1. Generation of TRPV2 KO mice
To evaluate the contributions of TRPV2 to thermal and mechanical sensation and to permit future tissue-specific TRPV2 gene disruption, we adopted a Cre-loxP-based KO strategy. Two loxP sites were inserted into the intronic DNA flanking four mouse TRPV2 exons encoding the 5th transmembrane, pore loop, and 6th transmembrane domains of TRPV2 (). Disruption of these segments should completely eliminate TRPV2 ion channel function and result in a frameshift downstream of the deletion site. For the current study, we analyzed only global TRPV2 gene-disrupted mice. After germline transmission of the floxed allele, TRPV2 loxP/+ mice were crossed with transgenic mice over-expressing Cre recombinase under the control of the CMV promoter. The resulting TRPV2 loxP/+; Cre transgenic offspring, in which one floxed TRPV2 allele was presumably recombined across most tissues, were bred with WT C57BL/6 mice to allow germline transmission of the disrupted TRPV2 locus and permit segregative loss of the Cre transgene. TRPV2 +/− (heterozygote) mice were intermated to generate TRPV2 −/− (KO) and TRPV2 +/+ (WT) littermates. Genotypes were assessed using Southern blotting () and PCR (not shown). RT-PCR confirmed the elimination of the targeted TRPV2 coding region in mRNA isolated from KO trigeminal ganglia (not shown). PCR primers corresponding to sequences upstream of the deleted segment amplified a band of normal intensity in KO trigeminal ganglia, suggesting a residual mRNA product. A much weaker band was obtained using one primer upstream and one downstream of the deleted segment. Sequencing of this latter product confirmed the generation of a truncated mRNA that was out-of-frame beyond the deletion, and therefore unable to encode either the pore or the C terminal portion of the channel protein. We recently reported a lack of TRPV2 function in macrophages derived from these TRPV2 KO mice (Link et al., 2010
Immunoblot analysis confirmed the absence of detectable TRPV2 protein from lysates of TRPV2 KO trigeminal ganglia (). Trigeminal ganglion expression of TRPV1 protein () was not altered by TRPV2 gene disruption. Consistent with our immunoblot findings, TRPV2-like immunofluorescence was virtually absent from TRPV2 KO trigeminal (not shown) or dorsal root ganglia (). In contrast, the prevalence of DRG neurons immunostained for markers of peptidergic (CGRP) or myelinated (NF200) neurons was not different between genotypes, suggesting that the absence of TRPV2 does not grossly alter sensory ganglion development ().
The availability of TRPV2 KO ganglia as a definitive control for antibody specificity also allowed us to examine the WT expression pattern of TRPV2 in greater detail than was possible in prior studies, where only strongly TRPV2 immunoreactive cell bodies (~16% of rat DRG neurons) could be reliably scored as positive (). We observed three classes of TRPV2 immunoreactive neurons in mouse L4 and L5 dorsal root ganglion: strong immunoreactivity that was uniform across the cell body but excluded from the nucleus was observed in 9.7 ± 0.4% of cells; strong immunoreactivity that was confined to the cell periphery was observed in 10.9 ± 0.1% of cells; and weak, uniform immunoreactivity above the KO background was observed in 34.3 ± 1.1% of WT cells (n = 3 mice, >300 neurons scored per mouse). Size analysis revealed that strong uniformly- or peripherally-stained neurons tended to have medium- to large-diameter somas, whereas the weakly TRPV2 positive neurons exhibited a size distribution more closely resembling that of the general population (). Together these immunoblot and immunofluorescence findings confirm the disruption of TRPV2 protein expression in KO mice, and provide support for reports that TRPV2 expression, while enriched in medium- to large-diameter neurons, occurs in many small neurons as well (Liapi and Wood, 2005
; Rutter et al., 2005
; Shimosato et al., 2005
Specific TRPV2 immunoreactivity was observed in fibers and cell bodies in the dermis and epidermis of both hairy and glabrous skin from WT mice. Much of this immunoreactivity was associated with epidermal and dermal phagocytes, which co-labeled with anti-F4/80, as described (Link et al., 2010
) (). However, we also observed a number of fiber-like structures that were TRPV2-immunoreactive but F4/80 negative. These included subepidermal fibers (), fibers in the interfollicular epidermis (), fibers within the epidermal neck of hair follicles (), and and palisade endings deeper within the hair follicles (). No such staining was observed in the skin of TRPV2 KO mice (). To determine whether any of the TRPV2-immunoreactive epidermal fibers were neurons, we stained for TRPV2 in skin from mice expressing green fluorescent protein in a subpopulation of nonpeptidergic nociceptive neurons under the control of the mas-related gene D promoter (mrgD-GFP, (Zylka et al., 2005
)). TRPV2 staining was observed in a subset of GFP-expressing fibers in the epidermis of both glabrous () and hairy () skin. TRPV2 immunoreactivity was not observed in Merkel cells. Immunoreactivity in associated neurites was inconclusive (data not shown). Although TRPV2 immunoreactivity was consistently observed in Meissner's corpuscles (14/14) (data not shown), occasional background staining of these structures in TRPV2 null mice prevented us from establishing definitively whether these cells express TRPV2. We also examined spinal cord TRPV2 immunoreactivity. Consistent with previous reports (Caterina 1999
, LeWinter and Basbaum 2004), we observed strong TRPV2 immunoreactivity in the superficial dorsal horn () that was absent from TRPV2 KO spinal cord (). Dorsal horn TRPV2 immunoreactivity was most intense in the most superficial portion of lamina I and in a region straddling inner lamina II (labeled with IB4, ) and lamina III (labeled with anti-PKCγ, ). Neither IB4 nor anti-PKCγ staining was visibly altered in TRPV2 KO spinal cords (data not shown).
