oligodendroglial and astroglial heterogeneity in mouse primary central nervous system culture as...

Developmental Brain Research, 36 (1987) 13-25 13 Elsevier

BRD 50607

Oligodendroglial and astroglial heterogeneity in mouse primary central nervous system culture as demonstrated by differences in

GABA and D-aspartate transport and immunocytochemistry

Richard Reynolds and Norbert Herschkowitz Department of Pediatrics, University of Berne, lnselspital, Berne (Switzerland)

(Accepted 24 February 1987)

Key words: Oligodendrocyte; Astrocyte; Immunocytochemistry; Autoradiography; [3H]v-Aminobutyric acid; D-[3H]Aspartate, Central nervous system culture; Serum-free medium

Using simultaneous autoradiography and immunofluorescence we have investigated the functional heterogeneity amongst oligodendrocytes and astrocytes in primary mouse central nervous system (CNS) culture as expressed by differences in their ability to accumulate y-[3H]aminobutyric acid ([3H]GABA) and D-[3H]aspartate. We have used a range of specific antibodies that identify oligodendrocytes and astrocytes, from precursor to fully mature cells, to address the question of whether all neuroglial cells are capable of expressing this function. Our results showing that A2B5-, 03-, and galactocerebroside-positive cells became heavily labelled with these two neuroactive amino acids, whereas cells expressing the myelin proteins 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) did not, demonstrate that this capacity is already present in oligodendrocytes at early developmental stages but may not extend to fully mature cells. Astrocytes in culture exhibited a large degree of variability with respect to their ability to transport GABA and D-aspartate. When grown in either serum-containing or serum-free hormone supplemented culture medium two morphologically distinct types of glial fibrillary acidic protein (GFAP)-positive astrocyte were identified, process-bearing and epithelioid. Process-bearing cells became heavily labelled with the amino acids under both growth conditions, whereas, data showed that although epithelioid astrocytes were not, or only lightly, labelled with either amino acid in serum-containing cultures, when grown in serum-free culture medium they became more heavily labelled. Thus the expression, in culture, by epithelioid astrocytes, of one of the functions attributed to these cells is largely dependent on growth conditions.

INTRODUCTION

The functional roles of astrocytes and oligoden-

drocytes in the central nervous system (CNS), with

the exception of myel inat ion, are as yet unclear. Of

the functions that have been a t t r ibuted to the neuro-

glia, their abil i ty to accumulate putat ive amino acid

transmitters has been one of the most widely studied.

Both biochemical and autoradiographical investiga-

tions have shown that glial cells, both astrocytes and

ol igodendrocytes , share with neurons the abili ty to

accumulate the inhibi tory amino acid G A B A , and

the putat ive excitatory t ransmit ters L-glutamate and L-aspartate 2'5-7'22'23'26'28. Pr imary dissociated cul-

tures of fetal or neonata l CNS tissue have been wide-

ly used in the investigation of the uptake of radiola-

belled transmit ters by different cell types and their

development . However , despi te the advantages of

such cultures, there is still uncer ta inty as to how

closely the functions of cells grown in culture reflect

those of their in vivo counterpar ts 9'29.

Immunocytochemical studies in pr imary disso-

ciated mixed cell cultures, p repa red from both neo-

natal and adult CNS tissue, using ant ibodies against

the astrocyte marke r glial f ibril lary acidic pro te in

(G FA P) , have revealed, in several laborator ies , two

morphological ly quite distinct types of astrocyte 3,8, 9,12,13,18,29. One type has a mult iple process-bear ing

morphology and the o ther is a large f lat tened epithe-

lioid cell. These two populat ions of morphological ly

Correspondence: R. Reynolds. Present address: Department of Biochemistry, Imperial College, London, SW7 2AZ, U.K.

0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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distinct GFAP-positive astrocytes can be also distin- guished by their surface antigenic properties 8,18, their response to epidermal growth factor (EGF) 15, and their expression of fl-adrenergic receptors 13. In addition the two types differed in their ability to accumulate [3H]GABA, the process-bearing cells becoming heavily labelled whilst the flat epithelioid cells accumulated lower levels of this amino acid 23,29. Howev- er, in contrast to this situation in culture, astrocytes in all layers of postnatal cerebellar slices, both protoplasmic and fibrous, accumulated [3H]GABA8.

