Ligands for expression cloning and isolation of GABA(B) receptors.

Outlined is the rationale behind the syntheses of radioligands [125I]CGP64213 and [125I]CGP71872, which led to the identification of cloned GABA(B) receptors 1a and 1b 17 years after the first pharmacological characterisation of native GABA(B) receptors by Bowery et al. [Nature 283 (1980) 92-94]. More recently it was shown that the N-terminal extracellular domains of GABA(B) receptors 1a and 1b contain the binding sites for agonists and antagonists [B. Malitschek et al., Mol. Pharmacol. 56 (1999) 448-454]. In order to isolate the extracellular domain(s) of GABA(B) receptors 1a (or 1b) and to purify and crystallise these proteins a third ligand [125I]CGP84963 was designed, which combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as photoaffinity moiety and 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion [W. Froestl et al., Neuropharmacology 38 (1999) 1641-1646].

Ligands for the isolation of GABA(B) receptors [corrected] .

Since the discovery that the most abundant inhibitory neurotransmitter in the mammalian brain, GABA (gamma-aminobutyric acid), interacts not only with ionotropic GABA(A) receptors, but also with metabotropic GABA(B) receptors (Bowery et al., 1980) much work has been devoted to the elucidation of the structure of GABA(B) receptors by either affinity chromatography purification or by expression cloning. In 1997 Kaupmann et al. succeeded in cloning two splice variants designated GABA(B) R1a (960 amino acids) and GABA(B) R1b (844 amino acids). Although the amino acid sequences are now known, precise information on the three-dimensional environment of the GABA(B) R1 binding site is still lacking. Recent experiments demonstrated that the amino acids of the seven transmembrane helices are not essential for ligand binding as a soluble GABA(B) receptor fragment is still able to bind antagonists (Malitschek et al., 1999). For the isolation and purification of the soluble N-terminal extracellular domain (NTED) of GABA(B) receptors potent ligands for affinity chromatography were synthesised with the aim of obtaining a crystalline receptor fragment-ligand complex for X-ray structure determination. The most promising ligand [125I]CGP84963 (K(D) = 2 nM) combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as a photoaffinity moiety separated by a spacer consisting of three GABA molecules from 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion.

Characterisation and partial purification of the GABA(B) receptor from the rat cerebellum using the novel antagonist [3H]CGP 62349.

The novel GABA(B) receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (K(d) = 0.9 nM, B(max) = 760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [3H]CGP 62349 binding (K(d) = 0.5 nM, B(max) = 1285 fmol/mg protein). Competition of [3HCGP 35348 = CGP 36742. The GABA(A) ligand isoguvacine did not displace [3H]CGP 62349 binding. Partial purification of [3H]CGP 62349 binding sites was obtained by sucrose density centrifugation and a predominant protein in the peak binding fraction was recognised by an anti-GABA(B) receptor antibody and had a molecular weight similar to the recombinant expressed GABA(B)R1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABA(B) receptor.

Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding.

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.

Morpholin-2-yl-phosphinic acids are potent GABA(B) receptor antagonists in rat brain.

