Knowledge Base

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 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 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.

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:

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, 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 may not always be 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 per variable.

Source: EthoVision XT 18 - Application Manual, Noldus Information Technology