Pupillary responses reflect dynamic changes in multiple cognitive factors during associative learning in primates.

Associative learning involves complex interactions of multiple cognitive factors. While adult subjects can articulate these factors verbally, for model animals such as macaques, we rely on behavioural outputs. In our study, we used pupillary responses as an alternative measure to capture these underlying cognitive changes. We recorded the dynamic changes in the pupils of three male macaques when they learned the associations between visual stimuli and reward sizes under the classical Pavlovian experimental paradigm. We found that during the long-term learning process, the gradual changes in the pupillary response reflect the changes in the cognitive state of the animals. The pupillary response can be explained by a linear combination of components corresponding to multiple cognitive factors. These components reflect the impact of visual stimuli on the pupils, the prediction of reward values associated with the visual stimuli, and the macaques' understanding of the current experimental reward rules. The changing patterns of these factors during interday and intraday learning clearly demonstrate the enhancement of current reward-stimulus association and the weakening of previous reward-stimulus association. Our study shows that the dynamic response of pupils can serve as an objective indicator to characterize the psychological changes of animals, understand their learning process, and provide important tools for exploring animal behaviour during the learning process.Significance Statement This study aimed to understand animal learning by observing their behaviour. By recording macaque monkeys' pupillary responses during a reward-based learning process lasting 2-3 months, researchers found that the dynamic changes in pupillary responses can be influenced by both external and internal factors, which can be explained by a linear combination of components corresponding to multiple factors. This study validates the effectiveness of using pupillary measurements to capture cognitive factors at different time scales, ranging from short term (hours) to long term (months), for the first time. This study provides valuable insights for a deeper understanding of animal learning processes and has significant implications for the application of pupillary responses in medicine, education, and scientific research.