Identification of Trigeminal Sensory Neuronal Types Innervating Masseter Muscle

Recordings from non-peptidergic IB4⁺ MM TG neurons. A, WGA-488⁺/IB4-555⁺ (marked with blue arrows), but not WGA-488⁺/IB4^– (marked with yellow arrows) were selected for recording non-peptidergic TG neurons innervating MM. B, Stimulus waveform (1 nA, 0.5 ms) indicated below trace generated a single AP in a WGA⁺/IB4⁺ TG neuron belonging to the S2 group (Table 1). AP width is duration at base, dB. AHP80 is the time required for the AHP (measured in mV) to decay by 80% to a RMP level. Characteristic AP deflection is indicated by black arrow. C, AP from a WGA⁺/IB4⁺ TG neuron belonging to the group S1 (Table 1). Distinctive AP feature, bow, is indicated by black arrow. Distance from RMP to a lowest point of AP, AHP peak, is measured as indicated with the green arrow. D, Current-evoked AP train from a WGA⁺/IB4⁺ TG neuron belonging to the S2 group. Current waveforms are below trace and are applied by steps from 50 to 550 pA with 100-pA increment. Depictured AP train is evoked by a 150-pA step lasting 1 s. E, Currents were generated from a WGA⁺/IB4⁺ TG neurons belonging to the S1 group by the indicated waveforms found below traces. The decay constant τ was derived from standard single exponential fits between points indicated by arrows for the outward portion of the final current trace (+20 mV).

Recordings from TRPV1⁺ MM TG neurons. A, In MM TG neurons belonging to the group S3, a single AP in WGA⁺/TRPV1⁺ neuron is generated by current pulse (protocol-1), which has characteristic hump on down stroke phase of AP. B, In MM TG neurons belonging to the group S4, a single AP in WGA⁺/TRPV1⁺ neuron has distinctive deflection on down stroke phase of AP. C, Current-evoked AP train from a WGA⁺/TRPV1⁺ TG neuron belonging to the group S4. Depictured AP train is evoked by a 50-pA step lasting 1 s. D, In WGA⁺/TRPV1⁺ TG neurons belonging to the group S3, currents were generated by waveforms from Figure 1E. E, In WGA⁺/TRPV1⁺ TG neurons belonging to the group S4, currents generated by waveforms from Figure 1E do not have inward component.

Recordings from CGRP⁺ MM TG neurons. A, Responsiveness of IB-4⁺, Nav1.8⁺, and Nav1.8^– MM TG neurons to MO (25 μm) and Ment (100 μm) measured with Ca²⁺ imaging. Each bar shows information on numbers of tested and responsive neurons. B, Representative AP from a MM TG CGRP⁺ neuron belonging to the S5 group. AP straight in S5 neurons is indicated with a blue arrow. Red dashed line demonstrates the AP shape. C, Comparison of AP shapes generated in MM TG CGRP⁺ neurons belonging to the M1 (red) and M2 (blue) groups. AP deflection in M1 neurons is indicated with a red arrow. M2 neuron’s AP does not display any deflection during the falling phase of AP. D, 5HT (30 μm)-induced current in MM TG CGRP⁺ S5 and M1 group neurons. The drug was applied for 5 s, and duration of application is indicated by a horizontal gray bar. E, Typical current (I) produced from MM TG CGRP⁺ neurons belonging to the S5 or M1 group. F, Typical I produced from MM TG CGRP⁺ M2 group neurons. Characteristic spike is indicated by green arrow. Names of neuronal groups are specified above traces. The magnitude (vertical) and time (horizontal) scale bars are presented for the B–F panels.

Recordings from Nav1.8^– and trkC⁺ MM TG neurons. A, Representative AP from a MM TG trkC⁺ neuron belonging to the M3 group. B, Representative AP from a MM TG trkC⁺ neuron belonging to the M4 group. C, Typical I produced from the M3 group neurons recorded either from a MM TG trkC⁺ or Nav1.8^– neuron. Characteristic spike is indicated by green arrow. D, Typical I produced from the M3 group neurons recorded from a MM TG trkC⁺ or Nav1.8^– neuron. Names of neuronal groups are specified above traces on the A–D panels. The magnitude (vertical) and time (horizontal) scale bars are presented for the A–D panels.

Expression of CGRP and TRPV1 in MM TG neurons. A, A’, Expression of CGRP (red) neurons in a TG section containing MM TG neurons (WGA; green). Objective is 10×. B, B’, Expression of CGRP (red) and trpV1 (blue) neurons in a TG section containing MM TG neurons (WGA; green). Objective is 20×. MM TG neurons lacking marker are marked with cyan arrows on the A–C panels. MM TG neurons containing both WGA and CGRP or trpV1 are marked with yellow arrows. MM TG neurons containing WGA, but not CGRP or trpV1, are marked with cyan arrows. MM TG neurons containing both CGRP and trpV1 are marked with white arrows on the B, B’ panels. TrpV1 neurons lacking CGRP and WGA are marked with brown arrows on the B, B’ panels. White horizontal bar shows 20-μm scale for each panel.

