A. Schematic of optogenetic withdrawal experimental design. Blue light (470 nm) LED stimulation was applied to the glabrous hindpaw for 1 s. Three different groups were tested; KOR-cre; ROSAlslChR2 mice, KOR-cre mice + FLEX.ChR2 (IP at P1), and the wild-type littermates (WT) + FLEX.ChR2 (IP at P1).
B. The frequency withdrawal upon optogenetic stimulation at three different LED intensities (1 mW, 3 mW, and 10 mW). Data are presented as mean ± SEM (n = 4 – 6 mice/group with 10 stimulus presentations per mouse).
C. Schematic diagram illustrating KOR agonists used in behavioral assays in D - J. Nalfurafine acts both centrally and peripherally, whereas ICI204,488 and FE200665 are peripherally restricted KOR agonists. Nalfurafine (Nalf; 20 μg/kg), ICI204,448 (ICI; 10 mg/kg), and FE200665 (FE; 12 mg/kg) were given IP 15 minutes prior to behavioral testing. All data are presented as mean ± SEM and colored symbols represent data points from individual animals.
D. Capsaicin-induced licking behavior (20 μL, intraplantar, 1.5%) was significantly reduced by Nalf, ICI, or FE (One-way ANOVA with Dunnett’s multiple comparison’s test, p < 0.001; n = 10 mice/group).
E. Acetic acid-induced licking behavior (20 μL intraplantar, 0.6%) was significantly reduced by Nalf, ICI, or FE (One-way ANOVA, Dunnett’s multiple comparison’s test, p < 0.05; n = 10 mice/group).
F. Chloroquine-induced scratching behavior (20 μL intradermal, 200 μg) was significantly reduced by Nalf, ICI, or FE (One-way ANOVA with Dunnett’s multiple comparison’s test, p < 0.001; n = 8 – 12 mice/group).
G. Paw withdrawal threshold (PWT) as measured by the von Frey test was not significantly changed by Nalf, ICI, or FE (One-way ANOVA, p = 0.8; n = 9 – 10 mice/group).
H. Nalfurafine significantly increased paw withdrawal latency (PWL) as measured by the Hargreaves’ test, but ICI and FE had no effect on PWL (One-way ANOVA with Dunnett’s multiple comparison’s test (* p < 0.05; n = 12 – 16 mice/group).
I. Mechanical hypersensitivity, measured 2 hours after an incision of the hindpaw, was significantly reduced by Nalf, ICI, or FE (One-way ANOVA, Dunnett’s multiple comparison’s test, p < 0.001; n = 7 – 9 mice/group). Mechanical sensitivity was recorded as the number of withdrawal responses (out of 10) to a single von Frey fiber (vF # 3.61, 0.4g) applied to the plantar surface of the paw adjacent to the incision. Data are normalized to a baseline measure recorded 24 hours prior to the incision.
J. Thermal hypersensitivity, measured 2 hours after an incision of the hindpaw, was significantly reduced by Nalf (One-way ANOVA with Dunnett’s multiple comparison’s test, *, p < 0.01), whereas ICI or FE had no significant effect (p > 0.05; n = 8 – 9 mice/group). Data are normalized to a baseline measure recorded 24 hours prior to the incision.