TITLE: Open Field
AUTHORS: Sabina Marciano, Roberta Marongiu

[DESCRIPTION]
Protocol for general locomotion in rodents using open field arenas and Ethovision video tracking.
Purpose: Assess cognitive, spontaneous motor activity, and anxiety
Note that there are many variations of this test. We perform this test during the mouse's dark cycle (i.e. their active phase).

[BEFORE_START]
Note that our lab performs this assay in the dark phase to reduce stress and to test animals during their active cycle. Our animals are kept on an inverted light cycle.

[STEPS]
SECTION: Protocol for spontaneous activity measurement:
1. Days 1-2 – training
The purpose of the training days is to reduce to the minimum the influence of cognitive and anxiety states on motor activity by acclimating the mice to the equipment. Additional training days can be added if preferred.
SECTION: Protocol for spontaneous activity measurement:
2. Acclimate mice to the behavior room for 1-2 hours before starting.
SECTION: Protocol for spontaneous activity measurement:
3. The mouse is gently carried and placed in the center of the open field (OF) box.
SECTION: Protocol for spontaneous activity measurement:
4. Let mouse explore the environment for 5-10 min (keep consistent across experiments).
SECTION: Protocol for spontaneous activity measurement:
7. Start with another mouse, etc…
SECTION: Protocol for spontaneous activity measurement:
6. At conclusion, return mouse to the home cage and clean OF boxes with water-EtOH-water sequence solutions. (Sequence is done to reduce strong odor of ethanol).
SECTION: Protocol for spontaneous activity measurement:
5. Record motor activity using a set of camera + Ethovision software.
SECTION: Protocol for spontaneous activity measurement:
8. Day 3 - testing
SECTION: Protocol for spontaneous activity measurement:
9. Acclimate mice to the behavior room for 1-2 hours before starting.
SECTION: Protocol for spontaneous activity measurement:
10. The mouse is gently carried and placed in the center of the OF box.
SECTION: Protocol for spontaneous activity measurement:
12. Record motor activity using a set of camera + Ethovision software.
SECTION: Protocol for spontaneous activity measurement:
11. Let mouse explore the environment for 30 min (keep consistent across experiments).
SECTION: Protocol for spontaneous activity measurement:
13. At conclusion, return mouse to the home cage and clean OF boxes with water-EtOH-water sequence solutions.
SECTION: Protocol for spontaneous activity measurement:
14. Start with another mouse, etc….
SECTION: Protocol for cognitive performance and anxiety:
16. Days 1-2 - AM
SECTION: Protocol for spontaneous activity measurement:
15. Main outcome measures:
Assess distance and time travelled/moved in the box within 30 min trial of day 3; resting time; vertical time and rearing episodes; ambulation in the periphery and center of the box; etc..
Comparison of movements (between periphery and center distance, time, center crosses) can be used as indication of anxiety behavior. We usually use the data from day #1.
 
SECTION: Protocol for cognitive performance and anxiety:
17. Acclimate mice to the behavior room for 1-2 hours before starting.
SECTION: Protocol for cognitive performance and anxiety:
18. The mouse is gently carried and placed in the center of the OF box.
SECTION: Protocol for cognitive performance and anxiety:
19. Let mouse explore the environment for 5-10 min (keep consistent across experiments).
SECTION: Protocol for cognitive performance and anxiety:
21. At conclusion, return mouse to the home cage and clean OF boxes with water-EtOH-water sequence solutions.
SECTION: Protocol for cognitive performance and anxiety:
20. Record motor activity using a set of camera + Ethovision software.
SECTION: Protocol for cognitive performance and anxiety:
22. Start with another mouse, etc…
SECTION: Protocol for cognitive performance and anxiety:
23. Days 1-2 – PM: After a trial interval time of~ 4 hours, repeat testing as above described.
SECTION: Protocol for cognitive performance and anxiety:
24. Main outcome measures:
Assess cognitive performance quantifying distance and time travelled/moved in the box within 5-10 min trials over the 4 trials. These should decrease over time in normal mice with no cognitive deficits as measure of learning capability.
Comparison of movements (between periphery and center distance, time, center crosses) can be used as indication of anxiety behavior. We usually use the data from trial #1, but performance over the 4 trials can also be compared.