Social Interaction Tests in Mouse Models

Human beings, and many other species exhibit a rich panoply of social behaviors. Social interactions common to a huge variety of different species include mating behaviors, co-operating to solve problems, disputes and aggression, and communication of important information. Being able to properly execute such behaviors is extremely advantageous, and can even be vital to an animal’s health and survival.

For human beings, it is almost impossible to live a normal life without being able to engage in many complex social behaviors. The negative impact on social skills caused by serious psychological disorders such as schizophrenia, bipolar disorder, obsessive compulsive disorder (OCD) and autism is part of what makes these disorders so important to study.

Animal models, including mice, are often used to investigate disorders of social skill. In this article, we will discuss how mice can serve as a model for human social interaction, and outline a number of different methods used to construct experiments with mouse models that can provide information on how to treat human psychological disorders.

Social Interaction and the Brain

Almost every part of the brain is linked with social interaction to some extent. Nonetheless, we can identify certain parts that are especially important for the maintenance of “normal” social behavior. Neuropsychological studies of individuals showing apparent deficiencies in social skills or abnormalities in social behavior have allowed us to pinpoint regions that contribute to this behavior such as the frontal lobe, amygdala and cingulate cortex.

Autism is perhaps the most prominent example of a disorder with severe social deficiencies. Abnormalities in Broca’s area and Wernicke’s area in autistic individuals have been linked with their communication difficulties.^[1] In addition, these individuals often show an enlarged frontal lobe;^[2] the frontal lobe is involved with sexual behavior,^[3] impulse control^[4] and empathy,^[5] amongst many other things. The famous neurological case-study Phineas Gage lost his ability to follow many social norms following severe frontal lobe damage.^[6]

The amygdala is most commonly associated with fear and aversive learning,^[7] but it also plays a key part in human social interaction.^[8] Individuals with severe amygdala damage seem to lack a concept of “personal space”,^[9] and lesions in this brain region are also linked with increased social trust^[10] and issues with recognizing facial expressions.^[11]

Activity in the cingulate cortex, near the middle of the brain, has been connected to the ability to properly evaluate one’s social situation.^[12] Human beings with a history of depression have been seen to have abnormally high activity in this region when making social judgments.^[13] Aberrant function of the cingulate cortex has also been associated with social anxiety, OCD, and schizophrenia.^[14]

Now that we have seen how different parts of the brain contribute to social behaviors, we shall turn to discussing the most important social interaction tests used with mouse models.

Social Memory Tests

A social memory test assesses each mouse’s ability to tell the difference between another mouse that it has seen before and one that it hasn’t. The test can involve five trials, or only two. In either case, the mouse is shown another mouse some number of times, before being shown a different mouse. Whether the subject mouse recognizes the second mouse as unfamiliar or not can be assessed based on how much the subject mouse chooses to physically investigate that mouse.

Social Memory in Humans and Mice

In both humans and mice, being able to distinguish between a stranger and a known individual is crucial to effective social interaction. Norms of behavior towards strangers are generally quite different from those exhibited towards familiars. Social memory is key for appropriate behavior within hierarchies, identifying and dealing with threats, and reproductive/mating behaviors.

Applications

Benefits

• Very easy to carry out
• No specialized equipment needed

Downside

• Take great care in set up to avoid confounding variables.^[17]

Things to Consider

1. Loud noises can distract the mice, and bright lights can heighten their anxiety, both of which can confound the results, so make sure to run the experiment in a quiet laboratory with dim lighting.
2. The experimenter should stand behind a curtain during the experiment to prevent his or her own presence from causing a distraction.
3. Never use the same cage more than once as any droppings or urine can affect the results.
4. Do not use adult mice or particularly aggressive strains to avoid a fight breaking out.^[17]

Tube Dominance Test

The tube dominance test (also known as the Lindzey test) is designed to assess aggression. Two mice simultaneously enter either end of a tube that is too narrow for the mice to be able to crawl past one-another. One mouse, the more aggressive one, must force the other to walk backward out of the tube the same way it came in. The test is repeated, and the number of times each mouse managed to dominate the other is measured and compared.

