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

Rats are social animals that use ultrasonic vocalizations (USV) in their intraspecific communication. Several types of USV have been previously described, e.g., appetitive 50-kHz USV and aversive short 22-kHz USV. It is not fully understood which aspects of the USV repertoire play important functions during rat ultrasonic exchange. Here, we investigated features of USV emitted by rats trained in operant conditioning, is a form of associative learning between behavior and its consequences, to reinforce the production/emission of 50-kHz USV. Twenty percent of the trained rats learned to vocalize to receive a reward according to an arbitrarily set criterion, i.e., reaching the maximum number of proper responses by the end of each of the last three USV-training sessions, as well as according to a set of measurements independent from the criterion (e.g., shortening of training sessions). Over the training days, these rats also exhibited: an increasing percentage of rewarded 50-kHz calls, lengthening and amplitude-increasing of 50-kHz calls, and decreasing number of short 22-kHz calls. As a result, the potentially learning rats, when compared to non-learning rats, displayed shorter training sessions and different USV structure, i.e. higher call rates, more rewarded 50-kHz calls, longer and louder 50-kHz calls and fewer short 22-kHz calls. Finally, we reviewed the current literature knowledge regarding different lengths of 50-kHz calls in different behavioral contexts, the potential function of short 22-kHz calls as well as speculate that USV may not easily become an operant response due to their primary biological role, i.e., communication of emotional state between conspecifics

Increased Vocalization of Rats in Response to Ultrasonic Playback as a Sign of Hypervigilance Following Fear Conditioning

We investigated the effects of prior stress on rats’ responses to 50-kHz (appetitive) and 22-kHz (aversive) ultrasonic playback. Rats were treated with 0, 1, 6 or 10 shocks (1 s, 1.0 mA each) and were exposed to playbacks the following day. Previous findings were confirmed: (i) rats moved faster during 50-kHz playback and slowed down after 22-kHz playback; (ii) they all approached the speaker, which was more pronounced during and following 50-kHz playback than 22-kHz playback; (iii) 50-kHz playback caused heart rate (HR) increase; 22-kHz playback caused HR decrease; (iv) the rats vocalized more often during and following 50-kHz playback than 22-kHz playback. The previous shock affected the rats such that singly-shocked rats showed lower HR throughout the experiment and a smaller HR response to 50-kHz playback compared to controls and other shocked groups. Interestingly, all pre-shocked rats showed higher locomotor activity during 50-kHz playback and a more significant decrease in activity following 22-kHz playback; they vocalized more often, their ultrasonic vocalizations (USV) were longer and at a higher frequency than those of the control animals. These last two observations could point to hypervigilance, a symptom of post-traumatic stress disorder (PTSD) in human patients. Increased vocalization may be a valuable measure of hypervigilance used for PTSD modeling

Percentage of short 22-kHz USV in the total number of USV in rats with 7 (AB) or 14 (CD) training days during training (AC) and test (BD) sessions.

A. Percent of short 22-kHz USV in rats with habituation and 7 training sessions. B. Percent of short 22-kHz USV emitted by 7-days-trained rats in test sessions. C. Percent of short 22-kHz USV in rats with 14 training sessions. D. Percent of short 22-kHz USV emitted by 14-days-trained rats in test sessions. Overall, the percentage of 22-kHz USV was lower in PL-SUM rats in comparison with NL rats. The line plots (A-D) represent the mean ± SEM. * PL-SUM vs. NL-SUM; # PL-SUM vs. NL-0; PL-SUM vs. NL-SUM/0, & NL-SUM/0 vs. NL-0; one character (*, #, or &) p S9 Table.</p

Session duration in NL and PL rats.

