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.
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