Knowledge Base

Experiment 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.

To detect freezing behavior, select Activity analysis under Analysis options. Click the video button next to it and watch the video tutorial to learn about Activity analysis.

If you work with rats or mice, you can let EthoVision XT automatically detect behaviors like Rearing, Grooming, and Sniffing. Select Behavior recognition under Analysis options and choose the options that apply to your experimental setup. The Behavior recognition option is only available if you have the Rat Behavior Recognition module or the Mouse Behavior Recognition module of EthoVision XT.

Manual Scoring Settings

Choose Setup > Manual Scoring Settings.

If you used the template for an open field experiment, two start-stop behaviors have been predefined in the manual scoring settings. These are the behaviors rearing and grooming. Depending on your research question, you can add other behaviors that can be manually scored, like defecating, which is a measure for anxiety, and urinating and vomiting. Note that with the Rat (or Mouse) Behavior Recognition module you can have EthoVision XT detect a number of behaviors automatically.

For more information, see Manual Scoring Settings and Behavior Recognition 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 of the walls so that the animal is still tracked when it rears, but exclude any bright reflective rims that might interfere with tracking. Make sure the label stays inside the arena.

Tip: If you create an experiment from scratch, the arena is not yet defined. Click 2. Select Shape and Draw Arena and choose the drawing tool. If you want to have the open field divided in quadrants, choose one of the subdivided tools. Depending on the shape of your open field, draw the outline of the arena and select the number of sectors. Each sector is automatically defined as a zone.

Zone Group "Borders"

Check that the 'Wall' zone covers only the wall of the open field. If necessary, adjust the center zone and the border zone.

Zone Group "Quadrants"

Check that the zones 'NW', 'NE', 'SE', and 'SW' divide the 'Floor' zone in four equal quarters.

Zone Group "Squares"

Check that the zones divide the arena in equal squares.

Arena Settings for Rat/Mouse Behavior Recognition

When you selected Behavior recognition in the Experiment Settings (only available when you have the Rat or Mouse Behavior Recognition module of EthoVision XT), defining a few special zones and points significantly increases behavior recognition accuracy.

• Feeder zones: To detect eating at the feeder.
• Drinking spout points: To detect drinking.
• Wall zones: To detect rearing to wall and to discriminate this from unsupported rearing.

For the exact procedure, see Zones and Points for Behavior Recognition in the EthoVision XT Help.

Trial Control Settings

In the Trial Control Settings you can define conditions for the start and stop of the data acquisition.

Choose Setup > Trial Control Settings.

Default Trial Control

The templates for the open field contain two Trial Control Settings profiles:

• Default: Track duration 10 (or 30) min — When you use this profile, tracking starts automatically 2 seconds after the animal has been placed in the arena. The track stops automatically after 10 or 30 minutes.

If you use Behavior Recognition or Activity Analysis, the default Trial Control rule is slightly different. See Trial Control in the EthoVision XT Help.

To Stop Acquisition After a Specific Time

Open a Trial Control Settings profile, click the Settings button in the Condition - Time (1) box, and set the time you require.

Traditionally, the open field test had a duration of between 2 and 10 minutes. This short length of time was chosen in the past because the data were acquired manually. It also emphasizes the exploratory behavior and response to novelty, rather than baseline activity of the rodents. Technology like video tracking with EthoVision XT allows for much longer periods of monitoring.

Detection Settings

Choose Setup > Detection Settings.

Sample Rate

Under Video, set the sample rate. Common values, expressed in number of images analyzed per second, are:

• For rats: 5 samples/s.
• For mice: 12.5 samples/s.
• For nose-tail tracking of all rodents: 25 to 30 samples/s.
• For adult zebrafish: 5 samples/s.

Remember that the optimal sample rate depends on a variety of factors, including the characteristics of the setup, and even the animal's strain and behavior. Faster animals need a higher sample rate. If the fish shows very rapid movements, set the sample rate to the maximum; in other cases a sample rate of 5 fps may be enough.

To Detect Freezing Behavior

Open the Activity section in the Detection Settings pane. This option is only available if you selected Activity analysis under Analysis options in the Experiment Settings. Enter the Activity threshold for detecting a change in the pixels in the arena. Start with the default value and check the purple-colored pixels in the video image. Adjust the threshold so that when the animal is completely still, those pixels are almost completely absent.

Rat or Mouse Behavior Recognition

If you selected Behavior Recognition in the Experiment Settings (only available when you have the Rat or Mouse Behavior Recognition module of EthoVision XT), remove the tail from the detected body contour. To do so, in the Detection Settings pane:

1. Under Subject Contour, select one or more pixels for the first Erosion field until the tail is no longer detected. The detected body area (shown in yellow) will appear smaller.
2. Increase the Dilation filter until the detected body area is the same size as before.

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