Procedures

Procedure 1: Drug administration

Definitions & Abbreviations: Oxycodone (OXY); saline (SAL)

Reagents and solutions

• Oxycodone hydrochloride (Sigma-Aldrich, St. Louis, MO, USA)

Steps

1. Oxycodone hydrochloride (OXY) is dissolved in sterilized saline (0.9% NaCl) and administered in an injection volume of 10 µL/g.
2. Saline (SAL) is injected in a volume-matched quantity.

Procedure 2: Conditioned Place Preference (CPP) test

Equipment, software, and supplies

• Conditioned Place Preference apparatus, two chambers (20 x 40 cm); left and right sides are distinct through different floor textures
• Sound attenuating chamber, to house CPP apparatus, unlit
• Video camera (Swann Security Inc., Santa Fe Springs, CA, USA)
• ANYMAZE tracking software (Wood Dale, IL, USA)

Steps

Introductory Comments: Testing and training starts at 1000h; all training and testing sessions are 30 min, and test apparatuses are housed in unit sound attenuating chambers; video captured with overhead cameras and graded using tracking software.

1. On day 1, initial preferences are assessed, where mice are administered saline (SAL), placed into the left side of the chamber, and provided open access to both chambers.
2. On days 2 and 4, mice are confined to the right side of the chamber and administered 1.25 mg/kg oxycodone (OXY) i.p. or SAL (volume matched 10 µL/g i.p.) depending on which treatment group the mice are in.
3. On day 8, drug-free preference is measured by administering SAL, placing the mice into the left chamber and providing access to both sides for 30 min.
4. On day 9, OXY state-dependent preference is measured where the OXY group receives 1.25 mg/kg and SAL group receives only saline prior to assessing preference for 30 min.
5. Preference is operationalized as the difference in time spent on the drug-paired (right) side between either day 8 or day 9 and day 1.

Procedure 3: Hotplate: cumulative dosing

Equipment, software, and supplies

• Hotplate, 52.5°C, within a 15 x 33 cm Plexiglas cylinder
• R/drc

Environmental Conditions

Testing Area

Acclimation Period: 1h

Steps

Introductory Comments: Strains are tested for cumulative dosing hotplate to determine antinociceptive tolerance, and sensitivity to OXY induced antinociception.

1. On days 15-18, mice receive once daily injections of either OXY (40 mg/kg, i.p.) or SAL at 1600h.
2. On day 19, 16h after last injection, hotplate testing begins.
3. First mice are habituated to the testing room for 1h without homecage water (0700-0800h).
4. Starting at 0800h, two baseline pain measurements are taken for each animal in both treatment groups, separated by 30 min.
5. Following baseline testing, cumulative dose testing is conducted in groups of 8-12 mice until testing is completed. Cumulative dose testing consists of 5 consecutive injections (0.65, 1.25, 2.5, 5, 10 mg/kg OXY or equal volumes of SAL, depending on the treatment group). Testing occurs 10 min after the administration of each dose and concurrently with administration of the following dose to account for the time it takes to experience OXY analgesia.
6. Hotplate is maintained at 52.5°C and a pain response is considered a hind paw lick or jump, and a cutoff of 60s is enforced (mice that reached max analgesia before the final dose (i.e., 60s on the hotplate), are not tested for pain responses further, but are administered the remaining doses to control OXY exposure.
7. Maximum possible effect is calculated as: (HP latency at current dose - Ave baseline latency) / (60s - Ave baseline latency)
8. Half maximal effective concentration (EC₅₀) for OXY and SAL groups is calculated in R/drc using the drc function.

Procedure 4: Elevated Plus Maze (EPM)

Equipment, software, and supplies

• Elevated Plus Maze apparatus, four arms 35 x 5 cm, with 5 x 5 cm center zone, elevated 50 cm from base
• Video camera (Swann Security Inc., Santa Fe Springs, CA, USA)
• ANYMAZE tracking software (Wood Dale, IL, USA)
• Red light, for illuminating room

Steps

Introductory Comments: Strains are tested for EPM to assess the emotional component of spontaneous withdrawal. A session lasts 5 min. Video is captured with overhead cameras and graded using tracking software.

1. On days 22-25, mice receive once daily injections of either OXY (40 mg/kg i.p.) or SAL at 1600h.
2. On day 26, 16h after last injection, EPM testing begins at 0800h. Mice are placed alone in holding cages for 5 min prior to testing and returned to the homecage immediately following testing.
3. For testing, mice are placed in the center zone of an EPM apparatus in a red light illuminated room and tested for 5 min.
4. Adjusted open arm time is defined as [open arm time - center time] / [Testing time (300s) - center time].