Fentanyl citrate salt

Fentanyl citrate salt, morphine sulfate salt, and (±)-trans-U50-488 methanesulfonate salt were purchased from Sigma-Aldrich. Fentanyl used for the neonatal pre-exposure was mixed in saline, given at a volume of 2.5 ml/kg, and injected subcutaneously (sc). Oral fentanyl was dissolved in distilled water or a sucrose solution. Morphine and U50-488 were mixed in saline and injected subcutaneously (sc) at a volume of 1 ml/kg. Drug doses were expressed in the forms listed above.

All drug doses were calculated according to the active component of the salt. All drugs [morphine sulphate (3.75–120 mg kg⁻¹; Hameln Inc., Hameln, Germany), fentanyl citrate (0.2–4 mg kg⁻¹; Rotexmedica, Trittau, Germany), and naloxone hydrochloride (2 mg kg⁻¹; Ratiopharm, Ulm, Germany), used to precipitate withdrawal] were freshly prepared prior to use and were injected subcutaneously in lightly restrained, unanaesthetized mice at a volume of 10 μl g⁻¹ body weight. For chronic infusion with Alzet osmotic minipumps (1007D), fentanyl citrate salt (2 mg kg⁻¹ day⁻¹) and morphine sulphate salt pentahydrate (17 mg kg⁻¹ day⁻¹) were obtained from Sigma-Aldrich (St. Louis, MO). Drugs were diluted in phosphate-buffered saline for acute injections or dissolved in sterile water and then diluted in phosphate-buffered saline for osmotic pump delivery. For electrophysiology studies, MK-801 was obtained from HelloBio (Princeton, NJ). [Met⁵]enkephalin (ME), bestatin and thiorphan were from Sigma-Aldrich.

Drugs were prepared as stock solutions and diluted to the final concentration immediately before use. Final concentrations and sources of the drugs were as follows: fentanyl citrate salt (0.01, 0.1, 1 μM; Sigma), picrotoxin (50 μM; Sigma), and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (CTOP, 10 μM; Sigma). Saline or dimethyl sulfoxide (DMSO) was used as vehicle control and the final concentration of DMSO control was less than 0.5% in all experiments. Incubation of hippocampal slices with drugs was performed in either a recovery chamber or interface-recording chamber as needed.