Selective uptake of neuroactive amino acids by both oligodendrocytes and astrocytes in primary dissociated culture: A posssible role for oligodendrocytes in neurotransmitter metabolism

Brain Research, 371 (1986) 253-266

253

Elsevier BRE 11619

Selective Uptake of Neuroactive Amino Acids by Both Oligodendrocytes and Astrocytes in Primary Dissociated Culture: a Possible Role for Oligodendrocytes in Neurotransmitter Metabolism RICHARD REYNOLDS and NORBERT HERSCHKOWITZ

Department of Pediatrics, University of Bern, Inselspital, CH-3010 Bern (Switzerland) (Accepted August 27th, 1985)

Key words: [3H]y-aminobutyric acid (GABA) - - [3H]l>aspartate - - amino acid uptake - - oligodendrocyte- astrocyte - - tissue culture - - autoradiography - - immunocytochemistry

CNS glia may be involved in the modulation of neuronal excitability through their capacity to accumulate and metabolize neuroactive amino acids. To investigate the possible role of oligodendrocytes in amino acid neurotransmitter metabolism, we have used light microscopic autoradiography, following the uptake of 3H-labelled amino acids by dissociated cultures of neonatal mouse brain, characterized immunocytochemically using cell-type specific markers. Oligodendrocytes, recognized by their characteristic galactocerebroside membrane staining, rapidly accumulated [3H]v-aminobutyric acid (GABA), becoming intensely labelled over cell body and processes after short incubations. In contrast, oligodendrocytes became only lightly labelled with [3H]L-glutamate and aspartate, which preferentially labelled astrocytes. [3H]o-aspartate, a non-metabolized analogue of L-glutamate, was avidly accumulated by oligodendrocytes, labelling cell bodies and processes after short incubations, to a similar extent as GABA. Thus, oligodendrocytes possess a transport mechanism for these excitatory amino acids, but rapidly metabolize them and release the metabolites. Not only the GC-positive cells but also the GC-negative undifferentiated oligodendrocyte precursors accumulated both GABA and D-aspartate, suggesting that this may be a function expressed early in the differentiation of oligodendrocytes. Net uptake of [3H]/3-alanine and [3H]glycine by oligodendrocytes was not observed under any conditions tested. A small number of oligodendrocytes were labelled with [3H]taurine after longer incubations, The uptake of certain neuroactive amino acids is thus a property shared by astrocytes and oligodendrocytes, the latter acting in a protective fashion around neuronal perikarya and axons.

INTRODUCTION The principal mechanism for the inactivation of the m a j o r inhibitory n e u r o t r a n s m i t t e r v-aminobutyric acid ( G A B A ) is thought to be by r e - u p t a k e into presynaptic nerve terminals, via a selective, high affinity transport mechanismS,23,26,27,29,54. H o w e v e r , certain types of glial cells have been shown to share this capacity to accumulate GABAI5,26,30,34,44,47,50,56, and although this u p t a k e is not as rapid as neuronal, it may still be quantitatively i m p o r t a n t 49. In contrast to G A B A , uptake of the putative excitatory amino acid transmitters, L-glutamate and L-aspartate, in vitro appears to occur p r e d o m i n a n t l y into glial cells9,16,21,35,38,49,55, although under certain conditions uptake by neurons has been observed25,35,52, 58. It has been suggested that the astroglial uptake rate for

L-glutamate is high enough to account for the rate of glutamate release from neurons 21. Thus, glial cells have been suggested to play an i m p o r t a n t role in the rapid inactivation of synaptically released neuroactive amino acids, and also in the control of overall or local neuronal excitability by the u p t a k e and subsequent metabolism of these transmitters at sites away from the synapse20,28,44, 51. The use of primary dissociated CNS culture offers significant advantages for the autoradiographical investigation of the localization of accumulated neurotransmitters in different cell typesS,36,40,54. H o w e v e r , due to the similar m o r p h o l o g y sometimes expressed by different cell types in culture, it is essential that cell populations are reliably identified. Specific antisera produced against established cell-type specific antigenic m a r k e r s now provide reliable identification

Correspondence: R. Reynolds, Neurochemie, Med. Kinderklinik, Inselspital, CH-3010 Bern, Switzerland. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

254 and therefore qualitative and quantitative knowledge of cellular composition of cultures 3a,43,44. Most studies on glial uptake of amino acid transmitters have emphasized the possible role of astrocytes in the control of the extracellular concentration of these amino acidsg,22,26,50, and few studies have investigated the role of other types of glial cells. However, we have recently shown that oligodendrocytes in mouse CNS primary dissociated culture share, with astrocytes, the ability to accumulate [3H]GABA, via a sodium-dependent, ouabain-sensitive, transport mechanism 44. In contrast to the previously reported inhibitor sensitivity of the glial transport system 15~28.5°, oligodendroglial [3H]GABA uptake was inhibited by diaminobutyric acid (DABA) but not by fl-alanine, and thus is similar to the neuronal GABA transport system. This has recently also been shown for astrocytes displaying a stellate morphology3656. Thus oligodendrocytes, in addition to astrocytes, may be involved in the modification of neuronal function by the uptake and inactivation of GABA. In the present investigation we have extended our previous study of GABA uptake by oligodendrocytes to other neuroactive amino acids, and in particular L-glutamic and L- and D-aspartic acids. We have studied the localization of 3H-labelled amino acids, using light microscopic autoradiography, following their uptake by dissociated CNS cultures prepared from neonatal mouse brain. The cell populations present in the cultures were defined by immunocytochemical localization of specific antigenic markers. Our results confirm the previous study that oligodendrocytes in culture actively accumulate [3H]GABA, and show that, whereas [3H]L-glutamic and [3H]L-aspartic acids only lightly labelled oligodendrocytes, [3H]D-aspartic acid, a non-metabolized analogue of L-glutamic acid 10produced a more pronounced labelling of oligodendrocytes. MATERIALS AND METHODS Cell cultures Cells were dissociated from whole brains of Swiss albino mice less than 12 h after birth, according to previously described procedures 5.44, and suspended in Dulbecco's Modified Eagle's Medium (Seromed), containing 10% fetal calf serum (Nabi), 30 mM D(+)-glucose, 7.4 mM L-glutamine and 100 units/ml

