This work was supported by The Human Frontier Science Program (HFSP grant RGP0019/2015), the Israel Science Foundation (ISF grants #1350/12, 1361/17), by the Milgrom Foundation and by the Ministry of Science, Technology and Space of Israel (Grant #3-12068).

All methods described have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Haifa.
1. Experimental Set-up
<ol>
	<li>Arena
	<ol>
		<li>Construct the experimental arena for mice (Figure 1A,D) by placing a white or black (depending on the animal&#39;s color) plexiglass box (37 cm x 22 cm x 35 cm) in the middle of an acoustic chamber (60 cm x 65 cm x 80 cm, made of 2 cm thick wood coated on the inside with 2 cm thick foam). For the light, remove a strip (2 cm wide, 10 cm below the ceiling of the chamber) of the foam around the acoustic chamber (besides the door), and attach an LED strip with either red or white light bulbs. Make sure the light is as uniform as possible around the arena to prevent reflections.</li>
		<li>Construct the arena for rats similarly to the one described above for mice, with different dimensions that appropriately fit the size of Sprague-Dawley (SD) rats (Figure 1G). Place a black plexiglass box (50 cm x 50 cm x 40 cm) in the middle of an acoustic chamber (90 cm x 60 cm x 85 cm, made of 2 cm thick wood coated on the inside with 2 cm thick foam).</li>
	</ol>
	</li>
	<li>Chambers
	<ol>
		<li>For mice, create two black or white (depending on the fur color) triangular chambers (12 cm isosceles, 35 cm height, with the floors closed) from 6 mm thick plexiglass. Locate them in two randomly selected opposite corners of the arena (Figure 1B,E). Stick a metal mesh (18 mm x 6 cm; 1 cm x 1 cm holes) in the lower part of each chamber using epoxy glue to allow direct interactions with the stimulus through the mesh (Figure 1C,F). Mark each chamber in a manner that allows discrimination from the others in a video without giving clues to the subjects (see Figure 1B,E for examples). 
		NOTE: Each chamber will eventually contain a social stimulus (mouse) or object (plastic toy, 5 cm x 5 cm x 5 cm, with a distinct shape and color; Figure 1C,I insets).&#160;Let the epoxy glue smell evaporate for at least a week before usage.</li>
		<li>For rats, create two black triangular chambers (20.5 cm isosceles, 40 cm height, made of 6 mm thick plexiglass, with the floors closed) and place them in two randomly selected opposite corners of the arena (Figure 1H), each with a metal mesh (25 cm x 7 cm; 2.5 cm x 1 cm holes) covering its lower portion (Figure 1I).</li>
	</ol>
	</li>
	<li>Place a high quality monochromatic camera, equipped with a wide-angle lens, at the top of the acoustic chamber and connect it to a computer to enable clear viewing and recording of the subject&#39;s behavior using commercial software (see Table of Materials for suggestions).</li>
</ol>
2. Behavioral Paradigm
NOTE: Steps 2.1-2.7 describe the behavioral paradigm for mice. See section 2.8 for specific instructions involving rats.
<ol>
	<li>Make sure that the cages of all animals (subjects: 2-4 month-old male mice; stimuli: 21-30 day-old juvenile mice) remain in the experimental room for at least 1 h before beginning the behavioral experiment.</li>
	<li>Following the acclimation period, insert two empty chambers into the arena randomly at two opposite corners. Place the subject in the middle of the arena for 15 min of habituation. During that time, place the two social stimuli, each in a different chamber located out of the arena for habituation. Place an object (a plastic toy, 5 cm x 5 cm x 5 cm, with a distinct shape and color) in another chamber.</li>
	<li>To perform the social preference (SP) test, start video recording and keep recording until the end of the test.</li>
	<li>Remove the two empty chambers and immediately insert the object and one of the social stimuli, each in a distinct chamber. Locate these chambers randomly in the opposite corners of the arena that were empty during habituation. Allow the subject to interact with the stimuli for the 5 min of the SP test. At the end of the test, stop recording.</li>
	<li>Following the SP test, remove the stimuli-containing chambers from the arena and leave the subject in the empty arena for 15 min.&#160;Clean the chambers from outside with 10% ethanol wipes.</li>
