Regional differences in the enhancement by GABA of [3H]zolpidem binding to ω1 sites in rat brain membranes and sections

134

Brain Research, 600 (1993) 134-140 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 18406

Regional differences in the enhancement by G A B A of [3H]zolpidem binding to to 1 sites in rat brain membranes and sections D. Ruano

a, j.

Benavides

b, A.

M a c h a d o a a n d J. V i t o r i c a a

a Departamento de Bioqufmica, Bromatolog{a y Toxicolog{a, Facultad de Farmacia, Unicersidad de Secilla, Secilla (Spain) and t, Department of Biology, Synthelabo Recherche (LERS), Bagneux (France) (Accepted 4 August 1992)

Key words: y-Aminobutyric acid A receptor complex; w I modulatory binding site; 7-Aminobutyric acid enhancement; [3H]Zolpidem binding; Rat central nervous system; Membrane; Autoradiography

The potential heterogeneity in the allosteric coupling between GABA and w I binding sites within the native GABA A receptor complex has been evaluated in the rat by measuring the enhancement by GABA of [3H]zolpidem binding to w 1 site in membranes from three rat brain structures (neocortex, cerebellum and hippocampus) and brain sections. The maximal stimulatory effect of GABA was significantly higher (265 +47%) in cortical membranes than in cerebellar (165 +48%) or in hippocampal (118_+ 17%) membranes. These differences are not due either to the presence of different amounts of residual GABA in the membrane preparations or to the labeling, in presence of GABA, of binding sites other than o) 1 since: (1) the pharmacological properties of the [3H]zolpidem binding sites were similar in the three regions; (2) the degree of allosteric enhancement was unrelated to the relative proportion of w I sites in each structure; and (3) GABA did not increase the Bmax for [3H]zolpidem. Regional differences in the effect of 100 /tM GABA on [3H]zolpidem binding were also observed by quantitative autoradiography. Regions where the strongest (3-4-fold) effects of GABA in [3H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large increase in [3H]zolpidem binding was also demonstrated in the cingulate and frontoparietal cortices with higher effects in deep (4.2-fold) rather than in superficial layers (3.3-fold). Heterogeneous subregional increases in [3H]zolpidem binding in the presence of GABA were quantified within the cerebellum, hippocampus and superior colliculus. In the cerebellum the effect of this neurotransmitter was larger in the molecular (3.8-fold) than in the granular (2.2-fold) layer. In the hippocampus the effect of GABA was also heterogeneous with larger increases in CA1 and CA2 fields (3.5-fold) than in CA3 field (2.2-fold) and dentate gyrus (2.5-fold). Finally in the deep layers of the superior colliculus GABA stimulation of [3H]zolpidem binding was greater than the superficial layer. In the other structures examined the GABA-induced increase in [3H]zolpidem binding was less than 3-fold. The smallest stimulations were quantified in the entorhinal cortex (2.1-fold), amygdala (2.4-fold) and nucleus accumbens (1.7-fold). These results suggest that [3H]zolpidem sites are associated to, at least, two GABA A receptor subtypes that can be differentiated by their allosteric interaction between GABA and [3H]zolpidem sites.

INTRODUCTION The benzodiazepines exert their anxiolytic/hypnotic actions through the activation of to (BZD) modulatory sites located within the GABA a receptor complex 17'31"32.This receptor complex is a multimeric membrane protein constituted by the association of a, /3, 7 a n d / o r ~ subunits 18. The heterogeneity of this receptor complex at the level of the protein composition3,~9,2°,33; mRNAs encoding for different subunits (see ref. 18 for review) and pharmacology of to sites 7'31'32 is now well established. The to modulatory sites have classically been classified in two pharmacologically different subtypes: to1 (Type 1 or BZ1) and to2 (Type II or BZ2) on the basis

of their differing affinities for the triazolopyridazine C1 218.872 28, for the imidazopyridine zolpidem 5'8'16 and for some /3-carboline derivates 3t. Each subtype has a differential regional distribution throughout the C N S 136. A third w site that possesses a very low affinity for zolpidem and a differential anatomical distribution has been recently identified in displacement studies of [3H]benzodiazepine binding to rat brain membranes 26 and sections 2 by a wide range of zolpidem concentrations. The diversity of the w modulatory site seems to be generated by the heterogeneity of the a subunits of the GABA A receptor complex 9'22'23. A heterogeneous allosteric interaction between the GABA and to recognition sites within the G A B A A receptor complex has also been demonstrated in native (by autoradiography 13'34 or in conventional membrane

Correspondence: J. Vitorica, Departamento de Bioqu~mica, Bromatolog~a y Toxicologia, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain. Fax: (34) (5) 423-3765.

