2.1.1. Self-Preservation Function

The emission of vocalization evolved to serve many functions, but its primary and most important role was associated with saving the individual's life and protecting the species since birth as we can observe today in altricial rat pups. The emission of infant vocalizations serve a self-preservation function, which is regulated by an innate, ancient emotional survival mechanism ascribed to the basic functions of the mammalian brainstem

limbic system [19–21]. The communication of mothers with their offspring with vocalizations is regarded as the phylogenetically oldest form of vocal communication in rats and in all mammals [22]. The emission of calls by rat infants in the ultrasonic range was probably caused by increased air pressure in the respiratory system with constricted vocal folds that evolved in response to the cold [23,24], and this range of sound frequencies appears to have been highly adaptive. It may be speculated that, initially, the crying of infants and juveniles emerged and it was paralleled by the vocal responses of the mothers [22] since mostly females were primary caregivers to the offspring due to nursing and additional critical care (grooming, licking, nest attendance, etc.), without which rat infants could not survive.

In broader sense, maternal behavior was termed epimeletic behavior (from Greek *epimeleteon*—caregiving), which also includes paternal and biparental behavior [25]. On the other hand, pup or young vocalizations directed to their parents were termed et-epimeletic behavior (from Greek, *aeteo*—to beg, + epimeletic), and this label also includes signals other than vocalizations [25]. These terms were introduced at the beginning of the 20th century to make studies on behavior unbiased, devoid of colloquialisms, and easier to compare across species. While maternal vocalizations directed to pups were not carefully studied, etepimeletic vocalizations of pups focused the extensive attention of researchers.

#### 2.1.2. Locating Function

The emission of juvenile broadband vocalizations contains a primary locating function. The infant's calls inform its mother as to where the pup is and help her to retrieve it back to her if it fell out of the nest, or to move it within the nest when the mother's body compresses too strongly on the infant's body [26]. The infant calls, termed sometimes as separation or isolation calls, or distress calls [27,28], provide mothers with critical information about the location of the vocalizing pup. Mothers orient toward the calling pup and approach it (phonotaxis) [29,30]. This maternal behavior is regulated not only by vocalizations but also by olfactory cues. In playback experiments, it was shown that mothers showed enhanced orientation toward the source of the infant's calls if a silent pup was placed under a speaker [31]. Ultrasonic calls provide critical directional information for mothers, while pup odors determine the urgency and speed at which mothers begin searching [32,33].

Initial calls of pups after birth are not well exercised, and infants learn to emit the separation calls in such a way that maximizes the locating of the calling infant. In successive days after birth, the isolation calls gradually become longer and more complex, and the sound frequency within each call has a fluctuating character across many frequencies in a fashion similar to an ambulance siren [34], and they may reach significantly deep fluctuations. This pattern of emitting vocalizations could appear by the natural vocal selection that was demonstrated in infant rats [35]. Although there are some differences in the number of emitted calls, peak frequency, and frequency modulation among male and female pups of the main laboratory strains of Wistars, Long–Evans, and Sprague Dawley rats [36,37], the principle of locating the calling pup remains the same.

Pups of 14 days of age and older, however, will markedly reduce their emission of these calls when an adult male rat is nearby [38,39]. If a pup was isolated from the nest for a short time and retrieved, the mother will spend more time with it and pay more attention to that pup as compared to pups that have always been in the nest [40]. Maternal care is conserved in evolution and it can be demonstrated from rats to humans [41]. It has been shown that maternal care and proper vocal communication with the offspring contributes to development of the infants' social brain and increases the offspring's survival and their future reproductive success [42].

The importance of communication by vocalizations in rat infants may be further demonstrated by experiments showing that pups with less maternal help than usual were more anxious and emitted more infantile ultrasonic vocalizations than controls when they were separated from their mother and litter [43]. Additionally, experimental daily 30 min maltreatment of pups caused increased emission of their isolation calls as compared to pups receiving expected maternal care, and this procedure caused detectable epigenetic changes in the development of the brains of the maltreated rats [44]. Moreover, rats selected for higher emotionality traits emitted more ultrasonic calls when isolated as compared to pups with lower emotionality [45]. With the prolonged separation of pups from their mother, the acoustic parameters of their vocalization changed, and the pups emitted a larger proportion of high sound frequencies than the controls [46,47].

