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)是美国持续存在的公共健康危机。尽管...的有效性 目前治疗OUD的药物，在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摄取和 寻求这一点可能会为未来的OUD治疗带来新的靶点。一种研究其影响的新兴文献 阿片类药物对人类和啮齿动物神经转录本的影响已确定在多发性硬化患者中基因表达的变化 大脑区域。然而，这些研究受到批量转录方法的限制，这阻碍了分配 表达改变为特定的神经细胞或神经胶质细胞类型。单核RNA测序(SnRNAseq) 这种方法有可能解决这一知识鸿沟，并识别特定细胞类型的转录 这些变化将促进我们对OUD的病理生理学的理解。最近的一项SnRNAseq研究 描述了急性实验注射吗啡对细胞类型特异性的影响 小鼠伏隔核(NAC)的转录本，这是一个在吸毒中起关键作用的大脑区域 和寻觅行为。我们的初步研究扩展了这些结果，使用SnRNAseq执行 急性吗啡后不同NAC细胞群体和亚型的差异基因表达分析 注射或重复注射吗啡。我们令人兴奋的结果首次揭示了新的细胞类型- NAC中与自愿服用阿片类药物的升级相关的特定转录变化。一个 该领域合乎逻辑的下一步是确定性别对单核转录本和轮廓的影响 在随后的禁欲期间基因表达的纵向变化。因此，这其中的一个目标 提案是描述急性吗啡暴露和意志力的性别和细胞类型特异性影响 大鼠NAC内吗啡摄取(目标1)。一组雌性SD大鼠将接受腹膜内注射 注射吗啡或生理盐水。第二组雌性大鼠将与带轭的生理盐水对照组配对， 允许自我注射吗啡10天，在此期间阿片类药物的服用逐渐升级。 然后，将使用SnRNAseq来区分性别特异性和性别无关的细胞类型特异性 在NAC中表达的基因。全面描述禁欲过程中基因表达的变化 在吗啡自我给药后，SNRNAseq将被用于分析性别和细胞类型特定的 急性(1天)和长期(10天)阿片类药物戒断后的转录变化。差异化的 来自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方法来鉴定性别和细胞类型，从而推进临床前OUD研究 与阿片类药物服用和戒断相关的特异性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的新药的开发。