Knowledge Base

The Social Interaction Test

Introduction

Measuring Social Interactions

The Social Interaction test is widely used as a rodent model of anxiety-like behavior, under the assumption that anxiety is incompatible with social behavior (File and Hyde, 1978; File, 1980, 1988). So, an increase in anxiety generally results in a decrease in time spent on social interaction. The Social Interaction test is used to measure i) sensitivity to both anxiogenic and anxiolytic stimuli, ii) sensitivity to pharmacological, physiological and environmental manipulations and iii) ethologically relevant behaviors that do not require confounding manipulations such as food deprivation, pain or conditioned fear. Until recently, it has been difficult to automate the test with tracking systems. The features in EthoVision XT make this possible. This document describes the general procedure of how to do this.

In most cases for a social interaction test you need the Social Interaction module. This module allows you to track two or more subjects in one arena. This module also includes the Deep learning option for individual recognition in a 2-subject interaction test.

Here below you find an overview of the ways you can implement a social interaction test in EthoVision XT:

Sociability Test (1-Subject Tracking)

If you intend to track a free-moving subject while a second subject is caged (social target) and barely visible from above, you can track only the free-moving animal and measure the behavior of that animal towards the cage defined as a zone. In that case you do not need the Social Interaction module.

Social Interaction - with Deep Learning-Based Recognition

This solution, new in EthoVision XT 18, is based on artificial intelligence which discriminates between two subjects in the arena. One of the two subjects must be marked, e.g. its back being partially shaved. For this solution you can use a monochrome digital camera.

Note: In order to apply artificial intelligence to animal tracking you need to have a compatible Graphics Processing Unit (GPU) installed on the EthoVision XT computer. See the EthoVision XT Help for more information.

Social Interaction - Marker-Based Tracking

This option assumes that you can color-mark your subjects that interact in the arena, and you have a color camera compatible with EthoVision XT. Most of this chapter is based on this solution.

Social Interaction - Live Mouse Tracker

With the Live Mouse Tracker add-on module you can import and analyze Live Mouse Tracker data. Live Mouse Tracker allows long-term analysis of behavior of socially-grouped mice. With Live Mouse Tracker you do not have to color-mark the subjects, however each subject must have a RFID chip implanted. For more information, see the EthoVision XT Help.

The Sample Experiment

To see an example of a social interaction test carried out in EthoVision XT, see also the sample experiment Social Interaction XT190 on the downloads section of the Noldus website (my.noldus.com). Download this file and save it on your computer. Next, in EthoVision XT, select File > Restore Backup and select this file. For more information, see the document Description of sample experiments of EthoVision XT.pdf.

References

• File, S.E., (1980) The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. Journal of Neuroscience Methods 2, 219-238.
• File, S.E., (1988) How good is social interaction as a test of anxiety? In: Simon, P., Soubrie, P., and Widlocher, D., Selected Models of Anxiety, Depression and Psychosis. Basel: Karger, 151-166.
• File, S.E., and Hyde, J.R.G., (1978) Can social interaction be used to measure anxiety?, British Journal of Pharmacology 62, 19-24.
• Lapiz-Bluhm, M.D.S., Bondi, C.O., Rodriguez, G.A., Bedard-Arana, T., and Morilak, D.A., (2008) Behavioural assays to model cognitive and affective dimensions of depression and anxiety in rats. Journal of Neuroendocrinology 20, 1115-1137.

For a list of selected publications on EthoVision XT and the Social Interaction test, see the results of Google Scholar.

Physical Setup

The following suggestions are aimed at optimizing video tracking of multiple, color-marked animals.

Lighting Conditions

• For general information on lighting conditions, see the section Physical Setup in the chapter The Open Field Test.
• Use a sensitive camera if possible. A low light intensity makes it difficult to separate different colors. When it is not possible to use a sensitive camera or strong illumination in your setup, try using fluorescent marker colors with UV lighting.
• For optimal color separation, illuminate your setup with lamps that approximate to day-light in color temperature, that is, have a wide spectrum range.

Marked vs. Unmarked Subjects

In some cases it is required that the identity of the animals that interact in the arena is known and followed, for example in resident-intruder or male-female interactions, or whenever one needs to separate behavioral endpoints (e.g. speed, zone visited) for each individual. In such situations, we recommend marking the animals (see below). EthoVision XT can also track unmarked animals, however identity switches may occur. It is not guaranteed that the correct identity of each unmarked animal is kept throughout the trial.

Markers for When Using the Deep Learning Technique

When using Deep learning, shave the back of one subject so that it looks different from the other. The shaved part should always be visible from the top. For more details, see the EthoVision XT Help.

