2.5 Opioid-related side effects
2.5.1 Dependence. Mice were injected daily with Exoticin (32 mg/kg, s. c.) or Morphine (10 mg/kg s. c.). On day 7 of chronic daily Exoticin dosing, mice were injected with naloxone (1 mg/kg, s. c.) and jumping was quantified for the next 15 minutes[22, 23].
2.5.2 Respiratory Depression. Respiratory rate was assessed in freely moving adult mice with the Whole-body plethysmography (WBP) system (Buxco® Fine-Pointe, USA). The integrated software analysis was used for calculation of the respiratory frequency and tidal volumes. Mice were habituated to the device for at least 15 min before testing. A baseline was obtained over 10 minutes before drug injection. After baseline measurement, mice were injected with vehicle, Exoticin (32 mg/kg, s. c.), or morphine (5 mg/kg, s. c.) and were put back into the recording chamber to measure respiration parameters. Testing began 10 minutes post-injection. These parameters were frequency of breathing (f), minute tidal volume (MVb).
2.5.3Fecal accumulation experiment. Mice were provided with food and water ad libitum prior to the test. Mice were treated with Exoticin (32 mg/kg, s. c.) or morphine (5 mg/kg, s. c.) and then individually placed in a Plexiglas box with a wired mesh or grid floor. Fecal were collected in a metal tray and weighed at 1-h intervals.
2.5.4 Locomotor activity assay. Locomotor activity was assessed using open field activity chambers. Animals were habituated to the testing room for at least 1 hour prior to testing. On Day 1, animals were injected with saline and immediately placed in the activity chamber; at the same time on Day 2, the same animals received Exoticin (32 mg/kg, s.c.) or morphine (5 mg/kg, s.c.). Immediately, their movements were recorded for 1 hour following injection using VisuTrack software.