*2.4. Functions of Vocalization in Intraspecies Agonistic Interactions* 2.4.1. Agonistic Function

Adult rats use vocalizations for the regulation of their social life, e.g., for the establishment of dominance hierarchies, during aggressive encounters (mostly with intruders), during sexual behavior, feeding, defending territory, and in other situations requiring significant emotional arousal [75]. In all these situations, the emission of ultrasonic calls plays an important communicative role. Even an overall, rough analysis of all emitted vocalizations in these situations shows significant changes in many acoustic parameters of calls, suggesting that rats emit, at least, some calls specific for a given situation and behavior [124].

Aggressive behavior focused the most attention in research and emissions of 50 kHz and 22 kHz type of vocalizations were recorded during the aggressive attacks and defensive actions of the rats [125,126]. Aggressive/defensive behavior is associated with the highest emotional and autonomic arousal because it may be associated with significant body damage. The general label "aggressive behavior" includes many behavioral patterns, such as threatening, aggressive sideway posturing, directed attack, wrestling, boxing, kicking, punching, jumping, submissive posturing, chasing, and flight, so both offensive and defensive elements. Each of these elements is associated with a potentially different type and combination of emitted vocalizations, which may further differ among different rat strains [127]; however, detailed studies about the role of particular types of calls in these components of behaviors have not been systematically conducted.

The 50 kHz vocalizations are emitted mostly by the attacker and the 22 kHz calls by the defeated rat [127,128]. In the intruder–resident interactions, the intruder emitted mostly 50 kHz vocalizations that were changed to 22 kHz calls after its defeat [128,129]. Audible squeals are frequently emitted during fighting and intermingled with ultrasonic calls. Study of vocalizations emitted in the resident/intruder situation with a wire mesh preventing physical contact confirmed that mostly intruders emitted ultrasonic calls [130]. Interestingly, the ultrasonic calls emitted by the intruder were decreased by systemic morphine, but audible vocalizations were not sensitive to morphine, suggesting that their function is different from ultrasonic calls [130].

The emission of the ultrasonic vocalizations in these offensive/defensive situations plays an agonistic function (from Greek *agonisticos*—combative). This term was first suggested by Scott and Fredericson in 1951, mostly in relation to the complex behavior of rats and mice, and it comprises offensive and defensive groups of behaviors, including withdrawal, avoidance, and escape [25,131,132]. In addition to that, agonistic function includes elements of territorial behavior and defense against intruders.

Rats live in large groups that need space for nesting, hiding, and foraging, and will defend this space against rats from neighboring groups. It was postulated that one of the important functions of the evolution of adult vocalizations was spacing among neighboring animal groups. Vocalizations that can be received from a distance serve this purpose very well [133]. The territories rats defend are rather small and there is no good evidence that rats are defending large and defined boundaries around their living burrows [132]. However, agonistic behavior and relevant calling was shown to play broader functions in controlling population density, group stability, and partner choice [75]. Ultrasonic vocalizations used during territorial defense were not studied in rats, but in a study on mice, several types of calls were demonstrated, which were important in territorial defense [134].

#### 2.4.2. Appeasement Function

Many researchers have observed that the emission of long 22 kHz vocalizations during agonistic encounters by the defeated rat may have an appeasement function, which is widely observed in animal behavior [135]. The emission of these calls would decrease or inhibit further attacks of the aggressive or dominant rat [126,127]. This effect probably does not occur immediately and requires some repeated calling, and the opposite might not be true, i.e., the lack of emission of appeasement calls will not necessarily increase attacks of the aggressor, as it was observed in studies with devocalized rats in which deprivation of ultrasonic signals failed to increase aggressive behavior of the attackers [136]. In this experiment, however, rats could not communicate by any type of calls. Appeasement 22 kHz calls may also be emitted to prevent attack in establishing a dominant–submissive relationship. It was observed that a face-to-face encounter with a dominant rat immediately induced the emission of 22 kHz vocalizations in the submissive rat (usually smaller in size) [137].

It was also suggested that during play behavior (both in juvenile and adult rats), the emission of 50 kHz calls may not only be a play signal but also an appeasement signal that de-escalates agonistic behavior during play and prevents aggressive outcomes, which can happen particularly in playing rats that are unfamiliar to each other [99,138].

