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.