Each morphine-treated subject was trained to come to the front of the enclosure for twice daily intramuscular (IM) injections (< 0.5 cc) of morphine sulfate (NIDA Drug Supply Program) dissolved in 0.9% saline. Injections were administered at 09:30 and 17:00 h. A gradual dosing escalation method (0.5 log unit increase every 3 days) was used to achieve the terminal dosage of 9 mg/kg/day (i.e., 4.5 mg/kg, BID). This dosage was selected to produce moderate opioid dependence124,125. All subjects received approximately 1500 mg of morphine over the 5–6-month period of chronic dosing. On the last day and approximately 3 h following the last IM morphine injection, each subject received an IM injection of ketamine (10 mg/kg) followed by 5.0 ml IV of a pentobarbital-based euthanasia solution (Beuthanasia-D).
