Zebrafish were released into an arena with one half of the tank surrounded by white corrugated plastic and the other half black corrugated plastic. Preference for either zone was calculated by cumulative time spent in each zone. Decreased time spent in the black zone was considered indicative of decreased anxiety. When the fish were released into the arena, the net was placed facing the long axis of the testing arena at the line dividing the black and white zones, to avoid biasing the fish to either side of the arena. The light/dark arena was 9.4 cm wide by 55 cm long and 9.5 cm deep and was filled to a water depth of 5 cm. The arena walls were surrounded by white and black corrugated plastic placed against the clear Plexiglas walls. White non-reflective corrugated plastic was attached to the bottom of the arena to provide contrast between the fish and the background (Fig. 1B). Each day, the arena was rotated 180 degrees after half of the fish had been tested to eliminate bias resulting from external visual stimuli due to the orientation of the arena. The light/dark test consisted of 5-min trials. Behaviours examined for analysis were duration of time spent in defined zones (light and dark half of the tank), average velocity, and time spent immobile.
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