tetanus toxin (b and d) and G F A antibody (f and h). Insets: epifluorescence of 7 DIV cultures in CDM (b) or SSM (d) stained for the D2 protein with BPM antiserum. Photographs were taken on the plastic culture dishes. Specific labelling of neurones by tetanus toxin or BPM antiserum was localized on the surface of both neuronal bodies and fibres. Note that GFA-positive astrocytes are more intensely labelled in chemically defined medium (f). Scale bars indicate 100 um.

20 croscope with either phase contrast or epiluminescence illumination (filter block H). For determination of the proportion of non-neuronal cell types cells were counted using an ocular graticule.

Scanning and transmission electron microscopy For scanning electron microscopy, cultures grown on either plastic surfaces or glass coverslips were fixed for 30 min in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer. After dehydration in ethanol, portions of the dishes cut out with scissors or whole coverslips were critically point dried, coated with gold and viewed in a JEM 35 scanning electron microscope. For transmission electron microscopy cultures were fixed for 30 rain in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 °C. After rinsing in phosphate buffer, cultures were osmicated for 1 h in 1% OsO 4 in sodium cacodylate buffer, pH 7.4, and following dehydration and embedding in Epon the plastic or glass was broken away from the polymerized resin. Ultrathin sections, cut parallel to the surface of the cultures with a diamond knife, were contrasted with uranyl acetate and lead citrate and viewed in a Jeol 100S electron microscope.

DNA estimation D N A was estimated using the method of Burton6; groups of 5 dishes were pooled for each measurement. Cells were washed in Ca2+,Mg2+-containing PBS (DAB) and harvested in ice-cold 0.4 M perchloric acid (PCA). D N A was extracted from the pelleted material (2000 g, 15 min) with 1 M PCA at 70 °C for 20 min. After centrifugation the supernatant was saved and the pellet reextracted with halfvolume of 0.2 M PCA. The two PCA supernatants were pooled and aliquots taken for D N A estimation. It was assumed that 6 #g of D N A were equivalent to 1 x 106 cells 20.

Examination of cell viability As an estimate of cell viability the release of 51Cr was determined after preincubating the cultures in 0.01 M Tris-buffered Krebs solution, pH 7.4, at first for 30 min in the absence and then for 1 h in the presence of 20/~Ci 51Cr (Amersham Int.) at 37 °C. After washing twice with the Krebs-Tris solution the cells were incubated for 3 h. The proportion of radioac-

tivity released during this time was determined by estimating the radioactivity content of both cells and medium using a gamma-counter (Tracor Analytic 1191). Additional information on the viability of cultures was also obtained by estimating the numbers of dead cells using the technique of Bodmer and Bodmer3. Cells were incubated for 15 min at room temperature in the presence of 10/~g/ml fluorescein diacetate followed by 2 min in the presence of 1/~g/ml ethidium bromide. After washing, dishes were examined by fluorescence microscopy with fluorescein or rhodamine optics. RESULTS

Morphology and composition of cultures The morphological development of nerve cells from dissociated rat cerebellum in serum-supplemented medium (SSM) has been described previously23 and observations in this study were consistent with earlier findings (see also e.g. refs. 7, 11 and 13). It was confirmed that the conditions described here select for the survival of cerebellar interneurones. Soon after seeding, these cells attached to the substratum and emitted fibres. The migration of cells, apparently along the fibres, resulted in the formation of small clumps which gradually increased in size. This was accompanied by the fasciculation of the fibres, which interconnected the clumps in thick, usually straight bundles. By about 7 days in vitro (DIV) the mature appearance of serum-supplemented cultures was established (Fig. lc, d, g). Cultures grown in CDM showed strikingly different organisation. Although the cells became attached to the substratum and grew fibres, cell migration, the formation of clumps and the fasciculation of fibres were limited. Over long incubation periods the appearance of these cultures remained more or less unaltered; the majority of the nerve cells remained dispersed and fine neurites formed a dense network over the surface (Fig. la, b, e). In contrast to cells grown in SSM, these fibres were irregularly curved. Neurite outgrowth could be seen particularly well in tetanus toxin-labelled preparations (Fig. lb): individual fibres predominated in serum-free cultures which could not be followed readily by phase contrast microscopy.

