Date of Completion

9-4-2019

Embargo Period

8-31-2029

Keywords

chemoreception, HCO3, pHi, ph-independent, brain slice, brainstem, retrotrapezoid nucleus

Major Advisor

Daniel K. Mulkey

Associate Advisor

Anastasios V. Tzingounis

Associate Advisor

Joseph J. LoTurco

Associate Advisor

Randall Walikonis

Associate Advisor

Joseph Crivello

Field of Study

Physiology and Neurobiology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+. The retrotrapezoid nucleus (RTN) is an important site of respiratory chemoreception. Mechanisms underlying RTN chemoreception involve H+‐mediated activation of chemosensitive neurons and CO2/H+‐evoked ATP‐purinergic signaling by local astrocytes, which activates chemosensitive neurons directly and indirectly by maintaining vascular tone when CO2/H+ levels are high. Although changes in CO2 result in corresponding changes in both H+ and HCO3− and despite evidence that HCO3− can function as an independent signaling molecule, there is little evidence suggesting HCO3− contributes to respiratory chemoreception. Therefore, the goal of this study was to determine whether HCO3− regulates activity of chemosensitive RTN neurons independent of pH. Cell‐attached recordings were used to monitor activity of chemosensitive RTN neurons in brainstem slices (300 μm thick) isolated from rat pups (postnatal days 7–11) during exposure to low or high concentrations of HCO3−. In a subset of experiments, we also included 2′,7′‐bis(2 carboxyethyl)‐5‐(and 6)‐carboxyfluorescein (BCECF) in the internal solution to measure pHi under each experimental condition. We found that HCO3− activates chemosensitive RTN neurons by mechanisms independent of intracellular or extracellular pH, glutamate, GABA, glycine or purinergic signaling, soluble adenylyl cyclase activity, nitric oxide or KCNQ channels. We also found that TASK-2, a known pH sensor in RTN neurons, was inhibited by the application of HCO3− when expressed in HEK-293T cells, lending evidence to the mechanism of HCO3− specificity for chemosensitive RTN neurons. Finally, when extracellular Cl- was equilibrated between our aCSF and HCO3− solutions, we found that the normally excitatory effect of HCO3− became an inhibition, which may signify a Cl- dependency or mechanism for HCO3−-mediated excitation. These results establish HCO3− as a novel independent modulator of chemoreceptor activity, and because the levels of HCO3− along with H+ are buffered by independent cellular mechanisms, these results suggest HCO3− chemoreception adds additional information regarding changes in CO2 that are not necessarily reflected by pH.

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Available for download on Friday, August 31, 2029

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