This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A.
Specific Aims The Specific Aims in the application have not changed and experiments have been conducted in accordance with these aims. We have progressed in our abilities to perform optical recordings of intracellular pH (pHi) from neurons in the spontaneously rhythmic in vitro tadpole brainstem preparation. B. Studies and Results Upon the completion of the BCECF calibration curve, we were then able to record emitted fluorescence rations from both chemo- and non-chemosensitive regions while simultaneously recording whole nerve respiratory activities. We report significant differences in resting pHi between neurons located in known chemosensitive areas and neurons from non-chemosensitive areas. In addition, we utilized the ammonium chloride prepulse technique to induce intracellular acidification while maintaining a control bath pH of 7.8. Finally, we were able to demonstrate a significant negative correlation between decreases in pHi in chemosensitive neurons and increases in lung burst frequency. We have successfully demonstrated a correlation between pHi and ventilation, as proposed in our Specific Aims, and are in the process of submitting these data for peer review publication. We also explored a potential new technique to induce intracellular acidosis rather than use the ammonium chloride prepulse technique, as the ammonium chloride prepulse technique induces intracellular acidification to all brainstem neurons. We hoped to use """"""""uncaging"""""""" of H+ via exposure to ultraviolet light, thereby allowing us to focally induce decreases in pHi in only those cells of interest.
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