Knowledge Base

The NOR Test in EthoVision XT

Create an experiment from a template (Ctrl+T). Choose Open field (round or square) as Arena template, and Novel object zones as Zone template.

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.

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. The PC must have a powerful graphics card which supports additional software (CUDA). See Deep Learning: Requirements and Limitations in the EthoVision XT Help.

Manual Scoring Settings

These settings enable you to record behaviors manually, using keystrokes. This is handy for example to record exploratory behaviors like sniffing.

Choose Setup > Manual Scoring Settings.

If you created the experiment from the template with novel object zones, the Manual Scoring Settings already includes one behavior, Sniffing object. This is defined as "start-stop", which means that you press a key (default: q) to score the start of sniffing, and then you press the same key once again to score the end of sniffing.

For more information, see Manual Scoring Settings in the EthoVision XT Help.

Note: With the Rat or Mouse Behavior Recognition add-on, you can have EthoVision XT detect sniffing and other behaviors, without the need to score them manually.

Arena Settings

Choose Setup > Arena Settings > Open Arena Settings 1.

To set up your Arena Settings:

1. Click the Grab Background Image button to grab an image of the empty enclosure from the camera image.
2. Click 1. Draw Scale to calibrate your arena (for details, see the EthoVision XT Help).

   Tip: Draw the double-arrow in such a way it points to two opposite walls of the open field, at the level where the animal moves (thus not at the top of the walls). When done, enter the distance in real world units between the tips of the arrow.

3. Click 2. Select Shape and Draw 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 the arena is not yet defined and you want to divide it into equal zones, click 2. Select Shape and Draw Arena. Choose the tool that applies depending on the shape of your open field and draw the outline of the arena.

4. If you used the Zone template Novel object zones, click 3. Select Shape and Draw Zones. Click the layer Zone group Objects. Check that the object 1 zone and object 1 boundary zone cover the object. If the predefined zones do not have the correct shape, delete the zones and draw new ones.
5. If necessary, draw additional zone groups/zones.

Additional Zones

• Zone group Floor and Wall: Check that the Floor zone covers the whole floor area of the open field.
• Zone group Halves: Check that the West and East zones divide the Floor zone into two equal halves. Make sure that the vertical line crosses the entire arena.

Trial Control Settings

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

Choose Setup > Trial Control Settings. The template contains the following profiles:

• Default: Tracking starts automatically two seconds after the animal has been placed in the arena. Tracking stops when you click the Stop trial button.
• Track duration 10 min: Tracking starts automatically two seconds after the animal has been placed in the arena. Tracking stops automatically after 10 minutes.

To Stop Tracking When Exploration Exceeds a Specified Time

You can also stop tracking when the subject has spent some time around an object. For example, when the protocol specifies that in pre-test trials the animal must explore an object for at least 30 seconds.

Make a new Trial Control Settings, and immediately before the Stop track box, insert a Condition box based on the variable In zone, or another variable, or a combination of variables using Multi condition. In the Condition settings, specify that Cumulative duration in the boundary zone for that object must be greater than N seconds, for the nose point.

If you use Multi condition, you can define "exploration" as a combination of variables. For example:

• In Zone for the nose point in the "object zone" is true
• Head directed to Zone "object" is true
• Velocity lower than 5 cm/s is true

The trial stops only when the cumulative time that the three criteria are met reaches the specified value.

To Stop Tracking Automatically When No Exploration Occurs

To stop tracking automatically even when the animal does not explore the object, add another condition in parallel with the first one, which specifies to stop tracking after some time (e.g. 10 minutes), and combine the two conditions with an Operator box of type ANY.

Detection Settings

Choose Setup > Detection Settings > Detection Settings 1.

Advanced Detection Settings

If detection of the subject is not optimal after using the Automated setup function, do the following:

1. Check that the sample rate is set to at least 25 samples/second when tracking the nose and tail.
2. Check that either Dynamic subtraction or Differencing is selected as the detection method. Differencing is the preferred option if you work with hooded animals.
3. When using Dynamic subtraction, move the slider to define the animal's contrast. The animal must be fully detected in all parts of the arena and the noise must be minimal. Specify the Current frame weight. A low value (for instance, 1 to 10) usually works well. A novel object test normally has a duration of a few minutes in which the background does not change much.

   When using Differencing, set the Sensitivity slider. The slider determines what difference in contrast from the background is detected.

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