2.8. Elevated Plus Maze (EPM)

This test assesses for signs of anxiety by considering the innate behavior of rats to prefer dark enclosed spaces over bright open spaces. An increase in open arm activity (duration and/or entries) reflects anti-anxiety behavior [32,34,35,39,43]. Like FST, EPM behavior could also be affected by baseline motor activity and fatigue experienced by the animal due to previous OP exposure [36,37,38].

The maze (Med Associates Inc., St. Albans, VT) was made of black polyvinyl chloride and consisted of four arms, 50 cm long × 10 cm wide, connected by a central square of 10 × 10 cm: two open without walls and two closed by 31-cm-high walls. All arms were attached to sturdy metal legs; the maze was elevated 55 cm above the floor level and was set in a dimly lit room. A video camera was suspended above the maze to record the rat movements for analysis. A video-tracking system (Noldus Ethovision XT 11) was used to automatically collect behavioral data. The procedure consisted of placing the rats at the junction of the open and closed arms, the center of the maze, facing the open arm opposite to where the experimenter was. The video-tracking system was started after the animal was placed in the maze so that the behavior of each animal was consistently recorded for 5 min. At the end of the 5 min test session, the rat was removed from the plus maze and returned to its home cage. The maze was cleaned with 70% ethanol and air-dried to remove any scent traces and allowed to dry completely before introducing the next animal in the arena. The time spent and entries made in the various arms of EPM were calculated.