Identification of Trigeminal Sensory Neuronal Types Innervating Masseter Muscle.

Understanding masseter muscle (MM) innervation is critical for the study of cell-specific mechanisms of pain induced by temporomandibular disorder or after facial surgery. Here, we identified trigeminal (TG) sensory neuronal subtypes (MM TG neurons) innervating masseter muscle fibers, masseteric fascia, tendons, and adjusted tissues. A combination of patch clamp electrophysiology and immunohistochemistry on TG neurons backtraced from reporter mouse MM found 9 distinct subtypes of MM TG neurons. Of these neurons, 24% belonged to non-peptidergic IB-4+/TRPA1- or IB-4+/TRPA1+ groups, while two TRPV1+ small-sized neuronal groups were classified as peptidergic/CGRP+ One small-sized CGRP+ neuronal group had a unique electrophysiological profile and were recorded from Nav1.8- or trkC+ neurons. The remaining CGRP+ neurons were medium-sized, could be divided into Nav1.8-/trkC- and Nav1.8low/trkC+ clusters, and showed large 5HT-induced current. The final two MM TG neuronal groups were trkC+ and had no Nav1.8 and CGRP. Among MM TG neurons, TRPV1+/CGRP- (somatostatin+), tyrosine hydroxylase+ (C-LTMR), TRPM8+, MrgprA3+ or trkB+ (Aδ-LTMR) subtypes have not been detected. Masseteric muscle fibers, tendons and masseteric fascia in mice and the common marmoset, a new world monkey, were exclusively innervated by either CGRP+/NFH+ or CGRP-/NFH+ medium-to-large neurons, which we found using a Nav1.8-YFP reporter, and labeling with CGRP, TRPV1, neurofilament heavy chain (NFH) and pgp9.5 antibodies. These nerves were mainly distributed in tendon and at junctions of deep-middle-superficial parts of MM. Overall, the data presented here demonstrates that MM is innervated by a distinct subset of TG neurons, which have unique characteristics and innervation patterns.Significance StatementIdentification of sensory neuron subtypes innervating masseter muscle (MM) will enable the study of cell-specific mechanisms of masticatory myofascial pain, including temporomandibular disorder and after restorative surgeries involving MM. Combining back tracing from MM, patch-clamp electrophysiology, and immunohistochemistry with sensory neuronal markers on mouse and nonhuman primate tissues, we identified trigeminal neuronal groups innervating MM (MM TG neurons). MM and adjacent tissues are innervated by 9 distinct types of TG neurons, some of which are substantially different from L3-L5 DRG neurons. However, MM fibers, tendon, muscle-tendon junction, and fascia in mice and common marmosets are exclusively innervated by medium-to-large neurons. These neurofilament heavy chain-positive sensory nerve fibers are mainly distributed in tendon and at junctions between deep, middle, and superficial parts of MM.