Molecular mechanisms of central C02 chemoreception

 描述(由申请人提供)：稳态呼吸性化学反射功能障碍可能导致人类多种呼吸相关疾病的发生或适应不良，但由于对负责检测和应对高碳酸血症的基本中枢神经系统机制缺乏了解，潜在的治疗受到阻碍。有多个中枢神经系统部位含有可能对CO2/pH具有内在敏感性的细胞，包括延髓中缝(MR)、5-羟色胺(5-HT)和PHOX2B表达的梯形后核(RTN)神经元。构成细胞二氧化碳/pH敏感性的分子对这些细胞的敏感性尚不清楚。此外，兴奋性神经调节剂如5-羟色胺、P物质和促甲状腺激素释放激素(TRH)对神经呼吸控制至关重要，但这些神经调节剂在二氧化碳化学反射中的重要性尚不清楚。为了解决这些知识差距，我们将通过完成三个具体目标来测试两个主要假设。我们假设：1)由于一个或多个pH敏感离子通道的选择性表达，PHOX2B RTN和MR 5-HT神经元亚群具有内在的化学敏感性；2)中缝衍生的RTN神经调制是哺乳动物CO2化学反射的主要决定因素。为了确定可能导致细胞二氧化碳/pH敏感性的分子，我们开发了一种独特的科学方法，利用荧光辅助细胞分类(FACS)和下一代RNA测序(RNAseq)在神经化学定义的脑干神经元亚群中识别差异表达的基因。我们已经证明了我们方法的可行性，并鉴定了两个基因(Kir4.1和Kir5.1)，它们可能是5-羟色胺神经元细胞二氧化碳化疗敏感性的基础。在目标1中，我们将使用这种方法，通过比较二氧化碳敏感和不敏感的5-羟色胺和RTN神经元，利用高碳酸血症诱导的c-Fos表达来识别二氧化碳敏感神经元，从而识别可能支持细胞二氧化碳敏感性的基因/分子。在目标2中，我们将使用膜片钳记录在体外和体内验证在基因组Kir4.1、Kir5.1和组合Kir4.1/5.1基因敲除大鼠中识别的基因，特别是Kir4.1/5.1K通道在体内的作用。为了进一步研究神经调节剂在呼吸性二氧化碳化学反射中的作用，我们将研究Brown挪威(BN)大鼠，这些大鼠在呼吸暂停、低氧和运动期间二氧化碳化学反射严重迟钝，但呼吸正常。这些对CO2不敏感的BN大鼠缺乏脑干5-HT和TRH，刺激5-HT或TRH受体可增强BN大鼠的CO2化学反射。因此，我们假设这些神经调节效应是通过对RTN的直接调节而发生的，我们将在目标3中通过微透析在二氧化碳不敏感的BN、高CO2敏感的盐敏感(SS)和SD(SD)大鼠的RTN中测试5-羟色胺、P物质和TRH受体的激动剂和拮抗剂。我们的创新研究将产生关于细胞二氧化碳化学接收和二氧化碳化学反射的基本机制的重要新数据，并为研究呼吸控制的其他组成部分的分子遗传学方法提供一个框架。

 DESCRIPTION (provided by applicant): Dysfunction of homeostatic ventilatory chemoreflexes likely contribute to the genesis of or maladaptation to multiple respiratory-related diseases in humans, but potential treatments are hampered by a poor understanding of the fundamental CNS mechanisms responsible for detecting and responding to hypercapnia. There are multiple CNS sites containing cells with presumed intrinsic CO2/pH sensitivity, including medullary raphe (MR) serotoninergic (5-HT) and phox2b-expressing retrotrapezoid nucleus (RTN) neurons. The identity of molecules that underlie cellular CO2/pH sensitivity to these cells remains unknown. In addition, excitatory neuromodulators such as 5-HT, substance P and thyrotropin-releasing hormone (TRH) are critical to neural respiratory control, but the importance of these neuromodulators in the CO2 chemoreflex is unclear. To address these knowledge gaps, we will test two major hypotheses by completing three Specific Aims. We hypothesize that: 1) sub-populations of phox2b+ RTN and MR 5-HT neurons are intrinsically chemosensitive due to the selective expression of one or more pH-sensitive ion channels, and 2) raphe-derived neuromodulation of the RTN is a major determinant of the mammalian CO2 chemoreflex. To identify molecules that may underlie cellular CO2/pH sensitivity, we have developed a unique scientific approach utilizing fluorescence-assisted cell sorting (FACS) followed by Next-gen RNA sequencing (RNASeq) to identify differentially-expressed genes among neurochemically-defined brainstem neuronal subpopulations. We have demonstrated the feasibility of our approach, and identified two genes (Kir4.1 and Kir5.1) that may underlie cellular CO2 chemosensitivity of 5-HT neurons. In Aim 1 we will use this approach to identify genes/molecules that may underlie cellular CO2 sensitivity by comparing CO2-sensitive and CO2-insensitive 5-HT and RTN neurons using hypercapnia-induced c-Fos expression to identify CO2 sensitive neurons. In Aim 2 we will functionally validate genes identified in Aim 1, and specifically the roles of Kir4.1/5.1 K+ channels in vitro using patch clamp recordings and in vivo in genomic Kir4.1, Kir5.1 and combined Kir4.1/5.1 knockout rats. To further address the role of neuromodulators in the ventilatory CO2 chemoreflex, we will study Brown Norway (BN) rats, which have a severely blunted CO2 chemoreflex but normal breathing during eupnea, hypoxia and exercise. These CO2-insensitive BN rats are deficient in brainstem 5-HT and TRH, and stimulation of 5-HT or TRH receptors augments the CO2 chemoreflex in BN rats. Accordingly, we hypothesize that these neuromodulatory effects occur through direct modulation of the RTN, which we will test in Aim 3 by microdialysis of agonists and antagonists of 5-HT, substance P and TRH receptors within the RTN of CO2-insensitive BN and highly CO2- sensitive Salt-sensitive (SS) and Sprague Dawley (SD) rats. Our innovative studies will generate important new data regarding fundamental mechanisms of cellular CO2 chemoreception and the CO2 chemoreflex, and provide a framework for a molecular genetics approach to study other components of ventilatory control.