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

- To begin, grow the embryos in 3 Petri dishes. The first one is a control plate that contains Hank's solution-- a balanced salt solution. The second and third plates have Hank's solution with a potentially neurotoxic compound at different concentrations.

The neurotoxic compound in the exposure solution penetrates the embryos' external membrane by passive diffusion and alters the nervous system's processes such as attention, coordination, and muscle activity. Once the embryos develop into larvae of the appropriate age, transfer each larva into an individual well of a 48 well plate. Keep track of the treatment condition for each animal.

Transfer the well plate on to a recording platform and pull down the cover. Let the larvae acclimate for 10 minutes. Set alternating cycles of light and dark periods using the software.

Zebrafish larvae normally exhibit more movement in the dark and less movement in the light. Record the total distance traveled by the larvae of the control and the exposure groups in each period. If the compound is neurotoxic, larvae show even more movement in the dark and in the light than control animals.

In the following protocol, we will perform behavioral tests on the zebrafish larvae exposed to neurotoxic compounds.

- At least 2 hours before performing an experiment, set the room temperature to 28 degrees Celsius and add 800 microliters of exposure solution to each well of a 48 well microplate. Then use a 1 milliliter pipette with a modified tip to transfer one larva in 200 microliters of exposure solution to each well of the microplate, in preparation for a locomotion and path angle experiment.

When all of the parameters have been set, turn down the lighting in the test room and allow the larvae to acclimate in the system for 10 minutes. At the end of the acclimation period, click Experiment and Execute, and create the folder in which the experimental files are to be saved. Then enter the result name and click Background and Start.

At the end of the experiment, click Experiment and Stop. Then return the 48 well microplate back to the light incubator for subsequent experiments.