TRPV2 immunoreactivity in skin and spinal cord
The first TRPV2 KO mice we analyzed functionally were on a randomly mixed C57BL/6 × 129S6 genetic background (see methods). We noticed that among the pups born to heterozygous parents on this background, there was a lower-than-expected yield of TRPV2 KO mice (15.2% of the total offspring genotyped at P0, 14.8% at 3 to 4 weeks, compared to 25% expected). The number of viable KO embryos was normal at E18.5, suggesting that loss of TRPV2 KO mice occurs perinatally. However, weights of KO embryos were significantly reduced compared to heterozygote (HE) or WT littermates, without any gross changes in anatomy (at E18.5, WT 1.32 ± 0.04 g, n = 4; HE 1.28 ± 0.02 g, n = 19; KO 1.02 ± 0.08 g, n = 8; WT vs. KO: p < 0.01, KO vs HE: p < 0.05, unpaired t-test). Reduced body weight was also observed in P0 TRPV2 KO pups (WT 1.43 ± 0.03 g, n = 11; KO 1.12 ± 0.06 g, n = 8; WT vs. KO: p < 0.001, unpaired t-test). No differences in lung inflation or presence of milk in the stomach were observed between genotypes at P0. Those KO mice that reached weaning age were apparently healthy, fertile, and normal in appearance, although their body weight was slightly less than that of WT littermates (~93% of WT in males, ~95% in females), during the period from weaning until 14 weeks of age (p < 0.001, Two-way ANOVA, data not shown). After 6 generations backcrossing onto a more homogenous C57BL/6 background, only 2.5% of the weaning age pups born from the heterozygote parents were KO animals. To obtain sufficient adult mice for analysis of nociceptive function, we therefore generated most of our experimental animals either on the randomly mixed background or as F1 hybrids, obtained by mating heterozygotes backcrossed 6 to 9 generations on a C57BL/6 background with heterozygotes backcrossed 2 to 9 generations on a 129S6 background. With this F1 hybrid breeding scheme, 15.5% of pups were KOs. Body weight of surviving KO animals was again ~7% lower in males and ~5% lower in females from 2.5 to 14 weeks of age (p < 0.001, Two-way ANOVA, data not shown), without other obvious differences in appearance. The specific genetic background used in a given experiment is indicated either in the text or in the figure legends.
2. Normal acute nociception in TRPV2 KO mice
Acute chemical nociception was examined using capsaicin and formalin (). In response to the application of a drop of capsaicin (0.5 mM in saline) on the corneal surface, TRPV2 KO mice showed a robust eye wipe response indistinguishable from that of WT littermates, suggesting that this channel is not required for normal TRPV1-mediated responses to capsaicin. Similarly, paw-licking behavior evoked by the injection of the TRPA1 activator, formalin (1.2%), into the hind paw was also unaltered in mice lacking TRPV2.
Acute nociception in mice lacking TRPV2
Acute mechanical sensitivity was measured by stimulating the glabrous skin of the hind paw with calibrated von Frey filaments (). The resulting stimulus-response profiles were not different between genotypes, with 50% response frequency observed at 0.830 g for WT (95% confidence interval 0.657 to 1.048 g), and 0.709 g for TRPV2 KO mice (95% confidence interval 0.522 to 0.964 g). This finding argues against a major role for TRPV2 in acute mechanical nociception.
