Photomotor Response Assay: A Method to Measure the Behavioral Response of Larval Zebrafish to a Sudden Change in Lighting Condition

- To begin, transfer 10 zebrafish embryos, with the help of a transfer pipette, to a beaker filled with 20 milliliters of water. Now, grow the embryos at 28 degrees Celsius, under the appropriate light dark cycle required for an experiment. Next, transfer the larvae into well plates. Make sure that each well contains one larva to give enough space for swimming.

Now completely fill each well with water. Place the well plate in the behavioral recording chamber and track larval movement using video tracking software. The larvae exhibit increased movement in the dark and decreased movement in the light when exposed to an abnormal light and dark cycle due to increased stress levels. Calculating the difference between the mean distance traveled during the last minute of an initial period and the first minute of the following period tells us the photomotor response of the fish.

In this protocol, we will examine the photomotor response in zebrafish and fathead minnow larvae exposed to caffeine. First, place the well plate containing the experimental fish in the behavioral recording chamber. Then, open the previously developed tracking protocol. In the video tracking viewer, ensure that all of the larvae are visible, that only one larva is present in each well, and that the wells are aligned with the defined observation areas.

Next, click on Experiment and Execute. Specify the name and save location of the data and click on the Several Live Images icon to highlight all of the predefined viewing areas. Finally, close the panel of the recording chamber and click Background followed by Start on the computer monitor.