Genetic encoding of an esophageal motor circuit

Abstract

Motor control of the striated esophagus originates in the nucleus ambiguus (nAmb), a vagal motor nucleus that also contains upper airway motor neurons and parasympathetic preganglionic neurons for the heart and lungs. We disambiguate nAmb neurons based on their genome-wide expression profiles, efferent circuitry, and ability to control esophageal muscles. Our single-cell RNA sequencing analysis predicts three molecularly distinct nAmb neuron subtypes and annotates them by subtype-specific marker genes: Crhr2, Vipr2, and Adcyap1. Mapping the axon projections of the nAmb neuron subtypes reveals that Crhr2^nAmb neurons innervate the esophagus, raising the possibility that they control esophageal muscle function. Accordingly, focal optogenetic stimulation of cholinergic Crhr2⁺ fibers in the esophagus results in contractions. Activating Crhr2^nAmb neurons has no effect on heart rate, a key parasympathetic function of the nAmb, whereas activating all of the nAmb neurons robustly suppresses heart rate. Together, these results reveal a genetically defined circuit for motor control of the esophagus.

Keywords: CP: Neuroscience; esophagus; larynx; medulla; motor neuron; nucleus ambiguus; parasympathetic; pharynx; single-cell RNA-seq; swallowing; vagus nerve.

Figure 1.. Molecular identification of nucleus ambiguus (nAmb) neuron subtypes

(A) Schematic of single-nuclei RNA sequencing (sNuc-seq) workflow. (B) Relatedness of neuron clusters, number of cells per cluster, and number of genes detected per cell. (C) Dot plot of nAmb regional genes (Chat, Phox2b, Isl1) and cluster marker genes. (D) UMAP (uniform manifold approximation and projection) visualization of clustered data. (E) Single-cell expression heatmap of top marker genes for each cluster. (F) Cluster-level average expression of transcription factor genes. (G) Cluster-level average expression of receptor genes. (H) Violin plots of Calca expression. (I) Fluorescence in situ hybridization of Calca, Chat, and Crhr2 mRNA in the “compact” (rostral) nAmb. Scale bar, 20 μm. (J) Fluorescence in situ hybridization of Calca, Chat, and Vipr2 mRNA in the “semi-compact” (caudal) nAmb. Scale bar, 20 μm. (K) Percentage of Calca-expressing Crhr2^nAmb neurons and Vipr2^nAmb neurons in FISH and sNuc-seq studies. nAmb neurons identified in FISH by systemic FluoroGold labeling and in sNuc-seq by Isl1 expression. The numbers of Calca⁺ cells as a fraction of the total cells in that group are shown in parentheses.

Figure 2.. Vipr2 and Crhr2 transcripts mark anatomically distinct subtypes of nAmb neurons

(A) Fluorescence in situ hybridization (FISH) of Chat, Crhr2, and Vipr2 transcripts in the compact, semi-compact, and loose nAmb, co-localized with systemically administered FluoroGold (n = 3 mice). Scale bar, 20 μm. (B) Venn diagram from FISH data showing lack of cellular colocalization between Vipr2 transcripts and Crhr2 transcripts. (C) Co-expression of Vipr2 and Crhr2 genes in neurons of the 3 nAmb neuron clusters in the sNuc-seq data. (D) Co-expression of Adcyap1 and Crhr2 genes in neurons of the 3 nAmb neuron clusters in the sNuc-seq data. (E) Co-expression of Adcyap1 and Vipr2 genes in neurons of the 3 nAmb neuron clusters in the sNuc-seq data. (F) Venn diagram of Crhr2, Vipr2, and Adcyap1 expression among neurons in the 3 nAmb neuron clusters, as detected by sNuc-seq. Expression counts of >1 were considered positive expression. (G) Venn diagram of Crhr2, Vipr2, and Adcyap1 mRNA as detected by RNA FISH (n = 3 mice). nAmb neurons were labeled by systemic injection of the retrograde tracer, FluoroGold. (H) Rostral to caudal distribution of Chat⁺/Vipr2⁺ and Chat⁺/Crhr2⁺ neurons throughout the extent of the nAmb (n = 3 mice). DVC, dorsal vagal complex; Sp5, spinal trigeminal nucleus; 12N, hypoglossal nucleus; LRN, lateral reticular nucleus; IO, inferior olivary nucleus; py, pyramidal tract. Compact nAmb: −6.47 mm through −6.75 mm from bregma, Intermediate nAmb: −6.83 mm through −7.10 mm from bregma, loose nAmb: −7.19 mm through −7.46 mm from bregma.

Figure 3.. Crhr2^nAmb neurons and Vipr2^nAmb neurons separately innervate esophagus and pharynx

(A) Schematic showing injection of AAV9-FLEX-PLAP into the nAmb of Chat-Cre (n = 6 mice), Vipr2-Cre (n = 5 mice), Crhr2-Cre (n = 5 mice), and wild-type (WT) mice (n = 3 mice). Portions of this figure were created with BioRender.com. (B) PLAP-stained axons in the upper throat after injection in the nAmb of Chat-Cre mice. (C) PLAP-stained axons in the cervical esophagus after injections in the nAmb of Chat-Cre, Vipr2-Cre, Crhr2-Cre, and the DMV of Chat-Cre mice. (D and E) PLAP-stained axons in the lower pharynx and upper esophagus after injection into the nAmb of Crhr2-Cre mice (D) and Vipr2-Cre (E) mice. (F) Low and high magnification images of PLAP-stained axon terminals in the mid-esophagus after injection into the nAmb of Crhr2-Cre mice. (G) High-magnification image of PLAP-stained axons in the pharyngeal muscles after injection into the nAmb of Vipr2-Cre mice. (H) PLAP-stained axon terminals in the larynx after injection into the nAmb of Chat-Cre, Vipr2-Cre, and Crhr2-Cre mice.

Figure 4.. Crhr2^nAmb neurons selectively control esophageal muscles

(A) Intersectional optogenetics approach for activating all nAmb neurons, Vipr2^nAmb neurons, and Crhr2^nAmb neurons. CaTCh-EYFP expression is dependent on the presence of both Cre and Flp recombinases. (B) Schematic of experimental setup for measuring esophageal function. The mouse is anesthetized and mechanically ventilated to expose the esophagus. A balloon is inserted in the esophagus to measure esophageal pressure and a fiber optic is placed above the esophagus for focal stimulation. (C) Blinded scoring of video data from optogenetic stimulation of esophagus in different genotype groups. Values represent the number of visible muscle contractions divided by the number of laser flashes observed during the video. (n = 3–4 mice per genotype, 2 mice for negative control). (D) Representative pressure recordings from balloon pressure transducer in esophagus during photostimulation of Crhr2-Cre:Chat-Flp:CaTCh mouse via optical fiber either implanted in nAmb (“central”) or placed over the esophagus. Esophageal response before and after administration of pancuronium, a competitive inhibitor of nicotinic acetylcholine receptors at the neuromuscular junction. (E) Heart rate before and after photostimulation of Chat-Cre:Phox2b-Flp:CaTCh (n = 6 mice), Vipr2-Cre:Chat-Flp:CaTCh (n = 6 mice), and Crhr2-Cre:Chat-Flp:CaTCh (n = 4 mice) mice via nAmb-implanted optical fiber (***p < 0.001 for baseline versus stimulation by Sidak’s multiple comparison test; F = [2,26] = 8.538; p = 0.0014).