Intracelluar pH (pHi) Measurements in the In Vitro Tadpole Brainstem:Direct Correlations between Changes in pHi and Ventilation

Abstract

Central respiratory chemoreceptors measure pH in the brain stem and are an integral part of the neural circuitry that modulates respiratory rhythmogenesis, specifically in response to hypercapnic acidosis. Central respiratory chemore- ceptor membrane potential and/or action potential firing rate are altered in response to changes in intracellular pH (pHi), which changes with the hydration of CO2 in both the extracellular and intracellular space, however the role of cellular changes in chemoreceptor properties on respiratory motor output has been difficult to identify. We studied whole nerve respiratory activity while simultaneously visualizing pHi dynamics using the pH-sensitive dye, BCECF, in the spontane- ously active in vitro tadpole brainstem. The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neu- ronsunder physiological conditions, where mammalian preparations do not. An ammonium prepulse was used to selec- tively induce a decrease in pHi. Our results show intracellular pH is regulated differently in cells located in chemosensi- tive and non-chemosensitive regions of the tadpole brainstem during hypercapnia. We were also able to show an inverse correlation between pHi in cells located in chemosensitive regions of the tadpole brainstem and whole nerve respiratory- related activity. Using this approach, the microenvironment of individual cells may be manipulated while monitoring real time changes in pHi, neuronal activity and ventilatory-related activity to elucidate the role of a variety of signals in elicit- ing changes in ventilation.