peptidergic nociceptive sensory neurons (TrkA; Figure 1A(a–c)) and the non-peptidergic nociceptive sensory neurons (IB4; Figure 1A(d–f)). Type III Nrg1 is also found in the population of nociceptive sensory neurons that expresses the thermoreceptor, TRPV1 (Figure 1A(g–i)). Additionally, we found punctate Type III Nrg1 expression along nociceptive peripheral nerve terminals innervating the plantar skin of the hindpaw as evidenced by Type III Nrg1 co-staining with both the peptidergic marker, CGRP (Figure 1B(a9–c9), white arrow) as well as TRPV1 (Figure 1B(d9– e9), white arrows). Heat pain and thermal hypersensitivity to pain are reduced in the Type III Nrg1+/2 mouse In order to examine the effect of reducing levels of Type III Nrg1 on pain sensation we tested WT and Type III Nrg1+/2 adult male mice in a variety of behavioral assays that probe the ability of the animals to respond to different modalities, as well as intensities, of sensory stimuli. We assessed response to noxious heat using the Hargreaves Paw Withdrawal Assay [24]. We set the intensity of the radiant heat source (used to heat the animals’ hindpaws over a thermal gradient) to a level where WT animals withdraw their paws from the stimulus at 8.0160.21 s and found that under those parameters the Type III Nrg1+/2 animals respond significantly more slowly (9.4560.39 s; p,0.01) (Figure 2A). These latencies Type III Nrg1, TRPV1 and Pain Sensation corresponded to the time it took to heat a thermal probe to 40u and 41uC, respectively. Interesting, these temperatures are in the range of the proposed gating temperature for TRPV1 [19]. Using additional behavioral assays, we demonstrated that this deficit in Type III Nrg1+/2 animals was specific to noxious heat. When we assessed response to noxious cold by placing the animals on a metal plate pre-cooled to 0uC, we found that the Type III Nrg1+/2 animals actually generated a nocifensive response significantly faster than their WT littermates (latency to respond: WT, 183637.0 s; Type III Nrg1+/2, 59.4626.1 s; p,0.01) and generated more nocifensive responses (WT, 261; Type III Nrg1+/2, 962; p,0.01) in the five minute time frame in which they were kept on the plate (Figure 2B). We did not observe any significant differences between genotypes in percent response to noxious mechanical stimulation with filaments of varying inten- sities in the von Frey Assay (Figure 2C), further emphasizing the thermal specificity of the behavioral deficit in the Type III Nrg1+/2 mice. In aggregate, our studies demonstrate that Type III Nrg1+/2 animals have selective deficits in noxious heat sensation. We also wanted to examine the effect of reducing levels of Type III Nrg1 on the development of inflammatory pain. To do this, we evaluated response latency in a modified version of the Hargreaves Paw Withdrawal Assay before and after the induction of inflammation in the hindpaw. We wished to increase the baseline Figure 2. Type III Nrg1+/2 animals have specific behavioral deficits in responding to noxious thermal stimuli. (A) Response in radiant paw heating assay. Latency to respond to radiant heat (set at 15% of maximum intensity) applied to the hindpaw was evaluated for WT and Type III Nrg1+/2 siblings. Response times from both paws were averaged by animal and compared by genotype. Type III Nrg1+/2 mice showed a significantly increased latency to respond relative to their WT littermates (WT, n = 16 animals; Type III Nrg1+/2, n = 14 animals; **p,0.01). (B) Response to noxious cold assessed with the cold plate test. Latency to respond to a 0uC stimulus was measured for WT and Type III Nrg1+/2 mice and compared by genotype. Type III Nrg1+/2 mice respond significantly faster than their WT littermates (WT, n = 16 animals; Type III Nrg1+/2, n = 14 animals; **p,0.01). The total number of nocifensive responses that each animal made in the 5 minute period they remained on the 0uC plate was measured and compared by genotype. Type III Nrg1+/2 mice made significantly more nocifensive responses than their WT littermates (**p,0.01). (C) Response in the von Frey assay of mechanosensation. Percent response for the various forces was evaluated for WT and Type III Nrg1+/2 mice. Data were compared by genotype for each test force. There were no significant differences between genotypes (WT, n = 15 animals; Type III Nrg1+/2, n = 14 animals). (D) Response in a modified version of the radiant paw heating assay pre- and post-capsaicin application. Latency to respond to radiant heat (set at 10% of maximum intensity) applied to the hindpaw was evaluated for WT and Type III Nrg1+/2 littermates before and after application of 15 ml of 0.075% capsaicin cream to the right hindpaw. Response latencies following capsaicin application were significantly less depressed in Type III Nrg1+/2 animals relative to WTs (WT, n = 16 animals; Type III Nrg1+/2, n = 13 animals **p,0.01). Similarly, the percent change (%D) in response latency for Type III Nrg1+/2 animals was significantly blunted relative to WTs (*p,0.05). All genotype comparisons were made using a Student’s t-test. All graphs show the mean6SEM. doi:10.1371/journal.pone.0025108.g002 PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25108