EthoVision XT - The Elevated Plus Maze Test
Last updated: Jul 25, 2026
The Elevated Plus Maze Test
The Elevated Plus maze experiment in EthoVision XT92
Introduction
the elevated plus maze test
The elevated plus-maze was designed to provide measures of anxiety that were relatively uncontaminated by changes in overall motor activity, and has been extensively validated by Pellow et al. (1985) using behavioral, physiological, and pharmacological measures. The open and closed arms are considered to evoke the same exploratory drive in the animals, therefore avoidance of the open arms is considered to be a result of the induction of higher levels of fear (Rodgers and Dalvi, 1997). It is thought that the aversion of animals to explore the open arms of the maze is caused by fear of open and/or elevated spaces. The plus-maze is sensitive to the anxiolytic effects of neurotoxic lesions of serotonergic neurons and to the anxiogenic effects of drugs (Pellow and File, 1986), drug withdrawal (File and Andrews, 1991), and Figure 5.1 An elevated plus maze. predator odor (Zangrossi and File, 1992). For a discussion of the additional, ethological measures that can be taken in this test, see Rodgers et al. (1995) and Fernandes and File (1996). Unlike the social interaction and light/dark tests, the elevated plus-maze does not rely on aversion to bright light, and it has been found repeatedly that behavior in the maze is independent of light level (e.g., Becker and Grecksch, 1996). For a review of the protocol, see Walf and Frye (2007).
What to measure?
Principal component analysis of the conventional plus-maze (File, 1991) has shown that the percentage of time spent on the open arms and the number of entries onto the open arms are the best measures of anxiety (these are increased by anxiolytic and decreased by anxiogenic treatments). The number of closed arm entries is the best measure of locomotor activity. There are marked strain differences in baseline scores and even between the scores of different batches of animals. If the scores are high it will be hard to detect an anxiolytic effect. If they are very low it will be difficult to detect an anxiogenic effect. The plus-maze was originally developed for male rats, but it can be used with female rats. However, while in male rats anxiety is the main factor measured, in females it is activity (Fernandes et al., 1999). The plus maze has also been validated for mice (Lister, 1987; Walf and Frye, 2007). For a discussion of the factors controlling measures of anxiety in the mouse, see File (2001).
Quantifying head dips
EthoVision XT can detect when the subject's nose is outside the open arms. However, you can also score head dips manually. First define head dips
in the Manual Scoring Settings. Next, during acquisition, press the
assigned keyboard key when a head dip occurs.
the sample experiment
To see how a plus maze test is carried out in EthoVision XT, see also the
sample experiment Elevated plus maze XT175 on the downloads section
of the Noldus website (my.noldus.com). Download this file and save it
on your computer. In EthoVision XT, choose File > Restore Backup and
select the file. For more information, see the document Description of
sample experiments of EthoVision XT.pdf.