21 TABLE I Cellular composition of cultures in chemically defined medium and serum-supplemented medium

Percentages of non-neuronal cells (+ S.E.M.) were determined by direct counting in the culture dish under an ocular graticule. Total cells were counted under phase contrast and cells labelled with the appropriate marker under epifluorescent illumination. A minimum number of 6 fields was counted in each dish, giving a total number of between 1000 and 1500 cells. Total cell number determinations were confirmed by comparison with the results of the DNA assay (Fig. 3). Cell type

Serum-supplemented medium Astrocytes Astrocytes Fibroblasts Endothelial cells Oligodendrocytes Chemically defined medium Astrocytes Astrocytes Fibroblasts Endothelial cells Oligodendrocytes

Marker (antiserum)

% of total cells 2 DIV

7 DIV

14 DIV

GFA vimentin Thy- 1 (MRC OX7) Chagas' serum Gal. C

3.0 + 0.2 4,0 + 0.5 <1

2.5 _+0.2 <1 <1 <1

2.0 _+0.4 -

GFA vimentin Thy-1 (MRC OX7) Chagas' serum Gal. C

3.0 + 0.3 4.0 + 0.5 1.5 + 0.3

3.0 ~ 0.3 not detectable 3.0 _+0.3 4.0 ___0.7

6.5 _+0.6 -

U n d e r both conditions the m a j o r i t y of the cells were neurones. This was b o r n e out by immunocytochemical studies in which the cells were stained for either tetanus toxin receptors or the D2 protein with anti-BPM serum (Fig. 1). Extensive investigations, including dual labelling of the p r e p a r a t i o n s , indicated that under the present e x p e r i m e n t a l conditions, G F A - p o s i t i v e cells which also showed the expression of these neuronal m a r k e r s could not be detected. Staining with anti-BPM serum confirmed the result obtained with tetanus toxin by showing that the organisation of neurones and fibres was different in the two culture conditions. The most a b u n d a n t non-neuronal cells in both types of cultures were G F A - p o s i t i v e astrocytes, constituting 2 - 6 % of the total cell n u m b e r (Table I). There were differences in the a p p e a r a n c e of glial cells in the two culture conditions (Fig. lf, h). A s t r o cytes in C D M a p p e a r e d to have m o r e and finer processes and, in particular, flat, astroblast-like cells were not usually e n c o u n t e r e d (see also ref. 9), although they constituted nearly 18% of the total of G F A - p o s i t i v e cells in ' m a t u r e ' serum-containing cultures23. W h e n cultures were examined soon after seeding (at 2 D I V ) vimentin-positive cells constituted only a slightly higher p r o p o r t i o n of the cells than G F A - p o s i t i v e astrocytes in both culture conditions (Table I). Endothelial cells, labelled with Chagasic serum,

were only rarely detected in SSM but in C D M their proportion was similar to that of astrocytes. These were usually e n c o u n t e r e d in small groups in C D M in which dividing cells were seen occasionally but their numbers did not rise above 3 - 4 % (Table I). In contrast, T h y - l - p o s i t i v e fibroblasts were consistently present in SSM but not in C D M cultures; no cell of this morphology was ever found to be G F A or tetanus toxin positive. Oligodendrocytes were detectable in C D M , about 4% at 7 D I V , but these cells were rare in SSM, Their m o r p h o l o g y was different in the two culture media; in C D M they a p p e a r e d less branched and more irregular in shape. The results obtained by direct counting of the identified non-neuronal cell types and estimating total cell numbers by D N A m e a s u r e m e n t confirmed that in both culture conditions m o r e than 90% of the cells were neuronal. It is worth mentioning that cells in the C D M were grown in the absence of an inhibitor of cell replication; in the presence of serum this would have resulted in an extensive proliferation of the nonneuronal cells. Scanning electron microscopy gave further information on the differences in the organisation of nerve cells under the two culture conditions. Fig. 2 shows cultures at 7 D I V , when the ' m a t u r e ' a p p e a r a n c e is already established. In the chemically defined medium (Fig. 2a), the p e r i k a r y a were flattened to the substratum, whereas they were different in shape

22

Fig. 2. Scanning electron micrographs of 7 DIV neuronal cultures in CDM (a) and SSM (c). Scale bars indicate 10 pro. Note the flattened appearance of the neuronal cell bodies and fibres in chemically defined medium compared with the serum-supplemented medium, the presence of neuropil-like structures in SSM (large arrow) and varicosities on the fibres (small arrows). Transmission electron micrographs showing the organisation of fibres in chemically defined (b) and serum-supplemented medium (d). Note the presence of varicosities (small arrows) and microtubules under both conditions. Scale bars indicate 1 pm. (ovoid) and clearly raised above the surface in the presence of serum (Fig. 2C). U n d e r the latter conditions big fibre bundles p r o d u c e d a neuropil-like underlay beneath the cell bodies which did not show any clear adhesion among themselves. In C D M the majority of fibres were small and curved and also appeared to be flattened onto the surface. Transmission electron microscopy showed that although fasciculation of fibres t o o k place in C D M , the number of neurites in the bundles always a p p e a r e d to be much lower than in the presence of FCS (Fig. 2b, d). U n d e r both conditions the neurites displayed varicosities, which often contained numerous vesicles and greatly o u t n u m b e r e d the classical synaptic structures. Although quantitative studies are still in progress, it would a p p e a r that the synapses are predominantly axo-axonic.