	<li>To perform the social novelty preference (SNP) test, start video recording and insert two chambers into the arena: one containing the same social stimulus used for the SP test (familiar stimulus), and the other containing the novel social stimulus. Place these chambers randomly in two opposite corners of the arena, making sure these&#160;locations&#160;were not used for the SP test. Allow the subject to interact with the stimuli for the 5 min of the SNP test.</li>
	<li>At the end of the SNP test, stop video recording, remove the subject and chambers from the arena, and place the subject back in its home cage. Leave the stimuli in the chambers for the next experiment (with another subject) or return them to their home cages. Clean the arena and chambers with running water followed by 10% ethanol and let dry.</li>
	<li>Behavioral paradigm for rats
	<ol>
		<li>For rats, repeat the behavioral paradigm described in steps 2.1-2.7, with two modifications: 1) handle the rat subjects and habituate the social stimuli to the chambers for 2 days (10 min every day) prior to the experiment; and 2) extend the SP test for 15 min to give the rats a longer period of exposure to social stimuli. Later, restrict the analysis of the SP test to the initial 5 min. 
		NOTE: At least one arena and five chambers are needed in order to run a single session.</li>
	</ol>
	</li>
</ol>
3. Using the TrackRodent GUI for Behavioral Analysis
NOTE: See the upper panel of Figure 2A for the TrackRodent GUI.
<ol>
	<li>Open MATLAB (tested with 2014a-2019a) and choose the TrackRodent folder.</li>
	<li>Add all subfolders to the working path by right-clicking on each folder and selecting Add to Path | Selected Folders and Subfolders.</li>
	<li>Type TrackRodent in the command window and press Enter.</li>
	<li>Upload a single file or multiple video files (AVI or MP4 format) by selecting Load session file (AVI).</li>
	<li>A movie inspector, allowing the inspection of the video clip frame-by-frame, will immediately be opened for the first file in the list (Figure 2A). Use it to examine the video clip and define the first and last frames of the segment to be analyzed. Record the numbers of these frames, which will be required later. Close the window when done.</li>
	<li>For inspecting additional video files, open the video inspector at any time by pressing Inspect movie and selecting a specific video file.</li>
	<li>Select the species tested (mouse or rat; mouse is the default).</li>
	<li>Exclude all the areas that may interrupt the tracking, in accordance with the colors of the subject and arena (black or white).
	<ol>
		<li>To exclude a given area, press Exclude area, and after the cursor changes to a cross shape, mark all corners of the area for exclusion. When done, right-click on the mouse, then double left-click the center of the marked area. The excluded area will become a shade of red on the screen. Repeat this procedure to exclude as many areas as needed.</li>
	</ol>
	</li>
	<li>To remove an area from exclusion, press Remove excluded area, then (using the crossed cursor) click on the area to be removed from exclusion.</li>
	<li>To define each chamber as a &#39;stimulus&#39; area, for automatic detection of its investigation by the subject, choose the shape of the &#39;stimulus&#39; area to be either polygon or elliptical by checking the appropriate box, then pressing Stimulus X (where &#34;X&#34; represents 1, 2, or 3). Mark the &#39;stimulus&#39; areas similarly to the excluded areas, which will then become yellow in color. For changing the location of a specific &#39;stimulus&#39; area, press Stimulus X again and mark the new area location (this will automatically update the location). 
	NOTE: Choose the different stimuli number in a consistent manner for all files (i.e. object as stimulus 1 for all the SP test files).</li>
	<li>To track the presence of the subject in a specific virtual compartment inside the arena, choose the shape of the &#39;compartment&#39; area (polygon or elliptical), then press Compartment X (where &#34;X&#34; represents 1, 2, 3, 4, or 5). Mark the &#39;compartment&#39; areas similarly to the excluded or stimuli areas, which will then become blue in color. For changing the location of a specific &#39;compartment&#39; area, press Compartment X again and mark the new area location (this will automatically update the location).</li>
	<li>Choose the desired algorithm (BlackMouseBodyBased was used for the video) from the list (see available algorithms in Figure 2B).</li>
