Regional effects of pertussis toxin in vivo and in vitro on GABAB receptor binding in rat brain

0306-4522/93$5.00+ 0.00 PergamonPress Lid IBRO

Neuroscience Vol. 52, No. 1, pp. 73--81,1993 Printed in Great Britain

REGIONAL EFFECTS OF PERTUSSIS TOXIN IN VIVO A N D IN VITRO ON GABAn RECEPTOR B I N D I N G IN RAT BRAIN C. KNOTT,* J. J. MAGUIRE,R. MORATALLAand N. G. BOWERY Department of Pharmacology, The School of Pharmacy, 29-39 Brunswick Square, London WCIN lAX, U.K. Al~raet--Agonist binding to GABAa receptors modulates the activity of the guanine nucleotide binding proteins, Go and Gi. These G proteins are ADP-ribosylated by pertussis toxin and this prevents them from coupling to the GABAe receptor resulting in a reduction in high-alfmity GABAB binding. GTP, which binds to a different site on the G protein ~t subunit, also reduces the affinity of the receptor for the G protein, and this can be used as a "marker" for G protein-GABAe receptor linkage. We have examined GABAe binding site distribution in rat brain after unilateral intrahippocampal pcrtussis toxin injection in vivo, and after incubating brain slices in pertussis toxin/n vitro, using the technique of receptor autoradiography. The effect of pcrtussis toxin was compared with that of GTPTS on GABAB binding. Intrahippccampal pcrtussis toxin administration reduced GABAe but not GABA^ receptor binding and the effects appeared to be limited by pertussis toxin diffusion. More widespread reductions in GABAB binding were seen after incubation of brain slices in vitro but the extent varied in different brain regions. No reduction was detected in the corpus striatum. GABAB binding was also reduced in membranes prepared from cerebral cortex, hippocampus and cerebellum but there was no sigificant reduction in the corpus striatum after pertussis toxin treatment. GTPyS reduced GABAs binding to a similar extent in all areas studied irrespective of their sensitivity to pertussis toxin suggesting that while GABAn binding sites are linked to G proteins throughout the rat brain, those in the corpus striatum may be predominantly pertussis toxin insensitive.

GABA is one of the major inhibitory neurotransmitters in the mammalian brain and binds to two different receptor subtypes with separate anatomical locations and different pharmacology, zlS,2s'3°'31,33The GABA^ subclass gates chloride channels, is sensitive to bicuculline and picrotoxin and is modulated by benzodiazepines and barbiturates. In contrast, GABAn receptors gate potassium, l° or calciun'l 9'11'19'20'26 channels indirectly via guanine nucleotide binding proteins ((3 proteins) and/or phosphatidyl inositol generating systems.8'9'13The GABAB receptor has been solubilized by Ohmori and colleagues26 and receptor mRNA extracted from rat cerebral and cerebellar cortices has been expressed in the Xenopus oocyte. However, the receptor expressed from cerebral cortex exhibited uncharacteristic pharmacological responses to (-)baclofen. 32 Receptor expression from cerebellar cortex mRNA appeared to be more analogous to the native receptor but was only evident in about 5% of the injected oocytes) 5 The existence of multiple GABAB receptor subtypes has been postulated based on functional *To whom correspondence should be addressed. 3-APA, 3-aminopropylphosphinic acid; EDTA, ethylenediaminetetra-acetate; EPSP, excitatory postsynaptic potential; GTPyS, guanosine 5'-O-(3-thiotriphosphate); G proteins, guanosine nucleotide binding proteins; NAD, nicotinamide adenine dinucleotide; PTX, pertussis toxin; TRIS, Tris-(hydroxymethyl)aminomethane.

Abbreviations:

