Molecular Mechanisms of Ethanol Reinforcement

描述（由申请人提供）：本申请的主要目的是表征代谢性谷氨酸受体亚型5受体（mGluR5）在酒精强化作用中的参与。初步证据表明，长期饮酒选择性地增加了大鼠伏隔核（NAcb）中mGluR5 mRNA的表达，这与酒精对大脑中一个区域的慢性受体抑制是一致的，该区域是成瘾发展的基础。本项目Aim 1的实验将更全面地描述mGluR5参与酒精强化的过程。这些研究将确定持续性酒精自我给药是否会改变mGluR5蛋白表达，以及mGluR5受体活性是否在功能上调节大鼠的乙醇强化。其他初步数据表明，在NAcb中微量注射mGluR5拮抗剂可降低乙醇的强化作用。Aim 2的研究将通过确定mGluR5受体活性在表达mGluR5的边缘脑区域的功能意义来扩展这一观察结果。实验将确定mGluR5拮抗剂在腹侧被盖区（VTA）、nab（核和壳）或内侧前额叶皮层（mPFC）特定部位输注对乙醇增强反应的影响。这些研究将确定mGluR5对酒精强化的调节是否具有脑区域特异性。有证据表明乙醇在体外通过pkc依赖机制抑制mGluR5的功能。同样，我们的初步数据表明，mGluR5阻断降低了野生型小鼠的乙醇自我给药，但对携带PKC-epsilon零突变的小鼠没有影响。因此，特异性Aim 3将表征mGluR5调控酒精强化的潜在细胞信号传导机制。使用PKC-epsilon敲除和野生型小鼠，实验将确定mGluR5阻断是否以PKC-epsilon依赖的方式降低乙醇强化。这些研究将检查mGluR拮抗剂对酒精和蔗糖增强反应的影响，以及对乙醇诱导的运动活动变化的影响，以确定行为特异性。最后，慢性乙醇暴露改变了大脑中的许多分子事件，包括与成瘾行为有关的基因转录因子环AMP-反应元件结合蛋白（CREB）。mGluR5的激活通过PKC依赖机制增加了p-CREB水平，而慢性乙醇饮用则降低了NAcb中的p-CREB水平。由于已知乙醇通过PKC依赖机制抑制mGluR5活性，这些研究结果表明，自我给药乙醇可能通过mGluR5/PKC（可能是PKCepsilon）依赖机制降低p-CREB。Aim 4的实验将确定乙醇诱导的mGluR5和p-CREB表达的变化是否依赖于PKCe，以及这种变化在多大程度上受到慢性操作性乙醇自我给药史的调节。另一项研究将确定乙醇是否以PKCepsilon依赖的方式抑制mglur5介导的p-CREB变化。这些研究考察了mGluR5和PKCepsilon之间的功能联系，在介导慢性乙醇对转录调节的影响中，这对成瘾的适应性变化具有重要意义。这些发现将有助于开发药物疗法来治疗与酒精中毒有关的问题。

DESCRIPTION (provided by applicant): The primary goal of this application is to characterize the involvement of metabotropic glutamate receptor subtype-5 receptors (mGluR5) in alcohol's reinforcing effects. Preliminary evidence indicates that chronic alcohol drinking selectively increases mGluR5 mRNA expression in the nucleus accumbens (NAcb) of rats, which is consistent with chronic receptor inhibition by alcohol in a brain region that is fundamental to development of addiction. Experiments in Aim 1 of this project will more fully characterize the involvement of mGluR5 in alcohol reinforcement. These studies will determine if operant alcohol self-administration alters mGluR5 protein expression and if mGluR5 receptor activity functionally regulates ethanol reinforcement in rats. Other preliminary data indicate that microinjection of an mGluR5 antagonist in the NAcb decreases the reinforcing effects of ethanol. Studies in Aim 2 will extend this observation by determining the functional significance of mGluR5 receptor activity in limbic brain regions that express mGluR5. Experiments will determine the effects of site-specific infusion of an mGluR5 antagonist in the ventral tegmental area (VTA), NAcb (core and shell), or medial prefrontal cortex (mPFC) on ethanol reinforced responding. These studies will determine if mGluR5 regulation of alcohol reinforcement is brain-region specific. Evidence indicates that ethanol inhibits mGluR5 function in vitro through a PKC-dependent mechanism. Similarly, our preliminary data indicate that mGluR5 blockade decreases ethanol self-administration in wildtype mice but has no effect in mice carrying a null mutation for PKC-epsilon. Thus, specific Aim 3 will characterize a potential cell- signaling based mechanism in mGluR5 regulation of alcohol reinforcement. Using PKC-epsilon knockout and wildtype mice, experiments will determine if mGluR5 blockade decreases ethanol reinforcement in a PKC-epsilon dependent manner. These studies will examine the effects of mGluR antagonists on alcohol and sucrose reinforced responding, and on ethanol-induced changes in locomotor activity to determine behavioral specificity. Finally, chronic ethanol exposure alters numerous molecular events in the brain including the gene transcription factor cyclic AMP- responsive element binding protein (CREB), which has been implicated in addictive behavior. Activation of mGluR5 increases p-CREB levels via a PKC dependent mechanism, whereas chronic ethanol drinking decreases p-CREB levels in the NAcb. Since ethanol is known to inhibit mGluR5 activity through a PKC-dependent mechanism, these findings suggest that self-administered ethanol may decrease p-CREB through an mGluR5/PKC (possibly PKCepsilon) dependent mechanism. Experiments in Aim 4 will determine if ethanol-induced changes in mGluR5 and p-CREB expression are dependent on PKCe, and the extent to which this is modulated by a history of chronic operant ethanol self-administration. Another study will determine if ethanol inhibits mGluR5-mediated changes in p-CREB in a PKCepsilon dependent manner. These studies, examine the functional linkage between mGluR5 and PKCepsilon in the mediation of chronic ethanol effects on transcriptional regulation, which has implications for adaptive changes in addiction. These findings will aid development of pharmacotherapeutics to treat problems associated with alcoholism.