135 b i n d i n g s t u d i e s 3'4'25'26'27) a n d in t r a n s f e c t e d r e c e p t o r s 24. It has b e e n p o s t u l a t e d t h a t w h i l e a s u b u n i t s a r e res p o n s i b l e for w site p h a r m a c o l o g y , 3' s u b u n i t s g o v e r n the efficacy of the allosteric coupling between w modul a t o r y sites a n d t h e G A B A b i n d i n g site 21'22'23'24. T h e e x i s t e n c e o f s e v e r a l 3' s u b u n i t s w o u l d p r e d i c t s e v e r a l d e g r e e s o f a l l o s t e r i c c o u p l i n g f o r a s i n g l e class o f o~ sites.

However,

displacement

of [3H]benzodiazepine

b i n d i n g by s e l e c t i v e ~0~ l i g a n d s f a i l e d to d e m o n s t r a t e s u c h d i f f e r e n c e s 26. I n t h e p r e s e n t s t u d y w e h a v e r e a d d r e s s e d this q u e s t i o n by s t u d y i n g t h e e f f e c t o f G A B A o n t h e b i n d i n g o f [3H]zolpidem (an

w l - s e l e c t i v e l i g a n d ) to m e m b r a n e s

p r e p a r e d f r o m rat c o r t e x , h i p p o c a m p u s a n d c e r e b e l l u m o r by q u a n t i t a t i v e a u t o r a d i o g r a p h y o f a l a r g e n u m b e r o f s t r u c t u r e s in rat b r a i n s e c t i o n s . T h e r e s u l t s o b t a i n e d demonstrate

an

allosteric

coupling

between

a n d [ 3 H ] z o l p i d e m b i n d i n g sites in m e m b r a n e s tions

stronger

than

that

observed

GABA a n d sec-

for non-selective

o m e g a l i g a n d s s u c h as [ 3 H ] f l u n i t r a z e p a m ( s e e ref. 26) a n d also c l e a r r e g i o n a l d i f f e r e n c e s in t h e e f f i c a c y o f t h e allosteric interaction between

to~ a n d G A B A

binding

sites. MATERIALS AND METHODS

-48°C and mounted onto a microtome chuck. Fifteen /zm thick coronal sections were then prepared, at -20°C, using a Leitz 1720 microtome-cryostat and were thaw-mounted onto chromalum-gelatin subbed microscope glass slides. Tissue sections were then dried rapidly under vacuum and frozen until the binding experiments. [ 3H]Zolpidem binding was assayed according to a modification of the technique described by Niddan et al. 16. Before binding experiments, sections were incubated for 5 min at room temperature in 120 mM NaCI, 5 mM KCI, 50 mM Tris-HC1 (pH 7.4) in order to wash out endogenous GABA. This incubation was sufficient to remove GABA from the slices since longer incubation times gave similar results (see below). Tissue sections were then incubated for 30 min at 4°C in 4 ml of the same medium containing 5 nM [3H]zolpidem and 10 /zM bicuculline or 100 /xM GABA for control and stimulated binding, respectively. Non-specific binding was determined in the presence of 2/xM flumazenil and represented less than 10% from total binding. The incubation was terminated by rinsing sections for 3 rain in cold incubation medium. Sections were then dipped briefly in cold distilled water and dried rapidly under a stream of cold air. In order to ensure the efficient removal of GABA from the cerebral sections, the [3H]zolpidem binding after different preincubation times was tested by wiping the tissue from the slides and determining the binding by convectional scintillation techniques. After 5 min preincubation, the [3H]zolpidem binding was reduced in 55 + 10%, n = 3, with respect to non-preincubated sections and no further reduction was observed with longer preincubation times (55 + 8% or 50_+6%, n = 3, of reduction after 15 or 30 min preincubation, respectively). Furthermore, after 5 min preincubation, the binding of [3H]zolpidem was only slightly reduced by the addition of 10 /xM bicuculline (10+ 5%, n = 3). Therefore, the basal [3H]zolpidem binding of the cerebral sections was determined after 5 min preincubation and in the presence of 10/zM bicuculline. Autoradiographs were prepared from the sections which were apposed, together with radioactivity standards to radiographic film (3H-Ultrofilm, Amersham, Buckinghamshire, UK) for 2 weeks 1.