Pups pay constant attention to their mother and to her proximity. It was demonstrated that the interaction of pups with their mother just before the pup's isolation further increased the pups' vocalizations to a subsequent isolation (named maternal potentiation). If male rat contributed to caring for infants, pups would also show paternal potentiation [48,49]. It was concluded that infant separation vocalizations express infantile anxiety, and these vocalizations could be pharmacologically decreased by numerous anxiolytics [50,51].

#### 2.1.3. Protective Function of Maternal Care

The expression of infantile anxiety with the emission of calls also represents a protective function, which secures continued maternal help and safeguarding and builds a bond between the mother and her infants. Although many cues contribute to the development of the mother–infant bond, a repeatable emission of calls is one of the important signals. As studied in mice, infants at Postnatal Day 17 and 21 were able to recognize their own mother in a two-choice tests and preferred their own mother to a foreign mother [52]. In addition, based on the emitted calls, mouse mothers located their own pups faster than a stranger pup [52]. These bonds are the precursor of adult social bonds and are established by the release of oxytocin both in mice and rats [53]. The development of this bond is important at the infantile age. The creation of new stable bonds in adult rat social groups seems to be difficult or not possible, as it was studied in pairs of adult female rats [54].

The same protective function applies to the vocalization of human infants; it was suggested that excessive human infant crying may express anxiety of being abandoned, and crying prevents the withdrawal of parental help and secures continued care [55]. Loss of contact with the mother and the nest environment seems to be the primeval aversive emotional state expressed vocally [56]. Ensuring the continuation of mother–infant contact is one of the evolutionarily oldest and fundamental functions of emitting vocalizations.

#### *2.2. Functions of Vocalization in Non-Agonistic Adult Social Interactions*

#### 2.2.1. Phatic Communication Function

The mother–infant relationship developed another related function of vocalization, termed phatic communication, that is mostly characteristic of the rats' adult life and seems to be appetitive. The term "phatic" was coined initially in anthropology as "bonding by language" [57], i.e., by emission of words (in humans) or vocalizations (in animals) that serve to create and maintain social bonds and closeness. The phatic communication in animals aims at maintaining connection between individuals, a reassuring proximal presence, and maintaining the cohesiveness of social groups in gregarious species [15]. The category of calls for establishing and maintaining contacts between adult members of the social group has been known for a long time. These vocalizations were later termed contact calls in classical ethological studies and were demonstrated in numerous species [58]. It has been even suggested that human humming may be regarded as the human equivalent of contact calls in social animals [59].

In rats, short-duration vocalizations, classified as flat 50 kHz calls, are used as contact calls and are emitted toward familiar conspecifics, even if these conspecifics are not present nearby [60–62]. Rats will particularly emit these calls when they detect fresh olfactory traces of other rats, and the more scent traces they detect, the more calls they emit, usually of a frequency-modulated type [63]. In the case of possible contact with many individuals, these calls may also play another, affiliative function (see below). It was also reported that rats may emit contact vocalizations in dyadic interactions or when being alone in a cage without detectable traces of other rats but shortly after separation from other companions [60,61].

Phatic communication has a character of the social announcement of presence, acceptance, and reassurance, and is not expected to be associated with approach. Phatic vocalizations may initiate similar reply calls from other rats but without further behavioral consequences. Such a mutual, infrequent calling is also evidence of social tolerance and potential social support, which would be associated with mutually positive emotional states. These calls may also have potential anxiolytic properties.

