Interactions between arousal state and CO₂ determine the activity of central chemoreceptor neurons that drive breathing.

The homeostatic regulation of pulmonary ventilation, and ultimately arterial PCO, depends on interactions between respiratory chemoreflexes and arousal state. The ventilatory response to CO is triggered by neurons in the retrotrapezoid nucleus (RTN) that function as sensors of central pH, which can be identified in adulthood by expression of Phox2b and Neuromedin B. Here we examine the dynamic response of genetically-defined RTN neurons to hypercapnia and arousal state in freely-behaving adult male and female mice using the calcium indicator jGCaMP7 and fiber photometry. We found that hypercapnia vigorously activates RTN neurons with a low CO recruitment threshold and with response kinetics that match respiratory activity whereas hypoxia had little effect. RTN activity increased transiently during wakefulness and respiratory-related arousals, rose persistently during REM sleep and their CO response persisted under anesthesia. Complementary studies using inhibitory optogenetics show that RTN activity supports eupneic breathing under anesthesia as well as during states of high arousal, but their activity is redundant for voluntary breathing patterns. Collectively, this study demonstrates that CO-activated RTN neurons are exquisitely sensitive to arousal state, which determines their contribution to alveolar ventilation in relation to arterial PCO Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial PCO and PO, thereby maintaining optimal gas exchange. Central chemoreceptor neurons expressing Phox2b and Neuromedin B in the Retrotrapezoid Nucleus (RTN) are required for the hypercapnia ventilatory response. However, the dynamic activity of RTN neurons in conditions of normal and elevated carbon dioxide has not been described in unanesthetized conditions. Here, we use a genetically-encoded calcium indicator to demonstrate that RTN neurons exquisitely sensitivity to hypercapnia and variations in arousal and sleep-state in freely-behaving mice. This work has implications for understanding the central control of breathing across arousal-states, particularly during sleep and anesthesia.