Data for [35S]GTPγS binding assays are reported as mean and standard error of at least four experiments, which were each performed in triplicate. Non-specific binding was subtracted from each sample. Net stimulated [35S]GTPγS binding is defined as agonist-stimulated minus basal [35S]GTPγS binding, and percent stimulation is defined as (net-stimulated/basal [35S]GTPγS binding) × 100%. Nonlinear iterative regression analyses of agonist concentration-effect curves were performed with Prism 4.0 (GraphPad Software, Inc., San Diego, CA). For mass spectrometry data in mice, mean and standard error were determined for anandamide (pmol) and 2-AG (nmol) per g of cerebellum for each condition. ANOVA, followed by Dunnett’s post hoc tests, was used to determine significant differences between basal and drug-induced concentrations of each endocannabinoid. Statistical analysis was performed using Sigma Stat, version 3.1 (Systat Software, Inc., San Jose, CA). Significance was defined as P < 0.05.
