Using bedding in a test environment critically affects 50-kHz ultrasonic vocalizations in laboratory rats

Rats utter distinct classes of ultrasonic vocalizations depending on their developmental stage, current state, and situational factors. One class, comprising the so-called 50-kHz calls, is typical for situations where rats are anticipating or actually experiencing rewarding stimuli, like being tickled by an experimenter, or when treated with drugs of abuse, such as the psychostimulant amphetamine. Furthermore, rats emit 50-kHz calls when exposed to a clean housing cage. Here, we show that such vocalization effects can depend on subtle details of the testing situation, namely the presence of fresh rodent bedding. Actually, we found that adult males vocalize more in bedded cages than in bare ones. Also, two experiments showed that adult rats emitted more 50-kHz calls when tickled on fresh bedding. Furthermore, ip amphetamine led to more 50-kHz vocalization in activity boxes containing such bedding as compared to bare ones. The analysis of psychomotor activation did not yield such group differences in case of locomotion and centre time, except for rearing duration in rats tested on bedding. Also, the temporal profile of vocalization did not parallel that of behavioural activation, since the effects on vocalization peaked and started to decline again before those of psychomotor activation. Therefore, 50-kHz calls are not a simple correlate of psychomotor activation. A final experiment with a choice procedure showed that rats prefer bedded conditions. Overall, we assume that bedded environments induce a positive affective state, which increases the likelihood of 50-kHz calling. Based on these findings, we recommend that contextual factors, like bedding, should receive more research attention, since they can apparently decrease the aversiveness of a testing situation. Also, we recommend to more routinely measure rat ultrasonic vocalization, especially when studying emotion and motivation, since this analysis can provide information about the subject's status, which may not be detected in its visible behaviour.