A. Schematic of TRPV2 KO strategy. From top: TRPV2 genomic locus, targeting construct, targeted TRPV2 allele, and Cre-recombined TRPV2 allele. Gray bars represent exons. Black bar represents Southern probe. B, BstEII; K, KpnI; S, SpeI; NEO, neomycin resistance cassette; tk, herpes simplex virus thymidine kinase; UHA, upstream homology arm; DHA downstream homology arm; LoxP, Cre recombination site; frt, Flippase recombination site. B. Southern blot of tail genomic DNA from mice of the indicated genotypes. WT band, 13 kb, disrupted band, 6.7 kb. C. Immunoblot of lysates from WT and TRPV2 KO trigeminal ganglia probed with antibodies against TRPV2, TRPV1, and β-actin. Upper band in TRPV2 blot likely indicates glycosylated form. D, E. Immunofluorescence staining of WT (D) and TRPV2 KO (E) L5 dorsal root ganglia with TRPV2 antibody. Representative cells exhibiting strong, uniform staining (filled arrowhead), peripheral staining (open arrowhead) and weak uniform staining (arrow) are shown. F. Prevalence of immunostaining for TRPV1, CGRP, and neurofilament (NF200) in L5 DRGs from WT (filled bars) vs. TRPV2 KO (open bars) mice. Data shown represent mean ± SEM from n = 3 to 7 mice per genotype, with >1000 cells scored per mouse. G. Size histogram of L5 DRG neurons obtained from WT mice immunostained for TRPV2. In both histograms, size distribution of all cells is indicated by gray bars. Left, strong uniformly stained cells (black bars) and peripherally stained cells (red bars) are indicated. Right, weak uniformly stained cells (black bars) are indicated.

A–C. TRPV2 immunoreactivity (red) and F4/80-immunoreactivity (green) in the hairy skin of WT mice. Open arrowheads indicate doubly immunoreactive structures, filled arrowheads indicate TRPV2 positive but F4/80 negative structures. Arrow indicates network of terminals in a hair follicle (hf). e, epidermis; d, dermis; sc, stratum corneum. Dashed line in panel 2C denotes dermal-epidermal junction. D. TRPV2 immunoreactivity in palisade endings (arrowheads) in a hair follicle. E. TRPV2 antibody staining of hairy skin from TRPV2 KO mouse. Dashed lines denote dermal-epidermal junction. F–K. TRPV2 double-labeling with anti-GFP in the glabrous (F–H) and hairy (I–K) skin epidermis of an mrgD-GFP mouse. Panels show TRPV2 staining (red, F,I), GFP staining (green, G,J) and merged image (H,K). Arrowheads indicate double-labeled epidermal fibers. L–N. Specific TRPV2 immunoreactivity in L5 spinal cord dorsal horn overlaps with both IB4-staining afferent projections (M) and PKCγ-immunoreactive spinal cord neurons (N). O. TRPV2 antibody staining of TRPV2 KO spinal cord dorsal horn. Genotype and labels used are indicated in each panel. Scale bars are 20 microns in panels A–K and 125 microns in panels L–O.

A. Number of eye wipes evoked over a 5 min period by application of capsaicin (0.5 mM) to the cornea. Mean ± SEM (n = 10 mice per genotype) are indicated for WT (filled bars) and TRPV2 null (open bars) mice. B. Time spent licking or shaking the hind paw following intraplantar injection of formalin (1.2%). Behavior was scored over the two indicated time periods, and is represented as mean ± SEM (WT, n = 11; KO, n = 12). C. Mechanical sensitivity, assessed in the von Frey filament assay in WT (filled squares) and TRPV2 null (open circles) mice. Mechanical force exerted by the filaments is indicated on the x-axis and the percent of challenges resulting in a response on the y-axis. Data shown represent mean + or − SEM (n = 11 per genotype). D, E. Acute thermal sensitivity measured in WT (filled bars) vs. TRPV2 KO (open bars) mice, using the tail immersion (D), hot plate (E) and radiant paw heating (F) assays. Data shown represent mean ± SEM from n = 11 to 12 mice per genotype. In panel F, lamp intensity is in arbitrary units. In the formalin assay, mice were on the F1 hybrid background. In all other assays, mice were on the mixed background.

