Protocol of morphine administration. Acutely treated mice (n = 9) were injected with a single dose of morphine (20 mg/kg, subcutaneously). Chronically treated animals (n = 9) were injected with increasing doses of morphine for 5 days (10, 20, and 40 mg/kg, subcutaneously; see Materials and methods). Control (n = 9) and acute morphine groups received injections of saline in the same time schedule as the chronic group received morphine. Animals were killed by decapitation 4 hours after the last injection. The time point used for tissue collection and gene expression analysis is indicated by the red arrow.