*2.3. Functions of Vocalization in Social Play and Teamwork*

#### 2.3.1. Ludic Function

The emission of vocalizations during social interactions within the nest evolved to perform another fundamental role during play behavior in juveniles and play-like behavior in adult rats [99]. The role of vocalizations aiding play is called ludic function or ludic behavior (from Latin *ludere*—to play), which is characteristic of immature animals [100]. The emission of 50 kHz calls is abundant during natural rough-and-tumble play (play fighting) in rats and occurs in anticipation of and during such play in juvenile rats [73,101,102]. The emission of 50 kHz vocalizations is associated with specific components of play behavior, and the calls function as play signals and signals maintaining playful mood and activity [99,103,104].

In general, the number of emitted 50 kHz calls was used as a quantitative index of the animal state both in juveniles and adults [15,105]. The number of emitted 50 kHz calls is not only the quantitative index of the magnitude of emotional arousal but the low levels of emission of these calls in infants and juveniles may inform about abnormal phenomena and may be indicative of potential prenatal damages to the limbic system as it was recently shown for prenatal exposure to valproic acid [106].

The emission of 50 kHz play calls can be also induced by a tickling procedure (heterospecific play with humans) that mimics natural rough-and-tumble play, and it has rewarding properties for rats [107–109]. The tickling procedure should follow two main events of rough-and-tumble play—dorsal contacts and pins separated by a short break and should be repeated daily for the full effect [90]. The repeated tickling procedure can select groups of rats that will emit a particularly large number of 50 kHz vocalizations, signaling the appetitive value of the play [110]. Rats that refuse to play will emit low numbers of 50 kHz vocalizations, and even some 22 kHz calls, at the beginning of the play. Restrain stress applied to rats before tickling sessions significantly decreased the number of tickling-induced 50 kHz calls afterward [111].

Although adults rarely play, the appetitive value of light tactile stimulation, which only partially resembles rough-and-tumble play, still retained rewarding play value in young adults, and tactile stimulation induced emission of 50 kHz calls in habituated animals [60]. Interestingly, other forms of tactile stimulation by human hand in different parts of the rat's body, which had low resemblance to natural rough-and-tumble play, or were entirely unnatural to rats (e.g., holding rats in a vertical position and touching their flanks), also induced the emission of 50 kHz calls, although at a lower rate than during natural play [60,112].

Tickling stimulation of adolescent and young adult rats is appetitive and has rewarding value, as it was demonstrated by the tickle-induced release of dopamine in the nucleus accumbens [113]. On the other hand, very light touching of the rats' skin that failed to induce calling was not associated with the release of dopamine 113]. Therefore, tickling is not a purely tactile phenomenon but a procedure inducing a positive emotional arousal. A recent study has confirmed that the number of emitted 50 kHz vocalizations induced by tickling is proportional to the magnitude of positive emotional arousal and is, indeed, a good quantitative measure of this arousal as compared to other measures [114].

Another recent study reported that rats can play with humans in a "hide-and-seek" game. Rats not only learned how to play "hide" versus "seek" but they were emitting 50 kHz vocalizations during play with predominance of flat and frequency-modulated 50 kHz calls (approx. 72% of all emitted calls with 32% of frequency modulated calls) [115]. Since the authors were using a brief, abdominal tickling procedure any time the rat found a person, or was found by a person, a question arises as to whether rats really were playing "hide-and-seek" game in the human sense or had just learned some rules to merely try to get the tickle-like experience. Somewhat similar behavior was observed during the daily tickling procedure when the rats were escaping from the human hand that was trying to pin them and were approaching and chasing the human hand before dorsal contact [90,108].

The emission of 50 kHz calls during juvenile play fighting, which occurs more frequently in males, facilitates and maintains play behavior [99] that is important for maletypical brain development [116]. The emission of 50 kHz vocalizations contributes to maintaining play, regulates play, and indirectly serves as preparation for the young organisms to develop aptitude for general sociability (gregariousness). Play develops motor skills, even strengthens the skeletal apparatus, develops exploration skills, establishes social ranks and dominance, and prepares juveniles for aggressive and sexual behaviors with always present vocal communication [100,117]. As emphasized by Berlyne (1960) [118], who first proposed the term ludic behavior, play consists of a multitude of functional components, such as perceptual, cognitive, and motor activities, and emotional arousal [118]. Play is a vigorous and highly emotional positive behavior with a crucial role of vocalizations.

#### 2.3.2. Conative Function

The emission of 50 kHz vocalizations during ludic behavior also has another function, a conative function. This is an intentional action of an animal to catch the attention of one or more of the social group members and eventually influence their behavior in a general, non-specific way, or to mobilize them to common action/play. The emission of vocalizations subserving this function in young individuals may play just a general activation role but may change, e.g., into invitation to play. This function is well known in mammals, particularly observed in the vocalizations of domestic animals living with humans, and in vocalizations of attention-seeking human infants, where they are often interpreted as excessive crying for the "manipulation of parents" [55,119,120]. In rats, conative function of calling may be also well illustrated in infants as maternal/paternal potentiation. The vocalization of the isolated pup is significantly increased when the pup has been in contact with its mother immediately before isolation [48]. In this situation, calling cannot be directly associated with a lack of food or other stimuli, but aims at rapid maternal/paternal attention in a general way.

#### 2.3.3. Cooperative Function

Along with the development of the social life in rats, a new function of vocalization appeared that is termed cooperative function. An interesting experiment demonstrated this behavior in rats [121]. To receive a sucrose reward, pairs of familiar (to each other) rats were trained to simultaneously nose poke the holes to receive the reward. Cooperative behavior gradually increased over 44 days of training along with an increased emission of 50 kHz vocalizations. When the pair of rats was separated by a partition, blocking acoustic signals, the cooperative success deteriorated but reappeared again when the rats were separated only by a wire mesh partition and could hear each other. Thus, direct physical contact between the rats was not needed but the rats needed to communicate by ultrasonic calls to achieve cooperative success [121]. The emission of some ultrasonic calls was also suggested as a form of cooperation during common play actions in juvenile rats [122] and in sexual interactions [123].
