Discovery of novel pharmacotherapeutic targets for opioid addiction

项目摘要/摘要 美国正处于阿片类药物滥用和吸毒过量的流行之中。羟考酮是最常用的处方药之一 止痛药，是许多人经历的第一个阿片类药物，具有使其能够 在大脑中积累的速度比其他阿片类药物高，这可能解释了它相当大的滥用潜力。 线索诱导恢复羟考酮寻求的机制。具体地说，我建议使用FosCreer小鼠 对由提示诱导的恢复激活的神经元集合进行荧光标记。然后我会用尖端的 转录学，以确定这些细胞系中与复发相关的基因，这些基因推动了复发。那我会的 确定并测试这些基因是否可以作为开发新药的底物 使用“巡回治疗”的方法防止复发。在目标1中，iDISCO，一种清除脂质的方法，将产生 关于复发激活的脑区的全脑数据。然后，我将使用ClearMap，一个已发布的Python包，来 高通量检测激活的神经元并将坐标注册到Allen大脑 阿特拉斯。然后，我将严格检查这个大型数据集，并测试恢复反应细胞 按优先顺序排列的结构中的集合有助于复发行为。在目标2中，我将重点介绍细胞群体 在大脑中与复发相关的行为所需的区域，并将再次标记由 在这些地点恢复。组织将被解剖，并将使用荧光激活的细胞分类 通过RNA-Seq分离这些激活的神经元以进行转译谱分析。这两个目标加在一起将产生 与羟考酮恢复的神经回路和分子生物学直接相关的大数据集 寻找。 对最有可能代表有效靶点的恢复反应基因进行优先排序 新的药物疗法。在完成培训阶段后，在目标3中，我将把这些调查结果扩展到 大鼠和小鼠的自我给药模型，并将验证这些转录适应发生 在不同物种之间，它们被检测为功能蛋白的变化。再一次，我会严格 根据翻译潜力确定这些目标的优先顺序。在目标4中，我将使用药物制剂来调节 确定蛋白质靶标的优先顺序，以确定它们是否可以阻止提示诱导的羟考酮恢复 寻找，重点放在可能能够迅速进入临床环境的化合物。增加的培训 这项提议将使我成为一名异常全面的独立调查员。在我的 独立职业生涯我将进行翻译研究，以开发基于电路的新疗法 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.