Knowledge Base

The Social Interaction Test

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 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.

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.

Notes

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 Social Interaction module. SeeThe Sociability Cage Test. 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 also 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., 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 20, 1115-1137.

EthoVision XT and the Social interaction test

For a list of selected publications, 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 condition, 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 need to separate behavioral endpoints (e.g. speed, zone visited) for each individual. In such situations, we recommend to mark 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. Figure 7.1 An example of color-marked rats in a social interaction test.

The Social Interaction test in EthoVision XT

In EthoVision XT, you can set up a social interaction test experiment by using a predefined template in the guided setup.

experiment settings

Choose Setup > Experiment Settings.

Under Video Source, and Tracked Features, make sure that the options

selected corresponds 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, 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

(see the figure below).

detection settings

Choose Setup > Detection Settings > Detection Settings 1.

important Follow the instructions in the order described below!

Figure 7.2 Trial control settings that specify a track duration of 10 minutes.

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 animal 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 (see Figure 7.3). 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 d 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. Figure 7.3 The color of the marker after fine-tuning the color settings. Most of the marker is now selected as indicated by the black outline.

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/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 part of one animal. When you

set the slider close to Noise, EthoVision considers separate smaller

parts not 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 big '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. Therefore, modeling quality is low. When you

set the slider to Modeling, EthoVision is allowed a longer time to

determined which part of the 'merged' blob belongs to which animal. 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.

Notes

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. Figure 7.4 An example of Trial List for a social interaction test.

Carrying out your trials

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 test an experimental animal paired with a 'neutral' stimulus animal, whose behavior is as constant as possible. See The Sociability Cage Test on page 107.

Habituation

The experimental animals are gently handled for 2 minutes on each of 3 days prior to the social interaction test. In addition, they are individually familiarized with the testing room and social interaction arena for 5 minutes on the 3 days prior to testing. The stimulus animals are habituated to the testing room and arena for 5 minutes per day for 3 days prior to testing. The stimulus animals are pre-exposed to the test arena in pairs, with a different, unfamiliar other stimulus animal on each pre-exposure day.

When using color marks

To control for the effect that the color of the marker might have on the behavior of the other animal, it is recommended to randomly assign the marker color to stimulus and experimental animals. To take into account the combinations of animal type and marker color, you can

either add a user-defined variable like Marker color to the Trial List or

define two Detection Settings, one for each combination. In the latter

case, make sure you set the appropriate Detection Setting as current before you start data acquisition.

Social memory test (1-min confrontation)

1. Let the focal animal establish a home-cage territory (e.g. 5 days).
2. Introduce the stimulus female into the home cage of a male mouse for a 1-min interaction. At the end of the 1-min trial, remove the stimulus animal.
3. Repeat step 2 a number of times, with a 10-min inter-exposure interval.
4. "Dishabituation" trial: Introduce a different stimulus female to the same male mouse for one minute. In EthoVision XT: If you use color markers, alternate different colors for the same subject (e.g. half resident mice marked orange, half marked green). If you use different colors for the resident animal, make different

detection settings in such a way that Subject 1 is always the

resident animal. Before acquisition, make sure that you use the detection settings that apply to that animal. Mark different types of trials with an independent variable in the Trial List. For example Type, with values C1, C2 ... for confrontation with the first stimulus female and D for the second stimulus female.

Scoring behaviors manually

If you want to score the subject's behavior manually, define them first

under Manual Scoring Settings (page 124). 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.

For more information, 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 with a value of

2 mm and option Direct selected.

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 (Filter) your tracks according to your independent

variable values (for example, Treated animals vs. Controls) and also

select parts of tracks (Nesting). Figure 7.5 shows a data selection to

compare Treated and Control animals. This way you create groups of tracks to obtain group statistics for each group. To create a group, click the button next to Common elements - Result. Connect the Start box to the new Result box. This creates a new group. Next, to specify a criterion for the new group, click an option under

Filter, and insert the resulting Filter box between Start and the new

Result box.

analysis profiles

The template experiment contains a number of analysis profiles, depending on which arena / zone template you selected in the template wizard:

Distance & Time - With the variables Distance Moved and Velocity,

the total distance moved and the mean velocity are calculated.

Movement - The variable Movement calculates how often and how

long the center-point was moving.

In zones - The variable In zone calculates how often and how long

the subjects visited each zone in the Arena Settings. The latency to the first visit in a zone is also calculated for each subject.

Figure 7.5 A data profile to compare tracks of Treated animals and Control

animals.

Behaviors - In this analysis profile, the frequency and duration of

Body elongation and the manually scored behaviors Rearing and

Grooming are calculated. For all statistics also the group means with standard errors are calculated.

social interaction in detail

The following variables are commonly used to study social interactions:

Distance between subjects - Use this dependent variable to

measure the average distance between the subjects. You can also use this dependent variable to determine the appropriate thresholds for the dependent variable Proximity.

Proximity - When the nose-point of the experimental animal is In

proximity relative to any of the body points of the stimulus animal, the experimental animal can be considered to be exploring or sniffing the other animal.

Body contact - You can use this variable to quantify body contact,

for example how often and for how long two animals are in contact with one another.

Relative movement - Use Relative movement to determine how

much time the experimental animal is actively seeking contact with the stimulus animal.

Net weighted movement - Net Weighted movement can be used as

an objective measure for the intensity of approach and avoidance behavior.

Weighted movement from - Weighted movement from can be used

as an objective measure for the intensity of avoidance.

Weighted movement to - Weighted movement to can be used as

an objective measure for the intensity of approach. See Dependent Variables in Detail in the EthoVision XT Help for details. Also see the analysis profile 'Social - Nose to Tail' in the sample

experiment Social Interaction XT170 which you can download from our

web site.

statistics and charts

Choose Analysis > Results > Statistics & Charts, then click Calculate.

The Trial Statistics tab shows the results per trial. The Group Statistics

tab shows the statistics and charts from the summarized results over all trials or the groups defined in your Data profile (see page 134).

visualizing data

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. You can select other data/analysis profile combinations from the lists on the tool bar. Choose Analysis > Results > Plot Heatmaps. There you can make heatmaps of the location of your animals during the test.

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