Co-labelling was performed with anti-phospho S6 ribosomal protein (Ser235/236, in green) and NeuN (in red, scale bars 30 Scr RB3 cocaine (n=5) p 0.001, Scr RB3 cocaine (n=5) RB3 cocaine (n=5) p 0.001. an inhibitory effect on cocaine-induced ERK activation and reward in mice. In particular, PD325901 persistently blocks S107 cocaine-induced place preference and accelerates extinction following cocaine self-administration. Thus, clinically relevant, systemically administered drugs that attenuate Ras-ERK signalling in the brain may be valuable tools for the treatment of cocaine addiction. DOI: http://dx.doi.org/10.7554/eLife.17111.001 Scr RB1 cocaine (n=3) p 0.001, Scr RB1 cocaine (n=3) RB1 cocaine (n=5) p 0.001. Two-way ANOVA: effect of RB3 F1,14 = 6.26, p 0.05, effect of cocaine F1,14 = 52.11, p 0.0001, effect of interaction F1,14 = 16.06, p 0.01; Bonferronis post-hoc, Scr RB3 saline (n=5) Scr RB3 cocaine (n=3) p 0.001, Scr RB3 cocaine (n=3) RB3 cocaine (n=5) p 0.01. **p 0.01, ***p 0.001. Data are shown as mean with SEM. DOI: http://dx.doi.org/10.7554/eLife.17111.005 Open in a separate window Figure 4. RB3, but not RB1 has an inhibitory effect on cocaine-induced S6 phosphorylation.RB1 (20 mg/kg, i.p.), RB3 (20 mg/kg, i.p.) or the scrambled peptides (Scr RB1 and Scr RB3, 20 mg/kg, i.p.) were administered to wild type mice 1 hr before an acute cocaine (25 mg/kg) or saline injection. After 20 min mice were perfused. Co-labelling was performed with anti-phospho S6 ribosomal protein (Ser235/236, in green) and NeuN (in red, scale bars 30 Scr RB3 cocaine (n=5) p 0.001, Scr RB3 cocaine (n=5) RB3 cocaine (n=5) p 0.001. RB1 does not exert any significant effect on cocaine-induced S6 activation. Two-way ANOVA: effect of RB1 F1,36 = 0.01, p 0.05, effect of cocaine F1,36 = 46.63, p 0.001, effect of interaction F1,36 = 1.65, p 0.5; Bonferronis post-hoc, Scr RB1 saline (n=10) Scr RB1 cocaine (n=10) p 0.0001, Scr RB1 cocaine (n=10) RB1 cocaine (n=10) p 0.001. ***p 0.001, ****p 0.0001. Data are shown as mean with SEM. DOI: http://dx.doi.org/10.7554/eLife.17111.006 Open in a separate window Figure 5. RB1 and RB3 have an?inhibitory effect on cocaine-induced H3 phosphorylation.RB1 (20 mg/kg, i.p.), RB3 (20 mg/kg, i.p.) or the scrambled peptides (Scr RB1 and Scr S107 RB3, 20 mg/kg, i.p.) were administered to wild type mice 1 hr before an acute cocaine (25 mg/kg) or saline injection. After 20 min mice were perfused. Co-labelling was performed with anti-phospho (Ser10)-acetyl (Lys14) histone H3 (in green) and NeuN (in red, scale bars 30 Scr RB1 cocaine (n=9) p 0.0001, Scr RB1 cocaine (n=9) RB1 cocaine (n=9) p 0.0001. Two-way ANOVA: effect of RB3 F1,14 = 9.90, p 0.01, effect of cocaine F1,14 = 14.84 p 0.01, effect of interaction F1,14 = 6.09, p 0.05, Bonferronis post-hoc, Scr RB3 saline (n=4) Scr RB3 cocaine (n=5) p 0.01, Scr RB3 cocaine (n=5) RB3 cocaine (n=5) p 0.01. **p 0.01, ****p 0.0001. Data are shown as mean with SEM. DOI: http://dx.doi.org/10.7554/eLife.17111.007 The MEK inhibitor Rabbit polyclonal to AGBL3 PD325901 is able to effectively block the Ras-ERK pathway in the brain via systemic injection In recent years, some MEK inhibitors and Raf/B-Raf inhibitors, able to block Ras-ERK signalling in vivo, have already been tested clinically for cancer therapy (Uehling and Harris, 2015; Wu and Park, 2015). Therefore, those drugs represent ideal candidates for repositioning studies to address efficacy also for neuropsychiatric disorders such as drug addiction. In order to verify whether these clinically relevant inhibitors pass the blood-brain barrier, we tested the MEK1/2 inhibitors PD325901, Trametinib (GSK1102212) and Selumetinib (AZD6244), and the Raf inhibitor Dabrafenib (GSK2118436). The doses of the inhibitors, as indicated below, were selected on the basis of their previously reported effects on tumour formation (Hennig et al., 2010; Gilmartin et al., 2011; Hofmann et al., 2012; King et al., 2013). For all compounds we followed the same procedure: the inhibitor was given (i.p.) 1 hr prior a cocaine injection (25 mg/kg), and 5 min after cocaine administration mice were transcardially perfused, brains were harvested, and ERK phosphorylation was subsequently determined. An acute administration of PD325901 (25 mg/kg) completely abolished ERK phosphorylation in the ventral striatum (Figure 6A). In contrast, Trametinib (5 mg/kg) and Selumetinib (50 mg/kg) were not effective (Figure 6BCC), while Dabrafenib (50 mg/kg) had a partial but not significant effect on ERK phosphorylation in the S107 ventral striatum (Figure 6D). Open in a separate window Figure 6. PD325901 prevents cocaine-induced ERK phosphorylation in vivo.Mice received an injection of different inhibitors or vehicle followed by cocaine (25 mg/kg, i.p.) or saline injection 1 hr later. 5 min after the stimulation, mice were perfused and ERK phosphorylation in the ventral striatum was determined. (A) PD325901 (25 mg/kg, i.p.) completely blocked ERK phosphorylation. Two-way ANOVA, effect of pre-treatment F1,80 = 125.76 p 0.0001, effect of cocaine F1,80 = 34.66 p 0.0001, effect of interaction F1,80 = 26.25 p 0.0001; Bonferronis post-hoc, PD Saline vs PD Cocaine: p 0.05, VEH Saline vs VEH Cocaine: p 0.0001. (B) Mice were pre-treated with Trametinib (GSK1102212) (5 mg/kg, i.p.), vehicle or PD325901 (25 mg/kg, i.p.) as a positive control for.