Endocannabinoid extraction and analysis

Drug naïve rats were killed after stress or homecage food restriction by rapid decapitation, and tissues were collected as described above. Lipid extractions were carried out as previously described (Qi et al., 2015). Briefly, frozen brain and gut tissue was weighed and then manually homogenized (with a glass rod) in borosilicate glass culture tubes containing 2 mL of acetonitrile with 5 nmol of d8‐2‐AG, 5 pmol of d8‐AEA, 40 pmol d4‐PEA and 40 pmol d4‐OEA. For serum endocannabinoid levels, 500 μL of serum was added directly to the acetonitrile with the same preparation of internal standard as the tissue samples. All other steps of processing for serum were identical to those for tissue (described below). All samples were sonicated for 30 min in an ice bath and incubated overnight at −20°C to precipitate proteins. The following day samples were centrifuged at 1500× g to remove particulates. The supernatant from each sample was transferred to a new glass tube and evaporated under nitrogen, the tube was then washed once with 350 μL acetonitrile (to recapture any lipids adhering to the glass wall) and the acetonitrile was dried under nitrogen gas again. After complete drying, the samples were re‐suspended in 200 μL of acetonitrile and stored at −80°C until analysis by LC–MS. Analysis by MS was performed exactly as previously described (Qi et al., 2015).