Survival and viability of cells in culture Cell numbers were estimated by D N A content after different times in culture (Fig. 3). In comparison with the number of plated cells, the values at 2 D I V were about 15% lower in SSM and 10% higher in CDM. Beyond that time, there was a m o d e r a t e decrease in cell numbers that was similar under the two conditions until about 14 D I V . T h e r e a f t e r cultures in C D M usually survived without much change in their overall appearance for up to 2 weeks longer. In contrast, nerve cells in SSM died rapidly after about 2 weeks in vitro (see also Fischer9). The retention of accumulated 51Cr was used as a means to assess the integrity of cells. The m e m b r a n e permeability of cells in the two types of cultures at 7 D I V was good and c o m p a r a b l e : only a very small proportion of the intracellular 51Cr (5.4% in SSM and

23

~SSM [~] CDM "1

?

::::>:,:,

i

X w

r

"ll m

i!!!!!!iiii!

iiiiiiiii!i!

i i i i i!i

iiiiiiiiiii

i i i i i~i~

iiiiiiiiiii

iiiiii~iii~i

5

7

14

DIV

Fig. 3. Cell numbers in cultures in SSM and CDM were calculated from mean DNA values determined in duplicate in three separate experiments. The data were analysed by analysis of variance with two-way classification; S.E.M. was 0.111. The difference in cell numbers between SSM and CDM was significant, (P < 0.01), and the decline in cell numbers with time in vitro was also significant (P < 0.01) in both conditions.

5.2% in CDM) was released over a 3 h period. Viability was also evaluated by means of the ability of cells to exclude ethidium bromide 3. In the case of the cultures in SSM this technique could only be used reliably before the formation of relatively big clusters of cells. At 2 DIV the proportion of cells which took up this marker was lower (3%) in SSM than in CDM (10-20% during the period 2 - 7 DIV). DISCUSSION

The aim of this study was to establish nerve cell-enriched cultures from dissociated cerebellum, which were never exposed in vitro to poorly defined supplements, such as serum or tissue extracts, and to characterize these cultures in terms of cellular composition, cell survival and morphological appearance. As a reference, we used cultures grown in the presence of FCS under conditions which had previously been found to select for the survival of cerebellar nerve cells (see ref. 23 and other references quoted therein). It was observed that the enrichment in nerve cells which constituted 90% or more of the total cells, was similar under the two conditions. Further similarities included the selective survival of the small interneu-

rones, most of which seem to be granule cells, since autoradiographic studies showed that the proportion of nerve cells which accumulate [3H]GABA and thus may be considered as inhibitory interneurones is relatively low (refs. 7, 13 and 15; also unpublished resuits from our laboratory). It should be emphasized, however, that in order to suppress the proliferation of non-neuronal cells an inhibitor of cell replication must be added to the SSM. This is not necessary under the serum-free conditions, which seem to limit the replication of GFA-positive cells and do not support the survival of Thy-l-positive fibroblasts (see also, refs. 5, 9 and 21), although the number of oligodendrocytes and endothelial cells is higher than in the reference cultures (Table I). Another point worth noting in connection with the comparison of the two culture conditions is the difference in the requirement for depolarizing concentrations of K ÷ ions. In order to maintain cerebellar nerve cells in the presence of small numbers of nonneuronal cells beyond a few days in vitro in the presence of FCS the concentration of K + ions must be increased above the physiological level (see ref. 23 and references therein), while this is not necessary under the serum-free conditions. The present conditions sustained the survival of the cerebellar nerve cells for 3 - 4 weeks in the d e fined medium, but only for about 2 weeks in the presence of serum. Similar improvement of neuronal survival in defined media has been reported previously for cultures of mouse cerebellar neurones 9 and of peripheral nerve cells 24, although it was noted that in the latter cultures the longer survival of nerve cells was accompanied by a marked increase in non-neuronal cell numbers. Besides the longer survival of nerve cells in the defined than in the serum-supplemented medium, it was a consistent finding that the number of cells found after 2 days incubation was significantly higher (by'about 10%) than that initially seeded, whereas it was about 20% lower in the reference cultures. Pilot studies, following [3H]thymidine incorporation into DNA, have indicated that differences in an initial wave of cell replication may be involved in this effect. The viability of the cells was also tested in both types of cultures. In terms of 51Cr retention, this was satisfactory and comparable under the two conditions. However, using the technique of Bodmer and