penicillin. The suspended cells were plated at 2.5 × 105 cells/cm2 (2.4 × 106 cells/dish), in 2 ml of medium, into 35 mm plastic dishes (Falcon Multiwell), each containing one 18 × 18 mm glass coverslip. The culture dishes were maintained at 37 °C, in a 5% CO2, 80% humid atmosphere and were fed by a complete change of medium initially after 4 days and every 3 days thereafter. 3H-A mino acid uptake Uptake experiments were performed as previously described 44. Cultures were rinsed once in a Tris-buffered Krebs medium (TM; pH 7.4) at 37 °C, and then preincubated for 3 min. Incubation at 37 °C was continued, after addition of 10 pl 3H-labelled amino acid solution, for 1-30 min. Final concentrations of the amino acids used were as follows: (a) [3H]GABA, 6 × 10-8 M (30.8 Ci/mmol); (b) [3H]L-glutamic acid, 5 × 10-8 M (44.1 Ci/mmol); (c) [3H]L-aspartic acid, 1.4 x 10-7 M (14.3 Ci/mmol); (d) [3H]D-aspartic acid, 1.4 x 10-7 M (14.0 Ci/mmol); (e) [3H]glycine, 5 x 10-8 M (44.3 Ci/mmol); (f) [3H]fl-alanine, 5 x 10-8 M (40 Ci/mmol); (g) [3H]taurine, 1.1 x 10-7 M (17.6 Ci/mmol). All 3H-labelled amino acids were obtained from New England Nuclear. The incubation was terminated by aspiration of the radiolabelled solution and 5 washes (2 ml, 2 min each) in Na ÷ free TM (4 °C), containing 1 mM unlabelled amino acid to minimize both specific and non-specific binding of the amino acids to membrane receptors. For measurement of the intracellular radioactivity 0.5 ml of 1 N NaOH was added to produce cell lysis, and after 2 h aliquots were added to 10 ml of Aquasure (NEN) for liquid scintillation counting. For experiments carried out in sodium-free medium, 118 mM lithium chloride was substituted for sodium chloride. Fixation and autoradiography The cultures incubated with 3H-amino acids were fixed in 2% glutaraldehyde (Fluka, EM grade) in isotonic phosphate-buffered saline (PBS, pH 7.4) for 20 rain, rinsed 4 times in PBS and air-dried. The coverslips were covered with Ilford K2 emulsion, exposed and developed as described previously44. The degree of retention of the 3H-labelled amino acids using this fixation procedure was similar for all amino acids tested.

255

Immunocytochemistry

Microscopy

Cultures were stained using antisera against the following markers: (a) galactocerebroside (GC)4,42; (b) myelin basic protein (MBp)4; (c) glial fibrillary acidic protein (GFAp)2; and (d) neuron-specific enolase (NSE) 39. The rabbit anti-GC serum was produced by priming rabbits i.m. with 1 mg bovine GC (Sigma), mixed with 1 mg methylated bovine serum albumin (BSA, Sigma), in complete Freund's adjuvant and boosting them 28 days later with 0.5 mg GC mixed with 0.5 mg BSA in isotonic aluminium phosphate solution. Animals were then bled 10 days after the booster injection. Obtained antisera were tested by ELISA and absorption of the antibodies with specific lipids and were found to show no cross-reactivity against sulfatide or glucocerebroside. The anti-MBP serum was produced as described previously60. The anti-GFAP and anti-fibronectin antisera were purchased from Dakopatts and Cappel Labs., respectively, and the anti-NSE serum was a gift from Dr. P. Marangos. For GC, fibronectin and GFAP localization the indirect immunofluorescence method was used. For the cell membrane markers, GC and fibronectin, living cultures were incubated sequentially with primary antiserum (30 min; diluted in Hanks balanced salt solution, HBSS) and fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit, or rabbit anti-goat, IgG (Cappel labs; 30 min; diluted in HBSS), and then fixed with 3.7% formaldehyde in PBS for 20 min. For the intracellular marker GFAP cultures were first fixed in 3.7% formaldehyde in PBS (15 rain) followed by 3.7% formaldehyde in PBS containing 0.2% Triton X-100 (5 rain), and then incubated sequentially with primary antiserum and FITC-labelled goat anti-rabbit IgG (30 min; diluted in PBS) as previously. MBP and NSE localization were investigated using the peroxidase-antiperoxidase method as described previously44, except that 4-chloro-1naphthol (0.06%) was used as chromagen in preference to diaminobenzidine. All incubation steps were separated by washing for 1 h with PBS or Tris-buffered saline. Control stainings were made with appropriately diluted normal rabbit sera. Coverslips were mounted in glycerol:PBS (9:1, v/v), containing 2.5% (w/v) 1,4-diazobicyclooctane (Aldrich) to retard FITC fading53.