Nscs2/~-o

73

evidence involving second messenger systems. Activation of the GABA a receptor by GABA or ( - ) b a c l o f e n in brain slices stimulates GTPase activity6,34 and can enhance the maximal accumulation of cAMP produced by G S coupled agonists such as noradrenaline~4'~7 or vasoactive intestinal peptide, 38 while forskolin-stimulated cAMP accumulation, which is mediated by direct activation of adenylate cyclase, is attenuated, a9 Interestingly, 3-aminopropylphosphinic acid (3-APA), an agonist at the GABAB receptor which has a five- to ten-fold higher affinity than (-)baclofen, ~8'29 has the same potency as (-)baclofen in inhibiting forskolin-stimulated cAMP production in rat cerebral cortex slices but resembles a partial agonist when enhancing the noradrenalinestimulated cAMP 29providing some evidence of receptor heterogeneity. However, there is no selective antagonist which supports this possibility. A toxin produced from Bordatella pertussis ADP-ribosylates23 and thereby uncouples Gi and Go proteins from the receptor reverting it to a low-affinity state. Radioligand binding studies in bovine cerebral cortex treated with pertussis toxin (PTX) have confirmed that high-affinity [3H]GABA binding to GABAa sites is reduced and low-affinity binding increased, but the total receptor number remained constant) Asano and colleagues334 have shown that both Gi and Go proteins are implicated in GABAB receptor binding, and GTPase activity can be stimulated by GABAe receptor activation in rat brain membranes.6'34

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Unilateral injection of PTX into rat hippocampus resulted in a reduction of baclofen-stimulated GTPase activity by 57% compared with sham-operated controls. 6'17 This was associated with a complete loss of baclofen inhibition of forskolin-stimulated c A M P production and, in our study but not in that of Wojcik et al.,4° P T X had very little effect on the potentiation of responses to noradrenaline by baclofen. 6'17 PTX has been shown to suppress GABAamediated postsynaptic hyperpolarizations resulting from increased potassium conductances 2:°,36 and to block postsynaptic responses in the rat dorsal raphe nucleus. 21 A reduction in GABAB, adenosine, 5-hydroxytryptaminelA, ~2-adrenoceptor or M2-muscarinic mediated hyperpolarizations with no effect on GABAA, high potassium, 5-hydroxytryptamine3, nicotinic or M : muscarinic or fl-adrenoceptor-mediated depolarizations has also been reported in the rat superior cervical ganglion. 25 More recently the postsynaptic action of baclofen and the depression of G A B A release from interneurons in organotypic hippocampal slice cultures treated with PTX for 48 h has been shown to be mediated by PTX-sensitive G proteins. 37 However, the effect of baclofen on isolated excitatory postsynaptic potentials (EPSPs) was never altered by PTX, indicating that presynaptic GABAB receptors on excitatory axons in this preparation were PTXinsensitive as previously shown in hippocampal slices.l° Interestingly, all the actions of GABAB receptor stimulation were reduced by phorbol 12, 13 dibutyrate which stimulates protein kinase C. 37 In contrast, pre-treatment of cultured cerebellar granule neurons with PTX completely inhibited the baclofen inhibition of potassium-stimulated glutamate release. 21 Furthermore, PTX pre-treatment of cultured hippocampal pyramidal cells has been shown to abolish the presynaptic inhibition produced by baclofen at inhibitory and excitatory synapses. 27 Taken together these data suggest that there may be sub-populations of GABAB receptors which are linked with G proteins that are sensitive or insensitive to PTX. G D P , or the hydrolysis resistant analogue GDPfl S, inhibits the binding of G T P to G proteins and antagonizes the actions of baclofen while GTP, and the non-hydrolysable analogues G T P y S or G p p ( N H ) p , enhance its action. 9 The addition of G T P or its analogues to G A B A binding assays is manifested as a reduction in high-affinity G A B A B binding 1'3'j6 and can be used as an indirect means of quantifying receptor-linked G proteins. We have therefore examined firstly whether the apparent separation between pre- and postsynaptic G A B A B sites with respect to PTX sensitivity could be demonstrated autoradiographically after in vivo or in vitro administration to rat brain, and secondly whether "PTX-insensitive" GABAB receptors are coupled to G proteins by assessing the ability of GTPy S (which acts as a " m a r k e r " for G protein linkage) to reduce [3H]GABA bound to these receptors.

EXPERIMENTAL PROCEDURES lntrahippocampal pertussis toxin injections in vivo

Four male Wistar rats (Bantin and Kingman 280-300 g) were anaesthetized with equithesin (0.3 ml/100 g) and placed in a Kopf stereotaxic frame with the incisor bars 5 mm above the ear bars. Pertussis toxin (4 #g) was injected into the fight dentate gyrus in 4/~1 of saline, using a Hamilton syringe connected to a 28-gauge needle. Stereotaxic coordinates were RC 3 and L 2 mm from bregma; DV 3.5 mm from the dorsal surface of the dura. Animals were allowed to recover and four days later were anaesthetized with sodium pentobarbitone (40mg/kg) and perfuse-fixed by intracardiac administration of 200 ml 0.1% paraformaldehyde in 0.01 M phosphate-buffered saline, pH 7.4. Brains were removed and frozen with isopentane at -40°C on to chucks for sectioning. Coronal frozen sections (10 ktm) were cut throughout the hippocampus and thaw-mounted on to glass microscope slides. The sections were stored at -20°C under desiccating conditions for at least 24 h before use. Incubation o f brain slices in pertussis toxin or vehicle in vitro

