F0]*100) (Figure 4). This response was eliminated by perfusion with 10 mM capsazapine, a selective TRPV1 antagonist (data not shown). In addition, no response to capsaicin was seen when similar experiments were done using cultures of DRG sensory neurons from TRPV12/2 mice (data not shown). Next, we compared the response to capsaicin along WT axons to that seen along sensory axons from Type III Nrg1+/2 mice. Axons from Type III Nrg1+/2 mice had a significantly reduced maximum change in fluorescence in response to focal application of 1 mM capsaicin compared to WT axons (WT, 15.9462.61%; Type III Nrg1+/2, 8.8261.63%; p,0.05) (Figure 4B). Interestingly, there was not a significant difference between the genotypes when comparing the maximum somal response to capsaicin (Figure 4C; WT, 72.3627.5%; Type III Nrg1+/2, 84.8625.3%; p = 0.75). Finally, there was no effect of genotype on the maximum change in fluorescence in response to depolarization with 56 mM KCl either along axons (Figure 4B; WT, 3.0960.95%; Type III Nrg1+/2, 2.5060.56%; p = 0.63) or within soma (Figure 4C, WT, 35.8626.3%; Type III Nrg1+/2, 66.4637.7%; p = 0.23), indicating that the decreased calcium signal in response to capsaicin seen in Type III Nrg1+/2 axons was not the result of changes in voltage gated calcium or tetrodotoxin (TTX)-resistant sodium channels. Cumulatively, these data show that the TRPV1 deficit in Type III Nrg1+/2 sensory neurons appears to be localized to axons, and does not appear to be indicative of abnormalities in other axonally-localized calcium-permeable ion channels. This deficit in functional TRPV1 along the axons of Type III Nrg1+/2 sensory neurons is consistent with the reduced capsaicin-induced thermal hypersensitivity to pain seen in these animals. Type III Nrg1, TRPV1 and Pain Sensation Acute activation of Type III Nrg1 back signaling enhances levels of functional TRPV1 receptors along WT sensory neuron axons Type III Nrg1 is a bi-directional signaling molecule. While Type III Nrg1 acting as a ligand can stimulate ErbB receptor tyrosine kinase signaling, Type III Nrg1 signaling as a receptor can be activated by interaction with ErbB receptor tyrosine kinases or by depolarization [1,22]. As Type III Nrg1 and TRPV1 are expressed in the same cell, we hypothesized that Type III Nrg1 was affecting TRPV1 by signaling as a receptor. Type III Nrg1 signaling as a receptor (Type III Nrg1 back signaling) can exert both transcrip- tion-dependent and transcription-independent effects [1,2,22,23]. To evaluate whether the deficit in functional TRPV1 along Type III Nrg1+/2 axons was due to transcription-dependent or transcription- independent effects of reducing levels of Type III Nrg1, we compared total TRPV1 protein in cultures from P21 male WT and Type III Nrg1+/2 mice (Figure 4E). As we found no difference in total TRPV1 protein between the genotypes, we concluded the effect of Type III Nrg1 signaling on functional TRPV1 along axons was most likely to be transcription-independent. To test whether Type III Nrg1 back signaling acutely affects levels of functional TRPV1 along sensory neuron axons, we used a calcium imaging paradigm with multiple applications of capsaicin where the fourth and fifth capsaicin applications were separated by a 12 minute rest period during which Type III Nrg1 back signaling could be acutely stimulated (Figure 5A). Repeated applications of capsaicin cause progressively smaller responses, most likely due to calcium dependent desensitization of TRPV1 channels [25]. When we treated WT sensory axons with 4 capsaicin applications spaced 4 minutes apart there was a Figure 5. Stimulation of Type III Nrg1 signaling enhances response to capsaicin in WT sensory axons. (A) Representative traces of intracellular calcium along WT sensory axons in response to repeated applications of 1 mM capsaicin followed by application of 56 mM KCl. The maximum percent change in intracellular calcium ([(F2F0)/F0]*100) in response to capsaicin decreased between the 4th and 5th capsaicin applications under control conditions (left), but increased when Type III Nrg1 signaling was stimulated by sErbB4-ECD application during that interval (right). (B) Quantification of percent change in maximum response to capsaicin between the 4th and the 5th capsaicin application ([(F52F4)/F4]*100) by treatment. WT sensory axons showed a significantly enhanced response to capsaicin when Type III Nrg1 signaling was stimulated with sErbB4-ECD (WT CON, n = 10 animals; WT B4, n = 7 animals; Student’s t-test, ***p,0.001). Graph shows mean6SEM. doi:10.1371/journal.pone.0025108.g005 PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25108