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（由适用提供）：稳态通气化学反射剂的功能障碍可能有助于人类中多种呼吸相关疾病的起源或疾病，但对基本CNS机制的基本机制的了解和响应造成了呼吸相关性疾病的多种呼吸相关疾病，从而阻碍了检测和响应高囊肿的基本机制。有多个包含具有固有CO2/pH敏感性的细胞的CNS位点，包括髓质Raphe（MR）5-羟色胺能（5-HT）和表达PHOX2B的逆转录曲霉核（RTN）神经元。细胞CO2/pH对这些细胞敏感的分子的身份仍然未知。此外，兴奋性神经调节剂，例如5-HT，物质P和甲状腺激素释放的马酮（TRH）对于神经培训对控制至关重要，但是CO2化学反射中这些神经调节剂的重要性尚不清楚。为了解决这些知识差距，我们将通过完成三个具体目标来检验两个主要假设。我们假设：1）PHOX2B+ RTN和MR 5-HT神经元的亚群体由于一个或多个pH敏感的离子通道的选择性表达而具有本质上的化学敏感性，而2）RAPHE衍生的RTN的RTN是RTN的RTN的主要决定性，是哺乳动物CO2 CO2 ChemoReflex的主要决定性。为了鉴定可能构成细胞CO2/pH敏感性的分子，我们使用荧光辅助细胞分选（FACS）开发了一种独特的科学方法，然后是下一代RNA测序（RNASEQ），以识别神经化定义的神经神经元神经元中神经化神经元中差异表达的基因。我们已经证明了方法的可行性，并鉴定了两个基因（Kir4.1和Kir5.1），这些基因可能是5-HT神经元的细胞CO2化学敏感性的基础。在AIM 1中，我们将使用这种方法来鉴定可以使用高capnia诱导的C-FOS表达来鉴定CO2敏感神经元的基因/分子，从而通过比较对CO2敏感和对CO2不敏感的5-HT和RTN神经元来识别CO2敏感和RTN神经元的基因/分子。在AIM 2中，我们将在AIM 1中验证在AIM 1中鉴定的基因，特别是Kir4.1/5.1 K+通道在体外使用贴片夹记录和基因组KIR4.1，KIR5.1和KIR4.1/5.1敲除大鼠组合的体外作用。为了进一步解决神经调节剂在通风CO2 Chemoreflex中的作​​用，我们将研究棕色挪威（BN）大鼠，这些大鼠具有严重钝化的CO2 ChemoreFlex，但在Eupnea，缺氧和运动过程中正常呼吸。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受体会增强BN大鼠中CO2 ChemoreFlex。根据以下内容，我们假设这些神经调节作用是通过直接调节RTN发生的，我们将通过5-HT的激动剂和拮抗剂，物质P和TRH受体的co2-nsiments敏感性BN的RTN和高度CO2敏感的盐敏感的（SSSSSSSSS）和Sprage（Sss）（SSSAge daw daw daw）在目标3中测试。我们的创新研究将生成有关细胞CO2化学感受器和CO2 ChemoreFlex的基本机制的重要新数据，并为分子遗传学方法提供了研究通风控制其他组件的框架。