	<li>Write the numbers of starting and ending frames for the analysis in the corresponding edit boxes of the GUI.</li>
	<li>Choose a threshold for detecting the subject body. 
	NOTE: Most algorithms use the &#34;Low&#34; threshold only, while the head directionality-based algorithms use the &#34;High&#34; threshold, as well. For the &#34;Low&#34; threshold, choose a level that includes the mouse/rat body without the tail (as much as possible), while the &#34;High&#34; threshold should also include the tail. In the case of using head directionality-based algorithms, the software will determine the head location as being opposite to the tail location. 
	NOTE: The software will later on ignores small objects detected using the chosen threshold.</li>
	<li>To evaluate automatic detection of the subject borders for a given threshold, insert a value to the relevant threshold field and press Enter on the keyboard.</li>
	<li>When choosing multiple files, move to the next file (using the Next button at the top) and select the appropriate definitions for each file. When finished, verify the parameters and area locations for all files by moving between each, using the Previous and Next buttons at the top of the GUI. 
	NOTE: The definitions of all areas and parameters are specific to a given file.</li>
	<li>For starting behavioral analysis of all selected files, move to the first file and press Start.</li>
	<li>At the end of the analysis, a results file (.mat file) is saved for each movie in the same directory of the movie files. 
	NOTE: If the slow (non-fast) version of the algorithm is used, it will also save a version of the movie with a white cross of the center of body mass, which changes its color every frame that is detected as investigatory, unless the Save analyzed movie toggle button of the GUI is unchecked. This version of the movie (saved in the same directory, with the same name as the original movie, with the suffix &#39;analyzed movie&#39;) can be used offline to evaluate the quality of the automatic detection performed by the system.</li>
</ol>
4. Using the TrackRodent GUI for Results Presentation
NOTE: See the lower panel of Figure 2A for results presentation.
<ol>
	<li>To inspect the results of each movie file, press Load results file and choose the .mat files generated by the behavioral analysis.</li>
	<li>Move between the toggle buttons to examine onscreen any of the following analyses: Mouse location trace (Figure 2A); Compartments occupation along session (if &#39;compartments&#39; were defined, not shown); Stimuli exploration along session (Figure 2C); Total time in compartments (if &#39;compartments&#39; were defined, not shown); and Total stimuli exploration time (Figure 2D). 
	NOTE: &#39;Stimulus&#39; areas are areas in which the software evaluates subject interaction, while &#39;Compartment&#39; areas are areas in which the software tracks the presence of the subject. Stopping the analysis using the Stop analysis button will automatically save the results generated up to the last analyzed frame. For most computers, it should be possible to upload and analyze as many as 20 movies at once (depending on computer performance).</li>
</ol>
5. Using the TrackRodentPopulationSummary GUI for Population Analysis (Figure 2E)
<ol>
	<li>Open MATLAB (tested with 2014a-2019a) and choose the TrackRodent folder.</li>
	<li>Type TrackRodentPopulationSummary in the command window and press Enter.</li>
	<li>Upload multiple TrackRodent results files (.mat format) by pressing Choose results files.</li>
	<li>Fill in the numbers of Last frame for analysis, Test name, Stimulus 1 name, and Stimulus 2 name.</li>
	<li>Choose the desired analyses from the list of optional analyses by checking all appropriate boxes.</li>
	<li>Choose the Export results to a speadsheet by checking the appropriate box to extract all the results of the checked analyses as a single spreadsheet file.</li>
	<li>Press Start and wait until analysis complete. 
	NOTE: This concludes the analysis. The software can be used to analyze the results of as many movie files as desired, given that they were all behaviorally analyzed using the TrackRodent software. The analysis performed by the software assumes video recording at a frame rate of 30 Hz. In the case that a different frame rate was used, multiply the time by 30 and divide by the frame rate used for recording to convert it to the correct value (s).</li>
</ol>

