analysis of recorded videos for behavioral testing of adult zebrafish

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

The zebrafish (Danio rerio) is a popular model vertebrate species for ecotoxicology, drug discovery, and safety pharmacology studies. Its low cost, well-established molecular genetic tools, and conservation of key physiological processes involved in the morphogenesis and maintenance of the nervous system make zebrafish an ideal animal model for neuroscience research, including neurobehavioral toxicology1,2. The main endpoint for evaluating the neurotoxicity of a chemical was, until recently, the presence of neuropathological effects. Lately, however, behavioral endpoints have been incorporated into neurotoxicological screening protocols, and these functional outcomes are now commonly used to identify and determine the potential neurotoxicity of chemicals3,4. Moreover, behavioral endpoints are highly relevant from an ecological point of view, as even a very mild behavioral change in fish could endanger the survival of the animal in natural conditions5.
One of the most used behavioral assays in adult zebrafish research is the novel tank test (NTT), which measures anxiety-like behavior6,7. In this assay, fish are exposed to novelty (fish are placed in an unfamiliar tank), a mild aversive stimulus and their behavioral responses are observed. NTT is used to assess basal locomotor activity, geotaxis, freezing, and erratic movements of fish, principally. Erratic8 is characterized by abrupt changes of direction (zigzagging) and repeated episodes of accelerations (darting). It is an alarm reaction and is usually observed before or after freezing episodes. Freezing behavior corresponds to a complete cessation of the fish&#39;s movements (except for opercular and ocular movements) while on the bottom of the tank, as distinguished from immobility caused by sedation, which causes hypolocomotion, akinesia, and sinking8. Freezing is usually related to a high state of stress and anxiety and is also part of submissive behavior. Complex behaviors are excellent indicators of the state of anxiety of animals. NTT has been shown to be sensitive to pharmacological and genetic manipulation9, making it a valuable tool for studying the neural basis of anxiety and related disorders.
Zebrafish are a highly social species, so we can measure a wide range of social behaviors. The shoaling test (ST) and the social preference test (SPT) are the most used assays to assess social behavior10. The ST measures the tendency of fish to group together11 by quantifying their spatial behavior and movement patterns. ST is useful for studying group dynamics, leadership, social learning, and understanding the social behavior of many fish species12. The SPT in adult zebrafish was adapted from Crawley&#39;s preference for social novelty test for mice13&#160;and quickly became a popular behavioral assay for the study of social interaction in this model species14. These two tests have also been adapted for use in drug screening assays and have shown promise for identifying novel compounds that modulate social behavior15,16.
In general, behavioral assays in adult zebrafish are powerful tools that can provide valuable information on the behavior mechanisms or the neurophenotypes of active compounds and abused drugs17. This protocol details how to implement these behavioral tools7 with basic material resources and how to apply them in toxicity assays to characterize the effects of a wide range of neuroactive compounds. In addition, we will see that the same tests can be applied to assess the neurobehavioral effects of acute exposure to a neuroactive compound (methamphetamine) but also to characterize these effects after chronic exposure to environmental concentrations of a pesticide (glyphosate).

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

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. A social 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 noninvasive tools fostering the neural and molecular mechanisms underlying behavior.Once a behavioral effort has been identified, the most challenging part is to determine the specific modes 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 the 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 kinematic of the gait-like movement or during an explosive movement like the acoustic evoked escape response, will be evaluated.

Watch this Scientific Journal Video about Analysis of Recorded Videos for Behavioral Testing of Adult Zebrafish at JoVE.com

Analysis of Recorded Videos for Behavioral Testing of Adult Zebrafish  

Open the Analysis Software for examining fish behavioral test data. To elaborate on a new template, click on New from template, then Apply a pre-defined template followed by From video file. Then select a video from the template set up.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.

analysis-of-recorded-videos-for-behavioral-testing-of-adult-zebrafish

Research

JoVE Journal

Neuroscience

156 Views.  Institute for Environmental Assessment and Water Research (IDAEA-CSIC). Open the Analysis Software for examining fish behavioral test data. To elaborate on a new template, click on New from template, then Apply a pre-defined template followed by From video file. Then select a video from the template set up.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 experim...