Open field test protocol for mice

The open field is a walled arena, square or round, with walls high enough to stop escapes and a floor large enough that its center feels exposed.¹ Rodents avoid large, brightly lit, open and unfamiliar places, so a mouse put in the center heads for the walls first.¹

One session gives two kinds of measure. Distance traveled and time spent moving show how active the animal is, and mice with weak muscles move less.² Time near the walls, called thigmotaxis, rises with anxiety-like behavior, so more time in the center is read the other way.¹

The first description of the test read two measures, activity and fecal boli, as signs of emotionality. Some later studies found the two can be unrelated.¹ Seibenhener and Wooten take this to mean emotionality in rodents has several dimensions, and that more than one measure makes a stronger case.¹

The open field is one of the most used tests of anxiety-like behavior. Prut and Belzung’s review of drug studies found it responds to some classes of anti-anxiety drugs and not others.³ Its authors suggest the test may not model features of anxiety disorders, so describe what it shows as anxiety-like behavior in the animal.³

This page follows two published protocols that use the arena in different ways. Your choice sets the session length, the measures and when you can test the same mice again.

The short design measures the response to a new place, so it uses each animal’s first visit to the arena. The long design takes the newness away first. Tatem and colleagues let the mice get used to the chambers and average 4 days of data, which they found cut the variation in their results.² Use the long design when you want steady activity levels, for example to follow muscle function through a study.

Seibenhener and Wooten use a box 50 cm long and wide with 38 cm walls, made of white, non-porous plastic.¹ The floor is textured for grip and the walls are smooth.¹ They run four boxes side by side, which lets one person test several mice at once.¹

Exploration depends on the light during testing and even the color of the floor, so keep both the same across the study.¹ Tatem and colleagues control the temperature and humidity of the room, light it evenly, and keep the arenas out of direct light, dark corners and shadow.² Temperature, humidity, light, noise and even odor can change the results.² Measure the light at the arena floor and record it in lux. Video tracking also needs the mouse to stand out from the floor, as a dark mouse does on white.

Activity in the open field varies with the time of day, so test at the same time each day, with the same people. It also varies with strain, age and sex, so match these across your groups.²

These steps follow the short design of Seibenhener and Wooten.¹

1. Clean the arena

   Wipe the arena with 95% ethanol before the first mouse and between mice, to remove the scent of the last one.¹ Let the ethanol evaporate completely, which can take 5 to 10 minutes.¹

2. Let the mice settle

   Bring the mice to the testing room in their home cages, and leave them for at least 30 minutes before the first test.¹

3. Set the recording

   Set the tracking to run for 10 minutes, with a start delay just long enough to place the mouse, so recording begins as you let go.¹

4. Place the mouse in the center

   Put the mouse in the center of the arena and let go as the tracking starts.¹ Most mice run straight to the walls, and the record should catch that first move.¹

5. Leave it alone for the session

   Let the mouse move freely and without interruption for the whole 10 minutes.¹

6. Count the boli, then clean

   Return the mouse to its home cage, count the fecal boli, and then remove them and any urine.¹ Spray the floor and walls with 95% ethanol, wipe them with a clean paper towel and let them dry before the next mouse.¹

With a 10-minute session and 5 to 10 minutes for the ethanol to dry, each box takes a new mouse every 15 to 20 minutes.¹ Several boxes side by side shorten the day for a large cohort.

Seibenhener and Wooten lay a 5 by 5 grid of 10 cm squares over the floor.¹ The 16 squares along the walls make the outer zone, and the 9 in the middle make the center zone.¹ Define your zones before the first mouse, and keep them for the whole study.

Look at distance before the zone measures. In their own study of two mouse lines, Seibenhener and Wooten compared time near the walls only after finding that the lines covered similar distances.¹ When groups differ in distance, a difference in time near the walls is harder to read.

Mice with damaged or missing whiskers lose touch with the walls and enter the center more readily, which lowers the measured anxiety-like behavior.¹ Before you test, check every mouse for whiskers chewed off by cage mates, known as whisker barbering.

In the long design, Tatem and colleagues add an extra day when the data were not recorded or a mouse slept through the session.²

Mice get used to the arena, so a second session no longer measures the response to a new place. In the short design, test each mouse once. To avoid habituation, Tatem and colleagues test the same mice no more than once a month. They run the chamber acclimation only before the first round of testing.²

The same arena can host other tests, such as novel object recognition, so plan the order of tests before the study starts.¹

• The tracking jumps or loses the mouse. Check that the light has not changed and the arena has not moved since you set up the tracking.¹
• One beam chamber gives odd counts. Run the beam check before each session. A failed check usually means the chamber or its center quadrant divider is out of line.²
• Groups differ in distance. Report distance beside the zone measures, and read time near the walls with care.¹
• Results shift from day to day. Test at one time of day with the same people, and keep light, noise and odor the same.²
• Mice from one cage behave alike. Spread each cage across groups and boxes, so cage and group are not confused.