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 a male and female intruders. 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 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-intruder test was also found in BALB/c males. Behavioral breakdown revealed the anogenital sniffing as a key behavioral feature that discriminates test male behavior toward intruders of different genders. At the same time, female preference 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. However, there was no correlation between female animal preference in the contact Two-intruder test and smell preference in the social odor preference test. We assume the Two-intruders test setup to be a useful tool to study the neurological basis of social communication in animal models. Combined with odor preference tests, this experimental paradigm can help to decipher neural circuits involved in social deficiency phenotypes in animal models of human diseases. Significance StatementThe Two-intruders test proves to be a highly reproducible and robust approach to assess social communication in mice. We demonstrate that the results obtained in this experimental setting replicate in different mouse groups and strains. This test is indispensable in studies assessing the competitive nature of male- and female-driven behaviors and the underlying neural mechanisms. Residents social interactions in the described here set up reflects odor processing and circulating testosterone - the key physiological drivers of animal communication with conspecifics. While easy to perform, this test provides a broad spectrum of behavioral patterns to study in the models of complex neurological diseases. Largely overseen in the literature as compared to the resident-intruder setup, the Two-intruders test can provide superior performance when used to understand the neural mechanisms of mating, aggression and social communication.