Materials [3H]zolpidem (58.0 Ci/mmol) was purchased from New England Nuclear. Clonazepam, diazepam and flumazenil were gifts from Hoffmann-LaRoche. Methyl-6,7-dimethoxy-4-ethyl-/3-carboline-3carboxylate (DMCM) was from RBI (USA). GABA was purchased from Sigma.

Membrane preparation Three-month-old male Wistar rats were killed by decapitation, and the different brain regions were rapidly dissected, frozen in liquid nitrogen and stored at -80°C until use. The membrane preparation and the removal of endogenous GABA was carried out according to the method of Mernoff et alJ 4 as we described in detail previously 2s'33.

Data analysis The dose-response curves for the GABA enhancement of [3H]zolpidem binding were fitted using the PAR-derivate free nonlinear regression analysis with the BMDP computer program as described previously 25. The Scatchard transformation of the saturation curves and displacement curves were adjusted using the program L1GAND is. The comparison between the means of different groups of data was evaluated by two-tailed Student's t-test or by one-way ANOVA or multifactor ANOVA followed by Tukey's test. Local tissue concentrations of 3H were determined by quantitative densitometric analysis with a computer-based densitometer and image analyzer (Biocom, Les Ullis, France), with a variable frame size s.

Membrane binding assays For all binding assays, 0.2-0.4 mg of membrane proteins were incubated in 0.2 ml (saturation or displacement experiments) or 0.4 ml (GABA enhancement experiments) of 50 mM Tris-HCl (pH 7.4) at 4°C for 35 min. Non-specific binding was determined in the presence of 5 /xM diazepam. Binding experiments were terminated by rapid filtration through Whatman G F / B fiber filters. The allosteric effect of GABA was assessed by measuring the effect of 8 different GABA concentrations, ranging from 0.01 to 100 /xM, on the binding of 3 nM [3H]zolpidem. Flumazenil, diazepam, clonazepam, DMCM and C1 218. 872 competition experiments were performed using 5 nM [3H]zolpidem and 10-15 different concentrations of each ligand in the presence or absence of 100 ~M GABA. The [3H]zolpidem saturation curves, in the presence or absence of 100 /xM GABA, were determined over a range of 1-20 nM [3H]zolpidem.

In vitro binding of [3H]zolpidem in rat brain sections Three-month-old male Fischer rats were killed by decapitation, the brain was rapidly removed, frozen in isopentane chilled at

RESULTS

Effect o f G A B A on [3H]zolpidem binding in membranes f r o m rat cortex, cerebellum and hippocampus G A B A e n h a n c e d t h e [ 3 H ] z o l p i d e m b i n d i n g to m e m b r a n e s f r o m rat c o r t e x , c e r e b e l l u m a n d h i p p o c a m p u s w i t h h i g h e f f i c a c y (Ema x > 1 0 0 % ) (Fig. 1). I n c o r t i c a l membranes ANOVA

GABA

had

F2, u = 13.86,

a

significantly

P = 0.0018;

g r e a t e r Ema x (265 + 4 7 % ,

Tukey

(one-way P < 0.03)

n = 4) t h a n in c e r e b e l l u m

(165 _+ 4 8 % , n = 4) o r h i p p o c a m p u s (118 _+ 1 7 % , n = 4) (Fig. 1, inset). T h e p o t e n c y o f G A B A [3H]zolpidem ANOVA,

binding

F2, u = 17.04,

was

at e n h a n c i n g

significantly

P = 0.0009;

Tukey

(one-way P < 0.01)

l o w e r in m e m b r a n e s f r o m h i p p o c a m p u s (1.8 _+ 0.6 ~ M ,

136 Z" 350 .£ -=

Emax

EC50

~ ........ "*,)

300

E

3CTX

~ 250 E 200 ....

