Sensory biology of respiratory control neurons in the vagus nerve

 描述(申请人提供)：迷走神经是正常呼吸所需的肺和脑之间的主要管道。在呼吸道内，迷走神经感觉神经元在潮气呼吸时探测到机械伸展的肺，与炎症和疾病有关的提示，以及在某些物种中引起咳嗽的刺激物。然而，迷走神经感觉神经元检测和编码呼吸刺激的分子机制仍然知之甚少。在初步数据中，我们使用分子和遗传学方法对呼吸道中的感觉传入进行分类，并采用遗传工具来绘制、成像、消融和功能控制迷走感觉神经元。我们从基因上标记了两个迷走神经传入的稀疏群体(P2RY1，NPY2R)，它们对呼吸产生了强大的相反的影响(Cell，2015)。P2RY1神经元主要是一种快速传导的纤维，这种纤维支配被称为神经上皮体的肺内分泌细胞团。对迷走神经P2RY1神经元的光遗传刺激停止呼吸，将动物困在呼气中，而不会对同样受迷走神经控制的心率或胃压产生明显影响。NPY2R神经元主要是辣椒素反应的C纤维，迷走神经NPY2R神经元的光遗传激活导致快速而浅的呼吸。根据这些结果，我们推测迷走神经P2RY1神经元介导Hering-Breuer吸气反射，而迷走神经NPY2R神经元参与肺防御。这些发现提出了一些基本的问题，涉及呼吸道中激活P2RY1和NPY2R神经元的感觉刺激，这些神经元是否是正常呼吸所必需的，以及这些神经元如何感知和传递呼吸道提示。我们将使用P2ry1-Ires-Cre和NPY2R-Ires-Cre小鼠，以及活体成像、神经元消融和细胞特异性基因敲除的遗传方法来探索迷走神经P2RY1和NPY2R神经元的感觉生物学。在目标1中，我们开发了一种新的迷走神经节体内成像范式，其中包括一个遗传编码的钙指示剂，并将使用这一技术来询问迷走神经节P2RY1和NPY2R神经元的特异性反应特性。在目标2中，我们将通过控制白喉毒素介导的细胞消融来选择性地消除P2RY1和NPY2R神经元，并确定其对呼吸生理的影响。在目标3中，我们将使用部分或全部迷走感觉神经元中缺乏Piezo2或P2RY1的基因敲除小鼠，探索特定细胞表面受体在迷走神经传入中的作用。Piezo2在一组支配呼吸道的感觉神经元中大量表达，是介导呼吸道机械感觉的一个方面的最佳候选者。总之，这些研究应该为外围线索激活和调节呼吸控制通路的潜在细胞机制提供洞察力。了解迷走神经中呼吸控制神经元的感觉生物学可能为呼吸道疾病干预提供治疗靶点。

 DESCRIPTION (provided by applicant): The vagus nerve is a major conduit between lung and brain required for normal respiration. Within the airways, vagal sensory neurons detect mechanical stretch of the lung during tidal breathing, cues associated with inflammation and illness, and irritants that in some species evoke cough. However, molecular mechanisms by which vagal sensory neurons detect and encode respiratory stimuli remain poorly understood. In preliminary data, we used a molecular and genetic approach to classify sensory afferents in the airways, and adapted genetic tools to map, image, ablate, and functionally control vagal sensory neurons. We genetically tagged two sparse populations of vagal afferents (P2RY1, NPY2R) that exert powerful and opposing effects on breathing (Cell, 2015). P2RY1 neurons are largely fast-conducting a fibers that innervate clusters of pulmonary endocrine cells termed neuroepithelial bodies. Optogenetic stimulation of vagal P2RY1 neurons stops breathing, trapping animals in exhalation, without acutely impacting heart rate or gastric pressure, which are also under vagal control. NPY2R neurons are largely capsaicin-responsive C fibers, and optogenetic activation of vagal NPY2R neurons causes rapid and shallow breathing. Based on these results, we hypothesize that vagal P2RY1 neurons mediate the Hering-Breuer inspiratory reflex, while vagal NPY2R neurons are involved in pulmonary defense. These findings raise basic questions regarding the sensory stimuli in the airways that activate P2RY1 and NPY2R neurons, whether these neurons are required for normal respiration, and how these neurons sense and transduce airway cues. We will use P2ry1-ires-Cre and Npy2r-ires-Cre mice, and genetic approaches for in vivo imaging, neuron ablation, and cell-specific gene knockout to probe the sensory biology of vagal P2RY1 and NPY2R neurons. In Aim 1, we developed a new in vivo imaging paradigm in vagal ganglia that involves a genetically encoded calcium indicator, and will use this technique to query the specific response properties of vagal P2RY1 and NPY2R neurons. In Aim 2, we will selectively eliminate P2RY1 and NPY2R neurons by controlled diphtheria toxin-mediated cell ablation and determine the impact on respiratory physiology. In Aim 3, we will explore the roles of particular cell surface receptors in vagal afferents using knockout mice that lack Piezo2 or P2RY1 in some or all vagal sensory neurons. Piezo2 is abundantly expressed in a cohort of airway- innervating sensory neurons, and is a prime candidate to mediate an aspect of airway mechanosensation. Together, these studies should provide insights into cellular mechanisms underlying activation and modulation of breathing control pathways by peripheral cues. Understanding the sensory biology of respiratory control neurons in the vagus nerve may provide therapeutic targets for airway disease intervention.