OKB and JRW wrote the manuscript. This work was supported by CIHR grant MOP 74711 (to JRW).

1. Equipment and Room Set Up:
<ol>
<li>The apparatus for Crawley's sociability and preference for social novelty test is comprised a rectangular, three-chamber box. Each chamber is 19 x 45 cm and the dividing walls are made from clear Plexiglas, with an open middle section, which allows free access to each chamber.</li>
<li>Use two identical, wire cup-like containers with removable lids that large enough to hold a single mouse. These are placed vertically inside the apparatus, one in each side chamber, and will contain the na&iacute;ve/unfamiliar mouse. Each container is comprised of metal wires to allow for air exchange between the interior and exterior of the cylinder but small enough to prevent direct physical interactions between an animal on the inside with one on the outside.</li>
<li>Behavioral testing should be performed between 9:00am and 6:00 pm.</li>
<li>General room lighting is 650 lux.</li>
<li>Employ Observer 6.0 (Noldus) program (or a suitable alternative) to monitor and record the observation and follow up parameters, mentioned in section 4.3.</li>
<li>After each trial, clean all chambers with 70% ethanol (between mice) and then with Clidox 1:5:1 (between the cages) to prevent olfactory cue bias and to ensure proper disinfection, respectively.</li>
<li>The person who makes the observation has to be at least 2 meters away from the apparatus.</li>
</ol>
2. 	Animal Preparation:
<ol>
<li>House three to five mice per cage in a room with a 12 hr light/dark cycle (lights on at 7:00 am) with ad libitum access to food and water, according to standards and requirements of local Animal Care Committee guidance and protocols.</li>
<li>Transfer all the cages containing mice into the behavioral room 30 minutes before the first trial begins.</li>
<li>There are two classes of mouse required for this experiment, one which acts as a control, na&iacute;ve or "unfamiliarized" animal and one that is the test subject. For the control mouse use a mouse of the same background (usually C57BL6), age (usually 8-12 weeks old), gender and weight, without any prior contact (not littermates) with the subject mouse. Two control mice are required per experiment, one is used for session I and another for session II. The same control mice may be used between trials.</li>
</ol>
3. 	Habituation (adaptation):
<ol>
<li>Isolate the right and left compartments by using the dividing Plexiglass walls.</li>
<li>Place empty wire containment cups in the middle of right and left chamber (one per each site).</li>
<li>Place the subject mouse at the center of the middle chamber for adaptation.</li>
<li>Habituate for 5 minutes.</li>
</ol>
4. 	Social Affiliation Aspect of the Test (session I):
<ol>
<li>Place one of the control mice ("Stranger 1") inside a wire containment cup that is located in one of the side chambers. The placement of Stranger 1 in the left or right side of the chamber is systematically altered between trials.</li>
<li>Remove the walls between the compartments, to allow free access for the subject mouse to explore each of the three chambers.</li>
<li>Immediately, begin monitoring and recording the following parameters:
<ol class="lalpha">
<li>duration and</li>
<li>number of direct (active) contacts between the subject mouse and the containment cup housing or not housing the Stranger 1 mouse, for each chamber individually. Direct contact between the subject mouse and the containment cup, or stretching of the body of the subject mouse in an area 3-5 cm around the cup is counted as an active contact;</li>
<li>duration and</li>
<li>number of other behaviors by the subject mouse in each compartment, including walking, self-grooming, lack of any body movements for more than 5 seconds ("freezing"), as well as unusual behavior, such as jumping, repetitive behavior, etc;</li>
<li>duration and</li>
<li>number of entries to each compartment. The mouse is considered to be in the chamber when its head and four paws have entered into the chamber.</li>
</ol></li>
<li>The duration of session I is 10 minutes.</li>
</ol>
5. 	Social Novelty/preference Session of the Test (Session II):
<ol>
<li>Place a second control mouse ("Stranger 2") inside an identical wire containment cup in the opposite side chamber (that had been empty during the Session I). Monitor the same parameters described in 4.3, differentiating the behaviors between the subject mouse in the presence of Stranger 1 compared with Stranger 2.</li>
<li>The duration of session II is 10 minutes.</li>
</ol>
6. Statistical Analysis:
<ol>
<li>By using the Origin 6.0 program (or a suitable alternative) analyze the following parameters, using the recorded data:
<ol class="lalpha">
<li>the total number of contacts and</li>
<li>total duration of contacts between experimental mouse and empty containment cup vs or cup housing Stranger 1 (in session I), or between experimental mouse and the cup housing Stranger 1 vs Stranger 2 (in session II);</li>
<li>mean duration per contact;</li>
<li>total number and duration of other behaviors (freezing, self-grooming, walking)</li>
<li>total time spent by the subject mouse in each compartment.</li></ol></li>
<li>Analyze the significant differences for each of the aforementioned parameters, by comparing the groups:
<ol class="lalpha">
<li>"Empty" containment cup vs "Stranger 1" for a wild type (WT) subject</li>
<li>"Empty" containment cup vs "Stranger 1" for the experimental mouse (e.g. a knockout, transgenic or drug-treated mouse, etc.).</li>
<li>"Stranger 1" vs "Stranger 2" for a WT subject</li>
<li>"Stranger 1" vs "Stranger 2" for the experimental mouse (knockout, transgenic, or drug-treated, etc.).</li></ol></li>
<li>Present each dataset of the aforementioned parameters as Session I: WT and experimental subjects; Session II: WT and experimental subjects, as shown in Figure 1(A and B) and 2(A and B), respectively.</li>
</ol>
It is also advisable to take into consideration other parameters, which help in standardizing this test, such as prior experience in recognizing specific mouse behavioral patterns, such as sniffing, grooming, rubbing, fighting, mounting, which can be largely dependent on subjective personal observation. The person acting as the recorder of the behavioral parameters during the test should be blind to the genotype or treatment conditions and this information decoded afterwards.

