Larval Locomotion Assay: High-Throughput Method to Study Neurobehavioral Toxicity of a Compound in Zebrafish Larvae

1. Preparation for the behavior test

1. Transfer 800 µL of exposure solution into every well of the 48 well microplate.
   NOTE: Use 16 wells for every group (i.e., the control solution, 5 µg/L, and 50 µg/L group).
2. Use a 1 mL pipette tip to transfer 200 µL of exposure solution with one larva from the glass Petri dish into one well of the 48 well microplate.
3. Transfer 4 mL of exposure solution into every well of the 6 well microplate.
   NOTE: Use one 6 well microplate for every group.
4. Use a 1 mL pipette tip to transfer 200 µL of exposure solution with two larvae into each well of the 6 well microplate.
   NOTE: Every group has six repeating groups.
5. Make sure the temperature of the test room is 28 °C 2 h before the test.
   NOTE: Behavioral tests are usually performed in the afternoon.

2. Locomotion and path angle test

1. Save the protocol and turn down the light of the test room.
2. Acclimate the larvae in the system for 10 min and click the "Experiment" "Execute" button in turn, then choose the folder where the experiment files are saved and enter the result name.
3. Click the "Background" "Start" button in turn to start the test.
4. Click the "Experiment" "Stop" button in turn to stop the experiment when the test ends.
   NOTE: The system shows the data tested when the system stops. The data include the tracked distance at three speed classes and path angle numbers at eight angle classes of every minute. For the locomotion test in the presented example, the total distance in every light period (10 min) is calculated and the difference between the control group and the treatment groups compared.
5. Transfer the 48 well microplate back to the light incubator for other experiments.