Type III Nrg1, TRPV1 and Pain Sensation Figure 9. Acute stimulation of Type III Nrg1 signaling in Type III Nrg1+/2 sensory axons does not rescue functional TRPV1 deficits. (A) Representative traces of intracellular calcium along Type III Nrg1+/2 sensory axons in response to repeated applications of 1 mM capsaicin followed by application of 56 mM KCl. The maximum percent change in intracellular calcium ([(F2F0)/F0]*100) in response to capsaicin decreased between the 4th and 5th capsaicin application in Type III Nrg1+/2 axons under control conditions (left) and did not increase when Type III Nrg1 signaling was stimulated by sErbB4-ECD application during that interval (right). (B) Quantification of percent change in maximum response to capsaicin between the 4th and the 5th capsaicin application ([(F52F4)/F4]*100) by treatment. Results from WT sensory axons are included for comparison (WT CON, n = 10; WT B4, n = 7; ***p,0.001). Type III Nrg1+/2 axons did not show a statistically significantly enhanced response to capsaicin when Type III Nrg1 signaling was stimulated (Type III Nrg1+/2 CON, n = 6 animals; Type III Nrg1+/2 B4, n = 8). All comparisons between genotypes and treatments were made using an ANOVA with a Holm-Sidak post-hoc test for multiple comparisons. Graph shows mean6SEM. doi:10.1371/journal.pone.0025108.g009 Type III Nrg1+/2 sensory axons neither activated PtdIns3K, nor enhanced functional TRPV1, is consistent with the idea that a Type III Nrg1-activated PtdIns3K signal is required to enhance functional TRPV1 in WT sensory axons. The absence of sErbB4-ECD-stimulated signaling in Type III Nrg1+/2 sensory neuron axons was unexpected. To determine if the heterozygous sensory neurons lacked axonal Type III Nrg1 we examined Type III Nrg1 expression along sensory axons using antibodies that specifically recognize Type III Nrg1 or the Nrg1- ICD. In cultures from P21 mice, Type III Nrg1 immunofluores- cent staining was punctate along WT and Type III Nrg1+/2 sensory neuron axons identified with the nociceptive-specific marker, peripherin (Figure 11). Quantification of the number of Type III Nrg1+ punctae per 100 mm of sensory axon demonstrat- ed that the number of Type III Nrg1+ punctae was reduced by fifty percent along Type III Nrg1+/2 axons (Figure 11B; WT, 2263 puncta/100 mm; Type III Nrg1+/2, 1363 puncta/100 mm; p,0.05). We found an even greater decrease in expression of Nrg1 proteins containing the ICD domain along Type III Nrg1+/2 sensory neuron axons relative to WT axons (Figure 12B; WT, 661 puncta/100 mm; Type III Nrg1+/2, 160.3 puncta/100 mm; p = 0.001). Therefore, although total levels of axonal Type III Nrg1 were reduced 2-fold in the heterozygotes, there was a 6-fold decrease in the level of signaling-competent axonal Type III Nrg1 in the heterozygotes. The preferential decrease in axonal Nrg1-ICD is likely to result from changes in the dynamics of proteolytic processing and targeting to axons, rather than altered splicing of TMc containing transcripts. In both WT and Type III Nrg1+/2 DRG ,90% of transcripts contained the TMc domain encoding exon, whereas less than 10% utilized the b3 splice choice that would result in expression of just the Type III Nrg1b3 isoform (data not shown). It is likely that the reduced level of Type III Nrg1 ICD- containing protein along Type III Nrg1+/2 sensory axons was insufficient to activate PtdIns3K or regulate functional TRPV1. Discussion Type III Nrg1 is involved in the perception of noxious temperatures, particularly under acute inflammatory conditions. Type III Nrg1+/2 mice display deficits in their response to noxious heat, and in their ability to develop thermal hypersensitivity to pain following capsaicin-induced inflammation of the hindpaw. Consistent with this behavioral phenotype we show that Type III Nrg1 is involved in the regulation of functional TRPV1 along sensory neuron axons. Type III Nrg1+/2 mice have reduced functional TRPV1 along their sensory axons. Additionally, acute stimulation of Type III Nrg1 back signaling in WT sensory axons enhances functional TRPV1 in a manner that is dependent on PtdIns3K activation. Acute stimulation of Type III Nrg1 back signaling in Type III Nrg1+/2 sensory axons, however, does not enhance functional TRPV1 or activate PtdIns3K, consistent with their blunted capsaicin induced thermal hypersensitivity. Togeth- er, these data provide support for a model whereby Type III Nrg1 back signaling in nociceptive sensory neurons helps regulate PLoS ONE | www.plosone.org 13 September 2011 | Volume 6 | Issue 9 | e25108