Regulation of alcohol intake by purinergic P2X4 receptors

描述（由申请人提供）：酒精使用障碍（AUD）具有惊人的社会经济影响，但目前的治疗策略在很大程度上是不够的，部分原因是缺乏关于乙醇作用的分子靶点的信息。目前的提案测试了P2X4受体（P2X4Rs）代表开发预防和/或治疗AUD药物的新靶点的假设。这一目标尚未探讨。我们已经组建了一个多学科的研究团队，他们将共同使用最先进的遗传，分子，化学和行为技术来系统地测试中脑边缘多巴胺（DA）系统中的靶点，这些靶点将适用于药物开发。拟议的研究将把实验室发现转化为发现和开发预防和治疗AUD的新疗法的机会。目的1研究将测试大脑多巴胺奖赏系统中的P2X4R调节乙醇摄入的假设。研究1，我们将评估组成型敲除p2rx4基因（P2X4KO）对酒精摄入量的影响（24小时接触和黑暗中饮酒[DID]测试）。研究2，使用慢病毒介导的短发夹RNA（shRNA）的靶向递送，我们将敲低丘脑核（NAc）和/或腹侧被盖区（VTA）中的P2X4R表达，以深入了解中脑边缘DA系统关于P2X4R减少/阻断的短期效应的具体贡献。这项工作也将有助于澄清的作用，P2X4 R在引起或调节乙醇的影响，而没有潜在的发展变化，可能伴随组成性科斯。我们将使用一种新的，选择性的P2X4R拮抗剂，5-BDBD和开发新的相关探针，作为第三种方法来确定P2X4Rs在中毒中的作用。P2X4Rs的成功药理学操作将为P2X4Rs作为未来AUDs治疗的可行靶点的重要性提供新的见解。研究三，运用分子生物学、脑片电生理学和快速扫描循环伏安法，开始研究P2X4受体在中脑边缘DA系统NAc和VTA中的作用。这项工作将提供有关中脑边缘DA系统中P2X4R的机制见解，以及它们如何相互作用以调节小鼠的乙醇摄入量和行为（研究1和2）。目的2研究将使用伊维菌素（IVM），一种P2X4Rs的正调节剂，与P2X4KO小鼠组合，以测试刺激P2X4Rs将减少乙醇摄入的假设。此外，我们将设计、合成和测试对P2X4R具有阳性、阴性或无效活性的新化学实体，这些化学实体可以作为药理学探针来询问P2X4R系统。总之，这项工作将进一步推动我们的长期目标，即确定控制酒精摄入的神经化学机制和关键大脑区域。此外，这项工作将确定新的靶点，并将开始开发新的化合物，用于预防和/或治疗AUD的治疗剂的转化开发。

DESCRIPTION (provided by applicant): Alcohol use disorders (AUDs) have a staggering socio-economic impact, yet current therapeutic strategies are largely inadequate, due, in part, to a lack of information regarding the molecular target(s) on which ethanol acts. The current proposal tests the hypothesis that P2X4 receptors (P2X4Rs) represent a novel target for the development of drugs to prevent and/or treat AUDs. This target has not yet been explored. We have assembled a multidisciplinary team of investigators that together will use state of the art genetic, molecular, chemical and behavioral techniques to systematically test for targets in the mesolimbic dopamine (DA) system that will be amenable for drug development. The proposed studies will translate laboratory findings into opportunities to discover and develop novel therapeutics for the prevention and treatment of AUDs. Aim 1 studies will test the hypothesis that P2X4Rs within the brain dopamine reward systems regulate ethanol intake. Study 1, we will evaluate the effects of constitutive knockout of the p2rx4 gene (P2X4KO) on alcohol intake (24 hr access and drinking in the dark [DID] tests). Study 2, using targeted delivery of lentiviral-mediated short hairpin RNA (shRNA), we will knockdown P2X4R expression in the nucleus accumbens (NAc) and/or ventral tegmental area (VTA) to gain insights into specific contributions of the mesolimbic DA system regarding short- term effects of P2X4R reduction/blockade. This work will also help clarify the role of P2X4Rs in causing or modulating the effects of ethanol without the potential developmental alterations that may accompany constitutive KOs. We will use a new, selective P2X4R antagonist, 5-BDBD and develop new related probes, as a third method to determine P2X4Rs role in intoxication. Successful pharmacological manipulation of P2X4Rs would provide new insights into the importance of P2X4Rs as a viable target for future therapies for AUDs. Study 3, using molecular biological, brain slice electrophysiology and fast scan cyclic voltammetry we will begin to investigate the role of P2X4Rs in the NAc and VTA of the mesolimbic DA system. This work will provide mechanistic insights regarding P2X4Rs in the mesolimbic DA system and how they interact to modulate ethanol intake and behavior in mice (Studies 1 and 2). Aim 2 studies will use ivermectin (IVM), a positive modulator of P2X4Rs, in combination with P2X4KO mice, to test the hypothesis that stimulation of P2X4Rs will decrease ethanol intake. In addition, we will design, synthesize and test new chemical entities with positive, negative or null activity on P2X4R that can serve as pharmacologic probes to interrogate the P2X4R system. Taken together, the work will further our long term goal of identifying neurochemical mechanisms and key brain regions that control alcohol intake. Moreover, this work will identify new targets and will begin to develo new compounds for the translational development of therapeutic agents to prevent and or treat AUDs.