Knowledge Base

Acquiring Data

Protocol

A typical NOR test consists of three phases:

1. Acclimation. The animal is placed in an empty arena for 10 minutes to habituate to the environment. Tracking in this phase is optional. After 10 minutes, the animal is taken out of the arena and is put back in its home cage.
2. T1. After 15 minutes in the home cage, the animal is put back in the arena in which two identical objects have been placed. The animal is put at a position midway between the objects and such that its nose points towards the wall. The animal is tracked for 3 minutes and is then taken out of the arena and put back in its home cage. Define a Trial Control Settings profile for this phase, then assign this profile to the trials of type T1.
3. T2. One of the objects in the open field is replaced with a novel object (sometimes the other object is also replaced with an identical one) and the animal is put in the arena again. Tracking is done for 3 minutes. Define a Trial Control Settings profile for this phase, then assign this profile to the trials of type T2.

Score Behaviors Manually

To score sniffing and other behaviors defined under Manual Scoring Settings, open the Manual Scoring tab on the Acquisition screen. There you find the key codes and the buttons for the behaviors.

To score an instance of the behavior or its end, either press the key or click the corresponding button on the screen.

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.

Data Analysis

Data Preparation

Track Editing

Choose Acquisition > Edit Tracks. You can fix tracking errors and swap back nose-points and tail-points that have been swapped in tracking. Normally you will not need to edit your data.

Track Smoothing

Choose Acquisition > Track Smoothing Profile > open MDM Filter 2 mm. In this profile, the Minimal Distance Moved filter is used (option set to Direct, with a threshold of 2 mm). When the animal sits still, the body points may still move slightly due to system noise. This results in an overestimation of, for example, the total distance moved. With the minimal distance moved filter, you measure the total distance moved over the time periods in which the animal was really walking. Adjust the threshold if necessary, based on your setup and sample rate.

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 Score Behaviors Manually in the EthoVision XT Help.

Selecting Data

Choose Analysis > Data Profile. You can select (Filter) your tracks according to your independent variable values (for example, Treated animals only) and also select parts of tracks (Nesting).

You can create groups of tracks to obtain group statistics for each group by using Filters to compare Novel Object and Familiar Object trials.

Analysis Profiles

The template experiment contains four analysis profiles:

• Touching Objects. This analysis profile contains an In zone variable Nose touching objects to calculate the frequency, duration, and latency of when the nose-point of the animal touches the objects. The variable Distance to objects is used to calculate the mean and standard error of the animal's nose-point to the objects. The manually-scored behavior Sniffing object calculates the frequency and duration, with standard errors, of the instances when you scored Sniffing object during the trials.
• In Halves. In this analysis profile, the In zone variable In Halves is used to calculate the frequency, duration and latency of when the center-point of the animal was in either half of the arena.
• Head Directed to Zone. This analysis profile contains two Head directed to zone variables, one for the novel object and one for the familiar object. For each object, the center plus a 0.10 cm radius is used as the Point of interest. The frequency and duration for Head direction to zone is calculated when the nose-point is in the zone of the corresponding object.
• Distance & Time. With the variables Distance Moved and Velocity, the total distance moved and the mean velocity are calculated.

All analysis profiles also contain settings to calculate the group mean and standard errors of the statistics for trial groups.

Exploratory Behavior

You can quantify exploratory behavior with the analysis profiles described above, or you can create your own analysis profiles.

There is usually variation between subjects in the time spent exploring an object. To account for this variation, you can select the part of each track up to when the animal explored an object for a specific time, and then analyze the behavior of the subject in that part. To do so, use the Free Interval function in the Data profile.

1. Choose Analysis > Data Profile > New.
2. Under Nesting, click the button next to Free Interval.
3. Select the following:
   • As a Start criterion: Time (select Track start).
   • As a Stop criterion: Dependent variable. Select the variable In zone, and the statistic Cumulative duration. In the settings, specify which zone the animal explored, and with which body point (for example, Novel object, nose point). Set the total exploration time.
4. Click OK and insert the resulting box as shown in the figure.
5. In the Analysis profile, choose the endpoints (distance moved, time spent in zones, etc.).
6. Choose Analysis > Results > Statistics and Charts. The results are calculated for the time until exploration reaches the duration specified.

For more examples, see Free Interval in the EthoVision XT Help.

Heatmaps

Heatmaps facilitate identification of "hotspots" and clustering of data points. Choose Analysis > Results > Plot Heatmaps. Next, click Plot Heatmaps on the toolbar.

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