The pharmacological properties of morpholin-2-yl-phosphinic acids were evaluated on GABA(B) receptors. In rat neocortical slices maintained in Mg2+-free Krebs medium, baclofen, a GABA(B) receptor agonist, produced a concentration-dependent depression of the frequency of spontaneous discharges with an EC50 of 14 +/- 5.5 microM, which was antagonised reversibly by the morpholin-2-yl-phosphinic derivatives. The order of potency was 3-[(3S,6R)-6-[(cyclohexylmethyl)hydroxyphosphinoylmethyl- morpholin-3-yl]benzoic acid (CGP 76290A) (pA2 = 7.1 +/- 0.05) > its enantiomer 3-[(3R,6S)-6-[(cyclohexylmethyl)hydroxyphosphinoylmethyl]-++ +morpholin-3-yl]benzoic acid (CGP 76291A) (pA2 = 6.8 +/- 0.1) > cyclohexylmethyl-[(2R',5S')-5-(3-nitrophenyl)-morpholin-2-++ +ylmethyl]phosphinic acid (CGP 71978) (pA2 = 6.5 +/- 0.05) > cyclohexylmethyl-[(2R,5S)-5-phenyl-morpholin-2-ylmethyl++ +]phosphinic acid (CGP 71980) (pA2 = 6.3 +/- 0.15) > its enantiomer cyclohexylmethyl-[(2S,5R)-5-phenyl-morpholin-2-ylmethyl++ +]phosphinic acid (CGP 71979) (pA2 = 5.8 +/- 0.1). An open chain analogue of CGP 76290A, CGP 56999A (3-[1(R)-[(3-cyclohexylmethyl-hydroxyphosphinoyl)-2(S)-hydro xypropyl-amino]-ethyl]benzoic acid lithium salt) gave a pA2 of 6.6 +/- 0.2. In GABA(B) receptor binding assays, CGP 71982 (the racemic mixture of CGP 76290A and CGP 76291A), CGP 76290A, CGP 76291A, CGP 71978, CGP 71980 and CGP 71979 had IC50 values against [3H]CGP 27492 binding of 8, 1.85, 69, 124, 326 and 1460 nM, respectively. In electrically-evoked [3H]GABA release from rat cortical slices, CGP 71982, CGP 71978, CGP 71980 and its enantiomer CGP 71979, antagonised GABA(B) autoreceptors with EC150 values of 2.5, 33, 181 and 474 nM, respectively. These compounds form a novel class of potent GABA(B) receptor antagonists.

Human gamma-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels.

gamma-Aminobutyric acid type B receptors (GABABRs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABABRs inhibit neurotransmitter release by down-regulating high-voltage activated Ca2+ channels, whereas postsynaptic GABABRs decrease neuronal excitability by activating a prominent inwardly rectifying K+ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABABRs, hGABABR1a (hR1a) and hGABABR1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABABRs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABABRs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABABR1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.

Developmental changes of agonist affinity at GABABR1 receptor variants in rat brain.

Recently, two N-terminal splice variants of the metabotropic receptor for GABA (gamma-amino-butyric acid) were cloned. Here, we describe an antiserum that recognizes the two receptor variants. We demonstrate that these proteins are identical with GABAB receptors that are photoaffinity labeled with [125I]CGP71872 in rat brain. The C-terminal epitopes recognized by the antiserum are conserved in several vertebrate species but not in chicken. No hints for the existence of additional closely related receptor subtypes or variants are found in double-labeling experiments with antibody and photoaffinity ligand. Western blot analysis reveals widespread expression of the GABABR1 receptor proteins in rat brain with the highest level of expression at early postnatal stages. The binding affinity of the GABAB receptor agonist L-baclofen at native R1a and R1b variants is similar. In early postnatal development the affinity at R1a and R1b is 10-fold lower than in adult brain and gradually increases with aging.

Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors.

GABA (gamma-amino-butyric acid), the principal inhibitory neurotransmitter in the brain, signals through ionotropic (GABA(A)/ GABA(c)) and metabotropic (GABA(B)) receptor systems. Here we report the cloning of GABA(B) receptors. Photoaffinity labelling experiments suggest that the cloned receptors correspond to two highly conserved GABA(B) receptor forms present in the vertebrate nervous system. The cloned receptors negatively couple to adenylyl cyclase and show sequence similarity to the metabotropic receptors for the excitatory neurotransmitter L-glutamate.

Binding characteristics of gamma-hydroxybutyric acid as a weak but selective GABAB receptor agonist.

