Fig. 3

Morphine enhances TRPM8 sensitivity to cold and menthol. a Representative whole cell current-clamp recording of menthol-evoked depolarization and AP firing (menthol: 100 μM) of DRG neurons collected from naïve mice and exposed to morphine (MS) overnight versus control. b Whisker plots of the membrane potential of menthol sensitive neurons exposed to vehicle or morphine, before (dark) or after (red) menthol stimulation (− 59.6 ± 1.4 mV vs. -27.6 ± 1.2 mV in control, n = 15; − 52.3 ± 1.2 mV vs. -40.2 ± 1.2 mV with morphine, n = 17), or menthol+AMTB (− 51.9 ± 1.9 mV; n = 7). c Whisker plot of the AP discharge, measured over a 1 min. Application of cold (0.95 ± 0.24 Hz in control (light blue) vs 3.6 ± 0.65 Hz with morphine (dark blue), n = 9 and 8, respectively) or menthol (2.4 ± 0.57 Hz in control (light green) vs 5.29 ± 0.39 Hz with morphine (dark green), n = 9 and 7, respectively), or menthol + AMTB (0.026 ± 0.01 Hz; n = 7). d Representative calcium imaging traces to two repeated applications of cold (20 °C, blue) or menthol (100 μM, green) in cultured DRG neurons exposed to morphine (500 nM) overnight. e Whisker plots showing the mean ratio (R2/R1) of the amplitude response to cold (52.85 ± 23.72% in control, light blue, vs 121.07 ± 45.89% with MS, dark blue; n = 60 and 54, respectively) or menthol (62.84 ± 17.87% in control, light green, vs 130.81 ± 37.48% with MS, dark green; n = 64 and 58, respectively) responses illustrated in (d). f Temperature-response curve measured by calcium imaging during cooling ramp on DRG neurons from naive (median temperature value 21.29 ± 0.53 °C, control, n = 36) versus morphine-treated mice (median temperature value, 26.23 ± 0.78 °C, in vivo MS, n = 41) or naïve cultured DRG neurons exposed to morphine overnight (median temperature value, 24.98 ± 0.57 °C, in vitro MS, n = 28). Sensitization of the temperature-response relationship is indicated by the shift toward warmer temperature. g Dose-response curve evoked by menthol, measured by calcium imaging on DRG neurons from naive (EC50 = 13.86 ± 1.16 μM, control, n = 34) versus morphine-treated mice (EC50 = 0.21 ± 0.09, in vivo MS, n = 23) or naïve culture DRG neurons exposed to morphine (EC50 = 0.33 ± 0.02 μM, in vitro MS, n = 51). The shift towards lower concentration indicates that morphine potentiates the sensitivity of TRPM8 to menthol. Statistical analysis was performed using one and Two-Way ANOVA followed by Bonferroni post hoc test (**p < 0.01). Data are mean values, error bars indicate ±SEM