Knowledge Base

Experiment Setup

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.

Under Analysis Options select Activity Analysis. Click the video button next to it and watch the video tutorial to learn about Activity Analysis. Activity is a useful feature that helps you detect changes in the movement patterns of the subject.

Manual Scoring Settings

EthoVision determines the behavior of your subjects automatically on the basis of the intensity of the subject's movement. This means that you do not have to manually score the subject's behavior.

However, if you want to compare the data scored by EthoVision XT with your own ratings, define behavioral categories under Manual Scoring Settings. Next, during acquisition watch the video and score those categories during your trial. You can also edit the data after acquisition.

Procedure: Choose Setup > Manual Scoring Settings.

1. To add a behavior, click the Add Behavior button. Enter a name for that behavior (for example, Struggling) and specify Mutually Exclusive as the behavior type. Specify the name of the behavior group (for example, Behavior Categories).
2. Add the remaining behaviors to the same group.

We advise you to create three categories for scoring the animal's mobility levels, which correspond to the three possible states of the Mobility dependent variable in EthoVision XT:

• Immobile (corresponding to EthoVision XT's Immobile state)
• Swimming (corresponding to Mobile)
• Struggling/Climbing (corresponding to Highly Mobile)

Arena Settings

1. Choose Setup > Arena Settings > Open Arena Settings 1.
2. Click 2. Select Shape and Draw Arena and check that the pre-defined arenas have the correct shape and size. If necessary, adjust the contour of the arena to fit the cylinders.

   Important: If you work with multiple cylinders and use Activity Analysis to quantify the animals' behavior, make sure that the arenas have the same size. You can check this under Shape Size and Position in the Arena Settings window.

3. Click 3. Select Shape and Draw Zones. Check that the zones (if you used a Zone template) have the correct shape and size. If necessary, draw additional zone groups or zones.

For details, see Arena Settings in the EthoVision XT Help.

Trial Control Settings

Choose Setup > Trial Control Settings and open one of the following profiles:

• Default: Tracking starts 1 second after the animal has been detected in the arena. Tracking stops manually (for example, when you click the Stop Trial button).
• Max Track Duration 15 Mins: Tracking starts 1 second after the animal has been detected in the arena. Tracking stops automatically after 15 minutes or when the animal has been in the Diving zone for over 1 second. You can use these Trial Control Settings for the pre-test session.
• Max Track Duration 5 Mins: This profile is similar to the previous one, but the maximum tracking duration is 5 minutes. You can use these Trial Control Settings for the test session.

If you need more time to put an animal in its cylinder, increase the number of seconds. This prevents detection of your hand or arm from starting tracking. The Start Condition works independently for each cylinder.

When you work with multiple arenas, trial control is applied to each arena separately. Therefore, tracking may start at different times in different arenas, depending on when the animal is released.

Detection Settings

Important Note: For stable detection, the extremities of the animal (paws and tail) must be either always included with the tracked body or always excluded from it. If the paws and tail are temporarily excluded and then included again during data acquisition, these changes affect the variables Mobility and Movement, while the animal might not actually be mobile or moving. The variable Activity is not affected by such changes, because it is based on the image change in the whole arena, not the change in the detected subject.

1. Choose Setup > Detection Settings > Detection Settings 1.
2. Under Video, choose a Sample Rate of 5 samples/second (for rats) or 12.5 samples/second (for mice) or higher.
3. Click Advanced.
4. Under Method, choose Dynamic Subtraction. Under Detection, verify you selected the correct contrast. Move the slider to define the animal's contrast. The animal's body must be fully detected in all parts of the arena and the noise must be minimal. Set the Current Frame Weight slider to a value between 1 and 5 to adjust the number of past images that contribute to the reference image.
5. If the limbs and tail are not included in the detected body and you want to include them, under Subject Contour select the Dilation filter, then the second Erosion filter. Leave the first Erosion filter at zero. Adjust Dilation and Erosion to achieve stable detection.
6. Under Subject Size, click Edit. Define a Minimum Subject Size to prevent small objects like droppings from being detected.
7. If you intend to use Activity Analysis to score the behavior of the subject automatically, click Activity. 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 immobile, those pixels are almost completely absent. Set Compression Artifacts Filter to On if you work with pre-recorded video.

Trial List

Choose Setup > Trial List.

Enter your independent variables such as rat ID, Session Type (with values Pretest or Test), Treatment (with values Drug vs. Control), Dose, Time of Drug Administration, Name of the Experimenter, etc. For variables which differ between subjects, select Subject as the scope.

You can pre-define all your trials by clicking the Add Trials button, or enter the independent variable values as you carry out the trials. You can also prepare the trials in Excel, randomize them, and then paste the values into your Trial List.

Furthermore, you can define a list of trials for batch acquisition. See Acquire a Series of Trials in the EthoVision Help.

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