Finally, it should be mentioned that audible squealing (sonic threat calls) in young rats that are emitted after bites as pain signals, have been also suggested in the past to have an appeasement effect [58] (p. 126).

#### *2.5. Functions of Vocalization in Reproductive Behavior*

#### 2.5.1. Mating Function

The emission of ultrasonic calls was well studied in the mating and reproductive behavior of rodents. This is a complex and partially ritualized behavior, so the emission of many types of calls were observed. These vocalizations play a mating function, i.e., they contribute to the regulation of the selection of partners, soliciting sexual contact, and initiating copulation. Fifty kilohertz vocalizations are emitted mostly during solicitation and mounting activity, while 22 kHz are emitted by males during the postejaculatory refractory period [73,123]. Male rats emit 50 kHz before successful mating, which may facilitate female responsiveness because females were less responsive when paired with a devocalized male [123,139]. Females also emit ultrasonic calls before copulation that were suggested to play a regulatory role in mating [140,141]. The emission of 50 kHz calls was dependent on female sex hormones because ovariectomized females exhibited few, if any, of the vocalizations [142] and their responses were graded depending on the hormonal condition [143].

Some recent studies, however, could not fully confirm the behavioral role of 50 kHz vocalization during mating [144,145]. Many factors could cause a lack of this response, such as rat strain, too-frequent repetition of tests, rat experience, or stress. Ultrasonic communication during mating is highly dependent on the gonadal status of both partners. For instance, it was found that females produced more 50 kHz ultrasonic vocalizations to intact males than to castrated males but produced similar numbers of calls to both relevant groups of females [146]. Using a playback paradigm, the role of ultrasonic calls in mating was studied in another laboratory, and it was concluded that female rats displayed high levels of social approach behavior in response to the playback of male, 50 kHz ultrasonic vocalizations and did not respond to amplitude-matched white noise [147]. The emission

of 50 kHz calls plays an important role in establishing social proximity [147], and it may be concluded that is important in the regulation of mating behavior.

#### 2.5.2. Social Detachment Function

Male postejaculatory 22 kHz vocalizations are particularly long calls [148]. However, unlike other types of 22 kHz vocalizations, they may have some limited frequency modulation, particularly in the medial and terminal fragments of the call. These calls were postulated to represent a different emotional state of the rat than during the emission of flat-type alarm 22 kHz vocalizations in other situations [149]. The emission of shorter 22 kHz calls than the postejaculatory calls may appear during mating and these calls are associated with unsuccessful intromissions or failed mountings [148], so aversive situations to males.

The postejaculatory 22 kHz vocalizations represent a state of behavioral inhibition with prolonged immobility, a withdrawn or socially depressed state, and an absolute refractory period with "desist-contact" function ([149–152]. During the postejaculatory calling state, rat males do not copulate [153]. It was suggested that the postejaculatory calls have function of keeping the females away [123,150]. It has been observed that experienced females leave the male during the emission of these calls [154]. Contrary to that, the prolonged presence of females together with males increased the duration of the emission of postejaculatory 22 kHz calls [152,155]. This notion that females would avoid male during emission of postejaculatory 22 kHz calls was, however, not always detected [156]. Many factors may influence this behavior and sexual experience is one of them. Nevertheless, the 22 kHz ultrasonic vocalizations in this situation may have a social detachment function or social disaffiliation function, i.e., a role opposite to the affiliative function. This function would be equivalent to the appeasement function in agonistic encounters, discussed above.

Acoustic analysis of the postejaculatory of 22 kHz vocalizations emitted by males during sexual contacts, or intended contacts, revealed that the calls are heterogenous and consist of long flat 22 kHz calls (20–35 kHz range) during the postejaculatory period, and anther class of 22 kHz calls. The other class of calls has higher sound frequency (23–45 kHz range) and some modulated frequency components that were observed during encounters of males with a female in a cage with a physical, perforated barrier, where animals could not have physical contact [157]. This last precopulatory category of calls would be compatible with a negative state of frustration caused by the presence of an inaccessible female, and not as a social detachment function [157] (for more details, see Sections 2.8 and 3.5).
