INTRACELLULAR PH RESPONSES OF CENTRAL CHEMORECEPTORS

描述(改编自申请者的摘要)：这个项目将测试 细胞内pH(Phi)是中枢神经系统刺激的假说 二氧化碳的化学感受，这是对呼吸最强烈的刺激之一。会的 采用了一种新的技术，即荧光成像显微镜 新生和幼年大鼠延髓脑片的神经元胞体 负载pH敏感的荧光染料(BCECF)，以研究phi的调节 在化学敏感和非化学敏感的中枢神经元。神经元是 通过对增加的二氧化碳有兴奋反应而被定义为化学敏感。 将使用尖锐的电极穿刺法或穿孔膜片钳来测量 单个神经元的电生理特性和标准方法 将采用PHI调节的检查(NH4Cl诱导的酸化， 用不同的外部离子替代物回收PHI PH调节的各种抑制剂)。人们的期望是， 化学感受器神经元会有明显的phi变化，并在低 缓冲能力和恢复率很低，而且电生理 响应将与Phi相关。四个具体目标是：1) 表征phi调节响应(缓冲能力、pho/phi PH调节转运体的关系、互补)，2)测定phi 对已知会影响产量的酸碱变化的反应，3)确定 在这些条件下的电生理输出，以及4)测量 同时进行phi和电活动。

DESCRIPTION (Adapted from the applicant's abstract): This project will test the hypothesis that intracellular pH (pHi) is the stimulus for central chemoreception of CO2, one of the strongest stimuli to breathing. It will employ a novel technique, that of fluorescence imaging microscopy of single neuronal cell bodies in neonatal and juvenile rat medullary brain slices loaded with a pH-sensitive fluorescent dye (BCECF), to study pHi regulation in chemosensitive and non-chemosensitive central neurons. Neurons are defined as chemosensitive by having an excitatory response to increased CO2. Sharp electrode impalement or perforated patch clamp will be used to measure electrophysiological properties of single neurons and standard methods for examination of pHi regulation will be employed (NH4Cl-induced acidification, pHi recovery with various external ion substitutes and in the presence of various inhibitors of pH regulation). The expectation is that central chemoreceptor neurons will have marked and maintained pHi changes with low buffering power and little recovery and that the electrophysiological responses will correlate with pHi. The four specific aims are: 1) characterize pHi regulatory responses (buffering power, pHo/pHi relationship, complement of pH-regulating transporters), 2) measure pHi responses to acid-base changes known to affect output, 3) determine electrophysiologic output under these conditions, and 4) measure simultaneously pHi and electrical activity.