Fig. 3

Profiling serine-hydrolases MAGL, FAAH and ABHD6 activity and endocannabinoids levels in mouse cortical samples. A Competitive gel-based activity-based protein profiling (ABPP) of MAGL, FAAH, and ABHD6 in mouse cortical samples. ABPP was performed on frozen mice cortical slices after the following ex vivo treatments: control (basal), high KCl (CSD), AKU-005 (AKU) preincubation, AKU preincubation followed by high KCl (AKU CSD). For the ABPP, samples were then incubated in vitro for one hour with the vehicle DMSO (basal) and the following treatments to identify specific enzymatic bands: dual MAGL/FAAH inhibitor AKU (100 nM, 500 nM, 1 µM), FAAH-inhibitor JZP327 A 1 µM, MAGL-inhibitor KML29 1 µM, ABHD6-inhibitors WWL70 1 µM and JZP430 1 µM; and then labelled with fluorescent probe TAMRA-FP. Complete description of the procedure in Material and Methods. B The basal activity of FAAH is lower than that of MAGL. C FAAH activity was not significantly changed in the 4 tested conditions (n = 6). The same basal data in panel B is shown in panels C-E. D MAGL activity in CSD condition was reduced in AKU CSD condition (n = 6, p = 0.003, Kruskal–Wallis with Dunn’s post-hoc test). E ABHD6 activity was not significantly changed in the 4 tested conditions (n = 5). F AEA levels were not affected by any ex vivo treatment of the cortical slices (n = 7) G 2-AG levels were increased after AKU preincubation, both in basal conditions (n = 7, p = 0.003, Kruskal–Wallis with Dunn’s post-hoc test) and during CSD (n = 7, p < 0.001, Kruskal–Wallis with Dunn’s post-hoc test)