Coronal and sagittal blocks of freshly dissected rat brain (250-300g) 250-400~m thick were sectioned along the midline into two halves. Each half was incubated for up to 24 h at room temperature in Krebs bicarbonate buffer of the following composition: KHSO 4 (1 mM), MgSO4 (1.2 mM), NaCI (118 mM), KC1 (9.2 mM), CaCI2 (1 mM), and containing nicotinamide adenine dinucleotide (NAD) (2 mM), ATP (1 mM), thymidine (10 mM), EDTA (1 mM), dithiothreitol (10 mM) and MgC12 (2.5 mM) and pertussis toxin (10 #g/ml Krebs; "treated") or vehicle (same volume; "control"). After incubation, individual blocks were mounted on cork chucks and frozen for the preparation of cryostat sections (10,um) as described above. Incubation o f rat brain membranes in pertussis loxin

Crude synaptic membranes were prepared from cerebral cortices, hippocampi, striata and cerebella pooled from 10-15 animals (250-300 g) per experiment according to the procedure of Bowery el al. 5 Membrane pellets, stored at -20°C, were thawed and washed three times in Tris-HCl (50 raM, pH 7.4) to remove endogenous GABA as described by Bowery et al. 5 and incubated in pre-activated PTX (7-20#g/mg membrane protein; Porton products), or an equal volume of vehicle for 30 min at 28-30°C. The reaction was stopped by adding l0 volumes of ice-cold Tris-HC1 (50 mM; pH 7.4) and the membranes pelleted by centrifugation at 10,000g for 10min. The pellets were washed immediately prior to assay for GABA B receptor binding. [3H]GABA binding

Frozen tissue sections were allowed to reach room temperature, dried and pre-incubated for 45 min in Tris-HC1 buffer (50 mM, pH 7.4) with or without the addition of CaCI2 (2.5 mM) for GABAB or GABAA sites respectively. Sections were then air-dried. The procedure for [3H]GABA binding to membrane fragments and to brain slices was essentially the same and involved incubation with 50 nM [3H]GABA with either isoguvacine (40#M) to saturate GABAA sites for GABA a receptor binding, or with (-)baclofen (100/t M) to saturate GABA Bsites for GABAA binding studies. The incubation was performed at 21~3°C for 20 min for slices and for 10 min for membranes. Nonspecific binding was defined as that binding not displaced by 100 #M isoguvacine or (-)baclofen for GABAAor GABA B receptors, respectively. For slices, the incubation was terminated by rapid aspiration of the labelled solution and two 3-s washes in fresh 50 mM Tris-HCl buffer at 4°C. The sections were then dipped in deionized water to remove salts and dried under a cold stream of air. For membranes the incubation was stopped by centrifugation at 10,000g for 3 min. The resulting pellets were washed superficially with

Regional effect of PTX on GABAA and GABAB binding ice-cold water, the tissue solubilized in Soluene and the radioactivity counted by liquid scintillation spectroscopy. In some experiments guanosine 5'-O-(3-thiotriphosphate) (GTI~S) was added to the incubation media. Protein was estimated according to the procedure of Bradford: Dry labelled sections with brain paste standards or Amersham microscales as calibration markers were exposed to LKB 3H-sensitive Ultrofilm for two tO three weeks. The films were developed using D-19 developer (Kodak, 3 min, 20°C) and fixed for 5 rain (Unifix, Kodak). The resulting autoradiograms were analysed using a Quantimet 970 Image analyser (Cambridge Research Instruments). Specific binding was determined by subtraction of background values for each area. All data are presented as mean + S.E.M.

