Molecular mechanisms of central C02 chemoreception

 描述（由申请方提供）：稳态代谢性化学反射功能障碍可能导致人类多种代谢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CNS机制理解不足，阻碍了潜在的治疗。有多个CNS部位含有具有假定的内在CO2/pH敏感性的细胞，包括延髓中缝（MR）胆碱能（5-HT）和phox 2b表达的后斜方核（RTN）神经元。这些细胞的CO2/pH敏感性的分子的身份仍然未知。此外，兴奋性神经调质如5-HT、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，但这些神经调质在CO2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）phox 2b + RTN和MR 5-HT神经元的亚群由于一个或多个pH敏感性离子通道的选择性表达而具有固有的化学敏感性，和2）RTN的中缝核源性神经调节是哺乳动物CO2化学反射的主要决定因素。为了鉴定可能导致细胞CO2/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选（FACS），然后进行下一代RNA测序（RNASeq），以鉴定神经化学定义的脑干神经元亚群中差异表达的基因。我们已经证明了我们的方法的可行性，并确定了两个基因（Kir4.1和Kir5.1），可能是5-HT神经元细胞CO2化学敏感性的基础。在目的1中，我们将使用这种方法来识别可能是细胞CO2敏感性的基础的基因/分子，通过比较CO2敏感性和CO2不敏感性5-HT和RTN神经元，使用高碳酸血症诱导的c-Fos表达来识别CO2敏感性神经元。在目标2中，我们将在功能上验证目标1中鉴定的基因，特别是Kir4.1/5.1 K+通道在体外使用膜片钳记录和体内基因组Kir4.1，Kir5.1和组合Kir4.1/5.1敲除大鼠中的作用。为了进一步阐明神经调节剂在呼吸性CO2化学反射中的作用，我们将研究Brown Norway（BN）大鼠，其具有严重钝化的CO2化学反射，但在正常呼吸、缺氧和运动期间呼吸正常。这些CO2不敏感的BN大鼠缺乏脑干5-HT和TRH，并且5-HT或TRH受体的刺激增强BN大鼠中的CO2化学反射。因此，我们假设这些神经调节作用是通过RTN的直接调节发生的，我们将在目标3中通过对CO2不敏感BN和高度CO2敏感盐敏感（SS）和Sprague道利（SD）大鼠RTN内5-HT、P物质和TRH受体的激动剂和拮抗剂进行微透析来测试。我们的创新研究将产生关于细胞CO2化学感受和CO2化学反射的基本机制的重要新数据，并为分子遗传学方法提供一个框架，以研究其他成分的免疫控制。

 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.