Slides were examined with a Leitz Orthoplan microscope equipped with an epi-illumination system for visualizing silver grains, and fluorescence (filter block I2) and Nomarski interference optics for observing immunocytochemical staining. Positively stained cells and cells that had accumulated 3H-labelled amino acids were counted in 20 randomly chosen fields, or over the whole surface of the coverslip when the frequency was low, using a 100 mm 2 lattice mounted in the ocular of the microscope. Photographs were taken with Ilford HP5 400 ASA black and white films. RESULTS

Immunocytochemistry Dissociated mixed cell cultures prepared from neonatal mouse brain displayed a multilayered structure similar to that described previously for cultures prepared from neonatal 5 and fetal mouse brain 44. From 7 days in vitro (DIV) a confluent layer of large flattened, GFAP-positive, protoplasmic astrocytes (Fig. la, b) formed the bottom layer of the culture. The density of this astroglial cell layer made it impossible to estimate the number of GFAP-positive cells in these cultures. Only very rarely were GFAP-positive fibrous astrocytes found growing on top of this layer of protoplasmic astrocytes, and only at later stages in culture (21 DIV). Only small discrete patches of fibronectin-positive fibroblasts (not shown) were identified in the cultures. Small numbers of ependymal cells were identified by their beating cilia and could still be identified after tissue fixation. Growing evenly distributed on top of the astroglial cell layer were a large number of small cell bodied (12-15 /,m), multiprocessed cells, that appeared dark in phase contrast. Approximately 50% (Table I) of these phase contrast dark cells were identified as oligodendrocytes by their characteristic intense surface labelling of cell body and processes with antigalactocerebroside serum (Fig. lc, d). A small number of oligodendrocytes growing on the surface of the astroglial layer expressed the myelin-specific marker, myelin basic protein, and represented 2% of the phase dark cells at 10 DIV, increasing to 6% at 21 DIV (Table I). MBP-positive cells had large irregu-

256

~,,,~,~,~!i~iii~' ~i,i;ii~i~i~iiiii~,,,~i~ ~~ ~,,~~ i ~ i i ,

~! ii

i~ i~

g

•

~

ii~

i~ • ~

i'*~i~

~

~,~ ~;~

~iilX~i~ .....

!ii~ili!

~'~

257 lar shaped cell bodies and had extended large areas of flat membranous processes giving the appearance of abortive myelin (Fig. le, f). MBP-positive cells have been shown previously to be also GC-positive in double immunofluorescence studies of similar cultures 4. Although the majority of neurons die shortly after plating in cultures produced using the methods described in this study, a small number of surviving neurons were identified by their intracellular staining with an anti-neuron specific enolase serum (Fig. lg, h), representing only 2% (Table 1) of the phase dark cells on the surface of the cultures both at 10 and 2l DIV. These neurons were usually relatively undifferentiated single isolated cells (Fig. lg) but were very occasionally observed in clumps (Fig. lh). The remaining small phase dark cells growing on the astroglial layer (approximately 50%) did not express any of the markers employed in this study and have been suggested in previous studies4S, 46 to be immature oligodendrocytes which differentiate into GC-positive cells.

[3H]GABA autoradiography Already after short incubations of 10 D I V cultures with [3H]GABA (1-5 min), virtually all the small cell bodied cells, growing on the surface of the astroglial layer, had become heavily labelled over the cell body and lightly over some processes (Fig. 2a). After 20 min incubation the distribution of the labelling was more uniform with all the labelled cells exhibiting lightly or heavily labelled processes (Fig. 2b). These G A B A accumulating cells represented 81% of the small phase contrast dark cells after 20 min incubation (Table I) and this value did not change significantly between 5 and 30 min incubation time. In older cultures (21 DIV), the accumulation of [3H]G A B A by oligodendrocytes was even more pronounced, cell bodies becoming heavily labelled after incubation times as short as 1 min, and after 5 rain all

labelled cells also had labelled processes (Fig. 2c). After 20 min cell processes became heavily labelled (Fig. 2d), often forming a network on the surface of the culture. In these older cultures the G A B A accumulating cells on the surface of the astroglial layer represented 99% of all the phase contrast dark cells (Table I). Whereas only approximately 50% of these small cell bodied cells expressed the myelin lipid GC on their cell surface (Table I), these cells represented a homogenous population with regard to their ability to accumulate [3H]GABA, showing that both immature GC-negative precursor cells and GC-positive oligodendrocytes share this ability. As would be expected in young cultures (8-10 DIV), a small number of cells with neuron-like morphology became heavily labelled over the cell body and several long branched processes (Fig. 2e) after short incubation times (1-5 min). However, after long incubation times, it became difficult to distinguish these labelled neurons from heavily labelled oligodendrocytes (compare Fig. 2d and e), and therefore no attempt has been made to quantify them. In cultures of all ages there was a uniform light labelling of the astroglial cell layer with [3H]GABA which did not increase appreciably after 5 min and was never as dense as the oligodendroglial labelling. In 21 D I V cultures a small number of cells growing on the surface of the astroglial cell layer resembling fibrous astrocytes 13, characterized by a large irregular shaped cell body and broad processes, became heavily labelled (Fig. 2f), to the same extent as oligodendrocytes. No labelled astroglial cells with a stellate morphology36 were observed in these cultures. Accumulation of [3H]GABA, either in the absence of sodium, or in the presence of 1 mM unlabelled G A B A , was reduced by 98% and 99%, respectively, autoradiograms revealing a complete absence of labelling of all cell types, demonstrating the absence of any significant amount of non-specific binding under the experimental conditions employed.

~-. Fig. 1. Immunocytochemical staining of primary dissociated neonatal mouse CNS cell cultures, a and b: anti-GFAP immunofluorescence of a 10 DIV culture and corresponding phase contrast picture. The cells on the bottom carpet layer of the culture are positively stained whilst the small phase contrast dark cells remain unlabelled, c and d: anti-GC immunofluorescence of a 10 DIV culture and corresponding phase contrast picture, showing the characteristic surface labelling of the cell body and processes of oligodendrocytes, e and f: anti-MBP PAP staining of a large oligodendrocyte and corresponding phase contrast picture, 10 DIV. Note the extensive areas of stained flat membranous processes, g and h: anti-NSE PAP staining of isolated (g) and clustered (h) neurons in 10 DIV cultures. Nomarski interference optics. Bar represents 30 ~m.

