The objectives of this study are to determine whether persistent alterations in the GABA receptor complex can provide a molecular explanation for the development of physical dependence on ethanol in an animal model of alcoholism. Chronic intermittent administration of ethanol (CIE) to rats has many features resembling human alcohol abuse behavior, including relevance to the long-lasting susceptibility to readdiction. The numerous episodes of ethanol induced depression of the CNS and the following rebound hyperexcitability have been shown to exert a kindling-like effect during the period of hyperexcitability. When ethanol is administered to rats under a regimen designed to promote kindling effects, the development of ethanol dependence is associated with a decreased seizure threshold to the convulsant pentylenetetrazol (PTZ), known to interact with the GABAA receptor-chloride ion channel complex. This hyperexcitability (kindling) to PTZ lasts for at least 40 days after cessation of ethanol. Neurochemical studies have been directed towards determining whether ethanol dependence and changes of seizure threshold can be correlated with alterations in the molecular properties of the GABAA receptor complex. PTZ receptors, assayed by the binding of [35S]TBPS, are increased in ethanol dependence, and benzodiazepine- insensitive binding sites for the alcohol antidote drug [3H]Rol5-4513 are decreased, while binding of other radioligands and allosteric modulators for the GABAA receptor complex such as neurosteroids are not changed. Polymerase chain reaction (PCR) measurements show that the ratio of receptor subunit mRNAs is altered, suggesting the production of novel receptor oligomeric subtypes. The subunit composition of these novel receptors will be determined by protein chemistry including~photoaffinity labeling and Western blotting with subunit-specific antibodies; their properties will be studied by electrophysiology on Xenopus oocytes expressing recombinant receptors of defined subunit composition. Further comparisons of alcohol-dependent and naive rats will study function of GABAA receptors using 36Cl- flux, radioligand binding/ autoradiography, and potential long-lasting protein modification by phosphorylation.
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