Ist AMTB alone did not alter the duration of IM-induced behavior
Ist AMTB alone did not alter the duration of IM-induced behavior (Figure 7c, p = 0.72). However, the effect of menthol was completely blocked by the co-application of AMTB on the dura at 1:1 molar ratio (Figure 7c), confirming that topical menthol at this concentration exerts anti-nociceptive effect through activation of TRPM8 channels. In mice receiving dural co-application of IM and WS-12, another more specific TRPM8 agonist (300 , [20]), the duration of nocifensive behavior wasRen et al. Mol Pain (2015) 11:Page 10 ofalso similar to that PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25679764 of the vehicle group in Figure 7c (99?111 of vehicle-induced behavior, n = 4 mice).Discussion In this study, we used TRPM8EGFPf/+ mice to investigate the postnatal changes of dural afferent fibers that express TRPM8 channels. Expression of EGFP protein corresponds well with endogenous TRPM8 expression [11]. Previous studies show that TRPM8 is predominantly expressed in a subpopulation of PANs in TG and DRG [12, 13]; only sparsely in nodose ganglion and not expressed in superior cervical ganglion neurons [44?6]. Thus, most, if not all, EGFP-positive fibers in the dura SIS3 side effects represent axons of PANs projecting from the TG. In P2 mouse dura, both the density and the number of branches of TRPM8-expressing fibers are comparable to those of CGRP-expressing fibers, whereas they are reduced by about 50 in adult mouse dura. This is consistent with a previous report of sparse innervation of TRPM8-expressing fibers in the dura of adult TRPM8EGFPf/+ mice [29]. This may also account for the failure to retrogradely-label TRPM8-expressing dural afferent neurons in adult mice in our previous study [28], as sparse innervation and lack of extensive axonal branches limit the likelihood and/or the amount of tracer taken up by individual TRPM8-expressing dural afferent neurons. Since we rely on EGFP-ir to identify TRPM8-expressing fibers, it is possible that the perceived reduction of axon density and branches is actually due to the decrease of EGFP expression that renders the EGFP-ir signal below PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 detection threshold. This, however, is unlikely. In TRPM8EGFPf/+ and TRPM8EGFPf/EGFPf mice, EGFP is expressed from TRPM8 loci but not fused to TRPM8 protein. Therefore, the expression of EGFP protein, but not its subcellular distribution, follows the pattern of the endogenous TRPM8 [11]. Since a differential half-life of somatic and axonal EGFP has not been reported, we assume that EGFP exhibits similar stability in soma and axon. Previous studies show that both the level of TRPM8 mRNA and the percentage of TRPM8-expressing PANs are stable in postnatal mouse PANs [46, 47]. Thus, the level of EGFP protein is likely stable in the soma as well as in the axon of postnatal mouse PANs. In rats, there is a massive regression of the TG fiber projecting to the middle cerebral artery between P5 and P55, as the result of both cell death and axon retraction [48, 49]. However, the percentage of TRPM8-expressing PANs does not decrease postnatally [46, 47]. The number of EGFP-positive fibers per mm2 dura is also stable from P2 to adulthood. This argues against a significant death of the TRPM8-expressing dural afferent neurons or the retraction of TRPM8-expressing fibers in mice.Conversely, the reduction of axon branches occurs earlier than the decrease of fiber density, suggesting that axon pruning at least partially accounts for the decrease of TRPM8-expressing fiber density in adult mouse dura. A thorough characterization of the postnatal cha.