Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors

Abstract

Mechanical pain contributes to the morbidity associated with inflammation and trauma, but primary sensory neurons that convey the sensation of acute and persistent mechanical pain have not been identified. Dorsal root ganglion (DRG) neurons transmit sensory information to the spinal cord using the excitatory transmitter glutamate, a process that depends on glutamate transport into synaptic vesicles for regulated exocytotic release. Here we report that a small subset of cells in the DRG expresses the low abundance vesicular glutamate transporter VGLUT3 (also known as SLC17A8). In the dorsal horn of the spinal cord, these afferents project to lamina I and the innermost layer of lamina II, which has previously been implicated in persistent pain caused by injury. Because the different VGLUT isoforms generally have a non-redundant pattern of expression, we used Vglut3 knockout mice to assess the role of VGLUT3(+) primary afferents in the behavioural response to somatosensory input. The loss of VGLUT3 specifically impairs mechanical pain sensation, and in particular the mechanical hypersensitivity to normally innocuous stimuli that accompanies inflammation, nerve injury and trauma. Direct recording from VGLUT3(+) neurons in the DRG further identifies them as a poorly understood population of unmyelinated, low threshold mechanoreceptors (C-LTMRs). The analysis of Vglut3(-/-) mice now indicates a critical role for C-LTMRs in the mechanical hypersensitivity caused by injury.

Figure 1. VGLUT3 is expressed by a unique subset of small- and medium-sized DRG neurons that project to dorsal horn lamina I and the inner part of lamina II

a, Immunostaining shows VGLUT3 in laminae I and innermost II (arrowheads) of the dorsal horn in WT but not VGLUT3 KO mice. Dorsal rhizotomy abolishes VGLUT3 immunoreactivity in ipsilateral laminae I and II. Calibration bars, 50 μm. b, VGLUT3 immunofluorescence (red) in the dorsal horn overlaps to a large extent with interneurons that express PKCγ (green), but little if at all with the binding to IB4 (green). c, Immunoperoxidase labeling of a DRG section from a VGLUT3 EGFP mouse shows the size and distribution of EGFP⁺ cells (size bar = 30 μm). d, In situ hybridization for VGLUT3 labels small- and medium-sized neurons in the trigeminal ganglion (TG) of WT mice. Hybridization with a sense probe confers no detectable signal. e, EGFP⁺ DRG neurons (green) colocalize strongly with peripherin (92%), to a small extent with IB4 (7%) but rarely with N52 and not at all with TRPV1, substance P or CGRP. Arrowheads indicate colocalization. Size bar in d and e, 30 μm.

Figure 2. VGLUT3 KO mice show a selective defect in acute mechanical pain sensation to intense noxious stimuli

a, Using a two-plate preference chamber to assess cold sensitivity, VGLUT3 KO and WT mice show the same preference for the 30°C plate over 24°, 18°or 10°C plates (n=6 mice for both genotypes). b, Using a hot plate to assess heat sensitivity, the latency of VGLUT3 KO mice to exhibit nocifensive behaviors is similar to that of WT littermates at both 48°C and 52°C (n=8). c, Both genotypes show a similar, biphasic response to 2% formalin injected into the hindpaw (n≥15). d, Mechanical thresholds measured using von Frey filaments do not differ between genotypes (n≥10). e, Measuring the acute mechanical pain sensitivity to tail clip, VGLUT3 KO mice show a significantly longer latency to respond than WT (16 ± 2 versus 11 ± 1; n≥17), indicating a reduced sensitivity to acute mechanical pain. p < 0.05, Mann-Whitney U test. f, VGLUT3 KO mice show a higher threshold than WT (10.2 ± 0.5 versus 8.1 ± 0.4; n= 9) to tail withdrawal on compression in the Randall-Selitto test. p<0.05, Mann-Whitney U test. g, Single unit extracellular recordings in the dorsal horn of the lumbar spinal cord show the response of WDR neurons to graded punctate mechanical stimulation of the plantar hindpaw receptive field. The genotypes differ specifically in response to 6 and 9 g. *p<0.05 by Fisher’s LSD post hoc test, n=50–53 cells. h, The same group of WDR neurons in VGLUT3 KO mice respond significantly less than those in WT to pinch (*p=0.026; students t-test), but not brush stimuli (p>0.05; student’s t-test) applied to the receptive field for 3 sec. n=10 mice for both genotypes (g,h). Error bars indicate SEM.

Figure 3. VGLUT3 KO mice show a profound, selective defect in the mechanical hypersensitivity produced by inflammation, nerve injury and trauma

a, VGLUT3 KO mice and WT littermates were injected with carrageenan in the hindpaw. Tested 24 hours later, both genotypes show a significant increase in sensitivity to radiant heat, but only WT mice show a significant decrease in mechanical threshold. (n≥10 mice) b, In the spared nerve injury model of neuropathic pain, WT mice exhibit a significant decrease in mechanical threshold (52%) three days after sciatic nerve section, but KO animals exhibit only a modest reduction (18%). (n≥9 for both genotypes) *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Tukey’s HSD post-hoc analysis for a and b. c, In WT mice, an incision made in the plantar hindpaw produces a robust decrease in mechanical threshold, but KO mice show only a mild reduction for up to 5 days. Response to radiant heat does not differ between genotypes at all times tested (n=6). d, In WT mice, capsaicin injection into the ankle produces a profound decrease in mechanical threshold measured in the hindpaw at 15, 30 and 60 minutes post-injection, whereas KO mice show only a modest decrease (n=7). ***p<0.001, one-way ANOVA with Newman-Keuls post-hoc analysis for c and d. Error bars indicate SEM.

Figure 4. VGLUT3 expression in DRG uniquely identifies C-LTMRs

a, An EGFP⁺ cell is stained by Alexa 555 from an intracellular microelectrode (red) while recording from an ex vivo somatosensory preparation. b, Electrical stimulation of the dorsal cutaneous nerve evokes a high amplitude, overshooting action potential. The first derivative (lower trace) reveals an inflected falling phase (arrow) typical for C-fiber spikes. c, Application of a 0.07 mN von Frey filament to the skin receptive field (duration indicated by bar) elicits a burst of spikes. d, Response to 25 mN force applied with a feedback-controlled stimulator (lower trace) shows adaptation in the continued presence of the force (middle trace). Upper trace shows the instantaneous frequency of spikes. e, Peak discharge rate of individual EGFP⁺ neurons (black lines) to increasing force (5–200 mN) shows a small increase with stronger stimuli (p<0.01 for 5–25 mN versus 200 mN by ANOVA with Bonferroni post hoc correction, n=5–9); red bars indicate mean ± SD. f, Temperature ramps performed with a Peltier device (middle trace) evoke firing in an EGFP⁺ neuron (lower trace) on cooling (left) but not heating (right), Upper trace (left) shows the instantaneous firing rate. g, Low power photomicrograph of central terminals arborizing in lamina IIi; dotted lines indicate the substantia gelatinosa (SG). h, Photomicrograph of different section at higher power shows arborization extending into lamina III (open arrows). i, Camera lucida reconstruction of arborization across four serial 50 μm sections, showing additional input to lamina I (arrow); the ventral border of SG is indicated by a dotted line, and the border between gray and white matter by a solid line. j, Staining of a DRG section for IB4 (blue, middle) shows that the Neurobiotin-labeled cell (red, left) is IB4-negative. Scale bars: 20 μm (a, h, j); 100 μm (g, i). Physiological calibration: 20 mV and 5 msec (b), 200 msec (c).