Molecular Disambiguation of Heart Rate Control by the Nucleus Ambiguus

The nucleus ambiguus (nAmb) provides parasympathetic control of cardiorespiratory functions as well as motor control of the upper airways and esophagus. A subset of nAmb neurons innervates the heart through the vagus nerve to control cardiac function at rest and during key autonomic reflexes such as the mammalian diving reflex. Yet, how these cardiovagal nAmb neurons differ from other nAmb neurons in the adult brain remains unclear. We therefore classified adult male and female mouse nAmb neurons molecularly, anatomically, and functionally. First, our integrated analysis of single-nucleus RNA-sequencing data predicted multiple molecular subtypes of nAmb neurons. Mapping the axon projections of one nAmb neuron subtype, Npy2r -expressing nAmb neurons, showed that they innervate cardiac ganglia but not the upper airways or esophagus. Chemogenetically stimulating Npy2r + nAmb neurons robustly decreased heart rate through peripheral muscarinic acetylcholine receptors. Finally, voluntary underwater diving decreases heart rate and activates Npy2r + nAmb neurons, consistent with a cardiovagal function for this nAmb subtype. These results together reveal the molecular organization of nAmb neurons and its control of heart rate. Significance statement The nucleus ambiguus of the brainstem controls heart rate through the vagus nerve, but the specific neurons involved have been difficult to identify because they are intermixed with neurons that regulate breathing and swallowing. Here we combine single-cell transcriptomics, anatomical tracing, and physiological experiments to identify a distinct subtype of nucleus ambiguus neuron, marked by Npy2r gene expression, that innervates the heart but not the esophagus or upper airways, and powerfully controls heart rate. We also show that these neurons become activated during voluntary underwater diving - a natural reflex that strongly slows the heart. These findings reveal the cellular organization of a key brain-heart circuit and a molecular entry point for studying its role in health and disease.