Light-Dark Preference Test: A Method to Study Anxiety-Related Behavior in Zebrafish

- First, create a three-sided enclosure around the testing area to minimize external stimuli that may affect fish behavior. Set up diffuse overhead lighting to avoid water surface reflections. Next, fill the arena, which is divided into dark and light zones, with habitat water maintained at 28 degrees Celsius.

Place the arena in the enclosure. Keep the habitat tank-- containing fish to be tested-- outside the enclosure. Let the fish acclimatize to reduce their stress.

Now, gently net a fish from the habitat tank and place it in the test tank. Position the fish perpendicular to the long sides of the arena to avoid bias toward either zone. Observe the behavior of the fish using recording software and repeat the test for the rest of the fish. Measure the average time spent by each fish in the light and dark zones.

Zebrafish tend to spend more time in the dark zone when they are anxious. In the example protocol, we will perform a light-dark test to observe the effect of ethanol exposure on zebrafish behavior.

- Obtain a light-dark arena 9 and 1/2 centimeters wide by 55 centimeters long and 9 and 1/2 centimeters deep with a white waterproof floor. Affix white and black waterproof, non-reflective paper to the inside walls of the arena using Velcro, with half of the arena covered in white and half covered in black. Fill the arena to a depth of 5 centimeters with habitat water at a temperature of 25 to 28 degrees Celsius. Maintain this temperature throughout testing.

Minimalize external visual stimuli by constructing a white three-sided enclosure for the arena to be placed in. Ensure the testing area has diffuse overhead lighting that does not cause reflections on the water surface, yet is sufficiently bright for the movement tracking software or post hoc manual quantification from video images. Then set the recording and movement analysis parameters of the behavior tracking software with a trial duration of 5 to 15 minutes, depending on the research question.

Transport the group of fish to be tested to the research area in the habitat tank and place them outside of the arena enclosure. Allow the fish to acclimate for 10 minutes. Gently net a fish from the appropriate group and place in the center of the light-dark arena, releasing each fish when it is positioned parallel to the long axis of the arena to avoid biasing the fish to the light or dark zone.

Begin recording behavior immediately after the animal is released. Watch for any software problems with tracking the fish or for fish jumping or freezing. After the trial has ended, gently net and remove the fish from the arena to a holding tank or habitat tank. Rotate the arena 180 degrees after half of the subjects have been tested to prevent any confounds due to biases resulting from which end of the arena is oriented toward the open end of the enclosure.

Begin analysis by examining the time spent in light versus dark zones. For each group and for each fish, obtain the relative time spent in the light and dark zones and analyze using a one sample t-test to determine if groups significantly prefer one area over the other. To compare preferences, calculate a preference index by subtracting the time spent in the light zone from the time spent in the dark zone and compare differences between groups using t-tests. Compare multiple groups with a one-way analysis of variance, utilizing Tukey's honest significant difference post hoc test where necessary.