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The three-chamber social interaction test is a newer variation of the one-chamber social interaction test that evaluates anxiety. It was designed to measure social affiliation, social memory, and preference for social novelty in rodents. This task has relevance to human disorders that are characterized by poor social interaction, for example autism spectrum disorders, schizophrenia, and bipolar disorder.
Jamain and colleagues (2008) used the three-chamber test to evaluate a genetically modified mouse model of autism. Autism is a neurodevelopmental disorder with high heritability, prompting major international efforts to identify candidate genes. Determining those genes would help to evaluate gene–environment interactions in the etiology of the disorder and provide a target for effective treatments. Since there are no identifiable biological markers for autism spectrum disorders, diagnosis is based solely on behavioral criteria. Since one core symptom cluster includes deficits in social interaction and reciprocal social communication, the social interaction test seemed to be an appropriate means to validate the animal model. Rodents are the natural choice of subjects because by nature they are sociable and will interact with and actively investigate a conspecific (an animal of the same species).
Numerous candidate genes have been implicated in autism, many of which regulate the formation and maturation of synapses. A good way to evaluate a gene’s importance is to create mice with selective mutations of the genome (described later in this chapter) and evaluate their behaviors, which in this case should resemble the symptoms of autism. The gene that Jamain and colleagues eliminated is Nlgn4. Its elimination produces neuroligin 4–deficient mice, called Nlgn4-knockout mice. Mutation in this gene in humans, NLGN4, is associated with autistic symptoms. Expression of NLGN4 is lowest prenatally and gradually increases until 3 weeks postnatally, a time that coincides with completed synapse formation. The Nlgn4-knockout mice and wild-type control mice were studied with a battery of behavioral measures, including the three-chamber social interaction test.
The task required a three-chamber box with retractable doors. The test mouse was first acclimated to the test box that was empty except for a wire mesh containment cage in each of the side chambers (Figure 1). For the social affiliation portion of the task, a novel mouse (stranger 1) was placed inside one of the wire containment cages, and the test mouse had access to the three chambers for 10 minutes. The spacing of the wire bars of the containment cages allowed olfactory, auditory, visual, and limited tactile interaction but prevented aggressive or sexual contact. The test mouse behavior was observed and scored for the duration and number of contacts, time spent in each chamber, and number of entries into each compartment, as well as other behaviors such as walking and grooming.
Figure 1 Experimental setup to measure social affiliation, social memory, and preference for social novelty in rodents. Subjects included wildtype and Nlgn4-knockout mice.
In Figure 2B, the wild-type mice spent more time in the chamber with stranger 1, whereas the Nlgn4-knockout mice spent equal time in both chambers. The wild-type mice had more interactions with stranger 1 than with the empty cage, as shown in Figure 2C. In contrast the Nlgn4-knockout mice showed no preference for spending time in the compartment with stranger 1 or interacting with it.
Figure 2 (B) Time spent in the compartment with stranger 1 versus the empty compartment. (C) Number of visits to the compartment of the stranger. (D) Time spent in the compartment with the familiar mouse (stranger 1) versus the novel stranger (stranger 2). (After Jamain et al. [2008]. Proc. Natl. Acad. Sci. USA, 105, 1710–1715.)
The next phase of the testing involved placing a new stranger mouse (stranger 2) in the previously empty cage, and the same behavioral evaluations were conducted. It is evident from Figure 2D that the wild-type mice remembered their previous experience with stranger 1 and interacted more with the novel mouse (stranger 2), demonstrating both memory of the previous social interaction and the preference for novel experiences. The Nlgn4-knockout mice did not show a similar preference for the new stranger. These results, along with other test battery results, led the researchers to conclude that Nlgn4-knockout mice were a viable model of autism and deserved further evaluation. For a detailed protocol of the procedure, including a video link, see Kaidanovich-Beilin and associates (2011).
References
Jamain, S., Radyushkin, K., Hammerschmidt, K., Granon, S., Boretius, S., Varoqueaux, F., et al. (2008). Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism. Proc. Natl. Acad. Sci. USA, 105, 1710–1715.
Kaidanovich-Beilin, O., Lipina, T., Vukobradovic, I., Roder, J., and Woodgett, J.R. (2011). Assessment of social interaction behaviors. J. Vis. Exp., 48, e2473.