Single-cell resolution analysis of chromatin accessibility and gene expression changes in a model of drug addiction

好了！ 项目摘要 每年，每天有100多名美国人死于过量服用阿片类药物。对阿片类药物上瘾，包括 处方药如羟考酮，以及违禁药品如S海洛因和芬太尼，是国家的危机 影响公共健康和经济。因此，迫切需要开发更好的鸦片类药物治疗方法。 上瘾，这需要更好地了解其生物学基础。我们建议的主要目标是 识别细胞类型和细胞类型特异的基因表达模式，与更高的易感性相关 以公正和定量的方式强制使用羟考酮。我们将通过以下方式实现这一目标： 单细胞测序分析测量数千名患者的基因表达和染色质可及性 单个实验中的单个细胞。我们将使用N/NIH异种系(HS)大鼠的大脑 接受延长使用羟考酮自我给药程序。我们关注HS大鼠是因为 它们在基因上是多样化的，并表现出非凡的行为能力。这些老鼠的特征是 基于对类似成瘾的高级分析的羟考酮强制摄入的易感性或抵抗力 行为特征，包括耐受性、依赖性、动机和强迫性药物摄入。这一行为 范式概括了在人类成瘾中观察到的许多关键的神经适应，并具有很高的面孔， 羟考酮使用障碍的预测性和构造性效度。这个项目利用了一个脑组织 HS大鼠的资料库，这些大鼠已经进行了基因分型，并被描述为易受强迫和抵抗强迫 羟考酮的使用。羟考酮生物库(www.OxycodonBioBank.org)将提供用于 这个项目。我们将重点关注伏隔核，这是一个参与从中度到中度转变的大脑区域 滥用药物。我们对大脑皮层单细胞分析的初步研究提供了一个令人信服的 策略研究阿片成瘾的生物学基础。我们建议：1)用单细胞rna-seq来鉴定 羟考酮易感或耐药HS大鼠脑内基因表达的变化 强制使用(具体目标1)；2)使用单细胞ATAC-SEQ来确定染色质可获得性的变化 和转录因子结合位点(特定目标2)；3)使用H3K27Ac- PLAC-SEQ将远端调控元件与羟考酮成瘾相关行为相关的靶基因联系起来 (具体目标3)。该项目将受益于多种专业知识，并将利用现有资源，包括 由羟考酮生物库(U01DA044451)和NIDA中心在近交系大鼠中提供的 (P50DA037844)。我们相信，拟议的研究有可能导致开创性的工作。 阿片成瘾的机制基础方面的发现。我们测序数据集的综合分析 将提供关于不同细胞类型对分子变化的贡献的相当新的见解 与成瘾相关的表型有关。 好了！ 好了！

! Project Summary Every year, more than 100 Americans a day die after overdosing on opiates. Addiction to opioids, including prescription drug such as oxycodone, and illicit drugs such a s heroin and fentanyl, is a national crisis that affects public health and the economy. Thus, there is an urgent need to develop better treatments for opiate addiction, which requires a better understanding of its biological basis. The primary goal of our proposal is to identify cell types and cell type-specific gene expression patterns associated with higher vulnerability to compulsive oxycodone use in an unbiased and quantitative way. We will accomplish this goal by implementing single-cell sequencing assays to measure gene expression and chromatin accessibility in thousands of individual cells in a single experiment. We will use brains of N/NIH heterogeneous stock (HS) rats that have undergone the extended access to oxycodone self-administration procedure. We focus on HS rats because they are genetically diverse and exhibit an exceptional behavioral repertoire. These rats are characterized as vulnerable or resistant to oxycodone compulsive intake based on advanced analysis of addiction-like behavioral traits, including tolerance, dependence, motivation, and compulsive drug intake. This behavioral paradigm recapitulates many of the key neuroadaptations observed in human addiction and has high face, predictive, and construct validity for oxycodone use disorders. This project takes advantage of a brain tissue repository of HS rats that have been genotyped and characterized as vulnerable and resistant to compulsive oxycodone use. The oxycodone biobank (www.OxycodoneBioBank.org) will provide the samples to be used in this project. We will focus on the nucleus accumbens, a brain region involved in the transition from moderate to excessive drug use. Our preliminary studies on single-cell analysis of the cerebral cortex provide a compelling strategy to study the biological basis of opiate addiction. We propose: 1) to use single-cell RNA-seq to identify gene expression changes in brains of HS rats that are characterized as prone or resistant to oxycodone compulsive use (Specific Aim 1); 2) to use single-cell ATAC-seq to identify changes in chromatin accessibility and transcription factors binding sites in the same population of HS rats (Specific Aim 2); 3) to use H3K27Ac- PLAC-seq to link distal regulatory elements to target genes involved in oxycodone addiction-related behaviors (Specific Aim 3). This project will benefit from multiple expertise and will leverage existing resources, including those provided by the oxycodone biobank (U01DA044451) and the NIDA center for GWAS in outbred rats (P50DA037844). We believe that the proposed studies have the potential to lead to groundbreaking discoveries in the mechanistic bases of opioids addiction. The integrative analysis of our sequencing datasets will provide considerable new insights concerning the contribution of distinct cell-types to molecular changes associated with addiction related phenotypes. ! !