High- and low-affinity GABA-receptors in cultured cerebellar granule cells regulate transmitter release by different mechanisms

Neurochem. Int. Vol. 19, No. 4, pp. 475~-82, 1991 Printed in Great Britain. All rights reserved

0197-0186/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc

HIGH- A N D L O W - A F F I N I T Y G A B A - R E C E P T O R S IN C U L T U R E D C E R E B E L L A R G R A N U L E CELLS R E G U L A T E T R A N S M I T T E R RELEASE BY D I F F E R E N T MECHANISMS Bo BELHAGE,~ INGE DAMGAARD,2 ELSE SAEDERUP,3 RICHARD F. SQUIRES3 and ARNE SCHOUSBOE2. ~PharmaBiotec Research Center, Department of Biochemistry A, Panum Institute, University of Copenhagen, Denmark 2PharmaBiotec Research Center, Department of Biological Sciences, Royal Danish School of Pharmacy, 2100 Copenhagen 0, Denmark 3The Nathan Kline Institute for Psychiatric Research, Orangeburg, New York, N.Y., U.S.A. (Received 13 March 1991 ; accepted 1 May 1991)

Abstract--The ability of high- and low-affinity GABAA-receptors, respectively to inhibit depolarization coupled transmitter release was studied in cultured glutamatergic cerebellar granule cells which, depending on the culture conditions, express either high-affinity GABAA-receptors alone or high-affinity receptors together with low-affinity receptors. In order to gain information about the coupling of these receptors to chloride channels the effect of picrotoxin and binding of [3sS]t-butylbicyclophosphorothionate, both of which interact specifically with such channels were studied. Moreover, the influence of Flunitrazepam on the GABA-mediated inhibition of transmitter release was investigated to see if the GABA-receptors are coupled to benzodiazepine binding sites. Under conditions where the granule cells express only highaffinity GABAA-receptors it was found that GABA was able to inhibit transmitter release elicited by mild depolarization induced either by 30 mM KCI or 25 #M glutamate. This effect of GABA could be enhanced by Flunitrazepam and blocked by picrotoxin. However, transmitter release from these neurons induced by a more pronounced depolarization (55 mM KC1) could not be inhibited by GABA. Under conditions where the neurons express both high- and low-affinity GABAA-receptors transmitter release elicited by 55 mM KC1 could be inhibited by GABA but this inhibitory effect of GABA could not be blocked by picrotoxin, nor could it be enhanced by Flunitrazepam. These results strongly suggest that while the action of the high-affinity GABAA-receptors is coupled to chloride channels and benzodiazepine binding sites, the physiological action of the low-affinity GABAA-receptors is not. This lack of coupling between the low-affinity GABAA-receptors and chloride channels is further supported by the finding that the KD and Bm,xvalues for [35S]TBPS binding to the granule cells were independent of whether or not the cells expressed low-affinity GABAA-receptors. While the results clearly show that the inhibitory action of GABA mediated by low-affinity GABAA-receptors is not coupled to chloride channels, the exact mechanism of action of these receptors still remains to be elucidated.

Membranes prepared from adult rat cerebellum exhibit at least two distinct binding sites for G A B A with KD values of around 7 and 500 nM, respectively (Meier and Schousboe, 1982). A large part of these receptors have been shown to be associated with the cerebellar granule cells (Simantov et al., 1976; Olsen and Mikoshiba, 1978 ; Palacios et al., 1980). Cerebellar granule cells in culture also have a high number of G A B A receptors but unlike cerebellum in vivo the cultured neurons only express the high-affinity binding site (Meier and Schousboe, 1982; Meier et al., 1991).

