Optical recording of intracellular pH in respiratory chemoreceptors

We studied the spontaneously active in vitro tadpole brainstem and recorded whole nerve respiratory activity while simultaneously visualizing intracellular pH (pHi) dynamics using the pH-sensitive dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF, AM). The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neurons. We generated a calibration curve to correlate the emitted fluorescence of BCECF to pHi. In addition, we demonstrated that the dye loading protocol that we established labeled an adequate number of cells and did not disrupt spontaneous respiratory rhythmogenesis or the respiratory response to central chemoreceptor stimulation. Validation of the use of the pH sensitive dye BCECF in this preparation will permit further characterization of the pH regulatory responses of central respiratory chemoreceptors and allow correlation between the changes in pHi in central chemoreceptors and respiratory motor output recorded from cranial nerves.