Project 2

项目概要/摘要-项目2 在阻塞性睡眠呼吸暂停（OSA）患者中，气流阻塞导致高碳酸血症和其他信号 增加通气量，扩张气道，同时也触发睡眠中的皮层觉醒。目前的治疗方法，如 因为CPAP专注于气道开放，但这些治疗的依从性很差，许多患者继续 白天嗜睡。由于睡眠中的反复觉醒会导致白天嗜睡和其他 OSA的后果，新的方法，保持睡眠在OSA而不中断通气将解决 这是阻塞性睡眠呼吸暂停综合症治疗中一个重要但尚未满足的需求。 在P01的最后一个周期中，Saper博士的小组（项目1）显示降钙素基因相关肽 臂旁外侧核的CGRP神经元是皮质觉醒所必需的， 高碳酸血症具体而言，PBCGRP神经元的失活实质上延迟或消除了脑皮层觉醒， 对高碳酸血症的反应，而不钝化呼吸反应。因此，PBCGRP神经元对于 驱动皮质觉醒，但它们不是高碳酸血症的解释性反应所必需的。我们假设 对PBCGRP神经元的抑制性输入的激活将延迟或消除高碳酸血症的皮层觉醒 而不改变解释性反应。 我们的目标是寻求确定这些输入和它们在PBCGRP神经元上的受体，最终目标是 选择性地降低PBCGRP神经元的活性，以防止皮质觉醒，同时保留对皮质的刺激。 应答该项目与项目1、项目3和项目4协同作用很好，项目1、项目3和项目4旨在加强对下列问题的澄清反应： 以及项目5，该项目旨在确定改善OSA的药理学方法， 人我们将首先使用条件和传统的追踪方法，以确定传入的PBCGRP 神经元，然后我们将使用视紫红质辅助电路映射（CRACM）建立突触 连通性。使用单细胞测序技术，我们将鉴定PBCGRP表达的受体， 神经元，并使用原位杂交和体外钙成像确认受体表达。然后我们将 使用fos和纤维光度测定法来确定哪些PBCGRP神经元的传入通路是睡眠活性的。 最后，我们将确定通过抑制性输入到PBCGRP神经元的信号传导是否延迟或消除 由短暂的高碳酸血症引发的皮层觉醒。我们将测量皮层唤醒的潜伏期， 高碳酸血症与对PBCGRP神经元的抑制性输入的光刺激组合，然后与 PBCGRP神经元的药理学抑制。 总的来说，这些多学科实验将确定关键的解剖学和神经化学输入， PBCGRP神经元，应该提供新的药理学机会，维持睡眠在OSA 而不抑制气道开放。

Project Summary/Abstract – Project 2 In people with obstructive sleep apnea (OSA), airflow obstruction results in hypercarbia and other signals that increase ventilation, dilate the airway, and also trigger cortical arousals from sleep. Current therapies such as CPAP focus on airway opening, but compliance with these therapies is poor, and many patients continue to have daytime sleepiness. As recurrent arousals from sleep contribute to daytime sleepiness and other consequences of OSA, new methods that maintain sleep in OSA without disrupting ventilation would address an important, unmet need in OSA treatment. In the last cycle of this P01, Dr. Saper’s group (Project 1) showed that calcitonin gene-related peptide (CGRP) neurons of the lateral parabrachial nucleus are necessary for cortical arousals in response to hypercapnia. Specifically, inactivation of PBCGRP neurons substantially delays or eliminates cortical arousals in response to hypercapnia without blunting ventilatory responses. Thus, the PBCGRP neurons are essential for driving cortical arousals, but they are not necessary for ventilatory responses to hypercapnia. We hypothesize that activation of inhibitory inputs to the PBCGRP neurons will delay or eliminate cortical arousals to hypercapnia without altering ventilatory responses. Our Aims seek to identify these inputs and their receptors on the PBCGRP neurons, with the ultimate goal of selectively reducing activity in the PBCGRP neurons to prevent cortical arousals while preserving ventilatory responses. This Project synergizes well with Projects 1, 3, and 4 that seek to enhance ventilatory responses to hypercapnia in mice, and Project 5 which seeks to identify pharmacological methods to improve OSA in people. We will first use conditional and conventional tracing methods to identify afferents to the PBCGRP neurons, and then we will use Channelrhodopsin-assisted circuit mapping (CRACM) to establish synaptic connectivity. Using single cell sequencing techniques, we will then identify receptors expressed by the PBCGRP neurons, and confirm receptor expression using in situ hybridization and in vitro calcium imaging. We will then use fos and fiber photometry to determine which afferent pathways to the PBCGRP neurons are sleep-active. Last, we will determine whether signaling through inhibitory inputs to the PBCGRP neurons delays or eliminates cortical arousals triggered by brief period of hypercapnia. We will measure the latency to cortical arousal after hypercapnia in combination with photostimulation of inhibitory inputs to the PBCGRP neurons and then with pharmacological inhibition of the PBCGRP neurons. Collectively, these multidisciplinary experiments will identify crucial anatomical and neurochemical inputs to the PBCGRP neurons that should provide new pharmacological opportunities for maintaining sleep in OSA without inhibiting airway opening.