Cellular/Molecular Mechanisms of Respiratory Neuronal Chemosensitivity

描述（由申请人提供）：中枢呼吸化学感受是一种众所周知的和非常敏感的稳态机制，通过该机制，大脑通过调节呼吸来维持生理学上适当的pH和PCO 2水平。中枢化学感受功能障碍涉及各种中枢呼吸障碍（例如，婴儿猝死、中枢性先天性通气不足），强调了这一系统的至关重要性。要从根本上理解这一重要的感觉反馈系统，就需要识别相关的传感器--包括相关的神经元和它们的分子探测器。在这方面，在延髓的后斜方核（RTN）中的离散神经元群体满足化学感受器神经元预期的关键标准。然而，关键问题依然存在。目前尚不清楚pH传感是否是这些神经元的固有特性，需要继续考虑作为化学传感器，RTN神经元pH敏感性的细胞/分子基础尚未阐明。为了解决这些基本问题，本资助申请中提出的研究在一种新的小鼠系中应用了复杂的分子和体外电生理技术，该小鼠系在化学敏感的RTN神经元中选择性表达绿色荧光蛋白（GFP）。支持特定目标1的假设是RTN神经元本质上是化学敏感的，并且不同类型的RTN神经元的特征pH响应反映了背景通道的不同补充。在一个子目标中，实验提供了脑干切片中记录的RTN神经元中pH敏感背景K+电流和TTX抗性漏Na+电流的详细表征，并且他们通过使用慢病毒介导的shRNA敲低来测试漏Na+电流是否由NALCN通道携带。 在第二子目标中，在解离的细胞系统中记录表达GFP的RTN神经元，以明确地测试pH敏感性是否是RTN神经元的固有特性。驱动特定目标2的假设是，负责RTN神经元化学敏感性的K+通道在静息膜电位下具有显著的组成性活性，并且它们对pH具有固有的敏感性，或者它们是pH激活的G蛋白偶联受体的下游效应子。三个子目标测试RTN神经元中表达的不同候选pH传感器的参与;这些包括电压门控（KV）和双孔结构域（K2 P）通道家族的不同K+通道，以及质子活化的G蛋白偶联受体。为了这个目的，在候选分子传感器的功能表达通过显性阴性或shRNA构建体的基因敲除或慢病毒介导的表达被破坏后，在表达GFP的RTN神经元中获得记录。 拟议的研究提供了关于这一重要的稳态调节系统的关键信息。 识别中枢呼吸化学感受的新分子底物可以为呼吸障碍的治疗干预提供新的靶点。 公共卫生相关性：一个重要的体内平衡系统调节呼吸以控制代谢CO2排泄，从而控制全身酸碱平衡（pH或H+）;该系统的功能障碍涉及潜在的致命综合征（例如，婴儿猝死、中枢性先天性换气不足）和CO2阈值/敏感性的重新设定可伴随并加重各种慢性呼吸障碍（例如，慢性阻塞性肺病）。众所周知，这种所谓的呼吸化学敏感性涉及尾侧脑干中感知CO2/H+的神经元，但尚未确定潜在的细胞和分子机制。在这项提案中进行的研究旨在澄清呼吸整合神经元的关键群体中的这些机制，从而为这一基本过程提供新的见解，这可能为治疗开发提供新的分子靶点。

DESCRIPTION (provided by applicant): Central respiratory chemoreception is a well known and exquisitely sensitive homeostatic mechanism by which the brain maintains physiologically appropriate levels of pH and PCO2 via regulation of breathing. Dysfunction of central chemoreception is implicated in various central disorders of breathing (e.g., sudden infant death, central congenital hypoventilation), underscoring the critical importance of this system. A fundamental understanding of this important sensory feedback system demands identification of the relevant sensors - both the neurons involved and their molecular detectors. In this regard, a discrete population of neurons in the retro-trapezoid nucleus (RTN) of the medulla oblongata fulfills key criteria expected for chemoreceptor neurons. However, key questions remain. It is not known if pH sensing is an intrinsic property of those neurons, as required for continued consideration as chemosensors, and the cellular/molecular basis for RTN neuronal pH sensitivity has not been elucidated. In order to address these fundamental issues, research proposed in this grant application applies sophisticated molecular and in vitro electrophysiological techniques in the context of a new line of mice that express green fluorescent protein (GFP) selectively in chemosensitive RTN neurons. The hypothesis underpinning Specific Aim 1 is that RTN neurons are intrinsically chemosensitive, and that characteristic pH responses for different types of RTN neurons reflect distinct complements of background channels. In one sub-aim, experiments provide detailed characterization of pH-sensitive background K+ current and TTX-resistant leak Na+ current in RTN neurons recorded in brainstem slices, and they test if the leak Na+ current is carried by NALCN channels by using lentiviral-mediated shRNA knockdown. In a second sub-aim, GFP-expressing RTN neurons are recorded in a dissociated cell system to test definitively if pH sensitivity is an intrinsic property of RTN neurons. The hypothesis driving Specific Aim 2 is that the K+ channel(s) responsible for RTN neuronal chemosensitivity have significant constitutive activity at resting membrane potentials and they are intrinsically sensitive to pH or they are downstream effectors for pH-activated G protein-coupled receptors. Three sub-aims test involvement of different candidate pH-sensors that are expressed in RTN neurons; these include distinct K+ channels of the voltage-gated (KV) and two-pore-domain (K2P) channel family, as well as proton-activated G protein-coupled receptors. For this aim, recordings are obtained in GFP-expressing RTN neurons after functional expression of candidate molecular sensors is disrupted by genetic knockout or lentiviral-mediated expression of dominant-negative or shRNA constructs. The proposed studies provide critical information regarding this important homeostatic regulatory system. Identification of novel molecular substrates that underlie central respiratory chemoreception could provide new targets for therapeutic intervention in disorders of breathing. PUBLIC HEALTH RELEVANCE: An important homeostatic system adjusts breathing to control metabolic CO2 excretion, and thus whole body acid-base balance (pH or H+); dysfunction of this system is implicated in potentially fatal syndromes (e.g., sudden infant death, central congenital hypoventilation) and re-setting of CO2 threshold/sensitivity can accompany and exacerbate various chronic disorders of breathing (e.g., chronic obstructive pulmonary disease). It is well known that this so-called respiratory chemosensitivity involves neurons in the caudal brainstem that sense CO2/H+, but the underlying cellular and molecular mechanisms have not been determined. The research undertaken in this proposal seeks to clarify those mechanisms in a critical population of respiratory integrative neurons - and thus to provide novel insights into this fundamental process that may suggest new molecular targets for therapeutic development.