Type III Nrg1, TRPV1 and Pain Sensation Figure 11. Type III Nrg1+/2 sensory axons have reduced numbers of Type III Nrg1+ punctae relative to WT sensory axons. (A) Conventional microscopic images of Type III Nrg1+ punctae (green) found along peripherin+ axons (blue) from WT and Type III Nrg1+/2 sensory cultures. Scale bars equal 10 mm. (B) Quantification of the average number of punctae per 100 mm axon length from 16 WT and 15 Type III Nrg1+/2 images. Comparison by genotype illustrates that Type III Nrg1+/2 sensory axons have significantly fewer Type III Nrg1+ punctae than WT sensory axons (Mann-Whitney Rank Sum test, *p,0.05). doi:10.1371/journal.pone.0025108.g011 nerve terminals innervating hindpaw skin, providing plausibility for our mechanism to occur in a physiologically relevant context in vivo. Stimulating Type III Nrg1 signaling in Type III Nrg1+/2 sensory neuron cultures had no effect on either functional axonal TRPV1 or PtdIns3K activation. Immunofluorescent detection of Type III Nrg1 and Nrg1-ICD protein revealed that Type III Nrg1+/2 animals have a two- and six-fold decrease in the number of Type III Nrg1+ and Nrg1-ICD+ punctae along their sensory axons, respectively, as compared to WTs. Therefore, it is likely that a threshold level of signaling-competent, ICD-containing Type III Nrg1 protein along axons is needed to achieve detectable effects on TRPV1 and PtdIns3K. These results also demonstrate that in vitro, there are two pools of Type III Nrg1 along nociceptive axons: one that is competent to engage in bi- directional signaling, the other that is not. In vivo, the latter pool is likely to provide the axonal Type III Nrg1 required by Schwann cells to organize axons into Remak bundles [27]. RT-PCR analyses of Type III Nrg1 expression in mouse DRG indicates that the majority of transcripts in both wild type and heterozygotes encode Type III Nrg1 proteins containing the c-terminal intracellular domain. Therefore the maintenance of the two pools is most likely controlled by rates of proteolytic processing. We propose that in vivo, the former, back signaling-competent pool is preferentially found at nerve terminals where it is available for acute regulation of receptors such as TRPV1 and a7*nAChRs [2,32]. We hypothesize that the endogenous activator of Type III Nrg1 back signaling at peripheral nerve terminals is either ErbB receptor expressed on keratinocytes or depolarization, as activity has been shown to activate Type III Nrg1 back signaling both in vitro [1] and in vivo [22]. The inability of Type III Nrg1+/2 cultures to acutely up-regulate levels of functional TRPV1 may explain why Type III Nrg1+/2 mice show profound reductions in their ability to develop capsaicin-induced inflammatory thermal hyper- sensitivity. Based on the strong association between the specificity of the pain phenotype seen in Type III Nrg1+/2 animals and the changes seen in sensory neurons from those animals, we conclude Figure 12. Type III Nrg1+/2 sensory axons have reduced numbers of Nrg1+ punctae that contain a signaling-competent intracellular domain (Nrg1-ICD) relative to WT sensory axons. (A) Conventional microscopic images of Nrg1-ICD+ punctae (green) found along peripherin+ axons (blue) from WT and Type III Nrg1+/2 sensory cultures. Scale bars equal 10 mm. (B) Quantification of the average number of punctae per 100 mm axon length from 14 WT and 15 Type III Nrg1+/2 images. Comparison by genotype illustrates that Type III Nrg1+/2 sensory axons have significantly fewer Nrg1-ICD+ punctae than WT sensory axons (Mann-Whitney Rank Sum test, ***p,0.001). doi:10.1371/journal.pone.0025108.g012 PLoS ONE | www.plosone.org 15 September 2011 | Volume 6 | Issue 9 | e25108