Figure 10. sErbB4-ECD stimulation of Type III Nrg1+/2 sensory neuron cultures fails to activate PtdIns3K along sensory axons. (A) Representative images of pAKT staining along WT and Type III Nrg1+/2 sensory axons stimulated for 15 minutes with either control or sErbB4-ECD media. All scale bars equal 10 mm. (B) Quantification of pAKT average fluorescence intensity (AFI) along WT and Type III Nrg1+/2 sensory axons under various treatment conditions. sErbB4-ECD stimula- tion significantly increased AFI levels for pAKT staining along P21 WT sensory axons (WT CON, n = 3 animals; WT B4, n = 3; *p,0.05) but not along sensory axons from Type III Nrg1+/2 cultures (Type III Nrg1+/2 CON, n = 3 animals; Type III Nrg1+/2 B4, n = 3; p = 0.9). All comparisons between treatments and genotypes were made using an ANOVA with a Fischer’s PLSD post-hoc analysis. Graph shows mean6SEM. doi:10.1371/journal.pone.0025108.g010 functional TRPV1 along sensory axons, thereby contributing to the ability to sense heat and to develop thermal hypersensitivity to pain under inflammatory conditions. Type III Nrg1+/2 animals have hypomyelinated peripheral nerves, disorganized Remak bundles and reduced conduction velocities [26,27]. Despite these general effects on peripheral Type III Nrg1, TRPV1 and Pain Sensation nerves, the Type III Nrg1+/2 animals have limited impairments in their response to noxious heat, an enhanced response to noxious cold and normal responses to mechanical stimuli. Over-expression of a dominant negative form of ErbB4 (DN-ErbB4) in non- myelinating Schwann cells results in decreased response to both noxious hot and cold stimuli that was attributed to a progressive loss of C-fiber sensory neurons [28]. These differences between our mice and the DN-ErbB4 mice indicate that the behavioral phenotype in the Type III Nrg1+/2 animals cannot be attributed to a loss of stimulation of Schwann cell ErbB receptors. Thus although altered Schwann cell function might contribute to the phenotype reported here, additional aspects of Type III Nrg1 signaling are likely to account for the sensory deficits. Type III Nrg1 heterozygotes also differ from mice in which all Nrg1 expression is disrupted specifically in a population of small- diameter C-fiber nociceptive sensory neurons that express the sodium channel, Nav1.8 [29]. These Nav1.8-specific Nrg12/2 animals showed no changes in response to thermal or mechanical stimuli, except for a deficit in response to tail pinch. Nav1.8 is not expressed in a population of peptidergic, TRPV1+ sensory neurons that are essential for heat pain sensation [30,31]. Thus, the most parsimonious explanation for the difference between our findings and those of Fricker et al [29] is that Type III Nrg1 signaling in TRPV1+/Nav1.82 nociceptive sensory neurons, contributes to heat pain sensation. Although the present study clearly shows an effect of Type III Nrg1 signaling on TRPV1, it is likely that Type III Nrg1 affects additional aspects of sensory neuron function that contribute to the sensory behavioral deficits seen. In fact, previous results from our laboratory demonstrate that Type III Nrg1 back signaling regulates functional surface a7* nAChRs [2,32], indicating that Type III Nrg1 modulates multiple ion channels that contribute to temperature and pain sensation. Type III Nrg1 is also expressed in other neurons that contribute to response to painful stimuli and peripheral nerves in the Type III Nrg1 heterozygotes are hypomyelinated with altered conduction velocities [26,27]. While we cannot rule out a contribution of hypomyelination or Type III Nrg1 signaling in other areas of the nervous system to the phenotype in the Type III Nrg1+/2 mice, the modality specific nature of the behavioral deficits in the Type III Nrg1+/2 mouse is consistent with a deficiency in Type III Nrg1 signaling in a specific population of sensory neurons. Consistent with our behavioral results, we present evidence that Type III Nrg1 acting along sensory axons can regulate levels of functional TRPV1. Under inflammatory conditions, rapid activa- tion of a variety of signaling pathways can acutely increase TRPV1 levels on the surface of sensory neurons, potentiate TRPV1 channel function and decrease the threshold for TRPV1 channel activation [33,34]. All of these mechanisms render sensory neurons more responsive to subsequent thermal stimulation, notably at temperatures at which they would have previously remained quiescent. We demonstrate that acute stimulation of Type III Nrg1 back signaling rapidly increases functional TRPV1 along cultured WT sensory neuron axons in a PtdIns3K- dependent manner. Previous studies linking PtdIns3K activation by NGF-TrkA signaling to acute insertion of new TRPV1 receptors into the cell membrane [4,5,6] relied on measurements made in either the soma of cultured mouse sensory neurons or heterologous expression systems. It was assumed that an analogous mechanism occurs along sensory axons and at peripheral nerve terminals, where this signaling would matter in vivo. Our data provide evidence that a PtdIns3K-dependent enhancement of TRPV1 can also occur along sensory axons in vitro. Furthermore, we show that Type III Nrg1 is present along TRPV1+ peripheral PLoS ONE | www.plosone.org 14 September 2011 | Volume 6 | Issue 9 | e25108