However, several recent studies have shown that G A B A acts as a modulator of neuronal development [cf Redburn and Schousboe (1987); Meier et al. (1991)]. Thus, exposure of cerebellar granule cells in culture to G A B A or G A B A agonists induces the synthesis of low-affinity GABAA-receptors (Meier et al., 1984; Belhage et al., 1986, 1990a) in addition to a morphological differentiation of the granule cells (Hansen et al., 1984 ; Meier et al., 1985). Tilese effects of G A B A are mediated by activation of the preexisting high-affinity GABAA-receptors and the crucial event following this activation appears to be a chloride channel mediated hyperpolarization (Belhage et al., *Author to whom all correspondence should be addressed. 1990b). This finding is in agreement with the general 475

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Bo BELHAGEet al.

assumption of the function of GABAa-receptors. Thus, it is expected that activation of the high-affinity receptor leads to a hyperpolarization and thereby an inhibition of neuronal activity [c/Curtis and Johnston (1974)]. The significance of the inducible low-affinity GABAA-receptors as mediators of physiologically important inhibitory signals has repeatedly been demonstrated in neurons in culture where the low-affinity receptors mediate the inhibitory effect of G A B A on transmitter release (Meier e t al., 1984; Belhage e t al., 1990a). This ability of the low-affinity GABAAreceptors to mediate an inhibitory signal is observed, even when the cell membrane potential has been clamped at about - 2 0 mV by 55 mM KCI [~f Schousboe and Hertz (1971)]. This notion has been confirmed in experiments using the lipophilic cation tetraphenylphosphonium to monitor the membrane potential (Meier e t al., 1987). It was demonstrated that in granule cells expressing low-affinity G A B A receptors, G A B A was unable to reverse the depolarization elicited by 55 m M KC1. It may therefore be hypothesized that the inhibitory effect of the lowaffinity GABA-receptors could be mediated by a mechanism not involving chloride channels, e.g. a closure of receptor operated voltage sensitive calcium channels. In contrast, the high-affinity GABAA-receptors do not seem to have any inhibitory action under conditions leading to such pronounced depolarization. Accordingly, it is of interest to study in more detail the inhibitory action mediated by respectively the high- and low-affinity GABA-receptors on granule cells during conditions leading to respectively mild or pronounced depolarizations. The present study was therefore undertaken in order to further investigate and characterize the inhibitory mechanisms of highand low-affinity GABA-receptors using chloride channel and GABA-receptor specific drugs such as picrotoxin and Flunitrazepam to modulate G A B A mediated inhibition of transmitter release under conditions where the cerebellar granule cells express either high-affinity GABA-receptors alone or low-affinity receptors together with high-affinity receptors. In addition to these functional studies of the GABA/benzodiazepine-receptor/chloride-channel complex, the expression of the chloride channels was monitored using [35S]TBPS (t-butylbicyclophosphorothionate) which binds specifically to the picrotoxin binding site in GABAa/benzodiazepine-receptor complexes that regulate chloride channels (Squires et al., 1983). It has been reported that exposure of cerebellar granule cells to T H I P during culture for 3 15 days did not influence [~sS]TBPS binding

(Squires e t al., 1990). Howeve,, since no kinetic analysis of TBPS binding was performed it cannot be excluded that treatment of the cells with T H I P could alter the affinity of the chloride channel for this ligand. In the light of this, it was decided to pertbrm a detailed kinetic analysis of [3SS]TBPS binding to cerebellar granule cells cultured for different periods of time in the absence or presence of THIP, i.e. conditions leading to a progressive expression of respectively high-affinity GABA-receptors alone or highand low-affinity receptors together.

EXPERIMENTAl, PROCEDURES

MateriaLs

Seven-day-old rats (Wistar) were obtained from the animal quarters of the Panum Institute. Plastic tissue culture flasks (80 cm 2) or Petri dishes (35 mm) were purchased from NUNC A/S, Denmark and fetal calf serum from Sera-Lab. Lid, Sussex, U.K. Poly-L-lysine (mol. wt >300,000), trypsin inhibitor, DNAse, trypsin, picrotoxin and amino acids were obtained from Sigma Chemical Comp., St Louis, MO, U.S.A., insulin from NOVO, Denmark and penicillin from Leo, Denmark. [35S]TBPS (sp. radioact. 60 Ci/mmol) and [3H]D-aspartate (sp. radioact. 35 Ci/mmol) were purchased from Du Pont-New England Nuclear Comp., Boston, MA, U.S.A. THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3ol) was synthesized by Professor P. Krogsgaard-Larsen, Department of Organic Chemistry, Royal Danish School of Pharmacy, Copenhagen, Denmark and Flunitrazepam was a gift from Dr W. Haefely, F. Hoffmann-La Roche & Co., Lid, Basel, Switzerland. ('ell culture

