Neurochemical Research, Vol. 12, No. 10, 1987, pp. 885-890

High-Affinity Uptake of /-[3H]Aminobutyric Acid by Isolated Mouse Oligodendrocytes in Culture*

Richard Reynolds , 1'2 Christ ine Steffen, 1 and Norbert Herschkowi tz I

(Accepted October 28, 1986)

Oligodendrocytes were isolated from mixed glial cultures of neonatal mouse forebrain and further grown in serum-free hormone supplemented culture medium. Cell populations were identified by indirect immunofluorescence using a range of specific antibodies, revealing a predominantly immature population of oligodendrocytes, the majority expressing the myelin glycolipids galactocerebroside and sulfatide on their plasma membrane. Astroglial contamination was found to be minimal. Simultaneous autoradiography and immunofluo- rescence demonstrated the presence of a transport system for the major inhibitory neu- rotransmitter GABA in the oligodendrocytes. The transport system was found to be energy, sodium and temperature dependent. Kinetic analysis revealed a high affinity system, with a Km of 6.27 p~M and a Vmax of 0.714 nmol/min/mg protein, which is comparable to that found previously for CNS neurons and astrocytes.

KEY WORDS: Oligodendrocytes; cell culture; immunocytochemistry; ~/-[3H]aminobutyric acid.

INTRODUCTION

Of the known functions of neuroglial cells in the mammalian CNS perhaps the most well estab- lished are the formation of the myelin sheath around neuronal axons by oligodendrocytes and the trans- port of K + and amino acid neurotransmitters by astrocytes. Recent studies in tissue culture have re- vealed that, in addition to their important function in myelination, oligodendrocytes are directly de- polarised by GABA and glutamate (7) and exhibit transport processes for both these neuroactive amino acids (21, 22), a property also shared by their PNS counterpart the Schwann cell (6). Previous au- toradiographic studies from our laboratory using mixed cell cultures have demonstrated qualitatively

* Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon. 1 Department of Pediatrics, University of Berne, Inselspital, CH-

3010 Berne (Switzerland). 2 Correspondence and present address, Dr. Richard Reynolds,

Department of Biochemistry, Imperial College, London, SW7 2AZ.

885

that galactocerebroside (GalC) expressing and also less differentiated oligodendrocytes actively accu- mulate GABA and D-aspartate (22, 24). It is note- worthy that the ability to transport GABA has also recently been reported to be present in a non-dif- ferentiated glial progenitor cell (15). However, stud- ies using mixed cell cultures cannot give us infor- mation on whether this glial uptake is of sufficiently high affinity and capacity to contribute to the in- activation of these amino acid transmitters in vivo.

Techniques are now available for the produc- tion of dissociated cultures, either, pure or highly enriched in all the major CNS cell types (12, 17, 25, 27) and such purified cultures of neurons and astro- cytes have previously been used to study the char- acteristics of the GABA transport system (14, 28, 30). In some of the stuudies using astrocyte cultures it has been suggested that glial uptake is of suf- ficiently high affinity and capacity to contribute to the inactivation of synaptically released GABA (10, 28).

To obtain more information on the character-

0364-3190/87/1000-0885505.00/0 �9 1987 Plenum Publishing Corporation

886 Reynolds, Steffen, and Herschkowitz

is t ics of G A B A up take by o l igodendrocy tes we have

i so la ted these cells f rom mixed glial cu l tures pre- p a r e d f rom n e o n a t a l m o u s e b ra in and g row n them

in s e rum- f ree h o r m o n e s u p p l e m e n t e d m e d i um.

I d e n t i f i c a t i o n of the cell popu la t ions p r e se n t was a c h i e v e d us ing a range of specific an t ibod ies and

o l igodendrog l ia l G A B A uptake was conf i rmed by c o m b i n e d au to rad iog raphy and i m m u n o f l u o r e s - cence . The resul ts show that i so la ted o l igodendro-

cy te s posses s an ac t ive sod ium d e p e n d e n t high af- f in i ty up t ake sys t em for G A B A with a grea ter

c a p a c i t y t han p r ev ious ly found for cu l tu red astro-

cy tes .

