MECHANISMS OF CO2/H+-SENSITIVITY IN BRAINSTEM NEURONS

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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.