St034307

Morphine (Sigma Chemical, St. Louis, MO, United States) was administered through a 100 µl subcutaneous injection in saline. For Morphine tolerance experiments, baseline mechanical paw withdrawal testing was performed before and after administration of 15 mg/kg of Morphine for 5 days (Liang et al., 2011 (link)). Baseline measurements were measured every morning before Morphine injection (Pre) to assess opioid-induced hyperalgesia, and 30 min post injection (Post) to assess tolerance. Escalating Morphine tolerance was performed similarly, except increasing doses of Morphine, starting at 10 mg/kg and increasing 10 mg/kg/day, were administered over 4 days. Day 5 and day 6 thresholds measured ∼18 and ∼42 h, respectfully, after last Morphine injection. In each model, Morphine was delivered twice per day (∼0800 and ∼1800 h).
ST034307 (6271, Tocris Bioscience, Minneapolis, MN, United States) was dissolved in 10% β-cyclodextrin with 5% DMSO in saline and administered in a 100 µl intraperitoneal injection. ST034307 or vehicle was administered 15 min after Morphine in tolerance experiments.
In separate experiments, Complete Freund’s Adjuvant (CFA, F5881, Sigma Chemical, St. Louis, MO, United States) was administered through an intraplantar injection (20 µl, undiluted) into the left hind paw.

AC1 was inhibited using the AC1 selective inhibitor ST034307 (Tocris, United Kingdom), which has been shown to demonstrate selectivity for AC1 over other AC isoforms at concentrations below 30 μM (Brust et al., 2017 (link)). In all experiments, ST034307 was dissolved in DMSO to make 3 mM stock prior to addition to experimental solutions at a final concentration of 1 µM and applied for at least 5 min for isolated cells, or 30 min for whole-tissue experiments in order to ensure sufficient tissue penetration.

ST034307 (6-Chloro-2-(trichloromethyl)-4H-1-benzopyran-4-one) was purchased from Tocris Bioscience and morphine sulphate from Spectrum Laboratory Products. Acetic acid, lactic acid, Tween 80, and formaldehyde were from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) and 0.9% sterile saline were from Fisher Scientific. ST034307 and morphine were prepared in a vehicle consisting of Dimethyl sulfoxide (DMSO), Tween 80, and milli-Q water (1:1:8). Specifically, ST034307 was first dissolved in DMSO and sonicated in a 50°C water bath for 15 min. Next, Tween 80 was added, the solution was vortexed, and the sonication was repeated. Warm (37°C) milli-Q water was added and the solution was vortexed immediately before injections. While morphine is also soluble in aqueous solutions, such as saline, we chose to dilute it in the same vehicle as ST034307 to avoid having to add an additional vehicle control condition to our experiments. These procedures reduced the number of animals required in all in vivo experiments performed in the study. Acetic acid, lactic acid, and formalin were diluted in 0.9% sterile saline.