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. 2020 Aug 19;40(34):6522–6535. doi: 10.1523/JNEUROSCI.0810-20.2020

Figure 7.

Figure 7.

Inhibition of C-Raf fully restores opioid sensitivity and reverses hyperexcitability after depolarization. A, DAMGO inhibition of Fsk responses in SCI cultured neurons pretreated for 30 min with vehicle (DMSO ≤0.1%), GW5074 (3 μm), or retigabine (10 μm), followed by 5 min stimulation with Fsk (3 μm) +/− indicated concentrations of DAMGO. Vehicle, n = 11; GW5074, n = 7 or 8; retigabine, n = 4. Treatment effects compared via two-way ANOVA, followed by Sidak's test. *p < 0.05. **,∧∧p < 0.01. Significance symbols: *Vehicle versus retigabine. ∧Vehicle versus GW5074. B, IC50 values from individual DAMGO dose–response curves for SCI groups treated as in A. Veh, n = 10; GW, n = 8; Ret, n = 4. Treatment effects compared using one-way ANOVA, followed by Dunnett's test. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. C, Representative traces of current-clamp recordings (I = 0) of sensory neurons isolated from Naive and SCI groups pretreated (30 min) with vehicle (DMSO 0.03%), C-Raf inhibitor (GW5074, 3 μm) or MEK inhibitor (UO126, 3 μm). APs are clipped at 0 mV so that subthreshold DSFs are more visible. D, A selective C-Raf inhibitor (GW5074) and MEK inhibitor (UO126) decrease the incidence (%) of neurons exhibiting ongoing firing at RMP (SA) or when held at −45 mV (OA). Comparisons of active neuron incidence by Fisher's exact test. n values and p values are indicated over each bar. For multiple comparisons, Bonferroni correction was applied and significance levels are provided on figure. E, Inhibition of C-Raf or MEK by GW5074 or UO126, respectively, hyperpolarizes RMP. The effects of C-Raf and MEK inhibitors on RMP were compared with Brown-Forsythe and Welch ANOVA, followed by Dunnett's test (p = 0.011 for GW5074 and p = 0.035 for UO126). F, Reduction of DSF amplitudes by GW5074 and UO126. Mean DSF amplitudes trended lower when recorded at RMP (p = 0.055 for each inhibitor) and were significantly lower at −45 mV (p = 0.033 for GW5074 and p = 0.013 for UO126). One-way ANOVA followed by Holm–Sidak's test at RMP and Kruskal-Wallis with Dunn's multiple comparison test at −45 mV. MP, Membrane potential. Detailed statistical information is provided in Table 1.