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, nonsocial, and a neutral middle compartment (Figure 1). Place inverted wire cups in each of the end compartments during sessions I and II (discussed below) and house the stimulus mouse during session II.</li>
	<li>Set up the camera in such a manner that the cage fills the field of view. Since lighting can interfere with mouse behavior, maintain a luminance of no more than 3.5 lux inside the cage, coming from indirect light from an incandescent light bulb and measured on the cage floor in upward direction. Also use an infrared light source, which mice cannot sense but the camera can detect. Record all movies in 720 x 480 standard definition.</li>
	<li>Record a single frame of an empty cage for later image processing (called the &#8220;reference frame&#8221;) and save as a grayscale image.</li>
	<li>Session I: Introduce test mouse into the cage and wave hand over the cage, indicating the start of session (compare Automated Analysis). Record movie for 10 min. At the end of 10 min, put the mouse back into its original cage.</li>
	<li>Session II: Put the stimulus mouse under one of the inverted cups (counterbalanced).</li>
	<li>Use the &#8220;Social left&#8221; or &#8220;Social right&#8221; button to select whether the stimulus mouse is in the inverted cup on the left or in the inverted cup on the right. 
	NOTE: The inverted cup with the stimulus mouse is called the &#8220;social cup&#8221;, whereas the other cup the &#8220;nonsocial cup&#8221;. The compartment in which the social cup is located is called the social compartment, the compartment with the nonsocial cup is the nonsocial compartment, and the middle compartment is the &#8220;neutral compartment&#8221;.</li>
	<li>Introduce the test mouse into the cage. Wave hand over the cage to indicate the start of the session. Record movie for 10 min. Importantly, keep mice in the sociability apparatus before removing the inverted cups to begin Session III.</li>
	<li>Session III: Remove the inverted cups, release both mice and allow the test and stimulus mice to interact freely with each other. Wave hand over the cage to indicate the start of the session. Record movie for 10 min. At the end of 10 min, put both mice back into their respective cages.</li>
</ol>
2. Automated Analysis
NOTE: The commands in section 2 are issued via a graphical user interface (see Figure 2) that controls our software. Here we explain the step-by-step processing that our software allows; the software is also capable of performing the analysis in batch mode without user input.
<ol>
	<li>For every movie, execute the following processing steps:
	<ol>
		<li>Load movie (click Load button).</li>
		<li>Select cage configuration, which tells the analysis program the location of cage boundaries, compartment boundaries, and cup locations. Select one of four saved configurations or manually adjust all coordinates. 
		NOTE: The configuration usually does not change from one experiment to the next and can therefore be reused.</li>
		<li>For each frame of the movie, perform the following processing steps (2.1.3.1-2.1.3.6).
		<ol>
			<li>Convert the frame to grayscale by clicking the &#8220;Grayscale&#8221; button.</li>
			<li>Digitally subtract the grayscale reference frame (see step 1.3) by clicking the &#8220;Subtract&#8221; button. NOTE: The goal of this step is to make the pixel values of the frame very close to zero everywhere except for the mouse position.</li>
			<li>Binarize the frame by setting all pixels below a certain threshold level to black, all pixels above that level to white (click the &#8220;Binarize&#8221; button). 
			Note: The idea is that since the mouse pixels generally have higher values than the remaining pixels, the mouse pixels will become white and all other pixels black. The optimal threshold level depends on the lighting condition and can be determined by assessing the mouse shape throughout a movie. If the threshold is chosen too low, the mouse appears too large, and pixels that do not belong to the mouse can be colored white. If the threshold is too high, the mouse becomes small and can disintegrate into disconnected pieces. In the recorded movies, the optimal threshold was always between 20 and 35 (on a scale from 0 to 255).</li>
			<li>Erode13 the binarized picture e times, then dilate13 2e times, then erode again e times, by pushing the button &#8220;Erod/Dil&#8221;. 
			Note: The purpose to this procedure is to remove the tail. For the resolution (720 x 480) that was used, e = 3 worked in all circumstances. 
			Note: The software checks automatically for the presence of a hand in the picture (during the recording of movies, hands are waved through the picture to indicate the beginning of a session). If a hand is present, there will be unusually many white pixels in the binarized frame because the hand is much larger than a mouse. The condition for the presence of a hand is that there are more than 10,000 white pixels in the binarized frame. The software determines the beginnings of sessions I, II, and III, by detecting the range of frames in which hand movements occur.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In sessions I and II, continue as follows:
	<ol>
		<li>Detect the largest connected component of the set of white pixels (Click &#8220;LComp&#8221;). Note: The purpose of this step is to eliminate white pixels that do not belong to the mouse, e.g., caused by the motion of the stimulus mouse in session II. The largest connected component is in almost all frames the mouse.</li>
		<li>Determine the two most distant white pixels (called the &#8220;ends&#8221; of the mouse) by clicking &#8220;Find Ends&#8221;. 
		Note: The software establishes the continuity of the two ends, i.e., for each of the ends in the first frame to which of the ends in the subsequent frames they belong. The general idea is that since frames are recorded at high rate, the ends cannot travel far from one frame to the next, so that for the correct continuation of the ends from one frame to the next is that which minimizes the sum of the distances that the ends move. See the Discussion for more detail.</li>
		<li>Determine which end of the mouse is the nose.
		<ol>
			<li>Determine the direction in which a mouse moves by observing how the center of gravity (COG) changes from frame to frame (the COG&#8217;s motion is the motion of the sum of all mouse pixels). 
			Note: The COG of a set of pixels p1, ... , pn&#160; with coordinates x1, .. , xn &#160; is defined by: 
			<img alt="Equation 1" src="/files/ftp_upload/52508/52508eq1.jpg" /> 
			Mice usually move forward (i.e., towards their nose). In a section of several hundred frames, there is always very high confidence in the head/tail detection. 
			Note: The software automatically determines towards which of the detected ends the mouse is moving; this end is identified as the nose and marked with a red circle in the movie. When the detected nose is marked with a red circle in every frame, the user can check the quality of the detection.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In session III, continue as follows:
	<ol>
		<li>Detect two largest components of white pixels.
		<ol>
			<li>If both are of acceptable size for a mouse and their centers of gravity (COGs) are compatible with the mouse COGs of the previous frame, consider these two components the two mice.</li>
			<li>Consider mouse sizes acceptable if they have not changed by more than 20% since the previous frame, and consider COGs of two consecutive frames as compatible if their difference is not larger than the distance a mouse can travel in the time between frames (this distance follows from the maximum mouse speed, which can be determined as part of the preparations of an experiment series by analyzing the motion of several individual mice (as in sessions I and II). 
			Note: If the two largest components are not acceptable sizes for a mouse, or the COGs are too different from those in the previous frame, the software automatically increases the binarization threshold until the two largest components are of acceptable size and their COGs are compatible with those of the previous frame.</li>
		</ol>
		</li>
		<li>Click the test mouse in the first frame. Use the analysis in 2.3.1 to identify both test and stimulus mouse in every frame of session III.</li>
		<li>Identify the frames in session III in which the one mouse socially approaches the other. Note: The software implements this step by automatically checking that the distance between the mice is less than the typical interaction distance of mice (we use 2 cm).</li>
		<li>When the software plays a short segment of the movie with the detected approach to the user, click a button to indicate whether this approach qualifies as a social approach by the test mouse, social avoidance by the test mouse, or neither. 
		Note: Step 2.3.4 can be automatized if it is known which mouse is the test mouse. Since this information gets lost when the mice get too close to each other (or climb on top of each other), and since we have not yet found a way to label the mice in a way that can be detected reliably by our software AND does not affect their behavior, we have reverted to manual classification for now.</li>
	</ol>
	</li>
	<li>To evaluate processed movies, load movie using Load button.
	<ol>
		<li>Click on Start Session 1 to display the first frame of session 1 (or Start Session 2 to display the first frame of session 2).</li>
		<li>Click on Play/Stop to play and stop movies. 
		Note: The processed movies will display mouse with head and tail marked with differently colored circles. All sociability measures are displayed on the screen and updated as the movie plays.</li>
		<li>Export sociability data in Excel format by clicking &#8220;Export&#8221;.</li>
		<li>Click on &#8220;Compile Data&#8221; to compile all the data of all movies in a folder into a single excel file.</li>
	</ol>
	</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+sociability+of+the+BALB/c+mouse,+an+animal+model+of+autism+spectrum+disorders,+is+attenuated+by+NMDA+receptor+agonist+interventions:+clinical+implications.">Impaired sociability of the BALB/c mouse, an animal model of autism spectrum disorders, is attenuated by NMDA receptor agonist interventions: clinical implications.</a> Comprehensive Book on Autism Spectrum Disorders. , 323-342 (2011).</li><li>Dunn, M. E., Burbine, T., Bowers, C. A., Tantleff-Dunn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderators+of+stress+in+parents+of+children+with+autism.">Moderators of stress in parents of children with autism.</a> Community Mental Health Journal. 37, 39-52 (2001).</li><li>Babineau, B. A., Yang, M., Berman, R. F., Crawley, J. 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The authors have nothing to disclose.

