response latency in the Hargreaves task in order to provide a larger window in which to evaluate the extent of hypersensitivity to pain developed as a result of an inflammation-inducing manipu- lation to one of the hindpaws. Therefore, we decreased the intensity of the radiant heat source (from 15% to 10% of maximum intensity) so that it took a longer time to elicit a withdrawal response. The new baseline response latencies for the WT and Type III Nrg+/2 animals were longer than what they had been (WT, 21.2261.05 s; Type III Nrg1+/2, 21.2061.35 s), and they did not differ significantly from one another (Figure 2D). At the reduced radiant heat source intensity used in these experiments, the response latencies of the animals corresponded to approximately 30uC. In the absence of inflammation, this temperature is generally not perceived as noxious. Therefore, at baseline in this experimental paradigm, it is possible that the animals were withdrawing their paws when they first perceived a warm stimulus, rather than a painful one. The different intensity of the thermal stimulus used in these experiments may also explain why we did not see any difference in response times at baseline between genotypes. Inflammation was induced by local capsaicin application to the right hindpaw, and 20 minutes later WT animals exhibited a robust reduction in response latency indicative of the development of thermal hypersensitivity to pain (WT post- capsaicin, 6.2161.06 s; p,0.001 v. WT pre-capsaicin). Type III Nrg1+/2 animals also exhibited a reduction in response latency following capsaicin application (Type III Nrg1+/2 post-capsaicin, 13.8562.31 s; p,0.01 v. Type III Nrg1+/2 pre-capsaicin), although the response latency for Type III Nrg1+/2 animals Type III Nrg1, TRPV1 and Pain Sensation post-capsaicin was significantly greater than WTs (p,0.01; Figure 2D). Thus, following capsaicin application, the thermal response latency of WTs decreased by 67% whereas the response latency of the heterozygous littermates decreased by only 33% (p,0.05; Figure 2D). Sensory neuron survival is not affected in Type III Nrg1+/2 lumbar DRG Type III Nrg1 is required for TrkA+ sensory neuron survival during late embryonic development [7]. Therefore, we undertook quantitative immunohistochemical analysis of L4/L5 DRG from adult WT and Type III Nrg1+/2 mice to assess the effect of reducing levels of Type III Nrg1 on sensory neuron survival. Sections were double-labeled with markers for sensory neuron subgroups (TrkA, IB4, NF200 and peripherin) as well as with a pan-sensory neuron marker (a cocktail of NF200 and peripherin), and the percentage of sensory neurons expressing each of the different markers was calculated. We found that reducing levels of Type III Nrg1 did not affect the percentages of any of these sensory neuron populations (TrkA: WT 30.461.9, Type III Nrg1+/2 25.363.4; IB4: WT 32.161.9, Type III Nrg1+/2 29.560.4; NF-200: WT 35.461.9, Type III Nrg1+/2 33.262.6; Peripherin: WT 65.061.6, Type III Nrg1+/2 66.462.9). Finally, the number of pan-sensory+ profiles per section from at least five sections evenly spaced throughout the DRG was averaged for each animal and animal averages were compared by genotype. There was no significant difference between the genotypes (Figure 3A; WT, 141619 profiles/section; Type III Nrg1+/2, 11469; Figure 3. Adult WT and Type III Nrg1+/2 animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1+/2 mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student’s t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1+/2 mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student’s t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1+/2 n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1+/2 n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes. doi:10.1371/journal.pone.0025108.g003 PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25108