CELL-TYPE SPECIFIC ROLE OF CIRCADIAN-DEPENDENT TRANSCRIPTION IN FENTANYL-INDUCED SYNAPTIC AND BEHAVIORAL PLASTICITY

项目摘要 建议是针对NHLBI/NIDA RFA-HL-19-028(睡眠和昼夜节律依赖机制)而设计的 促进阿片类药物使用障碍(OUD)和对药物辅助治疗的反应(MAT))作为 治愈计划。美国阿片类药物危机导致的年度成本打破了之前的估计。 像芬太尼这样的合成阿片类药物已经超过处方类阿片类药物，成为药物致死的主要原因 过量使用和非法使用芬太尼继续在OUD基本比率已经很高的地区继续存在。其中最多的 OUD患者的常见症状是严重且持续的睡眠和昼夜节律。 干扰。这些睡眠和昼夜节律的异常被认为是助长了阿片类药物的渴望和复发，尽管 直接证据有限。我们对导致阿片类药物依赖的机制缺乏基本的了解 以及它们与睡眠和生理系统的关系。我们发现了一种依赖昼夜节律的机制 可能通过脑部关键区域的细胞类型特异性作用调节芬太尼奖赏相关行为 伏隔核(NAC)。NAC内的大部分神经元是GABA能中棘神经元 它们主要表达多巴胺1或2受体(D1R-MSN或D2R-MSN)。这些MSN 亚群对与奖励相关的行为施加相反的、有时是互补的行为，从而 D1R-MSN的激活推动药物寻找和复发，而D2R-MSN则减弱这些行为。D1R-MSN 也参与了睡眠-觉醒周期的调节。我们的初步工作表明，人的昼夜兴奋性 NAC D1R-MSN及其对芬太尼的反应直接受昼夜节律转录因子NPAS2的调控。 我们还表明，NPAS2可能通过在NAC中的作用影响睡眠-觉醒周期。来自人类遗传学的发现 研究表明，NPAS2的变异与快感缺乏症和快感缺乏症的日变化有关 动机，表明NPAS2与奖励和动机的昼夜调节之间存在联系。我们确认了 NPAS2通过激活NAC的D1-MSN调节芬太尼奖赏的新作用。我们将使用组合 行为学(即自我给药和睡眠多导睡眠图)、切片电生理学和分子(即， 细胞类型特异性RNA-SEQ)方法：研究NPAS2在D1R-MSN中调节行为的作用 对芬太尼的反应(目标1)；评估芬太尼对D1R-MSN突触可塑性的影响并研究 NPAS2是否在特定的昼夜时相介导兴奋性突触的增强(目标2)；阐明 寻求芬太尼和复发行为的细胞类型特异性NPAS2依赖的转录机制(AIM 3)；以及，研究NPAS2救援和丁丙诺啡垫是否改善了芬太尼诱导的睡眠障碍 (目标4)。我们的建议将定义昼夜节律依赖的转录机制在关键奖赏回路中的作用 并发现NPAS2对阿片依赖和复发的治疗潜力。我们的研究 将为睡眠、昼夜节律和阿片类药物交叉点的机制提供新的见解。

PROJECT ABSTRACT Proposal is designed in response to NHLBI/NIDA RFA-HL-19-028 (Sleep and Circadian-Dependent Mechanisms Contributing to Opioid Use Disorder (OUD) and Response to Medication Assisted Treatment (MAT)) as part of the HEAL Initiative. Annual costs attributed to the opioid crisis in the United States have shattered previous estimates. Synthetic opioids like fentanyl have surpassed prescription opioids as the leading cause of death from drug overdose and illicit fentanyl use continues to ride in regions with already high base rates of OUD. Among the most common symptoms experienced by individuals suffering with OUD are severe and persistent sleep and circadian disruptions. These sleep and circadian abnormalities are speculated to foster opioid craving and relapse, although direct evidence is limited. We lack a basic understanding of the mechanisms contributing to opioid dependence and their relationship to sleep and circadian systems. We identified a circadian-dependent mechanism which modulates fentanyl reward-related behaviors potentially via cell-type specific action in a key brain region linked to OUD—the nucleus accumbens (NAc). A majority of the neurons in the NAc are GABAergic medium spiny neurons which predominantly express either dopamine 1 or 2 receptors (D1R-MSNs or D2R-MSNs). These MSN subpopulations exert opposing and sometimes complementary actions on reward-related behaviors, whereby activation of D1R-MSNs drives drug seeking and relapse, while D2R-MSNs attenuate these behaviors. D1R-MSNs are also involved in the regulation of sleep-wake cycles. Our preliminary work suggests the diurnal excitability of NAc D1R-MSNs and their response to fentanyl is directly modulated by the circadian transcription factor NPAS2. We also show NPAS2 impacts sleep-wake cycles, potentially via action in NAc. Findings from human genetics studies indicate variants of NPAS2 are associated with alterations in the diurnal variation of anhedonia and motivation, suggesting links between NPAS2 and the circadian modulation of reward and motivation. We identified a novel role for NPAS2 to regulate fentanyl reward via activity in D1-MSNs of the NAc. We will use a combination of behavioral (i.e., self-administration and sleep polysomnography), slice electrophysiology, and molecular (i.e., cell-type specific RNA-seq) approaches to: Investigate the role of NPAS2 in D1R-MSNs to modulate the behavioral responses to fentanyl (Aim 1); Assess the impact of fentanyl on synaptic plasticity at D1R-MSNs and investigate whether NPAS2 mediates the potentiation of excitatory synapses at specific diurnal phases (Aim 2); Elucidate the cell-type specific NPAS2-dependent transcriptional mechanisms of fentanyl-seeking and relapse behaviors (Aim 3); and, Investigate whether NPAS2 rescue and buprenorphine MAT improve fentanyl-induced sleep disturbances (Aim 4). Our proposal will define the role for circadian-dependent transcriptional mechanisms in key reward circuits of opioid reward and uncover the therapeutic potential of NPAS2 for opioid dependence and relapse. Our studies will provide novel insights into the mechanisms at the intersection of sleep, circadian rhythms, and opioids.