Granule cells were cultured from cerebella o[" 7-day-old rats essentially as described by Schousboe el al. (1989) and Drejer and Schousboe (1989). Cells were isolated according to Wilkin el al. (1976) by mild trypsinization [0,025% (w/v) trypsin, 15 min, 37C] followed by trituration in a DNAse solution (0.004% w/v) containing a trypsin inhibitor from soybeans (0.03% w/v). The cells were suspended at a concentration of 4×10" cells/ml in a slightly modified Dulbecco's minimum essential medium containing 10% (v/v) fetal calf serum (Schousboe et al., 1989). The cell suspension was subsequently inoculated into poly-L-lysine-coated culture flasks (15 ml/flask) or Petri dishes (2 ml/dish). The medium contained in addition to fetal calf serum 24.5 mM KCI, 30 mM glucose, 7 ~tM p-aminobenzoic acid, 50 ILM kainic acid and 100 mU/l insulin. After 48 h in culture, 20 tzM cytosine arabinoside was added. The addition of cytosine arabinoside prevents astrocytic proliferation and the presence of kainic acid selectively eliminates contaminating GABAergic neurons (Drejer and Schousboe, 1989). Cells were cultured for 4, 8 or 24 days before they were used for experiments. These cultured neurons are characterized by an intense stimulus-coupled glutamate release and by fine structural features of cerebellar granule cells (Hansen et al., 1984: Drejer and Schousboe, 1989). The cultures used lbr experiments regarding the neuromodulatory effects of THIP were exposed to THIP (150 itMI from day 2 in culture until

477

Inhibitory actions of high- and low-affinity GABA-receptors the day of experiments concerning either transmitter release (cfbelow) or binding of [35S]TBPS (cfbelow). Release experiments The experiments were carried out as detailed by Drejer et al. (1983, 1987) and Meier et al. (1984). In order to label the glutamate neurotransmission pool (Palaiologos et al., 1989) cells cultured for 8 days were loaded with [3H]D-aspartate (5 /aCi/culture) for 30 min followed by a rapid wash with HEPES-buffered saline (130 mM NaC1, 5 mM KC1, 1.2 mM MgSO4, 1.8 mM CaC12, 10 mM glucose, 10 mM HEPES; pH 7.4). The cultures were placed in a superfusion system (Drejer et al., 1987) and superfused at a flow rate of 2 ml/min with HEPES-buffered saline for 30 min. Placement of a nylon mesh on top of the cells facilitated a uniform dispersion of the superfusion medium on the surface of the cells in the slightly tilted culture dish. The superfusion system was connected to a fraction collector and fractions were collected every 30 s. The superfusion medium was changed for 30 s at 3.5 min intervals allowing brief exposures of the cells to buffers with different composition. The pulse-buffers were prepared from HEPES-buffered saline by addition of either 25 or 50 mM KC1 (with an equimolar reduction of NaC1) or 25 #M L-glutamate. In order to determine the Ca 2+dependent part of the evoked transmitter release, 1.8 mM COC12 replaced CaCI 2 in some of the experiments. In some experiments the pulse-buffers contained also 5 or I00 /~M GABA, 0.01-100 #M Flunitrazepam, 150 #M picrotoxin or a mixture of these compounds as specified. Immediately before the introduction of the depolarizing pulse-buffers containing these latter substances, a 15 s superfusion period with the HEPES-buffered saline (i.e. non-depolarizing) plus the substances was introduced. Membrane preparation and [35S]TBPS boldin 9 Granule cells cultured for 4, 8 or 24 days were harvested in icecold phosphate buffered saline [135 mM NaCI, 3.0 mM KCI, 1.0 mM CaCI~, 0.6 mM MgC12, 1.7 mM KH2PO4 ; 8.0 mM Na2HPO4, 6 mM glucose; pH 7.4 (PBS)], sedimented for 5 min at 10,000 g and frozen at -20°C. At the day of analysis, the frozen cells were weighed and homogenized in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA at a concentration of 4 mg tissue (wet wt) per ml. For determination of [3~S]TBPS binding, 1 ml of membrane suspension was carefully mixed with I ml of the assay buffer [1 M KBr, 5 mM Tris-HC1 (pH 7.5), 500 #M EDTA and 10 nM R5135]. Non-specific binding was determined in the presence of 100/~M picrotoxin. After incubation (100 min at 2Y'C) the membranes were trapped on Schleicher and Schuell No. 25 glass fiber filters, and subsequently washed twice with 5 ml icecold 200 mM NaC1, 5 mM Tris HCI buffer (pH 7.5) essentially as described previously (Squires et al., 1983).

