VGLUT2 controls heat and punctuate hyperalgesia associated with nerve injury via TRPV1-Cre primary afferents

Abstract

Nerve injury induces a state of prolonged thermal and mechanical hypersensitivity in the innervated area, causing distress in affected individuals. Nerve injury-induced hypersensitivity is partially due to increased activity and thereby sustained release of neurotransmitters from the injured fibers. Glutamate, a prominent neurotransmitter in primary afferents, plays a major role in development of hypersensitivity. Glutamate is packed in vesicles by vesicular glutamate transporters (VGLUTs) to enable controlled release upon depolarization. While a role for peripheral VGLUTs in nerve injury-induced pain is established, their contribution in specific peripheral neuronal populations is unresolved. We investigated the role of VGLUT2, expressed by transient receptor potential vanilloid (TRPV1) fibers, in nerve injury-induced hypersensitivity. Our data shows that removal of Vglut2 from Trpv1-Cre neurons using transgenic mice abolished both heat and punctuate hyperalgesia associated with nerve injury. In contrast, the development of cold hypersensitivity after nerve injury was unaltered. Here, we show that, VGLUT2-mediated glutamatergic transmission from Trpv1-Cre neurons selectively mediates heat and mechanical hypersensitivity associated with nerve injury. Our data clarifies the role of the Trpv1-Cre population and the dependence of VGLUT2-mediated glutamatergic transmission in nerve injury-induced hyperalgesia.

Figure 1. Trpv1-Cre show prominent activity at E15.5.

The onset of Trpv1-Cre expression was determined using the reporter line tdTomato. (A-D) Whole-embryo micrographs of Trpv1-Cre;tdTomato at the different developmental stages; E12.5, E13.5 and E15.5. No tdTomato expression was observed at E12.5, a few DRG cells expressed Trpv1-Cre at E13.5 and profound expression of tdTomato was observed at E15.5 in DRGs. Scale bars = 2000μm (A-B), 2500μm (C), 500μm (D). Auto fluorescence was observed in abdominal tissue (see S1 Fig. for details).

Figure 2. The Trpv1-Cre population co-express Vglut2 to a great extent.

(A-B) The Trpv1-Cre population constitutes 39.2% of the primary afferent neurons (n = 3/genotype, 1 DRG per animal, 77 sections/ Vglut2 ^f/f;Trpv1-Cre mice and 95 sections/ctrl). (C-D) Trpv1-Cre neurons overlap greatly with Vglut2 mRNA, 65.1±2.2% of Trpv1-Cre neurons express Vglut2 which corresponds to 73.8±2.1% of the Vglut2 population (n = 3, 35 number of sections). Scale bar = 33 μm.

Figure 3. Glutamate mediates heat, but not cold, hyperalgesia associated with peripheral nerve injury via the Trpv1-Cre population.

(A-B) Both control (n = 11) and R26 ^DTA/wt;Trpv1-Cre (n = 9) mice develop heat hyperalgesia but transgenes to a lesser extent. (C-D) Control (n = 7), but not Vglut2 ^f/f;Trpv1-Cre mice (n = 8), develop heat hyperalgesia following PSNL. (E-F) Both control (n = 11) and R26 ^DTA/wt;Trpv1-Cre (n = 9) mice develop cold hyperalgesia. (G-H) Similarly, both controls and Vglut2 ^f/f;Trpv1-Cre mice develop cold hyperalgesia. Baseline (white) = response before PSNL, after injury (grey) = average response after PSNL. Circles denotes controls, squares display R26 ^DTA/wt;Trpv1-Cre and triangles show Vglut2 ^f/f;Trpv1-Cre mice. * p<0.05, **p<0.01, *** p<0.001, ns p>0.05, Mann-Whitney two-tailed test.

Figure 4. Glutamate mediates punctuate hyperalgesia associated with peripheral nerve injury via the Trpv1-Cre population.

(A-B) Both control (n = 11) and R26 ^DTA/wt;Trpv1-Cre (n = 9) mice develop punctuate hyperalgesia but transgenes to a lesser extent. (C-D) Controls but not Vglut2 ^f/f;Trpv1-Cre mice develop punctuate hyperalgesia. Baseline (white) = response before PSNL, after injury (grey) = average response after PSNL. (E) Single-cell PCR analysis showed that out of 68 cells analyzed, 13 was positive for only MrgprD mRNA, 23 only for Trpv1-Cre mRNA, 11 expressed both markers, and 19 cells neither of the two markers. Cre-405 bp, MrgprD-271 bp. (F) The overlapping population corresponds to 50% of the MrgprD population and 32% of Trpv1-Cre population. Circles denotes controls, squares display R26 ^DTA/wt;Trpv1-Cre and triangles show Vglut2 ^f/f;Trpv1-Cre mice. **p<0.01, *** p<0.001, ns p>0.05, Mann-Whitney two-tailed test.