258 TABLE I

Quantitative correlation between immunohistochemistry and [3H]GABA and [3H]D-aspartic acid accumulation by oligodendrocytes Values are given as mean number of cells _+ S.E.M. per cm2 from 4 independent experiments at each age (n = 12). Values in parentheses represent the percentage of phase contrast dark cells that were positively stained or had accumulated 3H-amino acid.

Age of culture Phasecontrast (days) dark cells'*

GC-positive oligodendrocytes

MBP-positive oligodendrocytes

NSE-positive neurons

[3H]GABA [.~H]o-Asparticacid accumulating cells accumulatingcells

10

18550 _+ 1060

21

9980 _+890

8740 + 400 (47.1 _+2.1%) 4440 + 330 (44.5 _+3.3%)

440 _+80 (2.4 + 0.4%) 560 _+60 (5.5 _+0.6%)

350 _+20 (1.9 _+0.1%) 220 _+30 (2.2 _+0.3%)

15070 ___350 (81.2 ___1.9%) 9890 _+750 (99.1 _+7.5%)

13840 _+540 (74.6 _+3.2%) 8150 _+650 (81.7 + 6.4%)

* Phase contrast dark cells are those cells growing on top of the astroglial carpet cell layer that appear dark under phase contrast optics (see Fig. lb, d). [3H]GABA and [3H]aspartic acid accumulating cells were counted in cultures incubated for 20 min with the amino acid. Cells were counted as described in the Materials and Methods section.

[3H]L-glutamic and aspartic acid uptake In 10 D I V cultures there was a m o d e r a t e labelling of all astroglial cells in the carpet layer of the culture with [3H]L-glutamic acid, which did not increase in intensity with incubation times longer than 3 min (Fig. 3a, b). A few small phase d a r k cells were lightly labelled over the cell b o d y but the m a j o r i t y were completely unlabelled (Fig. 3a, b). In 21 D I V cultures the distribution of 3H-label was the same as at 10 DIV. Incubation of the cultures with [3H]k-aspartic acid produced a similar labelling pattern to that seen with L-glutamic acid. H o w e v e r , a population of small cell bodied oligodendroglial-like cells became m o r e heavily labelled than with L-glutamic acid (Fig, 3c, d). These cells were labelled p r e d o m i n a n t l y over the cell body, although a few cells had labelled processes in 21 D I V cultures. No labelled cells with a clear neuronal m o r p h o l o g y were seen. [3H]D-aspartic acid uptake In order to d e t e r m i n e whether this a p p a r e n t low level of uptake of the L-amino acids by oligodendrocytes was the result of their rapid metabolism and release of the labelled metabolites, we incubated the cultures with [3H]D-aspartic acid, a non-metabolized analogue of L-glutamic acid which shares the same

high affinity transport system 1. Oligodendrocytes in 10 D1V cultures became heavily labelled over the cell body and sometimes also over the cell processes, after incubation times as short as 1 - 3 min (Fig. 4a), to about the same extent as with G A B A . The labelling intensity increased with increasing incubation times up to 30 rain (Fig. 4b). As seen with G A B A the accumulation of D-aspartic acid by oligodendrocytes in 21 D I V cultures was even m o r e p r o n o u n c e d , m a n y cells becoming heavily labelled over cell body and processes after 1 min incubation (Fig. 4c). A f t e r longer incubation times ( 5 - 1 0 min), t h e s e cells were even m o r e intensely labelled (Fig. 4d) and sometimes seen with networks of interlinking labelled processes. The cells that had accumulated [3H]D-aspartic acid represented 75 and 82% of the small cell b o d i e d oligodendroglial-like cells in the cultures at 10 and 21 D I V , respectively (Table I), and although the n u m b e r was smaller than the n u m b e r of G A B A - l a b e l l e d cells, this still represented the m a j o r i t y of this cell type in the cultures. Astroglial cells in the carpet layer of the cultures became more heavily labelled with [3H]D-aspartic acid than with [ 3 H ] G A B A ( c o m p a r e Figs. 2b, d and 4b, d), and after longer incubation times in 21 D I V cultures the difference in labelling intensity between oligodendrocytes and astrocytes was not very great in

Fig. 2. Autoradiographs of [3H]GABA uptake by oligodendrocytes, neurons and astrocytes in 10 and 21 DIV cultures, a and b: 10 DIV cultures, labelling of oligodendrocytes, both perikarya and processes, after 5 rain (a) and 20 rain (b) incubations, c and d: 21 DIV cultures, oligodendrocyte labelling after 5 min (c) and 20 mifi (d) incubations. Note the intensity of the labelling occurring over oligodendrocytes compared to the astroglial cell layer, e: heavily labelled neuron in a 10 DIV culture after 5 min incubation, showing long branched, labelled, processes extending out of the visual field, f: heavy labelling of large irregular shaped astrocytes growing on the surface of the astroglial carpet cell layer in 21 DIV cultures, 20 min incubation. Bar represents 30 ~m.

259

260

Fig. 3. Autoradiographs of [3H]L-glutamic and aspartic acid uptake, a and b: [3H]L-glutamic acid, 5 min incubation, 21 DIV culture, bright field and phase contrast views, respectively. Oligodendrocytes are only very lightly labelled over the cell bodies, c and d: [3H]L-aspartic acid, 5 min incubation, 10 DIV culture, bright field and phase contrast views, respectively. Oligodendrocytes are lightly labelled over the cell bodies. Bar represents 30 j~m. some areas of the cultures (Fig. 4d). In 21 D I V cultures irregularly shaped astrocytes growing o n the surface of the carpet astroglial layer b e c a m e heavily labelled (Fig. 4e, f) after short i n c u b a t i o n times ( 1 - 5 min) a n d to a greater e x t e n t t h a n with [ 3 H ] G A B A .

