1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 2Toronto Centre for Phenogenomics, Mount Sinai Hospital, 3Department of Medical Biophysics, University of Toronto, 4Department of Psychology, University of Toronto, 5Department of Psychiatry, University of Toronto
Kaidanovich-Beilin, O., Lipina, T., Vukobradovic, I., Roder, J., Woodgett, J. R. Assessment of Social Interaction Behaviors. J. Vis. Exp. (48), e2473, doi:10.3791/2473 (2011).
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.
1. Equipment and Room Set Up:
2. Animal Preparation:
3. Habituation (adaptation):
4. Social Affiliation Aspect of the Test (session I):
5. Social Novelty/preference Session of the Test (Session II):
6. Statistical Analysis:
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-3α 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-3α 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.
Figure 1. Session I: Social affiliation and sociability. Mean length of time (±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 ≤ 0.0001 in WT group.
Figure 2. Session II: Social memory and novelty. Mean duration of time (±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 ≤ 0.0001 in WT group.
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).
No conflicts of interest declared.
OKB and JRW wrote the manuscript. This work was supported by CIHR grant MOP 74711 (to JRW).
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