MECHANISMS OF CO2/H+-SENSITIVITY IN BRAINSTEM NEURONS

中枢CO(2)/H+化学感受器提供重要的易化驱动 对心肺控制网络来说是必不可少的 大脑pH值的动态平衡。尽管他们在维持生命的过程中发挥了作用 系统，对神经底物知之甚少 CO(2)/H+去极化及其细胞机制 回应。这主要是由于缺乏适当的 细胞内电生理研究及定位困难 固有的化学敏感神经元，其中的细胞机制可以 学习。例如，延髓腹外侧区(VLM)中的神经元 通常被认为是唯一的化疗敏感部位，然而， 对VLM的大多数单一单位研究表明，大多数单位没有 在阻断突触传递的过程中保持对CO(2)/H+的敏感性。 此外，越来越多的证据表明，VLM可能不是唯一的 中枢化疗敏感部位，但这种化学感受可能是 通过一个比最初设想的更分散的系统来完成。 在这一点上，我们已经报道了某些神经元在纤维束核 孤束核(NTS)和迷走神经背侧运动核(DMNX) 心肺整合区，被CO(2)去极化 阻断体外突触输入。尽管我们不能明确地 证明在体外记录的NTS和DMNX中的CO(2)化学敏感神经元 参与中枢化学接收，它们确实提供了一个底物 CO(2)/H+致神经元去极化机制的研究 脑干。这份提案概述了研究这些机制的实验。 NTS和DMNX神经元以及附近的化疗敏感神经元 VLm.细胞内电生理技术在神经元中的应用 因为只有相关的细胞内测量才有意义 到目前为止，这些细胞是由神经胶质细胞制成的。拟议中的实验还将 确定其形态特征和传出指标 神经元对CO(2)/H+的敏感性测试试图关联 脑干化学药物敏感网络的结构和功能。 传统的细胞内记录、穿孔补丁记录和 全细胞记录在高碳酸血症和酸中毒期间进行 脑干切片(100-400微米厚)。药理和离子 对神经元细胞外环境的操纵被用来 确定潜在的细胞和突触机制 对化学药物敏感。此外，通过贴片的细胞内灌流- 在全细胞记录过程中，使用钳位移液管来研究这种影响 细胞内pH(Phi)对兴奋性的影响及其可能的作用 碳酸酐酶检测胞外CO(2)具体目标 建议研究的目的是：1)确定对化疗敏感的地点 脑干神经元；2)测定固有膜和突触 化疗敏感神经元的特性；3)区分细胞反应 对CO(2)和H+；4)测定化疗敏感性的轴突投射 体内逆行标记结合体外培养的神经元 细胞内记录和标记；5)确定碳酸酐酶 感知CO(2)的功能，以及；6)确定Phi对 神经元兴奋性。

The central CO(2)/H+ chemoreceptors provide important facilitatory drive to the cardiorespiratory control networks and are essential for homeostasis of brain pH. Despite their role in these life maintaining systems, little is known concerning the neural substrate that is depolarized by CO(2)/H+ and the cellular mechanisms underlying these responses. This is due primarily to the lack of appropriate intracellular electrophysiological studies and difficulty in locating inherently-chemosensitive neurons in which cellular mechanisms can be studied. For example, neurons in the ventrolateral medulla (VLM) are generally believed to be the sole site of chemosensitivity, however, the majority of single-unit studies of VLM have shown that most units do not retain sensitivity to CO(2)/H+ during blockade of synaptic transmission. Moreover, there is accumulating evidence that the VLM may not be the sole site of central chemosensitivity, but that chemoreception may be accomplished via a more distributed system than originally envisaged. In this regard, we have reported that certain neurons in nucleus tractus solitarii (NTS) and dorsal motor nucleus of vagus (DMNX), major cardiorespiratory integrative areas, are depolarized by CO(2) during blockade of synaptic input in vitro. Although we cannot unequivocally prove that CO(2)-chemosensitive neurons in NTS and DMNX recorded in vitro are involved in central chemoreception, they do provide a substrate for studying the mechanisms by which CO(2)/H+ depolarizes neurons in brainstem. This proposal outlines experiments to study these mechanisms in NTS and DMNX neurons as well as chemosensitive neurons in and near VLM. Applying intracellular electrophysiological techniques to neurons in VLM is significant since the only relevant intracellular measurements to date were made from glial cells. Proposed experiments will also determine the morphological characteristics and efferent targets of neurons tested for sensitivity to CO(2)/H+ in an attempt to correlate structure and function in the chemosensitive network in brainstem. Conventional intracellular recordings, perforated-patch recordings, and whole-cell recordings are conducted during hypercapnia and acidosis in brainstem slices (100-400 micro m thick). Pharmacologic and ionic manipulations of the neuron's extracellular environment are employed to identify the cellular and synaptic mechanisms underlying chemosensitivity. In addition, intracellular perfusion via the patch- clamp pipette during whole-cell recordings is used to study the effects of intracellular pH (pHi) on excitability and the proposed role of carbonic anhydrase in detection of extracellular CO(2). Specific aims of the proposed research are to 1) establish locations of chemosensitive neurons in brainstem; 2) determine intrinsic membrane and synaptic properties of chemosensitive neurons; 3) differentiate cellular responses to CO(2) and H+; 4) determine the axonal projections of chemosensitive neurons using in vivo retrograde labeling combined with in vitro intracellular recording and labeling; 5) determine if carbonic anhydrase functions in sensing CO(2), and; 6) identify the effects of pHi on neuronal excitability.