265±47 a

nH

~

l

0.6±0.1

0.6:LO. 1

T

~ ] I

/

b ['i[['[ [[[[i[ ['2:['[

1oo

.&

5010-10 10-9

~..,~" t 10-8

10-7 10-6 GABA (M)

10-5

10-4

Fig. 1. Regional differences in the enhancement by GABA of [3H]zolpidem binding to w I sites in rat brain membranes. The enhancement by GABA of [3H]zolpidem (3 nM) binding was determined in membranes from rat cerebral cortex (o), cerebellum (o) and hippocampus (zx). Results are m e a n + S . D , of 4 experiments performed in duplicate. Values are expressed as percent of stimulation of [3H]zolpidem binding in the absence of GABA. Specific [3H]zolpidem binding per assay (expressed in fmol) was 20.7_+7, 23.2+5 and 16.7_+5 for the cortex, cerebellum and hippocampus, respectively. The dose-response curves were fitted as described in Materials and Methods and the calculated parameters, mean_+ S.D., are shown in the inset. " Significant difference from cerebellun and hippocampus: one-way A N O V A /'2, n = 13.86, P=0.0018; Tukey P < 0.03. b Significant difference from cortex and cerebellum: one-way A N O V A F2j I = 17.04, P = 0.0009; Tukey P < 0.01. ~ Significant difference from cortex and cerebellum: one-way A N O V A /2, n = 6.017, P = 0.022; Tukey P < 0.05.

n = 4) than in cortex or cerebellum (0.6_+ 0.1 p,M, 0.4 _+ 0.2 IxM, n = 4, respectively). Hill slopes for these G A B A effects were similar in membranes from cortex and cerebellum, whereas it was significantly (one-way A N O V A Fz, jl = 6.02, P = 0.02; Tukey, P < 0.05) higher in hippocampus. This lower slope of the doseresponse curves in cortex and cerebellum raise the possibility that the allosteric interactions between w~ and G A B A binding sites were heterogeneous in these brain regions.

The regional differences in G A B A enhancement of the [3H]zolpidem binding were not due to different levels of residual G A B A since bicuculline (10 /xM) produced a very small and similar inhibition of basal [3H]zolpidem binding in the three structures (18 +_5%, 16 _ 6% and 18 +_ 7%, n = 3, in membranes from cortex, cerebellum and hippocampus, respectively). These regional differences in the GABA effects were not due either to the recruitment of additional [3H]zolpidem binding sites in some regions because the Bma x for this ligand (determined by Scatchard analysis of the saturation curves, not shown) was not significantly (less than 10%) modified by GABA in any of the three brain areas investigated. Finally, these differences in coupling were not due to the existence of pharmacologically distinct w I binding populations in these three rat brain structures because the affinity ( K d or K i) of a number of w site ligands for [3H]zolpidem binding, in the presence or absence of GABA, was similar in the three regions (Table I). The Hill coefficients for [3H]zolpidem saturation curves and for the inhibition by several ligands of [3H]zolpidem binding were close to the unity and similar in cortex, cerebellum and hippocampus (Table I). It is important to observe that the Hill coefficients for DMCM displacement of [3H]zolpidem binding were significantly (multifactor A N O V A /5,43 = 4.90, P = 0.002, Tukey; P < 0.05) lower from that of [3H]zolpidem or clonazepam.

Regional r'ariations in the allosteric interaction between the GABA A receptor and ~1 modulatory sites The effect G A B A on [3H]zolpidem binding to w] sites was also studied by quantitative autoradiography in rat brain sections. In the presence of 100 /xM

TABLE I Pharmacologic- characterization

of the 0)1 GABA A /benzodiazepine receptor in membranes from rat cortex, cerebellum and hippocampus

The affinity constant ( K d or K i) and the n H for each ligand were calculated from [3H]zolpidem saturation curves or from [3H]zolpidem (5 nM) displacement curves. The results are the mean + S.D. of the number of experiments given in parentheses. The affinities (K d or K i) are expressed in nM. Ligand

[3H]Zolpidem Clonazepam Diazepam Flumazenil DMCM +100#MGABA CL218-872 + 100/zM GABA

Cortex

Cerebellum

Affinity

nH

11.2_+ 3.5 1.7+_ 1.6 3.1_+ 3.1 1.5+ 0.1 3.6_+ 1.3 5.4_+ 2.1 58.7+15.1 ** 17.8+_ 11.0

1.0_+0.1 1.1+_0.1 0.9+_0.1 0.8+0.1 0.6+-0.2 0.8_+0.1 0.8+0.1 0.8+_0.1

(6) (4) (3) (3) (6) * (4) (3) (3)

Hippocampus

Affinity

nH

Affinity

nH

13.5_+6.1 3.8_+2.5 2.2+_0.8 1.3+0.8 3.6_+2.8 N.D. N.D. N.D.