It has been suggested that these in vitro astrocytic types, process-bearing and epithelioid, correspond in vivo to the fibrous and protoplasmic astrocytes respectively 9'14, and are developmentally distinct 14.

However, it has also been shown that the process- bearing astrocytes, both in adult rat CNS cultures

and neonatal rat cerebellar cultures, gradually undergo a morphological change, as confluency is ap- proached, to become epithelioid GFAP-positive cells 12'29. Epithelioid astrocytes also undergo morphological changes, becoming process-bearing, after addition of dibutyryl cyclic AMP (dBcAMP) to the culture medium 3. Therefore, considering that very little is known about the biochemical and functional differences between them in culture, the comparison with cell types in vivo is still unclear.

Oligodendrocytes in vivo have been segregated into 3 types on the basis of location, stainability, and size aS. Satellite oligodendrocytes are small, restricted to gray matter, and are closely applied to neuronal cell bodies, whereas interfascicular oligodendrocytes are larger and are interposed between myelinated nerve fibers in white matter. Intermediate oligodendrocytes are suggested to be potential satellite or myelinating types. It is doubted however, that there are any functional differences between the 3 types 3°. Oli- godendrocytes in primary dissociated culture cannot easily be compared with the in vivo classification above, and the majority of oligodendrocytes in dissociated cultures prepared from fetal or neonatal ro- dent CNS represent a relatively immature non-myelinating cell type 1'17'23. Interest in oligodendrocyte

function has been restricted to myelination and little is known about other functions and possible heterogeneity amongst these cells. We have recently shown that galactocerebroside (GC)-expressing and also more immature oligodendrocytes in culture are able

to actively accumulate several neuroactive amino acids 22'23, and it is suggested that this property is also present in the undifferentiated progenitor celO °.

In order to obtain more information about the functional heterogeneity amongst oligodendrocytes and astrocytes in culture we have investigated differences in their ability to accumulate [3H]GABA and o-[3H]aspartate. We have used the method of simultaneous autoradiography and immunofluorescence, using a range of specific antibodies, as described in the accompanying paper 24, to identify individual amino acid-accumulating cells. We have addressed the question of whether all neuroglial cells are capable of GABA and D-aspartate uptake, irrespective of developmental age, or are there specialized subpopulations of these cells which are adapted for the removal of extracellular amino acids. Our results show that oligodendrocytes in culture accumulated GABA and D-aspartate primarily at early developmental stages. Astrocytes in primary culture exhibited a large degree of heterogeneity with respect to their ability to accumulate these amino acids, process-bearing cells becoming heavily labelled and epithelioid cells only lightly labelled, although this was found to be highly dependent upon the culture conditions.

MATERIALS AND METHODS

Cell cultures Three different types of primary dissociated cul-

tures were used in these investigations: mixed glial bilayer cultures; epithelioid astrocyte cultures; and cultures enriched in process-bearing astrocytes. Mixed glial cultures were prepared from forebrains of neonatal mice (0-12 h) as described previously 23'24. Epithelioid astrocyte cultures were prepared by plating cells dissociated from neonatal mouse brain, as above, onto uncoated glass coverslips, at a 10-fold lower density (1 x 10 4 cells/cm 2) than for the mixed glial cultures. The cells were grown in Dulbec- co's Modified Eagle's Medium (DMEM) medium containing 10% fetal calf serum (FCS) for 3 weeks and the medium was changed after 4 days and there- after every 7 days. In several experiments the cultures were transferred after 2 weeks to DMEM-containing 0.25 mM dBcAMP for 1 week. After dBcAMP treatment cultures consisted almost entire- ly of large cell bodied process-bearing GFAP-posi-

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tive astrocytes, with the only contaminant cell type being patches of fibroblasts. No small process-bearing astrocytes as seen in mixed glial cultures were observed in dBcAMP-treated cultures.

Cultures enriched in process-bearing astrocytes were prepared from forebrains of embryonic day 15 mouse fetuses, as described in the accompanying paper for neuron enriched cultures 24. Cells were plated on poly-L-lysine-coated glass coverslips in 35- mm Multiwell dishes (1 x 105 viable cells/cm 2) in

DMEM/Hams F12, 1:1 (vol/vol), containing supple- ments as described previously 24. It was noted that after 14 days in vitro (DIV) in this medium neuronal degeneration became extensive until at 36 DIV the cultures consisted of large numbers of process-bearing GFAP-positive astrocytes amongst areas of epithelioid cells, with only few oligodendrocytes and virtually no neurons.