Aggression in Humans and Mice

Aggression is a central facet of both human and mouse social interaction. Both species live in hierarchies which are to a certain

Benefits

• Very reliable results
• Less stress caused to mice than in social defeat type experiment

Downsides

• Outcome does not solely depend on aggression but also physical strength

Things to Consider

To overcome confounding variables, it is best to use multiple dominance tests and compare the results, rather than just using the tube dominance test alone.

Standard Opponent Test

The standard opponent test is another test which is used to look at aggression and dominance. The procedure is very simple: two mice, who have not met before, are placed together in a cage, and their behavior is observed for a certain amount of time. Incidents of aggression committed by each mouse (sniffing, chasing biting etc) are recorded and can be used to give an overall evaluation of the social dominance of each mouse.

Applications

The applications for the standard opponent test are much the same as for the tube dominance test. As mentioned above, these tests are complementary: it is good practice to use multiple forms of test to assess aggression and then compare the results.

Benefits

• Very easy to carry out
• No specialized equipment needed

Downsides

• Outcome not only influenced by aggression but also age and weight
• Results highly qualitative, so open to much subjective interpretation

Things to Consider

1. The mice should be paired carefully, strains matched in order to elicit the most revealing behaviors.
2. The mice must be marked, or different enough to make it easy to track which is which during the aggressive behavior.
3. The test should be ended early if the violence committed risks permanent injury to one or both mice.
4. Experimenters should be concealed during the experiment to avoid risking their presence influencing the behavior.

Isolation Induced Fighting

This test is very similar to the standard opponent test, except that the mice used in the experiment are isolated for a long period (several weeks) prior to carrying out the experiment. Isolation increases the chances of the mice exhibiting aggressive behavior.

Resident-Intruder Test

The resident-intruder test is also very similar to the standard opponent test. This time, one mouse is a “resident” who lives in a “home cage”, which is then intruded upon by an unknown mouse in order to test the resident mouse’s dominance.

Things to Consider

1. The subject resident mouse will be more aggressive if living with a female and/or with his pups.
2. The pups and the female, if present, must be removed from the cage before the intruder enters to prevent them fighting with the intruder.

Three-Chamber Sociability and Social Novelty Test

The three-chamber sociability and social novelty test (also known as the Crawley test) is similar to the social memory test, in that it involves measuring a mouse’s interest in unfamiliar compared to familiar mice. The difference is that this test is used for assessing sociability and social novelty rather than memory.

The subject mouse is placed in an empty box, before being introduced to an unfamiliar mouse displayed under a see-through cup. It is next exposed to both the previous mouse and a new unfamiliar mouse, both displayed under different cups. Mice are considered more sociable if they spend more time investigating the first unfamiliar mouse than the empty cup, and more time investigating the second unfamiliar mouse than the first one.

Applications

Like social memory tests, this kind of test is useful for assessing mouse models of autism. Autistic humans are noticeably less sociable, tending to avoid social situations and exhibit very object-oriented behaviors. There is evidence that they are unable to categorically differentiate objects from persons. Being substantially less willing to engage in social situations prevents an individual from building the social networks that will allow them to benefit from societal structures.

Benefits

• Separation prevents physically aggressive behavior
• Enclosure is large, preventing stress
• Can be used to test a variety of hypotheses

Downsides

Time and effort required for set up is greater than for many other tests^[18]

Things to Consider

1. Make sure all the apparatus is properly set up and positioned before starting the experiment.
2. Stand at least 6 feet away during the experiment, to avoid distracting the mice.
3. The room must be lit reasonably brightly throughout.

Ultrasonic Vocalization Test

The ultrasonic vocalization test is used to examine child to mother communication in mouse pups. From a very young age, mice emit sounds above the hearable frequency range of human beings (ultrasonic vocalizations), which are used to communicate with their mother and convince her to attend to them. This communication mechanism is crucial to ensuring the mouse pup receives the parental care it needs to remain healthy during development.

Each young mouse is placed alone in an enclosure with walls coated with Styrofoam, and a microphone is fixed to the enclosure so that all noises made by the mouse can be recorded and analyzed. Researchers look at how loud, long and high-pitched the vocalizations are. Mice pups that deviate substantially from the norm are considered to have a deficit in communication ability.

