This project seeks to establish if ethanol affects GABAergic transmission, using an in vitro functional assay for measuring GABA responses and studying the effect of ethanol on the binding of radioligands to various sites on the oligomeric GABA receptor complex. The over all objective is to study the effect of ethanol on GABA function and on various binding sites on the oligomeric GABA receptor complex in the same preparation and using physiological conditions. The approach to be used to study the effect of ethanol on GABA-induced 36C1-influx in primary spinal cord cultured neurons. These neurons respond to GABA by increasing conductance to C1-, and this response is potentiated by benzodiazepines (BZ) and anesthetic barbiturates. Using these cultured neurons, we will do a pharmacological characterization of ethanol interactions with GABA responses. Studies will also be done to determine if ethanol-like pentobarbital has a direct effect on 36C1-influx in these neurons. Preliminary results indicate that these neurons take up (3H)GABA with the expected pharmacology, contain coupled GABA and benzodiazepine receptors, and GABAA agonists increase 36C1-influx. GABA-induced 36C1-influx in these neurons is potentiated by diazepam, pentobarbital and ethanol, and blocked by bicuculline and picrotoxin. Further, the cells will be grown in the presence of ethanol to determine if chronic presence of ethanol alters the properties and the function of GABA receptor-ionophore complex. This will be achieved by studying the effects of in vitro ethanol exposure on the binding of (3H)flunitrazepam (BZ receptor), (35S)t-butylbicyclophosphorothionate (picrotoxin receptor) and (3H)GABA and (3H)muscimol (GABA receptor) sites in intact spinal cord cultured neurons, and by studying the effect of chronic exposure of these cells with ethanol, on these binding sites and GABA-induced 36C1-influx. A thorough study of the pharmacological analysis of ethanol interactions with GABA gated C1- channels and various binding components in a defined preparation derived from the vertebrate CNS will add significantly to our understanding of the involvement of GABAergic system in the actions of ethanol.
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