Intracellular ph Responses of Central Chemoreceptors

二氧化碳增加(高碳酸血症)是增加呼吸和血压的主要刺激因素。这种心肺控制的途径涉及到特殊的中枢神经元，称为化学敏感神经元，可以感知高碳酸血症，但将高碳酸血症转化为神经元放电频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对至少两个化学敏感脑干区域(蓝斑和孤束核或延髓腹外侧核)和至少一个非化学敏感区域(下橄榄核或舌下核)的单个神经元进行研究。神经元膜电位(Vm)和细胞内pH(Phi)的同时测量将使用穿孔贴片记录或全细胞记录(WCR)结合pH敏感的荧光染料和荧光成像显微镜来实现。我们的第一个目标是确定将高碳酸血症转化为更高的放电频率的信号通路。它将由4个不同的目标组成：i)通过将神经元暴露在这些参数不同的溶液中，研究分子CO2、外部pH(Pho)和phi作为化学接收的最接近信号的作用；ii)研究“洗出”现象，即在WCR测量过程中，VM对高碳酸血症的反应丢失，以了解是否其他信号分子(如CaI、多胺或碳酸酐酶)也参与化学接收；iii)研究“缺氧悖论”(低氧诱导的酸化似乎不会增加化学敏感神经元的放电率)，看看是否由于缺乏额外的信号分子；以及iv)研究化学敏感性降低大鼠(由慢性高碳酸血症引起)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中确定的信号对不同K通道的影响，并确定每个通道在改变动作电位的形状和神经元放电频率中的作用。我们将研究三个K通道：i)内向整流性K通道，它对决定棘波去极化的斜率和神经元的放电率很重要；ii)钙激活的K通道，它对决定动作电位的形状和后超极化的大小很重要；iii)TASK相关的酸敏感K通道(TASK)，它对确定静息的Vm很重要。这项工作应该表明化疗敏感性的最接近信号的确切性质，阐明高二氧化碳刺激影响各种K通道的方式，并深入了解这些影响是如何整合在一起，导致最终的神经元反应。此外，通过比较两个化学敏感区在神经元中的发现，我们的发现应该有助于澄清为什么在脑干中有许多化学敏感区。这些研究将有助于我们理解呼吸系统疾病，这些疾病被认为是部分由中枢化学感受器功能障碍引起的，如婴儿猝死综合征(SID)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.