Antibodies Monoclonal antibody A2B5 (ascites fluid, dilution

1:500) was a gift from Dr. M. Noble (London) and has been shown to bind to glial precursor cells in mixed glial cultures 19. Monoclonal antibodies 03 and 04 (concentrated supernatants, dilution 1:10) were a gift from Prof. M. Schachner (Heidelberg) and have been shown to be specific for the myelin lipid sulfa- tide 25,27. Monoclonal anti-GC antibody 2° (culture su-

pernatant, dilution 1:5) was a gift from Dr. P. Deb- bage (Goettingen). Rabbit antiserum against the myelin protein 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; whole serum, dilution 1:30) was a gift from Dr. E. Carey (Sheffield). Rabbit anti-myelin basic protein (anti-MBP) serum was prepared by 7 weekly injections (s.c.) of 1 mg rabbit MBP with complete Freund's adjuvant (first injection), or in- complete Freund's adjuvant (subsequent injections). MBP isolated from rabbit brain was purchased from Calbiochem (purity greater than 95% by SDS- PAGE). The obtained antisera were further purified by affinity chromatography against MBP linked to Sepharose 4B, have been characterised in another paper 21, and were used for indirect immunofluorescence at a dilution of 1:50. Rabbit anti-GFAP (Ig fraction, dilution 1:50-1:100) was purchased from DAKO and TRITC-conjugated goat anti-rabbit and goat anti-mouse immunoglobulins (dilutions 1:150 and 1:100) were purchased from Cappel Labs.

Simultaneous immunofluorescence and autoradiogra-

phy The procedure was carried out exactly as

described in the accompanying paper 24. For the surface antigens A2B5, 03, 04, and GC, the cultures were incubated sequentially with primary and secondary antibodies before a 10-min incubation with [3H]GABA (6 x 10 -8 M) or D-[3H]aspartate (1.4 x

10 -7 M). Cultures were then fixed with 4% parafor-

maldehyde, 0.1% glutaraldehyde in 0.1 M sodium phosphate (pH 7.2) buffer. For the localization of the intracellular antigens MBP, CNP and GFAP the cultures were first incubated with [3H]GABA or I> [3H]aspartate for 10 min (20 min for epithelioid astrocyte cultures), and then fixed with 4% paraformalde- hyde, 0.1% glutaraldehyde followed by permeabili- zation with methanol (-20 °C). The cultures were then incubated sequentially with normal goat serum and primary and secondary antibodies.

The coverslips were processed for autoradiography as described previously 23. Slides were examined with a Leitz Orthoplan microscope using bright field and fluorescence optics, and x25 and x40 oil immer- sion objectives. Following the whole procedure of staining and autoradiography, phase contrast images were of very poor quality and therefore are not rep-

TABLE I

Quantitative estimation of the percentage of [3H]GABA - and D- [3 H] aspartate-accumulating cells positively stained for glial antigens

The data represent the percentage of process-bearing cells, growing on top of the astroglial carpet layer, in 11-DIV cultures grown in serum-containing medium, that were heavily labelled with [3H]amino acid (10 min incubation) and were also positively stained by immunofluorescence against the glial antigens. Epithelioid astrocytes growing in the carpet layer are not included. Values given are the mean percentage + S.E.M. of cells counted in two experiments (2 coverslips/experiment, 200-300 cells counted per coverslip, 20 fields counted per coverslip).

Antigen % [3H]-Labelled cells positively stained by immunofluorescence

[3H]GABA o-[3H]Aspartate

GC 39.5 + 2.7 38.6 + 4.6 03 68.8 + 3.1 56.9 + 5.0 A2B5 48.2 + 3.1 46.0 _ 5.6 CNP 0 0 MBP 0 0 GFAP 1.7 + 0.7 4.5 + 2.2

resented here. Photographs were taken with Ilford HP5 400 ASA black and white films.

RESULTS

In the present study [3H]GABA and D-[3H]aspar - tate accumulating cells could be reliably identified by the use of simultaneous autoradiography and immunofluorescence with a range of specific antibodies, as we have previously described 24.

