Regulation of Alcohol Intake by Purinergic P2X4 Receptors

我们已经确定了P2X4受体(P2X4Rs)是预防和/或治疗药物开发的靶点 酒精使用障碍(AUD)。这一假说源自令人信服的系统；遗传的、药理学的 行为证据表明，乙醇(Etoh)摄入量与P2X4R活性之间存在负相关关系。 支持这一假设的是，我们发现p2rx4基因敲除小鼠(P2X4KO)摄入的乙醇明显更多 而不是野生型(WT)产仔对照。我们和其他人发现伊维菌素(IVM)，一种正变构模-- P2X4Rs的ToR显著拮抗ATP门控的P2X4Rs对Etoh的抑制和减少Etoh的摄取 老鼠和老鼠。我们已经组建了一个多学科的团队，他们将共同利用遗传学、分子、 电生理、化学和行为技术系统地探索中脑边缘靶点 多巴胺(DA)系统，将服从于药物开发。拟议的研究将翻译成 实验室发现发现和开发新的预防和治疗方法的机会 AUD的治疗。目标1研究将检验DA奖赏系统内的P2X4Rs调节 乙醇摄入量。我们将使用：体内微透析结合P2X4R调制(研究1)和慢病毒- 短发夹状RNA(ShRNA)抑制伏核(NAC)和(或)P2X4R的表达 腹侧被盖区(VTA)(研究2和3)以深入了解P2X4Rs在大脑中的具体作用 中脑边缘DA系统对乙醇饮酒的影响。雄性和雌性P2X4KO和/或WT小鼠将使用两个 饮酒模式：(1)24小时畅饮，两瓶选择(自由选择)和(2)在黑暗中饮酒(DID；狂欢) 酒精饮酒)。AIM 2的研究将使用脑片电生理和敲除/敲除- Down技术来测试相互关联的假设，其中我们预测在P2X4R P2X介导的DA神经元放电抑制和乙醇对嘌呤能抑制的调节作用将减弱 减少或消除的。这项工作将提供关于P2X4Rs和Etoh相互作用的关键信息， 以及它如何影响中脑边缘多巴胺能神经元的嘌呤能和乙醇反应。AIM 3研究将测试 假设IVM可以被用作一个平台来识别和开发新的化合物 调节P2X4Rs，减少乙醇摄取。这一目标将通过设计和合成一个 半合成阿维菌素和米贝霉素类似物系列及其体外和体内评价。 通过这个结合了合成和生物评估的迭代过程，我们将识别出 有最大的戒酒作用，同时最大限度地减少IVM样毒性(即增加治疗指数)以 AUD治疗的进一步评估的候选人。综上所述，这些研究将进一步推动我们的长期- 学期目标：确定关键大脑奖赏区域内对调节重要的神经化学机制 乙醇摄入量。此外，这项工作还将为今后的翻译研究发展小说奠定基础 AUD的药物疗法。

We have identified P2X4 receptors (P2X4Rs) as a target for the development of drugs to prevent and/or treat alcohol use disorder (AUD). This hypothesis is derived from compelling systems; genetic, pharmacological and behavioral evidence reporting an inverse relationship between ethanol (EtOH) intake and P2X4R activity. Supporting this hypothesis we found that p2rx4 knock-out mice (P2X4 KO) consumed significantly more EtOH than wildtype (WT) littermate controls. We and others found that ivermectin (IVM), a positive allosteric modula- tor of P2X4Rs, significantly antagonized EtOH inhibition of ATP-gated P2X4Rs and reduced EtOH intake in mice and rats. We have assembled a multidisciplinary team that together will use genetic, molecular, electrophysiological, chemical and behavioral techniques to systematically explore 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 AUD. Aim 1 studies will test the hypothesis that P2X4Rs within the DA reward system regulate EtOH intake. We will use: in vivo microdialysis coupled with P2X4R modulation (Study 1) and lentiviral- mediated short hairpin RNA (shRNA) knockdown P2X4R expression in the nucleus accumbens (NAc) and/or ventral tegmental area (VTA) (Studies 2 & 3) to gain insights into specific contributions of P2X4Rs in the mesolimbic DA system to EtOH drinking. Male and female P2X4KO and/or WT mice will be tested using two drinking paradigms: (1) 24 hr access, two-bottle choice (free choice) and (2) drinking in the dark (DID; binge EtOH drinking). Aim 2 studies will use a combination of brain slice electrophysiological and knock-out/knock- down technologies to test interrelated hypotheses where we predict that under conditions in which P2X4Rs are reduced, P2X-mediated inhibition of DA neuron firing and EtOH regulation of purinergic inhibition will be reduced or eliminated. This work will provide key information regarding the interaction of P2X4Rs and EtOH, and how it may affect both purinergic and EtOH responses of mesolimbic DA neurons. Aim 3 studies will test the hypothesis that IVM can be used as a platform to identify and develop new compounds that positively modulate P2X4Rs and decrease EtOH-intake. This Aim will be accomplished by designing and synthesizing a series of semisynthetic avermectin and milibemycin analogs followed by their in vitro and in vivo evaluation. Through this iterative process combining synthetic and biological evaluation, we will identify molecules that have maximal anti-alcohol effects while minimizing the IVM-like toxicities (i.e., increased therapeutic index) to yield candidates for further assessment for treating AUD. Taken together, these studies will further our long- term goal of identifying neurochemical mechanisms within key brain reward regions important for regulating EtOH intake. Moreover, this work will set the stage for future translational studies to develop novel pharmacotherapies for AUD.