7. Representative Results:

An example of the two steps of the social interaction test is shown in Figures 1 and 2. The first session of the test allows estimation of social affiliation and motivation of the subject mouse. In this part of the test, the stranger mouse was enclosed in a circular wire cup that allowed nose contact, but prevented fighting between mice. These conditions ensure that social approaches are initiated by the subject mouse and are investigatory only, without direct physical contact. The mouse has the choice to spend more time around the containment cup containing the stranger mouse or to avoid any contact by moving to the compartment with the empty cup. Other parameters, such as number of individual contacts, can be used in additional to duration spent near to the stranger mouse.
Typically, a wild type animal will spend significantly more time in the compartment with Stranger 1 compared to the compartment with empty cup (Figure 1A), indicating normal sociability, social motivation and affiliation. In GSK-3&alpha; knockout mice, for example, there are no significant differences between the time spent with empty cup compared to the cup containing stranger 1 (Figure 1B), indicating impaired sociability in these animal model 6.
The second session of the test is designed to estimate social novelty and social memory. In this part, the subject mouse has a free choice between the first, already-investigated, unfamiliar mouse (Stranger 1), and a novel unfamiliar mouse (Stranger 2). Usually, a WT animal recalls its previous contact with Stranger 1, and, in this session, tends to spend more time with the newly encountered mouse (Stranger 2), indicating intact social memory and predilection for novel experiences (Figure 2A).
Unlike wild-type mice, GSK-3&alpha; mutant animals do not show preference for the chamber containing a newly introduced mouse (Stranger 2) over a chamber containing a now familiar mouse (Stranger 1) in session II (Figure 2B). Indifferent behavior of KO mice in this test is indicative of decreased social motivation and novelty.
 <img src="/files/ftp_upload/2473/2473fig1.jpg" alt="Figure 1" /> 
Figure 1. Session I: Social affiliation and sociability. Mean length of time (&plusmn;SEM) in the chamber with the stranger ("stranger side") compared to the opposite chamber ("empty side"). Unlike WT males (A), KO (B) animals failed to demonstrate a preference for social proximity by spending same time in both chambers. ***p &le; 0.0001 in WT group.
 <img src="/files/ftp_upload/2473/2473fig2.jpg" alt="Figure 2" /> 
Figure 2. Session II: Social memory and novelty. Mean duration of time (&plusmn;SEM) in the chamber with the unfamiliar mouse from the sociability phase ("Stranger 1") and in the opposite chamber with a new unfamiliar mouse ("Stranger 2"). Unlike WT males (A), KO (B) animals failed to demonstrate a preference for social novelty by spending same time in both chambers. ***p &le; 0.0001 in WT group.

