Single-nucleus transcriptomics in rat striatum following morphine administration and withdrawal

吗啡给药和戒断后大鼠纹状体的单核转录组学

• 批准号: 10571768
• 负责人: RICHARD C. CRIST
• 金额: $ 20.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10571768
• 关键词: Abstinence; Acute; Address; Adult; Anatomy; Behavior; Brain; Brain region; Buprenorphine; Cell Nucleus; Cells; Clinical Research; Corpus striatum structure; Country; Data; Data Set; Development; Disease; Dose; Drug Metabolic Detoxication; Drug Targeting; Effectiveness; FDA approved; Female; Fluorescent in Situ Hybridization; Functional disorder; Future; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Goals; Health Services Accessibility; Healthcare Systems; Home; Human; Individual; Injections; Intraperitoneal Injections; Knowledge; Literature; Methadone; Methods; Molecular; Morphine; Mus; Naltrexone; Neuroglia; Neurons; Nucleus Accumbens; Opioid; Opioid Analgesics; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Pilot Projects; Play; Population; Public Health; Rattus; Relapse; Research; Rodent; Role; Saline; Self Administration; Sex Differences; Sprague-Dawley Rats; Techniques; Time; Tissue-Specific Gene Expression; Tissues; United States; Withdrawal; addiction; antagonist; arm; base; cell type; cohort; differential expression; experimental study; innovation; insight; interest; long term abstinence; male; medication for opioid use disorder; morphine administration; mu opioid receptors; neural; neurobiological mechanism; neurochemistry; neuromechanism; new therapeutic target; novel; novel therapeutics; opioid exposure; opioid use disorder; opioid withdrawal; overdose death; pre-clinical; preclinical study; prescription opioid; prevent; prolonged abstinence; sex; single nucleus RNA-sequencing; therapy development; transcriptome; transcriptomics

Opioid use disorder (OUD) is an ongoing public health crisis in the United States. Despite the effectiveness of current medications to treat OUD, there is still a high rate of relapse following detoxification. Thus, there is a critical need for a better understanding of the neurobiological mechanisms contributing to opioid taking and seeking that could lead to novel targets for future OUD treatments. An emerging literature studying the effects of opioids on human and rodent neural transcriptomes has identified alterations in gene expression in multiple brain regions. However, these studies are limited by bulk transcriptomic approaches, which prevent assignment of expression changes to specific neuronal or glial cell types. Single nucleus RNA sequencing (snRNAseq) approaches have the potential to address this knowledge gap and identify cell type-specific transcriptomic alterations that will advance our understanding of the pathophysiology of OUD. A recent snRNAseq study characterized the effects of an acute experimenter-delivered morphine injection on cell type-specific transcriptomes in the mouse nucleus accumbens (NAc), a brain region that plays a critical role in drug-taking and -seeking behaviors. Our preliminary studies extended these results by using snRNAseq to perform differential gene expression analysis in distinct NAc cell populations and subtypes following acute morphine injection or repeated morphine self-administration. Our exciting results reveal, for the first time, novel cell type- specific transcriptomic changes in the NAc that are associated with the escalation of voluntary opioid taking. A logical next step for the field is to determine the effects of sex on single nuclei transcriptomes as well as profile longitudinal changes in gene expression throughout subsequent abstinence periods. Therefore, one goal of this proposal is to characterize the sex- and cell type-specific effects of acute morphine exposure and volitional morphine taking in the rat NAc (Aim 1). One cohort of female Sprague-Dawley rats will receive an intraperitoneal injection of morphine or saline. A second cohort of female rats will be paired with yoked saline controls and allowed to self-administer morphine for 10 days, a period in which escalation of opioid taking develops. snRNAseq will then be used to identify sex-specific and sex-independent cell type-specific differentially expressed genes in the NAc. To comprehensively profile changes in gene expression throughout abstinence following morphine self-administration, snRNAseq will be used to analyze sex- and cell type-specific transcriptomic changes following acute (1 day) and prolonged (10 days) of opioid abstinence. Differentially expressed genes from each aim will be validated by fluorescent in situ hybridization (FISH) and RNAscope. This proposal will advance preclinical OUD research by using a snRNAseq approach to identify sex- and cell type- specific NAc transcriptome alterations associated with opioid taking and withdrawal thereby identifying novel therapeutic targets that will help guide the development of new medications to treat OUD.

阿片类药物使用障碍（OUD）是美国一场持续的公共卫生危机。尽管有效 目前治疗 OUD 的药物，戒毒后复发率仍然很高。因此，有一个 迫切需要更好地了解导致阿片类药物服用和服用的神经生物学机制 寻求这可能会为未来的 OUD 治疗带来新的目标。研究影响的新兴文献 阿片类药物对人类和啮齿动物神经转录组的影响已发现多种基因表达的改变 大脑区域。然而，这些研究受到大量转录组学方法的限制，这阻碍了分配 特定神经元或神经胶质细胞类型的表达变化。单核 RNA 测序 (snRNAseq) 方法有可能解决这一知识差距并识别细胞类型特异性转录组 这些改变将增进我们对 OUD 病理生理学的理解。最近的一项 snRNAseq 研究 表征了实验者急性注射吗啡对细胞类型特异性的影响 小鼠伏隔核 (NAc) 中的转录组，这是一个在吸毒中发挥关键作用的大脑区域 和寻求行为。我们的初步研究通过使用 snRNAseq 来扩展这些结果 急性吗啡后不同 NAc 细胞群和亚型的差异基因表达分析 注射或反复自行注射吗啡。我们令人兴奋的结果首次揭示了新的细胞类型 - NAc 中特定的转录组变化与自愿阿片类药物服用的增加有关。一个 该领域合乎逻辑的下一步是确定性别对单核转录组和概况的影响 在随后的禁欲期间基因表达的纵向变化。因此，本次活动的目标之一是 该提案旨在描述急性吗啡暴露和意志力对性别和细胞类型的特异性影响。 吗啡摄入大鼠 NAc（目标 1）。一组雌性 Sprague-Dawley 大鼠将接受腹腔注射 注射吗啡或盐水。第二组雌性大鼠将与带轭的盐水对照配对， 允许自行服用吗啡 10 天，在此期间阿片类药物的服用量逐渐增加。 snRNAseq 然后将用于差异性地识别性别特异性和性别无关的细胞类型特异性 NAc 中表达的基因。全面分析禁欲期间基因表达的变化 吗啡自我给药后，snRNAseq 将用于分析性别和细胞类型特异性 急性（1 天）和长期（10 天）阿片类药物戒断后转录组的变化。差异化 每个目标的表达基因将通过荧光原位杂交 (FISH) 和 RNAscope 进行验证。这 该提案将通过使用 snRNAseq 方法来识别性别和细胞类型来推进 OUD 研究 与阿片类药物服用和戒断相关的特定 NAc 转录组改变，从而识别新的 治疗目标将有助于指导治疗 OUD 的新药物的开发。