Knowledge Base

Introduction

The Social Interaction Test

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:

• Sensitivity to both anxiogenic and anxiolytic stimuli
• Sensitivity to pharmacological, physiological and environmental manipulations
• 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.

To be able to automate the social interaction test, you need the Social Interaction module. This module allows you to track two or more subjects in one arena.

Note: 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. See The Sociability Cage Test.

Note: 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. 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 the sample experiment Social Interaction XT170 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 using 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 daylight 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 with color marks (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.

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 red and green and not red and orange.
• It may be wise to 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.

EthoVision XT Settings

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 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.

Note: Under Body point detection technique, the Deep learning option is not available for experiments with two or more subject roles. See Deep learning: Requirements and Limitations in the EthoVision XT Help.

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, 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.

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 and 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, 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 Advanced detection settings for color marks in the EthoVision XT Help. Also watch the video Identification Settings in the EthoVision XT video tutorial.

Set the Subjects' Contrast

Make sure you have selected the correct contrast and check the following:

• When you use Static Subtraction or 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. For Dynamic Subtraction, set the Current frame weight slider to a value between 1 and 5 to compensate for the changes in the background with time.
• When you use Differencing, adjust the position of the Sensitivity slider until the subjects are properly detected. From the Background changes list, select an option that reflects your conditions (usually, Medium slow works just fine).

Contour Erosion and Dilation

You need Contour erosion/dilation to get a smooth contour without the tail for accurate modeling and to remove individual pixels of noise.

To improve detection, open the Advanced section in the Detection Settings pane. Under Subject Contour, select one or more pixels for the first Erosion filter and the Dilation filter. Leave the second Erosion filter to zero. Increase the number of pixels for Erosion until the animal's tail is removed, then increase Dilation until the entire animal's body contour is detected.

Subject Size

Before you set the Subject Size, make sure the animals are properly detected and do not touch each other.

Click Subject Size, then click Advanced.

1. Under Modeled subject size, make sure the Apply to all subjects and Fix (for all subjects) options are selected.
2. Click the Grab button. This adjusts the Average size to the Current size.
3. Repeat clicking the Grab button until the body fill completely fits the animal.

Shape Stability and Modeling Effort

The Shape stability setting is used when you track animals whose body can be occluded by, for example, cage bars or part of the body of another animal.

When you set the slider closer to Occlusions, EthoVision considers separate objects that are close together to be part of one animal. When you set the slider close to Noise, EthoVision considers separate smaller parts not to be part of the animal.

The Modeling effort setting is used when two animals touch and EthoVision loses the separate shapes. At this point, EthoVision tries to determine which part of the large merged blob belongs to either animal, using a lot of processor load in the process.

When you set the slider close to Performance, EthoVision is only allowed a short time to determine which part of the merged blob belongs to which animal, and therefore modeling quality is low. When you set the slider to Modeling, EthoVision is allowed a longer time to determine which part of the merged blob belongs to which animal, and therefore modeling quality is good, but this costs a lot of processor load. If you track from video, video tracking slows down.

Trial List

Enter your independent variables such as rat ID, treatment (drug vs. control), dose, name of the experimenter, etc. If you want, you can predefine all your trials here, 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.

• When using color markers, add a Marker color variable to the Trial List to specify the marker color you assigned to each Subject role. This is useful when you have defined, for example, the Subject name Experimental and Stimulus, or Subject 1 and Subject 2, and you have randomly assigned the marker colors to the Subject roles.
• Furthermore, you can define a series of trials for batch acquisition. See Acquire a series of trials in the EthoVision XT Help.

Acquiring Data

Protocol

Animals

In many social interaction experiments, two animals from the same treatment group are tested together and scored as a single unit. According to Lapiz-Bluhm et al. (2008), this potentially can result in an artificial exaggeration of behavioral effects; that is, a treatment that only slightly impacts social behavior may have an apparently larger and more significant effect if the social behavior of both animals is affected. Their alternative approach tests an experimental animal paired with a neutral stimulus animal, whose behavior is as constant as possible. See The Sociability Cage Test.

Habituation

Source: EthoVision XT 17.5 Application Manual - The Social Interaction Test