The aim of this study was to reexamine the concept that gamma-hydroxybutyric acid (GHB) is a weak but selective agonist at gamma-aminobutyric acidB (GABAB) receptors, using binding experiments with several radioligands. Ki values of GHB were similar (approximately equal to 100 microM) in three agonist radioligand assays for GABAB receptors, [3H]baclofen (beta-para-chlorophenyl-gamma-aminobutyric acid), [3H]CGP 27492 (3-aminopropyl-phosphinic acid) and [3H]GABA, in the presence of the GABAA receptor agonist isoguvacine with rat cortical, cerebellar and hippocampal membranes. In competition experiments between GHB and the GABAB receptor antagonist, [3H]CGP 54626 (3-N [1-{(S)-3,4-dichlorophenyl}-ethylamino]-2-(S)-hydroxypropyl cyclo-hexylmethyl phosphinic acid), the IC50 values were significantly increased with 300 microM of 5'-guanyl-imidodiphosphate (Gpp(NH)p), which suggested that guanine nucleotide binding proteins (G-proteins) modulate GHB binding on GABAB receptors. The inhibition by GHB of [3H]CGP 27492 binding in cortical membranes was not altered in the presence of 0.3 or 3 mM of the two GHB dehydrogenase inhibitors, valproate and ethosuximide. Thus, GHB is not reconverted into GABA by GHB dehydrogenase. Taken together, the results of this study demonstrated that GHB is an endogenous weak but selective agonist at GABAB receptors.

Regional differences of the inhibition of GABAB ligand binding by the GTP analogue Gpp(NH)p.

In order to determine whether the interactions between GABAB receptors and G-proteins differ in several brain areas, we have used the reduction in high-affinity GABAB binding by the GTP analogue Gpp(NH)p as an internal assay marker for G-protein linkage to GABAB receptors. The results indicate that Gpp(NH)p inhibits the binding of the GABAB receptor agonist [3H]CGP 27492 (80 to 95%) in a biphasic manner between 0.1 nM and 1 mM. The IC50 for high-affinity sites is significantly higher in cerebellum (70 nM, 53% of binding sites) than in cortex, hippocampus, corpus striatum and thalamus (15-30 nM, 63-73% of binding sites). The IC50S of the low-affinity sites in hippocampus and cortex (170 microM and 210 microM, respectively) were significantly higher than the IC50S in cerebellum, thalamus and corpus striatum (18-39 microM). All these binding sites are sensitive to pertussis toxin (PTX; 7-15 micrograms/mg protein), implicating that they are linked either to Gi or to Gzero proteins. The two binding sites observed (high affinity, nM and low affinity, microM for Gpp(NH)p) and the regional dependence in affinity of these sites may originate either from different GABAB receptor subtypes, different G-proteins or different coupling mechanisms between G-proteins and GABAB receptors. Whereas the PTX site of G-protein linked to GABAB receptors changes with age [24], the GTP binding site does not differ between peripubertal rats (5-6 weeks) and adults rats (10-12 weeks).

Activity of 2-azaspiro[4.5]decane-6-carboxylates as GABA-uptake inhibitors.

Novel GABA analogous spirocyclic amino acid esters 7a-d and 8a-d were prepared and investigated for interaction with GABA-A and GABA-B receptors as well as the GABA uptake system. Starting from known bromoethyl lactones 1 or 2 and arylalkylamines spirocyclic hydroxyalkyl lactams 3a-d and 4a-d were obtained and reduced by LiAlH4 to yield spirocyclic hydroxymethyl pyrrolidines 5a-d and 6a-d. Oxidation by Jones reagent followed by subsequent esterification gave the title compounds 7a-d and 8a-d which represent conformationally restricted analogues of GABA. Whereas the new spirocyclic amino acid esters 7a-d and 8a-d showed no activity at GABA receptors they proved to be active as GABA uptake inhibitors. An examination of the relationship between structure and GABA uptake inhibition revealed a strong dependence of activity upon the length of the alkyl chain in N-arylalkyl substituents and upon the ring size of underlying spirocyclic system.

Effects of the putative P-type calcium channel blocker, R,R-(-)-daurisoline on neurotransmitter release.

