Elevated plus maze protocol for mice and rats

The maze is a plus-shaped platform raised off the floor, with two arms enclosed by walls and two open arms. The test relies on rodents being drawn to dark, enclosed spaces and fearing heights and open spaces, so the closed arms draw them and the open arms put them off.¹

This page follows two published protocols, Walf and Frye’s for rats and mice and Komada and colleagues’ for mice.^1,2

More time on the open arms, or more entries onto them, is read as less anxiety-like behavior.¹ In the study that validated the maze in rats, animals entered the open arms less often than the closed arms and spent less time on them.³ Rats confined to an open arm showed more anxiety-related behavior and more corticosterone in the blood than rats confined to a closed arm.³

The scores do not all track the same thing. In mice, Lister’s factor analysis of plus-maze and hole-board scores sorted them into three separate factors, read as anxiety, directed exploration and locomotion.⁴ Walf and Frye use closed-arm and total entries as a rough gauge of activity, while noting that arm entries may not be the best measure of it.¹

Published mazes differ in arm size, wall height and how high they stand, so record the dimensions of yours in your methods. These are the three mazes in the two protocols this page follows.

A lip on the open arms changes the test. Komada and colleagues add a 0.5 cm (5 mm) lip so fewer mice fall, and Walf and Frye note that a lip of 3 to 5 mm has been used to raise open-arm exploration.^2,1 In a survey of 65 groups that use the maze, anti-anxiety drug effects were harder to detect under less aversive conditions, such as a lip, or ledge, on the open arms.⁵ Decide on the lip before the study starts, and keep the same maze for every animal.

The surface matters for video tracking. Walf and Frye found tracking less accurate when contrast is poor, for example after an animal urinates or when its fur is close to the maze in color.¹ Komada and colleagues test black C57BL/6J mice on a white floor.²

Sources differ on how much light matters, so set it before the study and keep it the same for every animal. Walf and Frye note that light level can change behavior on the maze, and they place the maze where the open and closed arms get similar light.¹ Komada and colleagues test mice in a soundproof room held at 100 lux.²

The rat validation study found that illumination made no significant difference to behavior on the maze.³ The survey of 65 groups found that brighter light, a more aversive condition, made the test more sensitive to anti-anxiety drugs.⁵ Measure the light on the surface of an open arm and a closed arm, and report both values.

Behavior on the maze can change with the light cycle, so test every animal in the same phase of it.¹ Walf and Frye keep their animals on a reversed light cycle and test in the dark phase, when rodents are most active.¹

House animals from a vendor undisturbed for at least 1 week before testing, so the stress of shipping has passed.¹ On the day, Komada and colleagues move the mice into the testing room 30 minutes before the first trial.²

Handling, stress and injections before the test can all change how animals behave on the maze.¹ The same survey named handling before the test, and how aversive the test conditions are, as the factors that matter most.⁵ Handle every animal the same way, and keep the time each one spends out of its home cage brief and the same.¹

These steps follow Walf and Frye, and note where Komada and colleagues differ.^1,2

1. Prepare the maze and the record sheets

   Clean and dry the maze, and check that the tracking system is ready.¹ Fill in a sheet for each animal with its number, the date, its coded group and your initials, so the scorer never sees the group.¹

2. Bring the animal in

   Carry each animal into the testing room in its own transport cage, as Walf and Frye do.¹ Komada and colleagues bring all the mice in 30 minutes ahead instead.²

3. Place it in the center, facing the same type of arm each time

   Put the animal on the center square facing an open arm, as Walf and Frye do, or facing a closed arm, as Komada and colleagues do.^1,2 Behavior differs between the two start positions, so use one for every animal in the study.¹

4. Start recording at once

   Start the tracking and the timer as you let go of the animal, so every animal is recorded for the same time.¹ Walf and Frye record 5 minutes, the period in which early work found rats avoided the open arms most strongly.¹ Komada and colleagues record mice for 10 minutes, to give a difference between groups more chance to show.²

5. Stay still and quiet

   If you score live, avoid moving or making noise while the animal is on the maze.¹

6. Score arm entries by one rule

   Walf and Frye count an open-arm entry when all four paws are on the arm.¹ Komada and colleagues count an entry when the center of the mouse’s body enters the arm.² The two rules give different counts, so name yours in your methods.

7. Return the animal

   At the end of the session, lift the animal into its transport cage, then back into its home cage outside the room.¹

8. Clean the maze before the next animal

   Wipe the maze with disinfectant and dry it.¹ Komada and colleagues clean every arm and the center with hypochlorous water. It removes odor and leaves little of its own, so scent from one mouse does not bias the next.²

Each animal needs 5 minutes on the maze and 1 to 2 minutes of cleaning, so plan the day around 6 to 7 minutes per animal, plus the time to move cages.¹ Add 5 minutes per animal for a 10-minute session.

A head dip is a downward movement of the head toward the floor from an open arm. A stretched-attend posture is an animal standing still with its body stretched toward something.¹ Carobrez and Bertoglio’s review found that only a quarter of plus-maze studies scored these measures, though they may be more sensitive than open-arm time to drugs that change anxiety.⁶ The same review suggests scoring each minute as well as the whole session.⁶

Walf and Frye score each session with video tracking and a live observer at once, and the two agree more than 95% of the time.¹ Walf and Frye trace the disagreements to poor contrast in the video, so check a sample of sessions by hand.¹

For a sense of scale, Komada and colleagues report that C57BL/6J mice spend about 9% of a 10-minute session on the open arms, averaged over 914 mice.² Treat values like these as a rough check on your own controls, because strain, sex and the estrous cycle all shift them.¹

In Walf and Frye’s lab, time on the open arms was about 20 seconds for female rats in diestrus, 35 for males and 75 for females in proestrus.¹ Record the cycle stage of each female, or balance stages across groups.

Open-arm activity usually drops on a second exposure to the maze, so plan one test per animal.¹ Komada and colleagues give each mouse one trial.² If you must retest, Walf and Frye report that open-arm exploration holds when the tests are 3 weeks apart and the maze moves to a new room.¹

Some designs use the drop on purpose. Carobrez and Bertoglio’s review lists a test and retest design among the refinements worth adding.⁶

• Animals fall off the open arms. Decide before the study whether a fall ends the session, and report how many animals fell. A low lip on the open arms means fewer falls.²
• The tracking loses the animal. Raise the contrast between the animal and the maze, and keep an observer scoring or check the video by hand.¹
• Groups move different amounts. Report percent open-arm time and entries, alongside total entries.¹
• Results drift over the day. Test in one phase of the light cycle and balance the groups across the day’s testing order.^1,2
• A new maze or room gives odd baselines. Walf and Frye advise validating the maze in each lab that sets it up.¹ Run a pilot group of controls first, and compare their values with published ones.