references
Papers
Becker, A. and Grecksch, G. (1996). Illumination has no effect on rats' behavior in the elevated plus maze. Physiol. Behav., 59, 1175-1177. Fernandes, C. and File, S.E. (1996). The influence of open arm ledges and maze experience in the elevated plus-maze. Pharmacol. Biochem. Behav., 54, 31-40. Fernandes, C., Gonzalez, M.I., Wilson, C.A. and File, S.E. (1999). Factor analysis reveals that female rat behaviour is characterised by activity,
male rats are driven by sex and anxiety. Pharmacol. Biochem. Behav., 64,
Current Protocols in Neuroscience, Unit 8.3. DOI: 10.1002/
0471142301.ns0803s26 (online) Lister, R.G. (1987). The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology, 92, 180-185. Pellow, S., Chopin, P., File S.E., and Briley, M. (1985). Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods, 14(3), 149-167. Pellow, S. and File, S.E. (1986). Anxiolytic and anxiogenic drug effects in exploratory activity in an elevated plus-maze: A novel test of anxiety in the rat. Pharmacol. Biochem. Behav., 24, 525-529. Rodgers, R.J., Cole, J.C., Aboualfa, K., and Stephenson, L.H. (1995). Ethopharmacological analysis of the effects of putative "anxiogenic" agents in the mouse elevated plus-maze. Pharmacol. Biochem. Behav., 52, 805-813. Rodgers, R.J. and Dalvi, A. (1997). Anxiety, defense and the elevated plus-maze. Neurosci. Behav. Rev., 21, 801-810. Violle, N., Balandras, F., Le Roux, Y., Desor D., and Schroeder, H. (2009). Variations in illumination, closed wall transparency and/or extramaze space influence both baseline anxiety and response to diazepam in the rat elevated plus-maze. Behav. Brain Res., 203, 35 - 42. Walf, A.A. and & Frye, C.A. (2007). The use of the elevated plus maze as
an assay of anxiety-related behavior in rodents. Nature Protocols, 2, 322
- doi:10.1038/nprot.2007.44. Web:www.nature.com/nprot/journal/v2/n2/pdf/nprot.2007.44. pdf Zangrossi, H. and File, S.E. (1992). Behavioral consequences in animal tests of anxiety and exploration of exposure to cat odor. Brain Res. Bull., 29, 381-388.
Videos
https://www.youtube.com/watch?v=4rRsxf1U6-w http://www.dailymotion.com/video/x2e25p4tgndox16animals
Physical setup
The following suggestions are specifically to optimize video tracking: Place the plus maze in such a way that its apparent size is maximized. You can obtain this by rotating the plus maze (or the camera) until the closed arms lie along one of the diagonals of your video window (see Figure 5.2). The lighting should be as even as possible throughout the arena. Bright lights are not necessary but should you need to supplement the light, consider red (or infra-red) light, as the animals will be less sleepy. The camera should have a good view of the entire region the animal can be in. Center the plus maze in order to have the image of the arms as symmetrical as possible. This minimizes inaccuracy of calibration due to perspective.
Figure 5.2 Turn the plus maze to maximize its apparent size in the video
image from above. The lighting should be diffuse, so as not to cast strong shadows (which might be tracked instead of the animal; see Figure 5.3). Even, diffuse lighting is important to optimize tracking of the nose. A plus maze with transparent walls may help reducing shadows and make easier to observe closed arm rears. There is little practical difference between the two mazes in terms of their ability to detect differences in anxiety-related behavior. However, the transparent design may reduce the sensitivity for the detection of anxiolytic drug effects because it decreases the anxiogenic potential of the open arm, therefore leading to the reduction of anxiety-related behavioral baseline (Violle et al., 2009 and references therein). The background should contrast with the animal. If necessary use a different setup for light and dark colored animals. In the closed arms, the contrast between animal and background is generally lower. Take this into account when positioning lights and choosing the background color (Figure 5.4 and Figure 5.5).
Figure 5.3 Lighting should also adjusted to minimize the shadow made by
the animal in the arena, which in this example (indicated by the arrow) may result in incorrect detection.
Figure 5.4 In this example, the closed arm at the top-right corner receives
little light. This makes detection more difficult when the animal walks in that arm.
Figure 5.5 An example of good contrast between animal and background,
using a white mouse. Furthermore, the transparent walls also help reducing shadows. Make sure that the color of the floor is in good contrast with the color of the animal, and in minimal contrast with the arms. This makes it possible to track the animal's nose off the edge of the open arms (see Figure 5.6). Figure 5.6 Left: CORRECT. The gray floor contrasts with the black mouse. When the mouse dips its head off the edge of the open arms, its nose is still detected. Right: WRONG. When the floor is too dark, the head dip (indicated by the arrow) is not found.
