Naltrindole hydrochloride

The rat PC-12 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China and cultured and treated them as described previously [25 (link)]. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS. The differentiated cells were maintained in 6-well plates or 12-well plates and randomly allocated to normoxic, MPP⁺ and hypoxic groups. The cells in normoxic group were incubated at 37°C in a humidified incubator with 5% CO₂. To induce hypoxia, cells were transferred into a hypoxic chamber (Galaxy 48R, New Brunswick, Edison, NJ, USA) with the O₂ levels being kept strictly at 1% for 48hrs. To induce MPP⁺ injury, cells were exposed to 1.0mM of MPP⁺ for 24 hrs after cell passage. MPP⁺ or 1-methyl-4-phenylpyridinium was purchased from Sigma Chemical Co (Cat: 15H467, St. Louis, MS, USA) and used at 1μM concentration. UFP-512 is a highly specific and potent DOR agonist that was synthesized by our research group [24 (link), 25 (link), 30 (link), 42 (link), 60 (link)] and was used to treat PC12 cells at 5μM concentration. The DOR antagonist, Naltrindole hydrochloride, was obtained from Tocris Bioscience (Cat: 0740, Bristol, UK) and used to treat PC12 cells at 5μM concentration.

UFP-512, a specific and potent DOR agonist was produced by our research group. Naltrindole hydrochloride was purchased from Tocris Bioscience (Cat: 0740, Bristol, UK). MPP⁺ (1-methyl-4-phenylpyridinium), MTT powder for cell viability, and fetal bovine serum (FBS) were all purchased form Sigma Chemical Co. (Cat: D048, 15H467, M2128, respectively, St. Louis, MO, USA). LDH cytotoxicity assay kit was purchased from Beyotime Biotechnology (Cat: C0016, Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) for cell culture was purchased from Gibco^®, Thermo Fisher Scientific (Cat: 11995-065, Waltham, MA, USA). Anti-PINK1 antibody was purchased from Novus Biologicals (Cat: BC100-494, Littleton, CO, USA). Anti-β-actin antibody, anti-caspase 3-antibody were all purchased from Cell Signaling Technology (Cat: 4970, 4691, 4060, 9662S, respectively, Danvers, CO, USA).

Stock solutions of a broad-spectrum opioid antagonist (naloxone hydrochloride (Tocris, Bristol, UK)), a μ-receptor antagonist (naloxonazine dihydrochloride (Tocris)), a δ-receptor antagonist (naltrindole hydrochloride (Tocris)) and a κ-receptor antagonist (nor-binaltorphimine dihydrochloride (Tocris)) were dissolved in pyrogen-free saline (PFS). Pilocarpine (1 mg/μl, Sigma-Aldrich, St. Louis, MO, USA) was also dissolved in PFS. The stock solutions were stored at 4 °C until use. Our previous results and others have indicated that the appropriate microinjection dosage for naloxonazine, naltrindole and nor-binaltorphimine to selectively block μ-, δ- and κ-opioid receptors, respectively, without interaction with other opioid receptor subtypes, is within 20 μg [12 , 13 , 32 (link), 33 (link)]. In the current study, naloxone, naloxonazine, naltrindole and nor-binaltorphimine were microinjected at a dose of 10 μg/μl, which according to our previous studies efficiently exhibits pharmacological blockade [12 , 13 ]. The total volume for each injection was 1 μl and the duration of injection was 3 to 5 min. Our previous study has demonstrated that microinjection of 1 μl solution into the CeA does not cause CeA lesion [34 (link)].

Stock solutions of deltorphin II (100 μM; Phoenix Pharmaceuticals, Belmont, CA) were made in water and stored at 4°C. Working concentrations of the ligand were diluted in SIF, as needed. Naltrindole hydrochloride (Tocris, Ellisville, MO) was reconstituted in water to a stock solution of 100 μM and was stored at −20°C. Each drug solution was warmed to room temperature and saturated with oxygen prior to use.