Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus

- To begin, place five larvae in each lane of a six lane agarose plate. Add additional egg water, but don't overfill the lanes. Egg water contains methylene blue, which prevents mold growth. Let the larvae acclimatize for at least 10 minutes to reduce their stress.

Now place the well plate in an imaging chamber over a laptop screen. Focus the camera on the well plate. Fill the lane completely with egg water.

Next, record the behavior of the larvae placed on the blank background by taking time-lapse images. Display a moving red bar on the laptop screen, and take time lapse images of the larvae in response to the moving red bar.

The red shape is perceived as a threat, an aversive stimulus, and causes the larvae stress. In response, they avoid the bar and demonstrate positive thigmotaxis. Positive thigmotaxis is the fish's response to the stimulus seeking contact with the wall and swimming at the edge of the lane.

In the example protocol, we will analyze the avoidance and thigmotactic responses of zebrafish larvae treated with pharmaceutical compounds or environmental toxins.

- To begin image capture, carefully move the larvae from the Petri dish to the agarose lane to help reduce larvae stress. Up to 20 larvae can be placed in each lane, but five larvae per lane are typically used to facilitate the most accurate tracking of swim speed and to reduce the number of larvae that are needed per experiment.

Fill the lanes with egg water with or without pharmaceuticals or toxicants depending upon the experiment. Do not fill up the lanes all the way until they are placed in the imaging cabinets to prevent overflow. Allow 10 minutes for the larvae to acclimate. After the acclimatization period, position four plates by hand directly on top of the laptop screen.

At this time, the lanes can be topped off with egg water or chemical treatment so that it is level with the top of the lane, to eliminate shadows on the edges of the lanes in the images.

Program the computer for time lapse photography, taking pictures every six seconds for a total of 300 images per experiment. Set the camera at a lower resolution for imaging at video speed. While the lower resolution limits the recordings to a single multi well plate, the video recordings are appropriate for imaging rapid swimming events.

Use a PowerPoint presentation as an aversive stimulus for the larvae. In this demonstration, the PowerPoint starts with a blank white background for 15 minutes, followed by 15 minutes of a moving red bar on the top half of the plate. To avoid evaporation of the liquid within the agarose lanes, keep the maximal imaging time to below one hour.