RESULTS Release experiments

In cerebellar granule cells cultured in the absence of T H I P , d e p o l a r i z a t i o n with 55 m M KC1, led to a large, CaZ+-dependent evoked t r a n s m i t t e r release ([3H]Daspartate) a m o u n t i n g to 600% o f the basal (monitored at 5 m M KC1) efflux o f [3H]D-aspartate. Addition o f 100 /~M G A B A during exposure to 55 m M

KCI h a d n o effect o n the release of [3H]D-aspartate (Fig. 1). However, w h e n cells were cultured in the presence o f T H I P , a d d i t i o n o f G A B A (100 p M ) during exposure to 55 m M KC1 inhibited the evoked t r a n s m i t t e r release by 6 0 % (Fig. 1). The m a g n i t u d e o f the release evoked by 55 m M KC1 was similar to that observed in the cells which h a d n o t been cultured in the presence o f T H I P . Figure 2 shows the results o f a n a l o g o u s experiments in which the cells were depolarized by 30 m M KC1 instead o f 55 m M KC1. U n d e r these conditions, the m a g n i t u d e o f the release was smaller (400% of the basal efflux) t h a n t h a t observed after depolarization with 55 m M KC1. The evoked release from u n t r e a t e d cultures could be inhibited a b o u t 4 0 % by 100 # M G A B A , whereas in cells g r o w n in the presence o f T H I P , G A B A inhibited a b o u t 60%. It is also s h o w n in Fig. 2 t h a t the inhibitory effect of G A B A in cells g r o w n in plain culture media (i.e. w i t h o u t T H I P ) could be completely blocked by 150 /~M picrotoxin. However, in cells cultured in the presence of T H I P , picrotoxin h a d n o effect o n the ability of G A B A to inhibit p o t a s s i u m - s t i m u l a t e d [3H]o-

culture condition Control

+ 150~uM THIP

~." ~ loo ~ o ~

so

~

40

so

~ 2o _'~ Fig. 1. Effect of GABA (100/~M) on KCI (55 mM) stimulated, Ca2+-dependent [3H]D-aspartate release from cerebellar granule cells cultured for 8 days in the absence or presence of 150 pM THIP. Cells were loaded with [3H]oaspartate (5 ~tCi/culture) 30 min prior to the superfusion experiments which were performed as detailed in Experimental Procedures. The potassium-stimulated release in the absence of GABA (open columns) which amounted to 600% of the basal release (5 mM KC1) has been expressed as 100 arbitrary units (a.u.). The effect of the presence of 100/~M GABA during the superfusion is shown in hatched columns. Results are averages of 16 experiments with SEM values shown as vertical bars. The asterisk indicates a statistically significant difference from the control (P < 0.001 ; Student's t-test).

B o BELHAGE el a[.

478

the absence of T H I P whereas picrotoxin had no effect on the inhibitory effect of G A B A in cells grown in the presence of THIP. Picrotoxin added without G A B A did not have any effect on the evoked release of transmitter regardless of the culture condition (results not presented). To facilitate a comparison of the results obtained under the different depolarizing conditions the results of the experiments involving depolarization by 30 m M KCI have been summarized in Table 1 together with the results concerning glutamate mediated depolarization. In order to obtain i n f o r m a t i o n a b o u t a possible coupling between the G A B A - r e c e p t o r s and benzodiazepine-receptors, the effect of Flunitrazepam on the inhibitory action of G A B A on evoked t r a n s m i t t e r release was investigated. Figure 3 shows a dose response curve for e n h a n c e m e n t by Flunitrazepam of the inhibitory action of 5 tiM G A B A on [~H]Daspartate release evoked by 30 m M KCI. It is seen that F l u n i t r a z e p a m dose-dependently enhanced the inhibitory effect of G A B A in granule cells cultured in plain culture media. The EDs0 value could be estimated to be 0.14 tiM. At a G A B A concentration of 100 tiM, Flunitrazepam could not enhance the inhibition by G A B A , n o t did it exert any action of its own. In cells cultured in the presence of T H I P , Flunitrazepam had no effect on the inhibition by G A B A of evoked transmitter release regardless of the G A B A concentratitm used (results not presented).

