Analysis of behavioral flow resolves latent phenotypes
The nuanced detection of rodent behavior in preclinical biomedical research is essential for understanding disease conditions, genetic phenotypes, and internal states. Recent advances in machine vision and artificial intelligence have popularized data-driven methods that segment complex animal behavior into clusters of behavioral motifs. However, despite the rapid progress, several challenges remain: Statistical power typically decreases due to multiple testing correction, poor transferability of clustering approaches across experiments limits practical applications, and individual differences in behavior are not considered. Here, we introduce "behavioral flow analysis" (BFA), which creates a single metric for all observed transitions between behavioral motifs. Then, we establish a "classifier-in-the-middle" approach to stabilize clusters and enable transferability of our analyses across datasets. Finally, we combine these approaches with dimensionality reduction techniques, enabling "behavioral flow fingerprinting" (BFF) for individual animal assessment. We validate our approaches across large behavioral datasets with a total of 443 open field recordings that we make publicly available, comparing various stress protocols with pharmacologic and brain-circuit interventions. Our analysis pipeline is compatible with a range of established clustering approaches, it increases statistical power compared to conventional techniques, and has strong reproducibility across experiments within and across laboratories. The efficient individual phenotyping allows us to classify stress-responsiveness and predict future behavior. This approach aligns with animal welfare regulations by reducing animal numbers, and enhancing information extracted from experimental animals