129S1/Sv1mJ mice (The Jackson Laboratory, Bar Harbor, ME, USA) were injected with 1.0 mg/kg AM3506. Mice were killed via cervical dislocation and rapid decapitation immediately or 1 h, 1 day, 3 days or 10 days after injection. Brains were removed and the forebrain and cerebellum tissue quickly dissected on ice. Tissue was homogenized in 10 mm (pH 7.6) Tris-HCl buffer containing 1 mm ethylenediaminetetraacetic acid and centrifuged at 1000g to remove cell debris. Tissue was assayed for FAAH activity via measurement of the release of [3H]ethanolamine from [3H]anandamide labeled on the ethanolamine moiety.21 Tissue was also assayed for MAGL activity by analyzing the release of [3H]glycerol from 2-oleoyl-[3H]glycerol in the presence of 1 mm of the FAAH blocker URB597 (Cayman Chemical Company, Ann Arbor, MI, USA).22