#### 2.2.2. Affiliative Function

Related to phatic communication is the emission of ultrasonic calls that may play an affiliative function. This function includes assuring non-agonistic, close interactions, a signaling approach, promoting direct contacts among individuals, causing grouping, and huddling. Unlike the phatic role of vocalizations, the affiliative function is associated with the approach and even direct contact among rats [64,65]. There is some recent evidence suggesting that 50 kHz calls are emitted during grooming [66] so they may contribute to anti-stress and to close-contact social behaviors driven by rat-positive emotional arousal. In a recent study summarizing results from selective breeding, devocalization experiments, and playback studies, a general, broad-term conclusion was reached that 50 kHz ultrasonic calls "serve as situation-dependent socio-affective signals with important communicative functions" [67].

The affiliative function of calls may be observed in many situations. Rats spend most of the daytime in underground burrows where vocal communication with ultrasonic calls is needed and particularly effective [68]. However, subterranean social mole-rats evolved communication with low-frequency vocalizations of 1.6–6.3 kHz [69]. The rat emission of affiliative calls has a calming effect on approaching conspecifics and prevents unexpected, aggressive attack. During a non-aggressive, 'friendly' approach [18], particularly in dark tunnels, the approaching rat will emit 50 kHz calls, usually in a characteristic short series of three frequency-modulated vocalizations emitted in rapid succession, presumably announcing its movement toward the other rat(s) (unpublished observations).

On the part of receivers, there is ample evidence that, when rats hear abundant 50 kHz calls, they approach the emitter or the source of the calls (e.g., a loudspeaker [65,70,71]. This effect is dependent on previous social experience [64], call-specific (no approach to 22 kHz calls), stronger in juveniles and females [67,70], and species-specific, thus other rodents, for instance, bank voles, do not show an approach response to the rat's 50 kHz calls [72]. The perception of 50 kHz vocalizations and approach to them are clearly appetitive and rewarding responses to such an extent that rats can learn to self-administer the 50 kHz calls [73].

The extreme form of affiliative rat behavior is huddling. Huddling behavior, which is prevalent among infants, will also continue, to some extent, in adulthood [74,75]. While in infancy, one of the predominant roles of huddling is group thermoregulation [76], although other non-thermal stimuli are also important [77,78], adult huddling occurs less frequently and is associated with potential external danger or unfavored conditions, such as bright light or other dangers [79,80]. In these situations, rats have a tendency to crawl under other conspecifics, but this is not a blind behavior because males do not crawl under females but only under other males [81]. There are no studies recording the emission of vocalizations during this behavior.

#### 2.2.3. Passive Defensive Function

Ultrasonic vocalizations of infants, promoting huddling and nestling behavior, and calls serving affiliative function in juveniles and adults, that cause approaching and staying in close social groups have been jointly regarded as a form of "passive" defensive behavior of social groups and associations [82]. This basic defensive function is a form of primary defense, i.e., behavior without any detected presence of danger or predator (as distinguished from secondary defense, which appears when danger or a predator is present). This defensive behavior is a preventive form of behavior and is driven by an

ancient limbic mechanism, aiming at securing potential social support and protection while being among other conspecifics. It is dubbed "strength and safety in numbers", and it is present in all social mammals (and many other animal groups, e.g., fish and birds), including humans [83]. This defensive tendency must be a very old evolutionary development regulated by the oldest limbic mechanism.

### 2.2.4. Socio-Coordinating Function

In general terms, phatic, affiliative, and related defensive functions of the emission of ultrasonic vocalizations are associated with a social, regulatory function not only at the juvenile level but in later social life, particularly in larger groups. This function was termed socio-coordinating function and particularly flat 50 kHz calls are involved [43]. Specific 50 kHz ultrasonic vocalizations are not one-to-one related to individual movements but are associated with specific patterns of motor behaviors and are suggested to coordinate moment-to-moment during social interactions among rats [84]. Experiments with the newly developed *Cacna1c* haploinsufficiency rat model demonstrated the importance of the socio-coordinating function, particularly during rough-and-tumble play behavior and during female interactions [85,86]. The deletion of *Cacna1c* in rats reduced the number of emitted 50 kHz calls, reduced social approach behavior during the playback of 50 kHz vocalizations, and revealed general deficits in communication and coordination during social behavior [85]. In other mammals, the coordinating role of calls is important in the initiation of movement of animal groups, which was well documented for white-faced capuchin monkeys [87,88].

