TITLE: Collection and Analysis of Mouse Open Field Activity 
AUTHORS: Victoria Vance, Katerina Rademacher, Ken Nakamura

[DESCRIPTION]
The open field task is used to measure locomotor activity in mice. This protocol describes the setup of the task and the acquisition of open field data. It also covers the use of EthoVision Software to analyze recorded videos in order to calculate output velocity, distance traveled, time spent moving vs not moving, and clockwise vs counter-clockwise rotations.

[STEPS]
SECTION: Setup
1. 1 hour prior to recording video, bring mice to behavior room to habituate.
SECTION: Setup
2. Take two racks and place the clear acrylic sheet between them.
SECTION: Setup
3. Clip the acrylic to the racks using large binder clips.
SECTION: Setup
4. Tape 4 sheets of white paper together and wrap tightly around the outside of the cylinders and  tape in place.
SECTION: Setup
5. Place two cylinders on the acrylic. Use tape to mark locations of the cylinders to keep consistent  between trials. Check that the acrylic is flat.
SECTION: Setup
6. Roll up a paper towel and tape between cylinders to catch any urine between cylinders.
SECTION: Setup
7. Place camera on tripod under the acrylic sheet. Ensure both cylinders are in view and in focus,  are well lit, and that glares are minimized.
SECTION: Video acquisition: Procedure for each mouse
8. Write wheel numbers (or mouse IDs) on Post-It notes, and place in corners of acrylic next to each cylinder — check that this is visible in video frame.
SECTION: Video acquisition: Procedure for each mouse
9. Place one mouse into each cylinder and place white acrylic lid on top of cylinder. Do not run mice of the opposite sex together at the same time.
SECTION: Video acquisition: Procedure for each mouse
10. Record behavior for desired time.
SECTION: Video acquisition: Procedure for each mouse
11. After recording, return mice to their cages. Spray cylinder with 70% ethanol and thoroughly  clean.
SECTION: Video acquisition: Procedure for each mouse
12. If paper around cylinders or paper between cylinders are soiled with mouse urine, replace.
SECTION: Video acquisition: Procedure for each mouse
13. Repeat for as many trials as necessary.
SECTION: End of day
14. Spray cylinders with MB10/Vimoba, let sit for 10min, and thoroughly wipe down.
SECTION: Analysis
15. Record video data (see above).
SECTION: Analysis
17. Set the tracking settings on the 'Setup' menu

SECTION: Analysis
17.1. Experiment Settings:
Video Source: from video file
Number of Arenas: will change depending on experiment; e.g. 2 arenas for 2 cylinders
Tracked Features: Center-point detection or multipoint detection
All other settings: default
 
SECTION: Analysis
17.2. Skip Manual Scoring Settings
SECTION: Analysis
17.3. Arena Settings:
Check Arena Settings for each video in case cylinders have moved. You can make a new Arena Settings by duplicating the previous settings.
Select one video file and ‘Grab’ a background image; Ideally this image is taken when no mice are present, but it’s OK if mice are present
Highlight Arena from popup window and use ellipse tool to draw Arena(s)
Calibrate by clicking ‘Calibrate Scale’ and drawing a line across the diameter of the cylinder and entering the diameter of the cylinder in cm (e.g. 26 cm)
Click ‘Validate Arena Settings’ to ensure settings are valid
  

SECTION: Analysis
17.4. Trial Control Settings
  

SECTION: Analysis
17.5. Detection Settings (for black mice with white background)
Method: Gray scaling
Video sample rate should match frame rate from video (e.g. 30 fps)
Detection: Adjust range to highlight mouse in yellow and remove any orange  background noise
All other settings default
SECTION: Analysis
17.6. Trial List
Copy and paste metadata (mouse ID, sex, wheel, etc.)
Add appropriate number of trials.
Assign appropriate video file, arena settings, trial control settings, and detection settings  for each trial.
Assign videos you want analyzed as ‘Planned’
  

SECTION: Analysis
18. Run the video tracking using the 'Acquisition' tab
Select ‘Track all planned trials’
Check ‘Detection determines speed’
Click green button to start 
SECTION: Analysis
19.1. Data Profiles
If necessary, add a filter to reduce tracks so that only relevant trials are analyzed
Add a ‘Time’ nest to restrict analysis to a consistent time frame; This is necessary if you record a few extra seconds
If you want data broken into intervals (e.g. every 5 minutes), click ‘Result’ box, check  ‘Results per time bin’ and set length; check ‘ignore last time bin if incomplete’ and  ‘Apply to all results’  
These parameters can be easily changed and rerun once acquisition is complete
SECTION: Analysis
20. Analysis Profiles
Select outcome measures; ‘Distance moved’ and ‘Velocity’ are pre-selected.
SECTION: Analysis
21. Results
Statistics & Charts – click ‘Calculate’ to run analysis; click ‘Export Data’ to export Excel  sheet. Export trial statistics, not group statistics.
Track Visualization – can be used to see tracks in time bins; click camera icon to export  images.
SECTION: Analysis
16. Open EthoVision on PC.
SECTION: Analysis
19. Analyze tracks using the 'Analysis' tab