Identifying multi-omic signatures of opioid use and relapse

项目摘要 阿片类药物使用障碍对许多人来说是终身负担，造成高昂的个人，财务和健康成本。 即使在长期禁欲后，许多处于恢复期的人仍会继续复发，包括那些接受过 药物辅助治疗。重复的阿片类药物暴露通过产生 长期的分子变化，改变生理和支持继续使用药物。这些细胞适应性 与持续复发的脆弱性有关，但我们缺乏对是什么驱动他们的清晰理解。 持久性。更进一步缺乏关于改变的电路背后的精确分子适应的信息 功能，以及它们在哪些特定回路中起作用以促进复发。理解这个“谁，什么，什么时候， ”将是确定新治疗靶点的关键。在这里，我们将用一个多- 一种水平方法，允许我们对特定回路中的神经元进行排序、操作和记录 阿片类药物自我给药和复发的风险 我们的初步数据表明，腹侧神经元中基因不同和基因相同的神经元亚型， 在芬太尼自我给药后，被盖区（VTA）经历了不同的分子适应，我们 这种假设来自于特定回路中活性依赖性的转录变化。我们进一步假设 转录变化通过基因启动子的甲基化和去甲基化来维持。我们将首先 记录投射到延髓核（NAc）或杏仁核（AMY）的腹侧被盖区神经元中的钙活动- 预测已知是重要的药物摄入量和复吸。然后，在相同的神经元中， 同样的动物，我们将确定哪些基因网络在自我管理后发生了转录变化， 坚持到复发测试。接下来，我们将鉴定DNA甲基化标记， 表达，重点是对突触可塑性重要的基因。接下来，我们将使用CRISPR/dCas 9融合 构建在特定回路中我们识别的基因座上操纵甲基化状态。这项建议将使我们能够 定义支持阿片类药物摄入和复发的特定VTA回路，哪些基因网络支持 这些电路，以及DNA甲基化如何巩固转录景观以改变行为。这个奖项将 允许以前所未有的分辨率研究阿片类药物使用障碍的神经机制， 有可能改变我们如何研究物质使用障碍的遗传学。共同努力， 信息将有助于为预防复发的新治疗策略提供信息。

Project Summary Opioid use disorder is a life-long burden for many individuals, imposing high personal, financial, and health costs. Even after prolonged abstinence, many individuals in recovery will go on to relapse, including those that received medication-assisted treatments. Repeated opioid exposure usurps normal reward circuit function by producing long-lasting molecular changes that alter physiology and support continued drug use. These cellular adaptations have been implicated in sustained relapse vulnerability, but we lack a clear understanding of what drives their persistence. There is a further lack of information on the precise molecular adaptations underlying altered circuit function, and in which specific circuits they act to promote relapse. Understanding this “who, what, when, and where,” will be key to identifying new therapeutic targets. Here, we will answer these questions using a multi- level approach that allows us to sequence, manipulate, and record from neurons in specific circuits in the context of opioid self-administration and relapse. Our preliminary data show that both genetically-distinct and genetically-identical neuron subtypes in the ventral tegmental area (VTA) undergo differential molecular adaptations after fentanyl self-administration, which we hypothesize arises from activity-dependent transcriptional changes in specific circuits. We further hypothesize the transcriptional changes are sustained by methylation and demethylation at the gene promoters. We will first record calcium activity in VTA neurons that project to either the nucleus accumbens (NAc) or amygdala (AMY)— projections known to be important for drug intake and relapse, respectively. Then, in the same neurons from the same animals, we will identify which gene networks are transcriptionally changed after self-administration and persist until relapse testing. Next, we will identify the DNA methylation marks driving sustained differential expression, with an emphasis on genes important for synaptic plasticity. Next, we will use CRISPR/dCas9 fusion constructs to manipulate methylation states at our identified loci in specific circuits. This proposal will allow us to define the specific VTA circuits that support opioid intake and relapse, which gene networks support activity of these circuits, and how DNA methylation cements the transcriptional landscape to alter behavior. This award will allow research into neural mechanisms of opioid use disorder with unprecedented resolution, and has the potential to transform how we approach studying the genetics of substance use disorders. Together, this critical information will help inform new treatment strategies to prevent relapse.