Acute thermal nociception was evaluated using three different behavioral assays that differ in the anatomical location and geometric characteristics of the thermal stimuli, as well as in the nature of the behavioral avoidance response. In the tail immersion assay, the distal portion of the tail is immersed in heated water, and latency to the tail flick is measured (). In the hot plate assay, a mouse is place on a heated metal surface, and latency to escape behavior (jumping, hind paw licking or shaking) is measured (). In the radiant paw heating assay, a radiant heat source is applied to the glabrous skin of one hind paw of a mouse standing on a glass surface, and latency of paw withdrawal response is recorded (). In all the three assays, the response profile of TRPV2 KO mice was not different from that of WT littermates, over a broad range of thermal stimulus intensities. Together, these findings suggest that the thermal sensitivity of TRPV2 KO mice is not compromised in the basal state, even at temperatures above the threshold for in vitro activation of this channel.
3. Normal inflammatory and neuropathic thermal and mechanical hyperalgesia in TRPV2 KO mice
The number of TRPV2-expressing DRG neurons was reportedly increased upon induction of inflammation in the rat hind paw, suggesting the potential involvement of TRPV2 in inflammatory hyperalgesia (Shimosato et al., 2005
). To address this possibility, we injected complete Freund's adjuvant (CFA, 20 μl) into the glabrous hind paw skin of WT and TRPV2 KO mice on the F1 hybrid background, and evaluated inflammation as well as changes in thermal and mechanical sensitivities. CFA induced strong inflammation, as evidenced by paw edema, in both genotypes (changes from the baseline, 165.4 ± 4.2% for WT, 159.2 ± 2.8% for KO at 24 hr; 141.9 ± 2.4% for WT, 141.8 ± 2.0% for KO at 48 hr; n = 10, p
> 0.05 for both time points, unpaired student's t
-test). Baseline thermal response latencies, assayed using the radiant paw heating assay, were statistically indistinguishable between genotypes in either hind paw (, p
> 0.05, Two-way ANOVA with Bonferroni post-test), as previously observed in the mixed genetic background, though we did observe a very small difference between left and right paws in the TRPV2 KO mice in this experiment (p
< 0.05). Regardless, mice of both genotypes exhibited comparable degrees of thermal hypersensitivity. By 1 day after CFA, latencies in WT mice had declined to 34.9 ± 3.1% of baseline in the ipsilateral paw, while TRPV2 KO latencies had declined to 32.6 ± 2.6% of baseline. By 2 days, WT had partially recovered to 44.3 ± 3.0% of baseline and TRPV2 KO had recovered to 43.3 ± 3.2% of baseline (ipsilateral vs. contralateral: p
< 0.001 at 1 and 2 days in both genotypes; WT vs. KO: p
> 0.05 at 24 and 48 hr, n = 10). Baseline mechanical sensitivity, evaluated using the von Frey filament assay, and mechanical hypersensitivity evoked by CFA were also not significantly different between genotypes (; ipsilateral vs. contralateral: p
> 0.05 at baseline and p
< 0.001 at 1 and 2 days in both genotypes; WT vs. KO: p
> 0.05 at all time points; Two-way ANOVA with Bonferroni post-test). These results suggest that TRPV2 is dispensable for establishment of normal mechanical and thermal hyperalgesia during CFA inflammation.
