TITLE: Elevated plus maze test for mice
AUTHORS: Csilla Novák, Andrés Mauricio Jaramillo Flautero, Matthias Prigge

[DESCRIPTION]
The elevated plus maze is a classical test to screen anxiety-like behaviors in laboratory animals. This protocol is optimized to  mice. The protocol also describes the use of Biobserve Viewer, which is is a software to track online the movement of animals.
Generally, less time spent in the open arms of the maze is associated with higher levels of anxiety.

[STEPS]
SECTION: Biobserve Viewer
1. Open Biobserve Viewer.
SECTION: Biobserve Viewer
2. Make sure that the arena is well positioned in the middle of the view of the webcam.
SECTION: Biobserve Viewer
3. Go to Configuration and Experiment. Set “stop experiment after: “ to 10 minutes. If you are alone, it is best to tick “Delayed start”, so you can start the recording at the moment when the animal is put into the arena.
SECTION: Biobserve Viewer
4. At Camera, go to Capture settings and disable auto focus.
SECTION: Biobserve Viewer
5. At Filters and Objects, calibrate the camera. It is easiest if you highlight the center of the maze and tell the software its dimensions (5x5 cm).
SECTION: Biobserve Viewer
6. If you click on the Area configuration button, you can select the tracking area. Make sure that the whole arena is within the tracking area.
SECTION: Biobserve Viewer
7. You can make a background filter. With this, you can mask the walls and the background, so the tracking system will not confuse the mouse with dark areas.
SECTION: Biobserve Viewer
8. With a mouse which does not participate in your experiment, you can optimise the sensitivity of the background filter and the animal filter. Recommended values:
Background filter sensitivity: 20
Animal filter: 35
Min. animal size: 270 pixels
SECTION: Biobserve Viewer
9. Define the tracking zones in Zone Definition. I used the following zones:
center (5x5 cm)
closed arm north (5x25 cm)
closed arm south (5x25 cm)
open arm west (5x25 cm))
open arm east (5x15 cm)
SECTION: Biobserve Viewer
10. Reference point is the body.
SECTION: Biobserve Viewer
11. If you would like, you can define velocity classes.
SECTION: Biobserve Viewer
12. At Saving options, set the summary to 120 seconds (this will give you information about the mouse’s behaviour in 2-min bins).
SECTION: Biobserve Viewer
13. Tick “Record movie” and set the frame rate to 25 fps.
SECTION: Biobserve Viewer
14. Define your output folders. The initial data path is to the folder where you want to save the videos and the .vrf files, and the initial export data path is where you will save the csv files.
SECTION: Biobserve Viewer
15. Save your settings.
SECTION: Behaviour
16. Transport the mice to the behaviour room and put the mice in individual cages.
SECTION: Behaviour
17. Leave them in the lobby in darkness or with red light for at least 30 minutes.
SECTION: Behaviour
18. Turn on the red lights in the room.
SECTION: Behaviour
19. Open Biobserve Viewer and position the maze.
SECTION: Behaviour
20. Clean the maze with 70% ethanol.
SECTION: Behaviour
21. Turn on the white lights above the maze. Set the light intensity in the center of the maze to 50 lux with the luxmeter.
SECTION: Behaviour
22. 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: Behaviour
23. Place the animal in the center of the maze. Depending on your handling protocol, you can put it in by the tail or in the palm of your hand.
SECTION: Behaviour
24. Make sure to start the recording at the moment when you place the mouse on the maze. You can either do it by setting a delayed start in Biobserve, or with the help of another person.
SECTION: Behaviour
25. Make a recording of 10 min.
SECTION: Behaviour
26. Make sure to save the video file.
SECTION: Behaviour
27. After the video file is saved, go to Data analysis and click on the second icon on the left to save the results in .csv.
SECTION: Behaviour
28. Pick the mouse out of the maze and put it back in the cage.
SECTION: Behaviour
29. Count poop pellets and take a note of them.
SECTION: Behaviour
30. Clean the maze with 70% ethanol. If you feel the necessity, you can rinse it with distilled water.
SECTION: Data analysis
31. We use DLC to track the position of the mice at any time point

We calculate time spend (percentage) in closed arm as the proxy for anxiety-related behavior