Naloxone

For use in in vitro assays, drug stocks of DAMGO and fentanyl (Sigma‐Aldrich, Gillingham, Dorset, UK), morphine (MacFarlan Smith, Edinburgh, UK), alfentanil hydrochloride, sufentanil (Cayman Chemical, Ann Arbor, Michigan, USA) and β‐FNA (Tocris, Bristol, UK) were each dissolved in sterile de‐ionised water and stored at −20°C. Carfentanil (Cayman Chemical) was dissolved in a small amount of 100% DMSO before dilution in water. For cell signalling assays, drug stocks were further dissolved in phenol red‐free DMEM on the day of experiments. For electrophysiological experiments, all compounds or drugs were purchased from Sigma‐Aldrich except naloxone and Compound 101 (Hello Bio, Bristol, UK) and prazosin (Tocris). Drug stocks were prepared in de‐ionised water (or 100% DMSO for Compound 101) at 1000× the desired assay concentration and stored at −20°C. Drugs were diluted into the superfusing aCSF solution at 1:1000 ratio at the time of the experiment.

Naloxone and MK-801 were purchased from Hello Bio (Princeton, NJ). [Met⁵]enkephalin and CTAP were purchased from Sigma (St. Louis, MO). UK14304 tartrate and idazoxan were from Tocris (R and D system, Minneapolis, MN). Morphine alkaloid was obtained from National Institute on Drug Abuse, Neuroscience Center (Bethesda, MD) and was converted to HCl salt with 0.1 M HCl as a stock solution. All drugs were diluted to the tested concentrations in artificial cerebrospinal fluid (ACSF) and applied during superfusion. All salts used in electrophysiological experiments were purchased from Sigma.