Benzodiazepine agonist and inverse agonist actions on GABA(A) receptor-operated chloride channels. II. Chronic effects of ethanol

Mice were made tolerant to and dependent on ethanol by administration of a liquid diet. γ-Aminobutyric acid (GABA) receptor-dependent uptake of ³⁶Cl^- by mouse cortical microsacs was used to study the actions of benzodiazepine (BZ) agonists and inverse agonists. Chronic exposure to ethanol attenuated the ability of a BZ agonist, flunitrazepam, to augment muscimol stimulated uptake of ³⁶Cl^- and enhanced the actions of BZ inverse agonists, Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]- benzodiazepine-3-carboxylate) and DMCM (methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate), to inhibit GABA(A) receptor-operated chloride channels. Augmentation of chloride flux by pentobarbital was not reduced by chronic ethanol exposure. Attenuation of flunitrazepam efficacy was transient and returned to control levels within 6 to 24 hr after withdrawal from ethanol, but increased sensitivity to Ro15-4513 was observed as long as 8 days after withdrawal. Chronic exposure to ethanol did not alter [³H]SR 95531 ([2-(3'-carbethoxy-2'propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide] binding to low-affinity GABA(A) receptors or muscimol stimulation of chloride flux; and did not alter [³H]Ro15-4513 or [³H]flunitrazepam binding to central BZ receptors or allosteric modulation of this binding by muscimol (i.e., muscimol-shift). These results suggest that chronic exposure to ethanol reduces coupling between BZ agonist sites and the chloride channel, and may be responsible for the development of cross-tolerance between ethanol and BZ agonists. In contrast, coupling between BZ inverse agonist sites and the chloride channel is increased. This action may be involved in the development of tolerance to or dependence on ethanol and could contribute to the anxiety and seizure susceptibility observed during ethanol withdrawal.