Molecular characterization of expiratory breathing-related interneurons in mammals

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PROJECT SUMMARY Breathing is important behavior that ventilates the lungs for gas exchange, thus maintaining health and homeostasis. Breathing at rest consists of active inspiration (inhalation) but expiration (exhalation) is passive. Expiration becomes active to increase ventilation as respiratory demand increases. To understand the neural bases and control of breathing, we must be able to explain both the mechanisms that underlie active inspiration and those underlying active expiration. But there is a massive disparity at present: the mechanisms for inspiration are very well understood at multiple levels of analysis, but the mechanisms for active expiration remain almost entirely unknown. Neither the underlying rhythmogenic neurons nor their spatial organization have been investigated in any detail. All we do know at present is that expiratory rhythm probably emerges from a multifunctional region in the parafacial region of the rostral-lateral medulla. This project would bridge that knowledge gap and identify the neuron class giving rise to active expiration, map the borders of the expiratory rhythmogenic network, and establish their functionality definitively. Our project follows logical steps: first, sequence the transcriptomes of parafacial interneurons and identify highly expressed transcripts to define neuronal subtypes; second, map the borders of neurons whose subtypes were defined in step 1; and third, interrogate their expiratory function via cell population-specific photostimulation and photoinhibition experiments in awake intact adult mice. The upshot of this project will be a well-defined neural core for expiratory breathing movements, characterized at the molecular-genetic level of analysis, raw and annotated transcriptomes of parafacial interneurons disseminated freely in the public domain (via the Gene Expression Omnibus of the National Center for Biotechnology information), and a balanced understanding and explanation of the mechanisms underlying both active inspiration and active expiration. There are widely disparate mechanisms for whisking, chewing, and inspiratory breathing rhythms. This project would describe a fourth orofacial oscillation – active expiration – which would advance sensorimotor neuroscience. This present project would unravel whether expiratory rhythmogenic neurons are derived from Atoh1-expressing precursors that give rise to Phox2b-expressing central respiratory chemosensors, Krox20/Egr2-expressing progenitors, or another yet-to-be-identified cell class that develops in hindbrain rhombomeres 3 and 5 (r3/r5). This new knowledge would augment our current understanding of the development and assembly of the breathing central pattern generator (bCPG). Finally, whereas the entire bCPG is susceptible to opioid-induced respiratory depression (OIRD), the active expiratory oscillator is surprisingly opioid-insensitive. New knowledge in this project may be leveraged to provide acute treatments for opioid overdose and long-term treatment strategies that protect recovering opioid addicts from OIRD.