Cholinergic modulation of Descending Pain Control Pathways

在美国，慢性疼痛困扰着超过20%的成年人，这强调了需要更多的治疗。 全面了解疼痛控制机制和更好的疼痛治疗。这种需要被放大了 目前的阿片类药物危机在2018年造成47，600名美国人死亡。虽然阿片类药物 有效治疗急性和慢性疼痛，不良副作用，包括滥用倾向和耐受性， 重复使用的镇痛效果突出了对新的非阿片样物质疼痛疗法的需要。这种需要 激发了我们对乙酰胆碱（ACh）及其受体（AChRs）在疼痛调制中的研究。 腹外侧导水管周围灰质（vlPAG）。从外周到中枢神经系统的上行疼痛信号 由下行疼痛通路调节，包括vlPAG及其向吻侧腹内侧的投射 延髓（RVM）和蓝斑（LC）。这种下行疼痛通路是阿片类药物作用的关键部位， 内源性疼痛控制虽然对这一回路了解很多，但胆碱能神经的疼痛调节作用， 对vlPAG的输入未充分研究。我们的初步数据表明，胆碱能神经元的光遗传学刺激 从脚桥被盖核（PPTg）到vlPAG的投射在急性疼痛中是抗伤害感受的。 使用细胞和电路特异性光遗传学方法，我们将测试刺激胆碱能神经的作用。 在慢性神经病理性疼痛模型中，对vlPAG对伤害感受的投射，鉴定胆碱能受体 介导这些抗伤害效应，并探讨胆碱能信号和 阿片系统。 在目的1中，我们将使用光遗传学方法来测试刺激胆碱能神经元的抗伤害感受作用。 在经历慢性神经性疼痛的小鼠中投射到vlPAG的PPTg神经元。我们还将分析情感 使用实时位置偏好测定刺激这些投射的疼痛缓解。在目标2中，我们将确定 胆碱能受体介导PPTg和vlPAG神经元之间的突触通讯，使用高浓度的 通过荧光原位杂交试验观察毒蕈碱和烟碱的mRNA表达 vlPAG神经元上的AChR。在基于mRNA表达鉴定候选AChR后，我们将测试 这些受体在突触通信中的功能作用，使用切片电生理学和离体 光遗传学最后，我们将测试确定的AChRs在体内抗伤害感受中的因果作用， 神经性疼痛测定中的光遗传学和药理学。在目标3中，我们将探索可能的相互作用 这些胆碱能机制和阿片系统之间，通过测试胆碱能镇痛的功效， 在吗啡耐受小鼠和纳洛酮期间，从慢性吗啡治疗中沉淀戒断。 这些研究将采用组织学、电生理学、光遗传学和药理学方法， 推进我们对下行疼痛通路的胆碱能调节的知识，以最终帮助识别 用于治疗慢性疼痛的新的阿片样物质非依赖性靶点。

Chronic pain conditions plague more than 20% of adults in the United States, emphasizing the need for a more comprehensive understanding of pain control mechanisms and better pain therapies. This need is amplified further by the current opioid crisis, which killed 47,600 Americans in 2018. While opioids are remarkably effective treatments for acute and chronic pain, adverse side effects, including abuse liability and tolerance to the analgesic effects with repeated use, highlight the need for novel non-opioid pain therapies. This need motivates our investigations of pain modulation by acetylcholine (ACh) and its receptors (AChRs) in the ventrolateral periaqueductal gray (vlPAG). Ascending pain signaling from periphery to central nervous system is modulated by the descending pain pathway, including the vlPAG and its projections to rostral ventromedial medulla (RVM) and locus ceruleus (LC). This descending pain pathway is a key site of action of opioids and endogenous pain control. While much is known about this circuitry, the pain modulatory effects of cholinergic inputs to vlPAG are understudied. Our preliminary data show that optogenetic stimulation of cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to vlPAG is antinociceptive in acute pain. Using cell- and circuit-specific optogenetic approaches, we will test the effect of stimulating cholinergic projections to vlPAG on nociception in a chronic neuropathic pain model, identify the cholinergic receptors mediating these antinociceptive effects, and explore possible interactions between cholinergic signaling and the opioid system. In Aim 1, we will use optogenetic approaches to test antinociceptive effects of stimulating cholinergic PPTg neurons projecting to vlPAG in mice experiencing chronic neuropathic pain. We will also assay affective pain relief of stimulating these projections using a real-time place preference assay. In Aim 2, we will identify cholinergic receptor(s) mediating synaptic communication between PPTg and vlPAG neurons, using high- throughput fluorescence in situ hybridization assay to visualize mRNA expression of muscarinic and nicotinic AChRs on vlPAG neurons. After identifying candidate AChR(s) based on mRNA expression, we will test functional role of these receptors in synaptic communication, using slice electrophysiology and ex vivo optogenetics. Finally, we will test the causal role of identified AChRs in antinociception in vivo using optogenetics and pharmacology in neuropathic pain assays. In Aim 3, we will explore possible interactions between these cholinergic mechanisms and the opioid system, by testing the efficacy of cholinergic analgesia in morphine-tolerant mice and during naloxone precipitated withdrawal from chronic morphine treatment. These studies will employ histological, electrophysiological, optogenetic and pharmacological approaches to advance our knowledge of cholinergic modulation of descending pain pathways, to ultimately help identify novel opioid-independent targets for treating chronic pain.