Using a variant of the optomotor response as a visual defect detection assay in zebrafish
2Department of Biology, American University, 4400 Massachusetts Ave NW, Washington, DC 20016, USA
3Center for Behavioral Neuroscience, American University, 4400 Massachusetts Ave NW, Washington, DC 20016, USA
4Department of Chemistry, American University, 4400 Massachusetts Ave NW, Washington, DC 20016, USA
5Department of Psychology, American University, 4400 Massachusetts Ave NW, Washington, DC 20016, USA
6Department of Computer Science, American University, 4400 Massachusetts Ave NW, Washington, DC 20016, USA
- PsychoPy Psychophysics code package 
- Python-based stimulus code (source code available at github.com/MattLeFauve/OMRProject)
- Computer capable of running PsychoPy
- Standard Computer Monitor
- Larval Petri dish (100 mm diameter)
- Adult Dish (28 cm diameter)
- Video Recorder (Canon FS40 Handheld was used in this study)
- Closed Door Behavior Chamber/Cabinet (optional)
- Video playback software (VideoLan VLC Mediaplayer was used in this study)
- Repeating stopwatch
Assembling the OMR setup
Running the OMR setup
1.Install Psychopy on your computer (http://psychopy.org/installation.html).
2.Download the stimulus from GitHub. Please see Table 1 to determine how to modify the stimulus if necessary for your experimental setup.
3.Connect the monitor and the computer and test that the stimulus successfully fills the monitor screen. Prior to playing the stimulus, the monitor should be calibrated using the tools provided on a Windows or Mac computer (https://www.digitaltrends.com/computing/how-to-calibrate-your-monitor/). This will ensure the stimulus is presented at the optimal optical resolution.
4.Place a 20.32 × 25.4 cm (8 in × 10 in) transparent film/acetate on the monitor to prevent water damage. Prior to recordings, run the stimulus and position the experimental dish on the monitor to determine the optimal location for both the adult and larval dishes. The dish should be directly above the stimulus and positioned so the convergent point for the pinwheel is in the center of the dish.
5.Once the optimal dish position is determined, trace the outline of the bowl or make other markings, so you can consistently position the dish in the same location between trials.
6.Position the camera above the monitor, inside the behavior chamber (Fig. 1). Camera height is somewhat arbitrary (in our case ~45.72–60.96 cm above the dish) but should be high enough to allow movement/transferring of bowls and fish below it, but low enough so that the experimental dish fills the video screen. Recording within a behavioral chamber is optimal, as it prevents distraction of the fish and also allows control of external lighting while taking videos.
7.To prevent monocular visual distraction for the fish during stimulus presentation, place non-reflective tape around the outside of the bowl to the water line.
8.Place a physical barrier such as a small straight sided glass container over the central portion of the testing dish to prevent visual distraction at the point source of the stimulus as demonstrated in Figure 1B. To prevent visual distraction stemming from this barrier, the center barrier should also be covered with non-reflective tape. This provides a swimming arena for the fish that is an annulus, so the fish swims either clockwise or counterclockwise within the dish.
Presentation of the OMR
9.Fill the trial dish ~2/3 full with fresh system water. The larvae should be in 100 mm diameter dishes and the adults should be in 28 cm diameter dishes (see equipment list).
10.Place up to 10 larvae or 1–2 adult fish in the dish. Position the glass container (step 8 above) in the center of the dish.
11.Transfer the dish containing fish into the behavioral chamber and position it on the monitor, taking care to make sure it is in the center of the monitor and aligned with the position markers (steps 4–5 above) on the transparent cover. Allow the fish at least three minutes to acclimate to the testing chamber. Acclimation to the dish is performed within the recording chamber.
12.Turn the camera on and be sure the fish are visible.
13.Begin recording. Following the directions in the “OMR_Stimulus” code, project the stimulus below the fish. The stimulus is designed to rotate clockwise then counterclockwise with a blank gray screen in the middle. The default timing is 30 s for each direction and the blank screen, but timing can be altered by changing the second number in the TIME line. Altering the TIME line number will change the seconds of each stimulus direction, including the blank screen. The provided stimulus code has been written at the optimal speed and angular cycles to elicit an optimal response for each age group. The optimal speed and angular cycle number are presented in Table 1.
14.After allowing the stimulus to make as many full rounds of clockwise-gray-counterclockwise as necessary, stop the stimulus by pressing the spacebar and stop the video recording. Zebrafish tend to exhaust and need swimming recovery time after three to four full rounds of the stimulus when presented at 20 s intervals.
15.Once the behavior has been recorded, return fish back to the holding/stock tanks.
16.16. This process can be repeated as many times as necessary to behaviorally test each fish. Extinction of the OMR is possible however, so individuals should not be tested with more than 10 full rounds (clockwise, gray, counterclockwise) in a 48 h period.
|Time||Seconds that the stimulus and blank “recovery” screen are presented||30–60 s is the typical presentation time that reduces the potential for rapid exhaustion|
|Speed||Time (in seconds) that it takes for one angular cycle to go one full revolution. Number = rad/s||Larvae Stimulus: 1.04 Adult Stimulus: 1.033|
|Grating (angular cycles)||Number of angular cycles presented||Prime larval response: 16 angular cycles Prime adult response: 12 angular cycles Spatial resolution analysis tested 2–64 angular cycles|
|Grating (contrast)||Strength of the leading edges of the stimulus||The best OMRs were elicited by strong leading edges (0.9–1.0) (LeFauve, 2015, personal observation) We do not recommend changing this parameter in the stimulus code|
Analysis of the video, detection assessment for visuomotor defect discovery
17.Ensure fish are visible in the recorded video before proceeding with data analysis. Adjustment of the camera position may be necessary.
