Cellular/Molecular Mechanisms of Respiratory Neuronal Chemosensitivity

University Of Virginia, Charlottesville VA

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Abstract

PROJECT SUMMARY An interoceptive system within the CNS monitors levels of CO2 (or its proxy, H+) and regulates respiratory drive for rapid homeostatic control of blood gases and systemic acid-base balance; dysfunction of this central respiratory chem- oreception is cause or consequence of numerous hypoventilation syndromes. Despite recognition of this chemoreflex system since the early 1900s, and its importance for understanding respiratory (patho)physiology, the identity of the relevant sensory element(s) remains a point of significant controversy largely because none of the candidate cellular sensors and molecular detectors have yet fulfilled the requisite experimental criteria. Moreover, the molecular basis for CO2/H+ sensing, and how those are established developmentally and adapted to pathological conditions, remain matters of continuing scrutiny. Compelling evidence implicates a discrete group of developmentally specified and phenotypically characterized neurons located in the brainstem retrotrapezoid nucleus (RTN) as respiratory chemosen- sors, and suggests that CO2/H+ detection by RTN neurons is mediated by a pH sensitive G protein-coupled receptor (GPR4) and background K+ channel (TASK-2). However, the evidentiary record remains incomplete. In Aim 1, we address key remaining shortcomings and test the hypothesis that CO2/H+ sensing by GPR4 and TASK-2 in RTN neurons is both necessary and sufficient for CO2 stimulation of breathing. We propose to: [1.1] Determine if direct proton detection by TASK-2 accounts for its effects on RTN neuronal CO2/H+ sensitivity and CO2-stimulated breathing; [1.2] Examine CO2-sensitive breathing after re-expression of wild type and pH-insensitive GPR4 or TASK-2 in RTN neurons of knockout mice; and [1.3] Establish by in vivo photometry if CO2-induced modulation of RTN neuron activity requires GPR4 and TASK-2 in conscious mice. In Aim 2, we test the hypothesis that neuroadaptive mechanisms in RTN neurons account for changes in the respiratory chemoreflex associated with physiological and pathological chal- lenges. We propose to: [2.1] Examine mechanisms and consequences for birth-related PACAP expression in early postnatal RTN neurons; and [2.2] Characterize neuroadaptation of RTN neurons and the chemoreflex during persis- tent excessive chemosensory stimulation (chronic CO2 elevation, as in chronic obstructive pulmonary disease). For both aims, we apply multi-scale analyses in novel genetic and disease mouse models to examine: behavioral/func- tional outcomes at the whole animal and cellular levels by plethysmography, photometry, and in vitro electrophysiol- ogy; and we determine adaptive changes in gene expression using single neuron molecular analyses. Collectively, the proposed studies will: 1) interrogate the remaining criteria for RTN neurons to fulfill rigorous evi- dentiary requirements for their classification as bona fide respiratory chemosensors; and 2) define molecular and cellular mechanisms for RTN neuronal adaptation in response to (patho)physiological challenges, with relevance for identifying potential cellular and molecular targets for treating disorders of breathing.