EXPERIMENTAL PROCEDURE

Cell Culture. Mixed glial cell cultures were prepared from neonatal mouse forebrain as described previously (22), and grown in poly-L-lysine coated plastic flasks (Falcon, 75 cm2). These source cultures were grown in Dulbecco's Modified Eagles Me- dium (DMEM) containing 10% fetal calf serum (FCS) for 7 days and then for a further 6 days in DMEM containing 2.5% FCS, to enhance oligodendroglial differentiation (5). Oligodendrocytes were isolated from these source cultures essentially as described by the modified method of McCarthy and De Vellis (17, 25). Briefly source cultures were shaken at 150 rpm (37~ stroke diameter 2 cm) for 30 min to remove dividing astrocytes and dead cells, the medium changed (DMEM + 2.5% FCS), and then fur- ther shaken at 250 rpm for 17-18 hours. The isolated cells were pooled and passed through nylon sieves of 70 and 25 p~m pore size, centrifuged, resuspended in DMEM + 2.5% FCS, counted, and seeded on poly-L-lysine coated glass coverslips in 24-well culture plates (Falcon, 2.1 cm2/well) at a density of 105 cells per well. After 24 hours in culture the medium was changed to a serum-free medium, essentially as described previously (5), con- sisting of DMEM/Hams F12 (1:1) containing (final concentra- tions) 10 mM HEPES buffer, 12 mM NaHCO2, 30 mM D-glucose, 4.2 mM L-glutamine, 10 jxg/ml insulin, 50 ~g/ml transferrin, 100 txM putrescine, 30 nM sodium selenite, 20 nM progesterone and 15 nM triiodothyronine. Isolated oligodendrocytes were grown for a further two days in this medium until use. No attempt to culture the cells for longer periods was made because the aim of the experiments was to measure the capacity of oligodendrocytes for GABA uptake as observed in mixed cultures but in the ab- sence of the contaminating astrocytes.

Immunofluorescence. To identify cell types in culture in- direct immun0fluorescence was employed. The following anti- body preparations were used: mouse monoclonal 03 (dilution l:10, gift from M. Schachner, Heidelberg); rabbit anti-GalC (di- lution 1:10, prepared as described previously, 22); rabbit anti- myelin basic protein (MBP, diluted 1 : 100, prepared as described, 24); rabbit anti-2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP, dilution 1 : 100, gift from E. Carey, Sheffield); mouse mon- oclonal anti-GD3 (dilution 1:20, gift from R. Yu, New Haven); mouse monoclonal antibody A2B5 (dilution 1 : 100, gift from M. Noble, London); rabbit anfi-glial fibrillary acidic protein (GFAP, dilution 1 : 150, purchased from DAKO); rabbit anti-neuron spe- cific enolase (NSE, dilution 1:400, purchased from Polysci-

ences); and goat anti-fibronectin (dilution 1:50, purchased from Cappel Labs).

For surface staining (03, GalC, GD3, A2B5, and Fibronec- tin), live cells were incubated with the primary antibody diluted in DMEM/Hams F12 medium containing 5% normai goat serum, followed by TRITC conjugated goat anti rabbit/mouse lgG (Cap- pel Labs, diluted 1 : 150). The cells were fixed for 10 rain with 4% paraformaldehyde 0.05% glutaraldehyde in 0.1 M sodium phos- phate buffer (pH 7.2). For cytoplasmic staining (CNP, MBP, GFAP, and NSE), cells were first fixed for 10 rain in 4% para- formaldehyde in phosphate buffer as above, followed by 1 min in acetone (-20~ and incubated with primary and secondary antibodies diluted in culture medium containing 5% normal goat serum. Coverslips were mounted and viewed with a Leitz Or- thoplan microscope as described previously (22).

[3H]GABA Uptake and Autoradiography. To confirm the presence of the radiolabel in oligodendrocytes the method of si- multaneous immunofluorescence and autoradiography was em- ployed (13), exactly as described previously by us (22).

For the quantitative estimation of GABA uptake the cultures were washed once in DMEM/Hams F12 medium and then trans- ferred by gradual dilution to Hanks balanced salt solution con- taining 10 mM HEPES (pH 7.4, HBSS). After 10 rain preincu- bation in HBSS (37~ [3H]GABA was added at a final concentration of 10 -6 M (0.5 ~Ci/nmol) and the incubation con- tinued ibr 1-20 rain. The incubation was terminated by washing of the coverslips 3 times with HBSS at 4~ The coverslips were then transferred to scintillation vials and the cells solubilized in 0.5 N NaOH overnight. For the experiments on the sodium de- pendency of uptake, NaC1 was substituted by an equimolar con- centration of LiC1. For the determination of K,n and Vmax values the GABA concentration was varied from 0.1-40 p~M by addition of unlabeled GABA. Incubations for these determinations were for 5 min and the corresponding values for uptake at 4~ were subtracted from each value. Calculation of the Vm~x and Km val- ues was by a non-linear grouped least squares analysis (3) weighted for heteroscedastic data. Cell protein content was es- timated using a modified Lowry procedure (16) with crystalline BSA as standard.