Culture condition Control

+ 150juM THIP

10o

8o o

*> so g

i

4o

20 O

Fig. 2. Effects of GABA (100 itM) or GABA plus picrotoxin (150 /~M) on KCI (30 mM) stimulated, Ca2~-dependent [~H]D-aspartate release from cerebellar granule cells cultured lk~r 8 days in the absence or presence of 150 I*M TH1P. Cells were preloaded with [~H]D-aspartate (5 #Ci/culture)and subsequently superfused as described in the legend of Fig. 1. The potassium stimulated release in the absence of GABA or picrotoxin (open columns) which amounted to 400% of the basal release (5 mM KCI) has been expressed as i00 arbitrary units (a.u.). The effect of GABA is shown as dark-hatched columns and that of GABA plus picrotoxin as light-hatched columns. Results are averages of 44 experiments with SEM values shown as vertical bars. Asterisks indicate statistically significantly differences from controls (P < 0.001 : one way ANOVA).

aspartate release (Fig. 2). The inhibitory effect o f G A B A on evoked t r a n s m i t t e r release was also investigated using 25 itM g l u t a m a t e as the depolarizing agent. Table I shows that under these conditions, G A B A (100 itM) inhibited the evoked t r a n s m i t t e r release by ca 2 0 % regardless of the culture condition. This inhibitory effect of G A B A could bc reversed by picrotoxin (150/xM) when cells h a d been cultured in

Binding experiment.v Results of Scatchard analyses of [;~S]TBPS binding to m e m b r a n e s prepared from cerebellar granule cells cultured in the presence or absence o f T H I P ( 150 ILM) for different periods of time showed that regardless of the presence of T H I P in the culture media there was an increase in B ...... from 4 fmol/mg at day 4 in culture

Table I. Effect of G A B A or G A B A + picrotoxin on ew)ked release of [~H]I)-aspartate fi-om cerebellar granule cells cullared in the absence or presence o f 150 itM T H I P 25 t~M glutamalc ( o m p o s i t i o n o f superfusion media Control G A B A ( 100 ,uM ) G A B A (100 # M ) + picrotoxin (150 ,uM)

30 m M KCI

Untreated

THIP-treated

I ntreated

FHIP-tleated

100.0 + 5.0 84.6 + 3.9* 112.0+5.2"

100.0 + 4 9 78.7 ~ 1.3" 85.7 ± 4.6

100.0 ! 1.3 ~)3.{) 4 1.6"* t;'9.3 ~: 1.9"*

100.0 + 1.6 30.0 ~ 2.4** 32.34 1.5

('ells were loaded with [3H]D-aspartate (5 itCi,'culture) for 30 min belore the superfusion experiment was started. The superfusion was

pertbrmed as detailed in Experimental P~ocedures. Release evoked by 25/tM glutamate or 30 mM KCI amounted to respectively200 or 400% of the basal release (5 mM KCI) and these values have been expressed as 100 arbitrary units (a.u.). Inhibition by 100 ltM GABA or 100 ,uM GABA + 150 uM picrotoxin of the release evoked by 25 [~M glutamale or 30 mM K has been expressed relative to these values. Results are averages±SEM of 24 (glutamate stinmlations) or 44 (KCI stimulations) experiments. Asterisks indicate statistically significant differences from controls (effects of GABA) or t'ronl superfi~sions with GABA (effects of GABA + picrotoxin) using Student's t-test (*P < 0.05; **P < 0.001).

