Knowledge Base

Draw the Arena and the Zones

Tell EthoVision XT in what region in the video image the subject moves (the arena). If you created a template experiment, the arena has already been drawn in the correct shape. Change the size and/or position of the arena to match it with your video image.

To Obtain a Background Image

1. If you use video cameras for live tracking, before starting EthoVision XT power up and connect the video camera to the PC.
2. In EthoVision XT, choose Setup > Arena Settings. Open the default Arena Settings 1 or create a new one. If you do tracking from video files, click Browse and open the video file you want to use to draw the arena.
3. Click Grab. If possible, do so when the arena contains no animals.

Calibrating with the Noldus Calibration Card

1. The Grab Background Image dialog opens automatically when first accessing Arena Settings. You can also open it manually via Arena Settings > Arenas and zones > right-click on Background > Grab Background Image.
2. Place the Noldus Calibration Card inside the arena, ensuring the black rectangular outline on the card is clearly visible in the camera view. If you are using a dark arena, place a piece of white paper behind the card.
3. Click Calibrate. In the Calibration dialog that opens, review the result and accept or decline.

If the automatic calibration fails, continue with the steps below.

To Manually Calibrate and to Draw an Arena

1. Click 1. Draw Scale to Calibrate in the Arena Settings window.
2. Draw a line between two points of the arena that are at a known distance from each other. In the window that appears, type in the real world distance between the two points and click OK. Optionally repeat this procedure a few times.
3. Click 2. Select Shape and Draw Arena in the Arena Settings window.
4. Click one of the drawing tools and draw the outline of the region of the video image where the animal moves. Make sure that the label Arena points to inside this region.

To Draw a Zone

A zone is a region within the arena that you may want to use for analysis or for starting/stopping data acquisition. If you created a template experiment, zones are already defined. Change their size and/or position to fit the arena, delete zones or draw new ones.

1. Click 3. Select Shape and Draw Zones in the Arena Settings window.
2. Select one of the drawing tools.
3. Draw the contour of each zone. In this example, the platform in a water maze.
4. Give the zone a name.

• For more details, see Arena Settings in the EthoVision XT Help.
• For a DanioVision experiment, see the DanioVision DVOC-0041 - Reference Manual.

Control the Start and Stop of Data Acquisition

Specify the rules that control the start and stop of data acquisition.

Default Settings

Choose Setup > Trial Control Settings. Open the default Trial Control Settings 1.

• Each experiment has default Trial Control settings to start tracking when the subject is detected in the arena for one second and to stop tracking manually.
• If you created a template experiment, the Trial Control settings have been adjusted for that template. For instance, for the Morris water maze experiment, tracking stops either when the animal's center-point has been in the platform zone for more than five seconds, or when the animal has been swimming around for two minutes without finding the platform.

To Program the Start of Tracking

1. In the second box, click Settings.
2. If you want to start tracking when the animal is in a zone, click Settings, deselect Arena and select that zone.
3. If necessary, change the criterion to be used (for instance, Frequency instead of Duration).

To Program the Stop of Tracking

1. In the box immediately before Stop track, click Settings.
2. If you want to stop tracking after a specific time, choose After, and enter the time needed.

• The first box, Start Trial marks the moment when you click the Start Trial button. The Start track box marks the time when EthoVision XT starts tracking.
• For more complex start-stop rules, like "stop when the subject is on the platform", replace the Time condition with an In zone condition, and specify the platform zone. For more examples, see Trial Control in the EthoVision XT Help.
• Make sure that the Trial Control Settings you want to use is selected in the Acquisition settings window before you start your data acquisition.
• With the Trial and Hardware Control module, you can control hardware (lights, a pellet dispenser, etc.). For instance, turn on a light when the animal enters its shelter. It also allows you to create routines, for example in conditioning experiments.

Detect the Subject

1. Choose Setup > Detection Settings. Open the default Detection Settings 1.
2. If you work with video files, under Video click Select Video and choose a video file. Play the video up to when the subject appears. If you work with cameras, release the animal in the arena.
3. Click Automated Setup. Choose the type of subject you test, and click Next.
4. Wait until the subject is free from contact with objects or walls and walks with a normal posture. When ready, draw a rectangle around the subject. Do this for all subjects in the arena.
   • For rodents, try to leave the tail out of the rectangle. In all other cases, include the animal's tail.
5. If the subject's body is well detected, click Yes. Otherwise, try moving the Finetune slider and check if the yellow blob covers the entire animal's body (excluding the tail). If detection does not improve, try again a few times using another image from the video. If this does not help, click No and click Advanced. For details, see Configure Detection Settings > Advanced Setup in the EthoVision XT Help.

Tracking the Nose and Tail-Base

• In all cases, make sure that the body of the animal is well detected.
• If you use the Deep learning detection technique with one subject per arena, under Method click Define and select a box around the subject. Make sure that there is some space around the subject's body.

• Check that the subject is well detected in all parts of the arena. In the Detection Performance pane, check that the proportion of samples where the subject is not found is acceptable (for example less than 1%).
• If you selected Activity analysis in the Experiment Settings to detect freezing, click Activity. Adjust the settings until most of the purple pixels only appear when the animal moves. When the animal freezes, the number of purple pixels should be minimized.

Source: EthoVision XT 19 - Quick Start Guide