progressive decrease in responsiveness. Response to a 5th capsaicin application after a 12 minute rest period was on average 19.7865.71% smaller than the response to the 4th capsaicin application (Figure 5B). To stimulate Type III Nrg1 back signaling, we applied a solubilized form of the extracellular domain of the ErbB4 receptor tyrosine kinase (sErbB4-ECD). Stimulation of Type III Nrg1 back signaling during this 12 minute interval reversed the decrease in capsaicin responsive- ness, and in fact increased the amplitude by 24.1168.07% (Figure 5B). This increase in capsaicin responsiveness following stimulation of Type III Nrg1 back signaling was significantly greater than the decrease seen under control conditions (p,0.001). Importantly, stimulation of Type III Nrg1 back signaling did not affect response to depolarization with KCl (data not shown), indicating that the target of Type III Nrg1 back signaling was TRPV1 and not voltage-gated calcium or TTX- resistant sodium channels. Thus, acutely stimulating Type III Nrg1 back signaling along WT sensory axons enhances functional axonal TRPV1. PtdIns3K mediates signaling between Type III Nrg1 and TRPV1 Levels of functional, surface TRPV1 in sensory neuron soma or HEK-293 cells are known to be modulated by a number of Type III Nrg1, TRPV1 and Pain Sensation signaling cascades including PtdIns3K [4,5,6]. Type III Nrg1 back signaling also activates PtdIns3K in cultures from P21 WT sensory neurons (Figure 6A, B). 15-minute stimulation of Type III Nrg1 signaling with sErbB4-ECD resulted in an increase in the ratio of pAKT-AKT in whole cell lysates from P21 WT sensory neuron cultures, as assayed by immunoblot (Figure 6A(a); WT B4, 4.860.9-fold increase from WT CON; p,0.05). This effect of Type III Nrg1 signaling as a receptor is pathway specific as the same stimulation paradigm did not change the ratio of pERK- ERK (Figure 6A(b); p = 0.98). Additionally, 15-minute stimulation with sErbB4-ECD increased the fluorescence intensity of pAKT staining along axons of cultured sensory neurons from P21 WTs (Figure 6B; WT B4, 2.360.4-fold increase from WT CON; p,0.05), consistent with Type III Nrg1 activation of PtdIns3K signaling along sensory axons. We then investigated whether PtdIns3K activation mediates signaling between Type III Nrg1 and TRPV1. When PtdIns3K signaling was blocked by bath application of 50 mM LY294002 or 20 nM wortmannin during calcium imaging, there was no effect on baseline response to capsaicin (data not shown), but the effect of stimulating Type III Nrg1 back signaling on capsaicin responsiveness was abolished (6C; WT LY CON, 212.263.7% decrease between 4th and 5th capsaicin applications; WT LY B4, 220.061.7%; WT WM CON, 22.566.7%; WT WM B4, 217.469.5%). Figure 6. PtdIns3K activation is required for Type III Nrg1-induced enhancement of capsaicin responsiveness. (A) 15-minute stimulation of Type III Nrg1 signaling with sErbB4-ECD significantly increased the ratio of pAKT-AKT in whole cell lysates from P21 WT sensory neuron cultures (a: CON, n = 3; B4, n = 4; Student’s t-test, *p,0.05). This increase was blocked by pre-incubation with 20 nM wortmannin (WM), an inhibitor of PtdIns3K activity. (b) The same stimulation of Type III Nrg1 with sErbB4-ECD did not activate the MAPK pathway as illustrated by the lack of effect on the ratio of pERK-ERK. (B) Stimulation of Type III Nrg1 signaling with sErbB4-ECD significantly increased average fluorescence intensity (AFI) levels for pAKT staining along P21 WT cultured sensory axons (15 minutes; CON, n = 3; B4, n = 3; Student’s t-test, *p,0.05). Scale bars equal 10 mm. (C) Blocking PtdIns3K signaling with 50 mM LY294002 or 20 nM wortmannin blocked the Type III Nrg1-induced enhancement of functional TRPV1 (LY CON, n = 3 animals; LY B4, n = 2; WM CON, n = 10; WM B4, n = 7). Comparisons between CON and all treatment groups were made using an ANOVA with a Holm- Sidak post-hoc test for multiple comparisons. All graphs show mean6SEM. doi:10.1371/journal.pone.0025108.g006 PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e25108