Inhibitory actions of high- and low-affinityGABA-receptors

479

1981 ; Squires and Saederup, 1982, 1989) it could be demonstrated that these high-affinity GABA-recep~ 1(111 E tors are coupled to chloride channels as well as to benzodiazepine-receptor sites. This conclusion is based on the observation that the inhibition by GABA of transmitter release elicited by 30 mM KC1 could be 6o blocked by picrotoxin and enhanced by Flunitrazepam. The finding that Flunitrazepam per se had 40 no effect and did not alter the maximal response to GABA is in keeping with the classical concept of a functional association between GABA-receptors and I illllH I I IIIII I benzodiazepine-receptors (Polc et al., 1974; Costa 0.01 0.1 1 10 et al., 1975). It should be noted that at the more [Flunitrazepam] {~M) pronounced depolarization elicited by 55 mM KC1, Fig. 3. Dose-response curve for enhancement by Fluthe chloride channel coupled high-affinity GABAnitrazepam of GABA (5 #M) mediated inhibition of KC1 (30 mM) stimulated [3H]D-aspartate release from cerebellar receptors could not mediate an inhibition of evoked granule cells cultured for 8 days in plain culture media. transmitter release confirming previous reports (Meier Cells were loaded with [3H]D-aspartate (5 /~Ci/culture)and et al., 1984). Also when the excitatory amino acid subsequently superfused as described in the legend to Fig. 1. glutamate [cf Drejer et al. (1986)] was used as the Effects of increasing concentrations of FIunitrazepam on the ability of 5 ~M GABA to inhibit the KCI stimulated [3H]D- depolarizing agent it was found that at a conaspartate release are expressed as percentages of the maximal centration of 25 /~M, the glutamate induced transresponse seen at a Flunitrazepam concentration of 10 4 M. mitter release could be inhibited by GABA under Results are averages of 16 experiments_+SEM indicated by conditions where only high-affinity GABAa-receptors vertical bars. All points at Flunitrazepam concentrations >/10 s M were statistically significantly different from the are expressed. In contrast, it has been reported that inhibition by GABA in the absence of Flunitrazepam at a higher glutamate concentration leading to a more (P < 0.001, one way ANOVA). The curve was fitted to the pronounced depolarization, the evoked transmitter experimental points using the Enzfitter program from Else- release could not be inhibited by GABA or GABA vier/Biosoft. agonists unless the neurons also expressed low-affinity GABA-receptors (Abraham and Schousboe, 1989). In granule cells cultured in the presence of THIP, i.e. expressing both high- and low-affinity GABAato 20 fmol/mg at day 24. On the other hand, the KD receptors (Belhage et al., 1986), the evoked transmitter values (range 55-75 nM) did not change during the release could be inhibited by GABA regardless of the culture period. level of depolarization, i.e. at both 30 and 55 mM KCI. This finding is consistent with previous observations that under conditions where low-affinity DISCUSSION GABA-receptors are expressed on cerebellar granule It has been demonstrated repeatedly that cerebellar cells, evoked transmitter release can be inhibited by granule cells cultured under conditions where no GABA or GABA agonists acting on these receptors GABA is present only express high-affinity GABAa- (Meier et al., 1984; Abraham and Schousboe, 1989; receptors (Meier and Schousboe, 1982; Meier et al., Belhage et al., 1990a). It should be emphasized that 1984 ; Belhage et al., 1986, 1990a). When such neurons the inducible, low-affinity GABA-receptors pharmaare exposed to GABA or GABA agonists like THIP cologically have been characterized as GABAathey also express low-affinity GABAa-receptors receptors since Baclofen has no effect and THIP (Meier et al., 1984; Belhage et al., 1986, 1990a). mimics the effect of GABA (Meier et al., 1984). The Using granule cells cultured in plain culture medium finding that picrotoxin had no effect on the inhibitory accordingly allows functional studies of high-affinity effect of GABA on transmitter release evoked by GABAa-receptors to be performed. Using picrotoxin 30 mM KCI in the THIP treated cells shows that the which is a non-competitive blocker of GABAA-recep- low-affinity receptors are not likely to be linked to tors that regulate chloride channels (Olsen, 1981) and chloride channels. Moreover, the finding that FluFlunitrazepam which binds specifically to the benzo- nitrazepam had no effect in these cells suggests that diazepine binding site that is allosterically coupled to the low-affinity GABA-receptors may also not be GABAA receptors and picrotoxin binding sites (Olsen, associated with benzodiazepine binding sites. This is

480

Bo BELHAGEet al.

