Control of Heart Rate by Nucleus Ambiguus Cardiovagal Neurons

Abstract

Heart rate is a well-established indicator of overall health and well-being, yet little is known about the neurons that control it. Although the heart has its own pacemaker, heart rate is largely regulated by the interplay between sympathetic activities (via sympathoexcitatory neurons) and parasympathetic activities (via cardiac vagal preganglionic neurons (CVNs)). The nucleus ambiguus (nAmb), a region in the medullary reticular formation of the brainstem, is a major center of parasympathetic cardiac control and houses the majority of CVNs, along with other neurons that control respiratory functions (bronchoconstriction, bronchosecretion) and upper airway and esophageal muscles. Because of the diversity of neuron types in the nAmb, it is challenging to isolate CVNs for study, which greatly limits what we know about their gene expression, synaptic circuitry, and specific roles in cardiac function. To address these issues, we characterized cardiovagal neurons based on transcriptome-wide mRNA expression, synaptic circuitry, and sufficiency for heart rate control. Our single-cell transcriptomics analysis of 235 nAmb neurons pointed to three molecularly distinct neuron subtypes (marked by the genes Crhr2, Vipr2, and Tbx3) in the nAmb, which we validated and anatomically mapped by in situ detection of marker transcripts. We then used marker-driven genetic technology to fluorescently label the axon projections of Tbx3 nAmb neurons and imaged them in optically cleared heart samples. Our results show that these neurons innervate multiple sites in the atria and ventricles, consistent with multiple roles in cardiac function. Similar synaptic circuitry studies of the Crhr2 nAmb and Vipr2 nAmb neurons showed that they innervate the esophagus and larynx, respectively, but do not innervate the heart. Furthermore, using an intersectional optogenetics approach to specifically manipulate the activity of nAmb neuron subtypes in vivo, we have shown that activating the Tbx3 nAmb neurons in both freely behaving and anesthetized mice dramatically and immediately decreases heart rate (from 430 bpm to 160 bpm) as determined via EKG readings, whereas activating other subtypes of nAmb neurons did not affect heart rate. This effect on heart rate was abolished by administration of atropine, a muscarinic receptor antagonist. Together, these studies not only highlight the impact of Tbx3 nAmb neurons on heart rate, but also reveal a distinct molecular, anatomical, and functional organization within the nAmb.