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

阿片类药物使用障碍（OUD）是美国持续的公共卫生危机。尽管有效的 虽然目前治疗OUD的药物中，仍然有很高的复吸率戒毒后。因此，有一个 迫切需要更好地了解阿片类药物服用的神经生物学机制， 寻求这一点可能会为未来的OUD治疗带来新的靶点。一种新兴的研究 阿片类药物对人类和啮齿类动物神经转录组的影响已经确定了多种基因表达的改变， 大脑区域。然而，这些研究受到批量转录组学方法的限制，这妨碍了分配。 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） 这些方法有可能解决这一知识缺口，并确定细胞类型特异性转录组学。 这些改变将促进我们对OUD病理生理学的理解。最近的一项snRNAseq研究 表征了急性实验者递送的吗啡注射对细胞类型特异性 小鼠脑髓核（NAc）中的转录组，这是一个在吸毒中起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来执行 不同NAc细胞群和亚型在急性吗啡后的差异基因表达分析 注射或重复吗啡自我给药。我们激动人心的结果首次揭示了新的细胞类型- NAc中与自愿阿片类药物服用升级相关的特定转录组学变化。一 该领域合乎逻辑的下一步是确定性别对单核转录组的影响， 在随后的禁欲期间基因表达的纵向变化。因此， 建议是描述急性吗啡暴露和意志性的性别和细胞类型特异性的影响， 吗啡灌胃大鼠NAc（Aim 1）。一组雌性Sprague-Dawley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轭式盐水对照配对， 允许自我施用吗啡10天，在此期间阿片类药物的服用逐渐增加。 snRNAseq随后将用于鉴别性别特异性和性别非依赖性细胞类型特异性差异 在NAc中表达基因。为了全面分析整个禁欲过程中基因表达的变化， 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）阿片类药物戒断后的转录组学变化。差异 将通过荧光原位杂交（FISH）和RNAscope验证来自每个目的的表达基因。这 该提案将通过使用snRNAseq方法来识别性别和细胞类型， 与阿片类药物服用和戒断相关的特异性NAc转录组改变， 治疗目标，这将有助于指导新药物的开发，以治疗OUD。

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.