Oligodendrocytes The ability of oligodendrocytes at different devel-

opmental stages to accumulate [3H]GABA and D- [3H]aspartate was studied using mixed glial cultures, prepared from neonatal mouse CNS, whose cell com- position we have described previously 23. The monoclonal antibody A2B5 has been shown to bind to glial precursor cells in mixed glial cultures 19, in addition to other cell types 25. Of the cells in l l -DIV cultures that were heavily labelled with [3H]GABA 48% were also A2B5-positive (Table I) and the majority of these cells had the morphology of an undifferentiated cell type (Fig. la, b) with few processes. Virtually all (97%) of the 03-positive oligodendrocytes in the cultures had accumulated [3H]GABA (Fig. lc, d), these cells representing 69% of the [3H]GABA-accumulating cells (Table I). Similar results were obtained with the monoclonal antibody 04. Similarly, 95% of the GC-positive oligodendrocytes were heavily labelled with [3H]GABA (Fig. le, f), representing 39% of the [3H]GABA labelled cells (Table I). Those cells that were 03 and GC-positive but were not labelled with [3H]GABA were all highly differentiated cells, often with flattened myelin-like membrane extensions. In contrast to these results with surface antigens that identify relatively immature oligodendrocytes in culture, no [3H]GABA accumulating cells were seen that were positive for the myelin proteins MBP and CNP (Fig. 2). MBP and CNP immunoreactivity was only seen in the cells that appeared morphologically more differentiated.

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The identification of cells that had accumulated D- [3H]aspartate revealed a similar pattern (Table I); 46% of the heavily labelled cells were found to be A2B5-positive (Fig. 3a, b), 57% 03-positive (Fig. 3c, d) and 39% GC-positive (Fig. 3e, f). Again there were a small number of 03- and GC-positive cells that had not accumulated I>[3H]aspartate (e.g. Fig. 3c, d). Similar to the results with GABA, the more differentiated cells that were MBP- or CNP-positive had not accumulated D-[3H]aspartate (Fig. 4).

Astrocytes Although process-bearing GFAP-positive astro-

cytes were found only rarely on the surface of mixed glial bilayer cultures, as previously published 23, these cells were always heavily labelled with both [3H]GA- BA (Fig. 5a, b) and D-[3H]aspartate (Fig. 6a, b), representing only 1.7 and 4.5% of the process-bearing labelled cells respectively (oligodendrocytes and process-bearing astrocytes). The number of such cells in- creased between 11 and 21 DIV but still only repre- sented a small proportion of the labelled cells. These cells were generally small cell bodied with several fine processes (Fig. 5a).

In preliminary experiments it was found that when cultures prepared from fetal mouse CNS were grown in hormone-supplemented serum-free medium from 24 h onwards, there was a gradual enrichment in process-bearing GFAP-positive cells after approximate- ly 20 DIV. At 36 DIV there were large areas in the cultures with no or few epithelioid astrocytes that were filled with multiprocessed cells, the majority of which were GFAP-positive astrocytes. Only few of these process-bearing cells expressed any of the oligodendrocyte markers. Nearly all of these process- bearing cells became labelled after incubation with [3H]GABA or D-[3H]aspartate, the majority of them intensely labelled (Fig. 5c, d and Fig. 6c, d). These cells exhibited many more highly branched processes than those seen in mixed glial cultures. A small number of these GFAP-positive cells remained totally unlabelled, indicating some degree of heterogeneity

Fig. 1. Combined [3H]GABA autoradiography (10 min incubation) and immunofluorescence with monoclonai antibodies, A2B5 (a, b), 03 (c, d) and GC (e, f), against oligodendrocyte surface antigens in mixed glial cultures 11 D IV. a, b: an A2B5-positive cell with immature morphology is also heavily labelled with GABA. c, d: all 03-positive oligodendrocytes are also labelled over the cell body and some processes with GABA. e, f: two highly branched GC-positive GABA-labelled cells. Background grain density represents uptake into flat astrocytes (also in Figs. 2-4). Bar = 16ktm.

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Fig. 2. [3H]GABA autoradiography and immunofluorescence with antibodies against the myelin proteins MBP and CNP, in 11 DIV (a, b) and 21 DIV (c, d) mixed glial cultures. Oligodendrocytes, brightly MBP-positive (a) and CNP-positive (c), arrows indicating the cell bodies, are totally void of overlying silver grains (b, d), whilst other GABA-labelled cells are unlabelled with the antibodies. Bar = 26~m.

