Individual identity, not estrous state, diversifies mouse spontaneous behavior

Behavior is shaped by both the internal state of an animal and its individual behavioral biases. Rhythmic variation in gonadal hormones during the estrous cycle is a defining feature of the female internal state and has long been thought to increase overall variation in female behavior – an assumption that contributed to the sex-bias in biomedical research. However, there is little agreement on whether estrous state influences behaviors expressed by both sexes, such as spontaneous exploration. We address this question by longitudinally characterizing the open-field behavior of female mice across different phases of the estrous cycle, using unsupervised machine learning to decompose naturalistic behavior into its constituent elements. We find that each female mouse exhibits a characteristic pattern of exploration that uniquely identifies it as an individual across many experimental sessions; by contrast, estrous state only negligibly impacts behavior. Male mice also exhibit individual-specific patterns of behavior in the open field; however, male behavior was significantly more variable than that expressed by females both within and across individuals. These findings provide direct empirical support for the inclusion of both sexes in experiments querying spontaneous behaviors.