Dysfunction of homeostatic ventilatory chemoreflexes likely contribute to the genesis of or maladaptation to multiple respiratory-related diseases in humans, but potential treatments are hampered by a poor understanding of the fundamental CNS mechanisms responsible for detecting and responding to hypercapnia. There are multiple CNS sites containing cells with presumed intrinsic CO2/pH sensitivity, including medullary raphe (MR) serotoninergic (5-HT) and phox2b-expressing retrotrapezoid nucleus (RTN) neurons. The identity of molecules that underlie cellular CO2/pH sensitivity to these cells remains unknown. In addition, excitatory neuromodulators such as 5-HT, substance P and thyrotropin-releasing hormone (TRH) are critical to neural respiratory control, but the importance of these neuromodulators in the CO2 chemoreflex is unclear. To address these knowledge gaps, we will test two major hypotheses by completing three Specific Aims. We hypothesize that: 1) sub-populations of phox2b+ RTN and MR 5-HT neurons are intrinsically chemosensitive due to the selective expression of one or more pH-sensitive ion channels, and 2) raphe-derived neuromodulation of the RTN is a major determinant of the mammalian CO2 chemoreflex. To identify molecules that may underlie cellular CO2/pH sensitivity, we have developed a unique scientific approach utilizing fluorescence-assisted cell sorting (FACS) followed by Next-gen RNA sequencing (RNASeq) to identify differentially-expressed genes among neurochemically-defined brainstem neuronal subpopulations. We have demonstrated the feasibility of our approach, and identified two genes (Kir4.1 and Kir5.1) that may underlie cellular CO2 chemosensitivity of 5-HT neurons.
In Aim 1 we will use this approach to identify genes/molecules that may underlie cellular CO2 sensitivity by comparing CO2-sensitive and CO2-insensitive 5-HT and RTN neurons using hypercapnia-induced c-Fos expression to identify CO2 sensitive neurons.
In Aim 2 we will functionally validate genes identified in Aim 1, and specifically the roles of Kir4.1/5.1 K+ channels in vitro using patch clamp recordings and in vivo in genomic Kir4.1, Kir5.1 and combined Kir4.1/5.1 knockout rats. To further address the role of neuromodulators in the ventilatory CO2 chemoreflex, we will study Brown Norway (BN) rats, which have a severely blunted CO2 chemoreflex but normal breathing during eupnea, hypoxia and exercise. These CO2-insensitive BN rats are deficient in brainstem 5-HT and TRH, and stimulation of 5-HT or TRH receptors augments the CO2 chemoreflex in BN rats. Accordingly, we hypothesize that these neuromodulatory effects occur through direct modulation of the RTN, which we will test in Aim 3 by microdialysis of agonists and antagonists of 5-HT, substance P and TRH receptors within the RTN of CO2-insensitive BN and highly CO2- sensitive Salt-sensitive (SS) and Sprague Dawley (SD) rats. Our innovative studies will generate important new data regarding fundamental mechanisms of cellular CO2 chemoreception and the CO2 chemoreflex, and provide a framework for a molecular genetics approach to study other components of ventilatory control.

Public Health Relevance

We will use a unique genetics-based approach to identify molecules in specialized brainstem cells that enable them to detect changes in carbon dioxide (CO2) or pH. We will also determine the importance of specific neuromodulators within a brainstem region critical to the ventilatory CO2 chemoreflex. Understanding these fundamental neural mechanisms is critical to complete the understanding of respiratory-related diseases thought to result from altered chemoreception in humans, including sudden infant death syndrome (SIDS).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL122358-04
Application #
9462818
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Laposky, Aaron D
Project Start
2015-04-17
Project End
2020-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Physiology
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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Puissant, Madeleine M; Mouradian Jr, Gary C; Liu, Pengyuan et al. (2017) Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels. Front Cell Neurosci 11:34
Langer 3rd, Thomas M; Neumueller, Suzanne E; Crumley, Emma et al. (2017) State-dependent and -independent effects of dialyzing excitatory neuromodulator receptor antagonists into the ventral respiratory column. J Appl Physiol (1985) 122:327-338
Langer 3rd, Thomas M; Neumueller, Suzanne E; Crumley, Emma et al. (2017) Effects on breathing of agonists to ?-opioid or GABAA receptors dialyzed into the ventral respiratory column of awake and sleeping goats. Respir Physiol Neurobiol 239:10-25
Hodges, Matthew R (2017) Going to WAR: using a rat model of audiogenic seizure to uncover potential links to ventilatory dysfunction in epilepsy. J Physiol 595:617-618
Young, Jacob O; Geurts, Aron; Hodges, Matthew R et al. (2017) Active sleep unmasks apnea and delayed arousal in infant rat pups lacking central serotonin. J Appl Physiol (1985) 123:825-834
Palygin, Oleg; Levchenko, Vladislav; Ilatovskaya, Daria V et al. (2017) Essential role of Kir5.1 channels in renal salt handling and blood pressure control. JCI Insight 2:

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