Ashok K. Mehta

An update on GABAA receptors

Recent advances in molecular biology and complementary information derived from neuropharmacology, biochemistry and behavior have dramatically increased our understanding of various aspects of GABAA receptors. These studies have revealed that the GABAA receptor is derived from various subunits such as alpha1-alpha6, beta1-beta3, gamma1-gamma3, delta, epsilon, pi, and rho1-3. Furthermore, two additional subunits (beta4, gamma4) of GABAA receptors in chick brain, and five isoforms of the rho-subunit in the retina of white perch (Roccus americana) have been identified. Various techniques such as mutation, gene knockout and inhibition of GABAA receptor subunits by antisense oligodeoxynucleotides have been used to establish the physiological/pharmacological significance of the GABAA receptor subunits and their native receptor assemblies in vivo. Radioligand binding to the immunoprecipitated receptors, co-localization studies using immunoaffinity chromatography and immunocytochemistry techniques have been utilized to establish the composition and pharmacology of native GABAA receptor assemblies. Partial agonists of GABAA receptors are being developed as anxiolytics which have fewer and less severe side effects as compared to conventional benzodiazepines because of their lower efficacy and better selectivity for the GABAA receptor subtypes. The subunit requirement of various drugs such as anxiolytics, anticonvulsants, general anesthetics, barbiturates, ethanol and neurosteroids, which are known to elicit at least some of their pharmacological effects via the GABAA receptors, have been investigated during the last few years so as to understand their exact mechanism of action. Furthermore, the molecular determinants of clinically important drug-targets have been investigated. These aspects of GABAA receptors have been discussed in detail in this review article.

Synaptic and nonsynaptic localization of GABAA receptors containing the α5 subunit in the rat brain

The α5 subunit of the GABAA receptors (GABAARs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of α5 subunit‐containing GABAARs (α5‐GABAARs) both in culture and in the intact brain. A large pool of α5‐GABAARs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the α5‐GABAARs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of α5‐GABAARs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic α5‐GABAARs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex. J. Comp. Neurol. 499:458–470, 2006.

Chronic ethanol administration increases the binding of the benzodiazepine inverse agonist and alcohol antagonist [3H]RO15-4513 in rat brain

Chronic ethanol treatment which produced intoxication and physical dependence in rats, produced an increase in the specific binding of ethanol antagonist [3H]RO15-4513 in rat brain cerebral cortex and cerebellum, but not in hippocampus and striatum. The increase in both the regions was due to an increase in the number (Bmax) of receptor sites. These results suggest that the RO15-4513 binding sites on the oligomeric GABA receptor complex are altered following chronic ethanol administration, and support the notion of a unique role of RO15-4513 as an ethanol antagonist.

Role of N-methyl-D-aspartate (NMDA) receptors in experimental catalepsy in rats

N-methyl-D-aspartic acid (NMDA; 40 mg/kg, i.p.) did not elicit catalepsy, but it potentiated the cataleptic effect of haloperidol and GABAB receptor agonist, baclofen. MK-801 (0.2 mg/kg, i.p.), NMDA-receptor antagonist, reversed haloperidol- but not baclofen-induced catalepsy. MK-801 also potentiated the anticataleptic effect of scopolamine and bromocriptine against haloperidol-induced catalepsy. Dihydropyridine (DHP) calcium-channel antagonists such as nimodipine and nitrendipine (10 mg/kg, i.p.), reversed the anticataleptic effect of MK-801, and potentiated the cataleptic effect of haloperidol, as well as baclofen. These observations indicate the involvement of NMDA receptors in catalepsy, and suggest a potential clinical implication of NMDA-receptor antagonists in Parkinsons disease.