0.9_+0.1(3) 1.0_+0.4(2) 1.2+_0.4(2) 0.9+0.3(2) 0.7+-0.2(3) N.D. N.D. N.D.

9.1_+ 6.5 3.3+_ 2.5 3.4_+ 2.5 0.7+ 0.4 3.4+- 1.3 7.4_+ 4.0 58.8+22.1 ** 23.9+_ 12.2

0.9+_0.1 1.1+_0.2 0.9+0.1 0.9+-0.3 0.6_+0.3 0.7+-0.1 0.8_+0.1 0.9+_0.2

(4) (3) (2) (3) (5) * (4) (3) (3)

The K i or n u from cortex and hippocampus were subjected to multifactor analysis of variance followed by Tukey's test. A N O V A between ligands was significant for n H (/'5.43 = 4.90, P = 0.002) or K i (F5,43, P = 0.0001). No significant differences were found between cortex and hippocampus or between cortex and hippocampus and ligands for either K i or n H. Tukey: * P < 0.05 from zolpidem and clonazepam; • * P < 0.01 from all other ligands.

137 G A B A (saturation conditions, see Fig. 1) [3H]zolpidem binding was increased in all rat brain areas examined (Fig. 2). These regions where the strongest effect of G A B A in [3H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large G A B A induced increase in [3H]zolpidem binding was also seen in the cingulate and frontoparietal cortex where more pronounced increases in this p a r a m e t e r were seen in deep than in superficial layers (Table II). Intermediate increases in [3H]zolpidem binding in the presence of G A B A were quantified in the cerebellum, hippocampus and superior colliculus. In the cerebellum the effect of this neurotransmitter was larger in molecular than in the granular layer. In the hippocampus the effect of G A B A was also heterogeneous with larger increases in CA2 than in CA1 field than dentate gyrus and the smallest stimulation in the CA3 field (Fig. 3, Table II). Finally, G A B A stimulation of [3H]zolpidem binding was greater in the deep than in the superficial layer of the superior colliculus. In the other structures examined, the GABA-induced increase in [3H]zolpidem binding was lower than 3-fold (Table II).

DISCUSSION The present results demonstrate that the G A B A enhancement of [3H]zolpidem binding to w a sites has high efficacy. This observation is consistent with previous studies demonstrating a large GABA-induced increase in the potency of zolpidem at inhibiting [3H]benzodiazepine binding to o) 1 sites 26. This enhancement is much greater than that observed (in [3H]benzopiazepine displacement experiments) with o) sites with low ( ~ M ) affinity for zolpidem in which the stimulation never exceeded 4 0 % 26 . Furthermore, the present results demonstrate the existence of regional differences in the efficacy of the allosteric coupling between G A B A and w t sites within the G A B A A receptor complex in rat brain. Thus, while the pharmacological properties of w~ site in m e m branes from rat brain cortex, cerebellum and hippocampus seemed to be similar and consistent with the ~01 pharmacology H'29, the maximal stimulatory effect of G A B A on [3H]zolpidem binding was higher in cortical m e m b r a n e s than in cerebellar or hippocampal membranes (see Fig. 1). These results suggest that (,oI sites are associated to, at least, two G A B A A receptor subtypes that can be differentiated by the efficacy of the

ENHANCEMENT BY GABA OF 3H-ZOLPIDEM BINDING TO RAT BRAIN SECTIONS 5 nM 3H-zolpidem

5 nM 3H-zolpidem+ 100 laM GABA

Fig. 2. Enhancement by GABA of [3H]zolpidem binding to rat brain sections. Consecutive sections were incubated in the presence of 5 nM [3H]zolpidem with or without 100/xM GABA. Micrographs are representative of data obtained from 4-5 rats.

