Larval Locomotion Assay: High-Throughput Method to Study Neurobehavioral Toxicity of a Compound in Zebrafish Larvae
Transcription
– To begin, grow the embryos de 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 de 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 à 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 de the dark and less movement de the light. Record the total distance traveled by the larvae of the control and the exposure groups de each period. If the compound is neurotoxic, larvae show even more movement de the dark and de the light than control animals.
In the following protocol, we will perform behavioral tests on the zebrafish larvae exposed à neurotoxic compounds.
– At least 2 hours before performing an experiment, set the room temperature à 28 degrees Celsius and add 800 microliters of exposure solution à each well of a 48 well microplate. Then use a 1 milliliter pipette with a modified tip à transfer one larva de 200 microliters of exposure solution à each well of the microplate, de preparation for a locomotion and path angle experiment.
When all of the parameters have been set, turn down the lighting de the test room and allow the larvae à acclimate de the system for 10 minutes. At the end of the acclimation period, click Experiment and Execute, and create the folder de which the experimental files are à be saved. Then enter the result nom and click Background and Start.
At the end of the experiment, click Experiment and Stop. Then return the 48 well microplate back à the light incubator for subsequent experiments.
