Journal: Nature Communications

Article Title: TRPM8 contributes to sex dimorphism by promoting recovery of normal sensitivity in a mouse model of chronic migraine

doi: 10.1038/s41467-022-33835-3

Figure Lengend Snippet: a Wild-type and TRPM8 knockout males display similar acute mechanical hypersensitivity after nitroglycerin (left panel). Deletion of this receptor prevents recovery of baseline mechanical sensitivity after chronic nitroglycerin treatment in males (right). Acute and long-lasting hypersensitivity is similar in wild-type and knockout females. b Trigeminal TRPM8 mRNA expression is similar regardless of sex or nitroglycerin/vehicle treatment. c Calcium responses of trigeminal cultures from males and females chronically exposed to nitroglycerin or vehicle after exposure to TRPM8 agonist WS12 (500 nM). d Average size of calcium transients after WS12 is similar in neurons from males and females. Response sizes vs. respective KCl responses. e Female mice chronically exposed to nitroglycerin show increased mechanical sensitivity when compared to vehicle-treated mice. This sensitivity remained unchanged after 10 mg/kg WS12 i.p. f Nocifensive behavior in the acute phase of the formalin test (5 min) is significantly alleviated in females receiving 6 nmol WS12 i.pl. g TRPM8 antagonist AMTB (i.p.) unmasks latent mechanical pain sensitization in mice chronically exposed to nitroglycerin that already recovered their basal sensitivity. h 10 mg/kg AMTB administered 1 h after formalin injections induces significant reinstatement of licking behavior in males. Mean mechanical thresholds ( a , e , g ), expression ( b ), response size ( d ), nocifensive behavior ( f , h ) ± S.E.M. Datapoints without error bars represent values of individual animals. a ## p < 0.01 vs. baseline. ^^ p < 0.01 vs. vehicle two-way RM ANOVA n = 5 mice per condition. b Two-way ANOVA n = 6 samples from six different mice per condition, obtained after one behavioral experiment. d Two-way ANOVA n = 5 samples from five different mice per condition, obtained after three independent experiments. e *** p < 0.001 nitroglycerin vs. vehicle, three-way ANOVA n = 6(Veh + Veh), n = 5(NTG + Veh), n = 6(Veh + WS12), n = 7(NTG + Veh WS12) mice. f * p < 0.05 vs. vehicle. Multiple two-sided t -test n = 4(WS12) or 7(Vehicle) mice. g + p < 0.05 vs. vehicle. Two-sided Friedman test n = 6 mice. h * p < 0.05 vs. vehicle. Multiple two-sided t -test n = 6 mice per condition. ORX orchidectomy, NTG nitroglycerin, Veh vehicle. Source data are provided in Source Data file, statistical results in Supplementary Table .

Article Snippet: The potent and selective TRPM8 agonist WS12 ((1R,2S,5R)−2-Isopropyl-N-(4-methoxyphenyl)−5-methylcyclohexanecarboxamide, Tocris) was dissolved in DMSO and diluted in corn oil to reach 2.5% DMSO.

Techniques: Knock-Out, Expressing

Journal: Nature Communications

Article Title: TRPM8 contributes to sex dimorphism by promoting recovery of normal sensitivity in a mouse model of chronic migraine

doi: 10.1038/s41467-022-33835-3

Figure Lengend Snippet: a Orchidectomized and sham mice display similar acute hypersensitivity after nitroglycerin (left panel), however only orchiectomized mice remain sensitized on day 21 (right). b Left, all orchidectomized mice show similar acute hypersensitivity after a first nitroglycerin dose, whereas after the last dose, orchidectomized TRPM8KO mice develop stronger sensitization. Right, mice receiving testosterone supplementation show inhibition of the long-lasting hypersensitivity induced after chronic nitroglycerin. This attenuation allows recovery of baseline sensitivity in wild-type mice, but not in TRPM8KO mice. c Testosterone-supplemented wild-type mice that recovered normosensitivity reinstate sensitization after AMTB on day 21. This response is absent in TRPM8 knockouts d Sensitized wild-type females recover basal sensitivity after acute testosterone treatment on day 21. e Cultured trigeminal neurons showing calcium transients after 10 pM testosterone also respond to 500 nM WS12 whereas TRPM8 knockouts are unresponsive. Right, 7% of neurons from wild-type trigeminal cultures show calcium transients after 10 pM testosterone and 500 nM WS12. This is abolished in TRPM8 knockouts. f 10 pM testosterone elicits calcium transients in HEK293 cells when heterologously expressing rat or human TRPM8. Mean mechanical thresholds ( a – d ), response size ( e , f ) ± S.E.M. Datapoints without error bars represent values of individual animals. a *** p < 0.001 vs. vehicle, $$ p < 0.01 vs. sham, three-way RM ANOVA. n = 6 (Sham-Vehicle, Sham-NTG), n = 5 (ORX-Vehicle), n = 7 (ORX-NTG) mice. b ^^ p < 0.01, ^^^ p < 0.001 vs. TRPM8KO; & p < 0.05, && p < 0.01 vs. testosterone vehicle; ## p < 0.01, ### p < 0.001 vs. baseline, three-way RM ANOVA. n = 5 (WT-Veh), n = 6 (WT-Testos and TRPM8KO-Veh), n = 7 (TRPM8KO-Testos) mice. c ^^^ p < 0.001 vs. TRPM8KO; & p < 0.05, vs. testosterone vehicle; ## p < 0.01 vs. basal, three-way RM ANOVA n = 5 (WT Veh), n = 6 (WT Testos and TRPM8KO Veh), n = 7 (TRPM8KO Testos) mice. d ^^^ p < 0.001 vs. TRPM8KO, # p < 0.05 vs. basal, two-way ANOVA n = 7–9 mice per condition. e * p < 0.05 two-sided Mann–Whitney U n = 4 samples from four different mice per condition obtained in two independent experiments. f *** p < 0.001 vs. control, two-sided Mann–Whitney U n = 64–100 cells of two independent cultures per group. ORX orchidectomy, NTG nitroglycerin, Veh vehicle, Testos testosterone, TRPM8KO TRPM8 knockout. Source data are provided in Source Data file, statistical results in Supplementary Table .

