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/ Vglut2f/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 R26DTA/wt;Trpv1-Cre (n = 9) mice develop heat hyperalgesia but transgenes to a lesser extent. (C-D) Control (n = 7), but not Vglut2f/f;Trpv1-Cre mice (n = 8), develop heat hyperalgesia following PSNL. (E-F) Both control (n = 11) and R26DTA/wt;Trpv1-Cre (n = 9) mice develop cold hyperalgesia. (G-H) Similarly, both controls and Vglut2f/f;Trpv1-Cre mice develop cold hyperalgesia. Baseline (white) = response before PSNL, after injury (grey) = average response after PSNL. Circles denotes controls, squares display R26DTA/wt;Trpv1-Cre and triangles show Vglut2f/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 R26DTA/wt;Trpv1-Cre (n = 9) mice develop punctuate hyperalgesia but transgenes to a lesser extent. (C-D) Controls but not Vglut2f/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 R26DTA/wt;Trpv1-Cre and triangles show Vglut2f/f;Trpv1-Cre mice. **p<0.01, *** p<0.001, ns p>0.05, Mann-Whitney two-tailed test.