The alkaloid and medicinal herb constituent, R,R-(-)-daurisoline, was originally reported to be a N-type Ca2+ channel blocker, but newer evidence indicates that it is a blocker of P-type Ca2+ channels. To clarify its specificity with respect to N- and P-channels, we compared its effects on the electrically induced release of endogenous glutamate, 3H-GABA and 3H-noradrenaline, from brain slices with those of omega-agatoxin IVA and omega-conotoxin GVIA. Like omega-agatoxin IVA (but with about 1000-fold lower potency), and unlike omega-conotoxin GVIA, R.R-(-)-daurisoline inhibited the release of 3H-GABA and glutamate, with IC50 values of 8 and 18 microM. However, inhibition particularly of 3H-GABA release was more complete than by omega-agatoxin IVA, indicating interaction with one or more additional voltage-sensitive Ca2+ channels, possibly the Q-type. Its potency to inhibit glutamate release elicited either electrically, by veratrine or by high concentrations of K+ was similar, in contrast to sodium channel blockers. The effects of R,R-(-)-daurisoline on the release of 3H-noradrenaline, 3H-dopamine and 3H-acetylcholine were in agreement with previous knowledge from experiments with omega-agatoxin IVA suggesting an involvement of P-channels. A weak inhibition of 3H-noradrenaline release at 10 microM, similar to that by omega-agatoxin IVA at 0.03 microM, was occluded by alpha 2-antagonistic properties and could be unmasked in presence of rauwolscine. At 10 microM, it also inhibited electrically evoked 3H-dopamine and 3H-5-hydroxytryptamine release and caused a marked spontaneous release of all three monoamines in a reserpine-like manner. Spontaneous and evoked release of 3H-acetylcholine was inhibited by about 25% at 10 microM. In radioligand binding studies, R,R-(-)-daurisoline interacted with alpha 1- and alpha 2-adrenoceptors, 5-HT2 and muscarinic cholinergic receptors with IC50 values close to 1 microM, and with mu opiate receptors even with 0.18 microM. Atropine reduced the weak inhibitory effect of R,R-(-)-daurisoline on 3H-acetylcholine release somewhat, suggesting that it was brought about by both P channel blockade and cholinergic agonist activity. The effect on 3H-GABA release was unaffected by naloxone, indicating that the interaction of R,R-(-)-daurisoline with mu opiate receptors is antagonistic. The pattern of effects on neurotransmitter release observed with R,R-(-)-daurisoline resembles that of omega-agatoxin IVA and supports previous electrophysiological data suggesting that the compound blocks P-type voltage-sensitive Ca2+ channels. However, the more complete blockade of amino acid release by R,R-(-)-daurisoline suggests interaction with additional Ca2+ channel subtypes. Although it does also possess other pharmacological properties, we think that the compound is suitable to test whether blockade of glutamate release via voltage-sensitive Ca2+ channels is a viable concept to obtain novel neuroprotective and/or anticonvulsant compounds.

[Binding and electrophysiology of the muscarinic antagonist QNB in the mammalian retina]. / Bindung und Elektrophysiologie des muskarinischen Antagonisten QNB in der Säugetiernetzhaut.

PURPOSE: Based upon the extensive information from various laboratories on cholinergic enzymes and receptors in mammalian retina as well as cholinergic effects on retinal neurons we became interested in studying (1) retinal binding of a muscarinic antagonist, Quinuclidinyl benzilate (QNB) and (2), effects of the antagonist on retinal information processing. METHODS: Eyes from deeply anesthetized cats were used for homogenate of freshly isolated retina in the binding study, or they were arterially perfused for electrophysiology in vitro. The electroretinogram (ERG) and the compound action potential of the optic nerve (optic nerve response, ONR) were recorded under rod- and cone-stimulating conditions. QNB was infused intraarterially for 10-30 min, followed by washout (avoiding recycling or extraocular metabolism). RESULTS: 3H-QNB revealed a high affinity to muscarinic receptors with a dissociation constant KD of 0.27 nM and a relatively high density of muscarinic binding sites of 110 fmol per mg protein. QNB enhanced the amplitude of the ERG b-wave, but decreased dose-dependently and reversibly the components of the ONR. In addition, we observed a moderate vasoconstriction as indicated by a slight, dose-related decrease in flow rate of perfusion. CONCLUSION: The biochemical data on binding of 3H-QNB in connection with the marked electrophysiological changes induced by QNB suggest a substantial contribution of muscarinic cholinergic transmission in the cat retina.