A c c u m u l a t i o n of [3H]D-aspartic acid by the cultures was r e d u c e d by 84% in the a b s e n c e of s o d i u m , residual u p t a k e occurring into astroglial cells. In the presence of 1 m M u n l a b e l l e d D-aspartic acid u p t a k e was r e d u c e d by 9 6 % , a u t o r a d i o g r a m s revealing the ab-

Fig. 4. Autoradiographs of [3H]o-aspartic acid uptake by oligodendrocytes and astrocytes in 10 and 21 DIV cultures, a and b: heavy labelling of oligodendrocytes, both perikarya and processes, after 3 rain (a) and 30 min (b) incubations in 10 DIV cultures, c and d: heavy labelling of oligodendrocytes after 1 rain (c) and 5 rain (d) incubations in 21 DIV cultures. Oligodendrocyte processes are already heavily labelled after incubation as short as 1 min (c). Note the relatively intense labelling of the astroglial cell layer (d) on top of which the oligodendrocytes are growing, e and f: heavy labelling of large irregular shaped astrocytes growing on the surface of 21 DIV cultures after 3 min (e) and 5 rain (f) incubations. Both cells with numerous fine radial processes (e, f) and few broad processes (f) are heavily labelled. Bar represents 30/~m.

261

262

Fig. 5. Autoradiograph of [3H]taurine uptake by a 21 DIV culture, 10 min incubation, bright field (a) and phase contrast (b) views. Some of the oligodendrocytes are lightly labelled over the cell body. Bar represents 30/~m.

sence of any significant amount of non-specific binding of [3H]D-aspartic acid.

[-~H]fl-alanine, glycine and taurine uptake Net uptake of [3H]fl-alanine or [3H]glycine by oligodendrocytes was not observed under any of the conditions tested. At all culture ages and after incubation times up to 30 min these two amino acids labelled only astroglial cells in the carpet layer of the cultures. A small number of small cell bodied cells growing on the surface of the cultures became lightly labelled over the cell body with [3H]taurine (Fig. 5a, b), after longer incubation times (10-30 min) and in 21 DIV cultures. But these cells only represented a very small proportion of the oligodendroglial-like cells in the culture. DISCUSSION Using the cell-type specific markers described in this study, we found the presence of only a very small number of neurons in cultures prepared from neonatal mouse brain, in agreement with previous studies of similar cultures systems3, 53. Thus we were able, using a culture system consisting of differentiating oligodendrocytes and oligodendrocyte precursor. cells growing on a layer of protoplasmic astrocytes, to investigate the possible role of oligodendrocytes in neuroactive amino acid metabolism in the virtual ab-

sence of contaminating neurons. The present study confirms our previous results, obtained with primary cultures prepared from fetal mouse brain 44, that oligodendrocytes have a rapid sodium-dependent uptake system for G A B A . The ability of cultured oligodendrocytes to accumulate G A B A appeared greater in cultures prepared from neonatal brain compared to fetal brain, possibly due to a greater state of differentiation of the cells when initially isolated. The oligodendrocytes became heavily labelled over cell body and processes after short incubation times (3-5 min), similar to those routinely used for estimating the uptake of G A B A by neuronsS,26,40,54,58. Therefore caution should be exercised when assuming that uptake of G A B A occurring at low concentrations and after short incubation times is an indication of exclusively neuronal G A B A uptake. It is apparent from our study that two populations of astrocytes present in the cultures differed in their ability to accumulate G A B A and D-aspartic acid. The large astrocytes growing on top of the astroglial carpet layer, although few in number, accumulated both amino acids to a greater extent than those in the carpet layer. In agreement with this, recent studies have shown that stellate shaped rat cerebellar astrocytes in culture became heavily labelled with G A B A , whereas epithelioid polygonal astrocytes accumulated only low levels of this amino acid36. Thus it appears that there is a possible heterogeneity amongst

263 astrocyte subpopulations with respect to their ability to transport neurotransmitter amino acids. The structural characteristics of the GABA transport system described for cerebellar stellate astrocytes are similar to both those described as neuronal 36, and those described by us for oligodendrocytes44. This suggests that there may be little difference in the structural characteristics of the GABA transport system between these 3 CNS cell types, contrary to previous suggestions 2s. Contrary to suggestions that the uptake of [3H]fl-alanine may be used as a marker for the GABA transport system of all glial cells, including oligodendrocytesl2.40, we found no evidence that oligodendrocytes in culture accumulate this amino acid. Although astrocytes in our cultures became labelled with fl-alanine, it has been suggested that this is due to a passive mechanism distinct from the G A B A transport system 7. In contrast to CNS oligodendrocytes, Schwann cells in culture have a high affinity uptake system for GABA that is specifically inhibited .by fl-alanine rather than inhibitors of neuronal GABA re-uptake 15. It seems, therefore, that although this difference in inhibitor specificity between neuronal and glial GABA uptake applies to peripheral glial cells, such a difference is not so clear between CNS neurons and glia36. Our finding that not only the GC-positive and MBP-negative oligodendrocytes, but also the GCnegative undifferentiated oligodendrocyte precursor cells rapidly accumulate GABA and D-aspartic acid from the extracellular medium, suggests that this may be a function expressed relatively early in the differentiation of oligodendrocytes, and not related to membrane changes occurring after interaction with neurons. The uptake of these amino acids by oligodendrocytes is clearly not dependent on the presence of neurons in the cultures. During early CNS development, glia, both astrocytes and oligodendrocytes, may play an important role in stabilizing the extracellular environment for neuronal migration and differentiation. In agreement with this it has been suggested that during ontogenesis GABA may play several important functional roles before the typical synaptic processing of information is established 19,57. Whether GABA has any direct effects on the differentiation of oligodendrocytes is not known and is the subject of further study. We cannot totally exclude the possibility that some