Materials [3H]GABA (67-105 Ci/mmol) was obtained from Amersham International. (-)Baclofen was kindly supplied by CIBA Creigy Ltd and isoguvacine was a gift from Dr P Krogsgaard Larsen. All other chemicals were obtained from BDH or Sigma Ltd and were of Analar or equivalent grade. RESULTS

Effect of intrahippocampai pertussis toxin on GABAA and GABAs receptor binding GABAA binding sites were present in the pyramidal cell layers of CAI, CA2 and, at a lower density, in CA3 and CA4. They were also present in the dendrites of the pyramidal cells covering the stratum oriens and the stratum radiatum and, at a slightly lower density, in the stratum lacunosum moleculare. A higher binding density of GABAA sites was apparent in both the granular cell layer and the molecular cell layer of the dentate gyrus when compared with the pyramidal cell layers (Fig. 1). The density of G A B A B binding, although generally lower than that of GABAA sites in the hippocampal formation, was higher in the dentate gyrus, in particular in the molecular layer, compared with Ammon's horn. In C A I - 4 , GABAn sites were present on the dendrites in the stratum lacunosum moleculare and, at similar densities, in the stratum oriens and radiatum regions. Much less were present in the pyramidal cell layer as previously n o t e d : The highest binding density was observed in the stratum lacunosum moleculare at the intersection of fields CA2 and CA3 (Fig. 1). Quantitative analysis of GABAA and GABAB binding site densities in the hippocampus of animals pre-treated in vivo with P T X is shown in Table 1. At least six sections from each of four rat brains were used for each mean value. PTX injection into the dentate gyrus did not significantly affect G A B A A binding density (Table 1) whereas it produced a generalized decrease in GABAB binding in the hippocampus. This decrease was greatest in the molecular layer of the dentate gyrus (54.7%) while in Ammon's horn the decrease was greater in CA1 than in CA3 (Table 1). The effect of PTX on G A B A B binding was not only restricted to the area surrounding the site of injection but was also decreased in the ventral hippocampus, remote from the injection site (Fig. 1, lower panel).

75

Effect of pertussis toxin on whole brain slices in vitro To assess whether the limited effects of PTX we observed in vivo were due to regional differences within the hippocampus or because of restricted diffusion, we have compared GABAB binding densities in thick (1-2 mm) brain tissue blocks incubated in vitro in PTX (10 #g/ml) or vehicle for 24 h. Under these conditions G A B A a binding densities in vehicle were consistent with previously reported data. 7 Figure 2 shows autoradiograms of G A B A Bbinding in sagittal rat brain sections taken from approximately the same plane in each hemisphere in the presence (lower panel) or absence of PTX (upper panel). In the presence of PTX, GABAB binding densities were significantly reduced, but not abolished, in all brain areas examined with the notable exception of the corpus striatum (Table 2). The largest reductions in GABAB binding were found in the molecular layer of CA2 and in the cerebellum while more modest reductions were found in the thalamic nuclei. In the corpus striatum GABAB receptors appeared to be insensitive to PTX.

Effect of guanosine 5'-O-(3-thiotriphosphate) and pertussis toxin on GABAB binding in vitro G T P y S binds to all G proteins and reduces highaffinity binding to G A B A B receptors in rat cerebral cortex membranes with an ICs0 of approximately 4 n M in adult rat brain membranes. ~ W e have therefore used the reproducible reduction in G A B A B binding produced by GTPyS as an internal assay marker for G protein linkage to GABAB receptors with which the more variable effect of PTX could be compared. GABAB binding densities were measured in the presence and absence of GTPyS (2/zM) in rat brain slices prepared from nine animals. G A B A B binding density (means +- S.E.M.) in the cerebral frontal cortex was 18.0 + 1.8 nCi/mg in the absence and 4.39 _+ 0.4 nCi/mg in the presence of GTPyS (2/~ M). In the same animals GABAn binding in the corpus striatum was 6.7 +_ 6nCi/mg without and 1.85 _+ 0.25 nCi/mg with GTPyS (2/aM). GTI~,S also reduced G A B A B binding in the cerebellar molecular layer from 23.4 _+ 1.8 nCi/mg to 8.0 _ 1.8 nCi/mg, a reduction of 66.3+_6.5%. Thus, GTPyS reduced specifically bound GABAB to a similar extent in the cerebral cortex (74.6 __. 1.6%), cerebellum (66.3 + 6.5%) and corpus striatum (74.2 + 2.5%) suggesting that although approximately 66-74% of the high-affinity GABAB binding sites in all of these areas are linked with G proteins, those in the corpus striatum appear to be either insensitive to PTX or subject to greater tissue hindrance to the diffusion of PTX than the other areas examined. To assess this possibility we compared the effect of PTX with that of GTPyS on GABAB binding in membranes prepared from cerebral cortex, hippocampus and cerebellum, three areas which were highly responsive to the effects of PTX, and the

