TITLE: Open field locomotion test
AUTHORS: Hong-Yuan Chu

[DESCRIPTION]
This protocol describes open field locomotion test.

[STEPS]
SECTION: Set up
1. Place the open field boxes right below the camera attached to the ceiling.
SECTION: Set up
2. Connect the camera to the computer via USB.
SECTION: Set up
3. Clean the boxes with 70% ethanol.
SECTION: Set up
4. Open the ANY-maze software on computer.
SECTION: Set up
5. To create a new protocol, select “New Experiment”. 
SECTION: Set up
6. Add a new video source by selecting the green “+ (add item)” button in the header section. 
SECTION: Set up
7. Adjust the camera/open field box so that the whole boxes can be seen on the screen. 
 

SECTION: Set up
8. Add a new apparatus by selecting the green “+ (add item)” button in the header section.
SECTION: Set up
8.1. Add item > New apparatus > Name the apparatus based on which box it represents (e.g., “Top Left”) > Set the length of the ruler line based on the dimensions of the box (e.g., 500 mm) > Use the rectangle tool to draw the outline of the perimeter of the box.
SECTION: Set up
8.2. Add a grid over the box by selecting the “Grid settings” option in the header section. Select “Include a square grid spaved at (cm)” and set desired spacing (e.g., 5 cm).
SECTION: Set up
8.3. Repeat the above steps in “8.1” for each box.
SECTION: Set up
9. To create zones within each box, select the green “+ (add item)” button in the header section. 
 

SECTION: Set up
9.1. Add item > New zone > Name the zone (e.g., center) > select the squares within each apparatus that represent the zone. Optional: select “Highlight this zone during tracking when animal is in it” to be able to visualize when the animals are within each zone during testing.
SECTION: Set up
9.2. Go to ‘Set zone entry settings’ then check ‘use the centre of the animal’.
SECTION: Set up
9.3. Repeat for each desired zone.
SECTION: Set up
10. Set the animal colour under the “Animal colour” tab in “TRACKING AND BEHAVIOR”.
SECTION: Set up
11. If desired, select “detect peiods when the animal is immobile” and “detect period when the animal is freezing” under the “Immobility detections” and “Freezing detection” tabs, respectively.
SECTION: Set up
12. Set the test duration by selecting “Stages” > “First Stage” under the “TESTING” tab.
SECTION: Set up
13. Go to “Treatment groups” to set how ANY-maze should assign animals to treatment groups, or if treatment groups are not desired.
SECTION: Set up
14. If using the animals ID is needed, go to “Animal ID” below “Treatment groups” and select “Use my IDs to refer to animals”.
SECTION: Set up
15. Go to “EXPERIMENT” at the top and select “Add Animals” and the number of animals to add.
 

SECTION: Testing
16. Go to “TESTS” tab at the top.
SECTION: Testing
17. Untested animals should populate immediately and the apparatuses should be “Ready…”
 


SECTION: Testing
18. To begin testing, place an animal in its designated box and then press the green play button.
 


SECTION: Testing
19. Repeat step 18 for each animal.
SECTION: Testing
20. Either hide behind a curtain and remain silent or step out of the room so as not to distract/disturb the animals being tested.
SECTION: Testing
21. Once the session has ended, remove each animal from the box and return to its home cage.
SECTION: Testing
22. Clean each box with 70% ethanol before testing the next back of animals.
SECTION: Data retrieval
23. Go to the “PROTOCOL” at the top and scroll down to “Results, reports, and data” at the bottom under “ANALYSIS AND RESULTS”.
SECTION: Data retrieval
24. Select “Data page” and under “Apparatus measures” choose the desired parameters to be displayed.
SECTION: Data retrieval
25. Go to the “DATA” tab and the top and the chosen parameters will be displayed.
SECTION: Data retrieval
26. Either press “Save” to save results to the computer or select “Send as e-mail” to email a .CSV file with the results for analysis offline.
SECTION: Set up
5.1. Change the name of the protocol under the “APPARATUS” section.
SECTION: Set up
6.1. Add item > New video source > Select the camera from the drop-down menu.
SECTION: Set up
12.1.  Set the test duration to specified time (e.g., 10 min).