Molecular mechanisms of central C02 chemoreception

中枢CO2化学感受的分子机制

• 批准号: 9056583
• 负责人: Matthew Robert Hodges
• 金额: $ 38.44万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-17 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9056583
• 关键词: Acute; Address; Agonist; Air; Brain Stem; Breathing; Candidate Disease Gene; Carbon Dioxide; Cell Nucleus; Cell Separation; Cells; Chemoreceptors; Collecting Cell; Data; Development; Disease; Environmental air flow; Exercise; FOS gene; Fluorescence; Functional disorder; Gene Expression; Genes; Genetic; Genomics; HTR2A gene; Health; Human; Hypercapnia; Hypoxia; In Vitro; Injection of therapeutic agent; Ion Channel; Knock-out; Knowledge; Mammals; Measures; Microdialysis; Molecular; Molecular Genetics; Neuromodulator; Neurons; Norway; Phenotype; Population; Potassium Channel; RNA Sequence Analysis; Rattus; Rattus norvegicus; Respiratory Acidosis; Role; Serotonin; Site; Slice; Sorting - Cell Movement; Sprague-Dawley Rats; Substance P; Sudden infant death syndrome; Testing; Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone Receptors; Transgenic Organisms; Wakefulness; age related; awake; base; differential expression; enhanced green fluorescent protein; genetic approach; hormone analog; in vivo; inhibitor/antagonist; innovation; neuromechanism; neuroregulation; next generation; novel; patch clamp; postnatal; relating to nervous system; respiratory; response; reuptake; salt sensitive; selective expression; transcriptome; transcriptome sequencing; validation studies

 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.

 描述（由申请人提供）：稳态通气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响应高碳酸血症的基本中枢神经系统机制了解不足，潜在的治疗受到阻碍。有多个中枢神经系统位点含有假定具有内在 CO2/pH 敏感性的细胞，包括中缝髓 (MR) 血清素能 (5-HT) 和表达 phox2b 的后梯形核 (RTN) 神经元。构成细胞对这些细胞 CO2/pH 敏感性的分子的身份仍然未知。此外，5-HT、P物质和促甲状腺素释放激素（TRH）等兴奋性神经调节剂对于神经呼吸控制至关重要，但这些神经调节剂在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) phox2b+ RTN 和 MR 5-HT 神经元的亚群由于一个或多个 pH 敏感离子通道的选择性表达而具有内在的化学敏感性，2) RTN 的中缝衍生的神经调节是哺乳动物 CO2 化学反射的主要决定因素。为了识别可能导致细胞 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) 和下一代 RNA 测序 (RNASeq) 来识别神经化学定义的脑干神经元亚群中的差异表达基因。我们已经证明了我们方法的可行性，并确定了可能是 5-HT 神经元细胞 CO2 化学敏感性基础的两个基因（Kir4.1 和 Kir5.1）。在目标 1 中，我们将使用这种方法来识别可能构成细胞 CO2 敏感性的基因/分子，通过比较 CO2 敏感和 CO2 不敏感的 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 敏感神经元。在目标 2 中，我们将在功能上验证目标 1 中确定的基因，特别是使用膜片钳记录在体外以及在基因组 Kir4.1、Kir5.1 和组合 Kir4.1/5.1 敲除大鼠体内验证 Kir4.1/5.1 K+ 通道的作用。为了进一步探讨神经调节剂在通气 CO2 化学反射中的作用，我们将研究棕色挪威 (BN) 大鼠，该大鼠的 CO2 化学反射严重减弱，但在平静、缺氧和运动期间呼吸正常。这些对 CO2 不敏感的 BN 大鼠脑干 5-HT 和 TRH 缺乏，刺激 5-HT 或 TRH 受体会增强 BN 大鼠的 CO2 化学反射。因此，我们假设这些神经调节作用是通过直接调节 RTN 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 Dawley (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有关细胞二氧化碳化学感受和二氧化碳化学反射基本机制的重要新数据，并为研究通气控制其他组成部分的分子遗传学方法提供框架。