A discrete group of neurons located in the retrotrapezoid nucleus (RTN) that express the transcription factor, Phox2b provide a crucial excitatory drive to regulate downstream respiratory rhythm/pattern-generating circuits. The activity of these neurons is modulated by changes in CO2 (or its proxy, H+) and various other sensory and arousal-state inputs to control breathing; their dysfunction is implicated in various central disorders of breathing (e.g., sudden infant death, congenital central hypoventilation syndrome (CCHS)). The molecular and cellular mechanisms involved in CO2/H+ sensing by RTN neurons, and how those are established developmentally and adapted to pathological conditions, remain matters of continuing scrutiny. In two Aims, we address a receptor-mediated mechanism of pH sensitivity, and explore gene expression patterns that support developmental and adaptive RTN function.
In Aim 1, we use new mouse genetic models to identify mechanisms of pH sensitivity in RTN neurons that are mediated by proton-activated GPR4 modulation of a background K+ channel, exploring the hypothesis that GPR4 is expressed in RTN neurons, where its intrinsic pH sensitivity leads to inhibition of KNa1.1 (encoded by Kcnt1) to contribute to CO2 stimulation of breathing and arousal. We propose to: [1.1] Test whether direct detection of protons by GPR4 accounts for its effects on RTN neuronal sensitivity and CO2-stimulated breathing; [1.2] Test whether KNa1.1 (Slo2.2, Kcnt1) is a GPR4-inhibited K+ channel effector in RTN neurons; and [1.3] Define sites of GPR4 protein expression.
In Aim 2, we combine single cell RNA-Seq with gene manipulation and developmental/physiological challenges to test the hypothesis that Phox2b expression dictates a distinct molecular signature that supports critical physiological functions of RTN neurons, and that those gene expression patterns are malleable to developmental and physiological challenges in support of breathing. We propose to: [2.1] Determine consequences of Phox2b depletion on the RTN neuron transcriptome; [2.2] Determine effect of birth on RTN neuron transcriptome; and [2.3] Characterize developmental and adaptive gene regulation in RTN neurons. To accomplish these aims, we employ a variety of techniques at multiple levels of analysis. Specifically, we combine genetic and viral approaches for RTN neuron-specific manipulation of gene expression; perform electrophysiological and functional/behavioral studies at the cellular and organismal levels; and utilize molecular neuroanatomy and single neuron genetic analyses for phenotypic characterization and quantification of normal and adaptive gene expression profiles. Collectively, the proposed studies will provide novel information regarding molecular and cellular mechanisms that regulate the pH-dependent activity of RTN neurons, at critical periods during development and in response to physiological challenge, with relevance for identifying new therapeutic targets for disorders of breathing.
A population of Phox2b-expressing excitatory neurons in the retrotrapezoid nucleus (RTN) of the ventral medulla oblongata provide a CO2/H+-chemosensory and integrative role in regulating breathing; dysfunction of this neuronal system is implicated in potentially fatal syndromes (e.g., sudden infant death, congenital central hypoventilation) and re-setting of CO2 threshold/sensitivity can accompany and exacerbate various chronic disorders of breathing (e.g., chronic obstructive pulmonary disease). The cellular and molecular mechanisms that regulate pH sensitivity, and developmental or adaptive changes in those neurons have not been determined. Using mouse genetic models or viral transduction, the proposed work will incorporate single cell electrophysiological and molecular characterization of RTN neurons to uncover receptor and channel mechanisms of pH sensitivity, and to provide a transcriptome-wide examination of phenotypic features that support breathing in the vulnerable perinatal period or sustain function throughout life in the face of environmental or pathological challenge.
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