AM3506 has been shown to inhibit FAAH activity in recombinant human cells and increase anandamide in rat brain.19 Before behavioral experiments, we established the time course of FAAH inhibition by intraperitoneally injecting mice with vehicle or 1.0 mg/kg AM3506 and killing at time points from 1 h to 10 days post-injection. Forebrain tissue was extracted and processed for FAAH activity, as assayed by the release of [3H]ethanolamine from arachidonoyl-[3H]ethanolamine. AM3506 produced a near-complete inhibition of FAAH activity, relative to vehicle, within 1 h and produced >50% inhibition for up to 3 days before recovering to ∼80% by 10 days (ANOVA effect of time point: F5,12 = 410.34, P<0.01, followed by Newman–Keuls post hoc tests, n = 2–4) (Figure 1a). This long-lasting action of AM3506 is consistent with its profile as an irreversible FAAH inhibitor.19 FAAH inhibition was likely brain-wide, as there was a similarly potent and long-lasting inhibition of FAAH in cerebellar tissue extracts (Supplementary Figure S1). In the same samples, we also determined the activity of MAGL, by measuring the release of [3H]glycerol from 2-arachidonyl-[3H]glycerol. In contrast to FAAH
