MECHANISMS OF DEPENDENCE PATHOGENESIS

本提案的总体目标是阐明影响乙醇依赖发展的γ -氨基丁酸（GABA）A受体适应性改变的分子机制。乙醇在大脑中有几个作用部位，但直接或间接调节GABA/A受体可能是乙醇的行为作用。此外，长时间的乙醇消耗导致对乙醇的耐受性和依赖性的发展。退出乙醇，尤其是反复退出乙醇，可显著增加中枢神经系统的兴奋性和焦虑。大量证据表明，这些行为和神经适应涉及GABA/A受体药理特性的显著适应。此外，先前资助期的研究已经确定，GABA/A受体会随这些变化而适应，并且在大脑区域之间存在显著差异。我们计划关注PKCgamma和PKCepsilon在介导GABA/A受体适应中的作用。我们假设PKC与GABA/A受体的相互作用可能决定受体亚基适应，并可能是这些适应差异的区域差异的基础。特异性目的1将确定乙醇依赖性是否改变GABA/A受体与PKC同工酶的关联，从而改变乙醇诱导的GABA/A受体功能和癫痫易感性的适应。Specific Aim 2将利用这些小鼠来确定PKCgamma和PKCepsilon是否会不同地改变乙醇对膜表达和内化的影响，从而改变特异性GABA/A受体。最终目的将研究PKCgamma和PKCepsilon在GABA/A受体磷酸化状态中的作用，同样使用突变小鼠模型。通过载体介导的基因传递，在体内对PKCgamma或PKCepsilon进行组织特异性救援，将建立PKC的改变与随后对受体膜表达、内化和功能的影响之间的因果关系。PKCgamma或PKCepsilon的组织特异性拯救以及PKC拮抗剂的药理学挑战也将用于控制其他蛋白质的适应性导致PKCgamma或PKCepsilon基因缺失的可能性。我们预测这些实验将描述特定的GABA/A受体适应，涉及乙醇依赖诱导的癫痫易感性增强（双库兰癫痫阈值）和乙醇自我给药（与Clyde Hodge合作）。这些研究将为乙醇诱导的GABA/A受体适应影响乙醇耐受性和依赖性的分子基础提供重要的机制信息。

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.