R E S U L T S

Immunocy tochemis t ry . The c ompos i t i on of the cu l tures as def ined by i m m u n o f l u o r e s c e n c e wi th the

an t ibod ies desc r ibed a bove is g iven in Tab le I. Neu- rons and f ibroblas ts were on ly rare ly found in the cu l tures , as r epor ted p r ev ious ly (25). U s i ng G F A P , a m a r k e r which appears re la t ive ly ear ly in the dif- f e ren t i a t ion of a s t rocy te s (1), on ly a smal l p ropor- t ion (3.8%) of the cells expres sed an as t rocyt ic phe- no type , all of these be ing of the f ibrous p rocess bear ing type. The major i ty of the cells expres sed an o l igodendrogl ia l morpho logy which was conf i rmed us ing the marke r s 03 (Figure la ,b ) , a m o n o c l o n a l a n t i b o d y agains t the mye l in lipid sulfat ide (26), and GalC, the ma jo r m y e l i n glycol ipid and an ear ly cell surface m a r k e r specific for o l igodendrocy te s (20).

GABA Uptake by Oligodendrocytes

Table I. Immunocytochemical Characterization of Isolated Oligodendrocytes 3 Days After Isolation

Specific antibodies % Positive cells

03 (sulfatide) 62.4 _+ 2.0 Galc 54.1 _+ 3.8 CNP 10.6 _+ 1.3 MBP 1.2 _ 0.3 GD3 48.9 _+ 1.2 A2B5 10.6 -4- 3.1 GFAP 3.8 _+ 0.5 NSE <0.1 Fibronectin <0.1

Percentage of cells that were positively stained was determined by counting cells within 20 random fields or over the whole cov- erslip (100 mm2), Values are means -+ SEM of four separate experiments each with duplicate cultures.

887

Antibodies against these two glycolipids labeled 62% (03) and 54% (GalC) of the cells respectively (Table I). Those cells which did not express 03 and GalC exhibited a bipolar morphology reminiscent of the bipotential progenitor cells described by Raft et al. (19). In agreement with this some of these cells became labeled with antibodies against the gan-

Fig. 2. Simultaneous [3H]GABA autoradiography and 03 im- munofluorescence. 3 day isolated oligodendrocytes were labeled with 03 antibody and then incubated for 20 min with 1 p~M [3H]GABA. Two oligodendrocytes that have a bright surface staining with 03 are heavily labeled with [3H]GABA. Bar rep- resents 20 p~m.

Fig. 1. Immunofluorescent identification of 3 day isolated oli- godendrocytes with monoclonal antibodies against 03 antigen (a,b) and GD3 (c,d). Two highly branched oligodendrocytes are brightly surface labeled over the cell body and processes whereas several less differentiated remain unlabeled (a,b). Anti-GD3 an- tibodies have labeled two highly branched oligodendrocytes whilst another is unlabeled (c,d). An undifferentiated bipolar cell is also positive (arrow). Bar represents 30 p~m.

glioside GD3 (see Figure lc,d), which has been shown to be expressed by immature neuroectoder- mal cells that give rise to oligodendrocytes in culture (8). G D 3 immunoreactivity was also expressed on some of the more differentiated oligodendrocytes (Figure lc,d). Only relatively few cells expressed the myelin proteins CNP and MBP (11% and 1% respectively) emphasizing the immature nature of the oligodendroglial populations present in our cul- tures. It is clear from our results that CNP appears prior to MBP.

[ 3H]GABA Uptake. Combined immunofluores- cence and autoradiography revealed that virtually all of the 03-positive and GalC-positive oligoden- drocytes and A2B5-positive cells became heavily labeled after incubation with [3H]GABA (Figure 2a,b). Some of the more immature cells ( G D 3 - p o s -

itive) remained unlabeled. In agreement with our experiments using primary mixed glial cultures (23), the highly differentiated oligodendrocytes express- ing the myelin proteins CNP and MBP did not ac- cumulate [3H]GABA, suggesting that this capacity may not extend to fully mature cells. Uptake of 1 ~M [3H]GABA by the cultures remained linear for up to 20 min, and a time of 5 min was chosen to

888 Reynolds, Steffen, and Herschkowitz

Table II. Effect of Various Conditions on the Uptake of [3H]GABA by Oligodendrocytes in Culture

