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 心肺控制网络，是必不可少的 尽管它们在这些生命维持中起着重要作用， 系统，很少有人知道有关的神经基板， CO（2）/H+去极化及其细胞机制 应答 这主要是由于缺乏适当的 细胞内电生理研究和定位困难 内在化学敏感的神经元，其中细胞机制可以 研究了 例如，延髓腹外侧区（VLM）的神经元 通常认为是唯一的化学敏感性位点，然而， 大多数VLM的单单元研究表明，大多数单元不 在阻断突触传递期间保持对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 中心化学敏感性部位，但化学感受可能 通过比最初设想的更加分布式的系统来实现。 在这方面，我们已经报道，某些神经元在核束 孤束核（NTS）和迷走神经背侧运动核（DMNX），主要 心肺综合区，在CO（2）的作用下去极化。 在体外阻断突触输入。 虽然我们不能明确地 证明孤束核和DMNX的CO（2）化学敏感神经元在体外记录 参与中枢化学感受，它们确实为 研究CO（2）/H+使神经元去极化的机制， 脑干 该提案概述了研究这些机制的实验 在NTS和DMNX神经元以及在和附近的化学敏感神经元中， VLM。 将细胞内电生理技术应用于神经元 在VLM中是重要的，因为唯一相关的细胞内测量 迄今为止都是由神经胶质细胞制成的。 拟议的实验也将 确定的形态学特征和传出目标 测试神经元对CO（2）/H+的敏感性，试图将其与 脑干化学敏感网络的结构和功能。 传统的细胞内记录，穿孔斑记录， 在高碳酸血症和酸中毒期间进行全细胞记录， 脑干切片（100-400微米厚）。 药理学和离子 神经元的细胞外环境的操纵被用来 识别细胞和突触机制， 化疗敏感性 此外，通过贴片的细胞内灌注- 在全细胞记录过程中使用夹钳移液管来研究 细胞内pH（pHi）对兴奋性的影响以及 碳酸酐酶检测细胞外CO（2）。 具体目标 拟议研究的一项内容是：1）确定化学敏感的位置 脑干神经元; 2）确定固有膜和突触 化学敏感神经元的特性; 3）区分细胞反应 CO（2）和H+; 4）确定化学敏感的轴突投射 采用体内逆行标记结合体外 细胞内记录和标记; 5）确定碳酸酐酶是否 在感知CO（2）中的功能，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.