2.7. Precipitated withdrawal test

Immediately after the end of the FR1 self-administration session, each mouse received an intraperitoneal (IP) injection of naloxone (1 mg/kg, 10 mL/kg) and were placed in a transparent box in front of a mirror at a 45° angle for the improved visibility of behavior. The observation and recording period lasted 20 min. The videos were analyzed by an experienced scorer who was blind to group assignment. The number of paw tremors (“clapping”), jumps, and wet-dog shakes were recorded, and each occurrence was attributed one point. The presence of other”physical” signs, such as diarrhea, genital grooming, abnormal posture, ptosis, salivation, and teeth chattering, were also recorded, and attributed one point each, regardless of frequency. A precipitated global withdrawal score was calculated as the sum of points [36, 15].