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- Materials and methods
Mouse models of social dysfunction, designed to investigate the complex genetics of social behaviors, require an objective methodology for scoring social interactions relevant to human disease symptoms. Here we describe an automated, three chambered apparatus designed to monitor social interaction in the mouse. Time spent in each chamber and the number of entries are scored automatically by a system detecting photocell beam breaks. When tested with the automated equipment, juvenile male C57BL/6J mice spent more time in a chamber containing a stranger mouse than in an empty chamber (sociability), similar to results obtained by the observer scored method. In addition, automated scoring detected a preference to spend more time with an unfamiliar stranger than a more familiar conspecific (preference for social novelty), similar to results obtained by the observer scored method. Sniffing directed at the wire cage containing the stranger mouse correlated significantly with time spent in that chamber, indicating that duration in a chamber represents true social approach behavior. Number of entries between chambers did not correlate with duration of time spent in the chambers; entries instead proved a useful control measure of general activity. The most significant social approach behavior took place in the first five minutes of both the sociability and preference for social novelty tests. Application of these methods to C57BL/6J, DBA/2J and FVB/NJ adult males revealed that all three strains displayed tendencies for sociability and preference for social novelty. To evaluate the importance of the strain of the stranger mouse on sociability and preference for social novelty, C57BL/6J subject mice were tested either with A/J strangers or with C57BL/6J strangers. Sociability and preference for social novelty were similar with both stranger strains. The automated equipment provides an accurate and objective approach to measuring social tendencies in mice. Its use may allow higher-throughput scoring of mouse social behaviors in mouse models of social dysfunction.
Here we report a set of simple, easily automated mouse behavioral tasks that can be used to model symptoms of human disorders associated with social approach abnormalities. Autism, for example, is characterized by moderate to severe social interaction deficits, social communication abnormalities and ritualistic-repetitive behaviors (Folstein & Rosen-Sheidley 2001; Kanner 1943; Piven 2001; Schloper & Mesibov 1987). The cause of autism appears to be primarily genetic, with a heritability estimate of over 90% (Bailey et al. 1995). In contrast, William's syndrome, a disorder associated with a deletion of 1.5 Mb of chromosome 7, includes symptoms of very high levels of social approach behaviors. Children with William's syndrome typically have an ‘overfriendly’ personality along with strong language skills, but have deficits in visuospatial cognitive skills (Doyle et al. 2004; Laws & Bishop 2004; Morris & Mervis 2000). Social phobias and social anxiety are defined by avoidance of social situations (Bell et al. 1999; Marcin & Nemeroff 2003; Stein & Deutsch 2003; Tancer & Uhde 1997) and social dysfunction is a component of schizophrenia (Carpenter 1993; Dworkin 1992; Egan & Weinberger 1997; Morrison & Bellack 1987; Pinkham et al. 2003; Sams-Dodd et al. 1997; Tamminga 2003). Inbred strains of mice present an opportunity to evaluate social approach behaviors relevant to these human diseases and investigate the complex genetics of social behavior. Here we evaluate three inbred strains in two automated tasks, to begin to define the range of genetically influenced social behaviors in mice.
In a companion paper (Moy et al. 2004), we describe a three-chambered apparatus designed to quantitate preference for spending time with a conspecific mouse vs. an empty novel environment (sociability), as well as preference for a newly introduced mouse vs. a familiar mouse (preference for social novelty). This method employs human observation and data-entry on a computer. We subsequently designed a more automated version of this task, using photocells across the chamber doorways to record entries and time spent in each chamber. This report describes the components of the automated apparatus and a series of validation experiments, using three inbred strains of mice commonly used in behavioral genetics laboratories. The findings presented herein demonstrate comparable scores on measures of social tendencies, using the automated system vs. the observational hand scoring system. In addition, the present experiments describe the time course of the social approach behavior in both tasks, and the effect of the strain of the stranger mouse on the subjects' social behaviors.
- Top of page
- Materials and methods
The automated system for measuring sociability and preference for social novelty in mice yielded quantitative data comparable to data collected by human observers when scored simultaneously by the two methods. These results support an interpretation that the automated equipment yields data very similar to observer scoring in terms of detection of effects. Both automated and human observer scoring methods detected significant sociability in adult males of three inbred strains: C57BL/6J, DBA/2 and FVB/NJ. The magnitude and direction of effects were analogous to the sociability found in these strains as juveniles by the observer scored method, as described in the companion study (Moy et al. 2004). Similarly, preference for social novelty was detected by the automated equipment in juvenile male C57BL/6 and in independent cohorts of adult C57BL/6J and FVB/NJ, analogous to the preference for social novelty found in these strains by the observer scored methods (Moy et al. 2004). In addition, the low scores on time spent in the center chamber confirm that all three strains displayed high levels of general exploratory behavior.
