Genetic Engineering of Mammalian Sensory Circuits

光遗传学已成为控制神经元活动的有力工具，具有很高的时空精度。我们是第一个开发光遗传学策略来选择性地激活小鼠中涉及伤害感觉和疼痛的遗传定义的外周体感觉神经元的人（Daou et al., 2013）。在这项为期5年的研究计划中，我们建议设计和验证条件转基因模型，用于在体感觉回路中作用不明确的神经元亚群的功能询问。我们的主要科学目标是：***1)利用光遗传学在体内体感觉回路中询问遗传鉴定的神经元。初级感觉神经元位于背根、三叉神经节和结节神经节，其末梢和纤维支配皮肤和周围组织。它们的激活与体感模式（触觉、温度、瘙痒、伤害感受）和痛觉直接相关。我们之前使用了具有广泛神经元表达的“概念验证”Nav1.8-Cre驱动程序，但我们现在建议询问更具体的遗传定义神经元亚群：热肽能伤害感受器与TRPV1-Cre，触觉非肽能伤害感受器与MrgD-Cre，瘙痒感受器与MrgA3-Cre和机械感受器与thc - cre。一旦他们的ChR2的光学激活或Arch的抑制在体外和体内正常条件下的行为（触觉反应，热敏性，条件性场所厌恶）被证明，我们将使用行为分析和定量感觉测量来测试他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地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慢性疼痛的中枢机制中起着重要作用。我们已经证明，trpc3连接的gq偶联途径有助于伤害感受器的外周致敏（Alkhani等人，2014）。利用AAV病毒表达两种化学致动因子，即化合物CNO激活的DREADDs M3D （gq -偶联）和M4D （gi -偶联），我们拟在体外和体内研究CNO诱导的TRPV1+和MrgD+伤害感受器激活在转基因小鼠中的功能影响。我们假设M3D gq偶联通路的激活将促进致敏和炎症，而M4D gi偶联通路将保护DRG/TG神经元免受致敏和高兴奋性，激活类似于吗啡镇痛的细胞机制。***这些带有人工外周换能器/受体的新型转基因小鼠将使我们能够在体外解剖感觉神经元的信号多样性，并在体内研究在正常和致敏状态下特定神经元群对与疼痛相关的几种体感觉模式的贡献

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.**