Precision pharmacology by local control of opioid receptors in neural circuits

项目总结 这一提议寻求利用对阿片类药物细胞和亚细胞异质性的新理解。 这是治疗发现的信号。该项目开发了新的光遗传和化学遗传工具，以实现 在神经回路和亚细胞分辨率水平上对阿片受体活性和信号的局部控制。这个 该提案寻求开发一个通用的工具箱，并为“体内精确药理学”提供概念验证 还没有应用于阿片类药物系统。第一阶段(R61)是一项“高风险”技术 阶段，基于结合我们的协作团队在开发细胞内受体阻断方面的专业知识 具有已建立的方法来局部控制细胞质中的纳米体浓度的纳米体 和特定的膜结构域，使用光发生和化学发生策略，已经 经过验证但从未应用于阿片受体。R61阶段将开发这些工具(具体目标1) 并在神经元培养和急性切片制备中进行验证(特定目标2)。这些研究将提供 不仅是阿片受体体内精确药理学的概念验证，也是G蛋白偶联的概念验证 受体作为一类。我们设立了一系列具体的里程碑，以评估进展情况和评估可行性 以推进体内研究。如果成功达到里程碑，则第二阶段(R33)将应用新的 在活体神经回路中选择性靶向阿片信号的工具，侧重于止痛、行为 临床相关阿片类药物的强化和呼吸抑制。老鼠被用作久负盛名的 和相关模型(具体目标3)。这些研究利用了我们协作团队的综合专业知识， 并将为体内阿片类药物功能的研究建立强大的光遗传和化学遗传操作 这是第一次。因此，拟议的研究将提供一流的光遗传和 在体内对阿片系统进行化学遗传控制，他们将探索阿片类药物的精确药理学 基于神经回路中的位置。

PROJECT SUMMARY This proposal seeks to leverage the emerging understanding of cellular and subcellular heterogeneity in opioid signaling for therapeutic discovery. The project develops new optogenetic and chemogenetic tools to achieve local control of opioid receptor activity and signaling in neural circuits and at a subcellular level of resolution. The proposal seeks to develop a versatile toolbox and provide proof-of-concept for "in vivo precision pharmacology" that have not yet been applied to the opioid system. The first phase (R61) is a "high-risk" technology enabling phase, based on combining our collaborative team's expertise in developing receptor-blocking intracellular nanobodies with established methods for conferring local control of nanobody concentration in the cytoplasm and specific membrane domains, using optogenetic and chemogenetic strategies that have already been validated but never before applied to opioid receptors. The R61 phase will develop these tools (Specific Aim 1) and validate them in neuronal culture and acute slice preparations (Specific Aim 2). These studies will provide not only proof-of-concept for in vivo precision pharmacology of opioid receptors, but also for G protein-coupled receptors as a class. We have set a specific series of milestones to evaluate progress, and to assess feasibility for advance to in vivo studies. If the milestones are successfully met, the second phase (R33) will apply the new tools to selectively target opioid signaling in neural circuits in vivo, focusing on analgesia, behavioral reinforcement and respiratory depression by clinically relevant opioid drugs. Mice are used as a well-established and relevant model (Specific Aim 3). These studies leverage the combined expertise of our collaborative team, and will establish powerful optogenetic and chemogenetic manipulations for the study of in vivo opioid function for the first time. Accordingly, the proposed studies to provide the first-in-class application of optogenetic and chemogenetic control to the opioid system in vivo, and they will explore a precision pharmacology of opioids based on location in neural circuits.