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,&lt;br/&gt; can be adjusted according to needs.&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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...

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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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8.4K Views.  Old Dominion University. 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.

Video: Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

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Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Neuroscience

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data.

Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

Behavioral Tasks for Examining Identity Recognition In Mice

Social animals, like rodents, are able to recognize and differentiate between the identity of familiar individuals. Recognizing the identity of familiar individuals is important for developing social structures such as hierarchy, kinship, and family. However, mechanisms underlying the recognition of social identity remain unclear. Most rodent studies of social recognition are based on the propensity of rodents to interact with a novel social target, a phenomenon known as social novelty. However, behavioral tasks for examining social novelty cannot reveal the recognition of familiar conspecifics based on their identities. Presented here are behavioral tasks allowing for the examination of identity recognition in C57BL/6 mice by associating two familiar mice with or without a valenced experience. Subjects had interactions with two mice either without (neutral) or with a valenced experience (negative or positive) and became familiar with these mice. The negatively valenced mouse was associated with shocks, while the positively valenced mouse was associated with a food reward. Following training, the recognition of the identity of these familiar mice can be revealed in a social discrimination test, which is represented as the preference for the positively valenced mouse and avoidance of the negatively valenced mouse compared to the neutral mouse. Behavioral tasks for identity recognition could be useful in probing social memory mechanisms and the pathophysiology of disorders with impaired social cognition, such as autism spectrum disorder or schizophrenia.

By pairing negatively or positively valenced experiences with individual social targets, we developed behavior tasks for examining the identity recognition of C57BL/6 mice. These tasks allow for the study of mechanisms of social memory of individual social targets in healthy and disease-related mouse models with impaired social cognition.

Social animals, like rodents, are able to recognize and differentiate between the identity of familiar individuals. Recognizing the identity of familiar ...