24 B o d m e r 3 the p r o p o r t i o n of cells which take up ethidium bromide and are therefore considered as non-viable was found to be higher in the absence than in the presence of FCS. In addition to the observations mentioned above on cell survival and 51Cr retention, results in the accompanying p a p e r 10, which show that cells grown in serum-free m e d i u m are i m p e r m e a b l e to reagents affecting protein iodination, indicate that the exclusion of ethidium b r o m i d e cannot be taken as a measure of viability under the present experimental conditions. In spite of the similarities in cellular composition, the organisation of the two cultures showed m a r k e d differences (Figs. 1 and 2). In contrast to cultures in SSM, the migration of cells with consequent formation of clumps and the fasciculation of fibres were very limited in C D M . These results differ from those of Messer et al. 15 or Fischer 9 who cultured the cells in serum at first or in the presence of BSA. U n d e r the latter conditions we also found that cell clumping was more m a r k e d , although the size of the aggregates and the thickness of the fibre bundles were always smaller than in the reference cultures in FCS. F u r t h e r observations indicated that the failure of nerve cells to associate into clumps in C D M is not simply due to the absence of FCS; seeding the cells onto uncoated or collagen-coated dishes in C D M resulted in the formation of big aggregates and very thick fibre bundles, the a p p e a r a n c e of these cultures being similar to that in c o m p a r a b l e substrata in the FCS-reference cultures. These findings are consistent with the view

REFERENCES 1 Balazs, R., Regan, C. M,, Meier, E., Woodhams, P. L., Wilkin, G. P., Patel, N. J. and Gordon, R. D., Biochemical properties of neural cell types from rat cerebellum. In E. Giacobini, A. Vernadakis and A. Shahar (Eds.), Tissue Culture in Neurobiology, Raven Press, New York, 1980, pp. 155-168. 2 Barnes, D. and Sato, G. H., Serum-free cell culture: a unifying approach, Cell, 22 (1980) 649-655. 3 Bodmer, W. F. and Bodmer, J. G., Cytofluorochromasia for HLA A, B, C and DR typing. In The NIAID Manual of Tissue Typing Techniques, U.S. Dept. of Health, Education and Welfare, Washington, DC, 1979-1980, pp. 46-54. 4 Bottenstein, J. E. and Sato, G. H., Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 514-517. 5 Brunner, G., Lang, K., Wolfe, R. A., McClure, D. B. and Sato, G. H., Selective cell culture of brain cells by serumfree, hormone supplemented media: a comparative mor-

that serum factors may mitigate the attachment of the cells to the P L L - c o a t e d surface and this, in turn, facilitates adhesion among ceils and fibres. It is possible, however, that differences in cell surface properties are also involved in the distinct morphology of the two types of cultures. O n e of the integral cell surface molecules, implicated in nerve cell adhesion is the D2 protein 12 which is detected on the surface of rat cerebellar neurones cultured in the presence of serum (e.g. ref. 14). I m m u n o c y t o c h e m istry showed that D2 is also expressed throughout the whole neuronal surface of nerve cells grown in C D M (Fig. 1). Nevertheless, under the two culture conditions differences may exist in the a m o u n t of or in the d e v e l o p m e n t a l change in the molecular forms of this protein. Results in the accompanying p a p e r 10 showed that such alterations do indeed occur and that changes are also detectable in the organisation of proteins on the plasma m e m b r a n e when cells grown in the defined m e d i u m are c o m p a r e d with reference FCS-containing cultures. It appears therefore, that the organisation of cells in vitro is d e t e r m i n e d by a delicate interplay between the surface p r o p e r t i e s of the cells and influences m e d i a t e d by the nature of the substratum and chemical factors in the medium. ACKNOWLEDGEMENTS W e are i n d e b t e d to Mr. J. M c G o v e r n for skilled technical assistance. V . G . was s u p p o r t e d by a LongTerm E M B O Fellowship.

