Chronic Ethanol Effects on CNS Opiate Receptors

描述(由申请人提供)：慢性乙醇消费及其持续增加是一个持续存在的重大健康和社会问题。在中枢神经系统中，酒精摄入量的增加与作用于阿片受体的内源性阿片类药物的释放增加有关。一种非选择性阿片类拮抗剂药物纳曲酮已被批准用于人类减少酒精消耗、渴求和复发。在动物和人体试验中使用更具选择性的Delta受体拮抗剂Naltriben，表明Delta受体在酒精的使用和滥用中起着重要作用。然而，内源性阿片剂和阿片受体导致持续乙醇消费的机制仍不清楚。这项建议概述了使用组织化学和药理学技术来研究在大鼠动物模型中可能将慢性酒精消耗与增量阿片受体调节联系起来的机制。有待检验的主要假说是，在慢性酒精摄入过程中，前脑和中脑区域免疫反应性增量阿片受体的表达增加，并且这种表达的变化伴随着受体与G蛋白功能偶联的减少。Delta受体表达的变化可能会影响这些脑区神经元细胞内信号通路，以维持酒精消费。目的1：对慢性酒精暴露大鼠前脑伏隔核(NA)、中脑腹侧被盖区(VTA)及其他脑区的阿片受体亚型进行定位和定量。将使用共聚焦显微镜，结合计算机辅助定量，对免疫荧光标记的增量阿片受体进行检测，并与u受体表达进行比较。我们将比较对照组和饮用酒精的动物以及戒掉酒精的动物大脑区域的神经元。目的#2：确定慢性酒精摄入是否影响NA和VTA以及其他脑区神经元中Delta受体与第二信使系统的功能偶联，并与Mu受体偶联进行比较。我们将使用最近开发的一种方法[35S]-GTP-GammaS，通过该方法，可以通过放射自显影技术直接测量大脑部分区域的受体关联G蛋白激活。可以检查Delta或Mu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神经元中的G蛋白有关。通过使用定量免疫组织化学方法和功能偶联研究，研究慢性酒精摄入后动物体内Delta受体配体的相互作用，将有可能确定慢性乙醇的受体亚型特异性效应。还有可能研究停用后受体可能发生的功能变化。那么，未来的潜力可能是将Delta受体作为治疗慢性酒精中毒的靶点。

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.