Name: experimental assessment of mouse sociability using an automated image processing approach
Uploaded: 2016-05-15T14:00:00
Description: Watch this Scientific Journal Video about Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach at JoVE.com

This research was supported by grant funding from Virginia&#8217;s Commonwealth Health Research Board.

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
<ol>
	<li>Set up the cage, which is a black non-reflective Plexiglas rectangular box (52 x 25 x 23 cm3) divided into three compartments: social,...

<ol>
	<li>Brodkin, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BALB/c+mice:+Low+sociability+and+other+phenotypes+that+may+be+relevant+to+autism.">BALB/c mice: Low sociability and other phenotypes that may be relevant to autism.</a> Behavioural Brain Research. 176, 53-65 (2007).</li><li>Deutsch, S. I., Burket, J. A., Urbano, M. R., Herndon, A. L., Winebarger, E. E., Mohammadi, M. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+improves+sociability+in+the+BTBR+T(+)Itpr3(tf)/J+mouse+model+of+autism+spectrum+disorders.">Rapamycin improves sociability in the BTBR T(+)Itpr3(tf)/J mouse model of autism spectrum disorders.</a> Brain Research Bulletin. 100, 70-75 (2014).</li><li>Burket, J. A., Benson, A. D., Tang, A. H., Deutsch, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D-Cycloserine+improves+sociability+in+the+BTBR+T++Itpr3tf/J+mouse+model+of+autism+spectrum+disorders+with+altered+Ras/Raf/ERK1/2+signaling.">D-Cycloserine improves sociability in the BTBR T+ Itpr3tf/J mouse model of autism spectrum disorders with altered Ras/Raf/ERK1/2 signaling.</a> Brain Research Bulletin. 96, 62-70 (2013).</li><li>Burket, J. A., Herndon, A. L., Deutsch, S. 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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 (&#62; 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 ...

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&#39; ability to develop relationships and greatly affects patients, families, and caregivers2,3.
In an effort to better understand the mechanism underlying impaired sociability and develop treatments, several mouse models have been studied1,4&#8211;9. One such strain is the BALB/c mouse, an inbred strain that exhibits strikingly low sociability, resembling features seen in patients with ASD58&#8211;12. For example, the BALB/c mouse displays a lower frequency of social approaches and a usually negative response to social approaches by a stimulus mouse (e.g., social avoidance) than the Swiss Webster comparator strain10.
To test whether a proposed treatment is effective in increasing sociability, objective measures are needed. A widely accepted framework is to analyze the sociability in a standardized apparatus that has three compartments (see Figure 1).
The left and right compartments each contain an inverted wire cup which houses a socially salient stimulus mouse. The test mouse is released into the apparatus for three intervals of 10 min (sessions I-III); session I assesses the locomotor activity of the test mouse, while sessions II and III assess different aspects of the test mouse&#39;s sociability.
In session I, the test mouse is allowed to acclimate to the apparatus. In this session, the number of transitions that the test mouse makes between the different compartments is measured. Importantly, sociability measures obtained in the standard three-compartment apparatus are dependent upon locomotor activity, and reduced locomotor activity can confound interpretation of sociability data.
In session II, the test mouse is moving freely and a stimulus mouse is placed under one of the inverted cups (counterbalanced). The stimulus mice are 4-week old outbred male ICR mice, age- and sex-matched to the test mice. The stimulus mouse is enclosed in an inverted cup in the side designated as the social compartment, and an empty inverted cup is placed in the side designated as the nonsocial compartment. In session II, the sociability of the test mouse is quantified by comparing the time it spends near the &#8220;social&#8221; inverted cup (less than 2 cm away) vs. the time it spends near the &#8220;nonsocial&#8221; inverted cup, and by the time it spends in the social compartment vs. the time it spends in the nonsocial compartment. Swiss Webster comparator mice spend significantly more time in the social compartment and near the social inverted cup compared to BALB/c mice that show no significant preference for the social inverted cup or the social compartment12.
In session III, test and stimulus mice are allowed to interact freely (inverted cups are removed). Measures of sociability, stereotypic behaviors and transitions between compartments are reliably obtained in the third 10 min session of free interaction between test and stimulus mice and analyzed. In this report, the sociability of the test mouse is quantified via two measures: 1) the number of social approaches it makes toward the stimulus mouse, where a social approach is defined as moving forward (nose first) towards the stimulus mouse so that the nose is less than 2 cm away from the stimulus mouse; 2) the number of avoidances of social approaches by the stimulus mouse. The test mouse is considered to be avoiding the approach if it turns or moves away from the stimulus mouse, or if it temporarily stops moving (&#8220;freezing behavior&#8221;) after the stimulus mouse has approached the test mouse. BALB/c mice show a decrease in number of social approaches made towards the stimulus mouse and an increase in the number of negative responses to social approaches by the stimulus mouse, compared to the Swiss Webster comparator strain10.
The evaluation of the behavioral movies has traditionally been done manually, i.e., by watching the movie carefully (possibly at reduced speed) and activating stopwatches for the time the mouse spends in a certain compartment or near one of the inverted cups10. This method requires watching of the movie repeatedly and is, therefore, very time-consuming. Also, the accuracy is limited by the rater&#8217;s reaction time for the stopwatch measurements and ability to perceive whether the mouse is within 2 cm of the inverted cup. In recent work, two compounds, (D-serine and D-cycloserine) have proven effective in restoring sociability in BALB/c mice9,10,12. It is known that both components modulate the NMDA receptor, which is known to play a central role in social behavior2,12. A large array of compounds is known to modulate NMDA receptor activity in a variety of well-defined ways, and the potential for a breakthrough in ASD treatment makes the expedited evaluation of these compounds a priority and a fast automatic method for evaluation highly desirable.
Described below is an automatic analysis procedure developed for the assessment of mouse sociability. It uses image processing methods on all the frames of the mouse movies to identify the position of the mouse or mice. In sessions I and II, it further detects nose position and uses it to compute sociability measures. In session III, it automatically detects all approaches, but manual classification (social approach, social avoidance, or other) is required.

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

experimental assessment of mouse sociability using an automated image processing approach

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 &#34;stimulus mouse&#34;).
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.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
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.

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...

Watch this Scientific Journal Video about Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach at JoVE.com

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

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.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

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