Project 2

项目总结/摘要 – 项目 2 对于患有阻塞性睡眠呼吸暂停 (OSA) 的患者，气流阻塞会导致高碳酸血症和其他信号 增加通气，扩张气道，并引发睡眠中的皮质唤醒。目前的疗法如 由于 CPAP 专注于气道开放，但这些疗法的依从性很差，许多患者继续 白天嗜睡。由于反复从睡眠中醒来会导致白天嗜睡和其他 OSA 的后果，在不中断通气的情况下维持 OSA 睡眠的新方法将解决 OSA 治疗中一个重要的、未满足的需求。 在本次P01的最后一个周期中，Saper博士的小组（项目1）表明，降钙素基因相关肽 臂旁核外侧核 (CGRP) 神经元对于皮质唤醒做出反应是必需的 高碳酸血症。具体来说，PBCGRP 神经元的失活显着延迟或消除了 对高碳酸血症的反应而不减弱通气反应。因此，PBCGRP 神经元对于 驱动皮质唤醒，但它们对于高碳酸血症的通气反应不是必需的。我们假设 激活 PBCGRP 神经元的抑制性输入将延迟或消除高碳酸血症的皮质唤醒 而不改变通气反应。 我们的目标是识别这些输入及其 PBCGRP 神经元上的受体，最终目标是 选择性地减少 PBCGRP 神经元的活动，以防止皮质唤醒，同时保持通气 回应。该项目与旨在增强通气反应的项目 1、3 和 4 具有良好的协同作用 小鼠高碳酸血症，以及项目 5，旨在确定改善 OSA 的药理学方法 人们。我们将首先使用条件和传统追踪方法来识别 PBCGRP 的传入 神经元，然后我们将使用视紫红质通道辅助电路映射（CRACM）来建立突触 连接性。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。我们随后将 使用 fos 和光纤光度测定法来确定 PBCGRP 神经元的哪些传入通路是睡眠活跃的。 最后，我们将确定通过抑制性输入向 PBCGRP 神经元发出的信号是否会延迟或消除 短暂的高碳酸血症引发的皮质唤醒。我们将测量皮层唤醒的潜伏期 高碳酸血症与 PBCGRP 神经元的抑制性输入的光刺激相结合，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 神经元应该为 OSA 患者维持睡眠提供新的药理学机会 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.