Genetic engineering of mammalian somatosensory circuits

我们的团队研究了疼痛机制，并开创了转基因策略，以选择性和非侵入性地激活介导小鼠疼痛和体感的初级c纤维传入事件（Daou 2013, 2016; Beaudry 2017; Sharif 2019）。c -纤维位于背根（DRG）、三叉神经节（TG）和结节神经节（迷走神经节），它们的外周末梢传导有害、瘙痒、热或触觉刺激。它们的激活产生的相关信号传播到中枢神经系统，在那里产生体感知觉。我们建议将这项研究重点放在转基因小鼠模型上，以选择性激活介导光愉悦触摸和神经源性炎症的c传入事件。我们的两个主要目标是：1)评估c -触觉传入在愉快的轻度触摸和机械异常痛中的双重作用，越来越多的人类研究和啮齿动物临床前实验表明c -触觉传入的双重作用：它们在正常情况下介导轻微无害的触摸，在慢性疼痛条件下介导厌恶的机械异常痛。我们建议对小鼠中Nav1.8+ Th+ C低阈机械受体（C- ltmrs）群体进行功能表征。我们将使用交叉遗传策略选择性地针对这些神经元进行光遗传刺激和化学遗传致敏或沉默，以询问它们对情感愉悦触摸和疼痛触觉超敏的贡献。在正常和慢性疼痛条件下（CFA和SNI），我们将记录在光/化学发生干预之前，期间和之后对触觉刺激的行为反应和阈值。干预对c - ltmr活性的镇痛和情感（愉快）影响将使用位置偏好测定法进行测试，同时将进行机械和热敏性测定法。2)探讨神经源性炎症的细胞基础。外周致敏诱导c纤维痛觉感受器爆发放电。病理性致敏导致慢性疼痛和偏头痛的神经源性炎症。我们已经证明，与内源性或外源性代谢受体相连的gq偶联通路可以招募TRPC和Orai1通道来诱导外周敏化（Alkhani 2014）。我们观察到编码疼痛和瘙痒的MrgprA3+ c传入信号的光学刺激与代谢刺激引起的不同行为反应（Sharif 2019）。我们假设c -纤维的激活模式决定了它们对神经源性炎症的贡献，其特征是血管活性神经肽CGRP的释放和免疫细胞的浸润。我们将比较热敏TRPV1+和机械敏感MrgprD+伤害感受器的代谢诱导和光刺激诱导的体外和体内炎症反应。这项工作将使我们能够通过探索c传入事件在正常和患病状态下对触觉、疼痛和炎症的贡献来探索其模态多样性。

Our group investigates pain mechanisms and we pioneered transgenic strategies to activate selectively and non-invasively primary C-fiber afferents mediating pain and somatosensation in mice (Daou 2013, 2016; Beaudry 2017; Sharif 2019). C-fibers are located in the dorsal root (DRG), trigeminal (TG), and nodose (vagal) ganglia and their peripheral terminals transduce noxious, itch, thermal or tactile stimuli. Their activation generates the relevant signals propagated to the CNS where the somatosensory percepts are produced. We propose to focus this research program on transgenic mouse models for the selective activation of C-afferents mediating light pleasant touch and neurogenic inflammation. Our two main objectives are: 1) To assess the dual role of C-tactile afferents in pleasant light touch and mechanical allodynia Increasing evidence from human studies as well as rodent preclinical experiments point to a dual role of C-tactile afferents: they mediate light innocuous touch in normal conditions and aversive mechanical allodynia in chronic pain conditions. We propose to functionally characterize this population of Nav1.8+ Th+ C low-threshold mechanoreceptors (C-LTMRs) in mice. We will use intersectional genetic strategies to target selectively these neurons for optogenetic stimulation and chemogenetic sensitization or silencing in order to interrogate their contribution to affective pleasant touch and painful tactile hypersensitivity. In normal as well as chronic pain conditions (CFA and SNI), we will document behavioral responses and thresholds to tactile stimuli before, during and after opto/chemogenetic interventions. Analgesic and affective (pleasant?) effects of interventions on C-LTMRs activity will be tested using place preference assays, while mechanical and thermal sensitivity assays will be performed. 2) To investigate the cellular basis of neurogenic inflammation Peripheral sensitization induces burst firing of C-fiber nociceptors. Pathological sensitization leads to neurogenic inflammation at the source of chronic pain and migraine. We have shown that Gq-coupled pathways linked to endogenous or heterologous metabotropic receptors recruit TRPC and Orai1 channels to induce peripheral sensitization (Alkhani 2014). We observed distinct behavioral responses evoked by optical vs. metabotropic stimulation of MrgprA3+ C-afferents that encode both pain and itch (Sharif 2019). We make the hypothesis that the mode of activation of C-fibers determines their contribution to neurogenic inflammation, characterized by the release of the vasoactive neuropeptide CGRP and the infiltration of immune cells. We will compare in vitro and in vivo inflammatory responses induced by metabotropic vs. optical stimulation of thermosensitive TRPV1+ and mechanosensitive MrgprD+ nociceptors. This work will allow us to explore the modal diversity of C-afferents by probing their contribution to touch, pain and inflammation in normal and diseased states.