Knowledge Base

Trial Control Settings

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

• Default. In this profile, 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. In this profile, tracking starts 1 second after the animal has been detected in the arena. Tracking stops automatically after 15 mins 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. If you need more time to put an animal in its cylinder, increase the number of seconds. This way you prevent that detection of your hand or arm starts tracking. The Start Condition works independently for each cylinder.
• Max Track Duration 5 mins. This profile is similar to the previous one, but now the maximum tracking duration is 5 minutes. You can use these Trial Control Settings for the test session.

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: It is important for stable detection that the extremities of the animal (paws and tail) are either always included with the tracked body or always excluded from it. If the paws and tail are temporarily excluded and then included again in the detected subject during data acquisition, these changes affect the variables Mobility and Movement, while the animal might not actually be mobile or moving. On the other hand, 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 (for rats) or 12.5 samples/second (for mice) or higher.
3. Click Advanced.
4. Under Method, choose Dynamic subtraction. Under Detection, check 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 the 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 to 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 in such a way 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 a Scope.

If you want you can pre-define all your trials here (click the Add Trials button), or you can 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.

Acquiring Data

Protocol

Basic Protocol (Rats)

• Pre-test trial (day 1, before drug administration). The animal is placed in the cylinder for 15 minutes. No scoring of immobility is performed during the first session. This session is needed to acclimatize the rats to the experimental situation and to induce a stable, high level of immobility during the test session.
• Test (day 2). After drug administration, the animal is placed in the cylinder for 5 minutes. Score the duration of immobility by summing the time spent immobile; score as immobile minor movements strictly necessary to maintain the animal's head above water.

Alternate Protocol (Mice)

The mouse is placed in the cylinder. The trial lasts 6 minutes. Measure the latency to immobility from the start of the trial and the duration of immobility for the last 4 minutes.

See Castagné et al. (2011). Current Protocols in Neuroscience 8.10A.1-8.10A.14.

Score Behaviors Manually

If you want to score the subject's behavior manually, define them first under Manual Scoring Settings. After you have started the trial, watch the subjects in the video and in the Manual Scoring tab at the bottom of the screen, click the button to the category that applies. As soon as the behavior of the subject changes, click the button for the corresponding category.

• Important: If you do tracking from video, make sure you de-select DDS in the Playback Control window.
• You can also score behaviors after tracking. See Acquire Data > Score behaviors manually in the EthoVision XT Help.

Determining Mobility Thresholds

To find the optimal Highly mobile and Immobile thresholds, you can run a few test trials and check in the Analysis Results and Scoring pane (Dependent Variables tab) the values for Mobility state in relation to the behavior the animal displays. If the Mobility states do not correspond to the behavior of the animal, in the Analysis Results and Scoring pane click Dependent Variables, click the button under Mobility state and adjust the Highly mobile threshold and Immobile threshold.

For example, if the animal is struggling, its corresponding Mobility state is Highly mobile. If the current Mobility score is Mobile or Immobile, you probably need to reduce the Highly mobile threshold.

Note that you can also adjust the Mobility thresholds after acquisition. In the Analysis profile, add the Mobility state variable and visualize the data.

If you do not see the Mobility state variable on your screen, click Show/Hide on the toolbar and select Show Dependent variables, then Mobility state and Mobility.

Determining Activity Thresholds

In the same way as you defined thresholds for Mobility state, you can define thresholds for Activity state. Use Activity state if Mobility state does not give satisfying results, for instance when detecting thrashing of the animal's limbs which may not be detected properly by Mobility.

Note that when you work with multiple cylinders in the same video image, Activity state gives good results when the size of the arena is the same for all cylinders. In the Arena Settings, adjust the contour of the arenas to make them similar.

Data Analysis

Data Preparation

Track Editing

Choose Acquisition > Edit Tracks. You can fix tracking errors but normally you will not need to edit your data.

Track Smoothing

Choose Acquisition > Track Smoothing Profile > open MDM 0.2 cm. In this profile, the Minimal Distance Moved filter is used (option set to Direct, with a threshold of 2 mm). This filter filters out small movements which result from system noise.

Note that smoothing influences the track, that is, the coordinates of the center point of the subject, not the subject's detected area, and therefore does not influence the calculations of Mobility. It also has no effect on Activity analysis.

Review Video and Behaviors

If you have scored behaviors manually, you can review the video and if necessary edit the data. Choose Acquisition > Score behaviors manually. For details, see Acquire Data > Score behaviors manually in the EthoVision XT Help.

Selecting Data

Choose Analysis > Data Profile. You can select your tracks according to your independent variable values and also select parts of tracks (nesting). You can also create groups of tracks to obtain group statistics for each group.

Integrated Visualization

Choose Analysis > Results > Plot Integrated Data. Select a trial from the list on the tool bar. The video file and the Time Event Plot are displayed.

In the Time Event Plot tool bar you can choose which profiles to use for display of the variables. Play the video and check whether the states scored under Mobility state correspond to the behavior of the animal. If they do not, fine tune the Mobility (or Activity) thresholds in the Analysis profile. Then check the results of your changes again in the Integrated Visualization.

Analysis Profile

Mobility Analysis

In an Analysis Profile, you select the dependent variables and statistics for analysis. For the experiment made with the template for the Porsolt Test, a dependent variable Mobility is already present.

Choose Analysis > Analysis Profile > Open > Mobility.

1. Double-click the row for the Mobility state variable.
2. Under Outlier filter, the Averaging interval is the number of samples on which the running average mobility is based. With an Averaging interval of 1 sample, sudden changes in surface area caused by reflections are not filtered out.
3. Under Threshold, set the Highly mobile and Immobile thresholds to the values you have previously determined.
4. Under Calculate statistics for, select the states you want to analyze.
5. Click the Trial Statistics tab and select Duration and Frequency. Optionally select additional statistics in the Group statistics tab.
6. Click OK.

Comparing EthoVision Mobility Data with Your Ratings

If you have scored the animal's behavior manually, you can easily compare your data with those scored by EthoVision (Mobility). Make sure that your analysis profile includes the three Mobility states and the behaviors scored manually, then visualize the data.

If the EthoVision data do not match your ratings at an acceptable level, adjust the Mobility threshold values in acquisition and in analysis.

An Alternative to Mobility Analysis

You can use Activity state if the Mobility state variable does not give good results, for instance when detecting thrashing of the animal's limbs which may not be detected properly by Mobility state.

Activity analysis is based on the temporal change in pixel value in the whole arena. Because it is not based on the detected animal's surface area, it is independent of tracking.

1. In the Experiment Settings, under Analysis options select Activity analysis. Click the video button next to it and watch the video tutorial to learn about Activity analysis.
2. In the Detection Settings, 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 in such a way that when the animal is immobile, those pixels are almost completely absent.
3. Run the trial.
4. In the Analysis profile add Activity and Activity state.
5. Visualize the data (Plot Integrated Data). There you see the scored Activity states and the subject in the video. Adjust the Activity state settings in such a way that the states scored by EthoVision XT match the behavior of the subject at that time.

Source: EthoVision XT 19 - Application Manual