p = 0.27). Thus, although Type III Nrg1 is required for sensory neuron survival, the reduced levels of Type III Nrg1 expressed in the heterozygote are sufficient to support sensory neuron survival into and throughout adulthood. Sensory neuron cutaneous projections are not affected in Type III Nrg1+/2 mice Although we did not find any loss of sensory neuron soma in the Type III Nrg1+/2 mice, it was possible that these animals had a deficit in sensory cutaneous projections. To address this possibility, we quantified TrkA, Ret and TRPV1 protein in samples of galabrous hindpaw skin from WT and Type III Nrg1+/2 mice by immunoblot (Figure 3B, C). We did not see any loss of these proteins in the galabrous skin of Type III Nrg1+/2 mice, indicating that cutaneous projections in these animals were grossly normal. Type III Nrg1+/2 animals have a reduction in functional TRPV1 along sensory neuron axons The capsaicin receptor, TRPV1, is a cation-permeable ion channel found in nociceptive sensory neurons that contributes to noxious temperature sensation and thermal hypersensitivity Type III Nrg1, TRPV1 and Pain Sensation to pain under inflammatory conditions [3,19]. We had previously verified that Type III Nrg1 and TRPV1 are found in the same sensory neuron cell bodies in vivo by labeling transverse sections through L4/L5 DRG from adult WT mice with antibodies against TRPV1 and Type III Nrg1 (Figure 1A(g–i)). TRPV1 located along axons and at peripheral terminals is most physiologically relevant to temperature and pain sensa- tion. We verified that Type III Nrg1 and TRPV1 were co- expressed in the same sensory neuron axons by labeling sections from the plantar hindpaw skin of adult WT mice with antibodies against Type III Nrg1 and TRPV1 (Figure 1B (d9– f9)). Additionally, Type III Nrg1 and TRPV1 are co-expressed in vitro along the axons of sensory neurons from P21 WT male mice (Figure 4D). As TRPV1 is permeable to calcium, we measured functional TRPV1 along sensory axons by quantifying agonist-induced changes in intracellular calcium. For twenty seconds, 1 mM capsaicin was focally applied to cultures of DRG sensory neurons from P21 WT male mice. Capsaicin treatment resulted in a 16 percent increase in intracellular calcium from baseline ([(F2F0)/ Figure 4. Sensory axons, but not soma, from Type III Nrg1+/2 mice show reduced capsaicin responsiveness compared to axons from WT mice. (A) Representative traces of intracellular calcium along sensory axons in response to 1 mM capsaicin or 56 mM KCl. The change in intracellular calcium from baseline over time ([(F2F0)/F0]*100) is shown for WT (left) and Type III Nrg1+/2 (right) axons. Hatched diagonal lines indicate where the time course was non-continuous. (B) Quantification of the maximum change in intracellular calcium in response to application of 1 mM capsaicin or 56 mM KCl by genotype. Averages of 5 animals per genotype were compared using a Student’s t-test. Type III Nrg1+/2 axons showed a significantly decreased response to capsaicin (p,0.05), but not to KCl, relative to WTs. Graph shows mean6SEM. (C) Type III Nrg1+/2 sensory soma show normal response to capsaicin. Quantification of maximal change in fluorescence from baseline ([(F2F0)/F0]*100) in WT or Type III Nrg1+/2 sensory neuron soma in response to 1 mM capsaicin or 56 mM KCl. Average responses from 4 WT and 4 Type III Nrg1+/2 animals to application of capsaicin or KCl were compared by genotype using a Student’s t-test. There was no statistically significant difference between genotypes. Graphs show mean6SEM. (D) Type III Nrg1 (green) and TRPV1 (red) are co-expressed along P21 WT cultured sensory neuron axons identified with a pan-axonal (PA) marker (blue). White arrows indicate examples where Type III Nrg1 and TRPV1 are in close proximity. Scale bar equals 10 mm. (E) P21 WT and Type III Nrg1+/2 sensory neuron cultures have equivalent levels of total TRPV1 protein. Total TRPV1 protein measurement by immunoblot. The 95 kD TRPV1 band and the 35 kD GAPDH band are shown from a representative experiment comparing protein from P21 WT and Type III Nrg1+/2 cultures. Quantification of fold change in intensity of TRPV1:GAPDH normalized to WT average. There was no statistically significant change in the ratio of TRPV1 to GAPDH between genotypes (WT, Type III Nrg1+/2, n = 3 animals). Genotype comparisons were made using a Student’s t-test. Graph shows mean6SEM. doi:10.1371/journal.pone.0025108.g004 PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25108