Cellular/Molecular Mechanisms of Respiratory Neuronal Chemosensitivity

位于斜方体后核(RTN)的一组兴奋性神经元表达转录因子， PHOX2B，整合感觉输入和有关大脑状态的信息，以便传递到呼吸系统 节奏/图案产生电路。此外，RTN神经元还会根据二氧化碳的变化来调整其放电 (或H+)，并调整呼吸以维持生理上适当的pH和PCO2水平，这是一种动态平衡 这一过程被称为中枢呼吸化学接收。中枢化学接收功能障碍与多种疾病有关 睡眠中经常发生的中枢性呼吸障碍(例如，婴儿猝死、先天性中枢性呼吸障碍) 换气不足综合征(CCHS))。在上一个项目阶段，我们发现了表达PHOX2B的RTN 神经元是天生的化学敏感者，并在RTN中识别出两个独立的分子质子传感器 神经元-TASK-2，质子抑制的背景K+通道和GPR4，质子激活的G蛋白偶联 受体--这是二氧化碳刺激呼吸所必需的。然而，重要的问题仍然存在， 关于：基线激发特性的离子基础和唤醒状态依赖因素的调节； 在RTN神经元中参与GPR4下游的效应系统；以及拟议的星形胶质细胞的机制 在RTN介导的呼吸中，调节可以与GPR4和TASK-2的需求相结合 对化学药物敏感。该提案使用：新的条件性基因敲除小鼠系；病毒介导型 ShRNA敲除和/或拯救；单细胞电生理学和分子生物学；以及整个动物 呼吸功能和警觉状态的分析。支持特定目标1的假设是TTX- 耐受的亚阈值Na+通道NALCN和NaV1.9对RTN神经元的基础兴奋性有贡献 以及调节与觉醒状态依赖的脑核团相关的神经肽的促进作用。我们 阻断RTN神经元上这些通道的表达并确定对亚阈值Na+电流的影响 神经肽在体外调节放电，在体内唤醒状态依赖的呼吸性二氧化碳敏感性。 驱动特定目标2的假设是GPR4参与cAMP转导途径和背景 RTN神经元中细胞pH感知K+通道(独立于TASK-2)和星形胶质细胞扩增 呼吸化学反射的作用包括促进RTN神经元周围的局部pH变化。我们用 在单个RTN神经元中用药理学和转录学方法表征GPR4信号通路和 效应通道，我们破坏了髓质星形胶质细胞中调节pH的Na+-HCO3转运体NBCe1，以 测定对体内呼吸化学反射的影响。后者可能支持以下收敛理论 星形胶质细胞-神经元参与了这种高度敏感的化学反射，弥合了该领域的一个主要电流分歧。 总的来说，建议的研究提供了关于分子和细胞机制的关键信息。 它控制着RTN神经元的活动，并调节这个重要的稳态呼吸系统。 识别新的分子机制可能为呼吸障碍的治疗提供新的靶点。

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.