Intracellular ph Responses of Central Chemoreceptors

CO2增加（高碳酸血症）是呼吸和血压增加的主要刺激因素。 这种心肺控制途径涉及专门的中枢神经元，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增加的神经元放电率的细胞机制尚未得到很好的理解。 我们的工作将涉及从至少两个化学敏感的脑干区域（蓝斑和孤束核或延髓腹外侧）和至少一个nonchemosensitive区域（无论是下橄榄或舌下神经核）使用从新生大鼠大脑切片的个别神经元的研究。 将使用穿孔贴片记录或全细胞记录（WCR）结合pH敏感性荧光染料和荧光成像显微镜实现神经元膜电位（Vm）和细胞内pH（pHi）的同时测量。我们的第一个目标是确定使高碳酸血症增加放电频率的信号通路。 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中的每一个都不同的溶液来研究分子CO2、外部pH（pHO）和pHi作为化学感受的近端信号的作用; ii）检查“洗脱”现象，由此在WCR测量期间对高碳酸血症的Vm响应丢失，以查看是否存在额外的信号分子（例如Cai、多胺或碳酸酐酶）也参与化学感受; 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率），看看是否是由于缺乏额外的信号分子;和iv）研究来自具有降低的化学敏感性（由慢性暴露于高碳酸血症诱导）的大鼠的化学敏感性神经元中对高碳酸血症的Vm和pHi响应的变化。 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，并确定这些通道中的每一个在修改动作电位和神经元放电率的形状中的作用。 将研究三种K+通道：i）内向整流K+通道，其在确定尖峰间去极化的斜率以及由此确定神经元的放电速率方面很重要; ii）Ca 2+激活的K+通道，其在确定动作电位的形状和后超极化的幅度方面很重要;以及iii）TWIK相关的酸敏感K+通道（ASK），其在确定静息Vm方面很重要。 这项工作应表明化学敏感性的近端信号的确切性质，阐明高碳酸血症刺激影响各种K+通道的方式，并深入了解这些影响是如何整合，导致最终的神经元反应。 此外，通过比较来自2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呼吸系统疾病，认为部分是由于中央化学感受器功能障碍，如婴儿猝死综合征（SIDS）和中央肺泡通气不足综合征。

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.