Thermal and mechanical hyperalgesia in mice lacking TRPV2
We next asked whether TRPV2 was involved in neuropathic mechanical and thermal hypersensitivities caused by L5 spinal nerve ligation and transection, again on the F1 hybrid background. Before and after nerve injury, mechanical and thermal sensitivities were measured using the von Frey filament and radiant paw heating assays, respectively. Baseline thermal response latencies were again indistinguishable between genotypes (, upper panel; WT 10.7 ± 0.6 s, KO 9.6 ± 1.0 s; n = 9 per genotype; p
> 0.05; Two-way ANOVA with Bonferroni post-test). Nerve injury resulted in a comparable ~35% reduction in response latency in both genotypes by day 5 (ipsilateral vs. contralateral: WT, p
< 0.05; KO, p
< 0.01; WT vs. KO, p
> 0.05). Beyond day 5, the ipsilateral vs. contralateral difference was significant only at day 15 in KO. WT and KO latencies did not differ significantly from one another on any day. Because TRPV2 is most robustly expressed in medium- to large-diameter myelinated neurons, and because more rapid heating has been associated with selective activation of A-fiber nociceptors in rats (Yeomans and Proudfit, 1996
; Tzabazis et al., 2005
), we also assayed nerve-injured mice using a heat stimulus that resulted in an approximately 5 s baseline latency in both WT and TRPV2 KO mice (, lower panel; WT 4.8 ± 0.2 s, KO 4.9 ± 0.4 s; n = 9 per genotype; p
> 0.05, Two-way ANOVA with Bonferroni post-test). After nerve damage, response latency transiently decreased by ~20% in both genotypes (ipsilateral vs. contralateral: WT, p
< 0.001 at day 5 and p
< 0.01 at day 8; KO, p
< 0.05 at days 8 and 18), but again, no significant differences between genotypes were observed at any time point. Basal mechanical sensitivity and mechanical hyperalgesia were also similar between genotypes in this experiment. By 3 days after injury, the force eliciting a 50% response frequency dropped by approximately two-thirds in both genotypes and remained lower than that of the contralateral paw for at least two weeks (; ipsilateral vs. contralateral: p
< 0.05 to p
< 0.001 at days 3, 4, 7 and 14; WT vs. KO: p
> 0.05 at all time points; Two-way ANOVA with Bonferroni post-test, n = 9 per genotype). The apparently uncompromised mechanical and thermal sensitization observed after L5 spinal nerve ligation in TRPV2 KO mice indicates that this channel is unlikely to be a major contributor to the development or maintenance of hyperalgesia in this neuropathic pain model.
4. TRPV1 does not mask a thermosensory role for TRPV2
One possible explanation for the lack of apparent thermal nociceptive phenotype in TRPV2 KO mice is the presence of other heat-gated channels with lower thresholds for thermal activation. We sought to eliminate the possible masking effect of TRPV1, the most prominent heat-gated channel, by generating and analyzing TRPV1/TRPV2 double KO mice. An additional motivation for this experiment was the potential co-expression of these channel subtypes in some neurons (Liapi and Wood, 2005
; Rutter et al., 2005
). Ablated expression of both TRPV1 and TRPV2 proteins in TRPV1/TRPV2 double KO mice was confirmed by immunohistochemical staining of DRG (data not shown).
Our initial analysis was conducted on the randomly mixed background. Consistent with previous studies, TRPV1 single KO mice exhibited significantly longer withdrawal latencies to acute thermal stimuli, compared to WT mice, at ≥ 52.5°C in the hot plate assay () or at ≥ 50°C in the tail immersion assay (). TRPV1/TRPV2 double KO mice were indistinguishable from TRPV1 single KO mice in the hot plate assay (). On this background, we observed a slightly increased tail immersion latency at 54°C and 56°C in the double KO mice, compared with TRPV1 single KO mice. However, this small difference likely resulted from the variable genetic background among individual mice, rather than from the absence of TRPV2, since when we repeated this experiment on the more homogeneous F1 hybrid background, TRPV1/TRPV2 double KO mice exhibited response latencies indistinguishable from those of TRPV1 single KO mice across a range of intensities in both the tail immersion () and radiant paw heating () assays. These results demonstrate that the lack of effect of TRPV2 gene disruption on acute thermal nociception is not due to masking by TRPV1.
Because TRPV1 plays a major role in inflammatory thermal hyperalgesia, we also compared inflammatory thermal sensitization by CFA between TRPV1/TRPV2 double KO mice, TRPV1 single KO mice, and WT controls on the randomly mixed background. To maximize the likelihood of observing a TRPV2-mediated response, we assayed mice at high intensity stimulus (; WT latency = 2.0 ± 0.1 s; n = 9). Under these conditions, both TRPV1 single KO mice and TRPV1/TRPV2 double KO mice exhibited a two-fold longer baseline latency, compared with WT, but were not different from one another (WT vs. TRPV1 KO: p < 0.001; WT vs. TRPV1/TRPV2 KO: p < 0.001; TRPV1 KO vs. TRPV1/TRPV2 KO: p > 0.05; Two-way ANOVA with Bonferroni post-test; WT, n = 9; TRPV1 KO, n = 11; TRPV1/TRPV2 KO, n = 9). All three genotypes exhibited a similar reduction in withdrawal latency following CFA injection (ipsilateral vs. contralateral: p < 0.0001 in all three genotypes). Moreover, TRPV1 KO mice and TRPV1/TRPV2 KO mice were statistically indistinguishable at all times. Thus, while there clearly exists a TRPV1-independent component of inflammatory thermal hyperalgesia, this component does not appear to be mediated by TRPV2.
