A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

In the Dowling lab, we study the physiology and development of the retina in zebra fish in order to determine whether or not zebra fish mutants that are unable to detect motion can detect light and are not blind. We test their visual motor response or VMR to do this. We collect zebra fish at about four days post fertilization.

Place them in 96. Well plates prepare a suitable imaging rig, align the video tracking software with the plate, and then track the motor response of the fish following transitions from light to dark or dark to light, which is what we call the VMR. Hi, I'm Farida Imran from the laboratory of John Dowling and the Department of Molecular and Cellular Biology at Harvard University.

And I'm Jason Rio. I'm from Alexander Shear's Lab, also in the molecular and cellular biology department at Harvard University. Today we will show you the visual motor response in zebrafish to light increments and decrements.

So let's get started. To begin the visual motor response assay first grow wild type zebrafish larvae under a dark light cycle at 28 degrees Celsius until at least four days post fertilization. Our typical light dark cycle is 14 hours of lights on starting at 9:00 AM and 10 hours of lights off starting at 11:00 PM for the best behavioral results.

Avoid overcrowding the larvae. We usually keep no more than 50 larvae in a single Petri dish. After five days post fertilization, we transfer zebra fish to a 96 well plate with 650 microliter well size in order to give larvae a large swimming room.

Using a plastic transfer pipette gently transfer one larvae per well. After transferring fish into the wells, fill up each well with enough fish water such that the water surface is nearly flushed to the top of the wells. Be careful not to overfill or underfill the wells to avoid optical problems for the recording camera.

Also take care not to introduce bubbles into the wells. The fish are now ready to be monitored for their locomotive responses to light and dark. Let's take the plated zebrafish over to the recording apparatus.

So let's proceed by surveying the recording apparatus. The 96 well plate containing fish will be placed inside the recording chamber here in this white tray and held in place with a spring or rubber band. The camera is positioned in the back of the box here and is focused on the plate using mirrors off the top of the box located here.

The angle of these mirrors can be adjusted by turning the screws that hold the mirrors in place. The recording chamber is illuminated from the bottom by infrared LEDs. This allows the camera to record the fish even in the dark.

The larvae cannot detect IR light, so this constant IR illumination does not affect the experiment. White LEDs also illuminate the recording chamber from below. They're controlled separately from the IR lights for experiments that run for more than a few hours.

The chamber can be filled with gently running water to help maintain a constant temperature for the experimental duration. The water is pumped from a reservoir by a small aquarium pump and is heated to 28 degrees with a typical underwater aquarium heater. The water drains through a large drain hole and is recirculated into the reservoir to minimize stray vibration from the room.

The entire recording unit sits atop a heavy balance table. When the recording apparatus is ready, a box lid is used to cover the animals and further isolate them from light. Now let's proceed with the alignment of the 96.

Well plate with the computer grid of the video tracking software to do this first place, the 96 well plate containing the fish into the recording chamber. When using a water bath for long-term experiments, slowly place the plate in the water, giving the water level a chance to adjust without spilling onto the plate. In the viewpoint video track software check to be sure that all the larvae and the experiment are visible on the computer screen.

Using the controls of the software, align the grid of the video tracking software with the wells of the 96 well plate such that each fish is within a square of the grid. This step is very important for all your recordings. The tracking computer will be calculating the movement separately for each of these boxes.

So if you misalign the computer grid, some of the fish movements may be lost or even worse. Two adjacent fish will occupy the same area and be counted as one fish after the alignment. The timing for when the lights will go on and off is programmed into the computer.

We typically allow three hours of lighter dark adaptation in the box. This not only obtains a baseline activity level, but also gives the larvae an opportunity to calm down following the pipetting and handling. Next, we close the door of our recording chamber and start recording.

In practice, we record the activity of each fish per second, but the viewpoint software in fact records the frame by frame data. Here below the screen you can see a real time trace of a single fish. In terms of pixel movement, the white line indicates that the number of pixel changes is below a set threshold and the green indicates the pixel change is above the threshold setting.

The threshold value will depend somewhat on your particular camera and light setup. For our setup, we typically use a threshold of four pixels. That is, if fewer than four pixels are changing, it is considered background.

If more than four pixels are moving, it indicates the fish is moving. We empirically determined that this cutoff detects nearly all the larval swim and turn movements. Even though the recording boxes isolate the larvae fairly well, we still turn off the light in the room and take care to minimize interruptions with mechanical noise, such as closing and opening doors in the room, having a dance party or doing our exercise routines.

After the experiment is completed, transfer the collected data into an Excel sheet or into your favorite analysis suite. This is what the data looks like. It contains the time and seconds from the start of the experiment and the activity of each larvae per second.

Here is a graph showing the activity of a single larvae over the entire duration of the experiment with several on and off responses indicated by the arrows on the graph. This graph shows the average activity of 40 fish over the entire duration of the experiment with several on and off responses as indicated by the arrows. As you can see, the on and off responses are prominent and consistent.

This visual motor response experiment can also be performed on mutant fish. For these studies, we typically plate wild type fish and mutant fish on the same plate in a checkerboard outline for optimal control purposes. When using mutant fish on the same plate as wild type fish, make sure that you write down what type of fish was plated in each.Well.

Here is a suggestion on how to keep track of the fish plated in each.Well. Here are visual motor response results, which compare wild type and mutant larvae. Again, we see the prominent and consistent on and off responses from wild type larvae.

However, the chalk mutant larvae, which lack eye development, do not significantly increase their activity to either light increments or decrements and have a low baseline level of activity. These results compare wild type responses with the NO OKR mutants, also called NRC mutants. The NRC mutant was thought to be completely blind based on the OKR test.

Using the VMR test, we demonstrate that the NRC mutant has a normal off response and delayed and sluggish on response. Thus, the NRC mutant is not completely blind as had been previously thought. We've just shown you the visual motor response of zebrafish labate to light increments and decrements.

When doing this procedure. It's important to remember to do the experiment during the day as fish are more active and responsive to light intensity changes during the day versus during the night. So that's it.

Thank you for watching and good luck with your experiments.