18.Movement of the fish in response to each stimulus can be analyzed in three ways, described below. In general, as this method is designed to be implemented easily without costly behavioral software or time-costly coding setup, it is not well-suited to using currently available open-source computer vision tracking software such as PathtrackR .
18.1.Scan sampling—This can be applied to both larvae and adults but works best for larvae as the size of the Petri dish may preclude the larvae from making a full revolution during stimulus presentation. Scan sampling behavioral analysis can be used to assess group behaviors quickly. To assess OMR success, at 10 s intervals, perform a rapid clockwise sweep of the dish and count the individuals moving in the direction of the stimulus. All individuals moving with the direction of the stimulus at the time of the sweep are to be counted as demonstrating a positive OMR . Individuals not moving are therefore not showing an OMR and individuals moving in the opposite direction are showing a negative OMR. For ease of analysis, this study combined individuals showing no OMR and showing a negative OMR into a “non-positive OMR” group. If comparing treatment groups, it may be helpful to count the number of larvae moving during the break period when the stimulus is not being presented. Zebrafish have been shown to have motion aftereffect and so the first sweep of fish should not be counted using this method.
18.2.Counting the number of full revolutions—Adults are able to swim completely around the dish without exhaustion during the stimulus interval, so the number of full revolutions can be counted. Counting the number of times each fish makes one full revolution around the dish during the stimulus presentation is similar to counting individuals following the OMR stimulus when presented in a unidirectional pattern. This method works well for adult stimulus presentations.
18.3.Combination of observation and computer tracking. Open source behavioral software may be a viable option to track the fish in this behavioral setup. Easier to use options that the authors recommend include PathtrackR , ZebraZoom . Current open-source behavioral tracking software performs better when there is a constant background as most computer vision-based tracking is done using organism-background contrast differences. As a result, a high contrast pinwheel stimulus, presented below the fish, may result in automated tracking errors, as a dark fish above a dark portion of the stimulus would be ‘lost’ and not counted. Thus, the method described here may lend itself better to observer-based video tracking rather than computer-based tracking.
Analysis of the video, spatial frequency analysis
19.If necessary, repeat “Presentation of the OMR”. It is possible (and necessary when recording responses in larval vs. adult fish) to change the number of angular cycles presented to the fish, explanation of this can be found in Table 2. Angular cycles presented can be altered by changing the “angular cycles” number in the “grating” lines. Up to three angular cycle amounts can be tested in one recording session before the zebrafish will stop responding. If more angular cycle tests are needed for an individual, recovery time after bout swimming can take up to 15 min (LeFauve, 2015, personal observation). Recovery can be conducted in the testing apparatus with the monitor turned off. As stated above, zebrafish should not be tested with more than 10 full rounds (clockwise-gray-counterclockwise) in a 48 h period.
20.Repeat step 17.
21.Movement of the fish in response to each angular cycle number can be done using the same measurements provided in step 18. This will generate an optimal response curve (Fig. 2B). The optimal response curve indicates what angular cycle demonstrates the strongest response based on either the number of individuals exhibiting a positive OMR or the number of revolutions made by each individual in a treatment group.
22.The results are the proportion of time fish swim in one direction vs. either the frequency of grating (the number of angular cycles presented, as in Fig. 2B) or the visual angle subtended by one cycle of the grating on the fish retina. The visual angle can be calculated by the arctan of the distance from the fish to the screen (Y in Fig. 1A) divided by the distance covered by one angular cycle. In our example, the distance from the fish to screen was 40 mm for adults and 10 mm for larvae, the distance of the cycle depends on the fish’s radial position in the dish (as in the X1 and X2 values in Fig. 1B). The width of the angular cycles can be calculated by dish circumference divided by the number of angular cycles.
APPLICATION AND VALIDATION
|“Running OMR Setup”||Fish are jumping out of the OMR bowl||Fish may be startled by the light source below them||Letting the fish acclimate to the OMR setup may eliminate this problem, and after acclimation, the authors did not experience fish leaving the testing chambers. If this does not solve the issue, placing a clear piece of acrylic on top of the testing container may be necessary.|
|14||Larvae are remaining active during the blank ”control” period of the stimulus presentation||Fish are impacted by motion aftereffect motion as elicited by this stimulus||Do not count the number of larvae swimming during the first 10 s scan sample interval to allow them time to overcome the visual illusion of motion aftereffect OR only count the fish locomotion while the stimulus is being presented.|
|15||Testing visual acuity with variable angular cycle amounts, but needing speed to be consistent||Increased angular cycle amount will result in the stimulus increasing in speed based on stimulus code. This can be accounted for by changing the “SPEED” line.||
Below are the numbers to insert into the “SPEED” line for given angular cycles:
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