Multiomic profiling of cell types mediating opioid use disorder in rats

项目摘要 处方止痛药的滥用和滥用，如羟考酮，导致了前所未有的 阿片类药物在美国的流行。阿片类药物危机对公共卫生造成毁灭性后果，包括 阿片类药物滥用和相关过量使用激增。迫切需要研究开发更好的治疗方法 鸦片成瘾。 尽管对羟考酮在不同疾病中的药代动力学和行为影响有大量的了解 动物模型中，只有少数候选基因和神经解剖系统受到阿片类药物的影响 已经研究过了。单细胞基因组学领域的最新技术进步是 公正地发现和表征对阿片类药物有反应的脑细胞类型。 为了应对这种RFA，我们利用了一种创新的多组学方法(单细胞多组体 ATAC基因表达)来映射来自同一细胞的转录组和表观基因组 大脑中与阿片类药物暴露相关的区域的细胞。为此，我们将使用扩展的 获得羟考酮静脉自我给药概述了几个神经适应也观察到 患有阿片类药物使用障碍的人(OUD)。这种方法提供了一个难得的机会来系统地 探索阿片系统的细胞多样性，同时探讨调控的致病机制 基于表观遗传变化与靶基因表达之间关系的细胞状态 单个细胞。我们将把这种创新的多组学方法与严格的计算 探索多脑区、不同阶段阿片系统细胞组织的途径 OUD进展(初始接触、使用升级、急性戒断、长期禁欲和提示-- 诱发复发)。 我们已经提供了强有力的初步支持，支持我们提议的计划在以下方面的可行性 目标。在目标1中，我们将收集延长羟考酮静脉注射途径不同阶段的脑组织。 自我给药(IVSA)协议，我们将生成来自男性和女性的单细胞基因组数据 暴露于生理盐水或羟考酮的大鼠。在目标2中，我们将整合这些转录和 表观基因组数据集，以确定与以下相关的细胞状态、基因和上游调控因子的变化 羟考酮使用的不同阶段。这一办法将有助于确定独联体国家之间的联系。 调控元件和靶基因。在目标3中，我们将通过RNA-FISH验证关键细胞类型特定的发现 并确定用于功能验证的前3个靶基因。为此，我们将使用病毒介导的CRISPR- Cas9系统对羟考酮自我给药大鼠成瘾行为的调节作用。这样做的结果 这项研究将使未来的研究能够确定治疗和预防OUD的新靶点。

Project Summary The misuse and abuse of prescription pain relievers, such as oxycodone, contributed to the unprecedented opioid epidemic in the United States. The opioid crisis has devastating consequences on public health including a surge in opioid misuse and related overdoses. Research is urgently needed to develop better treatments for opiate addiction. Despite substantial knowledge of the pharmacokinetic and behavioral effects of oxycodone in various animal models, only a small number of candidate genes and neuroanatomical systems affected by opioids have been studied. Recent technological advances in the field of single cell genomics are promising avenues for the unbiased discovery and characterization of brain cell types that respond to opioids. In response to this RFA, we leverage an innovative multi-omics methodology (Single Cell Multiome ATAC + Gene Expression) to map the transcriptome and epigenome from the same cell across thousands of cells in brain regions relevant to the effects of opioid exposure. To this aim we will use a rat model of extended access to oxycodone intravenous self-administration that recapitulates several neuroadaptations also observed in humans with opioid use disorders (OUD). This approach provides an exceptional opportunity to systematically explore the cellular diversity of the opioid system and, at the same time, the causative mechanisms that regulate cellular states based on the associations between epigenetic changes and the expression of target genes in individual cells. We will integrate this innovative multi-omics methodology with rigorous computational approaches to explore the cellular organization the opioid system in multiple brain regions and different stages of OUD progression (initial exposure, escalation of use, acute withdrawal, prolonged abstinence, and cue- induced relapse). We have provided strong preliminary that support the feasibility of our proposed plan for the following aims. In Aim 1, we will collect brain tissues at different stages of the extended access to oxycodone intravenous self-administration (ivsa) protocol and we will generate single cell genomics data from both male and female rats that are exposed to either saline or oxycodone. In Aim 2, we will integrate these transcriptomic and epigenomic datasets to identify changes in cellular states, genes and upstream regulators that are associated with different stages of oxycodone use. This approach will facilitate the identification of linkages between cis- regulatory elements and target genes. In Aim 3, we will validate key cell type-specific findings by RNA-FISH and identify the top 3 target genes for functional validation. To this aim, we will use a viral-mediated CRISPR- Cas9 system to modulate addictive behaviors in rat models of oxycodone self-administration. The results of this study will enable future studies that may identify new targets for treatment and prevention of OUD.