consistent with the report that treatment of granule cells with THIP has no effect on [3H]Flunitrazepam binding (Squires et al., 1990). The observation that the high- and low-affinity GABAA-receptors are different from each other with regard to benzodiazepine-receptor coupling would suggest that the subunit composition of these receptors might be different. It has recently been reported that both high- and low-affinity GABA-receptors are recognized by the monoclonal GABAA-receptor antibody bd-17 (Hansen et al., 1991a, b) which reacts specifically with the//2- and//3subunits (Richards et al., 1987). This would indicate that at least one or more of the//-subunits are common to the two receptor complexes forming respectively high- and low-affinity GABAA-receptors. The presence of high-affinity benzodiazepine binding sites in GABAA-receptor complexes is dependent on the presence of a 7-subunit combined with ~- and/#subunits (Pritchett et al., 1989a, b; Von Blankenfeld et al., 1990). It is therefore possible that 7-subunits are missing from the low-affinity receptor complex. With regard to the lack of effect of picrotoxin on the inhibitory action of GABA mediated via the low-affinity receptors it should be noted that all GABAA-receptors made up of various ~- and /:~-subunits, alone or in combination are sensitive to picrotoxin (Levitan et al., 1988; Blair et al., 1988). The results obtained in the present study do not, however, exclude the possibility that the low-affinity receptors could be associated with picrotoxin sensitive chloride channels but such channels could not alone explain the inhibitory action of GABA. The finding that the synthesis and membrane insertion of the low-affinity GABA-receptors is dependent upon an intact apparatus for protein synthesis and post-translational modification and therefore cannot be explained by conversion of high-affinity receptors into low-affinity receptors (Belhage et al., 1990a) is consistent with the suggestion that highand low-affinity receptors could have different subunit compositions. The large number of different subunits that can be combined in various ways could result in a very large number of quite different GABA-receptor complexes. Since exposure of the cells to THIP induces primarily the low-affinity GABA-receptors, the number of [35S]TBPS binding sites would be expected to increase under these conditions if the lowaffinity receptors were coupled to chloride channels. It was, however, found that THIP treatment had no effect on Ko and Bm~ for [35S]TBPS binding regardless of the culture period. Again, this is consistent with the previous findings of Squires et al. (1990) although a detailed kinetic analysis was not performed by these investigators. It is, however, possible that a low-

affinity TBPS binding site may be present but that the binding assay may not have been able to detect such a site. The conclusion that the low-affinity GABAA-receptors which are able to mediate inhibition of evoked transmitter release from the granule cells even during pronounced depolarization are not functionally associated with chloride channels raises the question of the exact mechanism responsible for the inhibitory action of GABA L~ia these receptors. On the basis of the finding (Meier et al., 1987) that GABA under these conditions is unable to alter the degree of the depolarization of the membrane potential induced by 55 mM KCI it was suggested that the low-affinity GABA-receptors might be located presynaptically. Recently, the localization of GABA-receptors in cerebellar granule cells has been studied at the electron microscopic level using preembedding, immunogoldlabelling which allows a quantitative estimation of receptor density (Hansen et al., 1991a, b). It was shown that the increased density of GABA-receptors observed after treatment of the cells with THIP is much more pronounced in the processes than in the cell bodies (Hansen et al., 1991b). This finding is consistent with a preferentially presynaptic localization of the low-affinity GABA-receptors. It would thus seem reasonable to suggest that these low-affinity GABA-receptors could be coupled to voltage sensitive calcium channels which upon closure would prevent evoked transmitter release. Studies on the localization of such calcium channels using a specific antibody (Vandaele et al.,, 1987) are currently being performed in this laboratory and results from double labelling of GABAA-receptors and calcium-channels using the immunogold method have shown not only a parallel induction of GABA-receptors and calcium-channels but also a tight coupling between these macromolecules in granule cells cultured in the presence of THIP (Schousboe et al., 1991a, b). Acknowled.qements The financial support from the Danish Medical Research Council (12-9563) and the NOVO Foundation to Bo Belhage and from the Danish Biotechnology Program (1987 1990)and the Lundbeck Foundation to Arne Schousboe is gratefully acknowledged. Professor Povl Krogsgaard-Larsen, PharmaBiotec Research Center, Department of Organic Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark and Dr W. Haefely, F. Hoffmann-La Roche & Co., Ltd, Basel, Switzerland are cordially thanked for gifts of THIP and Flunitrazepam, respectively. REFERENCES Abraham J. and Schousboe A. (1989) Effects of taurine on

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