Fig. 3. Combined D-[3H]aspartate autoradiography (10 min incubation) and immunofluorescence with monoclonal antibodies, A2B5 (a, b), 04 (c, d) and GC (e, f), against oligodendrocyte surface antigens in mixed glial cultures, a, b: an A2B5-positive cell that has accumulated D-aspartate in a 22-DIV culture, c, d: of the 3 cells in this field of a 11-DIV culture that are 04-positive, one is heavily labelled with D-aspartate (arrow) whilst two are completely unlabelled (arrowheads). e, f: an oligodendrocyte in a 22-DIV culture that is both GC-positive and labelled with D-aspartate. Bar = 16/am.

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Fig. 4. D-[3H]Aspartate autoradiography and anti-MBP (a, b) and anti-CNP (c, d) immunofluorescence in ll-DIV mixed glial cultures. MBP and CNP-positive oligodendrocytes are totally unlabelled by D-aspartate (arrows). Bar = 33 ktm (a, b) and 20ktm (c, d).

even amongst this type of astrocyte. We have shown previously that the flattened epi-

thelioid GFAP-positive cell layer in mixed glial cultures becomes only lightly labelled with [3H]GABA and o-[3H]aspartate 23. The same results were ob-

tained in the present experiments using simultaneous immunofluorescence and autoradiography. To investigate this further we produced primary cultures containing only flattened epithelioid cells. These cultures contained no process-bearing glia and only small patches of fibroblasts. As expected the uptake

of both [3H]GABA (Fig. 5e, f) and D-[3H]aspartate

(Fig. 6e, f) by these GFAP-positive cells was very low, with no clear difference in labelling intensity between the two amino acids. The labelling pattern was also variable, some cells being totally unlabelled. Uptake of the amino acids by these cultures was investigated at several ages, representing both pre- confluent and confluent stages, but no differences were found. In order to investigate whether dBcAMP could induce a functional differentiation of these flattened astrocytes, the cultures were grown for the

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Fig. 5. Simultaneous [3H]GABA autoradiography and GFAP immunofluorescence, a, b: a process-bearing astrocyte in an 11-DIV mixed glial culture, with fine processes brightly GFAP-positive (arrows), is densely covered with silver grains (b) after 10 min incubation. c, d: a highly branched, heavily labelled, GFAP-positive astrocyte in a 36-DIV fetal mouse CNS culture grown in serum-free medium. e, f: a 21-DIV epithelioid astrocyte culture grown in serum-containing medium incubated with G A B A for 20 min. Note the very low density of silver grains over the cells, g, h: epithelioid GFAP-positive astrocytes, in a 36-DIV fetal CNS culture grown in serum free medium, are heavily labelled after a 10 min incubation with GABA. Bar = 16/~m (a, b), 20/~m (c, d) and 261zm (e -h) .

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Fig. 6. Simultaneous D-[3H]aspartate autoradiography and G F A P immunofluorescence. Process-bearing GFAP-posi t ive astrocytes in both l l - D I V mixed glial cultures (a, b) and 36-DIV fetal CNS cultures in serum-free medium (c, d), are heavily labelled with D-aspartate. Whereas epithelioid astrocytes grown in serum containing medium (e, f; 21 DIV) are only lightly labelled, those found in cultures grown in serum-free medium (g, h; 36-DIV fetal cultures) are densely covered in silver grains. Bar = 20/~m (a -d ) , 3 3 p m (e, f) and 40 p m (g, h).

third week in DMEM medium without FCS but containing 0.25 mM dBcAMP 26. Although a morphological change in the GFAP-positive cells, to process- bearing, was apparent, there was no increase in the labelling intensity after incubation with either amino acid (not shown). In contrast it was observed that the GFAP-positive epithelioid astrocytes that were present in the fetal cultures grown in hormone-supplemented serum-free medium exhibited a much greater uptake of both [3H]GABA (Fig. 5g, h) and D-[3H]as - partate (Fig. 6g, h), although a certain degree of heterogeneity was evident, with some cells being as heavily labelled as process bearing astrocytes whilst others were only lightly labelled.

