Genetic engineering of mammalian somatosensory circuits

我们的小组研究了疼痛机制，我们开创了转基因策略，以选择性和非侵入性地激活介导小鼠疼痛和躯体感觉的初级C纤维传入（Daou 2013，2016; Beaudry 2017; Sharif 2019）。C-纤维位于背根（DRG）、三叉神经（TG）和结状（迷走）神经节中，以及它们的外周末梢，这些末梢具有痛觉、瘙痒、热或触觉刺激。它们的激活产生相关信号，传播到产生躯体感觉的中枢神经系统。 我们建议将这项研究计划的重点放在转基因小鼠模型的选择性激活的C-传入介导轻愉快的触摸和神经源性炎症。我们的两个主要目标是：1）评估C-触觉传入在愉快的轻微触摸和机械异常性疼痛中的双重作用来自人类研究以及啮齿动物临床前实验的越来越多的证据指出C-触觉传入的双重作用：它们在正常条件下介导轻微无害的触摸，而在慢性疼痛条件下介导厌恶的机械异常性疼痛。我们建议在小鼠中对Nav1.8+ Th+ C低阈值机械感受器（C-LTMR）的这一群体进行功能表征。我们将使用交叉遗传策略来选择性地靶向这些神经元进行光遗传学刺激和化学遗传学敏化或沉默，以询问它们对情感愉悦触觉和疼痛触觉超敏反应的贡献。在正常以及慢性疼痛条件（CFA和SNI），我们将记录行为反应和触觉刺激的阈值之前，期间和之后的opto/chemogenetic干预。镇痛和情感（愉快？）将使用位置偏好测定来测试干预对C-LTMRs活性的影响，同时将进行机械和热敏感性测定。 2)目的：探讨神经源性炎症的细胞学基础。病理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，与内源性或异源性代谢型受体相关的Gq偶联途径募集TRPC和Orai 1通道以诱导外周致敏（Alkhani 2014）。我们观察到编码疼痛和瘙痒的MrgprA 3 + C-传入神经的光学刺激与代谢刺激诱发的不同行为反应（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，其特征在于血管活性神经肽CGRP的释放和免疫细胞的浸润。我们将比较由热敏TRPV 1+和机械敏感MrgprD+伤害感受器的代谢性与光学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测它们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.