Naltrexone

Naltrexone was obtained from Sigma Aldrich (N3136) and dissolved in water used for treatment and mixed in the NGM agar just before pouring. Worms were treated with compounds or water vehicles from the L4 stage onwards. Plates were changed at least once a week to ensure consistent exposure to the compound.

All peptides were prepared via fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid phase peptide synthesis and purified via reversed-phase high performance liquid chromatography-mass spectrometry (HPLC-MS) on an Agilent 6130 Quadrupole LC/MS system. Peptide hydrogels were prepared by weighing out the appropriate peptide mass in 2 mL-vials. After the requisite volume of phosphate-buffered saline (PBS) was added, the peptide-water mixture was shaken and sonicated for 10 s. The hydrogels were allowed to anneal for 24 h before they were used for further experiments. These procedures, along with the acquisition of precursors for peptide synthesis, were previously described in detail by us⁴¹ . Naltrexone, methotrexate, and doxorubicin were purchased from Sigma-Aldrich. For the drug elution studies, a stock solution of 20 mM in Millipore water of each of these drugs was prepared. methotrexate was neutralised with two equivalents of NaOH to produce the anionic form in solution. The stock solution was diluted to a final concentration of 2 mM in PBS prior to the preparation of the hydrogel for small-molecule drug elution studies (see below).

Guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS; 1,250 Ci/mmol) and [³H]naloxone (1 mCi/ml) were from PerkinElmer. Drugs and reagents were obtained from the following sources: β-chlornaltrexamine dihydrochloride (β-CNA), methionine enkephalin acetate salt hydrate (Met-Enk), dynorphin A (porcine), naltrexone, 6-β-naltrexol, nalmefene and materials for membrane preparation (all from Sigma); etorphine hydrochloride and morphine base pellets (from RTI); morphine hydrochloride (from Macfarlan Smith); nociceptin and naloxone hydrochloride (from Tocris).

Cocaine hydrochloride was received from the NIDA Drug Supply Program, nicotine bitartrate was purchased from Sigma Aldrich (St. Louis, MO, United States), and varenicline tartrate was purchased from Biotang (Lexington, MA, United States). Vehicle (0.97 mM HCl; salt equivalent of naltrexone HCl) and naltrexone HCl (N-3136; FW 377.9; Sigma-Aldrich) were used to pretreat animals prior to SOA preference testing. Benzaldehyde (#418099; 99.5%; Sigma-Aldrich; FW 106.12) was used to test for non-selective effects of naltrexone HCl. 2-nonanone (99%; CAS 821-55-6; FW 142.24; Arcos Organics) was used to show that animals could move away from the SOA target zone.

After 6 weeks of intermittent voluntary alcohol intake, Naltrexone treatment was initiated with three doses (0.03, 0.3, or 3 mg/kg) or saline based on previous studies (Daoura and Nylander, 2011 (link); Barson et al., 2013 (link)). Treatment was given in a Latin square design so that, at the end, all animals had received all doses and saline. Injections were given prior to the first alcohol session and followed by two wash out sessions each week for 4 consecutive weeks. Naltrexone (Sigma-Aldrich, Schenndorf, Germany) was dissolved in saline and administered subcutaneously (Williams and Broadbridge, 2009 (link)) at 1 ml/kg 30 min prior to alcohol access. Alcohol intake, by bottle weighing, was measured at 30 min, 2 h, and 24 h after alcohol access and data presented as Δ0-30, Δ30-2, and Δ0-24, respectively.

Naltrexone (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 0.9% saline and administered SC in a volume of 1 mL/kg. Commercially available sucrose was dissolved in tap water.