Finally, we examined the effects of functionally desensitizing neurons expressing TRPV1, using resiniferatoxin (RTX), an ultrapotent TRPV1 agonist. Because RTX has been shown to cause neurotoxicity in TRPV1-expressing neurons (Szallasi and Blumberg, 1989
; Neubert et al., 2003
; Shimosato et al., 2005
), this approach should, in theory, compromise both TRPV1-mediated thermal transduction and transduction by non-TRPV1 mechanisms in the same neurons. These experiments were conducted using a small group of WT and TRPV2 KO mice obtained at low efficiency from TRPV2 heterozygotes backcrossed 6 generations onto C57BL/6. As in the other backgrounds, baseline thermal sensitivities in the tail immersion () and radiant paw heating () assays were unaltered by the absence of TRPV2 (p
> 0.05, unpaired t
-test). After baseline analysis, mice were injected with RTX (10 μg/kg body weight, subcutaneously on back) on days 1 and 7 and reassayed for thermal nociception on days 14–21. Desensitization to vanilloid compounds was confirmed by loss of capsaicin-evoked hypothermia on day 33 (data not shown). Following RTX treatment, both WT and TRPV2 KO mice exhibited a significant increase in thermal response latency across a range of temperatures in both the tail immersion and radiant paw heating assays, consistent with desensitization of TRPV1-expressing neurons. However, the latencies were not longer in the TRPV2 KO mice than in WT mice, again strongly arguing against a role for TRPV2 in thermal nociception (p
> 0.05, unpaired student's t
-test at all stimulus intensities, n = 12 for WT, n = 7 for KO after RTX treatment).
5. Unimpaired responsiveness of skin afferents in TRPV2 KO mice to noxious thermal or mechanical stimuli
Behavioral endpoints do not necessarily reflect the threshold of primary nociceptors and are suboptimal indicators of suprathreshold coding properties of afferent fibers. Furthermore, abnormal properties in one subclass of primary afferents may not lead to behavioral changes. We therefore used neurophysiological recordings as a more direct method to determine whether the functional properties of peripheral afferent sensory neurons are altered in mice lacking TRPV2. We employed in vitro preparations of hairy and glabrous skin with the saphenous or tibial nerve attached to investigate single afferents innervating hairy or glabrous skin. From 30 WT (12 hairy, 18 glabrous) and 19 TRPV2 KO (11 hairy, 8 glabrous) mice in the F1 hybrid background, a total of 154 single Aδ or C-fiber recordings were obtained (WT: Aδ-fibers: 19 hairy, 29 glabrous; C-fibers: 25 hairy, 28 glabrous, KO: Aδ-fibers: 13 hairy, 10 glabrous; C-fibers: 18 hairy, 12 glabrous). There was no change in the conduction velocity in any subtype of the Aδ or C-fibers (mean ± SEM, unpaired t-test; Aδ-fibers hairy skin WT: 3.8 ± 0.4 m/s; KO: 5.5 ± 0.9 m/s; p > 0.05; Aδ-fibers glabrous skin WT: 6.8 ± 0.9 m/s; KO: 7.3 ± 1.3 m/s; p > 0.5; C-fibers hairy skin WT: 0.58 ± 0.04 m/s; KO: 0.56 ± 0.07 m/s; p > 0.3; C-fibers glabrous skin WT: 0.55 ± 0.09 m/s; KO: 0.58 ± 0.05 m/s; p > 0.3), indicating that the absence of TRPV2 does not alter this basic biophysical property.
