Characterizing the connectivity and molecular composition of opioid-sensitive neurons in the periaqueductal gray

项目摘要 每年有超过5000万美国人经历慢性疼痛，这导致阿片类药物处方增加 费率。虽然阿片类药物是有效的镇痛剂，但它会产生有害的副作用，如欣快感和身体依赖， 这导致了全国范围内的阿片类药物流行，以及对缺乏有害副作用的有效止痛药的需求 效果。了解阿片类药物如何影响神经系统对于区分神经回路很重要 驱动阿片类止痛药从潜在的不良副作用的电路。阿片类药物靶向Mu阿片受体 (MOR)，一种抑制性G蛋白偶联受体，由大脑各处的神经元表达。大脑中的神经元 中脑导水管周围灰质(PAG)表达高水平的吗啡，并在电刺激或吗啡刺激时产生 既有止痛药，又有奖励行为。阿片依赖的PAG环路与驱动镇痛的PAG回路的区别 奖励回路可能会揭示一种有效的、不会上瘾的止痛疗法。描述构图的特征 PAG的组织连接对于打击不同的阿片依赖回路是必要的。帕格 神经元在功能、位置和分子组成上各不相同，但尚未得到全面的研究 用单细胞方法表征的。PAG将感觉和情感信息往返传递 阿片类药物使用过程中的各种脑结构，但突触前输入和投射靶点的配置 阿片敏感的MOR+PAG神经元尚不清楚。了解阿片类药物的组成和组织- 敏感的神经回路对于辨别阿片类药物在哪里起作用产生止痛和回报作用很重要。 在初步的单细胞RNA测序(ScRNAseq)实验中，我从转录上鉴定了14个PAG 表达不同水平的MOR(OPRM1)基因的不同神经元亚型。在目标1中，我将使用 空间转录学用于确定OPRM1+PAG神经元的分布和细胞异质性。这就做 然后使用输入-输出电路映射来解析PAG中阿片敏感电路的体系结构。在AIM 2，我将首先确定PAG神经元是否可以根据它们的投射靶点进行遗传分类 使用追溯序号。我还将在感觉、情感和动机行为中使用抑制性化学遗传学 研究具有不同投射靶点的PAG神经元是否对特定的阿片类药物有贡献 诱导性行为。从这些实验中产生的分子、空间和电路组合数据将 提供了一种操纵特定PAG回路的方法，并揭示了哪些神经元对阿片类药物敏感。 此外，功能丧失行为分析的结果将证明特定的PAG回路 优先促进阿片类药物的感官或情感效应。将进行拟议的研究 在格雷戈里·谢勒博士的指导下。Scherrer博士有丰富的集成鼠标的经验 遗传学、功能神经解剖学和复杂的行为分析，以研究潜在的神经回路 阿片类药物行为。总的来说，这个项目将帮助我发展成为一名独立的研究人员，并为 拥有研究阿片类药物回路的资源的研究社区。

Project Abstract Over 50 million Americans experience chronic pain annually, which has led to increases in opioid prescription rates. While effective analgesics, opioids produce harmful side effects like euphoria and physical dependence, which has led to a nationwide opioid epidemic and a need for efficacious analgesics that lack harmful side effects. Understanding how opioids impact the nervous system is important to distinguish the neural circuits driving opioid analgesia from circuits underlying unwanted side effects. Opioids target the mu opioid receptor (MOR), an inhibitory G-protein coupled receptor expressed by neurons throughout the brain. Neurons in the periaqueductal gray (PAG) express high levels of MOR and, upon electrical or morphine stimulation, produce both analgesic and rewarding behaviors. Distinguishing opioid-dependent PAG circuits driving analgesia from rewarding circuits may reveal a powerful, non-addictive therapy for pain relief. Characterizing the composition and organizational connectivity of the PAG is necessary to target distinct opioid-dependent circuits. PAG neurons vary in function, location, and molecular composition, but have not been comprehensively characterized using single-cell approaches. The PAG relays sensory and affective information to and from various brain structures during opioid use, but the configuration of presynaptic inputs and projection targets of opioid-sensitive MOR+ PAG neurons is unknown. Understanding the composition and organization of opioid- sensitive neural circuits is important to discern where opioids act to produce analgesic and rewarding effects. In preliminary single-cell RNA-sequencing (scRNAseq) experiments I identified 14 PAG transcriptionally distinct neuron subtypes that expressed various levels of the gene encoding MOR (Oprm1). In Aim 1, I will use spatial transcriptomics to determine the distribution and cellular heterogeneity of Oprm1+ PAG neurons. I will then resolve the architecture of opioid-sensitive circuits in the PAG using input-output circuit mapping. In Aim 2, I will first determine whether PAG neurons can be genetically classified based on their projection target using retro-seq. I will also use inhibitory chemogenetics during sensory, affective, and motivational behavior assays to investigate whether PAG neurons with different projection targets contribute to specific opioid- induced behaviors. The combined molecular, spatial, and circuit data generated from these experiments will provide a means to manipulate specific PAG circuits and reveal which neurons are receptive to opioids. Furthermore, results from loss-of-function behavioral assays will demonstrate whether specific PAG circuits preferentially contribute to the sensory or affective effects of opioids. The proposed research will be conducted under the mentorship of Dr. Gregory Scherrer. Dr. Scherrer has extensive experience integrating mouse genetics, functional neuroanatomy, and complex behavioral assays to investigate the neural circuits underlying opioid behaviors. Collectively, this project will help me develop into an independent researcher and provide the research community with resources for investigating opioid circuits.