Discovery of novel pharmacotherapeutic targets for opioid addiction

项目总结/摘要 美国正处于阿片类药物滥用和过量流行之中。羟考酮是最常用的 镇痛药，是许多人经历的第一种阿片类药物，具有使其能够 阿片类药物在大脑中的累积速度高于其他类阿片，这或许可以解释其滥用潜力巨大的原因。 在这里，我试图进行高通量实验，以生成关于细胞和分子的全脑数据。 线索诱导恢复羟考酮寻求的机制。具体来说，我建议使用FosCreER小鼠 以荧光“标记”由线索诱导的恢复激活的神经元集合。我会用最先进的 转录组学来识别这些细胞集合中驱动恢复的复发相关基因。然后我将 优先考虑并测试这些基因是否可以为开发新药物提供底物， 使用“循环治疗”方法预防复发。在目标1中，iDISCO+，一种脂质清除方法，将产生 关于复发激活区域的全脑数据。然后，我将使用ClearMap（一个已发布的Python包）来 对激活的神经元进行高通量检测，并将坐标配准到艾伦大脑上 阿特拉斯然后，我将严格检查这个大数据集，并测试是否恢复响应细胞 优先结构中的集合有助于复发行为。在目标2中，我将重点关注细胞群 在大脑区域显示需要复发相关的行为，并将再次标记神经元激活， 在这些地方恢复。将解剖组织，并将使用荧光激活细胞分选术， 分离这些激活的神经元用于通过RNA-Seq进行转录组学分析。这两个目标结合起来， 与羟考酮复治的神经回路和分子生物学直接相关的大型数据集 寻找我将再次严格分析这些数据集，严格遵守预先制定的标准， 优先考虑最有可能代表有效靶点的恢复反应基因， 新的药物治疗。在培训阶段结束后，我将在目标3中将这些调查结果扩展到 大鼠和小鼠模型的自我管理，并将验证这些转录组适应发生， 在物种之间，它们被检测为功能蛋白质的变化。我再一次严格地 优先考虑这些目标的转化潜力。在目标4中，我将使用药理学试剂， 优先考虑蛋白质靶点，以确定它们是否可以阻断线索诱导的羟考酮恢复 寻找，专注于可能能够快速进入临床环境的化合物。培训内容包括： 这项建议将使我成为一名非常全面的独立调查员。在我 独立的职业生涯我将进行转化研究，开发新的基于电路的治疗方法， 预防复发。

PROJECT SUMMARY/ABSTRACT The US is in the midst of an opioid abuse and overdose epidemic. Oxycodone is one of the most prescribed analgesics, is the first opioid many people experience, and has physiochemical properties that allow it to accumulate in the brain at rates higher than other opioids, perhaps explaining its considerable abuse potential. Here, I seek to perform high-throughput experiments to generate brain-wide data on the cellular and molecular mechanisms of cue-induced reinstatement to oxycodone seeking. Specifically, I propose to use FosCreER mice to fluorescently `tag' neuronal ensembles activated by cue-induced reinstatement. I will then use cutting-edge transcriptomics to identify relapse-related genes in these cellular ensembles that drive reinstatement. I will then prioritize, and test, whether these genes may serve substrates for the development of novel medications to prevent relapse using a ”circuit therapeutic” approach. In aim 1, iDISCO+, a lipid clearing method, will produce brain-wide data on regions activated by relapse. I will then use ClearMap, a published Python package, to conduct high-throughput detection of activated neurons and registration of coordinates onto the Allen Brain Atlas. I will then rigorously examine this large data set, and test whether reinstatement-responsive cell ensembles in prioritized structures contribute to relapse behavior. In aim 2, I will focus on cellular populations in brain regions shown to be required for relapse-related behavior, and will again tag neurons activated by reinstatement in these sites. Tissue will be dissected, and fluorescence activated cell sorting will be used to isolate these activated neurons for trancriptomic profiling via RNA-Seq. Together, these two aims will yield large data sets directly related to the neurocircuitry and molecular biology of reinstatement of oxycodone seeking. I will once again rigorously analyze these data sets, with strict adherence to pre-established criteria, to prioritize reinstatement-responsive genes most likely to represent efficacious targets for development of novel pharmacotherapeutics. Following completion of the training phase, in aim 3 I will extend these findings to both rat and mouse models of self-administration, and will validate that these transcriptomic adaptations occur across species, and that they are detected as changes in functional proteins. Once again, I will rigorously prioritize these targets for translational potential. In aim 4 I will use pharmacological agents that modulate prioritized protein targets to determine whether they can block cue-induced reinstatement of oxycodone seeking, focusing on compounds that may be able to quickly move into clinical settings. The training added in this proposal will allow me to emerge as an exceptionally well-rounded independent investigator. During my independent career I will perform translational research to develop novel circuit-based therapeutics for prevention of relapse.