Pre-incubation with sErbB4-ECD enhances response to the second, but not the first, application of capsaicin along sensory axons The enhancement of functional TRPV1 seen following acute stimulation of Type III Nrg1 back signaling in WT sensory axons could be due to either insertion of new TRPV1 receptors or modifications of pre-existing receptors that alter channel func- tionality. To distinguish between these possibilities we pre-treated cultures with sErbB4-ECD for 12 minutes and then applied 2 pulses of capsaicin spaced 4 minutes apart, reasoning that the channels should be in a relatively un-desensitized state when we applied the first pulse of capsaicin, but not when we applied the second one. If stimulation of Type III Nrg1 back signaling increased the number of functional TRPV1 receptors on the axonal membrane, we would expect to see an increase in response to the first application of capsaicin in cultures pre-treated with sErbB4-ECD. In fact, stimulating Type III Nrg1 signaling resulted in a slight, not significant increase in response to the first capsaicin application (Figure 7B; WT B4, 1.27-fold increase, p = 0.17). However, if stimulation of Type III Nrg1 signaling affected receptor desensitization, we would expect to see an increase in the response to the second application of capsaicin (when the receptors are in a desensitized state) in cultures pre-treated with sErbB4- ECD. Indeed, the amplitude of the response to the second application of capsaicin was significantly greater in cultures where Type III Nrg1 signaling as a receptor had been stimulated by pre- incubation with sErbB4-ECD (Figure 7C; WT B4, 1.93-fold Type III Nrg1, TRPV1 and Pain Sensation increase, p,0.05). In both control and sErbB4-ECD treated cultures the response to the second application of capsaicin was smaller than the first, most likely due to calcium-dependent tachyphalaxsis. In control cultures, there was on average a 65.6663.36% decrease in responsiveness between the first and second capsaicin applications (Figure 7E). However, in cultures stimulated with sErbB4-ECD this decrease was reduced to 49.6764.30%, a statistically significant difference from control conditions (Figure 7E; p,0.05). There was no effect of pre- stimulation with sErbB4-ECD on responsiveness to depolarization with KCl (p = 0.38; Figure 7D). Therefore, the major effect of stimulating Type III Nrg1 back signaling would appear to be a change in the degree of desensitization. sErbB4-ECD stimulation does not affect functional TRPV1 by blocking signaling between endogenous Nrg1 and ErbB receptors It was possible that all the effects of applying sErbB4-ECD on capsaicin responsiveness were due to blockade of endogenous Nrg1 stimulation of ErbB3 receptors located on adjacent Schwann cells, rather than to direct stimulation of Type III Nrg1 back signaling. If this alternative scenario were true then endogenous ErbB receptor-mediated signaling should be tonically depressing levels of functional TRPV1. Thus, blocking ErbB receptor signaling should disinihibit this inhibition and enhance levels of functional TRPV1. To test this possibility, we decided to block ErbB receptor-mediated signaling by applying the ErbB kinase Figure 7. Pre-treatment with sErbB4-ECD enhances response to a second application of capsaicin along sensory axons. (A) Sensory neuron cultures from P21 WT mice were incubated with sErbB4-ECD 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. (B–C) Quantification of the maximum response to the first (B) or second (C) applications of 1 mM capsaicin were compared by treatment. Pre-treatment with sErbB4-ECD significantly increased the maximum response to the second pulse of capsaicin (WT CON, n = 8 animals; WT B4, n = 9 animals; *p,0.05). There was a trend towards an increase in response to the first pulse of capsaicin following sErbB4-ECD pre-treatment (p = 0.17). (D) Quantification of the maximum response to KCl by treatment. sErbB4-ECD pre-treatment had no effect on the maximum response to KCl (p = 0.38). (E) The percent change between the maximum response to the first and second capsaicin applications was calculated and axonal values were averaged by animal. Quantification of this percent change in response by treatment; Cultures pre- treated with sErbB4-ECD showed significantly less of a decrease in responsiveness to capsaicin between the first and second capsaicin applications (*p,0.05). All treatment comparisons were made using a Student’s t-test. Graphs show mean6SEM. doi:10.1371/journal.pone.0025108.g007 PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e25108