On completion of extinction training, mice were immediately killed via cervical dislocation and rapid decapitation. Brains were removed and the BLA, ventromedial prefrontal cortex and dorsal striatum were dissected on ice using 1- and 2-mm-diameter micropunches, respectively. Tissue was homogenized in 100 μl Tris (pH 8.0) buffer and protein concentrations determined using the Bradford assay with bovine serum albumin as a standard. Lipids were extracted and anandamide and 2-arachidonylglycerol levels quantified by liquid chromatography/tandem mass spectrometry, using multiple reactions monitoring, as described previously.33 The mass spectrometer was set for electrospray ionization operated in positive ion mode. The molecular ion and fragments for each compound measured were as follows: m/z 352.3 → 66.1 and 352.3 → 91 for [2H4] anandamide (CID energy: 12 and 56 V, respectively), m/z 348.3 → 62.1 and 348.3 → 91 for anandamide (CID energy: 12 and 48 V, respectively) and m/z 379.3 → 91 and 379.3 → 67.1 for 2-arachidonylglycerol (CID energy: 64 and 56 V, respectively). Analytes were quantified using MassHunter Workstation LC/QQQ Acquisition and MassHunter Workstation Quantitative Analysis (Agilent Technologies, Santa Clara, CA, USA).
