A group of excitatory neurons located in the retrotrapezoid nucleus (RTN) that express the transcription factor, Phox2b, integrate sensory inputs and information regarding brain state for transmission on to respiratory rhythm/pattern-generating circuits. In addition, RTN neurons adjust their firing in response to changes in CO2 (or H+) and adjust breathing to maintain physiologically appropriate levels of pH and PCO2, a homeostatic process called central respiratory chemoreception. Dysfunction of central chemoreception is implicated in various central disorders of breathing that often occur during sleep (e.g., sudden infant death, congenital central hypoventilation syndrome (CCHS)). In the last project period, we showed that Phox2b-expressing RTN neurons are intrinsically chemosensitive, and identified two independent molecular proton sensors in RTN neurons - TASK-2, a proton-inhibited background K+ channel and GPR4, a proton-activated G protein-coupled receptor - that are required for stimulation of breathing by CO2. Important questions remain, however, regarding: the ionic basis for baseline firing properties and modulation by arousal state-dependent factors; the effector systems engaged downstream of GPR4 in RTN neurons; and mechanisms by proposed astrocytic modulation can be integrated with the requirement for GPR4 and TASK-2 in RTN-mediated respiratory chemosensitivity. This proposal addresses these issues using: novel conditional knockout mouse lines; viral-mediated shRNA knockdown and/or rescue; single cell electrophysiology and molecular biology; and whole animal assays of respiratory function and vigilance states. The hypothesis underpinning Specific Aim 1 is that TTX- resistant subthreshold Na+ channels, NALCN and NaV1.9, contribute to baseline excitability of RTN neurons and mediate facilitatory effects of neuropeptides associated with arousal state-dependent brain nuclei. We disrupt expression of these channels in RTN neurons and determine effects on subthreshold Na+ currents, basal and neuropeptide-modulated firing in vitro, and arousal state-dependent respiratory CO2 sensitivity in vivo. The hypothesis driving Specific Aim 2 is that GPR4 engages a cAMP-transduction pathway and background K+ channel (independent of TASK-2) for cellular pH sensing in RTN neurons, and that astrocytic amplification of respiratory chemoreflexes involves boosting local pH changes around RTN neurons. We use pharmacological and transcriptomic approaches in single RTN neurons to characterize the GPR4 signaling pathway and effector channel, and we disrupt a pH-modulating Na+ -HCO3 transporter, NBCe1, in medullary astrocytes to determine effects on the respiratory chemoreflex in vivo. This latter may support a convergent theory for astrocyte-neuron contributions for this highly sensitive chemoreflex, bridging a major current divide in the field. Collectively, the proposed studies provide critical information regarding molecular and cellular mechanisms that control activity of RTN neurons, and regulate this important homeostatic respiratory system. Identification of novel molecular mechanisms may provide new therapeutic targets for disorders of breathing.

Public Health Relevance

An important homeostatic system adjusts breathing to control metabolic CO2 excretion, and thus whole body acid-base balance (pH or H+); dysfunction of this 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). It is well known that this so-called respiratory chemosensitivity involves neurons in the caudal brainstem that sense CO2/H+, but the cellular and molecular mechanisms that regulate basal excitability and pH sensitivity of those neurons have not been determined. The research undertaken in this proposal seeks to clarify those mechanisms in this critical population of respiratory integrative neurons ? and to provide novel insights into this fundamental process that may suggest new molecular targets for therapeutic development.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL108609-08
Application #
9646243
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Laposky, Aaron D
Project Start
2011-05-01
Project End
2020-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
8
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Virginia
Department
Pharmacology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Guyenet, Patrice G; Bayliss, Douglas A; Stornetta, Ruth L et al. (2018) Interdependent feedback regulation of breathing by the carotid bodies and the retrotrapezoid nucleus. J Physiol 596:3029-3042
Shi, Yingtang; Stornetta, Ruth L; Stornetta, Daniel S et al. (2017) Neuromedin B Expression Defines the Mouse Retrotrapezoid Nucleus. J Neurosci 37:11744-11757
Shi, Yingtang; Abe, Chikara; Holloway, Benjamin B et al. (2016) Nalcn Is a ""Leak"" Sodium Channel That Regulates Excitability of Brainstem Chemosensory Neurons and Breathing. J Neurosci 36:8174-87
Guyenet, Patrice G; Bayliss, Douglas A; Stornetta, Ruth L et al. (2016) Proton detection and breathing regulation by the retrotrapezoid nucleus. J Physiol 594:1529-51
Guyenet, Patrice G; Bayliss, Douglas A (2015) Neural Control of Breathing and CO2 Homeostasis. Neuron 87:946-61
Kumar, Natasha N; Velic, Ana; Soliz, Jorge et al. (2015) PHYSIOLOGY. Regulation of breathing by CO? requires the proton-activated receptor GPR4 in retrotrapezoid nucleus neurons. Science 348:1255-60
Vu, Michael T; Du, Guizhi; Bayliss, Douglas A et al. (2015) TASK Channels on Basal Forebrain Cholinergic Neurons Modulate Electrocortical Signatures of Arousal by Histamine. J Neurosci 35:13555-67
Bayliss, Douglas A; Barhanin, Jacques; Gestreau, Christian et al. (2015) The role of pH-sensitive TASK channels in central respiratory chemoreception. Pflugers Arch 467:917-29
Wang, Sheng; Shi, Yingtang; Shu, Shaofang et al. (2013) Phox2b-expressing retrotrapezoid neurons are intrinsically responsive to H+ and CO2. J Neurosci 33:7756-61
Wang, Sheng; Benamer, Najate; Zanella, S├ębastien et al. (2013) TASK-2 channels contribute to pH sensitivity of retrotrapezoid nucleus chemoreceptor neurons. J Neurosci 33:16033-44

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