Comparison of anticonvulsant effect of ethanol against NMDA-, kainic acid- and picrotoxin-induced convulsions in rats

The anticonvulsant effect of ethanol against N-methyl-D-aspartic acid-(NMDA), kainic acid-, and picrotoxin-induced convulsions was studied in rats. Ethanol (2 g/kg, ip) offered protection against these agents, and it was most effective against picrotoxin and least effective against kainic acid. MK801, NMDA receptor antagonist, also provided protection against these agents. However, it was most effective against NMDA and least effective against kainic acid. Ineffective doses of MK801 (0.1 mg/kg, ip) and ethanol (0.5 g/kg, ip), when administered concurrently, had a facilitatory anticonvulsant effect, thereby providing protection against mortality following severe convulsions induced by NMDA or picrotoxin, but not against kainic acid. The protective effect of ethanol against NMDA- and kainic acid-induced neurotoxicity, in contrast to picrotoxin-induced toxicity, was not reversed by imidazodiazepine, Ro 15-4513, an ethanol antagonist. Furthermore, Ro 15-4513 did not produce any proconvulsant effect with NMDA or kainic acid. These findings suggested that the anticonvulsant actions of ethanol may be attributed to its ability to antagonize NMDA-mediated excitatory responses and facilitate the GABAergic transmission.

Chronic GABA exposure down-regulates GABA-benzodiazepine receptor-ionophore complex in cultured cerebral cortical neurons

Cerebral cortical cultured neurons were characterized for GABA-benzodiazepine (BZ) receptor complex, and the effect of chronic exposure of cortical neurons to GABA on GABA-BZ receptor system was investigated. In the intact cells, the [3H]flunitrazepam binding was rapid and saturable, with an apparent Kd of 4.2 +/- 1.5 nM and Bmax of 776 +/- 54 fmol/mg protein. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by various BZ receptor ligands such as Ro15-1788, DMCM, Ro15-4513, clonazepam, alprazolam, diazepam and zolpidem, and enhanced by GABA, muscimol and pentobarbital. GABA induced enhancement of 36Cl-influx in a concentration-dependent manner (EC50 = 9 +/- 2 microM). Chronic exposure of the cultured neurons to GABA resulted in a reduced [3H]flunitrazepam, [3H]GABA, [3H]Ro15-1788, [3H]Ro15-4513 and [35S]TBPS binding, a reduced enhancement of [3H]flunitrazepam binding by GABA, and a reduced GABA-induced 36Cl-influx susceptible to reversal by concomitant exposure of the cultures to R 5135, a GABAA-receptor antagonist. These findings indicate that cerebral cortical cultured neurons provide an ideal model to study GABA-BZ receptor complex using binding and 36Cl-influx assays, and chronic exposure of cortical cultures to GABA leads to a down-regulation of GABA-BZ receptor system. It is a GABAA receptor-mediated slow process.

Chronic ethanol treatment alters the behavioral effects of Ro 15-4513, a partially negative ligand for benzodiazepine binding sites

Pentylenetetrazol (PTZ)-induced convulsion were studied in control, chronic ethanol-maintained, and ethanol-withdrawal rats. The convulsive doses of PTZ varied among the different groups of rats. Ethanol-maintained rats required higher doses of PTZ to produce convulsions, compared to control and ethanol-withdrawal rats. The partially negative ligands for benzodiazepine binding sites, Ro 15-4513 (2 mg/kg, i.p.) and FG 7142 (20 mg/kg, i.p.) produced proconvulsant effect in saline (control) and ethanol-withdrawal rats as they potentiated the effect of subconvulsive dose of PTZ. A higher dose of Ro 15-4513 (4 mg/kg, i.p.), but not FG 7142 (up to 80 mg/kg, i.p.), also produced proconvulsant effect in ethanol-maintained rats. Furthermore, Ro 15-4513 (5, 10 mg/kg, i.p.), but not FG 7142 (up to 80 mg/kg, i.p.), produced clonic-tonic seizures of short duration in ethanol-withdrawal rats. These effects of Ro 15-4513 and FG 7142 were reversed by diazepam (2 mg/kg, i.p.), as well as by the GABA-neutral Ro 15-1788 (10 mg/kg, i.p.), thereby, indicating the involvement of central benzodiazepine receptors in the action of Ro 15-4513 and FG 7142. These observations suggest that chronic ethanol treatment selectively alters the receptor sensitivity to Ro 15-4513, an ethanol antagonist and partially negative ligand for BZ sites, and this observation supports the notion that ethanol effects are more susceptible to reversal by the imidazobenzodiazepine as compared to other negative ligand for BZ binding sites.