138 allosteric interaction between the G A B A receptor and o)1 sites. As observed in membranes, the G A B A induced increase in [3H]zolpidem binding to rat brain sections was greater in the neocortex than in the cerebellum or hippocampus. It could be argued that these regional differences were due to a differential effect of bicuculline on the basal [3H]zolpidem binding in these brain regions 34. Although we have not determined the effect of bicuculline on different areas of the cerebral sections, this explanation seemed to be unlikely since: (i) bicuculline produced an identical effect (18% of decrement) on the basal [3H]zolpidem binding in membranes from cortex, cerebellum and hippocampus (see Results) and this effect was analogous to that observed in cerebral sections (see Methods); and (ii) the rank order of maximal G A B A stimulation of [3H]zolpidem binding in membranes from these three regions was similar to that observed by autoradiography. Therefore, the G A B A enhancement of [3H]zolpidem binding determined by quantitative autoradiography could be compared with that of membrane binding studies indicating that the regional heterogeneity observed in sections was faithfully reflecting the regional differences in G A B A efficacy. Besides confirming the regional differences seen in membranes, these autoradiographic studies also demonstrate a heterogeneity in this coupling within

subregions of the cortex, hippocampus and cerebellum. The allosteric interaction between G A B A and o)1 sites does n o t appear to be related to the relative proportion of o)1 sites in a given structure. Indeed, the coupling was stronger in the substantia nigra than in the cerebellum which contains a similar proportion of o) 1 sites than the former region 2'16. These results strongly indicate that the increases in binding observed are not due to the recruitment of binding sites other than o)1 sites. Since the regional distribution of the different GABA A receptor subunits is not yet well characterized it is difficult to correlate the regional differences in the strength of coupling with the presence of a particular subunit in a given structure. Moreover, these differences do not appear either to be related to a specific functional brain system (i.e. sensorimotor, limbic . . . . ). The pharmacological properties of the GABA A receptor complex are dependent on the subunit composition (see ref. 18 for review). An o)l-type pharmacology has been associated with the expression of a~ subunit 22'23 or with the presence of the P51 peptide 28'33. On the other hand, three different /3 subunits and three different 7 subunits have been cloned 18'35. It has been postulated that the potency of G A B A was determined by the a and the /3 subunits present in the receptor complex 3°. Therefore, the lower potency of G A B A at enhancing the [3H]zolpidem binding in hip-

E N H A N C E M E N T BY G A B A OF 3H-ZOLPIDEM B I N D I N G TO RAT BRAIN SECTIONS 5 nM 3H-zolpidem

5 nM 3H-zolpidem + 100 gM GABA

Fig. 3. Enhancement by GABA of [3H]zolpidembinding to the rat hippocampus. Consecutive sections were incubated in the presence of 5 nM [3H]zolpidemwith or without 100 txM GABA. Micrographsare representative of data obtained from 4-5 rats.

139 TABLE II

the lack of d e t e c t a b l e differences in the biochemical

Quantitati~'e autoradiographic assessment of the effect of GABA on [3H]zolpidem binding to rat brain sections

p r o p e r t i e s (glycosylation, p e p t i d e maps) for the P51

Consecutive sections were incubated with 5 nM [3H]zolpidem in the presence or in the absence of 100 ,aM GABA. Values are means with S.D. of values obtained on 3 rats. Structure

Cortex Anterior cingulate Cingulate Frontoparietal layer I-llI Frontoparietal layer IV Frontoparietal layer V-VI Entorhinal Olfactory tubercle Amygdala Nucleus accumbens Striatum Septum Hippocampal formation CA1 field CA2 field CA3 field Dentate gyrus Red nucleus Lateral geniculate Nucleus superior colliculi Superficial layer Deep layers Central grey Substantia nigra Cerebellum Molecular layer Granular layer Medulla-pons

Stimulation of [ 3H]zolpidem binding by 100 ,aM GABA (folds)

4.2 + 0.2 4.1 + 0.7 3.3 + 0.2 3.4 _+0.6 4.2 + 0.5 2.1 + 0.4 3.4 _+0.3 2.4 + 0.3 1.7 _+0.3 3.1 _+0.5 3.4_+0.6 3.5 _+0.6 3.5 _+1.5 2.2 + 0.3 2.5 +_0.3 4.3 _+0.4 4.2 + 1.1

