Photomotor Response Assay: A Method to Measure the Behavioral Response of Larval Zebrafish to a Sudden Change in Lighting Condition

1. Calibration of Video-tracking Parameters

1. Prior to behavioral measures, set observation and calibration parameters in the video track software (see Table of Materials).
   1. Place a well plate in the recording chamber with at least 1 larval fish in an individual well. Use the plate and associated fish as representations to set calibration parameters.
   2. In the video track software, click "File | Generate Protocol", which will open a “protocol creation wizard” dialogue box. In the "Location Count" field, enter the number of individuals wells of the well plate and then click "OK".
   3. At the top of the screen, click "View | Full Screen", which will prompt the system to display an overhead camera view of the well plate.
   4. Click the "Draw Areas" icon, which appears as three multicolored shapes. To the right of the well plate viewing area, select the circle icon in the field labeled "Areas".
   5. Use the cursor to delineate the circular video tracking area in the top left well of the well plate. Select "Top-Right Mark" and then outline the viewing area of the top right well. Then, select "Bottom Mark" to outline the bottom right well.
      NOTE: After drawing the circular outline, its position will likely need to be adjusted.  To adjust the position of the outline, click “select” and then use the cursor to move the outlined area. Also, outlines can be replicated by clicking "Copy" and then "Paste". 
   6. After the top left, top right, and bottom right well tracking areas have been defined, click "Build" to prompt the software to automatically delineate the viewing areas of the remaining wells.
   7. In the area labeled "Calibration", click "Draw Scale". Use the cursor to draw a horizontal line across the plate. Once the line is drawn, a dialogue box labeled “Calibration measurement” will appear. Enter the well plate length and click "OK".
   8. Exit the drawing manager by clicking the "Draw Areas" icon.
   9. Click the "Tiles" icon.  Using the cursor, highlight all the boxes that appear on the viewing screen so that each box is green.
      NOTE: The Tiles icon appears as a group of six individual small squares
   10. "Click View| Full Screen".  To the right of the plate viewing area, click "Bkg" in the box labeled "Detection Threshold". Use the threshold adjustment bar to set the pixel detection threshold. Once, the appropriate pixel detection threshold is selected, click "Apply to Group".
       NOTE: This protocol sets the detection threshold at 13 in black mode for zebrafish observations and at 110 in transparent mode for fathead minnow observations.
   11. In the box labeled "Movement Threshold", enter the desired movement speed tracking parameters. Once speed parameters are set, click "Apply to Group".
       NOTE: This protocol sets small/large movements at 20 mm/s and inactive/small movements at 5 mm/s. These selections program the software to track larval fish movement at three different speed levels: inactive (freezing) = <5 mm/s, small (cruising) = 5–20 mm/s, and large (bursting) = >20 mm/s.
   12. Click "Parameters | Protocol Parameters" from the drop-down menu. In the dialogue box, select the "Time" tab. Enter the observation time and the integration time. After parameters are entered click "Ok".
   13. To set the light/dark photoperiod times and light intensity for each photoperiod open the light driver settings dialogue box by selecting "Light Driving" from the "Parameters" drop down menu.
       NOTE: See protocol video for setting multiple light-dark photoperiods.
   14. After the video tracking parameters have been set, save the observation protocol.
       NOTE: This protocol observes fish behavior over a 50 min period that includes a 10 min acclimation phase followed by 4 altering light/dark phases consisting of two 10 min light periods and two 10 min dark periods. The integration time is set to measure behavior for each minute of the 50 min behavioral trial.

2. Observation of Larval Fish Locomotor and Photomotor Behavior

1. Place the well plate containing experimental fish in the behavioral recording chamber.
2. In the video tracking software, open the tracking protocol developed in the next step.
3. In the video tracking viewer, check to make sure that all larvae are visible on the computer screen, that only one individual larva is present in each well, and that individual wells are aligned within the observation areas that were defined in steps 1.1.5 and 1.1.6.
4. Click on "Experiment | Execute".
   NOTE: The system will prompt the user to provide a name and location to save the observation data.
5. Once the name and save location of the observation data have been specified, click on the "Several Live Images" icon to highlight all the pre-defined viewing areas
   NOTE: This icon is located at the top of the computer screen and appears as a box divided into four smaller squares. Clicking on this icon will highlight all the pre-defined viewing areas.
6. Close the panel of the recording chamber and click "Background | Start" on the computer monitor.