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型谷氨酸受体亚型-5受体（mGluR 5）参与酒精的强化作用。初步证据表明，长期饮酒选择性地增加mGluR 5 mRNA在大鼠的延髓核（NAcb）中的表达，这与酒精在大脑区域中的慢性受体抑制一致，这是成瘾发展的基础。本项目目标1中的实验将更充分地表征mGluR 5参与酒精强化的特征。这些研究将确定操作性酒精自我管理是否改变mGluR 5蛋白表达，以及mGluR 5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mGluR 5拮抗剂降低了乙醇的增强作用。目标2中的研究将通过确定表达mGluR 5的边缘脑区域中mGluR 5受体活性的功能意义来扩展这一观察结果。实验将确定在腹侧被盖区（VTA）、NAcb（核和壳）或内侧前额叶皮层（mPFC）中位点特异性输注mGluR 5拮抗剂对乙醇增强反应的影响。这些研究将确定mGluR 5对酒精强化的调节是否具有脑区域特异性。有证据表明，乙醇通过PKC依赖性机制在体外抑制mGluR 5功能。类似地，我们的初步数据表明，mGluR 5阻断减少了野生型小鼠的乙醇自我给药，但对携带PKC β无效突变的小鼠没有影响。因此，特异性目标3将表征mGluR 5调节酒精强化中基于潜在细胞信号传导的机制。使用PKC-Glu敲除和野生型小鼠，实验将确定mGluR 5阻断是否以PKC-Glu依赖性方式降低乙醇强化。这些研究将检查mGluR拮抗剂对酒精和蔗糖强化反应的影响，以及对乙醇诱导的自发活动变化的影响，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，包括基因转录因子环腺苷酸反应元件结合蛋白（CREB），它与成瘾行为有关。mGluR 5的激活通过PKC依赖性机制增加p-CREB水平，而慢性饮酒降低NAcb中的p-CREB水平。由于已知乙醇通过PKC依赖性机制抑制mGluR 5活性，因此这些发现表明，自我给药乙醇可能通过mGluR 5/PKC（可能是PKC β）依赖性机制降低p-CREB。目的4中的实验将确定乙醇诱导的mGluR 5和p-CREB表达的变化是否依赖于PKCe，以及这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PKC β依赖性方式抑制mGluR 5介导的p-CREB变化。这些研究，检查mGluR 5和PKC β在慢性乙醇对转录调控的调节作用的介导中的功能联系，这对成瘾的适应性变化有影响。这些发现将有助于药物治疗的发展，以治疗与酗酒有关的问题。

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.