Name: a system for tracking the dynamics of social preference behavior in small rodents
Uploaded: 2019-11-21T14:00:00
Description: Watch this Scientific Journal Video about A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents at JoVE.com

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 ...

<ol>
	<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 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 ...

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer independent manner. Our system allows a detailed investigation of social behaviors and their their dynamics in small rodent models and supports accurate comparisons between strains, conditions and treatments. This system may be used for any type of social discrimination tests, such as discriminating between males and females.Moreover, there are no limits to the sizes of the subjects. The analysis requires some experience for selecting the optimal parameters for tracking. For new users I recommend monitoring the tracking on the computer screen for the initial evaluation.At least one hour before the analysis place the home cages of all of the mice in the experimental room for acclimation. At the end of the acclimation period insert two empty chambers into the arena at two opposite corners of the arena. Then, locate the arena within the acoustic chamber and place the subject mice in the middle of the arena for 15 minutes of habituation.During this period, place the two social stimuli mice in different chambers located outside of the arena for habituation. And a 5x5x5 centimeter plastic toy with a distinct shape and color in another chamber. To perform the SP test, start recording with a video camera.Immediately remove the two empty chambers and place the social and object stimuli chambers in the corners of the arena that had been empty during the habituation. Allow the subjects to interact with the stimuli for five minutes before stopping the recording, and removing the stimuli containing chambers from the arena. Then, leave the subject in the empty arena for 15 minutes.To perform a social novelty preference test start the video recording, and insert the chamber containing the same social stimulus animals as used for the SP test and the chamber containing the novel social stimulus mouse into the two corners of the arena that were not used for the SP test. Allow the subjects to interact with the stimuli for five minutes before stopping the recording and removing the subjects and chambers from the arena. Then, clean the arena and chambers with running water and 10%ethanol.To use the track rodent graphic user interface to analyze the mouse behavior, open MATLAB and choose the Track Rodent Directory, right click to select Add to Path, Selected Folders and Subfolders to add all of the subfolders to the working path. Enter TrackRodent into the command window and press Enter. Select Load Session File to upload single or multiple video files.A movie inspector that allows a frame by frame inspection of the video clip will immediately be opened for the first file in the list. To inspect additional video files click Inspect Movie to open the video inspector and select a specific video file of interest at any time. Select the species tested if necessary and click Exclude Area to exclude all of the areas that my interrupt the tracking in accordance with the colors of the subject and arena.To define each chamber as a stimulus area for automatic detection of it's investigation by the subject, check the polygon or elliptical box and click Stimulus X to allow the stimulus areas to be marked. Then, select the desired algorithm from the list and enter the starting and ending frame numbers for the analysis in the corresponding edit boxes. To evaluate the automatic detection of the subject borders for a given threshold enter a value into the relevant threshold field and press Enter.For starting a behavioral analysis of all of the selected files, move to the first file and click Start. Finding the right threshold is critical and requires experience. It is recommended that new users use the non-fast algorithms and monitor the analysis online to ensure that they work properly.At the end of the analysis a results file will be saved for each movie in the same directory of the movie files. To inspect the results of each movie file click Load Results File and select the MAT files generated by the behavioral analysis. Move between the toggle buttons to examine the mouse location trace, stimuli exploration along session and total stimuli exploration time.For population analysis in the TrackRodent folder in MATLAB enter TrackRodentPopulationSummary as one word in the command window. Click Enter and choose Results Files to upload multiple TrackRodent results files. Fill in the numbers of the last frame for analysis, test name, stimulus one name, and stimulus two name and select all of the appropriate boxes for the desired analyses from the optional list.Check the Export box to extract the results to a spreadsheet in a single file. Then, click Start and wait until the program reports in the status line the analysis parameters and the data have been saved. In this experiment, the mean total investigation time of the population of subjects toward each of the two stimuli depicts a clear preference of the subjects toward the social stimulus.The social preference was maintained by the subject throughout the session although the preference seemed slightly stronger early in the session. The investigation time for each stimulus can be categorized according to the bout duration into short, medium and long bouts in addition to the total investigation time. As observed, there was no difference between the stimuli in the short bouts while the medium and long bouts showed clear social preferences, suggesting that in C57 Black 6J mice short bouts reflect curiosity, while long bouts reflect an interaction between the subject and the stimuli.In addition, heat maps of the investigation bouts toward the social stimulus and object during the course of the test can be generated for the whole population of subjects with the colors coding the bout duration. Importantly, the rate of transitions made by subjects from one stimulus to another was highest during the first two minutes of the test before gradually declining. The same tendency was apparent in the distribution of mean transition rate over time.This method can be used with any mouse connected to electrophysiological recording or optogenetic stimulating systems by a wire. For this, we use a special algorithm. Our system is currently in use by several laboratories worldwide for various paradigms requiring small rodent tracking.It may be used for assessing behavioral dynamics in any discrimination test.

Research

JoVE Journal

Behavior

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œ 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 ...

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 ...

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 ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...