The central chemoreceptors are one of the most important sources of feedback for respiration; however, the basic cellular mechanisms responsible for chemoreception and the location of the chemoreceptors have not yet been defined. CO2 also has significant effects on a number of other CNS functions, including cardiovascular control, cognition, and seizure threshold. The mechanisms of these effects on function are not known, in part because the effects of CO2 on individual CNS neurons are not well understood. Pacemaker neurons have recently been found in the rostral ventrolateral medulla (VLM) and medullary raphe that respond sensitively to small changes in CO2/pH by modulation of their pacemaker activity, some with excitation and others with inhibition (Richerson, 1992, 1993, submitted). This grant will examine a number of basic unanswered questions about the location and cellular mechanisms of these, and other CO2-sensitive neurons. 1) How extensive is the distribution of CO2-sensitive neurons in the CNS? 2) Is CO2 or pH the primary stimulus? 3) Which ion channels are modulated by CO2/pH? 4) Are the morphology and projections of CO2-sensitive neurons unique? A combination of whole-cell, perforated-patch, and cell-attached patch-clamp recordings will be made from thin (100 micromole) slices of the rat medulla. This preparation permits highly-stable recordings from individual neurons that are visualized at high power in fresh, non-enzymatically treated tissue. One set of experiments will map out the distribution of CO2-sensitive neurons, determine which of these are intrinsically CO2-sensitive by blocking synaptic transmission with high Mg++ / low Ca++ solution, and determine whether changes in CO2 or pH are required for the response. Another set of experiments will examine the mechanisms responsible for pacemaker activity in regions that contain CO2-sensitive neurons. Recordings will then be made from neurons in those areas found to have intrinsically CO2-sensitive neurons, to determine which ion currents are modulated by CO2. Biocytin will be used in all electrodes and neuronal morphology will be analyzed after recording. In some experiments, recordings will be made from slices several days after injection of rhodamine beads to determine neuronal projections. The approach described should be technically feasible, because most of the techniques proposed in this grant are already in routine use in the laboratory of the principal investigator, and the preparation has been developed to the point that stable recordings can be routinely obtained. The proposed experiments provide a way of linking electrophysiologic data with anatomical data, and should help determine the distribution of CO2-sensitive neurons and the cellular mechanisms of the response. Disturbances of breathing are common in many human diseases, and disorders of chemoreception significantly contribute to mortality in a number of them, including chronic obstructive pulmonary disease and sleep apnea. Effects of CO2 and pH on neuronal activity also contribute to the pathophysiology of a number of other diseases, including stroke and epilepsy. Understanding the basic mechanisms involved in modulation of neuronal activity by CO2 and pH may help provide successful treatment for these diseases.
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