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	<li>Insel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+translation+in+social+neuroscience:+a+review+of+oxytocin,+vasopressin,+and+affiliative+behavior.">The challenge of translation in social neuroscience: a review of oxytocin, vasopressin, and affiliative behavior.</a> Neuron. 65 (6), 768-779 (2010).</li><li>Moy, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sociability+and+preference+for+social+novelty+in+five+inbred+strains:+an+approach+to+assess+autistic-like+behavior+in+mice.">Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice.</a> Genes, Brain and Behavior. 3 (5), 287-302 (2004).</li><li>Carr, W. J., Yee, L., Gable, D., Marasco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+recognition+of+conspecifics+by+domestic+Norway+rats.">Olfactory recognition of conspecifics by domestic Norway rats.</a> Journal of Comparative and Physiological Psychoogyl. 90 (9), 821-828 (1976).</li><li>Ferguson, J. N., Aldag, J. M., Insel, T. R., Young, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+in+the+medial+amygdala+is+essential+for+social+recognition+in+the+mouse.">Oxytocin in the medial amygdala is essential for social recognition in the mouse.</a> Journal of Neuroscience. 21 (20), 8278-8285 (2001).</li><li>Nadler, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+apparatus+for+quantitation+of+social+approach+behaviors+in+mice.">Automated apparatus for quantitation of social approach behaviors in mice.</a> Genes, Brain and Behavior. 3 (5), 303-314 (2004).</li><li>Page, D. T., Kuti, O. J., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computerized+assessment+of+social+approach+behavior+in+mouse.">Computerized assessment of social approach behavior in mouse.</a> Frontiers in Behavioral Neuroscience. 3, 48 (2009).</li><li>Sankoorikal, G. M., Kaercher, K. A., Boon, C. J., Lee, J. K., 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=A+mouse+model+system+for+genetic+analysis+of+sociability:+C57BL/6J+versus+BALB/cJ+inbred+mouse+strains.">A mouse model system for genetic analysis of sociability: C57BL/6J versus BALB/cJ inbred mouse strains.</a> Biological Psychiatry. 59 (5), 415-423 (2006).</li><li>Martin, L., Sample, H., Gregg, M., Wood, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+operant+social+motivation+paradigms+using+BTBR+T+tf/J+and+C57BL/6J+inbred+mouse+strains.">Validation of operant social motivation paradigms using BTBR T+tf/J and C57BL/6J inbred mouse strains.</a> Brain and Behavior. 4 (5), 754-764 (2014).</li><li>Noldus, L. P. J. J., Spink, A. J., Tegelenbosch, R. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EthoVision:+A+versatile+video+tracking+system+for+automation+of+behavioral+experiments.">EthoVision: A versatile video tracking system for automation of behavioral experiments.</a> Behavior Research Methods Instruments &amp; Computers. 33 (3), 398-414 (2001).</li><li>Sams-Dodd, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automation+of+the+social+interaction+test+by+a+video-tracking+system:+behavioural+effects+of+repeated+phencyclidine+treatment.">Automation of the social interaction test by a video-tracking system: behavioural effects of repeated phencyclidine treatment.</a> Journal of Neuroscience Methods. 59 (2), 157-167 (1995).</li><li>Netser, S., Haskal, S., Magalnik, H., Wagner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+system+for+tracking+social+preference+dynamics+in+mice+reveals+sex-+and+strain-specific+characteristics.">A novel system for tracking social preference dynamics in mice reveals sex- and strain-specific characteristics.</a> Molecular Autism. 8, 53 (2017).</li></ol>

