Fig. 2

Profiling serine-hydrolases MAGL, FAAH and ABHD6 activity and endocannabinoid levels in rat cortical samples. A Competitive gel-based activity-based protein profiling (ABPP) of MAGL, FAAH and ABHD6 in rat cortical samples. ABPP was performed on frozen rat cortical slices after the following ex vivo treatments: control (basal), high KCl (CSD), AKU-005 (AKU) pre-incubation, AKU preincubation followed by high KCl (AKU CSD). For the ABPP, samples were incubated in vitro for one hour with the vehicle DMSO (basal) and the following treatments to identify specific enzymatic bands: MAGL/FAAH/ABHD6 inhibitor AKU 100 nM, FAAH-inhibitor JZP327 A 1 µM, MAGL-inhibitor JJKK- 048 100 nM; and then labelled with fluorescent probe TAMRA-FP. B Basal FAAH activity is slightly lower than MAGL one. ABHD6 basal activity is lower than both MAGL and FAAH basal activities (n = 43, p < 0.001, Kruskal–Wallis with Dunn’s post-hoc test). The same basal data in panel B is shown in panels C-E. C FAAH basal activity (basal) was unchanged following CSD. FAAH activity levels in AKU condition were reduced compared to the basal ones (n = 43 in basal and n = 11 in AKU, p = 0.0001, Kruskal–Wallis with Dunn’s post-hoc test). FAAH activity was also reduced in AKU CSD condition compared to CSD (n = 43 in CSD and n = 11 in AKU CSD, p = 0.0001, Kruskal–Wallis with Dunn’s post-hoc test). D MAGL basal activity (basal) was increased during CSD (n = 43, p = 0.0145, Kruskal–Wallis with Dunn’s post-hoc test). Compared to basal levels, MAGL activity levels in AKU condition were reduced (n = 11, p = 0.0006, Kruskal–Wallis with Dunn’s post-hoc test). Increased MAGL activity in CSD condition was reduced in AKU CSD condition (n = 11, p < 0.001, Kruskal–Wallis with Dunn’s post-hoc test). E ABHD6 basal activity (basal) was unchanged following CSD. ABHD6 activity in CSD condition was reduced in AKU CSD condition (n = 43 in CSD and n = 11 in AKU CSD, p < 0.0001, one-way ANOVA with Tukey post-hoc test). F AEA levels increased following CSD (n = 44, p = 0.006, one-way ANOVA with Tukey post-hoc test). AKU-005 pre-treatment lead to high increase of AEA basal levels (AKU, n = 11, p < 0.001, one-way ANOVA with Tukey post-hoc test), and AEA levels after CSD (AKU CSD, n = 11, p < 0.001, one-way ANOVA with Tukey post-hoc test). G 2-AG basal levels did not change after CSD. AKU-005 pre-treatment increased 2-AG levels in AKU conditions (n = 39 for basal and n = 11 for AKU, p < 0.001, Kruskal–Wallis with Dunn’s post-hoc test) and AKU CSD conditions (n = 41 for CSD and n = 11 for AKU, p < 0.001, Kruskal–Wallis with Dunn’s post-hoc test)