of the small cell bodied cells growing on the astroglial layer were 'O-2A' bipotential progenitor glial cells, as described by Raft and coworkers a3, which develop into oligodendrocytes or type-2 fibrous astrocytes, depending on the composition of the culture medium. However, we did not observe either large numbers of type-2 fibrous astrocytes, or that any of these small cells expressed GFAP, as would be expected in cultures grown in serum-containing medium. The small phase contrast dark GC-negative cells may already be committed to differentiating into GC-positive oligodendrocytes. The inactivation of synaptically released L-glutamic and aspartic acids, and maintenance of their low extracellular concentrations, is proposed to occur mainly via uptake into surrounding glial cells rather than neurons 9,21. Many studies using tissue cultures have found that uptake of the excitatory amino acids into cells other than astrocytes is very low or negligible9,16,35,55. Our results showing only low levels of oligodendroglial labelling, but labelling of astrocytes, with L-glutamic and aspartic acids seemed to agree with these previous results. However, glial metabolism of [3H]L-glutamic and aspartic acids has been shown to result in [3H]water production and thus loss of the label from the cells 16. Therefore, the cultures were incubated with the metabolically stable [3H]D-aspartic acid 10, which is taken up by the same uptake mechanism 1.~4with similar kinetic parameters H. We found that D-aspartic acid was rapidly accumulated by oligodendrocytes via a sodium-dependent mechanism, resulting in heavy labelling over cell body and processes after only 3 min incubation time, similar in intensity to the labelling seen with GABA. Thus, knowing from previous studies that L-glutamic and L- and D-aspartic acids are accumulated by the same transport mechanism, and the only difference is the metabolic stability of D-aspartic acid, we conclude that oligodendrocytes do indeed have a transport system for the putative excitatory amino acid transmitters and also possess the necessary enzymatic machinery for their rapid metabolism. This finding raises the important question of which enzymes are responsible for glutamate metabolism in oligodendrocytes. The most important glutamate metabolizing enzyme in astrocytes appears to be glutamine synthetase, which is generally considered to be an astrocyte marker enzyme 49, due to its exclusive

264 astrocytic localization 41. Oligodendrocytes, both in CNS slices 41 and in primary cultureslL have been shown not to contain detectable amounts of glutamine synthetase. Therefore, the most likely routes of glutamate metabolism in oligodendrocytes are conversion to a-ketoglutarate, either as a transamination by glutamate-oxaloacetate aminotransferase, or as an oxidative deamination catalyzed by glutamate dehydrogenase. Both of these enzymes have been shown to be present in cultured astrocytes 2~.-s9, but there is no information available concerning their presence and activity in oligodendrocytes. Glycine has been suggested as an inhibitory neurotransmitter in several CNS regions in addition to its established role in the spinal cord 37. In agreement with several studies on the uptake of [3H]glycine by CNS cultures 24,34,54, we found that astrocytes became heavily labelled with glycine. Although it is possible that oligodendrocytes accumulate glycine and rapidly metabolize it so that the metabolites are not retained, our results suggest that they do not accumulate glycine to any great extent above that required for protein synthesis, which is supplied via a low affinity transport system, shared by other small amino acids, that is present in all cells. Several studies have demonstrated that glial cell lines or glial primary cultures exhibit sodium-dependent high affinity uptake of the amino acid taurine via a mechanism distinct from that for G A B A 6.17.48~51. Very little information is available concerning taurine metabolism in specific cell types in the brain 48. Our results agree with the previous studies showing uptake of taurine by astrocytes in primary culture 6,48, and show that, although a small number of oligodendrocytes became lightly labelled after a 10 rain incubation, taurine uptake does not appear to be a dynamic property of oligodendrocytes in

REFERENCES 1 Balcar, g.J. and Johnston, G.A.R., The structural specificity of high affinity uptake of L-glutamate and L-aspartate by rat brain slices, J. Neurochem., 19 (1972) 2657-2666. 2 Bignami, A. and Dahl, D., Specificity of the glial fibrillary acidic protein for astroglia, J. Histochem. Cytochem., 25 (1977) 466-469. 3 Bologa, L., Joubert, R., Bisconte, J.C., Margules, S., Deugnier, M.A., Derbin, C. and Herschkowitz, N., Development of immunologically identified brain cells in culture:

culture. The uptake of certain neuroactive amino acids is thus a property shared by astrocytes and oligodendrocytes, but whereas astrocytes play an important role at the nerve terminal regions by removing neuronally released transmitters from around the synapse, oligodendrocytes probably act in a protective fashion by reducing the concentration of the amino acids around the neuronal cell body, in the case of perineuronal satellite oligodendrocytes, and also around axons, in the case of interfasicular oligodendrocytes. In view of the need to maintain the extracellular concentration of such neuroactive substances, and in particular G A B A and glutamate, at low levels 9,33, glial cells are envisaged to play an important role in brain homeostasis by stabilizing and protecting the extracellular environment of neurons20.21 ~33.51. In conclusion, oligodendrocytes should now not only be viewed with regard to their ability to produce myelin sheaths, their recognized role in the CNS, but also with regard to their ability to actively accumulate 44, and to respond to 32, certain neuroactive amino acids. ACKNOWLEDGEMENTS This work was supported by Grant 3.493.83 from the Swiss National Research Foundation, a grant from the Swiss Multiple Sclerosis Society, and a grant from the Swiss Foundation of Encouragement of Research in Mental Retardation. The authors are grateful to B. Angst (Berne) for the preparation of the anti-MBP serum and to Dr. P.J. Marangos (Bethesda) for the gift of rabbit anti-NSE serum. We thank Mrs. C. Steffen and Mr. M. Stoller for technical assistance and Miss B. Moser for secretarial assistance.

