β funaltrexamine β fna

BU08070 was synthesized at the Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom as described previously (Cami-Kobeci et al., 2011 (link)). J-113397 and β-funaltrexamine (β-FNA) were purchased from Tocris Bioscience (Ellisville, MO, USA). Tumour necrosis factor- α (TNF-α) and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Zeocin was obtained from Invivogen (San Diego, CA, USA). Cell culture media and supplements were from Sigma-Aldrich Chemicals (St. Louis, MO, USA) or Invitrogen (Paisley, UK). All other reagents, unless otherwise stated, were purchased from Sigma-Aldrich (Poznan, Poland).
For animal studies, BU08070 was dissolved in dimethyl sulfoxide (DMSO), further diluted with 0.9% NaCl solution to the final concentration of 5% DMSO. Animals without treatment received vehicle alone. BU08070 was injected intraperitoneally (i.p.) at the dose of 1 mg/kg in the final volume 100 μl. The dose of BU08070 used in all experiments was selected based on preliminary studies. β-FNA and J-113397 were injected i.p. (at the dose of 1 mg/kg and 12 mg/kg, respectively) 15 min before BU08070.

MP1104 and naltrindole (>98% purity) (Váradi et al., 2015a (link)) were provided by Dr Susruta Majumdar (Center of Clinical Pharmacology, St. Louis College of Pharmacy and Washington University School of Medicine, USA). MP1104, DOP antagonist naltrindole, MOP antagonist β-Funaltrexamine (β-FNA) (Tocris Bioscience), and morphine were dissolved in a vehicle of dimethyl sulfoxide (DMSO), tween 80 (Sigma-Aldrich) and physiological saline in a ratio 2:1:7. The KOP antagonist nor-binaltorphimine (nor-BNI) (Tocris Bioscience) was diluted in physiological saline. The pre-treatment time was 15 min for naltrindole (15 mg/kg), whereas β-FNA (10 mg/kg) and nor-BNI (10 mg/kg) were injected 24 h prior to testing via subcutaneous (s.c.) injection. All drugs and solutions were stored in the dark at 4 °C. For the tolerance and withdrawal experiments, the naloxone, morphine pellets and placebo pellets were provided by the National Institute on Drug Abuse. Paclitaxel (Paclitaxel Ebewe; Sandoz Pty Ltd., NSW, Australia) was commercially available in concentrated liquid form (6 mg/mL). Paclitaxel was diluted to 0.4 mg/mL to give a mixture of absolute ethanol, Kolliphor EL (Sigma-Aldrich), and 0.9% saline in 1:1:18 ratio.

Ethanol was purchased from Scharlau (Madrid, Spain). β-funaltrexamine (β-FNA; an irreversible antagonist of the MORs) was obtained from Tocris (Bristol, UK). Stock solutions of β-FNA were prepared by dissolving the compound in the correct volume of distilled water to obtain 13.6 mM concentration of β-FNA. Aliquots of these solutions were then kept frozen at −20°C until use. Prior to use, aliquots of the stock solutions were conveniently diluted with artificial cerebrospinal fluid (aCSF) solution to the appropriate concentration (8.3 mM) (Sánchez-Catalán et al., 2009 (link)). Ethanol was also freshly dissolved in aCSF solution (from 87.5 to 750 mM) prior to intra-pVTA administration. The aCSF solution was prepared and stored at −20°C. Its composition was: 120.0 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25.0 mM NaHCO₃, 1.2 mM CaCl₂, 100 mM d-glucose, and 0.2 mM ascorbate, pH adjusted at 6.5 (Hipólito et al., 2010 (link))
All the other reagents used were of the highest commercially available grade.