Disambiguating control of the diving reflex by the nucleus ambiguus

Heart rate at rest and during cardiac reflexes is set primarily by cardiovagal neurons (CVNs) in the nucleus ambiguus (nAmb), which innervate the heart via the vagus nerve. These CVNs are thought to control the diving reflex, a powerful and highly conserved mammalian autonomic reflex that robustly decreases heart rate during voluntary underwater diving to preserve oxygen levels in the heart and brain. However, the identity of the CVNs mediating diving reflex bradycardia remains unknown. Previous studies in our lab uncovered three molecularly distinct subtypes of nAmb neurons, one of which innervates the heart, is capable of robustly decreasing heart rate, and is marked by its expression of the gene Npy2r (Npy2rnAmb). Accordingly, we hypothesize that Npy2rnAmb neurons are responsible for the decrease in heart rate during the diving reflex. To test this hypothesis, we first asked whether Npy2rnAmb neurons are activated by voluntary diving. Over the course of 6 days, mice (n=17) were trained to voluntarily dive about 0.5 meters underwater in a custom-built tank, with each dive lasting about 2 seconds. Upon completion of training, mice were injected intraperitoneally with 0.05 mL of the retrograde tracer Fluorogold to fluorescently label all nAmb neurons. On the day of testing, the experimental group of dive-trained mice (n=9) underwent 15 minutes of consecutive voluntary dives, about 40 dives each. After diving, mice recovered in a warm cage for 30-40 minutes and were then anesthetized and transcardially perfused with fixative. For controls, the same procedure was performed on another group of dive-trained mice (n=8), except that the dive chamber only contained enough water to wet their paws (no diving occurred). After tissue fixation, the medulla was extracted, coronally sectioned, and processed for RNA fluorescence in situ hybridization (RNA FISH) to visualize transcripts for Npy2r and the immediate early gene, Fos. There was a significant increase in the proportion of Fluorogold+ Npy2r+ nAmb neurons that expressed Fos after diving compared to controls (45 ± 6% vs. 16 ± 4% triple positive, n=7 & 6, P=0.0047, Mann Whitney test), indicating that Npy2rnAmb neurons are activated during voluntary diving. These data raise the possibility that Npy2rnAmb neurons are CVNs that mediate the diving reflex. The authors gratefully acknowledge Maisie Crook for her technical assistance throughout this project. Funding was provided by the UVA Biology Schwager Summer Research Scholarship and UVA Harrison Undergraduate Research Award to V.G.; National Institutes for Health (NIH) F31 HL158187 to T.C.C.; NIH R01 HL148004 to S.B.G.A.; and an American Diabetes Association Pathway to Stop Diabetes Initiator Award 1-18-INI-14 and NIH R01 HL153916 to J.N.C. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.