DISCUSSION

The present study underlines the usefulness of the method of combined autoradiography and immunofluorescence in investigating the existence of transport systems in immunologically identifiable cell sub- types. A2B5 antigen has been shown to be present on membranes of oligodendrocyte and astrocyte precur- sors in culture 1°A9, and these cells, when grown in serum-free medium, are capable of accumulating GABA before becoming GC-positive 1°. We have confirmed that A2B5-positive glia can accumulate GABA and D-aspartate. However, in contrast to Levi et al. 1°, who found that in serum-containing medium the majority of GABA-accumulating cells were GFAP-positive, we found very few cells grown in serum-containing medium labelled with both GABA and GFAP. Thus the majority of A2B5 cells present in our cultures were either uncommitted precursor cells or cells already committed to an oligodendrocyte phenotype.

The oligodendroglial nature of the majority of the GABA- and D-aspartate-accumulating cells was confirmed by the presence of 03 antigen 25'27, and GC on their plasma membranes. Thus our results, showing that A2B5-positive, 03-positive and GC-positive cells became heavily labelled with these two neuroactive amino acids, together with the results of Levi et al. 1°, demonstrate that this ability is already present in oligodendrocytes at early developmental stages. Our results using antibodies against the myelin proteins MBP and CNP, although suggesting that more mature oligodendrocytes do not accumulate these

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amino acids, are not conclusive evidence because these cells in dissociated culture never express the true in vivo phenotype of the myelinating cell, and therefore may also lose their amino acid transport capabilities. Further studies utilizing these methods and tissue sections are required to determine whether myelinating or only immature: and non-myelinating oligodendrocytes are capable of accumulating GABA and D-aspartate.

The finding that even undifferentiated glial progenitor cells exhibit this ability suggests that even at early developmental stages it is important that the extracellular levels of these amino acids be kept very low. Further studies of regional and developmental differences in the ability to accumulate these neuroactive amino acids using in situ preparations are required to determine whether this function is a general property of all glial cells at all stages of development or that it is dependent upon the surrounding environ-

ment. It is clear from our results with astrocytes in culture

that this cell type exhibits a large degree of variability with respect to its ability to accumulate GABA and D-aspartate. This variability exists not only between the two types seen in culture, epithelioid and process- bearing, but also within these two populations, and this is dependent to a large extent on culture conditions. Epithelioid type astrocytes, although never becoming heavily labelled with either GABA or D-aspartate when grown in serum containing medium, exhibited far greater uptake of these two amino acids when grown in serum-free hormone supplemented medium, whilst still retaining a similar morphology. The lack of labelling of epithelioid astrocytes in serum-containing medium with o-aspartate is at vari- ance with a previous study 29 and, although an expla- nation is not obvious, it may be due to the different species used. Not all process-bearing astrocytes, both in serum-containing and serum-free medium, accumulated GABA and D-aspartate. This heterogeneity amongst the process-bearing cells has been noted in other studies 11. These results are in total contrast to a more recent study of Levi et al.10 who found that none of the GFAP-positive cells in cultures grown in serum-free medium accumulated GABA, irrespective of morphology. The reason for this difference in results is not clear but may lie in the different culture conditions used and the use of

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mouse CNS (our results) vs rat CNS (Levi et al. 10). The effects on the morphology of astrocytes of

growth in serum-free medium, i.e. the enrichment in

process-bearing cells, is in agreement with a previous

study 16. However, our observations have extended

this to show that the expression, in culture, of one of

the functions attributed to astrocytes, that of neuro-

transmitter transport, is also largely dependent on

the growth conditions. We suggest that further work

in this direction should also investigate the effect of

various growth factors on the functional differentia-

tion of these cells rather than simply morphological

changes. The low capacity of astrocytes to take up

G A B A found with some astrocyte culture prepara-

tions 4'26 is probably not representative of the situa-

tion in vivo and we suggest that our results obtained

using serum-free hormone-supplemented medium

more closely reflects this situation. This is supported

by observations in tissue sections showing that astro-

cytes, both fibrous and protoplasmic, in all regions of

the cerebellum became heavily labelled after incuba-

tion with G A B A s.

ACKNOWLEDGEMENTS

This work was supported by grants from the Swiss

National Science Foundat ion (3.493.83), Swiss

Multiple Sclerosis Society, and a grant from the Swiss

Foundat ion of Encouragement of Research in Men-

tal Retardation. The authors wish to thank Prof. M.

Schachner (Heidelberg), Dr. M. Noble (London) ,

Dr. P. Debbage (Gott ingen), and Dr. E.M. Carey

(Sheffield) for their gifts of antibodies, and Mrs. C.

Steffen for technical assistance.

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