Effect of ethanol on dopamine receptor trafficking

描述（由申请人提供）：多巴胺受体（D1）将用绿色荧光蛋白（GFP）标记，并插入辛德毕斯病毒中。这种病毒将被注射到器官型培养物中，其中包括中脑边缘通路，腹侧被盖区（VTA）到丘脑核（NAcc）的投射，这是一种被认为在滥用药物（包括酒精）的强化特性中很重要的通路。具体而言，将D1受体注射到NAcc中。在这种构型下，D1受体将如体内一样位于突触后。这些外植体将以两种方式暴露于乙醇：急性和慢性。对于急性乙醇暴露，将使用多光子显微镜来寻找表达我们的GFP标记的多巴胺受体的神经元，同时在外植体上灌注aCSF，然后将乙醇添加到灌注液中。在急性乙醇暴露后，应该看到膜表达的GFP标记的多巴胺受体的差异。在长期暴露的情况下，外植体将在含有乙醇的培养基中孵育。与对照相比，这些外植体可能显示出致敏作用，其中膜上表达的标记受体较少。此外，利血平预处理将用于消耗多巴胺，然后进行急性乙醇暴露，以确定乙醇是否仅通过多巴胺释放引起受体内化。

DESCRIPTION (provided by applicant): Dopamine receptors (D1) will be tagged with green fluorescent protein (GFP), and inserted into the sindbis virus. This virus will be injected into organotypic cultures that include the mesolimbic pathway, the projections of the ventral tegmental area (VTA) to the nucleus accumbens (NAcc), a pathway believed to be important in the reinforcing properties of drugs of abuse, including alcohol. Specifically, D1 receptors will be injected into the NAcc. With this configuration D1 receptors will be located post-synaptically as in vivo. These explants will be exposed to ethanol in two ways: acutely and chronically. For acute ethanol exposure, multi-photon microscopy will be used to find neurons that are expressing our GFP tagged dopamine receptors while aCSF is perfused over the explant, then ethanol will be added to the perfusate. A difference in membrane expressed GFP tagged dopamine receptors should be seen after acute ethanol exposure. With chronic exposure the explants will be incubated in medium that contains ethanol. These explants may show a sensitization effect with less tagged receptors expressed on the membrane, compared to control. Additionally pretreatment with reserpine will be used to deplete dopamine, then acute ethanol exposure will be conducted to determine if ethanol is working only through dopamine release to cause internalization of receptors.