Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Our protocol provides a clear process for studying the neurobehavioral effects of the substances and the pollutants of interest on zebrafish larvae and for revealing the potential neurotoxicity of these agents. The technique can be performed using high throughput testing of neurobehaviors in the zebrafish model. This method can be applied to evaluate allele-neurobehavioral effects of pollutants to demonstrate potential neurotoxicity on us humans, as zebrafish genome has a high homology with humans, so it can provide indications for chemical management and the public health problems caused by pollutants.

As zebrafish has the advantage of high throughput screening, this method provides insight into both development of psychotropic drugs and environmental toxicology research. Don’t be intimidated about studying the first experiment. If you follow the protocol, you will have a successful outcome.

At least two 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 one-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. To prepare a six-well microplate for social behavior experiments, add four milliliters of exposure solution to each well of the six well microplate and transfer two larvae in 200 microliters of exposure solution to each well.

Before beginning the test, open the high throughput monitoring software program and select the tracking rotations path angles module. Transfer the 48-well microplate to the recording platform and pull down the cover. Click File and Generate Protocol to begin generating a new protocol and enter 48 into the Location count position.

Click Parameters, Protocol Parameters, and Time, set the Experiment duration to one hour and 10 minutes, and the Integration period to 60 seconds. Using the elliptical shape tool, draw a circle around the top left well. Select the circle and click Copy, Top Right Mark, Paste, and Select.

Use the mouse to drag the copied circle to the top right well and click Copy, Top Right Mark, Paste, and Select again. Then, drag the copied circle to the bottom right well and click Build and Clear Marks. The system will automatically draw a circle over every other well of the plate.

Next, click Draw Scale and draw a calibration line on the screen. Enter the length of the line, set the unit, and click Apply to Group. Set the animal color to black and set the detection threshold to 16 to 18 to allow detection of the animals, set the inactive to small speed to 0.5 centimeters per second and the small to large speed to 2.5 centimeters per second.

Set the path angle classes from minus 180 to plus 180 as indicated. To set the light conditions, click Parameters, Light Driving, Uses one of the 3 triggering methods below, and Enhanced stimuli to set the light conditions. Click Edge and set a dark period of 10 minutes, followed by three cycles of alternating 10-minute light and dark periods.

Then, save the protocol. 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.

Because every group can include 16 animals, the system can be used to perform high throughput tests of the locomotion and path angle under different treatment conditions in a single 48-well microplate. The social activity of the larvae can be tested using six-well plates. In this representative experiment, the highest concentration group of BDE-47 demonstrated a pronounced hypoactivity during the dark period, while no changes due to BDE-47 exposure were observed during the light periods.

Further, the high concentration group of 6-hydroxy-BDE-47 performed fewer routine and average turns at five days post-fertilization. However, more responsive turns were induced in the 6-methoxy-BDE47 exposure groups. The social activity of the zebrafish was stimulated by chlorinated paraffin-70 and the short-chain chlorinated paraffin-52b, while the long-chain chlorinated paraffin-52a shortened the duration for contact of the larvae.

It’s important to make sure that the transferred larvae for the test should have a normal swimming ability and no malformations. Other variables, such as color preference, light-dark preference, all darkness, tail movements of zebrafish, can be included to answer additional questions about neurobehavioral toxicity of environmental pollutants. The BDE-47 exposure solutions are neurotoxic and have endocrine disruption potential, so be sure to avoid direct contact with skin.