Twenty-four hours after the last extinction session (day 13), rats were tested for foot shock-induced reinstatement. During this session, rats were exposed to 15 min of intermittent foot shock stress (0.5 mA, 0.5 s ON, a mean OFF period of 40 s) and then immediately monitored for lever-pressing in extinction conditions for 3 hours. Forty-eight hours later (day 15), rats were tested for heroin-induced reinstatement. No additional extinction was given between the two tests. For this second test, rats received an injection of heroin (0.25 mg/kg s.c.), and following a 10-min interval, lever-pressing in extinction conditions was monitored for 3 h. Twenty-four hours following the heroin reinstatement session (day 16, 9 days after the last self-administration session), the experiment was concluded by exposing the rats to 15 min of intermittent foot shock stress (0.5 mA, 0.5 s ON, a mean OFF period of 40 s) in the operant chambers. Shortly after (45 min) this stress “challenge,” rats were sacrificed by decapitation following brief exposure to CO2 (approximately 15 seconds), and specific brain and pituitary regions, as well as plasma were collected for subsequent mRNA and hormone analyses.
