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 (TMDs) or after facial surgery. Here, we identified trigeminal (TG) sensory neuronal subtypes (MM TG neurons) innervating MM fibers, masseteric fascia, tendons, and adjusted tissues. A combination of patch clamp electrophysiology and immunohistochemistry (IHC) on TG neurons back-traced from reporter mouse MM found nine distinct subtypes of MM TG neurons. Of these neurons, 24% belonged to non-peptidergic IB-4¹/TRPA1^– or IB-4¹/TRPA1¹ groups, while two TRPV1¹ small-sized neuronal groups were classified as peptidergic/CGRP¹. One small-sized CGRP¹ neuronal group had a unique electrophysiological profile and were recorded from Nav1.8^– or trkC¹ neurons. The remaining CGRP¹ neurons were medium-sized, could be divided into Nav1.8^–/trkC^– and Nav1.8^low/trkC¹ clusters, and showed large 5HT-induced current. The final two MM TG neuronal groups were trkC¹ and had no Nav1.8 and CGRP. Among MM TG neurons, TRPV1¹/CGRP^– (somatostatin¹), tyrosine hydroxylase (TH)¹ (C-LTMR), TRPM8¹, MrgprA3¹, or trkB¹ (Ad-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 CGRP¹/ NFH¹ or CGRP^–/NFH¹ 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.