Microglia and Opioid Withdrawal: Mechanisms of Negative Reinforcement

小胶质细胞和阿片类药物戒断：负强化机制

• 批准号: 10313923
• 负责人: John F Neumaier
• 金额: $ 33.72万
• 依托单位: SEATTLE INST FOR BIOMEDICAL/CLINICAL RES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10313923
• 关键词: 3-Dimensional; Abstinence; Analgesics; Animals; Anxiety; Award; Behavior; Behavioral; Behavioral Model; Biosensor; Brain; Calcium; Cells; Cessation of life; Chronic; Complex; Complication; Confocal Microscopy; Corpus striatum structure; Cyclic AMP; Decision Making; Dependence; Disease; Disincentive; Emotional; Emotions; Epidemic; Face; Fentanyl; Fluorescence Resonance Energy Transfer; Goals; Grant; Harvest; Hyperalgesia; Immune; In Vitro; Individual; Inflammatory; Injections; Investigation; LoxP-flanked allele; Measures; Mediating; Messenger RNA; Methods; Microglia; Microscopy; Modeling; Molecular; Morphine; Morphology; Mus; Naloxone; Narcotics; Nature; Negative Reinforcements; Neuroglia; Neurons; Opiate Addiction; Opioid; Opioid Receptor; Oral; Overdose; Pharmaceutical Preparations; Phase; Procedures; Process; Purinoceptor; RNA; Receptor Signaling; Relapse; Reporter; Rest; Rewards; RiboTag; Ribosomes; Role; Self Administration; Severities; Signal Transduction; Slice; Substance Withdrawal Syndrome; Symptoms; Testing; Time; Transgenic Mice; Translations; Withdrawal; Withdrawal Symptom; base; brain research; cell motility; common symptom; confocal imaging; designer receptors exclusively activated by designer drugs; drug craving; mu opioid receptors; negative emotional state; neuroinflammation; novel therapeutics; opiate tolerance; opioid abuse; opioid epidemic; opioid overdose; opioid use; opioid withdrawal; overdose death; receptor; recruit; response; time use; transcriptome sequencing; translatome; two-photon

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.

阿片类药物过量的当前流行病已被非法和开处方麻醉疼痛推动 药物。大量的OIOID使用和反复禁欲增加了严重撤离和 通过负面强化手段将继电器的脆弱性永存。负面情绪价值 戒断的持续很长时间是在最初的戏剧性物理迹象之后持续的，涉及持久的负面情绪 状态，渴望毒品和退休的可能性很大。这些组合的符号通常称为 是“生病的”。上瘾的人通常更喜欢继续毒品而不是面对戒断。存在 “浓汤”具有严重炎症状态的许多属性，这使我们调查了参与 小胶质细胞，驻留在大脑中的先天免疫细胞，阿片类药物的耐受性和戒断，这是 由CEBRA R21赠款（R21-DA044757）支持。 CEBRA R21赠款导致了我们对核糖体结合mRNA的巨大变化的发现 - “ Translateme” - 使用Ribotag纯化的小胶质细胞RNA的RNA测序的小胶质细胞。这些结果 为我们提供了该提案基础的潜在客户。许多与循环放大器有关的更改 发现了信号及其下游靶标，并发现了GI/O信号的实验化学发生模拟 实际上会加重阿片类药物的提取。认识到神经胶质是可塑性的合作伙伴，我们怀疑 阿片类药物依赖性的复发/恢复性质可用于小胶质细胞和状态小胶质细胞，改变了撞击 从初始阶段的回火提取到加剧戒断和寻求阿片类药物。 宽容和戒断的周期。这是对神经胶质在戒断中作用的调查可能会提供新的 治疗途径。我们建议使用芬太尼和最近开发的转基因小鼠的三个目标 允许有条件和小胶质细胞特异性的CRE和TDTOMATO表达，而不会破坏小胶质细胞功能。在 目标1我们将分析一个与五个循环的阿片类药物后，小胶质细胞不断变化的轨迹 依赖和赞助戒断。在AIM 2中，我们将检查身体和行为的 一个与五个周期的阿片类药物依赖和戒断的后果，以探索间歇性的想法 依赖和戒断的循环加剧了退出的负面后果。然后我们会 研究嘌呤能受体P2Y12和P2X7参与小胶质细胞反应的假设 在阿片类药物提取的初始和延迟阶段。在AIM 3中，我们将使用2-的体外脑切片模型 小胶质细胞的光子共聚焦成像。我们将使用时间 - 衰减显微镜。我们将使用基于FRET的生物传感器和 使用GCAMP6的钙动力学。这三个目的整合了临时，行为和分子 阿片类药物依赖性和戒断过程中小胶质互动的后果。