<ol>
	<li>Berry, R. J., Bronson, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+history+and+bioeconomy+of+the+house+mouse.">Life history and bioeconomy of the house mouse.</a> Biological reviews of the Cambridge Philosophical Society. 67, 519-550 (1992).</li><li>Robinson, G. E., Grozinger, C. M., Whitfield, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sociogenomics:+social+life+in+molecular+terms.">Sociogenomics: social life in molecular terms.</a> Nature reviews. 6, 257-270 (2005).</li><li>Silverman, J. L., Yang, M., Lord, C., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+phenotyping+assays+for+mouse+models+of+autism.">Behavioural phenotyping assays for mouse models of autism.</a> Nat Rev Neurosci. 11, 490-502 (2010).</li><li>Clapcote, S. J., Lipina, T. V., Millar, J. K., Mackie, S., Christie, S., Ogawa, F., Lerch, J. P., Trimble, K., Uchiyama, M., Sakuraba, Y., Kaneda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotypes+of+Disc1+missense+mutations+in+mice.">Behavioral phenotypes of Disc1 missense mutations in mice.</a> Neuron. 54, 387-402 (2007).</li><li>Labrie, V., Lipina, T., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+with+reduced+NMDA+receptor+glycine+affinity+model+some+of+the+negative+and+cognitive+symptoms+of+schizophrenia.">Mice with reduced NMDA receptor glycine affinity model some of the negative and cognitive symptoms of schizophrenia.</a> Psychopharmacology. 200, 217-230 (2008).</li><li>Kaidanovich-Beilin, O., Lipina, T. V., Takao, K., Eede, M. v. a. n., Hattori, S., Laliberte, C., Khan, M., Okamoto, K., Chambers, J. W., Fletcher, P. J., Macaulay, K., Doble, B. W., Henkelman, M., Miyakawa, T., Roder, J., Woodgett, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+in+brain+structure+and+behavior+in+GSK-3alpha+mutant+mice.">Abnormalities in brain structure and behavior in GSK-3alpha mutant mice.</a> Molecular brain. 2, 35-35 (2009).</li><li>Moy, S. S., Nadler, J. J., Perez, A., Barbaro, R. P., Johns, J. M., Magnuson, T. R., Piven, J., Crawley, J. N. <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, 287-302 (2004).</li><li>Carter, C. S., Williams, J. R., Witt, D. M., 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=Oxytocin+and+social+bonding.">Oxytocin and social bonding.</a> Annals of the New York Academy of Sciences. , 652-6204 (1992).</li><li>Miczek, K. A., Maxson, S. C., Fish, E. W., Faccidomo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggressive+behavioral+phenotypes+in+mice.">Aggressive behavioral phenotypes in mice.</a> Behavioural brain research. 125, 167-181 (2001).</li><li>Arakawa, H., Arakawa, K., Blanchard, D. C., Blanchard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+test+paradigm+for+social+recognition+evidenced+by+urinary+scent+marking+behavior+in+C57BL/6J+mice.">A new test paradigm for social recognition evidenced by urinary scent marking behavior in C57BL/6J mice.</a> Behavioural brain research. 190, 97-104 (2008).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+mouse+behavioral+tasks+relevant+to+autistic-like+behaviors.">Designing mouse behavioral tasks relevant to autistic-like behaviors.</a> Mental retardation and developmental disabilities research reviews. 10, 248-258 (2004).</li><li>Miyakawa, T., Leiter, L. M., Gerber, D. J., Gainetdinov, R. R., Sotnikova, T. D., Zeng, H., Caron, M. G., Tonegawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+calcineurin+knockout+mice+exhibit+multiple+abnormal+behaviors+related+to+schizophrenia.">Conditional calcineurin knockout mice exhibit multiple abnormal behaviors related to schizophrenia.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 8987-8992 (2003).</li><li>Yamasaki, N., Maekawa, M., Kobayashi, K., Kajii, Y., Maeda, J., Soma, M., Takao, K., Tanda, K., Ohira, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-CaMKII+deficiency+causes+immature+dentate+gyrus,+a+novel+candidate+endophenotype+of+psychiatric+disorders.">Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.