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

项目总结 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有限的了解 在大脑中：我们仍然不知道哪种类型的细胞表达每种阿片受体(u、Delta、kappa、nociceptin)。 调节内源性和外源性阿片类药物的影响，也不是这些细胞中存在的其他分子。 在没有这种知识的情况下，理解阿片类药物是如何改变电路中的活动以产生行为效应的 难以捉摸。这一认识上的差距阻碍了识别分子靶点以加强阿片类药物的镇痛和 减轻阿片使用障碍(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 (scRNA-seq)数据库，由Allen Institute for Brain Science(&gt；340万细胞)生成 作为大脑倡议细胞普查网络(BICCN)努力的一部分。使用这个数据库，我们将 建立一个全面的目录，列出贯穿始终表达每个阿片受体和多肽的所有细胞类型 大脑，以及调节或调节阿片类药物活动的基因网络的共同表达，包括其他 阿片受体的G蛋白偶联受体和细胞效应器(目标1)。此外，我们将利用我们的油井- 建立了高通量的单细胞rna-seq管道，以表征特定细胞类型的分子。 在慢性阿片类药物暴露、戒断和戒断期间发生的适应，使用临床相关的 手术后疼痛模型，通常开阿片类药物(目标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实验来剖析阿片类药物的作用机制 关键区域--前额叶皮质阿片受体表达细胞类型的功能成像研究 阿片类止痛和成瘾(目标3)。 我们的变革性工作将是第一个在 分子、回路和神经整体水平，包括高通量scRNA-seq，新的病毒株 改善了跨突触传输并降低了电路标测的毒性，水晶头骨和微型望远镜- 微棱镜光学方法用于在体脑状态的广视场成像和记录脑电动力学 自由活动小鼠在阿片类止痛和成瘾范例中的分子定义神经元类型。 我们拥有一支非凡的跨学科调查团队，在以下领域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环路中的分布和功能 分子和解剖脑结构，大规模细胞类型表征和电路映射，以及 应用于神经回路研究的高度创新的脑成像方法。 总体而言，这项研究的目的是为阿片类药物领域(将公开进行)创造一个特殊的资源 可通过NIDA资助的SCOCH数据协调中心获得)来解释阿片类药物如何改变大脑， 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.