Profiling mouse behavior with computational tools to assess age-dependent differences in associative learning.

Second-order conditioning (SOC) enables animals to form associations between stimuli without direct reinforcement. In this study, we present a behavioral analysis pipeline that combines a light-tone SOC paradigm in mice with tools such as DeepLabCut, Keypoint-MoSeq, and DeepOF to evaluate responses across sex and age. Our results show that responses to the second-order stimulus (CS2) specifically stem from its association with the first-order stimulus (CS1). While CS1 triggers behavioral syllables related to immobility, CS2 elicits distinct behavioral responses, including immobility- and escape-related actions, suggesting SOC reorganizes, rather than replicates, first-order responses. These data-driven insights surpass the resolution of simple traditional measures (e.g., immobility). Lastly, we identified age-related deficits: older mice maintained responses to CS1 but exhibited impaired responses to CS2, regardless of sex. These findings uncover the complexity of SOC, its susceptibility to aging, and the value of data-driven tools in behavioral neuroscience.