Intracellular ph Responses of Central Chemoreceptors

二氧化碳增加（高碳酸血症）是呼吸和血压增加的主要刺激因素。这种控制心肺功能的途径涉及到感知高碳酸血症的特殊中枢神经元，称为化学敏感神经元，但将高碳酸血症转化为神经元放电率增加的细胞机制尚不清楚。我们的工作将涉及使用新生大鼠大脑切片研究来自至少两个化学敏感脑干区域（蓝斑核和孤束核或延髓腹外侧核）和至少一个非化学敏感区域（橄榄下核或舌下核）的单个神经元。同时测量神经元膜电位（Vm）和细胞内pH （pHi）将实现使用穿孔贴片记录或全细胞记录（WCR）结合pH敏感荧光染料和荧光成像显微镜。我们的第一个目标是确定将高碳酸血症转化为放电速率增加的信号通路。它将包括4个独立的目标：i)通过将神经元暴露于这些参数不同的溶液中，研究分子CO2，外部pH （pHO）和pHi作为化学接受的近似信号的作用；ii)检查“冲洗”现象，即在WCR测量期间Vm对高碳酸血症的反应丢失，以查看是否其他信号分子（例如Cai，多胺或碳酸酐酶）也参与化学接受；Iii)研究“缺氧悖论”（缺氧诱导的酸化似乎不会增加化学敏感神经元的放电速率），看看这是否由于缺乏额外的信号分子；iv)研究化学敏感性降低大鼠（慢性高碳酸血症诱导）化学敏感神经元Vm和pHi对高碳酸血症反应的变化。我们的第二个目标是研究目标1中确定的信号对各种K+通道的影响，并确定每个通道在改变动作电位形状和神经元放电率方面的作用。三种K+通道将被研究：i)向内整流K+通道，这对于确定峰间去极化的斜率很重要，从而确定神经元的放电速率；ii) Ca2+活化的K+通道，对决定动作电位的形状和超极化后的大小很重要；iii) twik相关的酸敏感K+通道（TASK），在确定静息Vm中很重要。这项工作应该表明化学敏感性近似信号的确切性质，阐明高碳酸刺激影响各种K+通道的方式，并深入了解这些影响是如何综合起来导致最终的神经元反应的。此外，通过比较来自两个化学敏感区域的神经元的发现，我们的发现应该有助于澄清为什么脑干中有许多化学敏感区域。这些研究将有助于我们理解部分由中枢化学感受器功能障碍引起的呼吸系统疾病，如婴儿猝死综合征（SIDS）和中枢性肺泡低通气综合征。

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.