Color Marker Characteristics

• Use a color scale (for example from a paint company) to find out which colors are most easily recognized by EthoVision in your setup and lighting conditions. Do this before applying color markers to your animals.
• Use colors that have different hue values. For example, use orange and green, pink and yellow, not red and orange. Avoid using red for marking, since it looks like blood.
• Note that marking your animals may stress them, and therefore affect their behavior. If necessary, ensure that you select a marking method that lasts for a longer period of time.
• Make sure that the marker is as round as possible. This will ensure that the relative movement of the center of gravity of the marker is the same in all directions when the edges of the marker change due to posture changes or otherwise.
• Make sure the marker is not too big; the marker can interfere with proper detection of the body contour. For example, make sure that a dark marker on a white animal does not cover the complete width of the animal because it can cause the body to be split in two during tracking in EthoVision XT.
• See also Tips for Color Tracking in the EthoVision XT Help.

The Social Interaction Test in EthoVision XT

Create an Experiment

For Live Mouse Tracker:

1. Choose File > New and in the dialog that opens select Live Mouse Tracker experiment.
2. For the rest of the procedure, see the EthoVision XT Help.

For all other cases:

Choose File > New From Template and choose a predefined template in the guided setup.

Experiment Settings

Choose Setup > Experiment Settings.

• Under Video Source, click the video icon in the camera row and adjust the camera settings if necessary.
• Under Subjects, specify the Subject roles (for example Resident or Intruder, or Subject 1 and Subject 2). Do not enter the ID of the individual animals.
• Under Tracked Features, select the option that corresponds to your needs. In most cases that is Center-point, nose-point and tail-based detection.
• Under Body Point Detection Technique, select Contour-based if you use color-marked animals, or Deep learning if your subjects look different (e.g. the back of one individual has been partially shaved).

Note: You can use Deep learning to track two subjects per arena. The Deep learning technique works if you have a recent graphics card and the driver is up to date.

Manual Scoring Settings

Choose Setup > Manual Scoring Settings.

Here you can define behaviors that you score manually, for instance by pressing a keyboard key. In the template experiment, two start-stop behaviors, Rearing and Grooming, have been defined. Define other behaviors if needed, for example Sniffing and Boxing.

For more information, see Set Up an Experiment > Manual Scoring Settings in the EthoVision XT Help.

Trial Control Settings

Choose Setup > Trial Control Settings > New.

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

• Starting condition. For instance, 'Start tracking 5 seconds after both animals are detected in the arena'. If you are tracking live, this 5-second delay enables you to put both animals in the arena and to take out your hand before tracking starts (5 seconds might not be enough; check beforehand how much time you need).

  Tip: To make sure that tracking starts when both individuals are in the arena, not before, click Settings in the Condition box before the Start track box. Select Current Duration from the Statistic list, then click Settings and then Actors. Select both subject names and select All selected subjects from the list.

• Stopping condition. In order to be able to compare the tracks in the analysis, they must have the same length. Either define a Maximum Trial duration or a Stop track condition based on time.

Detection Settings

Choose Setup > Detection Settings > Detection Settings 1.

Important: Follow the instructions in the order described below!

If you selected Deep learning in the Experiment Settings, set the sample rate (below), and skip the next steps. The software is able to recognize the subjects automatically so you do not need to adjust subject contrast, size, contour filters etc. Go to Trial List.

Sample Rate

Click Video. Select a high sample rate: 25 (for PAL analog cameras) or 30 (for NTSC analog cameras and digital cameras) samples per second.

Automated Setup

Once the animals are in view, click Automated Setup. Select the type of animal you are going to test. Click Next and draw a rectangle around each subject. Make sure that you do this when the animals are not in close contact.

If detection of the subject is not optimal after using the Automated Setup function, see Configure Detection Settings in the EthoVision XT Help for details on the advanced detection settings.

Subject Identification for Color-Marked Subjects

If you mark animals with colors, under Subject Identification make sure that Marker-assisted identification is selected.

1. Put the marked animals in the arena or play the video. Make sure to select a point in the video where the animals do not touch each other. Alternatively, place the animals in the arena one at a time.
2. In the Subject Identification section, select one of the subjects and click the Identification button. As a result, the Identification [Subject name] window opens.
3. Move the mouse pointer to the Video window so the pointer becomes an eyedropper. Move the eyedropper over the color marker of the subject you want to identify and click the left mouse button.
4. Fine-tune the color settings by adjusting the Hue, Saturation and Brightness in the Identification [subject role name] window. Change the range of color settings by changing the numbers or by resizing the Hue box on the vertical color bar, or resizing/moving the box in the color map (horizontally to adjust Saturation, vertically to adjust Brightness). As a result, the outline covers (almost) the complete marker.
5. Next, play the video to see in the Video window whether the marker is detected correctly in different parts of the arena. If the marker 'dances' then your color settings are too sensitive. Go back to step 4 and make the box larger.
6. When you are finished fine-tuning the color settings, you continue with setting the Minimal marker size: increase the Minimal marker size until, first, noise is not detected anymore and, second, the marker is not detected anymore (as a result, the outline in the Video window disappears). Now enter a value for the Minimal marker size that is somewhere in between.
7. Click OK when you are done and repeat steps 1-6 for the other subjects.

For details, see Configure Detection Settings in the EthoVision XT Help.

Source: EthoVision XT 19 - Application Manual