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Rat ultrasonic vocalizations have been suggested to be either a byproduct of physical movement or, in the case of 50-kHz calls, a means to communicate positive affect. Yet there are up to 14 distinct types of 50-kHz calls, raising issues for both explanations. To discriminate between these theories and address the purpose for the numerous 50-kHz call types, we studied single juvenile rats that were waiting to play with a partner, a situation associated with a high number of 50-kHz calls. We used a Monte-Carlo shuffling procedure to identify vocalization-behavior correlations that were statistically different from chance. We found that certain call types (“split”, “composite” and “multi-step”) were strongly associated with running and jumping while other call types (those involving “trills”) were more common during slower movements. Further, non-locomotor states such as resting and rearing were strongly predictive of a lack of vocalizations. We also found that the various sub-types of USVs can be clustered into 3–4 categories based on similarities in the way they are used. We did not find a one-to-one relationship between any movements and specific vocalizations, casting doubt on the motion byproduct theory. On the other hand, the use of specific calls during specific behaviors is problematic for the affect communication hypothesis. Based on our results, we suggest that ultrasonic calls may serve to coordinate moment-to-moment social interactions.

Specific 50-kHz vocalizations are tightly linked to particular types of behavior in juvenile rats anticipating play