The authors have nothing to disclose.

The experimental system described here, which was designed as an alternative to the three-chamber apparatus2,5, allows performance of the same tests while solving some of its limitations. The use of triangular chambers, which are located in two opposite corners of the rectangular arena limits the subject-stimulus interaction area to a well-defined plane, thus enabling precise automated analysis of investigation behavior. One advantage is the use of the analysis s...

Revealing the biological mechanisms underlying neurodevelopmental disorders (NDDs) is one of the main challenges in the field of neuroscience1. Addressing this challenge requires behavioral paradigms and experimental systems that typify the behavior of rodents in a standard and unbiased manner. An influential study published more than a decade ago by Moy and colleagues2 presented the three-chamber test. Since then, this test has been widely used to investigate social behavior in rodent models of NDDs. This test evaluates two innate tendencies of rodents: 1) to stay in the proximity of a social stimulus over an object (sociability, also termed social preference [SP]), and 2) to prefer the proximity of a novel social stimulus over a familiar one (social novelty preference [SNP])3,4. Several subsequent studies suggested methods of automated analysis of the three-chamber test using computerized methods5,6.
This test still suffers from several caveats. First, it principally examines social place preference rather than the motivation of the subject to directly interact with a social stimulus, although some groups also measure olfactory investigation (sniffing) time, either manually7 or using commercial computerized systems8,9,10. Second, the three-chamber test is mostly used to measure the total time spent by the subject in each chamber, and it neglects behavioral dynamics. Finally, it relies on only one aspect of the social behavior, which is the time spent by the subject in each chamber (or sniffing time, if measured).
Here we present a novel and affordable experimental system that is an alternative to the three-chamber apparatus. It also allows performance of the same behavioral tests while solving the abovementioned caveats. The presented behavioral system automatically and directly measures the investigative behavior of a rodent towards two stimuli. Additionally, it analyzes the behavioral dynamics in an observer-independent manner. Moreover, this system measures multiple behavioral parameters and analyzes these at both individual and population levels; thus, it supports a rigorous analysis of social behavior and its dynamics during each test. Furthermore, random repositioning of the chambers in opposite corners of the arena during the various test stages neutralizes any effects of spatial memory or preference. This system may also be used for other discrimination tests, such as sex discrimination. The custom apparatus is easy to produce, and the analysis system is publicly accessible as an open-source code, thereby allowing its use in any laboratory. We demonstrate the ability of this system to measure multiple parameters of social behavior in rodent strains with distinct fur colors during the social preference and social novelty preference tests. We also validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations, such as whisker trimming.
TrackRodent software: three algorithms were written in MATLAB (2014a-2019a) to track the experimental subject and its interactions with the stimuli. All algorithms were deposited in GitHub, found at &#60;https://github.com/shainetser/TrackRodent&#62;. The main aim of all four algorithms is to track the contours of the subject&#39;s body to detect any direct contact with the stimuli areas.
Body-based algorithm: this algorithm has three versions that track the contours of an unwired dark mouse on a white background (BlackMouseBodyBased), a white mouse on a dark background (WhiteMouseBodyBased), or a white rat on a dark background (WhiteRatBodyBased). The graphical user interface (GUI) of the software requires that the experimenter chooses an experiment using either mice or rats and then selects the correct code. For each version of the algorithm, there are two optional codes: one that presents the tracking process on the screen while it performs the analysis, and one that does not (hence, it runs faster and is termed &#34;fast&#34;). For example, the names of the relevant codes for the BlackMouseBodyBased algorithm are: &#34;BlackMouseBodyBased23_7_14&#34; and &#34;BlackMouseBodyBased23_7_14_Fast&#34;. All algorithms ending with &#34;fast&#34; do not show the tracking online, and users must directly save the data to the results file (.mat file). All body-based algorithms require setting a single threshold (&#34;low threshold&#34; in the software&#39;s GUI) to detect the body of the subject.
Head-directionality based algorithm: the second algorithm, which is available only for black mice, is based on the body-based algorithm, in addition to determining head directionality. This algorithm detects the interactions of subject&#39;s head with the &#34;stimuli&#34; areas, thus avoiding false positives that can arise from random contacts of the subject&#39; with these areas. For this algorithm, two detection thresholds of mouse body contours are defined: high threshold, which includes the brighter tail of black mice, and low threshold, which includes the body without tail. Thereafter, the algorithm fits an ellipsoid to the detected boundaries using the lower threshold and defines the location of the mouse head and tail (with no distinction between the two). The final discrimination between the tail and head is based on the boundaries defined by the higher threshold.
Wired animal algorithm: the third algorithm aims to minimize artifacts resulting from cables (i.e., electrical wire or optical fiber) connected to the animal, allowing analysis of the animal&#39;s behavior while connected to a cable. This algorithm has codes only for black mice and white rats. The code for rats requires the experimenter to define both low and high thresholds, while the mouse code requires only a low threshold.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Flea3 1.3 MP Mono USB3 Vision</td>
			<td>FLIR (formerly PointGrey)</td>
			<td>FL3-U3-13Y3M-C</td>
			<td>Monochromatic Camera</td>
		</tr>
		<tr>
			<td>FlyCap 2.0</td>
			<td>FLIR (formerly PointGrey)</td>
			<td>FlyCapture 2.13.3.61X64</td>
			<td>Video recording software</td>
		</tr>
		<tr>
			<td>Home 5 minute Epoxy glue</td>
			<td>Devocon</td>
			<td>20845</td>
			<td>For gluing the metal mesh to the Plexiglas stimuli chambers</td>
		</tr>
		<tr>
			<td>Matlab 2014-2019</td>
			<td>MathWorks</td>
			<td>R2014a - R2019a</td>
			<td>Programming environment</td>
		</tr>
		<tr>
			<td>Plexiglas boards (6 mm thickBlack or white)</td>
			<td>Melina (1990) LTD, Israel</td>
			<td>NaN</td>
			<td>For arena and stimuli chambers construction</td>
		</tr>
		<tr>
			<td>Red led strips (60 leds per meter) connected to a 12V power supply</td>
			<td>2012topdeal eBay supplier</td>
			<td>NaN</td>
			<td>For illumination of the acoustic chamber</td>
		</tr>
	</tbody>
</table>

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.