Figure 5.7 An example of good contrast between floor and animal
when testing white rats. Both the plus maze arms and the floor should not be reflective. If necessary, place an opaque rubber mat on the floor. If there are barriers above the closed arms, remove them. If that is not possible, in the Detection Settings use the Dilation-Erosion filter (see page 97) to prevent EthoVision to "see" the animal cut in two. See also Contour Settings in the EthoVision XT Help. Depending on circumstances, pools of urine can cause problems with the tracking. An absorbent base can help. Always place the apparatus in the same position in the room, cues from overhead might influence the behavior of the animal. For the same reason the experimenter should not be visible to the animal during the trial (automated tracking makes this possible). If you want to use Deep learning technique to track the subject's nose, note that there are additional requirements and limitations.
Among other things, you need a powerful graphics card. See Deep
learning: Requirements and Limitations in the EthoVision XT Help.
The Elevated Plus maze experiment in EthoVision XT
Create an experiment. For details, see Chapter 1 of this manual, or
in EthoVision XT press F1 and see Setup an Experiment in the
EthoVision XT Help.
experiment settings
Choose Setup > Experiment Settings.
Under Video Source, and Tracked Features, make sure that the options
selected corresponds to your needs. To adjust the camera settings, click the video icon in the camera row. If you want to use Deep learning technique to track the subject's nose,
under Body point detection technique choose Deep learning. Note that
in order to use this technique there are additional requirements.
Among other things, you need a powerful graphics card. See Deep
learning: Requirements and Limitations in the EthoVision XT Help.
manual scoring settings
Choose Setup > Manual Scoring Settings. If you used the template for an open field experiment, two start-stop behaviors have been defined: Head dipping, to record manually the head dips over the edge of the open arms. Rearing, to record manually the posture of the animal with forelimbs lift off the floor. Note that EthoVision XT does not detect rearing automatically in an Elevated plus maze. You must score rearing manually in order to record it. You can define more behaviors, like scanning. Stretched posture can in principle be detected automatically by using the Elongation variable in EthoVision.
Notes about Head dipping
By head dipping we mean here protruding the head over the ledge of an open arm and down towards the floor. This response can occur while the animal's body is in the closed arms, central square or open arms. You can also let EthoVision count the number of times or the total time that the nose point is found within the head-dipping area (provided that this has been chosen in the template). This also quantifies the risk-assessment behavior. For a more reliable scoring of head dips, you could place a camera in front of each open arm, to film the arm's side-view. You can record video from multiple cameras using the Media Recorder software. If you film the side-view of the open arm, you can in principle record head-dips automatically with EthoVision by defining a zone immediately below the arm's floor. For these videos you need to create additional Arena Settings and Detection Settings.
For more information, see Set Up an Experiment > Manual scoring
settings in the EthoVision XT Help.
arena settings
Choose Setup > Arena Settings > open Arena Settings 1.
We assume that you followed the procedure in arena settings on page
15.
Arena
Check that the arena covers the whole area in which you want to track the animal. Remember to include enough space around the open arms (so that the animal is still tracked when it dips its head off the edge of those arms) and the inner side of the walls (to track the entire animal when it rears). Exclude any bright reflective rims that might interfere with tracking. Make sure the label Arena 1 stays inside the arena. Figure 5.8 Arena Settings. To adjust the contour of the arena to the plus maze in the video image, do the following:
Click the Normal mode button on the tool bar and drag the
arena contour until it is centered on the plus maze.
Click the Point edit mode button. Click one of the corners of
the arena and drag the mouse to move the corner to the position you require. If you use video files, select the aspect ratio correction that applies. For
more information, see Adjust the video aspect ratio in the EthoVision
XT Help. If you use the live camera image or the video was recorded with EthoVision XT in the same experiment, you can skip this step.
Zone group Arms
Check that each arm zone covers the corresponding sector of the plus maze. To make sure that a zone is limited to the actual arm, move the label of that zone until the arrow points outside the zone. The color of the zone should change. If the color changes for a wider area of the plus maze, it means that the zone label also pointed to that area. Use the edit functions to move and reshape the zone.
For details, see Move, rotate and resize a shape in the EthoVision XT
Help. Do not forget to place the arrow of the zone label back in original position. If the zones "closed arms" predefined in the template overlap with the open arms of the plus maze, rotate the entire arena and zones. To do so, drag around all the shapes, so they are selected. Next,
click the Rotation mode button. Click in the middle of the
arena and drag the mouse to rotate it. Click the mouse pointer icon on the toolbar to exit the rotation mode.
Zone group Open arms, Closed arms, Head dip zone
These are cumulative zones. They are defined automatically from the sum of the open arms, the closed arms and the head dip zones, respectively.
trial control settings
Choose Setup > Trial Control Settings. In the Trial Control Settings you can define conditions for the start and stop of the track. The template contains two trial control settings profiles: Default.
Track duration 5 mins - When you use this profile, tracking starts
automatically 2 seconds after the animal has been placed in the plus maze. The track stops automatically after five minutes.
detection settings
Choose Setup > Detection Settings.
In the Detection Settings window, check in the Video Section that the
sample rate is set to: Rats: 5 samples/second. Mice: 12.5-15 samples/second. Nose-tail tracking: 25-30 samples/second.
See Configure Detection Settings in the EthoVision XT Help for details
on the advanced detection settings. If you use hooded animals, use the detection method Differencing. If the plus maze has barriers above the arms, the animal is not always well detected, like in this example.
To improve detection, open the Advanced section In the Detection
Settings pane. Under Subject Contour, select one or more pixels for the
Dilation filter and the second Erosion filter. Leave the first Erosion filter
to zero. Select more pixels for Dilation than for Erosion, for example 3
vs. 1, until the entire animal's contour is detected.
trial list
Choose Setup > Trial List. Do one of the following:
Click Add Variables and enter your independent variables such as
Animal ID and Treatment (with possible values Treated, Control, Sham etc.), Dose, Name of the experimenter, etc. Each trial will receive only one value of each of those variables; for example, one trial must be either Control or Treated or Sham etc.
Making a list of trials
If you want, you can pre-define all your trials here. Click the Add Trials
Independent variables
You can also enter the independent variable values as you carry out the trials. You can edit the independent variables for trials already acquired, for example to enter a-posteriori data, like whether the animal in that trial entered the open arms or not. This way you can quickly create groups of tracks based on the result of the trial, and use such groups in analysis. You can add a new independent variable at any time. Figure 5.9 An example of the Trial List with twelve planned trials.
Acquiring data
protocol
The subject is placed at the junction of the four arms of the maze, facing an open arm. Each subject is generally tested once for 5 minutes. For extensive protocol information, see Walf, A.A. & Frye C.A. (2007). The use of the elevated plus maze as an assay of anxiety-related
behavior in rodents. Nature protocols 2(2): 322-328. doi:10.1038/
nprot.2007.44 See also acquiring tracks on page 21.
score behaviors manually
To score head dipping and other behaviors defined under Manual Scoring Settings, open the Manual Scoring tab on the Acquisition screen. There you find the key codes for the behaviors. To score an instance of the behavior or its end, either press the key or click the corresponding button on the screen.
Notes
important If you do tracking from video, make sure you de-select
DDS in the Playback Control window.
You can also score behaviors after tracking. See Acquire Data >
Score behaviors manually in the EthoVision XT Help.
Data analysis
data preparation
Data editing
Choose Acquisition > Edit Tracks. You can fix tracking errors and swap
back nose-points and tail-points that have been swapped in tracking. Normally you will not need to edit your data.
Smoothing data
Choose Acquisition > Track Smoothing Profile and open MDM Filter 0.2
cm. In this profile, the Minimal Distance Moved filter is used with a
value of 2 mm and option Direct.
Review video and behaviors
If you have scored behaviors manually, you can review the video and if
necessary edit the data. Choose Acquisition > Score behaviors
manually. For details, see Acquire Data > Score behaviors manually in
the EthoVision XT Help.
selecting data
Choose Analysis > Data Profile. You can select your tracks according to
your independent variable values (for example, Treated animals vs. Controls) and also select parts of tracks (data nesting). Figure 5.10 shows the Data Profile Treated vs. Control from the template to compare 'Treated animals' and 'Control animals' trials. This way you create groups of tracks to obtain group statistics for each group. For more information and to create your own data selection, see the EthoVision XT Help.
Figure 5.10 An example of data selection to compare two data sets in your
experiment, based on the independent variable "Treatment". Two groups are formed: Treated and Control. Trial Statistics will be displayed per trial, and Group Statistics will be displayed per group of trials, according to the treatment level assigned to them. If you have more treatment groups (e.g. Sham), to create more treatment groups click the Common Elements - Result button in the Components pane, and place the new box somewhere on your screen. Create a new branch by connecting the first (Start) box to this new box. Click the button next to "Treatment" and select the new treatment level. Insert the box in the new branch.
visualizing data
You can visualize your tracks in three ways:
Plot tracks. You can view your tracks on a still image of the
background. Tracks can be shown in different colors according to the values of independent variables (for example, blue for animals treated with saline and red for drug-treated animals). Sample points can be shown in different colors according to the values of dependent variables, for example red when the animal was moving fast.
Plot integrated data. You can look at a track with the video file in
the background. When you plot integrated data you can also view Time Event plots of your independent variables. Just like with plotting tracks, you can show tracks or sample points in different colors.
Heatmaps. You can make heatmaps of the location of your animals
during the test.
analysis profiles
The template experiment contains three analysis profiles:
Time in arms - This analysis profile contains two In zone variables:
- In closed arms to calculate the frequency and duration of when the animal was in the closed arms.
- In open arms to calculate the frequency, duration and latency of when the animal was in the open arms. Here, latency (the time to the first visit in any open arm) is used as an indication of anxiety. For all variables, the animal is considered to be in a zone when all its body points are found in the zone simultaneously.
Behaviors - In this analysis profile, four variables have been
defined. - Head dipping and Rearing provide statistics of the behaviors manually scored. - Body elongation is used to quantify the time that the animal shows stretching behavior. - Nose in Head Dip Area is an In zone variable to calculate the frequency and duration of when the nose point of the animal was in the head dip areas. The last two variables are present when you set the experiment to track the three body points or you select the zone template with the head dip area.
Velocity and distance - With the variables Distance Moved and
Velocity, the total distance moved and the mean velocity and the
group means and their standard errors are calculated. Movement is based on a velocity threshold, and quantifies the time that the animal has moved significantly. For more information, see the EthoVision XT Help.
False positives in arm entry statistics
Sometimes the center point of the animal fluctuates around the border line between the center and the arm zones. This may be caused by jitter or exploration behavior, and results in false positives when calculating the number of zone entries. To prevent this from happening, set the Zone exit threshold for the In zone variable.
To set a Zone exit threshold
- In the Analysis profile, add the In Zone variable. Specify the arm zones you are interested in, and the body points that define a zone entry (when tracking the center, nose and tail base points).
- Under Threshold, enter a value for Zone exit threshold. This is the distance from the border of the arm zone that the animal must be in order to be considered outside the zone. Use the Zone exit threshold to filter out small movements of the body points that result from jitter or exploration behavior.
- Visualize the data (Analysis > Results > Plot Integrated Data).
Example
In the following example we see that slight movements of the mouse's center point result in multiple open arm entries. In the Analysis profile, In zone is set with Zone exit threshold = 0 cm. Left: Mouse enters the open zone. Middle: The center point is detected out of the zone. Right: The center point is inside the zone; a second arm entry is scored. To remove false positives of zone entries, in the Analysis profile, In zone is set with Zone exit threshold = 2 cm. This means that when the center point is outside the zone by less than 2 cm from the border, it is still considered in the zone. Result: This time, one arm entry is scored.
For more information on the Zone exit threshold, see In zone in the
EthoVision XT Help.
Source: EthoVision XT 17.5 Application Manual, The Elevated Plus Maze Test