Cellular/Molecular Mechanisms of Respiratory Neuronal Chemosensitivity

一组位于后斜方核（RTN）的兴奋性神经元表达转录因子， Phox 2b，整合感觉输入和有关大脑状态的信息，以传输到呼吸系统， 节奏/模式生成电路。此外，RTN神经元会根据CO2浓度的变化调整其放电 (or H+）和调节呼吸，以维持生理上适当的pH值和PCO 2水平， 称为中枢呼吸化学感受。中枢化学感受功能障碍涉及各种 在睡眠期间经常发生的中枢性呼吸障碍（例如，婴儿猝死，先天性中枢性 低通气综合征（CCHS））。在上一个项目期间，我们发现表达Phox 2b的RTN 神经元具有内在的化学敏感性，并在RTN中确定了两个独立的分子质子传感器 神经元-ASK-2，质子抑制的背景K+通道和GPR 4，质子激活的G蛋白偶联的 受体-这是刺激呼吸所需的二氧化碳。然而，重要的问题仍然存在， 关于：基线放电特性的离子基础和唤醒状态依赖因素的调制; 在RTN神经元中参与GPR 4下游的效应器系统;以及提出的星形胶质细胞 在RTN介导的呼吸系统中，调节可以与对GPR 4和ASK-2的需求整合。 化疗敏感性该提议使用以下方法解决这些问题：新型条件性敲除小鼠系;病毒介导的 shRNA敲除和/或拯救;单细胞电生理学和分子生物学;和整个动物 呼吸功能和警觉状态的测定。支持具体目标1的假设是TTX- 耐受性阈下Na+通道NALCN和NaV1.9参与RTN神经元的基线兴奋性 并介导与唤醒状态依赖性脑核团相关的神经肽的易化作用。我们 破坏RTN神经元中这些通道的表达，并确定对阈下Na+电流、基础Na+电流、 和体外神经肽调节的放电，以及体内唤醒状态依赖的呼吸CO2敏感性。 驱动特异性目的2的假设是GPR 4参与cAMP转导途径和背景 RTN神经元中细胞pH传感的K+通道（独立于ASK-2），以及星形胶质细胞扩增 呼吸化学反射的变化涉及促进RTN神经元周围的局部pH值变化。我们使用 在单个RTN神经元中的药理学和转录组学方法来表征GPR 4信号通路， 效应器通道，我们破坏了髓质星形胶质细胞中的pH调节Na+ -HCO 3转运蛋白NBCe 1， 确定对体内呼吸化学感受器反射的影响。后者可能支持一个收敛理论， 星形胶质细胞-神经元对这种高度敏感的化学反射的贡献，桥接了该领域的主要电流鸿沟。 总的来说，拟议的研究提供了有关分子和细胞机制的关键信息 控制RTN神经元的活动，并调节这一重要的自我平衡呼吸系统。 新的分子机制的鉴定可能为呼吸障碍提供新的治疗靶点。

A group of excitatory neurons located in the retrotrapezoid nucleus (RTN) that express the transcription factor, Phox2b, integrate sensory inputs and information regarding brain state for transmission on to respiratory rhythm/pattern-generating circuits. In addition, RTN neurons adjust their firing in response to changes in CO2 (or H+) and adjust breathing to maintain physiologically appropriate levels of pH and PCO2, a homeostatic process called central respiratory chemoreception. Dysfunction of central chemoreception is implicated in various central disorders of breathing that often occur during sleep (e.g., sudden infant death, congenital central hypoventilation syndrome (CCHS)). In the last project period, we showed that Phox2b-expressing RTN neurons are intrinsically chemosensitive, and identified two independent molecular proton sensors in RTN neurons - TASK-2, a proton-inhibited background K+ channel and GPR4, a proton-activated G protein-coupled receptor - that are required for stimulation of breathing by CO2. Important questions remain, however, regarding: the ionic basis for baseline firing properties and modulation by arousal state-dependent factors; the effector systems engaged downstream of GPR4 in RTN neurons; and mechanisms by proposed astrocytic modulation can be integrated with the requirement for GPR4 and TASK-2 in RTN-mediated respiratory chemosensitivity. This proposal addresses these issues using: novel conditional knockout mouse lines; viral-mediated shRNA knockdown and/or rescue; single cell electrophysiology and molecular biology; and whole animal assays of respiratory function and vigilance states. The hypothesis underpinning Specific Aim 1 is that TTX- resistant subthreshold Na+ channels, NALCN and NaV1.9, contribute to baseline excitability of RTN neurons and mediate facilitatory effects of neuropeptides associated with arousal state-dependent brain nuclei. We disrupt expression of these channels in RTN neurons and determine effects on subthreshold Na+ currents, basal and neuropeptide-modulated firing in vitro, and arousal state-dependent respiratory CO2 sensitivity in vivo. The hypothesis driving Specific Aim 2 is that GPR4 engages a cAMP-transduction pathway and background K+ channel (independent of TASK-2) for cellular pH sensing in RTN neurons, and that astrocytic amplification of respiratory chemoreflexes involves boosting local pH changes around RTN neurons. We use pharmacological and transcriptomic approaches in single RTN neurons to characterize the GPR4 signaling pathway and effector channel, and we disrupt a pH-modulating Na+ -HCO3 transporter, NBCe1, in medullary astrocytes to determine effects on the respiratory chemoreflex in vivo. This latter may support a convergent theory for astrocyte-neuron contributions for this highly sensitive chemoreflex, bridging a major current divide in the field. Collectively, the proposed studies provide critical information regarding molecular and cellular mechanisms that control activity of RTN neurons, and regulate this important homeostatic respiratory system. Identification of novel molecular mechanisms may provide new therapeutic targets for disorders of breathing.