Because the strongest expression of TRPV2 is observed in medium- to large-sized DRG neurons, our first objective was to investigate whether heat sensitivity of A-fiber myelinated nociceptors was altered in the absence of this channel. These myelinated nociceptors were distinguished from C-fiber nociceptors and A-fiber low-threshold mechanoreceptors by their conduction velocity, von Frey filament threshold, receptive field size, and coding properties to suprathreshold stimuli (Koltzenburg et al., 1997
). We initially tested mechanically sensitive A-fiber nociceptors, all conducting within the Aδ range, with a standard heat ramp. Out of 19 units recorded from WT hairy skin, only 1 responded to heat with 4 action potentials at a threshold of 43.0°C, and none of the 13 Aδ fibers recorded from KO hairy skin were activated by noxious heat. Similarly, none of the 29 Aδ units recorded from glabrous skin in WT mice were excited by heat and only one out of 10 Aδ units recorded from KO mice responded with 8 action potentials at a threshold of 41.7°C. This low prevalence of heat responsiveness among Aδ units prevented us from making a meaningful statistical comparison of their thermal responsiveness between genotypes. In nonhuman primates, type I heat-sensitive A-fiber nociceptors typically have higher thermal thresholds and longer receptor utilization times compared to type II heat-sensitive A-fiber nociceptors (Meyer et al., 2005). In order to rule out the possibility that the low prevalence of heat sensitivity was the consequence of a insufficient stimulus intensity, we used a plateau heat stimulus of 50°C at the corium and up to 30 s duration to test 37 Aδ-fibers that had not responded to the standard heat ramp. This plateau heat stimulus used is of sufficient intensity to differentiate type I heat-sensitive A-fiber nociceptors from heat-insensitive A-fiber nociceptors in nonhuman primate skin (Ringkamp et al., 2001;Treede et al., 1998). In hairy skin, none of the 9 units from WT or 8 units from mutant mice responded to heat. Similarly, in glabrous skin, none of the 12 units from WT or 8 units from KO mice was excited.
Given our observation of low-level TRPV2 expression in small diameter unmyelinated neurons, we also investigated whether lack of TRPV2 alters heat responsiveness. In WT hairy skin, 8 out of 23 mechanosensitive C-fibers were excited by heat (2 mechanosensitive C-fibers were not tested for heat responsiveness), whereas 7/18 responded to heat in KO mice (p > 0.5, Chi-squared). In glabrous skin, there was also no significant difference in the proportion of heat-sensitive units between WT (10/28) and KO (5/12) preparations (p > 0.5, Chi squared). Furthermore, there was no significant difference in the threshold between genotypes. In hairy skin the thresholds (mean ± SEM) were 36.9 ± 1.5 °C in the WT and 37.5 ± 2.4 °C in the KO (p > 0.5, unpaired t-test) whereas in the glabrous skin they were 35.8 ± 1.1 °C in the WT and 36.0 ± 0.8 °C in the KO (p > 0.3, unpaired t-test). Inspection of the average responses to suprathreshold stimuli among heat-responsive C-fibers further showed that lack of TRPV2 did not result in a reduction of the response magnitude. There was no significant difference in the average (± SEM) total number of action potentials evoked by the standard heat ramp stimulus of C-fiber nociceptors innervating the hairy skin (WT: 27 ± 5; KO: 35 ± 8; p > 0.1, unpaired t-test) or glabrous skin (WT: 64 ± 15; KO: 87 ± 18; p > 0.1, unpaired t-test). There was also no significant difference in the percentage of cold sensitive nociceptors.
Analysis of A- and C-fiber nociceptors to mechanical stimulation also revealed little change in the threshold or the response to suprathreshold stimuli. This was true for both punctate mechanical stimuli applied with calibrated von Frey filaments and suprathreshold stimuli applied with a feedback-controlled mechanical stimulator. The median (first, third quartile) of the von Frey hair threshold of A-fiber nociceptors of the hairy skin was 8 (5.6, 11.2) in the WT and 8 (5.6, 22.4) in the KO (p > 0.5, U-test). In the glabrous skin it was 8 (5.6, 16) in the WT and 8 (8, 16) in the KO (p > 0.5, U-test). The corresponding values for the C-fibers from hairy skin were 11.2 (8, 22.4) in the WT and 16 (5.6, 22.4) in the KO (p > 0.5, U-test) and in glabrous skin 11.2 (5.6, 22.4) in the WT and 16 (11.2, 22.4) in the KO (p > 0.5, U-test).
There was no significant difference in the average (± SEM) total number of action potentials evoked by the standard mechanical ramp stimulus between A-fiber nociceptors innervating the hairy skin (WT: 101 ± 12; KO: 118 ± 23; p > 0.3, unpaired t-test) or glabrous skin (WT: 129 ± 22; KO: 106 ± 27; p > 0.5, unpaired t-test). This also applied to C-fibers innervating the hairy skin (WT: 59 ± 14; KO: 83 ± 18; p > 0.1, unpaired t-test) or glabrous skin (WT: 95 ± 19; KO: 52 ± 21; p > 0.3, unpaired t-test).
In aggregate, the neurophysiological analysis of heat and mechanical sensitivity of myelinated and unmyelinated nociceptive sensory neurons from TRPV2 KO mice fully concurs with their lack of impairment in thermally and mechanically evoked nociceptive behaviors.