</a> Molecular brain. 1, 6-6 (2008).</li><li>Crawley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's Wrong with My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. , (2000).</li><li>Holmes, A., Kinney, J. W., Wrenn, C. C., Li, Q., Yang, R. J., Ma, L., Vishwanath, J., Saavedra, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galanin+GAL-R1+receptor+null+mutant+mice+display+increased+anxiety-like+behavior+specific+to+the+elevated+plus-maze.">Galanin GAL-R1 receptor null mutant mice display increased anxiety-like behavior specific to the elevated plus-maze.</a> Neuropsychopharmacology. 28, 1031-1044 (2003).</li><li>Matochik, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+main+olfactory+system+in+recognition+between+individual+spiny+mice.">Role of the main olfactory system in recognition between individual spiny mice.</a> Physiology &#38; behavior. 42, 217-222 (1988).</li><li>Bluthe, R. M., Dantzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+vomeronasal+system+in+vasopressinergic+modulation+of+social+recognition+in+rats.">Role of the vomeronasal system in vasopressinergic modulation of social recognition in rats.</a> Brain research. 604, 1-2 (1993).</li><li>Tanda, K., Nishi, A., Matsuo, N., Nakanishi, K., Yamasaki, N., Sugimoto, T., Toyama, K., Takao, K., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+social+behavior,+hyperactivity,+impaired+remote+spatial+memory,+and+increased+D1-mediated+dopaminergic+signaling+in+neuronal+nitric+oxide+synthase+knockout+mice.">Abnormal social behavior, hyperactivity, impaired remote spatial memory, and increased D1-mediated dopaminergic signaling in neuronal nitric oxide synthase knockout mice.</a> Molecular brain. 2, 19-19 (2009).</li><li>Scattoni, M. L., Crawley, J., Ricceri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+vocalizations:+a+tool+for+behavioural+phenotyping+of+mouse+models+of+neurodevelopmental+disorders.">Ultrasonic vocalizations: a tool for behavioural phenotyping of mouse models of neurodevelopmental disorders.</a> Neuroscience and biobehavioral reviews. 33, 508-515 (2009).</li><li>Couture, S. M., Penn, D. L., Losh, M., Adolphs, R., Hurley, R., Piven, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+social+cognitive+functioning+in+schizophrenia+and+high+functioning+autism:+more+convergence+than+divergence.">Comparison of social cognitive functioning in schizophrenia and high functioning autism: more convergence than divergence.</a> Psychological medicine. 40, 569-579 (2010).</li><li>Zisook, S., Shuchter, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depression+through+the+first+year+after+the+death+of+a+spouse.">Depression through the first year after the death of a spouse.</a> The American journal of psychiatry. 148, 1346-1352 (1991).</li><li>Trainor, B. C., Workman, J. L., Jessen, R., Nelson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+nitric+oxide+synthase+signaling+dissociates+social+investigation+and+aggression.">Impaired nitric oxide synthase signaling dissociates social investigation and aggression.</a> Behavioral neuroscience. 121, 362-369 (2007).</li><li>Volke, V., Soosaar, A., Koks, S., Bourin, M., Mannisto, P. T., Vasar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=7-Nitroindazole,+a+nitric+oxide+synthase+inhibitor,+has+anxiolytic-like+properties+in+exploratory+models+of+anxiety.">7-Nitroindazole, a nitric oxide synthase inhibitor, has anxiolytic-like properties in exploratory models of anxiety.</a> Psychopharmacology. 131, 399-405 (1997).</li><li>Griffin, M. G., Taylor, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norepinephrine+modulation+of+social+memory:+evidence+for+a+time-dependent+functional+recovery+of+behavior.">Norepinephrine modulation of social memory: evidence for a time-dependent functional recovery of behavior.</a> Behavioral. 109, 466-473 (1995).</li></ol>