76

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LEFT

RIGHT

Fig. 1. Autoradiograms of [3H]GABA binding to (top right) GABA^ and (bottom) GABA e sites in rat brain slices. PTX (4/zg) in 4 #1 saline was injected into the right dentate gyrus in four male Wistar rats under equithesin anaesthesia. After four days rats were killed and the brains perfusc-fixed with 0.1% parafomaldehyde in 0.01 M phosphate-buffered saline, pH 7.4. Brains were processed for receptor autoradiography as described in Experimental Procedures.

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corpus striatum which appeared to be PTX-insensitive. The data are presented in Fig. 3. Preliminary experiments demonstrated that there was no effect of incubation at 29°C for 30 min on GABAB receptor binding in any brain region. In membranes that had not been treated with PTX, the amount of [3H]GABA that was specifically bound to GABAB sites (expressed as fmol/mg protein) was 5 2 5 _ 69 in the cortex (55.7 _ 6.1% of total binding), 150.9 -t- 22.2 in the corpus striatum (31.7 _ 3.4% of total), 199.9 + 51 in the hippocampus (48.2+ 5.5% of total) and 344.1 + 47.8 in the cerebellum (47.4 ___6.4% of total). PTX treatment reduced specific GABAB binding by 22.8 ___12.4% (to 385.4 + 55.9 fmol/mg) in the cortex (n = 6), by 36.4 + 14.6% (to 127.7 + 43.8 fmol/mg) in the hippocampus (n = 3) and by 4 2 . 4 _ 13.2% (to 217 + 62 fmol/mg) in the cerebellum (n = 6). No significant reduction was seen in the striatal membranes from 150.9 _ 22.2 to 142.3 _ 27.2 fmol/mg (n = 6). The addition of GTPy S reduced GABAB binding in all brain areas by approximately the same percentage (77-88%) irrespective of the response to PTX pretreatment (Fig. 3).

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The distribution of GABAA and GABAB binding sites in the rat hippocampal formation observed in the present study are in accordance with previous results. 7 We have shown that in vivo administration of PTX resulted in a marked reduction in GABAB binding in the injected hippocampus compared with the contralateral side. In contrast, GABAA binding was not significantly altered indicating that the effect of PTX was specific. When whole brain blocks were incubated in the presence of PTX we observed that the reduction in GABAB binding was not restricted to the hippocampus but was more generalized throughout the brain. The largest reduction in GABAB binding was found in the stratum lacunosum moleculare of the hippocampus, the cerebral cortex and molecular layer of the cerebellum, while the corpus striatum appeared to be predominantly insensitive to PTX. This was surprising because in this region baclofen exhibits a marked effect on adenylate eyelase activation by forskolinY Since pertussis toxin is thought to selectively inactivate Gi/Go proteins by transferring an ADP-ribose unit from N A D to the ct subunit of the G protein to prevent its association with the receptor, 3,24 and the baclofen inhibition of adenylate cyclase activation is sensitive to the toxin, 41 a decrease in binding might be expected. A wealth of electrophysiological1°'22and biochemical 3,9 evidence exists for a reduction of GABAB function by PTX. Wojcik and colleagues4° have injected PTX (6/~g) i.c.v, into rats and four days later found that G proteins in the 40,000--41,000 mol. wt band prepared from cerebral cortex and hippocampus were ADP-ribosylated by 38% and 52%, respectively. This is in accord with our previous

78

C. KNOTT et al.

CONTROL

PERTUSSIS TOXIN

Fig. 2. Autoradiograms of [3H]GABA binding to GABAa sites in rat brain: upper panel control and lower panel PTX. Sagittal blocks of freshly dissected rat brain were incubated for 24 h at room temperature in Krebs bicarbonate buffer containing PTX vehicle (control) or PTX (10#g/ml). Tissue blocks were processed for autoradiography as described in Experimental Procedures. findings of reduced G A B A a binding and GTPase activity by approximately 54% and 57%, respectively, in rat hippocampus. 6,17 Similarly, Asano and colleagues found that when bovine cerebral cortex

membranes were incubated in the presence of 50/lg/ml PTX for 30 min /n vitro, GABAB binding was reduced by approximately 600/0. 3 These authors obtained a greater reduction of G A B A B binding by