quantitative aspects, Exp. Brain Res., 53 (1983) 163-167. 4 Bologa, L., Siegrist, H.P., Z'Graggen, A., Hofmann, K., Wiesmann, U., Dahl, D. and Herschkowitz, N., Expression of antigenic markers during the development of oligodendrocytes in mouse brain cell cultures, Brain Research, 210 (1981) 217-229. 5 Bologa, L., Z'Graggen, A., Rossi, E. and Herschkowitz, N., Differentiation and proliferation: two possible mechanisms for the regeneration of oligodendrocytes in culture, J. Neurol. Sci., 57 (1982) 419-434. 6 Borg, J., Ramaharobandro, N., Mark, J. and Mandel, P.,

265 Changes in the uptake of GABA and taurine during neuronal and glial maturation, J. Neurochem., 34 (1980) 1113-1122. 7 Cummins, C.J., Glover, R.A. and Sellinger, O.Z., Betaalanine uptake is not a marker for brain astroglia in culture, Brain Research, 239 (1982) 299-302. 8 Currie, D.N. and Dutton, G.R., [3H]GABA uptake as a marker for cell type in primary cultures of cerebellum and olfactory bulb, Brain Research, 199 (1980) 473-481. 9 Currie, D.N. and Kelly, J.S., Glial versus neuronal uptake of glutamate, J. Exp. Biol., 95 (1981) 181-193. 10 Davies, L.P. and Johnston, G.A.R., Uptake and release of D- and baspartate by rat brain slices, J. Neurochem., 26 (1976) 1007-1014. 11 Drejer, J., Larsson, O.M. and Schousboe, A., Characterization of uptake and release processes for D- and L-aspartare in primary cultures of astrocytes and cerebellar granule cells, Neurochem. Res., 8 (1983) 231-243. 12 East, J.M., Dutton, G.R. and Currie, D.N., Transport of GABA, fl-alanine and glutamate into perikarya of postnatal rat cerebellum, J. Neurochem., 34 (1980) 523-530. 13 Fedoroff, S., White, R., Neal, J., Subrahmanyan, L. and Kalnins, V.I., Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin and GFP-containing intermediate filaments, Dev. Brain Res., 7 (1983) 303-315. 14 Fonnum, F., Glutamate: a neurotransmitter in mammalian brain, J. Neurochem., 42 (1984) 1-11. 15 Gavrilovic, J., Raff, M.C. and Cohen, J., GABA uptake by purified Schwann cells in culture, Brain Research, 303 (1984) 183-185. 16 Gordon, R.D. and Balazs, R., Characterization of separated cell types from the developing rat cerebellum: transport of glutamate and aspartate by preparations enriched in Purkinje cells, granule neurons and astrocytes, J. Neuro-, chem., 40 (1983) 1090-1099. 17 Haber, B. and Hutchison, M.T., Uptake of neurotransmitters and precursors by clonal cell lines of neural origin, Adv. Exp. Med. Biol., 69 (1976) 179-198. 18 Hallermayer, K. and Hamprecht, B., Cellular heterogeneity in primary cultures of brain cells revealed by immunocytochemical localisation of glutamine synthetase, Brain Research, 295 (1984) 1-11. 19 Hansen, G.H., Meier, E. and Schousboe, A., GABA influences the ultrastructural composition of cerebellar granule cells during development in culture, Int. J. Dev. Neurosci., 2 (1984) 247-257. 20 Henn, F.A., Neurotransmission and glial cells: a functional relationship?, J. Neurosci. Res., 2 (1976) 271-282. 21 Hertz, L., Functional interactions between neurons and astrocytes. I. Turnover and metabolism of putative amino acid transmitters, Prog. Neurobiol., 13 (1979) 277-323. 22 Hertz, L., Wu, P.H. and Schousboe, A., Evidence of net uptake of GABA into primary cultures - - its sodium dependence and potassium independence, Neurochem. Res., 3 (1978) 313-323. 23 H6kfelt, T. and Ljungdahl, A., Application of cytochemical techniques to the study of suspected transmitter substances in the nervous system, Adv. Biochem. Psychopharmacol., 6 (1972) 1-36. 24 H6sli, E. and H6sli, L., Autoradiographic studies on the uptake of [3H]noradrenaline and [3H]GABA in cultured rat cerebellum, Exp. Brain Res., 26 (1976) 319-324. 25 H6sli, E. and H6sli, L., Uptake of L-glutamate and L-aspar-