Are GABAB receptors involved in the pharmacological effects of ethanol

The interaction of ethanol with GABAB-receptor system and the selectivity of phaclofen for GABA-receptor subtypes were investigated by employing an in vitro model of 36Cl-influx assay in mammalian cultured neurons and also in vivo models of picrotoxin- and NMDA-induced convulsions in rats. Ethanol (20 mM), without having any effect per se, potentiated the effect of GABA on 36Cl-influx, whereas at concentration 50 mM, ethanol activated Cl(-)-channels directly in mice spinal cord cultured neurons. In contrast, (-)baclofen (100 microM) did not modify the effects of GABA or ethanol on 36Cl-influx. Similarly, phaclofen (500 microM), as well as pertussis toxin (140 ng/ml, overnight incubation) did not modify these effects. Interestingly, phaclofen (200 micrograms i.c.v.) reversed the anticonvulsant effect of ethanol, but not that of pentobarbital or diazepam or progabide, against picrotoxin-induced convulsions in rats. However, phaclofen failed to modify the anticonvulsant effect of ethanol against NMDA-induced convulsions. These observations indicate that phaclofen is devoid of GABAA-receptor blockade property, and the anticonvulsant effect of ethanol against picrotoxin may be mediated through the activation of both GABA-receptor subtypes.

Quantitative autoradiographic analysis of the new radioligand [3H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6h-benzo[a] [7]annulen-6-ylidene) ethanoic acid ([3H]NCS-382) at γ-hydroxybutyric acid (GHB) binding sites in rat brain

(2E)-(5-Hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid (NCS-382) is an antagonist for gamma-hydroxybutyric acid (GHB) at GHB receptor sites. Advantages of using [(3)H]NCS-382 over [(3)H]GHB in radioligand binding studies are that unlike GHB, NCS-382 does not appear to bind to, activate, or interfere with the functioning of GABA(B) or GABA(A) receptors, either directly or indirectly. Herein we establish a protocol for use of [(3)H]NCS-382 by quantitative autoradiography. GHB was used to define non-specific binding, since it displaced [(3)H]NCS-382 to an extent equivalent to NCS-382. Among many areas of brain examined, two regions in which high specific binding of [(3)H]NCS-382 occurred were the hippocampus and cerebral cortex. Areas such as the striatum and nucleus accumbens exhibited intermediate levels of specific binding. No or very low binding was observed in other areas such as the cerebellum and dorsal raphe nucleus. The distribution of GHB binding sites as defined by [(3)H]NCS-382 suggests that GHB may play a role in neuromodulation or neurotransmission in frontal brain areas.

Comparison of anticonvulsant effect of pentobarbital and phenobarbital against seizures induced by maximal electroschock and picrotoxin in rats

Pentobarbital and phenobarbital exhibited anticonvulsant effect against maximal electroshock (MES) and picrotoxin-induced seizures in rats. Bicuculline, a GABAA receptor antagonist, reversed the anticonvulsant effect of pentobarbital, but not of phenobarbital, at a dose having no effect per se. Although picrotoxin (2 mg/kg, IP) potentiated MES seizures, it did not reverse the anticonvulsant effect due to either pentobarbital or phenobarbital. GABAB receptor antagonists such as delta-amino-n-valeric acid and homotaurine failed to modify the anticonvulsant effect due to pentobarbital or phenobarbital. Furthermore, GABAA agonist muscimol but not baclofen, a GABAB receptor agonist, exhibited the anticonvulsant effect against MES-induced seizures. However, baclofen when combined with sub-effective dose of pentobarbital or phenobarbital offered protection against MES seizures. Pentobarbital and phenobarbital were effective in almost equivalent doses against MES, as well as against picrotoxin-induced seizures. These observations indicated that pentobarbital exhibits anticonvulsant effect against MES seizures through the involvement of GABAA receptors, and activation of GABAB receptors alone does not seem to play any significant role in MES seizures and in the anticonvulsant effect of pentobarbital. However, activation of GABAB receptor does potentiate the facilitatory effect of barbiturates on GABAAergic transmission and in their anti-MES effect. Moreover, these results also suggest that the anticonvulsant effect of barbiturates against MES-seizures may involve other mechanisms in addition to GABAAergic transmission.