Regional effect of PTX on GABAA and GABAB binding

79

Table 2. Effects of pertussis toxin on GABAB binding in rat brain regions (nCi/mg) Brain region

Control

Cortex: lamina I-II Cortex: lamina Ill-IV Corpus striatum CAI: stratum oriens CAI: stratum radiatum CA2: lacunosum moleculare CA3: stratum oriens CA3: stratum radiatum Dentate gyrus Dorsolateral geniculate Mediolateral geniculate Cerebellar molecular layer Corpus callosum

16.1 + 2.30 12.4 _ 2.10 4.96 _ 0.46 7.47 + 1.08 7.65 __+1.12 13.5 + 2.10 7.34 + 1.05 8.04 _ 1.13 11.4 + 1.75 16.2_ 1.60 15.9 + 1.50 23.1 + 3.10 1.34 __+0.17

Pertussis toxin I1.7 +__1.30" 8.40 + 0.77** 4.71 +__0.27 5.10 + 0.76** 5.83 ___0.74* 7.65 _ 1.67"* 5.96 + 1.06 7.13 _ 0.80 8.57 + 1.33" 13.4 _+ 1.00"* 12.5 + 1.90" 14.4 + 4.10" 1.4 + 0.20

Regional effect of PTX on GABA Bbinding density in rat brain sections taken from tissue blocks incubated in activated PTX (10 #g/ml) for 24h. Data are mean_S.E.M, from >five sections taken from eight animals. Significant difference compared to control: *P < 0.05; **P < 0.02. Student's paired t-test, two-tailed. P T X than we have found in the present study which may be due to their higher dose and possibly greater activity of PTX, or may reflect incomplete A D P ribosylation by P T X in our experiments. P T X incorporates N A D covalently into G proteins. 23 It may be more appropriate therefore to express the P T X " d o s e " as # g P T X / m g tissue protein, in spite of the fact that this does not take into account variations in its biological activity. Furthermore the effect of PTX is both time and concentration depend e n t ) ,23 Because the GABAB receptor becomes progressively inactivated with increasing temperature above 30°C and with incubation times exceeding 600

500

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30 min 3 we optimized our assay for the effect of PTX and for the stability of GABAB binding by incubating the membranes for 30 min at 28-30°C in the presence of 1 5 . 7 _ 5 . 3 # g P T X / m g cerebral cortex, 10.3 + 1.9 # g P T X / m g corpus striatum, 7.3 + 1.9/tg P T X / m g hippocampus and 10.5 + 1.4/~g P T X / m g cerebellum. The relative insensitivity of the corpus striatum to the effects of PTX in vitro led us to examine the effect of GTP),S alone and in combination with PTX on G A B A B receptor binding in these membranes. The use of G T P y S was based on observations that [3H]Gpp(NH)p, a non-hydrolysable analogue of G T P which has been used to autoradiographically localize G proteins in rat brain, showed a high binding density in the corpus striatum 12 (also personal observations). It had also been noted that G T P y S reduced the high-affinity component of [3H]GABA binding to GABAB sites to a maximum value of 82--85% 3'16'26 which is in accord with our own findings. We reasoned, therefore, that only PTX-insensitive, n o n - G protein linked lowaffinity GABAB sites should remain in PTX-treated membranes after GTPTS displacement.

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Cortex Striatum

Hippocampus Cerebellum

Fig. 3. The effect of PTX (7-20/ag/mg protein; hatched bars) on GABAB binding in membranes prepared from cerebral cortex, striatum, hippocampus and cerebellum pooled from 10 animals per experiment (n = 6 experiments). Membranes were incubated in pre-activated PTX (10-30/zg/mg) or vehicle for 30min at 28-30°C. GABAB binding was performed in the presence or absence of GTPyS (2/zM) in triplicate as described in Experimental Procedures.

In accordance with previous findings our data confirmed that the addition of GTP~,S reduces G A B A a binding in all areas studied irrespective of the tissue sensitivity to PTX. This would suggest that G A B A e receptors in the corpus striatum may be linked predominantly to PTX-insensitive G proteins while in other adult rat brain areas GABAB receptors may be linked to PTX-sensitive and to PTX-insensitive G proteins.

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