50 ml plastic tube

The acute, complex stress consists of concurrent restraint, bright lights, loud noise, physical jostling and awareness of peer discomfort [as described in (Hokenson et al., 2020 )], and has been previously shown to elicit a robust stress response that are greater than a single stressor alone (Maras et al., 2014 ). Briefly, adult male mice were put in a restrainer made from a 50-mL plastic tube (Corning, Corning, NY, USA), placed next to unfamiliar conspecifics (social stress) on a laboratory shaker, and jostled in a brightly lit room with loud rap music playing (dB level = 95) for 1 h. Small blood samples (~100 μL) were collected from the facial vein of each mouse at baseline, 30 min and 60 min after stress initiation, and serum was collected after clotting for ~30 min at room temperature and centrifugation at 1,200×g for 15 min. Serum was stored at −20°C until later assayed for ACTH and CORT using commercially available enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions (ACTH kit: cat# EK-001–21, Phoenix Pharmaceuticals, Burlingame, CA; CORT kit: cat# 501320, Cayman Chemical, Ann Arbor, MI). Due to the rapid release of ACTH relative to CORT, commencing upon the moment we disturbed the cage, the baseline ACTH samples were somewhat elevated in all groups (and not significantly different from each other), and thus were not included in Figure 4.