582-P: Disambiguating the Nucleus Ambiguous with Single-Cell Transcriptomics

The nucleus ambiguous (nAmb), a hindbrain region which degenerates in diabetes models, is the major parasympathetic input to the heart but also innervates the larynx and esophagus to control swallowing. These disparate functional roles are likely mediated by distinct nAmb neuron subtypes. Yet, the molecular identities of these subtypes are mostly unknown, limiting access to them for functional studies. Therefore, we classified mouse nAmb neurons using single-nuclei RNA-Seq (sNuc-Seq). First, we labeled nAmb neuron nuclei by injecting an adeno-associated viral (AAV) vector which Cre-dependently expresses a nuclear-localized mCherry protein into the nAmb of adult male Chat-IRES-Cre mice, in which all nAmb neurons (and few nearby ones) express Cre recombinase. We later dissected the nAmb, isolated its cell nuclei, and sorted the H2b-mCherry+ ones singly into microplate wells with fluorescence assisted cell sorting (FACS). We then used SmartSeq2 to generate cDNA sequencing libraries from the single-nuclei poly-adenylated RNA samples and deeply sequenced them to near-saturating depths for gene detection. Clustering the samples based on expression of their highly variable genes revealed six molecularly-distinct neuron subtypes, four of which localized to nAmb based on in situ detection of their marker transcripts. Comparing their gene expression revealed candidate transcriptional markers for each neuron subtype (Crhr2; Uts2; Vipr2; and Tbx3). In light of previous studies, three nAmb populations (Crhr2 neurons, Uts2 neurons, and Vipr2 neurons) are likely motor neurons innervating the larynx and esophagus, as they expressed high levels of CGRP (Calca) but little to no PACAP (Adcyap1). The opposite was true for the fourth subtype, Tbx3 neurons, raising the possibility that they are the cardiovagal nAmb neurons. Additional studies are underway to identify (1) tissues innervated by each marker-defined nAmb neuron type and (2) their role in controlling heart rate, laryngeal or esophageal muscle tone. Disclosure T. Coverdell: None. R. Ivison: None. J. Tao: None. J. Campbell: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-18-INI-14 to J.C.)