inhibitor, PD158780. At 1 mM, this drug completely blocked ErbB receptor-mediated signaling, as indicated by its ability to block soluble Nrg1 (sNrg1)-induced stimulation of ErbB receptors and activation of ERK (Figure 8A). We then incubated our sensory neuron cultures in this concentration of ErbB receptor inhibitor for 15 minutes prior to applying 2 pulses of capsaicin, reasoning that if sErbB4-ECD was exerting its effects by blocking endogenous ErbB receptor signaling then directly blocking ErbB receptor signaling should mimic the effect of sErbB4-ECD application. Instead, we found that blockade of ErbB receptor signaling had no effect on subsequent capsaicin responsiveness (Figure 8B & C). This result provided further evidence that acutely stimulating Type III Nrg1 back signaling along WT sensory axons enhances functional axonal TRPV1. Stimulation of Type III Nrg1 back signaling with sErbB4- ECD fails to rescue deficits in functional TRPV1 along Type III Nrg1+/2 sensory axons As acute stimulation of Type III Nrg1 back signaling enhances functional TRPV1 along sensory axons, and Type III Nrg1+/2 sensory axons have reduced levels of TRPV1, we attempted to rescue levels of functional TRPV1 along Type III Nrg1+/2 axons by acutely stimulating Type III Nrg1+/2 sensory neuron cultures with sErbB4-ECD. Using the capsaicin application paradigm described above (for Figure 5), we found that Type III Nrg1+/2 sensory axons showed the same degree of desensitization to repeated capsaicin applications as WT axons. In Type III Nrg1+/2 sensory axons a 5th capsaicin application after a 12 minute rest period was on average 17.666.4% smaller than the response to the 4th capsaicin application, which was not significantly different than the 19.7865.71% decrease seen in WT axons (p = 0.8; Type III Nrg1, TRPV1 and Pain Sensation Figure 9B). Unexpectedly, we found that acute stimulation of Type III Nrg1 back signaling along Type III Nrg1+/2 sensory axons during this 12 minute interval did not enhance the response to capsaicin as was seen in WT axons (Figure 9B; Type III Nrg1+/2 B4, 29.666.21%; for reference, WT B4, +24.1168.07%). Thus, sensory axons with reduced Type III Nrg1 were deficient in both basal levels of functional TRPV1 and in the ability to acutely up- regulate functional TRPV1 in response to exogenous stimulation of Type III Nrg1 back signaling. Stimulation of Type III Nrg1 back signaling with sErbB4- ECD does not activate PtdIns3K signaling along Type III Nrg1+/2 sensory axons Axons from Type III Nrg1+/2 animals fail to up-regulate functional TRPV1 in response to stimulation of Type III Nrg1 back signaling. We wondered if this impairment resulted from an inability to activate PtdIns3K signaling. To test this, we stimulated P21 WT and Type III Nrg1+/2 cultures with sErbB4-ECD for 15 minutes and then quantified the intensity of pAKT staining along sensory axons. When stimulated with sErbB4-ECD, cultures from WT animals showed a significant increase in axonal pAKT (1.9-fold increase; p,0.05), but cultures from Type III Nrg1+/2 animals did not (p = 0.97) (Figure 10B). Both P21 WT and Type III Nrg1+/2 sensory neurons showed a robust activation of PtdIns3K signaling in response to a five-minute stimulation with 100 ng/ml NGF (data not shown; WT NGF, 1.860.28-fold change from WT CON; p,0.05; Type III Nrg1+/2 NGF, 2.2560.54-fold change from WT CON; p,0.05). Thus, the deficit in PtdIns3K activation observed in Type III Nrg1+/2 sensory axons was specific to stimulation of Type III Nrg1 back signaling. That exogenous stimulation of Type III Nrg1 back signaling along Figure 8. Blockade of ErbB signaling does not enhance response to capsaicin in WT sensory axons. (A) Incubation of P21 WT sensory neuron cultures with soluble Nrg1 (sNrg1) stimulates ErbB signaling and activates ERK (measured as a ratio of pERK to GAPDH). This sNrg1-induced activation of ERK can be blocked by incubation with 1 mM PD158780, an ErbB kinase inhibitor (n = 3 animals per condition; *p,0.05). (B–D) Blockade of ErbB signaling does not enhance response to application of capsaicin or KCl. Sensory neuron cultures from P21 WT mice were incubated with 1 mM PD158780 or control media for 12 minutes before two 1 mM pulses of capsaicin were applied (spaced 4 minutes apart), followed by application of 56 mM KCl. Within axons that responded to both capsaicin and KCl, the percent change in fluorescence from baseline ([(F2F0)/F0]*100) in response to both applications of capsaicin as well as KCl was calculated. Axonal responses were averaged by animal and normalized to the WT CON average. Quantification of the maximum response to the first (B) or second (C) applications of 1 mM capsaicin or KCl (D) were compared by treatment. Blockade of ErbB signaling with 1 mM PD158780 did not affect response to the first or second application of capsaicin, or to KCl (WT CON, n = 7 animals; ErbB Inh, n = 7 animals). doi:10.1371/journal.pone.0025108.g008 PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e25108