GABA uptake (pmol/mirdcult) % Control

Control 1.147 - 0.055 100 AOAA (10 txM) 1.271 -+ 0.122 111 Ouabain (0.2 mM) 0.185 _+ 0.020 16 4~ 0.019 -+ 0.005 2 Sodium free 0.003 _+ 0.001 0.3

Values are the means -4- SEM from 3 independent experiments each with triplicate cultures. Cultures were incubated for 5 min with [3H]GABA.

represent the initial uptake rate for kinetic analysis. Uptake was highly temperature and sodium-depen- dent and was inhibited by 84% in the presence of 0.2 mM ouabain (Table II). Preincubation with l0 ~M aminooxyacetic acid, a GABA transaminase in- hibitor, produced only a small non-significant in- crease in net uptake.

For kinetic analysis of [3H]GABA uptake the results from three independent experiments were combined, with 4 cultures for each GABA concen- tration in each experiment. The initial velocities of GABA uptake were analysed using the method of least squares described by Wilkinson (30), and mod- ified for grouped values of uptake (3). Fine adjust- ment of provisional estimates of Km and Vmax and the standard errors of the adjusted values were ob- tained by fitting the Michaelis-Menten function di- rectly in the hyperbolic form, as described by Wilk- inson (30). To test for the presence of more than one component of uptake, the statistical significance of deviations from the Michaelis-Menten equation was assessed using the ratio (F) of the deviation mean square to the residual mean square (see Cleaver et al., 3). Uptake was saturable and no de- viations from the predicted hyperbolic relationship for a single transport component were observed (Figure 3). Uptake was high affinity with a Km of 6.27 + 0.2 ~zM and a maximum velocity of 0.714 ___ 0.100 nmol/min/mg protein.

DISCUSSION

The immunocytochemical characterization of the cultures described in this study reveals the pres- ence of a predominantly immature population of oli- godendrocytes with very little contamination with

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~ 0.4

>

0.2

1 2 qs (pM)

I I [ I

; lo S( .uM) ~o 30

Fig. 3. Kinetics of [3H]GABA uptake by isolated oligodendro- cytes in culture. Cells were incubated for 5 min with different concentrations of [3H]GABA. Each point is the mean _+ SEM of 4 independent values, Km: 6.27 -+ 0.2 p.M, Vm~x: 0.714 -- 0.100 nmol/min/mg protein, from 3 independent experiments.

GFAP-positive astrocytes, which is in agreement with several other studies using similar culture methods (2, 18). As the object of this study was to analyse the properties of the GABA transport sys- tem we found previously to be present in oligoden- drocytes growing in mixed glial cultures (21, 22), no attempt was made to culture the isolated oligoden- drocytes for longer periods. Therefore, it is not pos- sible to say whether the isolated oligodendrocytes would become fully differentiated under the present culture conditions. The large difference in the expression of MBP between this study and that of Saneto and DeVellis (25) is probably due to the spe- cies difference and differences in the composition of the culture medium and length of culture.

Our results show conclusively that immature oligodendrocytes in culture possess an active so- dium-dependent high affinity transport system for the major inhibitory neurotransmitter GABA, con- firming our previous qualitative studies (21, 22). This is in agreement with another study finding that immature oligodendrocytes grown in serum-free medium have the ability to accumulate GABA (15) although no quantitative data was presented. Inter- estingly this has been shown to be a differentiated property of Schwann cells in culture (6). Whether or not the ability to accumulate GABA extends to mature oligodendrocytes in vitro and in vivo re-

GABA Uptake by Oligodendrocytes 889

quires further experiments, although a previous study suggests that it does (11).

Comparison of the data presented here with that for the uptake of GABA by astrocytes and neu- rons reveals that the affinity and capacity of the oligodendroglial system is comparable to that for CNS neurons, both in culture (14) and bulk isolated (4). Astroglial uptake of GABA, as demonstrated in culture, is of lower affinity and capacity (9, 28), al- though it is suggested that this does not truly rep- resent GABA uptake by differentiated astrocytes (24, 29). However, data for the uptake of GABA by bulk isolated astrocytes (4) is comparable with that for oligodendrocytes presented here, and for neu- rons (14). Thus, although the present study cannot tell us whether the ability of oligodendrocytes to accumulate GABA is of physiological importance, we have shown that the transport system is com- parable to that present in other neural cell types in terms of affinity and capacity.

ACKNOWLEDGMENT

This work was supported by grants from the Swiss National Science Foundation (3.493.83) and the Swiss Multiple Sclerosis Society. The authors wish to thank M. Schachner, E. Carey, R. Yu and M. Noble for gifts of antibodies.

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