A, B. Thermal and mechanical hyperalgesia produced by intraplantar injection of complete Freund's adjuvant (CFA, 20 μl). Thermal sensitivity (A) was evaluated using the radiant paw heating assay in both the injected paw (ipsilateral WT, filled triangles, solid lines; ipsilateral KO, open circles, dashed lines) and in the contralateral paw (contralateral WT, filled squares, solid lines; contralateral KO, open diamonds, dashed lines). These symbols apply to all four panels (A–D). Data represent mean ± SEM measured at baseline or at the indicated times after CFA injection. n = 10 mice per genotype. Mechanical sensitivity (B) was evaluated in WT and TRPV2 KO mice at the indicated time points using the von Frey filament assay. Data shown represent mean ± 95% confidence interval of the force evoking a response on 50% of the trials. C, D. Thermal and mechanical hyperalgesia produced by L5 spinal nerve ligation and transection. Thermal and mechanical sensitivities were evaluated as for CFA at baseline and at the indicated time points after surgery. Thermal sensitivity was evaluated at two different lamp intensities, to produce different baseline values. n = 9 mice per genotype.

A. Baseline hot plate latencies in WT (gray bars, n = 9), TRPV1 KO (black bars, n = 11), or TRPV1/TRPV2 double KO (open bars, n = 9) mice on the mixed background. B. Baseline tail immersion latency in WT (gray, n = 9), TRPV1 KO (black, n = 11), or TRPV1/TRPV2 double KO (white, n = 9) mice on the mixed background. In (A) and (B), * p < 0.05, ** p < 10⁻³, ***p < 10⁻⁴ for indicated comparisons. C. Baseline tail immersion latency in TRPV1 KO (black bars) vs. TRPV1/TRPV2 double KO (open bars) mice on the F1 hybrid background. n = 14 to 15 mice per genotype. D. Baseline response latency in the radiant paw heating assay in TRPV1 KO (black bars) vs. TRPV1/TRPV2 double KO (open bars) mice on the F1 hybrid background. n = 12 mice per genotype. E. CFA induced thermal hyperalgesia. Thermal responsiveness was measured using the radiant paw heating assay at baseline and at the indicated times after injection of CFA into the hind paw in WT (squares), TRPV1 KO (triangles), or TRPV1/TRPV2 double KO (circles) mice on a mixed background. Data shown represent mean + or − SEM measured in the ipsilateral (solid lines and symbols) and contralateral (dashed lines and open symbols) paws. WT, n = 9; TRPV1 KO, n = 11; TRPV1/TRPV2 KO, n = 9. F, G. Effect of systemic resiniferatoxin desensitization on thermal response latencies in the tail immersion (F) and radiant paw heating (G) assays in WT (n = 12) and TRPV2 null (n = 7) mice on a C57BL/6 background. Latencies were assayed prior to (WT, black bars; KO, open bars) or after (WT, gray bars; KO, hatched bars) subcutaneous injection of RTX into the back. Comparisons in (F) and (G) are pre vs. post RTX in a given genotype; using paired student's t-test. (In (F), * p < 0.001, ** p < 10⁻⁴, *** p < 10⁻⁵; In (G), * p < 0.05, ** p < 0.01, *** p < 0.001). For bar graphs, data shown represent mean + SEM.

A–B. Representative examples of individual WT C-fibers innervating the hairy skin in response to a mechanical (A) or heat (B) ramp. C–D. Average responses of WT and TRPV2 KO δ- (C) and C-fibers (D) in response to the standardized mechanical ramp stimulus. E. Average responses of WT and TRPV2 KO C-fibers to the standardized heat ramp stimulus. Recordings from hairy skin are on the left and those from glabrous skin are on the right.