phological study, Develop. Brain Res., 2 (1982) 563-575. 6 Burton, K., A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of DNA, Biochem. J., 62 (1956) 315-323. 7 Currie, D. N. and Dutton, G. R., 3[H]GABA uptake as a marker for cell type in primary cultures of cerebellum and olfactory bulb, Brain Res., 199 (1980) 473-481. 8 Faivre-Baumann, A., Rosenbaum, E., Puymirat, J., Grouselle, D. and Tixier-Vidal, A., Differentiation of fetal mouse hypothalamic cells in serum-free medium, Develop. Neurosci., 4 (1981) 118-129. 9 Fischer, G., Cultivation of mouse cerebellar cells in serumfree, hormonally defined media: survival of neurons, Neurosci. Lett., 28 (1982) 325-329. 10 Gallo, V., Balazs, R. and Jorgensen, O. S., Cell surface proteins of cerebellar interneurones and astrocytes cultured in chemically defined and serum-supplemented media, Develop. Brain Res., 17 (1985) 27-37. 11 Gallo, V., Ciotti, M. T., Coletti, A., Aloisi, F. and Levi, G., Selective release of glutamate from cerebellar granule

25 cells differentiating in culture, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 7919-7923. 12 Jorgensen, O. S., Delouvee, A., Thiery, J. and Edelman, G. M., The nervous system-specific protein, D2, is involved in adhesion among neurites from cultured rat ganglia, FEBS Lett, 111 (1980) 39- 42. 13 Lasher, R. S. and Zagon, I. S., The effect of potassium on neuronal differentiation in cultures of newborn rat cerebellum, Brain Res., 41 (1972) 482-488. 14 Meier, E., Regan, C. M., Balazs, R. and Wilkin, G. P., Specific recognition of the neuronal cell surface by an antiserum raised against plasma membrane preparation of immature rat cerebellum, Neurochem. Res., 7 (1982) 1031-1043. 15 Messer, A., Maskin, P. and Mazurkiewicz, J. E., Effects of using a chemically defined medium for primary rat monolayer cerebellar cultures: morphology, GABA uptake and kainic acid sensitivity, Brain Res., 184 (1980) 243-247. 16 Messer, A., Mazurkiewicz, J. E. and Maskin, P., Growth of dissociated rat cerebellar cells using serum-free supplemented media and varied transferrin concentrations, Cell. molec. Neurobiol., 1, 1 (1981) 99-114. 17 Morrison, R. S. and de Vellis, J., Growth of purified astrocytes in a chemically defined medium, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 7205-7209. 18 Patel, A. J, Hunt, A., Gordon, R. D. and Balazs, R., The activities in different neural cell types of certain enzymes associated with metabolic compartmentation glutamate, Develop. Brain Res., 4 (1982) 3-11. 19 Ranscht, B., Clapshaw, P. A., Price, J., Noble, M. and Seifert, W., Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 2709-2713. 20 Satake, H. and Abe, S., Preparation and characterisation

of nerve cells perikaryon from rat cerebral cortex, J. Biochem. (Jap.), 59 (1966) 72-75. 21 Skaper, S. D., Adler, R. and Varon, S., A procedure for purifying neuron-like cells in cultures from central nervous tissue with a defined medium, Develop. Neurosci., 2 (1979) 233-237. 22 Sternberger, L. A., Immunocytochemistry, Prentice Hall, New Jersey, 1974. 23 Thangnipon, W., Kingsbury, A., Webb, M. and Balazs, R., Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurons and astrocytes, Develop. Brain Res., 11 (1983) 177-189. 24 Wakade, A. R., Edgar, D. and Thoenen, H., Substrate requirement and media supplements necessary for the longterm survival of chick sympathetic and sensory neurons cultured without serum, Exp. Cell Res., 140 (1982) 71-78. 25 Wilkin, G. P., Balazs, R., Wilson, J. E., Cohen, J. and Dutton, G. R., Preparation of cell bodies from the developing cerebellum: structure and metabolic integrity of the isolated cells, Brain Res., 115 (1976) 181-199. 26 Wilkin, G. P., Woodhams, P. L. and Ribeiro dos Santos, R., Rat cerebellar cells in tissue culture. I. An immunological marker for brain vascular endothelial cells. Antibodies from the sera of patients with Chagas' disease, Develop. Neurosci., 4 (1981) 296-306. 27 Woodhams, P. L., Basco, E., Hajos, F., Csillag, A. and Balazs, R., Radial glia in the developing mouse cerebral cortex and hippocampus, Anat. Embryol., 163 (1981) 331-343. 28 Woodhams, P. L., Cohen, J., Mallet, J. and Balazs, R., A preparation enriched in Purkinje cells identified by morphological and immunocytochemical criteria, Brain Res., 199 (1980) 435-442.