tate in neurons and glial cells of cultured human and rat spinal cord, Experientia, 32 (1976) 219-222. 26 H6sli, L. and HOsli, E., Action and uptake of neurotransmitters in CNS tissue culture, Rev. Physiol. Biochem. Pharmacol., 81 (1978) 135-188. 27 Iversen, L.L., Role of transmitter uptake systems in synaptic neurotransmission, Brit. J. Pharmacol., 41 (1971) 571-591. 28 Iversen, L.L. and Kelly, J.S., Uptake and metabolism of GABA by neurons and glial cells, Biochem. Pharmacol., 24 (1975) 933-938. 29 Iversen, L,L. and Neal, M.J., The uptake of [3H]GABA by slices of rat cerebral cortex, J. Neurochem., 15 (1968) 1141-1149. 30 Iversen, L.L. and Schon, F.E., The use of autoradiographic techniques for the identification and mapping of transmitter specific neurons in CNS. In A. Mandell (Ed.), New Concepts in Neurotransmitter Regulation, Plenum Press, New York, 1973, pp. 153-193. 31 Kennedy, P.G.E., Neural cell markers and their applications to neurology, J. Neuroimmunol., 2 (1982) 35-53. 32 Kettenmann, H., Gilbert, P. and Schachner, M., Depolarisation of cultured oligodendrocytes by glutamate and GABA, Neurosci. Lett., 47 (1984) 271-276. 33 Krnjevic, K., Some functional consequences of G A B A uptake by brain cells, Neurosci. Lett., 47 (1984) 283-287. 34 Lasher, R.S., The uptake of [3H]GABA and differentiation of stellate neurons in cultures of dissociated postnatal rat cerebellum, Brain Research, 69 (1974) 235-254. 35 Levi, G., Aloisi, F., Ciotti, M.T. and Gallo, V., Autoradiographic localization and depolarisation induced release of acidic amino acids in differentiating cerebellar granule cell cultures, Brain Research, 290 (1984) 77-86. 36 Levi, G., Wilkin, G.P., Ciotti, M.T. and Johnstone, S., Enrichment of differentiated, stellate astrocytes in cerebellar interneuron cultures as studied by GFAP immunofluorescence and autoradiographic uptake patterns with [3H]D-aspartate and pH]GABA, Dev. Brain Res., 10 (1983) 227-241. 37 McGeer, P.L., Eccles, J.C. and McGeer, E.G., Molecular Neurobiology of the Mammalian Brain, Plenum Press, New York, 1978, pp. 225-231. 38 McLennan, H., The autoradiographic localization of L-glutamate in rat brain tissue, Brain Research, 115 (1976) 139-144. 39 Marangos, P.J., Zomzely-Neurath, C., Luk, D.C.M. and York, C., Isolation and characterisation of the nervous system specific protein 14-3-2 from rat brains, J. Biol. Chem., 250 (1975) 1884-1891. 40 Messer, A., The maintenance and identification of mouse cerebellar granule cells in monolayer culture, Brain Research, 130 (1977) 1-12. 41 Norenberg, M.D. and Martinez-Hernandez, A., Fine structural localization of glutamine synthetase in astrocytes of rat brain, Brain Research, 161 (1979) 303-310. 42 Raff, M.C., Fields, K.L., Hakomori, S., Mirsky, R., Pruss, R.M. and Winter, J., Cell type specific markers for distinguishing and studying neurons and the major classes of glial cells in culture, Brain Research, 174 (1979) 283-308. 43 Raft, M.C., Miller, R.H. and Noble, M., A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium, Nature (London), 303 (1983) 390-396. 44 Reynolds, R. and Herschkowitz, N., Uptake of [3H]GABA

266

45

46

47

48

49

50

51

52

by oligodendrocytes in dissociated brain cell culture: a combined autoradiographic and immunocytochemical study, Brain Research, 322 (1984) 17-31. Sarlieve, L.L., Delaunoy, J.P., Dierich, A., Ebel, A., Fabre, M., Mandel, P., Rebel, G., Vincendon, G., Wintzerith, M. and Yusufi, A.N.K., Investigations on myelination in vitro. III. Ultrastructural, biochemical and immunohistochemical studies in cultures of dissociated brain cells from embryonic mice, J. Neurosci. Res., 6 (1981) 659-683. Schachner, M., Kim, S.K. and Zehnle, R., Developmental expression in central and peripheral nervous system of oligodendrocyte cell surface antigens (O-antigens) recognized by monoclonal antibodies, Dev. Biol., 83 (1981) 328-338. Schon, F. and Kelly, J.S., Autoradiographic localization of [3H]GABA and [3H]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. Schousboe, A., Glutamate, GABA and taurine in cultured, normal glia cells. In E. Schoffeniels, G. Franck. L. Hertz and D.B. Tower (Eds.), Dynamic Properties of Glial Cells, Pergamon Press, Oxford, 1978, pp. 173-182. Schousboe, A., Transport and metabolism of glutamate and GABA in neurons and glial cells, Int. Rev. Neurobiol., 22 (1981) 1-45. Schousboe, A., Hertz, L. and Svenneby, G., Uptake and metabolism of GABA in astrocytes cultured from dissociated mouse brain hemispheres, Neurochem. Res., 2 (1977) 217-229. Schrier, B.K. and Thompson, E.J., On the role of glial cells in the mammalian nervous system, J. Biol. Chem., 249 (1974) 1769-1780. Storm-Mathisen, J. and Iversen, L.L., Uptake of [3H]glutamic acid in excitatory nerve endings: light and electron microscopic observations in the hippocampal formation of

the rat, Neuroscience, 4 (1979) 1237-1253. 53 Walker, A.G., Chapman, J., Bruce, C.B. and Rumsby, M.G., Immunocytochemical characterisation of cell cultures grown from dissociated 1-2 day postnatal rat cerebral tissue, J. Neuroimmunol., 7 (1984) 1-20. 54 White, W.F., Snodgrass, S.R. and Dichter, M., Identification of GABA neurons in rat cortical cultures by GABA uptake autoradiography, Brain Research, 190 (1980) 139-152. 55 Wilkin, G.P., Garthwaite, J. and Balazs, R., Putative acidic amino acid transmitters in the cerebellum. II. Electronmicroscopic localization of transport sites, Brain Research, 244 (1982) 69-80. 56 Wilkin, G.P., Levi, G., Johnstone, S.R. and Riddle, P.N., Cerebellar astroglial cells in primary culture: expression of different morphological appearances and different ability to take up [3H]D-aspartate and [3H]GABA, Dev. Brain Res., 10 (1983) 265-277. 57 Wolff, J.R., Rickmann, M. and Chronwall, B.M., Axoglial synapses and GABA accumulating glial cells in the embryonic neocortex of the rat, Cell Tiss. Res., 201 (1979) 239-248. 58 Yu, A.C.H. and Hertz, L., Uptake of glutamate, GABA and glutamine into a predominantly GABA-ergic and a predominantly glutaminergic nerve cell population in culture, J. Neurosci. Res., 7 (1982) 23-35. 59 Yu, A.C., Schousboe, A. and Hertz, L., Metabolic fate of a4C-labelled glutamate in astrocytes in primary cultures, J. Neurochem., 39 (1982) 954-960. 60 Zurbriggen, A., Vandevelde, M., Steck, A. and Angst, B., Myelin-associated glycoprotein is produced before myelin basic protein in cultured oligodendrocytes, J. Neuroimmunol., 6 (1984) 41-49.