MECHANISMS OF DEPENDENCE PATHOGENESIS

这项建议的总体目标是阐明影响酒精依赖发展的伽马氨基丁酸(GABA)A受体适应性变化的分子机制。乙醇在大脑中有多个作用部位，但GABA/A受体的直接或间接调节可能是乙醇的行为作用。此外，长期摄入乙醇会导致对乙醇的耐受性和依赖性的形成。戒酒，尤其是反复戒酒，会显著增加中枢神经系统的兴奋性和焦虑感。大量证据表明，这些行为和神经适应涉及GABA/A受体的药理特性的显著适应。此外，过去一段时期的研究已经证实，GABA/A受体提交的适应伴随着这些变化，并且在不同的大脑区域之间存在显著差异。我们计划将重点放在PKCGamma和PKCepsilon在介导GABA/A受体适应中的作用。我们推测，PKC与GABA/A受体的相互作用可能决定了受体亚单位的适应性，并可能是这些适应性差异的地区差异的基础。具体目的1将确定酒精依赖是否改变了GABA/A受体与PKC同工酶的联系，从而改变了乙醇诱导的GABA/A受体功能和癫痫敏感性中的适应性蛋白。特殊目的2将利用这些小鼠来确定PKCGamma和PKCepsilon是否差异地改变了乙醇对膜表达和内化的影响，从而改变了特定的GABA/A受体。最终目的将再次使用突变的小鼠模型来研究PKCGamma和PKCepsilon在GABA/A受体的磷酸化状态中的作用。载体介导的基因传递用于体内组织特异性拯救PKCGamma或PKCepsilon将被用来建立PKC变化与随后对受体膜表达、内化和功能的影响之间的因果关系。对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对PKCGamma或PKCepsilon基因缺失的影响的可能性。我们预测，这些实验将描绘特定的GABA/A受体适应，参与酒精依赖诱导的癫痫敏感性(荷包牡丹碱癫痫阈值)和乙醇自我给药(与克莱德·霍奇合作)的增强。这些研究将为酒精诱导GABA/A受体适应的分子基础提供重要的机制信息，从而影响酒精耐受和依赖的发展。

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.