Fig. 3

IDO1 in the ACC regulates pain hypersensitivity in chronic migraine mice. (A) Procedure for evaluation of IDO1 protein expression in various chronic mouse models of migraine using western blotting. (B) Increased IDO1 protein levels in the ACC of NTG-treated mice. (C) Quantitative analysis of the data in (B). The IDO1 band density was normalized to the loading control GAPDH (n = 6). (D-E) Schematic of viral injection, and representative image of viral injection site in the ACC. Scale bar, 200 μm. (F) The silencing effects of three IDO1 shRNAs targeted to different sequences, analyzed with western blotting. (G) Quantitative analysis of the data in (F). The IDO1 band density was normalized to control GAPDH (AAV-CAG-mCherry) (n = 3). (H) Schematic of the experiment used to evaluate pain sensitivity in WT mice injected with AAVs. (I-J) Basal and acute mechanical paw withdrawal thresholds measured by von Frey filament in response to mechanical stimuli in WT mice treated with mCherry and shRNA (n = 12). (K) Example traces of 10 s recordings of sEPSCs from ACC neurons among groups, as indicated. Scale bars, 20 pA, 2 s. (L-M) Cumulative probability plots of sEPSC inter-event interval and amplitude, with additional summary graphs demonstrating the frequency and amplitude of sEPSCs recorded from ACC neurons among groups (n = 8–19 cells from 4–5 mice). (N) Example traces of 10 s recordings of sIPSCs from ACC neurons among groups, as indicated. Scale bars, 20 pA, 2 s. (O-P) Cumulative probability plots of sIPSC inter-event interval and amplitude, with additional summary graphs demonstrating the frequency and amplitude of sIPSCs recorded from ACC neurons among groups (n = 8–19 cells from 4–5 mice). The data are shown as mean ± SEM, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^**** or ^####p < 0.0001; Student’s t test in (C), one-way ANOVA in (G, L, M, O, P), two-way ANOVA in (I, J)