Microglia and Opioid Withdrawal: Mechanisms of Negative Reinforcement

目前阿片类药物过量的流行是由非法和处方麻醉剂疼痛推动的 药物治疗大量使用阿片类药物和反复戒断增加了严重戒断的可能性， 通过消极强化的方式使复发的脆弱性永久化。消极情绪效价 在最初的、戏剧性的身体迹象之后，戒断症状可能会持续很长时间， 状态，药物渴望，以及复发的高可能性。这些综合症状通常被称为 “毒瘾”吸毒者往往宁愿继续吸毒，也不愿面对戒断。被 “毒瘾”具有严重炎症状态的许多属性，这使我们调查了 小胶质细胞，一种存在于大脑中的先天免疫细胞，在阿片类药物耐受和戒断中， 由CEBRA R21资助（R21-DA 044757）支持。 CEBRA R21的资助使我们发现了核糖体结合的mRNA发生了巨大变化， “translatome”-使用RiboTag纯化的小胶质细胞RNA的RNA测序在小胶质细胞中。这些结果 为我们提供了构成这一提议基础的线索。许多变化与环磷酸腺苷有关 信号及其下游靶点，并发现Gi/o信号的实验化学遗传刺激 实际上会加重阿片类药物戒断症状。了解到胶质细胞是可塑性的伙伴，我们怀疑， 阿片类药物依赖的复发/缓解性质有助于引发和调节小胶质细胞， 从最初阶段的缓和戒断到多次戒断后的加剧戒断和阿片类药物寻求 容忍和退缩的循环。因此，对胶质细胞在戒断中作用的研究可能会提供新的 治疗途径我们提出了三个目标，使用芬太尼和最近开发的转基因小鼠， 允许条件性和小胶质细胞特异性Cre和TdTomato表达，而不破坏小胶质细胞功能。在 目的1我们将分析阿片类药物治疗1个周期和5个周期后小胶质细胞翻译组的变化轨迹 依赖性和自发性戒断。在目标2中，我们将检查身体和行为 一个与五个阿片类药物依赖和戒断周期的后果，探讨间歇性 依赖和戒断的循环加剧了戒断的负面后果。然后我们将 研究嘌呤能受体P2 Y12和P2 X7参与小胶质细胞反应的假设 在阿片类药物戒断的初始和延迟阶段。在目标3中，我们将使用体外脑切片模型， 小胶质细胞的光子共聚焦成像。我们将使用时间来研究小胶质细胞的微观结构和运动性- 激光显微镜我们将使用基于FRET的生物传感器测量环AMP的实时变化， 钙动力学与GCaMP 6。这三个目标整合了时间，行为和分子 阿片类药物依赖和戒断期间小胶质细胞参与的后果。

The current epidemic of opioid overdoses has been propelled by both illicit and prescribed narcotic pain medications. Extensive opioid use and repeated abstinence increases the likelihood of severe withdrawal and perpetuates the vulnerability to relapse via means of negative reinforcement. The negative emotional valence of withdrawal can last long after the initial, dramatic physical signs, involving a protracted negative emotional state, drug craving, and a high likelihood of relapse. These combined symptoms are commonly referred to as being “dope sick”. Addicted individuals often prefer to continue drugs rather than face withdrawal. Being “dope-sick” has many attributes of a severe inflammatory state and this led us to investigate the involvement of microglia, the innate immune cells that reside in the brain, in opioid tolerance and withdrawal, and this was supported by a CEBRA R21 grant (R21-DA044757). That CEBRA R21 grant resulted in our findings of dramatic changes in ribosome-bound mRNAs—the “translatome”—in microglia using RNA sequencing of RiboTag purified microglial RNAs. Those results provided us with the leads that form that basis for this proposal. Many of the changes related to cyclic AMP signaling and its downstream targets, and experimental chemogenetic stimulation of Gi/o signaling was found to actually worsen opioid withdrawal. With the understanding that glia are partners in plasticity, we suspect that the relapsing/remitting nature of opioid dependence serves to prime and condition microglia, shifting the impact from tempering withdrawal during initial stages to exacerbating withdrawal and opioid seeking after multiple cycles of tolerance and withdrawal. Thus, investigations into the role of glia in withdrawal may provide new therapeutic avenues. We propose three Aims using fentanyl and a recently developed transgenic mouse that allows conditional and microglia-specific Cre and TdTomato expression without disrupting microglia function. In Aim 1 we will analyze the trajectory of the changing microglial translatome after one vs. five cycles of opioid dependence and spontaneous withdrawal. In Aim 2 we will examine the physical and behavioral consequences of one vs. five cycles of opioid dependence and withdrawal, to explore the idea that intermittent cycles of dependence and withdrawal exacerbate the negative consequences of withdrawal. We will then investigate the hypothesis that the purinergic receptors P2Y12 and P2X7 are involved in microglial responses during initial and delayed phases of opioid withdrawal. In Aim 3 we will use an in vitro brain slice model with 2- photon confocal imaging of microglia. We will study the microstructure and motility of microglia using time- lapse microscopy. We will measure real-time changes in cyclic AMP using a FRET-based biosensor and calcium dynamics with GCaMP6. These three Aims integrate the temporal, behavioral, and molecular consequences of microglial engagement during opioid dependence and withdrawal.