Neuromedin B-Expressing Neurons in the Retrotrapezoid Nucleus Regulate Respiratory Homeostasis and Promote Stable Breathing in Adult Mice

Abstract

Respiratory chemoreceptor activity encoding arterial Pco₂ and Po₂ is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomic evidence suggests that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN^Nmb neurons are necessary for the CO₂-dependent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN^Nmb neurons causes compensated respiratory acidosis because of alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN^Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN^Nmb neurons. Interestingly, ventilation following RTN^Nmb-lesion was unresponsive to hypercapnia, but behavioral responses to CO₂ (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN^Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests that RTN^Nmb neurons are dedicated to the respiratory effects of arterial Pco₂/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.

SIGNIFICANCE STATEMENT Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial Pco₂ and Po₂, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO₂ on breathing. Our functional and anatomic data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO₂-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO₂- and O₂-sensing mechanisms in respiratory homeostasis of mammals.