Molecular mechanisms of central chemoreception in breathing

描述(由申请人提供)：对中枢神经系统中二氧化碳或pH水平敏感的化学感受器对心肺稳态的调节至关重要。它们的功能障碍导致与一些疾病相关的发病率和死亡率，如先天性中枢低通气综合征、慢性阻塞性肺疾病、睡眠呼吸暂停和婴儿猝死综合征(SIDS)。尽管中枢化学感受器在心肺功能中的重要性，但特定的化学感受器位置与呼吸调节的相关性，甚至是服务于这一功能的特定细胞类型(如神经元或神经胶质细胞)仍然存在争议。孤束核(NTS)和斜方后核(RTN)是中枢化学感受的最佳支持部位。为了确定这些位置对中枢化学接收的相对贡献以及它们活跃的条件，我们将解决两个基本问题：1)候选脑干神经元化学接收的分子/生物物理基础是什么？2)它们向控制呼吸的中枢回路提供输入的途径是什么？该项目将采用体内和体外互补的电生理方法，结合神经解剖学和分子方法来确定NTS和RTN细胞内化疗敏感性的分子/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和切片记录。急性切片中的双光子钙成像将提供有关化疗敏感神经元反应曲线的信息。这些脑干区域的神经元将被分离，以便仔细地描述细胞外酸化的化学敏感性反应的电生理特征。细胞内酸化的影响将使用双吸管膜片钳记录和细胞内灌流来确定。体内记录的化学敏感细胞将被并列标记，以确定它们的躯体树突组织和局部轴突树枝。他们的脑干靶标将通过逆行标记来确定。体内记录的填充神经元与体外鉴定的化疗敏感细胞的同源性将通过比较它们相关的pH敏感离子通道的含量、细胞形态(包括轴突投射)和相关的神经化学标志物来确定。特定的化学敏感神经元类型对高碳酸血症反应的影响将在药物阻断/刺激存在特定拮抗剂/激动剂的靶离子通道(例如，我们初步数据中所涉及的TRPV1通道)的过程中进行评估。这些实验将阐明动物生理学的一个核心问题，并可能为药物治疗提供新的药理靶点。 与公共健康相关：呼吸受到动脉和大脑二氧化碳部分压力的微调，这种调节失败可能会导致严重的后果，如婴儿猝死综合症(SIDS)或先天性中枢低通气综合征(CCHS)；然而，这种调节背后的机制尚不清楚。特别是，中枢神经系统化学接收的回路和分子机制仍然是主要争论的主题。这一建议综合了电生理、分子和组织化学技术，以确定参与中枢化学接收的神经元类型，并剖析其潜在的分子机制。

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. PUBLIC HEALTH RELEVANCE: Breathing is finely tuned by the partial pressures of arterial and brain carbon dioxide and failure of this tuning may lead to dramatic consequences such as the sudden Infant Death Syndrome (SIDS) or congenital central hypoventilation syndrome (CCHS); yet, the mechanisms underlying this regulation are not well understood. In particular, the circuitries and molecular mechanisms of CNS chemoreception remain the subjects of major debate. This proposal marshals electrophysiological, molecular and histochemical techniques to identify the neuronal types involved in central chemoreception and dissect the underlying molecular mechanisms.