Brain regions were subjected to a lipid extraction process as described previously (Patel et al., 2003). Tissue samples were weighed and placed into borosilicate glass culture tubes containing two ml of acetonitrile with 84 pmol of [2H8]anandamide and 186 pmol of [2H8]2-AG. Tissue was homogenized with a glass rod and sonicated for 30 min. Samples were incubated overnight at -20°C to precipitate proteins, then centrifuged at 1,500 × g to remove particulates. The supernatants were removed to a new glass tube and evaporated to dryness under N2 gas. The samples were resuspended in 300 μl of methanol to recapture any lipids adhering to the glass tube, and dried again under N2 gas. Final lipid extracts were suspended in 20 μl of methanol, and stored at −80°C until analysis. The contents of the two primary endocannabinoids AEA and 2-AG within lipid extracts in methanol from brain tissue were determined using isotope-dilution, liquid chromatography–mass spectrometry as described previously (Patel et al., 2005a).
