Chronic Ethanol Effects on CNS Opiate Receptors

描述（由申请人提供）：长期的乙醇消费及其持续的强化是一个持续的主要健康和社会问题。在中枢神经系统中，乙醇摄入的增强与内源性阿片受体作用的阿片释放的增强有关。一种非选择性阿片类拮抗剂药物，纳曲酮，被批准用于人类减少乙醇消耗，渴望和复发。在动物和人体试验中使用选择性更强的丁型受体拮抗剂纳曲本，表明丁型受体在酒精的使用和滥用中起重要作用。然而，内源性阿片和阿片受体导致持续乙醇消耗的机制尚不清楚。本提案概述了在大鼠动物模型中使用组织化学和药理学技术来研究可能将慢性乙醇消耗与三角洲阿片受体调节联系起来的机制。要验证的主要假设是，在慢性乙醇摄入期间，前脑和中脑区域的免疫反应性δ阿片受体表达增加，并且表达的变化伴随着受体与G蛋白功能偶联的减少。受体表达的变化可能会影响这些脑区维持乙醇消耗的神经元胞内信号通路。目的1：在长期暴露于乙醇的大鼠中，定位和量化前脑伏隔核（NA）、中脑腹侧被盖区（VTA）以及其他大脑区域的三角洲阿片受体亚型水平。将使用共聚焦显微镜，结合计算机辅助定量，对免疫荧光标记的δ阿片受体，与mu受体的表达进行比较。我们将比较对照组和摄入乙醇的动物，以及停止使用乙醇的动物大脑区域的神经元。目的2：通过与受体偶联的比较，确定慢性乙醇消耗是否会影响NA和VTA神经元中delta受体与第二信使系统的功能偶联，以及其他脑区域。我们将使用最近开发的[35S]-GTPgammaS方法，通过该方法，受体连接的g蛋白激活可以通过对大脑区域切片的放射自显影技术直接测量。可以检测阿片配体的直接作用，以确定它们是否在功能上与选定脑区的神经元中的g蛋白相连。通过定量免疫组织化学方法和功能偶联研究，研究慢性乙醇消耗后动物体内δ受体配体的相互作用，将有可能确定慢性乙醇对受体亚型特异性的影响。这也将有可能研究可能发生的功能变化，受体退出消费后。未来可能会以delta受体为目标治疗慢性酒精中毒。

DESCRIPTION (provided by applicant): Chronic ethanol consumption and its continued reinforcement is an ongoing major health and societal problem. In the Central nervous system, the reinforcement of ethanol intake has been linked to enhanced release of endogenous opiates which act at opiate receptors. A non-selective opiate antagonist drug, naltrexone, is approved for humans to reduce ethanol consumption, craving and relapse. Use of the more selective delta receptor antagonist, naltriben, in animals and human trials, suggests that delta receptors are important in use and abuse of alcohol. However, mechanisms by which endogenous opiates and opiate receptors lead to continued ethanol consumption remain unclear. This proposal outlines the use of both histochemical and pharmacologic techniques to study mechanisms which may link chronic ethanol consumption to modulation of the delta opiate receptor, in a rat animal model. The major hypothesis to be tested is that immunoreactive delta opiate receptor expression in the forebrain and midbrain regions is increased during chronic ethanol intake, and the change in expression is accompanied by a reduction in functional coupling of the receptor to G proteins. Changes in delta receptor expression may affect neuronal intracellular signaling pathways in those brain areas to maintain ethanol consumption. Aim #1: To localize and quantify levels of delta opiate receptor subtype in the nucleus accumbens (NA) of the forebrain, and the midbrain ventral tegmental area (VTA), as well as other brain regions, in rats chronically exposed to ethanol. Confocal microscopy will be used, together with computer-assisted quantification, for immunofluorescent-labeled delta opiate receptors, with comparisons to mu receptor expression. We will compare neurons in brain areas of control and ethanol-consuming animals, and in animals which have been withdrawn from ethanol. Aim #2: To determine if chronic ethanol consumption affects functional coupling of delta receptors to second messenger systems in neurons of the NA and VTA, as well as other brain areas, with comparisons to mu receptor coupling. We will use a recently developed method with [35S]-GTPgammaS, whereby receptor-linked G-protein activation can be measured directly with autoradiographic techniques on sections of the brain areas. The direct effects of delta or mu opiate ligands can be examined to determine if they are functionally linked to G-proteins in the neurons of the selected brain areas. By using both quantitative immunohistochemical methods, and functional coupling studies, to examine the interactions of delta receptor ligands in animals after chronic ethanol consumption, it will be possible to determine receptor subtype-specific effects of chronic ethanol. It will also be possible to study the functional changes that may occur in the receptors after withdrawal from consumption. A future potential may be then to target delta receptors for treating chronic alcoholism.