The social nature of the time spent in the chamber containing a novel conspecific was confirmed by the strong correlation between time spent in the chamber and time spent sniffing the wire cage containing the stranger mouse. This finding supports the interpretation that time was spent in close proximity to the stranger, rather than elsewhere in the chamber containing the stranger. All three strains spent more time sniffing the wire cage containing the stranger than the empty wire cage. This supports the interpretation that sniffing of the wire cage reflects social approach behavior rather than non-specific exploration of a novel object. The wire cages allowed substantial olfactory, auditory, visual and tactile contact between test subjects and stranger animals, including nose-to-nose and nose-to-tail sniffing. Containment of the stranger in the wire cage prevented fighting that may have been anticipated using males as subjects and strangers.
Number of entries appeared to be independent of time spent in the chambers and number of sniffs directed toward the wire cages. Instead, the entries parameter provided useful control information about general locomotor and exploratory activity. High or low baseline exploratory activity could confound the interpretation of a social deficit in this task. In this regard, it is interesting to note that FVB/NJ mice were significantly more active in terms of number of entries, but showed levels of sociability and preference for social novelty that were similar to C57BL/6J mice. DBA/2J displayed lower levels of activity in terms of number of entries, but retained a significant sociability score and showed a trend toward preference for social novelty. Differences in general exploratory activity therefore did not appear to directly affect sociability and social novelty preference in this initial evaluation of these three inbred strains of mice in the automated equipment.
Time course analysis found that the majority of the social approach behaviors occurred within the first five minutes of the sociability test. The amount of time spent on the side with the stranger animal is high in the first five-minute time bin and steadily decreases as the test continues. In contrast, the amount of time spent in the empty chamber did not vary significantly across the four time bins. The statistical significance of the sociability test is therefore due solely to the change in amount of time spent with the stranger animal. The amount of time spent sniffing the wire cage containing the stranger mouse was similarly highest in the first five-minute time bin and declined over the course of the 20-minute session. There was also a significant increase in the amount of time spent in the center chamber in the last time bin. Further, total number of entries was reduced in the last time bin. Taken together, these results suggest that by the end of the test, subject mice were less engaged in sniffing the wire cages and exploring the side chambers, while spending more time in the center start chamber. The time course obtained justifies the use of 10-minute time bins to capture the majority of the social approach behaviors in this task.
Similar to sociability, the time course of the test of preference for social novelty showed the majority of social approach behavior occurring in the first five-minute time bin. Amount of time in the side with the now-familiar stranger 1 did not vary across the time bins, while there was a significant difference between the time spent with the novel stranger 2 in the first time bin compared to the other three. Significantly more time was spent in the center chamber in the final two time bins of the test. Time spent sniffing followed the same time course, supporting an interpretation that the majority of the social approach behavior occurred in the first five-minute time bin. Entries also dropped off significantly in the fourth time bin of the novelty preference test, indicating reduced exploratory activity. These time course results further support the use of 10-minute test sessions, as the majority of the preference for social novelty occurred during the first two five-minute time bins of the 20-minute test session.
Using a different strain for the stranger animal had no apparent effect on the sociability and preference for social novelty of test subjects. It is reasonable to suppose that the odors, appearance, behaviors and responses of the stranger will influence the social behaviors of the test subject. We examined this potential effect by substituting the C57BL/6J strangers with A/J animals, a strain which has been previously reported to display higher anxiety-like behaviors, lower exploration and decreased sociability and social novelty preference (Bouwknecht & Paylor 2002; Cohen et al. 2001; Mathis et al. 1994; Mathis et al. 1995; Moy et al. 2004; van der Staay & Steckler 2001). Replacing C57BL/6J strangers with A/J strangers produced no significant difference on either social task. This result suggests that the social approach behavior observed in the tests of sociability and preference for social novelty may be inherent in the subject strain, rather than a response to a particular stranger strain.
A major advantage of the automated equipment is the elimination of the labor-intensive and tedious aspects of hand-scoring. The automated apparatus is also likely to minimize observer fatigue and increase the consistency of results across experiments and across laboratories. In addition, automating the basic scoring leaves the observer free to score more interesting and complex behaviors of the test subjects.
The automated apparatus described herein is proposed as a simple, accurate approach to quantitate social behaviors in various strains of mice. A mouse line displaying selective and robust differences in tendencies to initiate social approach behaviors may offer construct validity towards modeling aberrant social approach behaviors in neuropsychiatric diseases such as autism, William's syndrome, social phobias and schizophrenia. Availability of inexpensive automated equipment opens the possibility of higher-throughput experiments that accurately score the tendencies of mice to initiate or avoid social approach. This automated equipment may be useful in future large-scale investigations into the genetic basis of social behavior.