Expression of MrgprD in MM TG neurons. A, A’, Expression of MrgprD (mrgD; red) neurons in a TG section containing MM TG neurons (WGA; green). Objective is 20×. B, B’, Expression of MrgprD-cre/tdTomato (mrgD-cre; red) neurons in a TG section from mice back-traced from MM with WGA-488 (WGA; green). Objective is 20×. MM TG neurons containing both mrgD or mrgD-cre and WGA are marked with yellow arrows. MM TG neurons containing WGA, but not mrgD or mrgD-cre or trpV1, are marked with cyan arrows. White horizontal bar shows 20-μm scale for each panel.

Expression of calbindin-D28 and parvalbumin mechanoreceptor markers in MM TG neurons. A, A’, Expression of calbindin D28 (Calb; red) in a TG section containing MM TG neurons (WGA; green). B, B’, Expression of parvalbumin (PV; red) in a TG section containing MM TG neurons (WGA; green). MM TG neurons lacking Calb or PV are shown with cyan arrows. MM TG neurons containing Calb or PV are shown with yellow arrows. For all panels, objective is 20×. White horizontal bar shows 20-μm scale for each panel.

Expression of trkC and TH mechanoreceptor markers in MM TG neurons. A, A’, Expression of trkC (trkC; red) in a TG section containing MM TG neurons (WGA; green). B, B’, Expression of TH (red) in a TG section containing MM TG neurons (WGA; green). MM TG neurons lacking a sensory neuronal marker are shown with cyan arrows on the A, B panels. MM TG neurons containing a sensory neuronal marker are shown with yellow arrows on the A, A’ panels. For all panels, objective is 20×. White horizontal bar shows 20-μm scale for each panel. C, Percentages of WGA⁺ TG neurons labeled with a variety of indicated sensory neuronal markers. Cell counting is from four animals, three to five sections each.

Location of CGRP⁺ and NFH⁺ TG sensory neuronal afferent fibers in MM. A, A’, Expression of CGRP (green) and NFH (blue) fibers in a main trunk of the masseteric nerve located near MM. Objective is 40×. CGRP⁺/NFH^– nerves are marked with yellow, CGRP⁺/NFH⁺ with cyan, and CGRP^–/NFH⁺ with white arrows. Red arrow shows MM fibers. B, Expression of CGRP (red) and NFH (green) nerves in muscle fibers and MM tendon. Objective is 10×. Red arrow shows muscle fibers and blue points on tendons. Nerve bundles are marked with yellow arrows and individual nerves with cyan arrows. C, Expression of CGRP (red) and NFH (green) nerves in MM. Objective is 2×. A red arrow shows muscle fibers. C’, High magnification (objective is 20×) of the image within a white rectangle on the panel C. On the panels C, C’, nerve bundle is marked with a yellow arrow and individual nerves with cyan arrows. D, Expression of Nav1.8-Ai32-YFP (green), CGRP (red), and NFH (blue) nerves in MM fascia area, which is indicated by white double arrowed line. Objective is 10×. White horizontal bar shows 20- or 100-μm scale for each panel.

MM TG neuron types innervating mouse MM fibers and tendons. A, A’, Expression of CGRP (red) and NFH (green) nerves in MM tendon. Objective is 20×. CGRP⁺/NFH⁺ nerves are marked with cyan and CGRP^–/NFH⁺ with white arrows. B, B’, Expression of CGRP (red) and NFH (green) nerves in masseteric muscle fibers. Objective is 10×. CGRP⁺/NFH⁺ nerves are marked with cyan and CGRP^–/NFH⁺ with white arrows.

MM TG neuron types innervating MM in mice and common marmosets. A, A’, Expression of pgp9.5 (red) and NFH (green) in male mouse masseteric muscle fibers. Objective is 10×. Pgp9.5⁺/NFH⁺ nerves are shown with cyan arrows. B, Expression of pgp9.5 (red) and NFH (blue) in male mouse MM tendon. Objective is 10×. Muscle fibers are shown by a red arrow, and tendons by a green arrow. Pgp9.5⁺/NFH⁺ nerves are shown with cyan arrows. C, Expression of pgp9.5 (red) and NFH (blue) in male mouse masseteric fascia. Objective is 20×. Muscle fibers are shown by a red arrow, and fascia layer by a double-headed white arrow. Pgp9.5⁺/NFH⁺ nerves are shown with cyan arrows, and a Pgp9.5^–/NFH⁺ nerve with a yellow arrow. D, D’, Expression of pgp9.5 (green) and NFH (red) in 11-year-old male marmoset masseteric muscle fibers. Objective is 10×. Pgp9.5⁺/NFH⁺ nerves are shown with cyan arrows, and Pgp9.5⁺/NFH^– nerves with white arrows. White horizontal bar shows 20-μm scale for each panel.