p e p t i d e i m m u n o p u r i f i e d from bovine cortex or cerebellum 2°, n o differences in the p h a r m a c o l o g y of co t sites has b e e n f o u n d b e t w e e n rat cortex, c e r e b e l l u m or h i p p o c a m p u s . Thus, it is t e m p t i n g to suggest that the differences in allosteric coupling b e t w e e n cortex and c e r e b e l l u m or h i p p o c a m p u s are d u e to the p r e s e n c e (in different p r o p o r t i o n s ) in the G A B A a r e c e p t o r complex to which w 1 sites are associated with y subunits, o t h e r t h a n the Yl variant. O n the o t h e r hand, although the majority of the G A B A A r e c e p t o r complex c o n t a i n s only a single type of a s u b u n i t 6'12, a small p r o p o r t i o n of a 3 - c o n t a i n i n g receptors a p p e a r to c o n t a i n an a l s u b u n i t tool0'l< T h e G A B A A receptor complex constructed by the expression of c~l, a 3, /32 a n d Y2 subu n i t s displayed a very high p o t e n t i a t i o n of G A B A - i n d u c e d C1- c u r r e n t by d i a z e p a m (see T a b l e I in ref. 30). T h e r e f o r e , we c a n n o t completely rule out the possibility that the high degree of coupling b e t w e e n w 1 and G A B A b i n d i n g sites in rat cortical m e m b r a n e s is due to the p r e s e n c e of a G A B A a receptor complex con-

2.4 _+0.3 3.6 _+0.6 3.5 + 0.4 4.4_+ 1.1

t a i n i n g a I a n d any o t h e r a variants. I n conclusion, the p r e s e n t results d e m o n s t r a t e that, despite the fact that w~ p h a r m a c o l o g y is similar in all the rat b r a i n structures tested, the allosteric i n t e r a c t i o n

3.8 _+1.3 2.1 _+0.6 2.6 _+0.1

b e t w e e n w I a n d G A B A b i n d i n g sites are high b u t not u n i f o r m t h r o u g h o u t the rat brain. T h e s e results add a n e w degree of complexity to native G A B A A receptor subtypes.

p o c a m p a l m e m b r a n e s could reflect the relative differe n t a b u n d a n c e of the /3 s u b u n i t messages in rat hipp o c a m p u s with respect to cortex or c e r e b e l l u m 7. O n the o t h e r h a n d , the y s u b u n i t v a r i a n t p r e s e n t in the G A B A A r e c e p t o r complex seems to i n f l u e n c e the b i n d i n g to o m e g a sites a n d to govern the allosteric effect of G A B A a n d o m e g a sites 2~. It has b e e n recently r e p o r t e d that G A B A A r e c e p t o r c o n s t r u c t e d with Yl s u b u n i t s display a low degree of coupling b e t w e e n the G A B A a n d ~o sites 24 a n d an atypical b e n z o d i a z e p i n e p h a r m a c o l o g y 36. T h e a l , /31, ")/1 construct exhibited a 10-fold r e d u c e d affinity, as c o m p a r e d with a~, /31, or Y2 for several b e n z o d i a z e p i n e a n d n o n - b e n z o d i a z e p i n e ligands a n d this Yl s u b u n i t c o n t a i n i n g r e c e p t o r lacks affinity for a n t a g o n i s t s (i.e. flumazenil) or inverse agonists (i.e. D M C M ) 36. However, [3H]zolpidem b i n d i n g sites in the neocortex, in the c e r e b e l l u m or the hipp o c a m p u s display a high affinity (low K i) for flumazenil a n d D M C M , thus, suggesting that the Yl s u b u n i t is absent, or in very low p r o p o r t i o n , in the G A B A A r e c e p t o r complex c o n t a i n i n g ~oI b i n d i n g sites p r e s e n t in m e m b r a n e s from these structures. As p r e d i c t e d by

Acknowledgements This work was supported by a grant from Fundacion Ramon Areces and by a fellowship from Junta de Andalucia, F.P.I. for D.R. We thank Drs. Scatton and B. Zivkovic for critical reading.

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N o t e a d d e d in p r o o f While this report was under revision, the pharmacologic properties of al, Y3 and /32 contruct were reported (Herb et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 1433-1437). This receptor construct displayed very low affinity for zolpidem (5.5 p~M) and low zolpidem-induced potentiation. In our experiments the [3H]zolpidem concentration never exceeded 5 nM (20 nM in saturation experiments). Therefore, under these experimental conditions, it is very unlikely that [3H]zolpidem binds significantly to the oq, Y3, and /32 containing receptors.