Influence of drug abuse on neuronal nuclear and chromatin architecture

描述（由申请人提供）：阿片类药物对急性和持续性疼痛的管理至关重要，但其长期使用与显著的不良反应有关，包括耐受性和依赖性。现在有相当多的信息与慢性阿片类药物使用相关的生理和分子变化，但缓解问题的治疗是有限的，一般无效。毫无疑问，有助于耐受性发展和戒断表现的长期持续的细胞变化至少部分地由阿片受体信号传导期间直接参与的途径诱导的基因表达的稳定变化所控制。另一方面，由于慢性阿片类药物使用的表现，包括心理依赖，可能在阿片类药物摄入终止后持续很长时间，我们假设有额外的适应不良的表观遗传机制，在去除初始刺激后保留转录反应。在这里，我们建议不仅要确定与耐受性和戒断相关的转录变化，而且要确定我们假设诱导和维持它们的表观遗传变化。我们将使用吗啡耐受和戒断的小鼠模型，我们将集中分析两个大脑区域，蓝斑（LC）和腹侧被盖区（VTA），这两个区域都与长期阿片类药物使用的关键特征有关。我们将对FAC-sorted纯神经元群体的转录组（RNA-seq）和表观遗传景观（ChIP-seq）的变化进行全基因组，下一代基于序列的分析。重要的是，我们将我们的分析扩展到表观基因组的三维组织，正如现在所认识到的，提供了一个额外的表观遗传调控层，可能比染色质和DNA翻译后修饰更稳定。为了实现从LC和VTA分离的原代神经元的高分辨率、三维和定量成像，我们将使用软X射线断层扫描（SXT）。正如我们最近所证明的，SXT是研究原代神经元核结构的理想方法，并提供了最灵敏的成像方法，用于无偏地识别与慢性阿片类药物（吗啡）使用相关的染色质组织和致密化变化。该方法将通过全基因组染色质构象捕获方法（Hi-C）和DNA FISH实验来补充。因此，我们提出的实验不仅将映射完全“未知”的神经元群体的核和表观遗传景观，但也将有可能提供新的和基本的见解与耐受性和依赖性的表观遗传过程。我们的研究结果将为开发新的药理学干预措施奠定基础，这些干预措施不仅可以改善阿片类药物对疼痛的管理，还可以减少不良副作用的发生率，包括阿片类药物滥用。

DESCRIPTION (provided by applicant): Opioids are essential to the management of acute and persistent pain, but their chronic use is associated with significant adverse effects, includin tolerance and dependence. There is now considerable information as to the physiological and molecular changes associated with chronic opioid use, but treatments that mitigate the problems are limited and generally ineffective. Unquestionably, the long lasting cellular changes that contribute to the development of tolerance and the manifestations of withdrawal are governed, at least partly, by stable changes in gene expression that are induced by pathways directly engaged during opioid receptor signaling. On the other hand, as manifestations of chronic opioid use, including psychological dependence, may persist long after the termination of opioid intake, we hypothesize that there are additional, maladaptive epigenetic mechanisms that retain transcriptional responses after the removal of the initial stimulus. Here, we propose t identify not only the transcriptional changes associated with tolerance and withdrawal, but also the epigenetic changes that we hypothesize induce and sustain them. We will use mouse models of morphine tolerance and withdrawal and we will focus our analysis on two brain regions, the locus ceruleus (LC) and the ventral tegmental area (VTA), both of which have been implicated in critical features of long term opioid use. We will perform genome wide, next generation sequence-based analysis of changes in the transcriptome (RNA-seq) and the epigenetic landscape (ChIP-seq) of FAC-sorted, pure neuronal populations. Importantly, we will extend our analysis to the 3-dimensional organization of the epigenome, which as is now recognized, provides an additional layer of epigenetic regulation that is likely more stable than chromatin and DNA post-translational modifications. To achieve the high-resolution, 3-dimensional and quantitative imaging of primary neurons isolated from the LC and VTA, we will use soft X-ray tomography (SXT). SXT, as we have recently demonstrated, is ideal for the study of nuclear architecture in primary neurons, and provides the most sensitive imaging method for generating an unbiased identification of the changes in chromatin organization and compaction associated with chronic opioid (morphine) use. This approach will be complemented by a genome wide chromatin conformation capture approach (Hi-C) and DNA FISH experiments. Thus, our proposed experiments will not only map the nuclear and epigenetic landscape of completely "uncharted" neuronal populations but will also have the potential to provide novel and fundamental insights into the epigenetic processes associated with tolerance and dependence. Together our findings will lay the foundation for the development of novel pharmacological interventions that may not only improve the management of pain by opioids, but also reduce the incidence of adverse side effects, including opioid abuse.