A comprehensive dissection of cell types, circuits and molecular adaptations during opioid use

项目摘要 阿片类药物研究领域的一个主要障碍是我们对阿片系统的组织结构了解有限 在大脑中：我们仍然不知道哪些细胞类型表达每种阿片受体（μ，δ，κ，伤害感受素）， 介导内源性和外源性阿片类药物的作用，也没有什么其他分子存在于这些细胞。 如果没有这些知识，了解阿片类药物如何改变回路中的活动以产生行为效应仍然是 难以捉摸。这种知识上的差距阻碍了对增强阿片类镇痛作用的分子靶点的鉴定， 减轻阿片类药物使用障碍（OUD）的有害影响，包括成瘾和呼吸抑制。 为了填补这一知识空白，我们建议利用独特的大规模单细胞RNA测序， 在由艾伦脑科学研究所生成的scRNA-seq）数据库中（整个脑组织中> 340万个细胞）， 小鼠大脑）作为BRAIN Initiative Cell Census Network（BICCN）努力的一部分。利用这个数据库，我们将 建立一个全面的目录，列出所有表达每种阿片受体和肽的细胞类型。 大脑，以及介导或调节阿片类药物作用的基因网络的共表达，包括其他 G蛋白偶联受体和阿片受体的细胞效应物（Aim 1）。另外，我们会用我们的好- 建立了高通量单细胞RNA-seq管道，以表征细胞类型特异性分子 在慢性阿片类药物暴露、戒断和禁欲期间发生的适应，使用临床相关的 手术后疼痛模型，通常处方阿片类药物（目的2）。最后，我们将利用这本小说 通过执行先进的电路映射和体内解剖阿片类药物的作用机制的知识 功能成像研究表明，在前额叶皮层（PFC）中表达阿片受体的细胞类型是一个关键区域， 阿片类镇痛和成瘾（目标3）。 我们的变革性工作将是第一个将联合收割机几项高度创新的技术结合在一起的工作。 分子、电路和神经系统集成水平，包括高通量scRNA-seq，新的病毒株， 改善了跨突触传递，降低了电路映射、水晶头骨和微型镜的毒性， 微棱镜光学方法，用于脑状态的体内宽场成像和用于记录 分子定义的神经元类型在经历阿片类镇痛和成瘾范例的自由活动小鼠中。 我们拥有一支非凡的跨学科研究团队，他们在以下方面具有高度互补的专业知识： 阿片类药物的神经生物学及其受体在疼痛和成瘾回路中的分布和功能， 分子和解剖大脑结构，大规模细胞类型表征和电路映射，以及 高度创新的脑成像方法应用于神经回路的研究。 总的来说，这项研究的目的是为阿片类药物领域创造一个特殊的资源（公开发表）。 可通过NIDA资助的SCORCH数据协调中心获得）来解释阿片类药物如何改变大脑， 并发现新的治疗方法来预防和治疗OUD。

PROJECT SUMMARY A major barrier in the field of opioid research is our limited understanding of the organization of the opioid system in the brain: we still do not know which cell types express each opioid receptor (mu, delta, kappa, nociceptin) to mediate the effects of endogenous and exogenous opioids, nor what other molecules are present in these cells. Without this knowledge, understanding how opioids alter activity in circuits to produce behavioral effects remains elusive. This gap in knowledge prevents the identification of molecular targets to potentiate opioid analgesia and mitigate the deleterious effects of opioid use disorder (OUD), including addiction and respiratory depression. To fill this gap in knowledge, we propose to leverage the uniquely massive single-cell RNA sequencing (scRNA-seq) database generated by the Allen Institute for Brain Science (>3.4 million cells throughout the entire mouse brain) as part of the BRAIN Initiative Cell Census Network (BICCN) effort. Using this database, we will establish a comprehensive catalog of all the cell types that express each opioid receptor and peptide throughout the brain, as well as the co-expression of gene networks that mediate or regulate opioid actions, including other G protein-coupled receptors and cellular effectors of opioid receptors (Aim 1). Further, we will use our well- established high-throughput single-cell RNA-seq pipelines to characterize the cell-type-specific molecular adaptations that occur during chronic opioid exposure, withdrawal, and abstinence, using a clinically relevant model of post-surgical pain for which opioids are typically prescribed (Aim 2). Finally, we will leverage this novel knowledge to dissect the mechanisms of action of opioids by performing advanced circuit mapping and in vivo functional imaging studies of cell types expressing opioid receptors in the prefrontal cortex (PFC), a region critical to both opioid analgesia and addiction (Aim 3). Our transformative work will be the first to combine several highly innovative technologies at the molecular, circuit, and neural ensemble levels, including high-throughput scRNA-seq, new viral strains with improved transsynaptic transfer and decreased toxicity for circuit mapping, the crystal skull and miniscope- microprism optical approaches for in vivo wide-field imaging of brain state and for recording dynamics of molecularly defined neuron types in freely moving mice undergoing opioid analgesia and addiction paradigms. We have an extraordinary interdisciplinary team of investigators with highly complementary expertise in the neurobiology of opioids and the distribution and function of their receptors in pain and addiction circuits, the molecular and anatomical brain architecture, large-scale cell type characterization and circuit mapping, and highly innovative brain imaging methods as applied to the study of neural circuits. Overall, this research aims to generate an exceptional resource for the opioid field (to be made publicly available through the NIDA-funded SCORCH data coordination center) to explain how opioids change the brain, and discover novel therapeutic approaches to prevent and treat OUD.