Chronic use of ethanol leads to tolerance and physical dependence. The exact molecular mechanisms by which ethanol produces behavioral effects, tolerance, and physical dependence have yet to be defined. However, several lines of evidence implicate GABA and NMDA receptor systems in these events. The present proposal will examine the effect of chronic, and intermittent, ethanol treatments on these receptors. The proposed studies will be conducted in mammalian cultured cortical neurons, under controlled conditions, independent of pharmacokinetic variability, and by using a comprehensive approach that involves investigation of binding, coupling interactions, functional assays, and the expression of specific GABA/A and NMDA receptor subunit mRNA, and polypeptide levels, and immunoprecipitation studies. A major advantage of the cultured neurons is that they reflect the diversity of cell types of intact CNS neurons, and provide an ideal in vitro model system to study the effect of chronic drug treatment. Our reason for using cultures from cerebral cortex is based on the presence of high density of GABA/A and NMDA receptors in this region, and the importance of this region in mediating the actions of ethanol. Since GABA/A and NMDA receptors exist in several isoforms, it is feasible that chronic, and intermittent, ethanol treatments may produce a differential alteration of receptor subunits, with increases in some, decreases in some, and/or no changes in other subunits. This could result in altered receptors with or without an apparent change in the ligand binding. The following specific aims will test this hypothesis: I) compare and characterize the effects of chronic, and intermittent, ethanol treatment on the binding of ligands to the GABA/A and NMDA receptor complex; II) does chronic, and intermittent, ethanol treatments alter the coupling between various sites associated with the GABA/A and NMDA receptors; III) does chronic, and intermittent, ethanol treatments alter the efficacy of GABA/A and NMDA receptor-mediated transmission; IV) determine the effects of chronic, and intermittent, ethanol treatment on GABA/A and NMDA receptor subunit mRNAs; and V) determine the effects of chronic, and intermittent, ethanol treatment on GABA/A and NMDA receptor subunit polypeptide levels using Western blot and immunoprecipitation studies.
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