Intracellular ph Responses of Central Chemoreceptors

中枢化学感受器的细胞内 ph 反应

• 批准号: 6527092
• 负责人: Robert W Putnam
• 金额: $ 24.31万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6527092
• 关键词: acid base balance; acidity /alkalinity; action potentials; blood pressure; brain stem; carbonate dehydratase; central neural pathway /tract; chemoreceptors; fluorescent dye /probe; hypercapnia; hypoglossal nucleus; hypoventilation; hypoxia; intracellular; intravenous administration; laboratory rat; locus coeruleus; neurons; neurotransmitters; potassium channel; respiratory acidosis

Increased CO2 (hypercapnia) is a major stimulus for increased respiration and blood pressure. This pathway for cardiorespiratory control involves specialized central neurons, called chemosensitive neurons, that sense hypercapnia, but the cellular mechanisms that transduce hypercapnia into an increased neuronal firing rate are not well understood. Our work will involve the study of individual neurons from at least two chemosensitive brainstem areas (locus coeruleus and either nucleus tractus solitarius or ventrolateral medulla) and at least one nonchemosensitive area (either inferior olive or hypoglossal nucleus) using slices from neonatal rat brains. Simultaneous measurements of neuronal membrane potential (Vm) and intracellular pH (pHi) will be achieved using perforated patch recordings or whole cell recordings (WCR) combined with pH- sensitive fluorescent dyes and fluorescence imaging microscopy. Our first aim is to identify the signal pathways that transduce hypercapnia into an increased firing rate. It will consist of 4 separate aims: i) study the roles of molecular CO2, external pH (pHO) and pHi as the proximate signal of chemoreception by exposing neurons to solutions that vary in each of these parameters; ii) examine the phenomenon of "washout", whereby the Vm response to hypercapnia is lost during WCR measurements, to see if additional signal molecules (e.g. Cai, polyamines or carbonic anhydrase) are also involved in chemoreception; iii) study the "hypoxia paradox" (hypoxia-induced acidification does not appear to increase firing rate in chemosensitive neurons) to see if it is due to the lack of additional signal molecules; and iv) study the changes in the Vm and pHi response to hypercapnia in chemosensitive neurons from rats with reduced chemosensitivity (induced by chronic exposure to hypercapnia). Our second aim is to study the effects of the signals, identified in Aim 1, on various K+ channels and determine the role of each of these channels in modifying the shape of the action potential and neuronal firing rate. Three K+ channels will be studied: i) inward rectifying K+ channels, important in determining the slope of the interspike depolarization and thereby the firing rate of the neuron; ii) Ca2+-activated K+ channels, important in determining the shape of the action potential and the magnitude of the after hyperpolarization; and iii) TWIK-related acid sensitive K+ channels (TASK), important in determining the resting Vm. This work should indicate the precise nature of the proximate signal of chemosensitivity, elucidate the way in which hypercapnic stimuli affect various K+ channels and give insight into how these effects are integrated to result in the final neuronal response. Further, by comparing the findings in neurons from 2 chemosensitive areas, our findings should help clarify why there are numerous chemosensitive regions in the brainstem. These studies will contribute to our understanding of respiratory diseases thought to be due in part to central chemoreceptor dysfunction, such as sudden infant death syndrome (SIDS) and central alveolar hypoventilation syndromes.

二氧化碳增加（高碳酸盐）是增加呼吸和血压的主要刺激。 心肺控制的这一途径涉及专门的中枢神经元，称为化学敏感性神经元，这种神经元可以感觉到过度capnia，但是将过度capnia转移到升高的神经元放电速率中的细胞机制尚不清楚。 我们的工作将涉及从至少两个化学敏感的脑干区域（凝固层和小核Tractus solitarius或腹侧髓质）和至少一个非化学敏感区域（使用下橄榄核）的研究。 将使用带状斑块记录或全细胞记录（WCR）与pH-敏感的荧光染料和荧光成像显微镜相结合，同时测量神经元膜电位（VM）和细胞内pH（PHI）。我们的第一个目的是确定将高碳酸盐转化为发射速率提高的信号途径。 它将由4个单独的目的组成：i）研究分子二氧化碳，外部pH（pHO）和PHI的作用，作为化学感受的近端信号，通过将神经元暴露于这些参数中的每个参数中变化的溶液中； ii）检查“洗涤”现象，在WCR测量过程中，VM对高碳酸血症的反应丢失，以查看是否还参与化学感受性的其他信号分子（例如CAI，多胺或碳酸赤霉酶）是否也参与。 iii）研究“低氧悖论”（缺氧诱导的酸化似乎不会增加化学敏感性神经元的发射率），以查看是否由于缺乏其他信号分子而引起的； iv）研究来自化学敏感性降低的大鼠化学敏感性神经元中VM和PHI对高碳酸高症的反应的变化（通过长期暴露于高碳酸血症引起的诱导）。 我们的第二个目的是研究AIM 1中确定的信号对各种K+通道的效果，并确定每个通道在修改动作电位和神经元放电速率的形状中的作用。 将研究三个K+通道：i）向内整流K+通道，对于确定尖刺斜率去极化的斜率以及神经元的发射速率很重要； ii）Ca2+激活的K+通道，对于确定动作电位的形状和后超极化的大小很重要；和iii）与TWIK相关的酸敏感K+通道（任务），对于确定静止VM很重要。 这项工作应表明化学敏感性近端信号的确切性质，阐明过度capnic刺激影响各种K+通道的方式，并深入了解这些效果如何整合以导致最终的神经元反应。 此外，通过比较来自两个化学敏感区域的神经元的发现，我们的发现应有助于阐明为什么脑干中存在许多化学敏感区域。这些研究将有助于我们理解被认为是由于中央化学感受器功能障碍的部分原因，例如猝死综合征（SIDS）和中央肺泡低衰减综合征。