Infant Vocalization in Humans and Mice

Although human beings do not emit ultrasonic vocalizations, infant humans do cry out at a high pitch in order to communicate their desire for attention to their parents. Abnormalities in the pitch and consistency of cry acoustics are a predictor of autism in human infants.^[19] In addition, excessive crying during infancy is linked with the development of deficits in emotional and behavioral regulation a few years later.^[20]

Benefits and Downsides

The key advantage of the ultrasonic vocalization test is how, unlike many other test paradigms, it allows vocal behaviors to be considered in a genuine social context rather than in isolation. However, it is worth bearing in mind that the experimental set up is still highly artificial, and so it is quite possible that the patterns of vocalization exhibited by the mice in the enclosure do not reflect how they would behave under normal circumstances.^[21]

Things to Consider

1. It is essential to keep the test conditions standardized in order to prevent confounding environmental variables from altering the pups’ vocalizations.
2. Isolating the pups for a certain period before carrying out the test is a good way to increase their motivation to engage in social interaction.
3. Make sure to include littermate controls to prevent confusing genetic effects with litter effects.^[21]

Social Olfactory Dishabituation

The social olfactory habituation and dishabituation test look at mouse memory, sense of smell and interest in social behaviors. It has an initial non-social phase and then a later social phase.

In the social phase of the experiment, the mouse is shown urine samples from two different female mice with their ovaries removed. The mouse should get used (become habituated) to the urine they have smelt before and spent less time sniffing it, and they should spend more time sniffing new urine samples. Abnormal habituation and dishabituation to the urine samples are taken as a sign of social deficit.

Social Olfaction in Humans and Mice

In terms of resemblance to human behavior, this test seems rather distant; human beings are generally not interested in the smell of one another’s urine, and urine does not play a positive role in social interaction for the vast majority of humans. Nonetheless, responding to social novelty is key for effective sociability, and this test does allow the assessment of mouse models of disorders such as autism which is often associated with decreased interest in social novelty.

Benefits and Downsides

Out of all the tests detailed here, the social olfactory dishabituation test is the least specific to assessing social interaction. However, it is quite easy to carry out.

Things to Consider

1. It is important to avoid contaminating the tool that is used to present the odor sample, to make sure additional unwanted odors are not included.
2. One pitfall often encountered in performing this experiment is the tendency of the mice to attempt to grab or bite the sample applicator. This can be solved by trying to present the odor in a way that the mouse cannot make contact with the sample.^[22]

Conclusion

Mouse models have proven a useful tool in the study of disorders of social interaction in humans. The tests detailed here provide a number of experimental paradigms involving the effective use of mouse models in this line of research. Taking note of the information here should allow you to choose, design and implement experiments suited to your research aims.