No conflicts of interest declared.

Mus musculus is a social species that engages in high levels of reciprocal social interactions, communal nesting, sexual and parenting behaviors, territorial scent marking and aggressive behaviors 8-10. Several tests have been developed to examine social behavioral in mice (reviewed in 3). Here, we present a widely employed test to evaluate sociability in mice. Crawley's sociability and preference for social novelty test was originally developed by Crawley and colleagues 7, 11. There are several advantages to the three-chamber paradigm, which improve data reliability for examination of sociability, social affiliation, social memory and preference, using similar procedures. The main specificity of this test is the design in which a mouse, previously unencountered by the subject animal, is placed under a wire cup and unable to move freely. This prevents direct physical contact, eliminating fighting and/or aggressive behavior but allows sensory interactions (smell, sight, sound, taste). Under these conditions, the experimental mouse initiates and terminates any interactions, always having a choice between unfamiliar mouse vs empty container (in session 1), or between familiar mouse (Stranger 1) vs the novel mouse (Stranger 2). For example, in one-chamber social interaction tests, the subject mouse is exposed to a stranger in the same chamber, and since both mice are able to move freely the aforementioned active physical contact tends to be dominant and cannot be eliminated. The three-chamber apparatus is much larger than a typical single chamber device, which allows the subject mouse to remain in proximity to or move far away from the Stranger mouse. Thus, the stress level of Crawley's sociability test is low and mostly caused by placing the experimental mouse into a novel environment, which allows repetition of this test over time, for example at different ages of the same mouse cohort: juvenile vs adult. By comparing a young group of animals with the same but older cohort, it is possible to follow up and discriminate between impairment in social recognition vs social memory and/or cognitive impairments, which can be observed in Session 2, and be related to age-dependent physiology.
This test is relatively time-consuming (~40 minutes per mouse), which is a disadvantage. However, multiple repertoires of specific parameters can be monitored and recoded (manually and/or video), permitting robust analysis. For example, some of additional parameters, such as freezing and/or self-grooming behavior, may indicate increased anxiety and decreased motivation for social behaviour. By using the same protocol, mice can be retested in the next day with the same Stranger 1 mouse to rule out an effect of social anxiety. Additional parameters (such as tail rattling, biting of the wire cup where Stranger 1 is located) can be helpful in distinguishing aggressive versus unusual friendliness behavior towards the novel animal. Observed anxiety can be due to inappropriate adaptation of the mice to the experimental room, excessive room lighting, poor mouse health, or as a result of genetic modification and/or neophobia (in some KO models). Moreover, impaired habituation may be interpreted as a cognitive impairment, and may affect the results of second session. Before subjecting mice to this test, it is important to subject animals to general health and neurological screening, including body temperature and weight, coat examination, and the series of following reflex examinations: Whisker, Righting, Ear-Twitch, Reaching and Key Jangling (described in 12-15). Additional behavioral tests can be helpful in discriminating specific responses. An increased number of contacts and/or an increase in the total duration of active contacts maybe associated with hyperactive behavior of specific mouse stains, which can be confirmed by using an open field test (to measure locomotor activity). Decreased duration and/or number of contacts may be associated with depressive- and/or anxiety-like behavior, which can be evaluated by using FST, TST or EPM, zero-maze, light/dark tests, respectively (described in 6, 13, 15).
Once appropriate conditions are applied and significant differences observed, there is a requirement to perform follow-up tests, in order to confirm the findings. Olfaction plays a key role in social recognition in rodents, since either chemically induced anosmia or removal of the vomeronasal organ blocks individual recognition 16, 17. Mice tend to sniff a novel odour and then quickly habituate to its novelty. The olfactory bulb test can be used for this purpose (described in 6, 7).
There are also modifications and alternative usage of three-compartment chamber sociability test, such as estimation of maternal behavior, by examining the time spent by a subject female proximal to a cup containing her pups (reflecting maternal motivation), as well as sexual motivation, by assessing the response of a tested male/female to a female/male under cup instead of Stranger 1 in session 1 7, 11.
Further tests can be used to examine sociability in mice by using alternative approaches/paradigms, such as social interaction in the home cage and/or in a novel environment (one-chamber social interaction test), resident intruder, partition test, social approach test, reciprocal social interaction, etc (described in 3, 18). Vocalizations in the ultrasonic and sonic ranges, visual cues, gustatory and tactile modalities may also contribute to communication of information and to social bonding (reviewed in 19).
Impaired sociability and dysregulated social behavior has been implicated as a hallmark for many psychiatric disorders. For example, autism spectrum disorder and schizophrenia are both characterized by marked deficits in social cognition and social behavior 20, and disruptions of social relationships result in an increased risk of depression 21. Thus, the described here test can be used in animal models of the aforementioned conditions. Moreover, this test is applicable for assessing potential effects of pharmacological compounds on sociability (reviewed in 3, 22-24).

