This R01 renewal project aims to explain the neural origins of breathing behavior at the cellular and synaptic level. It advances understanding of the brainstem pre-Btzinger complex (preBtC), which is acknowledged to be the principal site driving respiration in humans and all terrestrial mammals so far studied. Also, this project examines interneurons of the intermediate reticular formation, adjacent to the preBtC, which may give rise to respiratory premotor neurons. The intellectual driving force for this project is te discovery by the PI's team - and French colleagues - that the key rhythmogenic preBtC interneurons in perinatal mice are derived from embryonic precursors that express transcription factor Dbx1 (i.e., Dbx1 preBtC neurons). This project exploits this new knowledge and by coupling Dbx1 Cre-driver mice with six different flox-STOP reporter strains to perform a spectrum of experiments in vivo and in vitro such as patch-clamp recordings, cell-specific laser ablations with physiological monitoring, and optogenetic manipulations that interrogate network properties.
Aim 1 uses juvenile and adult mice (in vivo and in vitro) to examine whether Dbx1 preBtC neurons are rhythmogenic beyond embryonic and neonatal stages of development.
Aim 2 uses embryonic and neonatal mice in vitro in conjunction with cell-specific laser ablation methods to test whether preBtC neurons with bursting-pacemaker properties are obligatory for respiratory rhythm generation, offering a fresh approach to a 24-year-old unsolved problem regarding `pacemaker' driven preBtC rhythms.
Aim 3 uses perinatal mice in vitro to characterize synaptic interconnections among Dbx1 neurons and quantify the input-output relationship. These experiments elucidate recurrent synaptic excitation in Dbx1 preBtC neurons, which is also putatively rhythmogenic.
Aim 4 uses perinatal through adult mice (in vivo and in vitro) to examine whether Dbx1 neurons in the adjacent intermediate reticular formation serve as the first layer of premotor neurons for respiratory movements of the tongue (genioglossus) and pharynx. Dysfunctions in respiratory control circuits cause significant health problems including obstructive and central apneas, as well as respiratory failure and death. These conditions afflict premature infants, children, adults, and patients with neurodegenerative disorders. This project is significant because it characterizes the cellular and synaptic mechanisms that animate the key genetic class of neurons (i.e., Dbx1) at the core of the respiratory oscillator, which represents a transformative advance in our understanding that would inform new prevention and treatment strategies to combat respiratory pathologies. The PI is the ideal scientist for this job because of his track record as a leader in respiratory neurobiology, who - with French colleagues - first characterized the role of Dbx1 neurons in the preBtC and now is poised to further discover their detailed properties and downstream premotor counterparts. If this project succeeds, neuroscience would finally know the cellular and synaptic origins of a significant central pattern- generating circuit in a mammal and the point of origin for an important behavior, breathing.

Public Health Relevance

This project advances understanding of the neural control of breathing. It characterizes the genetic class of interneurons that putatively generate respiratory rhythm and express motor output (i.e., breathing movements) in humans and likely all mammals. This new knowledge elucidates a physiologically significant regulatory brain function with considerable relevance to human health, providing a basic neuroscience foundation for prevention and treatment of a range of diseases from apnea of prematurity, to obstructive and sleep-related apnea, to respiratory failure that occurs in neurodegenerative disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Study Section
Special Emphasis Panel (ZRG1)
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Laposky, Aaron D
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College of William and Mary
Schools of Arts and Sciences
United States
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Phillips, Wiktor S; Del Negro, Christopher A; Rekling, Jens C (2018) Dendritic A-Current in Rhythmically Active PreBötzinger Complex Neurons in Organotypic Cultures from Newborn Mice. J Neurosci 38:3039-3049
Kottick, Andrew; Martin, Caroline A; Del Negro, Christopher A (2017) Fate mapping neurons and glia derived from Dbx1-expressing progenitors in mouse preBötzinger complex. Physiol Rep 5:
Akins, Victoria T; Weragalaarachchi, Krishanthi; Picardo, Maria Cristina D et al. (2017) Morphology of Dbx1 respiratory neurons in the preBötzinger complex and reticular formation of neonatal mice. Sci Data 4:170097
Hayes, John A; Kottick, Andrew; Picardo, Maria Cristina D et al. (2017) Transcriptome of neonatal preBötzinger complex neurones in Dbx1 reporter mice. Sci Rep 7:8669
Vann, Nikolas C; Pham, Francis D; Hayes, John A et al. (2016) Transient Suppression of Dbx1 PreBötzinger Interneurons Disrupts Breathing in Adult Mice. PLoS One 11:e0162418
Phillips, Wiktor S; Herly, Mikkel; Del Negro, Christopher A et al. (2016) Organotypic slice cultures containing the preBötzinger complex generate respiratory-like rhythms. J Neurophysiol 115:1063-70
Song, Hanbing; Hayes, John A; Vann, Nikolas C et al. (2016) Functional Interactions between Mammalian Respiratory Rhythmogenic and Premotor Circuitry. J Neurosci 36:7223-33
Song, Hanbing; Hayes, John A; Vann, Nikolas C et al. (2015) Mechanisms Leading to Rhythm Cessation in the Respiratory PreBötzinger Complex Due to Piecewise Cumulative Neuronal Deletions eNeuro 2:
Kottick, Andrew; Del Negro, Christopher A (2015) Synaptic Depression Influences Inspiratory-Expiratory Phase Transition in Dbx1 Interneurons of the preBötzinger Complex in Neonatal Mice. J Neurosci 35:11606-11
Revill, Ann L; Vann, Nikolas C; Akins, Victoria T et al. (2015) Dbx1 precursor cells are a source of inspiratory XII premotoneurons. Elife 4:

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