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-r