Using the system for the social preference test in C57BL/6J mice 
Figure 1 shows three versions of the experimental set-up. The first version (Figure 1A-C) is designed for mice with dark fur colors, such as C57BL/6J mice. The second (Figure 1D-F) is planned for mice with bright fur colors, such as BALB/c or ICR (CD-1) mice. The third is larger (...

Watch this Scientific Journal Video about A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents at JoVE.com

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

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 ...

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7.5K Views.  University of Haifa. 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.

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

The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression and defensive behavior in a semi natural setting. The most important behavioral elements performed by the resident and the intruder are demonstrated in the video and illustrated using artistic drawings. The use of the resident intruder paradigm for acute and chronic social stress experiments is explained as well. Finally, some brief tests and criteria are presented to distinguish aggression from its more violent and pathological forms.

This video shows the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression, defensive behavior and violence in a semi-natural setting. The use of the paradigm for social stress experiments is explained as well.

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to ...

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children &#8211; Working Memory (CABC-WM)

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of working memory in young school-age children. Working memory deficits have been identified in children with language-based learning disabilities, including dyslexia1,2 and language impairment3,4, but it is not clear whether these children exhibit deficits in subcomponents of working memory, such as visuospatial or phonological working memory. The CABC-WM is administered on a desktop computer with a touchscreen interface and was specifically developed to be engaging and motivating for children. Although the long-term goal of the CABC-WM is to provide individualized working memory profiles in children, the present study focuses on the initial success and utility of the CABC-WM for measuring central executive, visuospatial, phonological loop, and binding constructs in children with typical development. Immediate next steps are to administer the CABC-WM to children with specific language impairment, dyslexia, and comorbid specific language impairment and dyslexia.

Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children.

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

Use of a Video Scoring Anchor for Rapid Serial Assessment of Social Communication in Toddlers

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency&#8212;which is foundational for both social learning and social competency&#8212;is hypothesized to be disrupted in autism spectrum disorder (ASD). The ability to quantify the full range of RSB during the toddler period, when core symptoms of ASD often arise, is pivotal for evaluating early risk for ASD, characterizing social development, and tracking response to early interventions. However, important parameters of variation in RSB&#8212;especially prior to the development of verbal language&#8212;can be nuanced and difficult to characterize using questionnaire-based methods. To address this challenge, we developed a system for measuring quantitative variation in RSB in toddlers (ages 18 - 30 months) that incorporated not only standard questionnaire data from caregivers but also a novel set of video-referenced items, through which a respondent compares the behavior of a subject to that observed in a short video of a young child manifesting a highly competent level of social communication. Testing of this measure in a general population sample of twins confirmed that both the video-referenced items and the RSB Total Score (video-referenced items plus non-video-referenced items) displayed unimodal, continuous distributions, strong internal consistency, marked preservation of individual differences, and extremely high heritability. In addition, video-referenced items were particularly sensitive to quantifying incremental changes in social communication, a major element of RSB, over the course of early childhood development. Scores on the vrRSB clearly differentiated children with and without ASD and these data comprise an initial validation of this promising method for quantifying early RSB&#8212;cross-sectionally, over time, and as a function of early intervention.

Here we present a protocol for administration of the video-referenced rating of reciprocal social behavior (vrRSB) using a paper or online version. This survey quantifies RSB, a prerequisite for social competence, in toddlers through &#34;video-referenced&#34; items whereby a subject is compared to a reference child shown in a three-minute video.

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency&#8212;which is foundational for both social learning and ...

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants&#39; fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

We present a visual guide to disease defense behaviors in leaf-cutting ants, with individual clips and accompanying definitions, illustrated in an experimental infection scenario. Our main aim is to help other researchers recognize key defensive behaviors and to provide a common understanding for future research in this field.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies ...