Knowledge Base

Introduction

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 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, 731-738.
• File, S.E. (1991). Animal models of anxiety. In Biological Psychiatry (G. Racagni, N. Brunello, and T. Fukuda, eds.), 596-599. Elsevier, New York.
• File, S.E. (2001). Factors controlling measures of anxiety and responses to novelty in the mouse. Behav. Brain Res., 125, 151-157.
• File, S.E. and Andrews, N. (1991). Low but not high doses of buspirone reduce the anxiogenic effects of diazepam withdrawal. Psychopharmacology, 105, 578-582.
• File, S.E., Lippa, A.S., Beer, B., Lippa, M.T (2004). Animal Tests of Anxiety. 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, 322-328. doi:10.1038/nprot.2007.44.
• 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/x2e25p_4tgndox16_animals

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.
• 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.
• The lighting should be diffuse, so as not to cast strong shadows (which might be tracked instead of the animal). Even, diffuse lighting is important to optimize tracking of the nose.
• A plus maze with transparent walls may help reducing shadows and make it 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.
• 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.
• 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. WRONG: When the floor is too dark, the head dip is not found.
• 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 to prevent EthoVision from seeing 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 the 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.

EthoVision XT Settings

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 correspond to your needs. To adjust the camera settings, click the video icon in the camera row.

If you want to use the 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 elevated plus maze 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 lifted 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.

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.

To adjust the contour of the arena to the plus maze in the video image, do the following:

1. Click the Normal mode button on the toolbar and drag the arena contour until it is centered on the plus maze.
2. 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 its 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:

1. Drag around all the shapes so they are selected.
2. Click the Rotation mode button. Click in the middle of the arena and drag the mouse to rotate it.
3. Click the mouse pointer icon on the toolbar to exit the rotation mode.

Zone Groups: Open Arms, Closed Arms, and 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.

Source: EthoVision XT 17.5 Application Manual - The Elevated Plus Maze Test