外周神经元的驱动在几乎所有疼痛状态中都是至关重要的，但通过药理学上沉默这些神经元以实现镇痛效果一直是一个棘手的问题。可逆基因治疗使用长效化学遗传学方法是一个有吸引力的选择。我们使用基因激活的氯离子通道PSAM4-GlyR来研究小鼠疼痛通路。利用重组AAV9载体的传递给感觉神经元，我们发现通过使用varenicline激活PSAM4-GlyR，在体外和体内的GCaMP成像中，急性疼痛行为逆转和神经元活动减弱。还观察到炎症性热性过敏和奥沙利铂诱导的寒冷性痛觉异常有显著减少。重要的是，没有影响运动协调，但无害的von Frey感觉被抑制。我们生成了一个转基因小鼠，该小鼠在Rosa26位点下游表达受CAG驱动的FLExed PSAM4-GlyR，需要Cre重组酶使PSAM4-GlyR和tdTomato的表达成为可能。我们使用NaV1.8 Cre来研究主要感受性NaV1.8+神经元在癌症诱导的骨痛（CIBP）和慢性压迫伤导致的神经痛中的作用。PSAM4-GlyR在NaV1.8阳性神经元中通过varenicline激活逆转CCI驱动的机械、热和寒冷敏感性。此外，给患有CIBP的小鼠进行varenicline处理，在PSAM4-GlyR表达在NaV1.8+感觉神经元中时，通过负重检测方法逆转了癌症疼痛。此外，当这些小鼠接受急性疼痛测试时，检测到对有害机械和热刺激的撤退阈值升高，但无害机械感觉保持不受影响。这些研究证实了PSAM4-GlyR化学遗传学沉默在慢性疼痛状态中的实用性，用于机制分析和潜在的未来治疗利用。 重要性陈述：慢性疼痛是一个严重的问题。在许多慢性疼痛病况中，外周神经阻滞是有效的，表明外周驱动在慢性疼痛中的重要性。我们使用基于修改的配体门控氯离子通道PSAM4-GlyR的化学遗传工具，在体内和体外沉默脊神经节神经元。这种方法减少急性和慢性疼痛模型中的疼痛行为，包括像神经病性疼痛或癌症诱导的骨痛这样的难治性疼痛。我们制备了一种以Cre依赖的方式表达PSAM4-GlyR的小鼠系，为解决不仅是疼痛状态中的伤害感觉感受性神经元的作用，还包括在正常和病理条件下神经系统中的基因定义的神经元族群的功能提供了有用的研究工具。 版权所有 © 2023 Haroun et al.

Drive from peripheral neurons is essential in almost all pain states, but pharmacological silencing of these neurons to effect analgesia has proved problematic. Reversible gene therapy using long-lived chemogenetic approaches is an appealing option. We used the genetically-activated chloride channel PSAM4-GlyR to examine pain pathways in mice. Using recombinant AAV9-based delivery to sensory neurons, we found a reversal of acute pain behavior and diminished neuronal activity using in vitro and in vivo GCaMP imaging upon activation of PSAM4-GlyR with varenicline. A significant reduction in inflammatory heat hyperalgesia and oxaliplatin-induced cold allodynia was also observed. Importantly, there was no impairment of motor coordination, but innocuous von Frey sensation was inhibited. We generated a transgenic mouse that expresses a CAG-driven FLExed PSAM4-GlyR downstream of the Rosa26 locus that requires Cre recombinase to enable the expression of PSAM4-GlyR and tdTomato. We used NaV1.8 Cre to examine the role of predominantly nociceptive NaV1.8+ neurons in cancer-induced bone pain (CIBP) and neuropathic pain caused by chronic constriction injury (CCI). Varenicline activation of PSAM4-GlyR in NaV1.8-positive neurons reversed CCI-driven mechanical, thermal, and cold sensitivity. Additionally, varenicline treatment of mice with CIBP expressing PSAM4-GlyR in NaV1.8+ sensory neurons reversed cancer pain as assessed by weight-bearing. Moreover, when these mice were subjected to acute pain assays, an elevation in withdrawal thresholds to noxious mechanical and thermal stimuli was detected, but innocuous mechanical sensations remained unaffected. These studies confirm the utility of PSAM4-GlyR chemogenetic silencing in chronic pain states for mechanistic analysis and potential future therapeutic use.Significance StatementChronic pain is a massive problem. Peripheral nerve block is effective in many chronic pain conditions, demonstrating the importance of peripheral drive in chronic pain. We used chemogenetic tools based on the modified ligand-gated chloride channel PSAM4-GlyR to silence dorsal root ganglion neurons in vitro and in vivo This approach reduces pain-like behavior in acute and chronic pain models, including resistant pain conditions like neuropathic pain or cancer-induced bone pain. We generated a mouse line that expresses PSAM4-GlyR in a Cre-dependent manner, providing a useful research tool to address not only the role of nociceptive sensory neurons in pain states but also the function of genetically defined sets of neurons throughout the nervous system in normal and pathological conditions.Copyright © 2023 Haroun et al.