Semi-Automated Training of Rat Ultrasonic Vocalizations

Abstract

Rats produce ultrasonic vocalizations (USVs) for conspecific communication. These USVs are valuable biomarkers for studying behavioral and mechanistic changes in a variety of diseases and disorders. Previous work has demonstrated operant conditioning can progressively increase the number of USVs produced by rats over multiple weeks. This operant conditioning paradigm is a useful model for investigating the effects of increased laryngeal muscle use on USV acoustic characteristics and underlying central and peripheral laryngeal sensorimotor mechanisms. Previous USV operant conditioning studies relied on manual training to elicit USV productions, which is both time and labor intensive and can introduce human variability. This manuscript introduces a semi-automated method for training rats to increase their rate of USV production by pairing commercially available operant conditioning equipment with an ultrasonic detection system. USV training requires three basic components: elicitation cue, detection of the behavior, and a reward to reinforce the desired behavior. With the semi-automated training paradigm, indirect exposure to the opposite sex or an olfactory cue can be used to elicit USV production. The elicited USV is then automatically detected by the ultrasonic acoustic system, which consequently triggers the release of a sucrose pellet reward. Our results demonstrate this semi-automated procedure produces a similar increase in USV production as the manual training method. Through automation of USV detection and reward administration, staffing requirements, human error, and subject behavioral variability may be minimized while scalability and reproducibility are increased. This automation may also result in greater experimental flexibility, allowing USV training paradigms to become more customizable for a wider array of applications. This semi-automated USV behavioral training paradigm improves upon manual training techniques by increasing the ease, speed, and quality of data collection.

Rats that learn to vocalize for food reward emit longer and louder appetitive calls and fewer short aversive calls

“…As a result of the above-mentioned general difficulties, arguably, there are only a few USVbased operant conditioning protocols. Some researchers have used USV as stimuli to respond to, to distinguish between [e.g., 10,11,44,45] and finally as rewarded operant responses [13,14]. Notably, lengthy training of weeks or even months was needed to produce acceptable results.…”

“…To our best knowledge, in the current literature there is only one functioning operant conditioning protocol that reinforces USV emitted by rats [e.g., 13,14]. However, we encountered numerous discrepancies when scrutinizing the publications using this protocol.…”

“…Firstly, the studies were only performed on male rats that vocalized when introduced to females in estrus, which is an impractical precondition. Moreover, 20-50% of these rats did not vocalize and were excluded from the training procedures [14]. Finally, even after weeks of daily training, the effectiveness of the protocol remains difficult to assess.…”

“…Across many publications using the same operant conditioning protocol, the stated key changes in rat behavior supporting evidence of learning are not properly supported by statistical analysis. This makes it difficult to assess whether "the rate of vocalizations can be increased" [13]; "all trained rats progressively increased their performance each week during the vocal training" [13,14,25]; "USV production steadily increased", and "the semi-automated procedure resulted in a similar increase in the number of USVs produced over the first 4 weeks of training" [14] are actually statistically conclusive results. Also, several aspects of the protocol are only vaguely described and inconsistent.…”

“…To our knowledge, there is only one published operant conditioning protocol that rewards rats for USV emission, which is based on initially exposing a male rat to an estrus female as an elicitation cue to evoke USV. The protocol has both manual [13] and semi-automated versions [14]. However, apart from an unpractical requirement of estrus females, there are documentation flaws (described in Discussion), and finally, it is focused mainly on the overall and unspecified increase in the number of USV emitted.…”