Employing a pH Sensitive Fluorophore to Measure Intracellular pH in the In Vitro Brainstem Preparation of Rana catesbeiana

We developed an in vitro tadpole brainstem preparation in order to investigate the development of central respiratory chemoreception and rhythmogenesis. pH sensitive fluorescent dyes have been utilized to record intracellular pH (pHi) optically in central respiratory chemoreceptive regions in mammals. Our goal in this study was to develop the ability to record pHi optically while simultaneously recording respiratory motor output in the superfused tadpole brainstem preparation. We developed a dye-loading protocol that demonstrated our ability to adequately load the majority of brainstem neurons. The presence of the dye was not disruptive to ongoing respiratory rhythmogenesis or the respiratory response to central respiratory chemoreceptor stimulation. The tadpole brainstem is an excellent model to study the development of the neural control of respiration, as it is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neurons. Validating of the use of the pH sensitive dyes to record pHi optically in central respiratory chemoreceptors in this preparation will permit further characterization of the pH regulatory responses of central respiratory chemoreceptors.