GABA and glutamate release affected by GABAB receptor antagonists with similar potency: no evidence for pharmacologically different presynaptic receptors.

1. The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]-GABA in the absence or presence of 10 microM of the GABAB agonist, (-)-baclofen and of endogenous glutamate in the presence of (-)-baclofen were compared. 2. The concentrations of the compounds half maximally increasing [3H]-GABA release (EC50's) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]-CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A > or = CGP 55845 A > CGP 52432 > or = CGP 56433 A > CGP 57034 A > CGP 57070 A > or = CGP 57976 > CGP 51176 > CGP 35348). 3. Likewise, the concentrations causing half-maximal increases of [3H]-GABA in the absence or presence of (-)-baclofen, and of endogenous glutamate in the presence of (-)-baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (-)-baclofen's effects on glutamate over [3H]-GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model. 4. Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies. 5. In particular, our results with CGP 35348 and CGP 52432 do not support the hypothesis that GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings represent subtypes with different pharmacology.

The actions of orally active GABAB receptor antagonists on GABAergic transmission in vivo and in vitro.

The goal of this report is to present the results obtained with three new GABAB receptor antagonists. CGP 54062 has an IC50 in a GABAB binding test of 0.013 microM which is roughly 2500-fold lower than one of the most potent blockers known so far, CGP 35348 (IC50 = 34 microM). CGP 46381 and CGP 36742 have IC50s of 4.9 and 36 microM respectively. The latter two compounds are the first orally active GABAB receptor antagonists. All three compounds bind to the GABAB receptor selectively, and are inactive in a number of binding tests assessing the compounds' affinity to various other receptor sites. The effect of these blockers on GABAergic transmission was investigated in the CA1 area of hippocampal slices. The Schaffer collateral/commissural fibers were stimulated and the evoked postsynaptic potentials were recorded intracellularly in pyramidal neurons. The three antagonists blocked the late inhibitory postsynaptic potential with the following rank order of potency CGP 54062 > 46381 > 36742 approximately 35348. These findings support the hypothesis that these potentials are mediated by GABAB receptors. Orally administered CGP 36742 and CGP 46381 block the neuronal depression induced by iontophoretically applied baclofen in anaesthetised rats. Up to a dose of 10 mg/kg i.v. CGP 54062 was inactive and thus does not appear to cross the blood-brain barrier at this dose. In anaesthetised rats the effects of the three new GABAB antagonists and of CGP 35348 were investigated on the paired-pulse inhibition of the population spikes evoked in the CA1 area of the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Brofaromine: a monoamine oxidase-A and serotonin uptake inhibitor.

Brofaromine is a tight-binding, reversible inhibitor of monoamine oxidase-A (MAO-A), with concomitant serotonin (5-HT) uptake-inhibiting properties. In psychopharmacologic investigations, the compound shows the properties expected of an MAO inhibitor, antagonizing the effects of reserpine, tetrabenazine, and 5-hydroxytryptophan in rats and mice, and suppressing rapid eye movement sleep in cats. Brofaromine showed antidepressant-like activity in a rat social conflict test. In radioligand binding assays, brofaromine exhibited weak or no interaction with alpha 1- and alpha 2-noradrenergic, 5-HT1, 5-HT2, 5-HT3, cholinergic, histamine H1 and H2, mu-opiate, GABAA, benzodiazepine, adenosine, neurotensin, and substance P receptors. Comparison of in vitro and in vivo potencies to inhibit 5-HT uptake with those of reference drugs, and direct evidence in patients and volunteers suggest that 5-HT uptake inhibition plays a role in the clinical profile of brofaromine.

Experimental absence seizures: potential role of gamma-hydroxybutyric acid and GABAB receptors.

We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R(-)-baclofen are mediated by the GABAB receptor, whereas only the induction of SWD by GHB is dependent on GABAB receptor mediation, the decrease in cGMP being only partially so. Taken together with the observations of Marescaux et al. (1992), these results indicate that GABAB receptors are of primary importance in experimental absence epilepsy and that GABAB receptor antagonists may represent a new class of anti-absence drugs.