Rats are social animals that use ultrasonic vocalizations (USV) in their intraspecific communication. Several types of USV have been previously described, e.g., appetitive 50-kHz USV and aversive short 22-kHz USV. It is not fully understood which aspects of the USV repertoire play important functions during rat ultrasonic exchange. Here, we investigated features of USV emitted by rats trained in operant conditioning, is a form of associative learning between behavior and its consequences, to reinforce the production/emission of 50-kHz USV. Twenty percent of the trained rats learned to vocalize to receive a reward according to an arbitrarily set criterion, i.e., reaching the maximum number of proper responses by the end of each of the last three USV-training sessions, as well as according to a set of measurements independent from the criterion (e.g., shortening of training sessions). Over the training days, these rats also exhibited: an increasing percentage of rewarded 50-kHz calls, lengthening and amplitude-increasing of 50-kHz calls, and decreasing number of short 22-kHz calls. As a result, the potentially learning rats, when compared to non-learning rats, displayed shorter training sessions and different USV structure, i.e. higher call rates, more rewarded 50-kHz calls, longer and louder 50-kHz calls and fewer short 22-kHz calls. Finally, we reviewed the current literature knowledge regarding different lengths of 50-kHz calls in different behavioral contexts, the potential function of short 22-kHz calls as well as speculate that USV may not easily become an operant response due to their primary biological role, i.e., communication of emotional state between conspecifics.</div

Call rate (USV/min) in PL and NL rats.

Rats are social animals that use ultrasonic vocalizations (USV) in their intraspecific communication. Several types of USV have been previously described, e.g., appetitive 50-kHz USV and aversive short 22-kHz USV. It is not fully understood which aspects of the USV repertoire play important functions during rat ultrasonic exchange. Here, we investigated features of USV emitted by rats trained in operant conditioning, is a form of associative learning between behavior and its consequences, to reinforce the production/emission of 50-kHz USV. Twenty percent of the trained rats learned to vocalize to receive a reward according to an arbitrarily set criterion, i.e., reaching the maximum number of proper responses by the end of each of the last three USV-training sessions, as well as according to a set of measurements independent from the criterion (e.g., shortening of training sessions). Over the training days, these rats also exhibited: an increasing percentage of rewarded 50-kHz calls, lengthening and amplitude-increasing of 50-kHz calls, and decreasing number of short 22-kHz calls. As a result, the potentially learning rats, when compared to non-learning rats, displayed shorter training sessions and different USV structure, i.e. higher call rates, more rewarded 50-kHz calls, longer and louder 50-kHz calls and fewer short 22-kHz calls. Finally, we reviewed the current literature knowledge regarding different lengths of 50-kHz calls in different behavioral contexts, the potential function of short 22-kHz calls as well as speculate that USV may not easily become an operant response due to their primary biological role, i.e., communication of emotional state between conspecifics.</div

Changes in 50-kHz USV duration (A-D) and amplitude (E-H) in rats with 7 (ABEF) or 14 (CDGH) training days during training (ACEG) and test (BDFH) sessions.

A. 50-kHz USV duration in 7-days-trained rats during habituation and training. B. 50-kHz USV duration during test sessions in 7-days-trained rats. C. 50-kHz USV duration in 14-days-trained rats. D. 50-kHz duration in 14-days-trained rats during test sessions. Values for non-learning rats are presented in two ways: all non-learning rats are depicted as white squares (NL-SUM), additionally, this group is divided into NL-0 rats (dark gray squares) that never received the maximum number of rewards and NL-SUM/0 rats (light gray squares). In PL-SUM rats, the duration of 50-kHz USV increased and was longer than in NL groups. E. 50-kHz USV amplitude in 7-days-trained rats during habituation and training. F. 50-kHz USV amplitude during test sessions in 7-days-trained rats. G. 50-kHz USV amplitude in 14-days-trained rats. H. 50-kHz USV amplitude in 14-days-trained rats during test sessions. In 14-days-trained PL-SUM rats, the amplitude of 50-kHz USV was higher than in NL-SUM/0 rats. The line plots represent the mean ± SEM. * PL-SUM vs. NL-SUM, # PL-SUM vs. NL-0, PL-SUM vs. NL-SUM/0, & NL-SUM/0 vs. NL-0; one character (*, #, or &) p S7 and S8 Tables.</p

Percentage of rewards obtained and duration of training sessions in rats trained with several protocols of instrumental learning with USV emissions or nosepokes as rewarded responses.

Percentage of rewards obtained and duration of training sessions in rats trained with several protocols of instrumental learning with USV emissions or nosepokes as rewarded responses.</p

Percentage of rewards obtained (A-F), call rate (GH), and session duration (IJ) in rats grouped by performance in USV-training.