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ethanol 70% (commercial Alcohols)</td>
<td>&nbsp;</td>
<td>MSH</td>
<td>19901</td>
<td>&nbsp;</td>
<tr>
<td>Clidox 1:5:1 Base Activator</td>
<td>&nbsp;</td>
<td>Pharmacal</td>
<td> 96130F 95130F</td>
<td>1 part Base to 5 parts water to 1 part Activator</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Assessment of Social Interaction Behaviors at JoVE.com

Assessment of Social Interaction Behaviors

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 ...

assessment-of-social-interaction-behaviors

Research

JoVE Journal

Neuroscience

92.6K Views.  Mount Sinai Hospital. Qui descriviamo un protocollo dettagliato per l'esame di socialità nei topi utilizzando socialità Crawley e la preferenza per il test novità sociale. Descriviamo i vantaggi e le possibili applicazioni per questa procedura, compresi i dettagli critici importanti per una corretta interpretazione dei risultati.

Video: Assessment of Social Interaction Behaviors

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Electroencephalographic, Heart Rate, and Galvanic Skin Response Assessment for an Advertising Perception Study: Application to Antismoking Public Service Announcements

The evaluation of advertising, products, and packaging is traditionally performed through methods based on self-reports and focus groups, but these approaches often appear poorly accurate in scientific terms. Neuroscience is increasingly applied to the investigation of the neurophysiological bases of the perception of and reaction to commercial stimuli to support traditional marketing methods. In this context, a particular sector or marketing is represented by public service announcements (PSAs). The objective of this protocol is to apply electroencephalography (EEG) and autonomic signal analysis to study responses to selected antismoking PSAs. Two EEG indices were employed: the frontal alpha band EEG asymmetry (the Approach Withdrawal (AW) index) and the frontal theta (effort index). Furthermore, the autonomic Emotional Index (EI) was calculated, as derived from the Galvanic Skin Response (GSR) and Heart Rate (HR) signals. The present protocol describes a series of operational and computational steps required to properly estimate, through the aforementioned indices, the emotional and cerebral reaction of a group of subjects towards a selected number of antismoking PSAs. In particular, a campaign characterized by a symbolic communication style (classified as "awarded" on the basis of the prizes received by specialized committees) obtained the highest approach values, as estimated by the AW index. A spot and an image belonging to the same PSA campaign based on the "fear arousing appeal" and with a narrative/experiential communication style (classified as "effective" on the basis of the economical/health-related improvements promoted) reported the lowest and highest effort values, respectively. This is probably due to the complexity of the storytelling (spot) and to the immediateness of the image (a lady who underwent a tracheotomy). Finally, the same "effective" campaign showed the highest EI values, possibly because of the empathy induced by the testimonial and the explicitness of the message.

The following protocol describes a series of operational and computational steps required to properly estimate the emotional and cerebral reaction of a group of subjects towards a selected number of Public Service Announcements (PSAs) against smoking, aired in the USA and Europe during the period between 1998 and 2015.

The evaluation of advertising, products, and packaging is traditionally performed through methods based on self-reports and focus groups, but these ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...