Precision pharmacology by local control of opioid receptors in neural circuits

项目摘要 该提案旨在利用对阿片类药物中细胞和亚细胞异质性的新认识， 用于治疗发现的信号。该项目开发了新的光遗传学和化学遗传学工具， 阿片受体活性的局部控制和神经回路中的信号传导以及亚细胞水平的分辨率。的 一项提案旨在开发一个多功能工具箱，并为“体内精确药理学”提供概念验证 尚未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，基于结合我们的合作团队在开发受体阻断细胞内 具有用于赋予细胞质中纳米抗体浓度的局部控制的已建立方法的纳米抗体 和特定的膜结构域，使用光遗传学和化学遗传学策略， 这是一个验证，但以前从未应用于阿片受体。R61阶段将开发这些工具（具体目标1） 并在神经元培养物和急性切片制备物中验证它们（特定目标2）。这些研究将提供 这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 受体作为一个类。我们已设定一系列具体的里程碑，以评估进展情况和可行性 用于推进体内研究。如果成功达到里程碑，第二阶段（R33）将采用新的 选择性靶向体内神经回路中阿片类信号传导的工具，重点是镇痛，行为 临床相关阿片类药物的强化和呼吸抑制。小鼠被用作公认的 具体目标（3）。这些研究利用了我们合作团队的综合专业知识， 并将建立强大的光遗传学和化学遗传学操作，用于研究体内阿片功能 第一次因此，提出的研究提供了光遗传学的一流应用， 化学遗传控制阿片类药物系统在体内，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.