References

1. Ha, Sungji et al. “Characteristics of Brains in Autism Spectrum Disorder: Structure, Function and Connectivity across the Lifespan.” Experimental Neurobiology 24.4 (2015): 273–284. PMC. Web. 6 Apr. 2018.
2. Brun, Caroline et al. “Mapping Brain Abnormalities in Boys with Autism.” Human brain mapping 30.12 (2009): 3887–3900. PMC. Web. 6 Apr. 2018.
3. Baird, Amee D et al. “Neurological Control of Human Sexual Behaviour: Insights from Lesion Studies.” Journal of Neurology, Neurosurgery, and Psychiatry 78.10 (2007): 1042–1049. PMC. Web. 6 Apr. 2018.
4. Sebastian, Alexandra et al. “Frontal Dysfunctions of Impulse Control – A Systematic Review in Borderline Personality Disorder and Attention-Deficit/Hyperactivity Disorder.” Frontiers in Human Neuroscience 8 (2014): 698. PMC. Web. 6 Apr. 2018.
5. Hillis, A. E. (2014). Inability to empathize: brain lesions that disrupt sharing and understanding another’s emotions. Brain, 137(4), 981–997.
6. O’Driscoll, K., & Leach, J. P. (1998). “No longer Gage”: an iron bar through the head : Early observations of personality change after injury to the prefrontal cortex. BMJ : British Medical Journal, 317(7174), 1673–1674.
7. Ohman, A. (2005). The role of the amygdala in human fear: automatic detection of threat. Psychoneuroendocrinology. 2005 Nov;30(10):953-8.
8. Adolphs, Ralph. “What Does the Amygdala Contribute to Social Cognition?” Annals of the New York Academy of Sciences 1191.1 (2010): 42–61. PMC. Web. 6 Apr. 2018.
9. Kennedy, D. P., Gläscher, J., Tyszka, J. M., & Adolphs, R. (2009). Personal Space Regulation by the Human Amygdala. Nature Neuroscience, 12(10), 1226–1227.
10. Koscik, T. R., & Tranel, D. (2011). The human amygdala is necessary for developing and expressing normal interpersonal trust. Neuropsychologia, 49(4), 602–611.
11. Baxter, M. G., & Croxson, P. L. (2012). Facing the role of the amygdala in emotional information processing. Proceedings of the National Academy of Sciences of the United States of America, 109(52), 21180–21181.
12. Anastasia E Rigney, Jessica E Koski, and Jennifer S Beer. (2018). The functional role of ventral anterior cingulate cortex in social evaluation: disentangling valence from subjectively rewarding opportunities. Social Cognitive and Affective Neuroscience. Jan; 13(1).
13. Dedovic, K., Slavich, G. M., Muscatell, K. A., Irwin, M. R., & Eisenberger, N. I. (2016). Dorsal Anterior Cingulate Cortex Responses to Repeated Social Evaluative Feedback in Young Women with and without a History of Depression. Frontiers in Behavioral Neuroscience, 10, 64.
14. Yücel, M., Wood, S. J., Fornito, A., Riffkin, J., Velakoulis, D., & Pantelis, C. (2003). Anterior cingulate dysfunction: Implications for psychiatric disorders? Journal of Psychiatry and Neuroscience, 28(5), 350–354.
15. Diane L.Williams. GeraldGoldstein. Nancy J. Minshewa. (2002). Impaired memory for faces and social scenes in autism: clinical implications of memory dysfunction. Archives of Clinical Neuropsychology. Volume 20, Issue 1, January 2005, Pages 1-15
16. Evelien M Barends, Marc PH Hendriks, Jacobus FA Jansen, Walter H Backes, Paul AM Hofman, Geert Thoonen, Roy PC Kessels and Albert P Aldenkamp. (2013). Working memory deficits in high-functioning adolescents with autism spectrum disorders: neuropsychological and neuroimaging correlates. Journal of Neurodevelopmental Disorders. 5:14.
17. Jacobs, SA. Huang, F. Tsien, JZ. (2016). Social Recognition Memory Test in Rodents. Bio-protocol. 6(9). e1804.
18. Kaidanovich-Beilin, O., Lipina, T., Vukobradovic, I., Roder, J., & Woodgett, J. R. (2011). Assessment of Social Interaction Behaviors. Journal of Visualized Experiments : JoVE, (48), 2473.
19. Sheinkopf, S. J., Iverson, J. M., Rinaldi, M. L., & Lester, B. M. (2012). Atypical Cry Acoustics in 6-Month-Old Infants at Risk for Autism Spectrum Disorder. Autism Research : Official Journal of the International Society for Autism Research, 5(5), 331–339.
20. Kim, J. S. (2011). Excessive crying: behavioral and emotional regulation disorder in infancy. Korean Journal of Pediatrics, 54(6), 229–233.
21. Ferhat, A.-T., Torquet, N., Le Sourd, A.-M., de Chaumont, F., Olivo-Marin, J.-C., Faure, P., … Ey, E. (2016). Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication. Journal of Visualized Experiments : JoVE, (112), 53871.
22. Yang, Mu, and Jacqueline N. Crawley. “Simple Behavioral Assessment of Mouse Olfaction.” Current protocols in neuroscience / editorial board, Jacqueline N. Crawley … [et al.] CHAPTER (2009): Unit–8.24. PMC. Web. 6 Apr. 2018.