Genetic engineering of mammalian somatosensory circuits

我们的团队研究疼痛机制，我们开创了转基因策略，以选择性和非侵入性地激活初级C纤维传入，介导小鼠的疼痛和躯体感觉(Daou 2013,2016；Beaudry 2017；Sharif 2019)。C纤维位于背根神经节(DRG)、三叉神经节(TG)和结状神经节(迷走神经节)，它们的外周终末传递伤害性、瘙痒、温度或触觉刺激。它们的激活产生相关的信号，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的重点放在转基因小鼠模型上，以选择性地激活C-传入，介导光、愉悦的触摸和神经源性炎症。 我们的两个主要目标是： 1)评估C-触觉传入在愉悦的光触觉和机械性痛觉异常中的双重作用 来自人类研究和啮齿动物临床前实验的越来越多的证据表明，C-触觉传入具有双重作用：它们在正常情况下调节轻微的无害触摸，在慢性疼痛条件下调节厌恶的机械性异位痛觉。我们建议在小鼠中对这一群体的Nav1.8+Th+C低阈值机械受体(C-LTMR)进行功能表征。我们将使用交叉遗传策略来选择性地针对这些神经元进行光遗传刺激和化学发生敏化或沉默，以询问它们对情感愉悦触摸和痛苦触觉超敏的贡献。在正常和慢性疼痛条件下(CFA和SNI)，我们将记录在光/化学干预之前、期间和之后对触觉刺激的行为反应和阈值。止痛药和情感(愉快？)干预对C-LTMR活性的影响将使用位置偏好分析来测试，同时将进行机械和热敏感性分析。 2)探讨神经源性炎症的细胞基础 外周敏化可诱发C纤维伤害性感受器的爆发式放电。病理致敏导致慢性疼痛和偏头痛的根源神经源性炎症。我们已经证明，与内源性或异源代谢性受体相连的GQ偶联通路招募TRPC和Orai1通道来诱导外周敏化(Alkhani 2014)。我们观察到对同时编码疼痛和瘙痒的MRgprA3+C-传入的光刺激与代谢性刺激引起的不同的行为反应(Sharif 2019)。我们假设C纤维的激活方式决定了它们在神经源性炎症中的作用，其特征是血管活性神经肽CGRP的释放和免疫细胞的渗透。我们将在体外和体内比较温度敏感型TRPV1+和机械敏感型MRgprD+伤害性感受器的代谢性和光性刺激引起的炎症反应。 这项工作将使我们能够通过探索C-传入在正常和疾病状态下对触摸、疼痛和炎症的贡献来探索C-传入的模式多样性。

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.