Pharmacological Validation of Lipase Assays

AM6545, a peripherally-restricted CB₁R neutral antagonist, was given by IP injection at 10 mg per kg (Northeastern University Center for Drug Discovery, Boston, MA, USA). Devazepide (Tocris, Bristol, UK), a CCK_A receptor antagonist, was given IP at 0.3 mg per kg. Both drugs were dissolved in vehicle consisting of 7.5% DMSO, 7.5% Tween80, and 85% sterile saline, and warmed in a water bath to ensure solubility. All control conditions were matched, using vehicle in place of drugs and injections occurred 1 h prior to behavior recording (1,600 h). A 72-h washout period was allowed between drug treatments. JZL184 (Tocris, Bristol, UK), a potent inhibitor of monoacylglycerol lipase (MGL), was used to prevent monoacylglycerol hydrolysis in the diacylglycerol lipase (DGL) assay and to validate our MGL assay (described below). Tetrahydrolipstatin (Tocris, Bristol, UK), a lipase inhibitor used routinely to study DGL activity (Gregg et al., 2012 (link); Jung et al., 2012 (link)), was used to validate our DGL assay.