Knowledge Base

The Elevated Plus Maze Test

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. 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. 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.

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 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 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 software Noldus MediaRecorder.
• 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 described earlier in this manual.

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 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. 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 set 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.

Making a List of Trials

If you want, you can pre-define all your trials here. Click the Add Trials button and specify the number of trials (recording sessions of one animal), and the values of the independent variables for each trial.

You can also prepare the trials in Excel, randomize them and then paste the values into your Trial List.

Furthermore, you can define a series of trials for batch acquisition. See Acquire a series of trials in the EthoVision XT Help.

• Test animals only once, unless the intention is specifically to study the different form of anxiety evoked by a second trial. If the animal falls off, it is best to exclude its scores.

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.

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

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). The Data Profile Treated vs. Control from the template compares 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.

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.

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Source: EthoVision XT 19 - Application Manual