U50,488H (U50,488 methanesulfonate), morphine sulfate, β-Funaltrexamine (β-FNA), Norbinaltorphimine (norBNI) and heroin HCl was provided by the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). U50,488H and morphine sulfate were dissolved in water. NCP was synthesized as described previously (Wang et al., 2021) and dissolved in saline or deionized water at 0.3-0.5 mg/ml as a stock solution, aliquoted and frozen and before use diluted with saline or deionized water, respectively. In mice, compounds were injected s.c. or i.p. at 10 µl/g body weight. Solutions for rat IV administration (heroin HCl and NCP) were passed through a 0.22-micron sterile filter (Millex GV, Millipore Sigma, Burlington, MA) before injection. Naloxone-d 5 , a reference standard, was purchased from Cerilliant Corp (Round Rock, TX). Paraformaldehyde and compound 48/80 were purchased from Sigma-Aldrich (St. Louis, MO). Formalin (5%) were prepared with Paraformaldehyde (1.85%). Other commonly used chemicals were obtained from Sigma-Aldrich or ThermoFisher Scientific.