TITLE: Light-dark box test for mice
AUTHORS: Csilla Novák, Andrés Mauricio Jaramillo Flautero, Matthias Prigge

[DESCRIPTION]
This protocol describes the setup and execution of the light-dark box test for mice, a behavioral experiment used to assess anxiety-related responses. The protocol involves preparing a light-dark box in a controlled environment, utilizing specific lighting conditions and camera settings for optimal observation and recording.

[STEPS]
SECTION: Pylon Viewer settings
1. Connect the camera to the computer and to a power source.
SECTION: Pylon Viewer settings
2. Open the Pylon Viewer and select the camera.
SECTION: Pylon Viewer settings
3. At AOI controls, crop the image, so that you only see the setup. You can only change the width and the height of the image when there is no continuous shot or recording.
SECTION: Pylon Viewer settings
4. With the help of a test mouse, you can set the image quality. On the camera, you can set the aperture and the focus. In Pylon Viewer, you can play around with Analog Controls > Gain (Raw), Black level (Raw), and Acquisition Controls > Exposure time (Raw) to achieve the best quality.
SECTION: Pylon Viewer settings
5. Go to Window and select Recording Settings. Set the Output format to .mp4, the fixed playback speed to 67 fps, and the Recording buffer size to 100 frames. Record a frame every 15 milliseconds. Stop the recording after 10 min. Set the output folder.
SECTION: Behavior
6. Transport the mice to the behaviour room and put the mice in individual cages.
SECTION: Behavior
7. Leave them in the lobby in darkness or with red light for at least 30 minutes.
SECTION: Behavior
8. Turn on the red lights in the room. Turn on the IR lights below the table.
SECTION: Behavior
9. Place the light/dark box on the table.
SECTION: Behavior
10. Clean the box and the table with 70% ethanol.
SECTION: Behavior
11. Hang the big light filter above the arena. This is to have a homogenous background.
SECTION: Behavior
12. Turn on the white lights above the arena. Have 310-320 lux of light intensity in the light box. Probably you will need an additional lamp to achieve this intensity.
SECTION: Behavior
13. Open Pylon Viewer and check settings (see below).
SECTION: Behavior
14. Bring a mouse to the behaviour room. Open the cage and gently get the animal out. Do not chase it around in the cage.
SECTION: Behavior
15. Block the opening between the two compartments of the arena.
SECTION: Behavior
16. Place the animal in the dark compartment. Depending on your handling protocol, you can put it in by the tail or in the palm of your hand.
SECTION: Behavior
17. Close the dark compartment and wait 3 seconds before you unblock the opening.
SECTION: Behavior
18. Make sure to start the recording at the moment when the opening is free.
SECTION: Behavior
19. Make a recording of 10 min.
SECTION: Behavior
20. The video file is saved automatically. Rename it, so that you can easily identify your recording.
SECTION: Behavior
21. Pick the mouse out of the arena and put it back in the cage.
SECTION: Behavior
22. Count poop pellets and take a note of them.
SECTION: Behavior
23. Clean the arena and the table with 70% ethanol. If you feel the necessity, you can rinse the area with distilled water.
SECTION: Data analysis
24. We use DeepLabCut to measure locomotion and the time spend in each compartment