#### 2.2.5. Social Buffering Function

It has been observed that the repeated tickling of rats (heterospecific play with a human hand) that was associated with the emission of vocalizations (mostly 50 kHz vocalizations) had a buffering effect on anxiety caused by handling or by the intraperitoneal injection of saline in these rats [89,90]. Although tactile stimulation during play and other cues are responsible for the buffering effect, it has been noticed that vocalization itself can also have a buffering function in other mammalian species [91]. It is conceivable that the emission of 50 kHz calls during grooming [66] may, jointly with tactile stimuli, have a buffering effect and may reduce stress and anxiety by the release of oxytocin. It was recently shown that juvenile and young rats that received repeated tactile stimulation with a human hand increased the emission of 50 kHz calls and showed the activation of oxytocin neurons in the hypothalamic paraventricular nucleus [92,93].

The abovementioned huddling behavior in rats also has an acute emotional buffering effect [80]. It is not known, however, what call types that rats emit in this situation, if any. However, the results with self-administration of 50 kHz calls by adult rats without any tactile stimulation may support the notion that the calls themselves could have a social buffering effect [73].

#### 2.2.6. Investigative Function

Rats are known to have well-developed responses to novelty, although not all individuals are high responders [94]. High responders to novelty have increased locomotor activity in the new environment and an enhanced level of dopaminergic activity in the nucleus accumbens, and the novel stimuli are rewarding for them [94,95]. In such novel situations, rat will emit vocalizations that play a positive, investigative role and are associated with rewarding novelty-seeking behavior and positive expectation. However, this response is dependent on the rat's preliminary or pre-existing assessment of the environment or novel object. If the new environment seems to be rewarding, the rat will emit vocalizations, mostly 50 kHz calls. If, however, the new environment seems to be dangerous, the rat will not vocalize. This animal's initial bias in its evaluation of the new environment or novel stimuli has been shown experimentally. Rats were initially trained to respond differently to acoustic stimuli as being positive or negative stimuli. Then, upon hearing the playback

of 50 kHz calls or 22 kHz calls, the rats were presented with a new, ambiguous cue that was neither positive nor negative. The rats responded positively to the ambiguous cue when they heard 50 kHz calls, but responded to the same cue negatively when they heard 22 kHz calls [96].

In a recent experiment in a semi-novel environment, i.e., in a cage that had holes in the walls and was familiar to the rats but the lighting was changed so that the illumination of the cage had a novel element, rat ultrasonic vocalizations were recorded. Any time the experimental animal nose-poked the hole, the light was switched off for 5 s. The yoked control group was unable to switch the light off, but the on–off lights were controlled by the experimental group of animals [97]. It was found that rats that could control lighting performed more nose-pokes and emitted more vocalizations than the yoked rats. Long, alarm 22 kHz calls were not emitted; however, the experimental rats had significantly more nose-pokes with the emission of 50 kHz calls (with predominance of flat calls) and more nose-pokes with the emission of short 22 kHz calls (approx. 10–20 ms in duration) as compared to the control yoked group. Although the behavioral situation was not a typical novel environment, there was an element of novelty and novel exploration, and it was associated with calling [97]. It was concluded that 50 kHz calls and short 22 kHz calls could be associated with the investigative function and novelty seeking. The role of ultrasonic calls emitted by rats in novelty situation needs, however, more studies.

The role of call emission, particularly 50 kHz vocalizations, in novel situations could be interpreted as signaling a rewarding novelty-seeking behavior but it is situation dependent. This conclusion is supported by studies on mice tested in novel or familiar environments. Mice emitted significantly more high-frequency ultrasonic calls and of longer duration in dimly lit novel environments than in the brightly illuminated novel environment. In the bright-light conditions (aversive stimulus) of the novel environment, more calls were emitted with lower sound frequencies [98].
