Investigating social communication in mice: A two-intruders test approach

Understanding the complex dynamics of social communication behaviors, such as exploration, communication, courtship, mating, and aggression in animal models, is crucial to reveal key neural and hormonal mechanisms underlying these behaviors. The two-intruders test is designed to investigate residents' behavior toward both male and female intruders within the home cage of the test male. During this test imitating natural conditions, several aspects of social interaction were investigated: Exploration, courtship, mating, and aggressive behavior. As mating and aggression involve overlapping neural circuits, the behavioral setup testing both behaviors is best at reflecting their competitive nature. Our findings demonstrate that resident male mice exhibit strong preference to communicate with a female intruder, which correlates with baseline testosterone levels of test males. Relevant female preference in the two-intruders test was also found in BALB/c males. Behavioral breakdown revealed the anogenital sniffing as a key behavioral feature that discriminates resident male behavior toward intruders of different sex. Furthermore, resident male interaction with female intruder was accompanied by neuronal activation in the ventromedial hypothalamus. We demonstrate that odor recognition underlies preference toward females in male residents, as experimental anosmia reduced communication with a female intruder. We conclude the two-intruders test setup to be a useful tool to study the neurological basis of social communication in animal models, which provides detailed analysis of various aspects of the laboratory animals’ social behavior in the most natural conditions.