MECHANISMS OF DEPENDENCE PATHOGENESIS

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The overall goal of this proposal is to elucidate the molecular mechanisms that underlie alterations in gamma-aminobutyric acid (GABA)A receptor adaptations that influence the development of ethanol dependence. Ethanol has several sites of action in the brain, but direct or indirect modulation of GABA/A receptors may behavioral actions of ethanol. Moreover, prolonged ethanol consumption results in the development of tolerance and dependence upon ethanol. Withdrawal from ethanol, and particularly repeated withdrawals from ethanol, produce marked increases in CNS excitability and anxiety. Substantial evidence suggests that these behavioral and neural adaptations involve marked adaptations in the pharmacological properties of GABA/A receptors. Furthermore, research over the previous funding period has established that GABA/A receptor submit adaptations accompany these changes and differ markedly across brain regions. We plan to focus on the role of PKCgamma and PKCepsilon in mediating GABA/A receptor adaptations. We hypothesize that PKC interactions with GABA/A receptors may determine receptor subunit adaptations and may underlie the regional differences in these differences in these adaptations. Specific Aim 1 will determine if ethanol dependence alters the association of GABA/A receptors with PKC isozymes in alter ethanol-induced adaptatins in GABA/A receptor function and seizure susceptibility. Specific Aim 2 will utilize these mice to determine if PKCgamma and PKCepsilon differentially alter the effects of ethanol on membrane expression and internalization to alter specific GABA/A receptors. The final aim will investigate the role of PKCgamma and PKCepsilon in the phosphorylation state of GABA/A receptors, again using mutant mouse models. Vector-mediated gene delivery for tissue specific rescue of PKCgamma or PKCepsilon in vivo will be used to establish a cause and effect relationship between the alterations in PKC and subsequent effects on receptor membrane expression, internalization and function. Tissue specific rescue of PKCgamma or PKCepsilon as well as pharmacological challenge with PKC antagonists will also be used to control for the possibility that adaptations of other proteins contribute to the effects of genetic deletion of PKCgamma or PKCepsilon. We predict that these experiments will delineate specific GABA/A receptor adaptations involved in ethanol dependence-induced enhancement of seizure susceptibility (bicuculline seizure threshold) and ethanol self-administration (in collaboration with Clyde Hodge). These studies will provide important mechanistic information on the molecular basis of ethanol-induced adaptations in GABA/A receptors that influence the development of ethanol tolerance and dependence.