Knowledge Base

Introduction

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. See The Sociability Cage Test on page 107.

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 XT180 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, 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, 19–24.
• Lapiz-Bluhm, M.D.S., Bondi, C.O., Rodriguez, G.A., Bédard-Arana, T., and Morilak, D.A., (2008) Behavioural assays to model cognitive and affective dimensions of depression and anxiety in rats. Journal of Neuroendocrinology, 1115–1137.

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 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.

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