Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Summary

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Abstract

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).

The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called "stimulus mouse").

In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.

The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.

Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

Introduction

Impaired sociability is one of the primary symptom domains in a range of neurodevelopmental disorders, including autism spectrum disorders (ASDs)^1,2. It severely limits the patients' ability to develop relationships and greatly affects patients, families, and caregivers^2,3.

In an effort to better understand the mechanism underlying impaired sociability and develop treatments, several mouse models have been studied^1,4–9. One such strain is the BALB/c mouse, an inbred strain that exhibits strikingly low sociability, resembling features seen in patients with ASD5^8–12. For example, the....

Protocol

The protocol for the ease of explanation has been divided into 2 parts: 1) Experimental procedure and 2) Automated analysis. All animals were treated according to the Guide for the Care and Use of Laboratory Animals¹⁵, and all procedures were approved by the Institutional Animal Care and Use Committee.

1. Experimental Procedure

1. Set up the cage, which is a black non-reflective Plexiglas rectangular box (52 x 25 x 23 cm³) divided into three compartments: social, nonsocial, and a neutral middle compartment (Figure 1). Place inverted wire cups in each of the end compartments during sessions I a....

Results

Figure 3 shows a color frame of a behavioral movie with an empty cage. The mouse has not been introduced into the cage yet. The position of the cups, (social and nonsocial) and the compartment boundaries are superimposed. The color frame of empty cage is converted to 8 bit grayscale as shown in Figure 4 and used as a reference frame. The reference frame is subtracted from every other frame of the captured movie.

Figure 5 shows one of the frame.......

Discussion

By combining videotaping mice and automatic movie analysis, we have created an affordable, reliable high-throughput screening technique.

The accuracy of the automated analysis was compared with the results of manual analysis for more than 100 movies. In the overwhelming majority of frames (> 99%) of frames, the nose of the mouse is identified with good precision. Most of the (few) misdetections have no effect on the sociability measures, so that around 80% of the movies do not require any c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plexiglas cage	Norva Plastics, Norfolk, VA	custom made	Dimensions and layout described in manuscript, can be adjusted according to needs. Use non-reflective plexiglas for to facilitate image processing
Wire cups	Kitchen plus	315	Use one to house stimulus mouse, one empty
Video camera	SONY	HDR-PJ790	Can be replaced by any camera with Comparable specifications
OpenCV (Image processing library)	Willow Garage	N/A	Any modern image processing library can be used.