SPARC: The structure of vagal nociceptive nerve fibers in the mouse esophagus

Abstract

The esophagus is innervated by nociceptive sensory afferent nerve fibers originating from the vagus nerve. The nature of the vagal nociceptive fiber innervation, specifically which esophageal layers, however, remains unknown. We hypothesized that the vagal nociceptive TRPV1+ afferent fibers innervate the esophageal mucosa. We evaluated our hypothesis by using genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of separated esophageal mucosa and muscle were immunostained for appropriate fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/RC: FLTG mice(N=8) in which TRPV1‐expressing cells express tdTomato the TRPV1+ fibers with varicose appearance densely innervated both the mucosa and the muscle. In the mucosa the TRPV1+ fibers are located just beneath the epithelium, run in parallel registers of two or more fibers, have craniocaudal orientation and branch repeatedly. In the muscle the TRPV1+ fibers form a dense network in the myenteric plexus innervating virtually all myenteric ganglia forming intraganglionic varicose endings (IGVEs). Many TRPV1+ fibers connecting IGVEs run in parallel to the fibers of the outer and inner striated muscle. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (the marker of esophageal nociceptors derived from neural crest) the esophageal innervation by TRPV1+/Tac1+ fibers mimicked the dense innervation by TRPV1+ fibers. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice(N=4) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of esophageal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the esophagus. Consistent with this finding, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=2) in which GFP is selectively expressed in vagal P2X2+ fibers, the nodose TRPV1‐negative tension mechanosensor nerve terminals (intraganglionic laminar endings) were visualized in the esophageal muscle. Finally, in the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP virus into vagal ganglia(N=3), in which only the vagal TRPV1+ fibers express GFP, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with lower density and patchy distribution. Our data indicate that vagal nociceptive TRPV1+ fibers innervate both the esophageal mucosa and myenteric ganglia and originate from neural crest‐derived jugular portion of the vagal jugular‐nodose ganglia complex.