Control mice or stressed mice were killed by decapitation, the brain rapidly removed, and a 2 mm block containing the amygdala was made using a coronal brain matrix. Blocks were placed on a glass dish on top of dry ice-cooled metal block. Less than 90 s elapsed between decapitation and tissue freezing. Once frozen, 1 mm brain punches were made of the amygdala. Tissue was stored at −80° C until used for tissue extraction. Lipid extraction from amygdala micropunches was carried out exactly as described earlier (Patel et al, 2005b), except that the homogenization volume was spiked with 50 pmol d8-2-AG, and 500 pmol d8-SAG, and 1000 pmol d8-AA per sample. Immediately before LC-MS analysis, the samples were reconstituted in 200 µl of 9:1 methanol:water (v:v), vortexed, and transferred to autosampler vials.
