SPARC: The Structure and Location of Vagal Nociceptive Nerve Fibers in the Mouse Trachea

Abstract

Vagal afferent innervation of the airways and lungs comprises of afferent fibers from vagal nodose neurons (developmentally derived from epibranchial placodes) and vagal jugular neurons (derived from neural crest). Here we evaluated the structure and the location in tracheal wall of afferent nerve fibers innervating the trachea focusing on the nociceptive TRPV1‐positive fibers (putative C‐fibers). We used genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of the trachea were immunostained for fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (in the vagal system the marker of TRPV1‐positive fibers derived from neural crest) we observed dense innervation of the trachea. In the anterolateral tracheal wall the TRPV1+Tac1+ fibers were located beneath the epithelium in the mucosa above tracheal ligaments (which connect tracheal rings) running transversally (i.e. in parallel with tracheal rings). These fibers ramified richly and repetitively. In the dorsal (membranous) tracheal wall the TRPV1+Tac1+ fibers were also located beneath the epithelium in the mucosa above tracheal smooth muscle, running craniocaudally and often branching. These fibers occasionally penetrated through the tracheal smooth muscle. Some TRPV1+Tac1+ fibers were also observed in the epithelium in the sublaryngeal region of the trachea. In the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP or AAV9‐FLEX‐tdTomato virus vector into vagal ganglia (N=4), in which only the vagal TRPV1‐positive fibers express GFP or tdTomato, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with expected lower density and patchy distribution. Similarly, in the Tac1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP vector into vagal ganglia (N=3) in which only the vagal Tac1‐positive fibers express GFP had the same pattern. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of vagal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the trachea. However, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEXEGFP vector into vagal ganglia (N=2) in which GFP is selectively expressed in vagal P2X2− positive fibers, the P2X2+ fibers had distinct distribution from TRPV1+Tac1+ and often located in the tracheal smooth muscle and tracheal epithelium. Our data indicate that vagal afferent nociceptive (TRPV1+) nerve fibers (putative C‐fibers) innervating the mouse trachea originate almost exclusively from jugular ganglion neurons. In contrast, vagal nodose ganglia project only TRPV1‐fibers into the trachea. Our results are in agreement with electrophysiological studies in other species.

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