Genetic Engineering of Mammalian Sensory Circuits

光遗传学已经成为一种强大的工具，以控制神经元的活动与高时空精度。我们是第一个开发光遗传学策略以选择性地激活参与小鼠的伤害感受和疼痛的遗传上定义的外周体感神经元的人（Daou等人，2013年）。在这个为期5年的研究计划中，我们提议设计和验证条件转基因模型，用于对躯体感觉回路中作用不明确的神经元子集进行功能询问。我们的主要科学目标是：*1）使用光遗传学在体内询问躯体感觉回路中遗传鉴定的神经元。初级感觉神经元位于背根、三叉神经和结状神经节中，而它们的外周末梢和纤维支配皮肤和外周组织。它们的激活与躯体感觉方式（触觉、温度、瘙痒、伤害感受）和疼痛感知直接相关。我们以前使用了具有广泛神经元表达的“概念验证”Nav1.8-Cre驱动器，但我们现在建议询问遗传定义的神经元的更具体的子集：TRPV 1-Cre的热肽能伤害感受器，MrgD-Cre的触觉非肽能伤害感受器，MrgA 3-Cre的瘙痒传感器和TH-Cre的机械传感器。一旦在正常条件下（触觉反应、热敏感性、条件性位置厌恶）在体外和体内行为上证明了它们与ChR 2的光学激活或与Arch的抑制作用，我们将使用行为分析和定量感觉测量来测试它们对炎症或神经性损伤后慢性疼痛的贡献。2)利用化学遗传学在体内选择性调节两种主要类型的伤害感受器。伤害感受器的外周敏化在触发慢性疼痛的中枢机制中起主要作用。我们已经表明TRPC 3连接的Gq偶联途径有助于伤害感受器中的外周敏化（Alkhani等人，2014）。使用AAV病毒表达的两个化学发生致动器，DREADDs M3 D（Gq偶联）和M4 D（Gi偶联）激活的化合物CNO，我们建议调查的功能后果的hoc诱导的调制TRPV 1+和MrgD+伤害感受器发射在转基因小鼠在体外和体内。我们假设M3 D Gq偶联通路的激活将促进致敏和炎症，而M4 D Gi偶联通路将保护DRG/TG神经元免于致敏和过度兴奋，招募类似于吗啡镇痛的细胞机制。* 这些新的转基因小鼠与人工外周传感器/受体工程将使我们能够在体外解剖感觉神经元的信号多样性，以及在体内研究特定的神经元群体的贡献的几种形式的躯体感觉相关的疼痛，在正常和敏化状态。

Optogenetics has emerged as a powerful tool to control neuronal activity with high spatio-temporal precision. We were the first to develop optogenetic strategies to activate selectively genetically-defined peripheral somatosensory neurons involved in nociception and pain in mice (Daou et al., 2013). In this 5-year research program, we propose to design and validate conditional transgenic models for the functional interrogation of neuronal subsets with unclear roles in somatosensory circuits.Our main scientific objectives are:***1) To interrogate genetically-identified neurons in somatosensory circuits in vivo using optogenetics. Primary sensory neurons are located in the dorsal root, trigeminal, and nodose ganglia while their peripheral terminals and fibers innervate the skin and peripheral tissues. Their activation is directly linked to somatosensory modalities (touch, temperature, itch, nociception) and to pain perception. We previously used the "proof-of-concept" Nav1.8-Cre driver with broad neuronal expression but we propose now to interrogate more specific subsets of genetically-defined neurons: thermal peptidergic nociceptors with TRPV1-Cre, tactile non-peptidergic nociceptors with MrgD-Cre, itch sensors with MrgA3-Cre and mechanosensors with TH-Cre. Once their optical activation with ChR2 or inhibition with Arch is demonstrated in vitro and in vivo behaviorally in normal conditions (tactile response, thermal sensitivity, conditioned place aversion), we will test their contribution to chronic pain following inflammation or neuropathic injury using behavioral analysis and quantitative sensory measurements.***2) To modulate selectively the two main types of nociceptors in vivo using chemogenetics. Peripheral sensitization of nociceptors plays a major role in triggering central mechanisms of chronic pain. We have shown that TRPC3-linked Gq-coupled pathways contribute to peripheral sensitization in nociceptors (Alkhani et al., 2014). Using AAV viral expression of two chemogenetic actuators, the DREADDs M3D (Gq-coupled) and M4D (Gi-coupled) activated by the compound CNO, we propose to investigate the functional consequences of CNO-induced modulation of TRPV1+ and MrgD+ nociceptor firing in transgenic mice in vitro and in vivo. We make the hypothesis that activation of M3D Gq-coupled pathways will promote sensitization and inflammation, while M4D Gi-coupled pathways will protect DRG/TG neurons from sensitization and hyperexcitability, recruiting cellular mechanisms analogous to morphine analgesia. ***These novel transgenic mice engineered with artificial peripheral transducers/receptors will allow us to dissect in vitro the signaling diversity of sensory neurons as well as to investigate in vivo the contribution of specific neuronal populations to several modalities of somatosensation relevant to pain, in normal and sensitized states.**