N transducers of noxious heat and cold, respectively, responding to temperatures above 43uC or below 17uC [7,50], and sensitization of these channels have been reported as crucial for thermal hyperalgesia in pathological conditions [51]. Indeed, some reports have shown that treatment with TRPA1 antisense oligodeoxynucleotides reduces behavioral hypersensitivity to cold after CFA-induced inflammation or Title Loaded From File sciatic nerve injury, and TRPA1 knockout mice exhibits impaired behavioral responses to a cold plate maintained at 0uC [19,45]. Furthermore, cold stimulus was found to potentiate the activation of TRPA1 caused by allylisothiocyanate and 4-hidroxynonenal, which is consistent with the assumption of cold hypersensitivity being driven by TRPA1 under inflammatory conditions [18,44]. 24195657 However, although several reports point to TRPA1 as important channels for cold sensation [7,9,13,19,44,45,48], their role on cold nociception is still controversial, since there are several other studies showing no impairment in cold sensation on TRPA1 deficient mice [6,11], suggesting the presence of other sensors for colder temperatures, in addition to TRPA1. Indeed, the transient receptor potential melastatin 8 (TRPM8) has also been proposed to act as this cold sensor. Recent studies have demonstrated that TRPM8 is important for the detection of both cooling sensation and noxiousS-(+)-Dicentrine Induces AntinociceptionFigure 6. Effect of S-(+)-dicentrine (DCTN) administered by oral route (10 – 100 mg/kg, panels A and B) or by intraplantar route (10 100 mg/paw, panels C and D), or the TRPA1 antagonist camphor by subcutaneous (7.6 mg/kg) or intraplantar (3.8 mg/paw) routes on cinnamaldehyde-induced nociception. Panels A and C represents the spontaneous nociception (licking time) and panels B and D represents the hypersensitivity to cold (latency time to paw withdrawal). Each bar represents the mean 6 S.E.M. of 6 – 8 animals, being column C indicative of control values (cinnamaldehyde i.pl. injection) and column V indicative of group receiving only vehicle i.pl. injection. Significance levels are indicated by ***p,0.001 when compared to vehicle (V) group and ##p,0.01 and ###p,0.001 when compared to respective control (C) groups, and the values above the symbols represent the percent of inhibition (one-way anova and Student-Newman-Keuls post hoc test). doi:10.1371/journal.pone.0067730.gcold, since TRPM8 knockout mice have impaired behavioral responses in models such as sensitivity to acetone and cold plate in inflammatory and neuropathic conditions [52?5]. Taking this into account, it is Title Loaded From File reasonable to think that the antihypersensitivity effect of S-(+)-dicentrine in the CFA model may be mediated by TRPs. To test this hypothesis, and considering that TRPV1 and TRPA1 are highly involved in the CFA-induced mechanical hypersensitivity, we evaluated the antinociceptive effect of S-(+)-dicentrine against specific activators of these two channels. Capsaicin, a selective activator of TRPV1, induced a nociceptive behavior that was reversed by AMG9810, a selective blocker of TRPV1, but not by S-(+)-dicentrine. This finding is in line with the results 23977191 on CFA model, when dicentrine did not reverse the heat hypersensitivity, suggesting that S-(+)dicentrine do not interact with TRPV1 channels. On the other hand, when cinnamaldehyde (a selective activator of TRPA1) was used, S-(+)-dicentrine was able to reverse the licking time indicative of nociception and also increase the late.N transducers of noxious heat and cold, respectively, responding to temperatures above 43uC or below 17uC [7,50], and sensitization of these channels have been reported as crucial for thermal hyperalgesia in pathological conditions [51]. Indeed, some reports have shown that treatment with TRPA1 antisense oligodeoxynucleotides reduces behavioral hypersensitivity to cold after CFA-induced inflammation or sciatic nerve injury, and TRPA1 knockout mice exhibits impaired behavioral responses to a cold plate maintained at 0uC [19,45]. Furthermore, cold stimulus was found to potentiate the activation of TRPA1 caused by allylisothiocyanate and 4-hidroxynonenal, which is consistent with the assumption of cold hypersensitivity being driven by TRPA1 under inflammatory conditions [18,44]. 24195657 However, although several reports point to TRPA1 as important channels for cold sensation [7,9,13,19,44,45,48], their role on cold nociception is still controversial, since there are several other studies showing no impairment in cold sensation on TRPA1 deficient mice [6,11], suggesting the presence of other sensors for colder temperatures, in addition to TRPA1. Indeed, the transient receptor potential melastatin 8 (TRPM8) has also been proposed to act as this cold sensor. Recent studies have demonstrated that TRPM8 is important for the detection of both cooling sensation and noxiousS-(+)-Dicentrine Induces AntinociceptionFigure 6. Effect of S-(+)-dicentrine (DCTN) administered by oral route (10 – 100 mg/kg, panels A and B) or by intraplantar route (10 100 mg/paw, panels C and D), or the TRPA1 antagonist camphor by subcutaneous (7.6 mg/kg) or intraplantar (3.8 mg/paw) routes on cinnamaldehyde-induced nociception. Panels A and C represents the spontaneous nociception (licking time) and panels B and D represents the hypersensitivity to cold (latency time to paw withdrawal). Each bar represents the mean 6 S.E.M. of 6 – 8 animals, being column C indicative of control values (cinnamaldehyde i.pl. injection) and column V indicative of group receiving only vehicle i.pl. injection. Significance levels are indicated by ***p,0.001 when compared to vehicle (V) group and ##p,0.01 and ###p,0.001 when compared to respective control (C) groups, and the values above the symbols represent the percent of inhibition (one-way anova and Student-Newman-Keuls post hoc test). doi:10.1371/journal.pone.0067730.gcold, since TRPM8 knockout mice have impaired behavioral responses in models such as sensitivity to acetone and cold plate in inflammatory and neuropathic conditions [52?5]. Taking this into account, it is reasonable to think that the antihypersensitivity effect of S-(+)-dicentrine in the CFA model may be mediated by TRPs. To test this hypothesis, and considering that TRPV1 and TRPA1 are highly involved in the CFA-induced mechanical hypersensitivity, we evaluated the antinociceptive effect of S-(+)-dicentrine against specific activators of these two channels. Capsaicin, a selective activator of TRPV1, induced a nociceptive behavior that was reversed by AMG9810, a selective blocker of TRPV1, but not by S-(+)-dicentrine. This finding is in line with the results 23977191 on CFA model, when dicentrine did not reverse the heat hypersensitivity, suggesting that S-(+)dicentrine do not interact with TRPV1 channels. On the other hand, when cinnamaldehyde (a selective activator of TRPA1) was used, S-(+)-dicentrine was able to reverse the licking time indicative of nociception and also increase the late.