Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

Our main research aims to identify the behavioral effects induced by environmental pollutants and drugs in fish. Also trying to understand the mechanisms leading to these effects. Asocial and anxiety-like behavior cannot be conveniently addressed using embryos.

We conduct studies targeting these behaviors in the adult zebrafish model. This protocol details how to implement behavioral tools with basic material resources to facilitate the reproducibility of tests and harmonize the results obtained within the scientific community. The assays presented in this protocol have proven to be highly reproducible and sensitive to genetic and pharmacological manipulations, making them variable and non-invasive tools fostering the neural circuit and molecular mechanisms underlying behavior.

Once a behavioral effort has been identified, the most challenging part is to determine the specific mode of action leading to the observative efforts. Starting by the molecular initiating event where predictions can provide some clues on the molecular initiating event, we are still far from obtaining accurate predictions. We are currently interested in determining the phase validity of the chemical models of Parkinson's disease developed in embryonic and adult zebrafish by using MPTP and rotenone, with the same, the kinematics of the gait-like movement or during an explosive movement like the acoustic evoked escape response, will be evaluated.

To begin, assemble the square experimental tanks, cameras, and computers for individual behavior assays of zebrafish. Fill the tank with seven liters of 28 degrees Celsius well-oxygenated fish water. Adjust the position of the tank in front of the camera to avoid distorted images.

Using the LED backlight, check the illumination setup and turn on the camera. Next, open the camera manager to check the availability of the jiggy camera on the computer. Launch the jiggy camera controlling software and open the camera option.

Then select Monochrome Mode and adjust the image size. Open Camera Properties, and under Camera, set the pixel clock to maximum and frame rate to 30 frames per second. Then adjust the exposure.

Now, under Image, set the gain to zero and adjust the black levels. In the Size tab, adjust the window dimensions to the region requiring engraving. Once done, close the camera properties.

Next, create a general folder for the experiment session to save the camera settings and videos. Then click File, Save Parameters, and To File to save the settings. After configuring all cameras, open Record Video Sequence and select Create to save as a new video file.

Select Max Frames and enter 10, 800 in the frame box. Then select Calc Frame Rate. Introduce the fish one by one at the bottom of the experimental tanks for behavioral tests, and click Record to start the recording.

For the shoaling test, introduce the shoal at the bottom of the experimental tank. For the social preference test, introduce the fish one by one at the bottom of the different experimental tanks. Once a chat box with a maximum number of frames achieved appears on the screen, click Accept.

Then select Close to conclude the recording. Finally, remove the fish recorded for behavioral tests from the experimental tank and separate them from naive fish. Open the analysis software for examining fish behavioral test data.

To elaborate on a new template, click on New from template, then Apply to predefined template, followed by From video file. Then select a video from the template setup. In the parameters window, select the model fish to adult zebrafish, the arena to open field square, to one arena, and the number of subjects per arena.

Then choose the type of detection by center point and adjust the frame rate to 30 FPS. Name the experiment as a template and store it in the same folder as the rest of the videos. Under Experiment Settings, review the configured setup.

In Arena Settings, right click on the center of the screen and select Grab. From File in the display, select a good quality video image and click Accept to capture this image for the background settings. Using the 19 centimeters width of the tank as a scale, calibrate the captured image to create a calibrated rule.

Then draw the arena, and using the frame function, delineate the shape zones. For the novel tank test and shoaling test, draw two equal horizontal boxes on the tank. Draw three equal vertical boxes for the social preference test.

Under Trial Control Settings, put an infinite time condition and remove the other components. Next, under Detection Settings, select the video for analysis from the video file and check the detection quality. Click on Auto detect to adjust the detection and refocus the animal.

Under Trial Settings, input one trial. Then under Data Profiles, create results dialogue windows. And under Analyze Profiles, select the parameters to analyze.

To copy and use the template, open the template file with behavior analysis software. Then go to File and click Save As to create a new identical file. Alternatively, select New from template, then apply to custom template.

Next, go to Arena Settings to readjust the template. Under Acquisition, select DDS. Then click Ready to Start to process the video.

Once the acquisition is complete, go to Track Editor to check the processed video. Finally, in Statistics, click on Calculate, then Export Data. Anxiety behavior assessed in a novel tank test of zebrafish exposed to methamphetamine exhibited a decrease in distance traveled, exploration distance, and time, and number of visits to the upper zone of the tank.

The latency time before the first visit to the upper zone increased significantly. Freezing behavior increased also significantly after three hours of methamphetamine exposure. Social behavior assessed in the shoaling test of the methamphetamine-treated fish displayed a behavioral phenotype of social isolation, with an increased average and farthest distances between individuals.

Social behavior assessed in the social preference test of the methamphetamine-treated fish showed a significant decrease in time spent and distance traveled in the conspecific zone.