Adult male ICR mice were injected i.p. with vehicle (1:1:18), 5 mg/kg NAM, or 1 mg/kg URB597 one hour before decapitation or with 16 mg/kg JZL184 two hours prior to decapitation. After decapitation, the cerebellum was harvested and rapidly cooled by immersion in liquid nitrogen. Tissue was stored at −80°C until use. Anandamide and 2-AG were then extracted using a methanol/chloroform extraction (Burston et al., 2008; Hardison et al., 2006). Samples were homogenized on ice in 2 mL chloroform: methanol (2:1, v/v). The internal standards, 1 pmol anandamide-d8 and 2 nmol 2-AG-d8, were added to each sample, calibrator or control. Samples were mixed and centrifuged after the addition of 0.2 mL of a 0.73% sodium chloride solution. The chloroform was collected and evaporated to dryness with nitrogen. The extracts were reconstituted with 100 μL methanol and placed in autosampler vials for analysis. The injection volume was 20 μL and the auto sampler temperature was set at 5°C. The chromatographic separation of anandamide and 2-AG was performed on a Discovery® HS C18 column 15cm × 2.1mm, 3μm (Supelco: Bellefonte, PA) kept