Article Snippet: The potent and selective TRPM8 agonist WS12 ((1R,2S,5R)−2-Isopropyl-N-(4-methoxyphenyl)−5-methylcyclohexanecarboxamide, Tocris) was dissolved in DMSO and diluted in corn oil to reach 2.5% DMSO.

Techniques: Inhibition, Cell Culture, Expressing, MANN-WHITNEY, Control, Knock-Out

Journal: Nature Communications

Article Title: TRPM8 contributes to sex dimorphism by promoting recovery of normal sensitivity in a mouse model of chronic migraine

doi: 10.1038/s41467-022-33835-3

Figure Lengend Snippet: a Current density (pA/pF) vs. voltage relationship evidences increase in outward rectifying current 10 pM testosterone in HEK293 cells expressing human TRPM8 (hTRPM8). 10 µM AMTB inhibits testosterone effect ( n = 5 cells from two independent cultures. Each cell received a testosterone pulse followed by a pulse of AMTB with testosterone). b 10 pM testosterone increases basal TRPM8 current at +80 mV ( n = 7 cells from two independent cultures; p < 0.017 vs. Basal current) and 10 µM AMTB reverses its effect ( n = 5 cells from two cultures, p < 0.009 vs. 10 pM testosterone). c hTRPM8 HEK 293 cells (hTRPM8 HEK) transfected with small interfering RNA (siRNA) against androgen receptor (AR) ( n = 7 from two cultures) and control hTRPM8 HEK transfected with scrambled siRNA ( n = 6 from two cultures) show similar testosterone-induced outward rectifying current. This current is absent in wild-type HEK293 cells (WT HEK, n = 6 from 1 culture) lacking TRPM8. d At +80 mV, testosterone-evoked current is similar in anti-AR siRNA or scrambled siRNA-transfected hTRPM8 HEK cells, and this current is absent in wild-type HEK ( p < 0.046 vs. anti-AR siRNA, p < 0.005 vs. scrambled siRNA). e Western blot of AR (110 kDa) and β-tubulin loading control (50 kDa) shows disrupted AR expression in anti-AR siRNA-transfected hTRPM8 HEK cells. WT HEK and scrambled siRNA-transfected hTRPM8 HEK cells display similar AR expression. f 15 nM AMTB (IC50 for WS12 inhibition, Supplementary Fig. ) shifts to the right the dose-response curve for testosterone-induced currents. EC50 for testosterone (7pM, 95% CI 6–8) increases in presence of AMTB (34 pM, 95% CI 16–61, n = 9 cells from three independent cultures for both dose-response curves). “ n ” indicates number of registered cells. b , d Data were subjected to Shapiro–Wilk normality tests, then Kruskal–Wallis followed by Mann–Whitney U tests were applied. ** p < 0.01, * p < 0.05. Data expressed as mean ± SEM, dots on bar charts represent individual cells. EC50s were estimated fitting data to a sigmoidal dose-response curve with constraints (Top = 100, Bottom = 0, GraphPad Prism Software). e is a representative western blot of two independent transfections. Source data are provided in Source Data file, statistical results in Supplementary Table .

Article Snippet: The potent and selective TRPM8 agonist WS12 ((1R,2S,5R)−2-Isopropyl-N-(4-methoxyphenyl)−5-methylcyclohexanecarboxamide, Tocris) was dissolved in DMSO and diluted in corn oil to reach 2.5% DMSO.

Techniques: Expressing, Transfection, Small Interfering RNA, Control, Western Blot, Inhibition, MANN-WHITNEY, Software