A. NL-0: Rats that did not obtain the maximum number of rewards in any training session (NL-0, non-learning “zero”, n = 34). B. NL-D1: Rats that obtained the maximum number of rewards on day 1 of training but not in the last 3 training sessions (NL-D1, non-learning “day one”, n = 17). C. NL-SGL: Rats that obtained the maximum number of rewards in only one training session, but not the first (NL-SGL, non-learning “single”, n = 5). D. NL-CEN: Rats that obtained the maximum number of rewards in a minimum of two consecutive training sessions, but not in the first two or last two (NL-CEN, non-learning” center”, n = 5). E. PL-PROG: Rats that obtained the maximum number of rewards during each of the last 3 training sessions, but not during other training sessions (PL-PROG, potentially learning “progress”, n = 10). F. PL-MAX: Rats that obtained the maximum number of rewards in all training sessions (PL-MAX, potentially learning “maximum”, n = 5). G. Call rate in rats from experiments with 7, 10, or 14 training sessions divided into six groups. H. Call rate in rats from experiments with 7, 10, or 14 training sessions divided into two groups: NL-SUM (all non-learning rats,) and PL-SUM (all potentially learning rats). Note that the call rate of PL-SUM rats is higher than that of NL-SUM rats in almost all training and test sessions. I. Session duration in all rats divided into six groups (as in G). J. Duration of a training session in PL-SUM and NL-SUM rats. Session time was shorter in PL-SUM rats than in NL-SUM rats from the second training session onward. The bars and line plots represent the mean ± SEM. The dots represent individual values for each rat. **p S3 and S4 Tables.</p

Duration of 50-kHz USV.

Rats are social animals that use ultrasonic vocalizations (USV) in their intraspecific communication. Several types of USV have been previously described, e.g., appetitive 50-kHz USV and aversive short 22-kHz USV. It is not fully understood which aspects of the USV repertoire play important functions during rat ultrasonic exchange. Here, we investigated features of USV emitted by rats trained in operant conditioning, is a form of associative learning between behavior and its consequences, to reinforce the production/emission of 50-kHz USV. Twenty percent of the trained rats learned to vocalize to receive a reward according to an arbitrarily set criterion, i.e., reaching the maximum number of proper responses by the end of each of the last three USV-training sessions, as well as according to a set of measurements independent from the criterion (e.g., shortening of training sessions). Over the training days, these rats also exhibited: an increasing percentage of rewarded 50-kHz calls, lengthening and amplitude-increasing of 50-kHz calls, and decreasing number of short 22-kHz calls. As a result, the potentially learning rats, when compared to non-learning rats, displayed shorter training sessions and different USV structure, i.e. higher call rates, more rewarded 50-kHz calls, longer and louder 50-kHz calls and fewer short 22-kHz calls. Finally, we reviewed the current literature knowledge regarding different lengths of 50-kHz calls in different behavioral contexts, the potential function of short 22-kHz calls as well as speculate that USV may not easily become an operant response due to their primary biological role, i.e., communication of emotional state between conspecifics.</div

Amplitude of 50-kHz USV.

Rats are social animals that use ultrasonic vocalizations (USV) in their intraspecific communication. Several types of USV have been previously described, e.g., appetitive 50-kHz USV and aversive short 22-kHz USV. It is not fully understood which aspects of the USV repertoire play important functions during rat ultrasonic exchange. Here, we investigated features of USV emitted by rats trained in operant conditioning, is a form of associative learning between behavior and its consequences, to reinforce the production/emission of 50-kHz USV. Twenty percent of the trained rats learned to vocalize to receive a reward according to an arbitrarily set criterion, i.e., reaching the maximum number of proper responses by the end of each of the last three USV-training sessions, as well as according to a set of measurements independent from the criterion (e.g., shortening of training sessions). Over the training days, these rats also exhibited: an increasing percentage of rewarded 50-kHz calls, lengthening and amplitude-increasing of 50-kHz calls, and decreasing number of short 22-kHz calls. As a result, the potentially learning rats, when compared to non-learning rats, displayed shorter training sessions and different USV structure, i.e. higher call rates, more rewarded 50-kHz calls, longer and louder 50-kHz calls and fewer short 22-kHz calls. Finally, we reviewed the current literature knowledge regarding different lengths of 50-kHz calls in different behavioral contexts, the potential function of short 22-kHz calls as well as speculate that USV may not easily become an operant response due to their primary biological role, i.e., communication of emotional state between conspecifics.</div