The Elevated Plus Maze is a widely used assay for anxious behavior in rodents. The assay leverages rodent’s natural behaviors to provide a useful experimental analog for anxiety. A typical rodent prefers to be in a cozy space. That is to say, if you let a mouse loose in your living room, the mouse will spend most of its time running alongside the wall, sitting in the corner of the room, or exploring underneath the furniture. Only the boldest mouse would be found hanging out in the center of your living room, away from any sort of shelter or hiding place.
This natural behavior is known as thigmotaxis, which describes motion along something, like a wall or furniture. It’s a survival mechanism for rodents in the wild; a mouse is much more likely to be scooped up by a bird in the middle of an open field than in a spot with lots of rocks to hide under. For this reason, rodents love the feeling of a nearby wall or other protection when they’re exploring. Assays like the elevated plus maze are designed so that parts of the maze are inherently more comfortable for the rodent, just like in the great outdoors. Two arms of the maze are enclosed, which provides a nice, safe environment for the rodent to explore, never straying too far from the safety of a wall. The other two arms of the maze have no walls, so a rodent exploring those areas does not have any reassuring barrier between itself and a fall to the ground (no need to worry, though – rodents have good balance and almost never fall off).
When running the elevated plus maze, the rodent is placed in the center of the plus-shaped maze. They’ll then be given some amount of time (usually around 7 minutes) to explore the four arms of the maze. The rodent’s behavior can be assessed in real time by a researcher or by specialized software and is frequently taped so that the behavior can be reanalyzed if needed. To assess the relative anxiety displayed by an animal, the following behaviors are scored:
- Open Arm Entries: This is the number of times a rodent enters the open arms of the maze (those with no walls). A more anxious rodent is less likely to do this.
- Closed Arm Entries: This is the number of times a rodent enters the closed arms of the maze (those with walls). A more anxious rodent will probably have more of these than open-arm entries. Rodents without an anxiety-like phenotype may also be keen to explore these arms (thus, this measure is not very useful on its own).
- The ratio of Time Spent in Each Arm: This is calculated as the amount of total session time spent in the open arm relative to the amount of time spent in the closed arm. This relative measure is essential. Rodents without an anxiety-like phenotype may be happy to explore the whole maze and divide their time pretty evenly, or even spend more time with open arms. Rodents with an anxiety-like phenotype, however, should demonstrate a significantly skewed ratio in favor of time spent in the closed arms.
- Unprotected Head Dips/Stretches/Rearing: This is the number of times that a rodent either dips its head/the top half of its body below the open arm to see what’s going on under the maze or simply stands up while on the open arm (a behavior known as rearing). A more anxious rodent is not likely to do this often, if at all, as it makes the rodent more unsteady.
- Closed Arm Rearing: This is the number of times that a rodent stands on its hind legs in closed arms. Compare this to unprotected rearing behavior. Rodents rear while exploring a lot, but a more anxious rodent may save that behavior for the closed arms.
An interesting question in the elevated plus maze studies is what qualities of the open arm make them more stressful. Is it that the rodents can see that they are unprotected from falling, or is it that they cannot feel a wall protecting them? Or perhaps both? Many neuroscientists argue that a rodent’s tactile sensation in their vibrissae (aka whiskers) is more important for driving thigmotaxis behavior. That said, behavioral assays have been shown to be sensitive to visual cues like the brightness of the room (i.e. rodents are less likely to explore brighter spaces), so vision cannot be ruled out of playing a role in the elevated plus maze behavior.
A study by Filgueiras and colleagues (2014) examined this question specifically within the elevated plus maze. Their study utilized three different types of the elevated plus maze: one conventional, one whose open arms were replaced with transparent walls, and one whose open arms were replaced with opaque walls. They then divided a cohort of rats (n=71) into three groups, one for each type of maze. Within each group, some rats had their whiskers trimmed before testing, and others did not.
The study ultimately showed that rats using a conventional maze (that still had its normal open arms) demonstrated the greatest anxiety-like behavior overall. The behavior of rats in the modified mazes, however, did not differ significantly. Whether the “open” arms were fitted with transparent or opaque walls, they explored them similarly, regardless of whether or not they had their whiskers intact. Overall, the study suggests that rodents in the EPM do rely on tactile sensation to guide their behavior and that when their whiskers have been cut, they are able to rely on other senses beyond vision to guide the way.
Overall, the elevated plus maze is a really good essay for anxiety-like behavior assessment in rodents. It’s a relatively low-stress behavioral test, so it can be combined easily with other assays as part of a battery of tests. It’s also a relatively quick test, since you don’t have to account for any habituation sessions and can frequently be scored by software (although it’s never a bad idea to manually score a bit to make sure your software is behaving). Be sure to mind the lighting of the room to not create additional stressors or confounds!
- Filgueiras GB, Carvalho-Netto EF, & Estanislau C (2014). Aversion in the elevated plus-maze: role of visual and tactile cues. Behavioural processes, 107, 106-11 PMID: 25151938
- Wall PM, & Messier C (2001). Methodological and conceptual issues in the use of the elevated plus-maze as a psychological measurement instrument of animal anxiety-like behavior. Neuroscience and biobehavioral reviews, 25 (3), 275-86 PMID: 11378181