Molecular mechanisms of central chemoreception in breathing

描述（由申请方提供）：对中枢神经系统中CO2或pH水平敏感的化学受体对心肺稳态的调节至关重要。它们的功能紊乱导致与一些疾病有关的发病率和死亡率，如先天性中枢性通气不足综合征、慢性阻塞性肺病以及睡眠呼吸暂停和婴儿猝死综合征。尽管中央化学感受器在心肺功能中的重要性，但特定化学感受器位点与呼吸调节的相关性，甚至服务于此功能的特定细胞类型（例如神经元或神经胶质细胞）仍然存在争议。中枢化学感受的最佳支持部位是孤束核（NTS）和后斜方核（RTN）。为了确定这些网站的相对贡献，中央化学感受和条件下，他们是活跃的，我们将解决两个基本问题：1）什么是化学感受的候选脑干神经元的分子/生物物理基础？2）它们通过什么途径向控制呼吸的中枢回路提供输入？拟议的项目将采用互补的体内和体外电生理方法，结合神经解剖学和分子方法来定义NTS和RTN细胞内化学敏感性的分子/生物物理基础，以及它们与脑干呼吸回路的直接或间接联系。体外记录将利用分离的神经元以及切片记录。双光子钙成像在急性切片将提供信息的化学敏感的神经元的反应曲线。这些脑干区域的神经元将被分离，以允许对细胞外酸化的化学敏感反应进行仔细的电生理表征。将使用双移液管膜片钳记录和细胞内灌注测定细胞内酸化的影响。体内记录的化学敏感细胞将被标记以确定其体树突组织和局部轴突分支。它们的脑干靶点将通过逆行标记来确定。通过比较体内记录的填充神经元与体外鉴定的化学敏感细胞的相关pH敏感离子通道的含量、细胞形态（包括轴突投射）和相关神经化学标志物，确定体内记录的填充神经元与体外鉴定的化学敏感细胞的同源性。在存在特异性拮抗剂/激动剂的靶离子通道（体外鉴定）的药理学阻断/刺激期间（例如，我们的初步数据中涉及的TRPV 1通道），将评估特定化学敏感神经元类型对高碳酸血症反应的影响。这些实验将阐明动物生理学的一个中心问题，并可能为药物治疗提供新的药理学靶点。

DESCRIPTION (provided by applicant): Chemoreceptors sensitive to the levels of CO2 or pH in the central nervous system are critical to the regulation of cardiorespiratory homeostasis. Disturbances in their function contribute to the morbidity and mortality associated with a number of diseases such as congenital central hypoventilation syndrome, chronic obstructive pulmonary disease, as well as sleep apnea and sudden infant death syndrome (SIDS). Despite the importance of central chemoreceptors in cardiorespiratory function, the relevance of specific chemoreceptor sites to respiratory regulation, and even the specific cell types serving this function (eg neurons or glia) remain controversial. Among the best supported sites for central chemoreception are the nucleus of the solitary tract (NTS) and the retrotrapezoid nucleus (RTN). To define the relative contributions of these sites to central chemoreception and the conditions under which they are active we will address two essential questions: 1) What are the molecular/biophysical bases of chemoreception for the candidate brainstem neurons? and 2) What are the pathways by which they provide input to central circuits controlling breathing? The proposed project will employ complementary in vivo and in vitro electrophysiological approaches, combined with neuroanatomical and molecular methods to define the molecular/biophysical basis of chemosensitivity within NTS and RTN cells, as well as their direct or indirect connections with brainstem respiratory circuits. In vitro recordings will take advantage of isolated neurons as well as slice recordings. Two-photon calcium imaging in acute slices will provide information on the response profiles of chemosensitive neurons. Neurons in these brainstem areas will be dissociated to allow careful electrophysiological characterization of the chemosensitive response to extracellular acidification. Effects of intracellular acidification will be determined using dual pipette patch clamp recordings with intracellular perfusion. Chemosensitive cells recorded in vivo will be juxtacellularly labeled to define their somatodendritic organization and local axonal arborization. Their brainstem targets will be determined by retrograde labeling. Homology of the filled neurons recorded in vivo with chemosensitive cells identified in vitro will be determined by comparing their content of the relevant pH sensitive ion channels, cell morphology including axonal projection, and related neurochemical markers. The impact of specific chemosensitive neuron types on the response to hypercapnia will be assessed during pharmacological blockade/stimulation of the target ion channels (identified in vitro) where specific antagonist/agonists exist (eg for TRPV1 channels implicated in our preliminary data). These experiments will shed light on one central question of animal physiology and may suggest new pharmacological targets for drug therapy.