Ultrasonic Vocalizations in Adult C57BL/6J Mice: The Role of Sex Differences and Repeated Testing

Abstract

Ultrasonic vocalizations (USVs) are a major tool for assessing social communication in laboratory mice during their entire lifespan. At adulthood, male mice preferentially emit USVs toward a female conspecific, while females mostly produce ultrasonic calls when facing an adult intruder of the same sex. Recent studies have developed several sophisticated tools to analyze adult mouse USVs, especially in males, because of the increasing relevance of adult communication for behavioral phenotyping of mouse models of autism spectrum disorder (ASD). Little attention has been instead devoted to adult female USVs and impact of sex differences on the quantitative and qualitative characteristics of mouse USVs. Most of the studies have also focused on a single testing session, often without concomitant assessment of other social behaviors (e.g., sniffing), so little is still known about the link between USVs and other aspects of social interaction and their stability/variations across multiple encounters. Here, we evaluated the USVs emitted by adult male and female mice during 3 repeated encounters with an unfamiliar female, with equal or different pre-testing isolation periods between sexes. We demonstrated clear sex differences in several USVs' characteristics and other social behaviors, and these were mostly stable across the encounters and independent of pre-testing isolation. The estrous cycle of the tested females exerted quantitative effects on their vocal and non-vocal behaviors, although it did not affect the qualitative composition of ultrasonic calls. Our findings obtained in B6 mice, i.e., the strain most widely used for engineering of transgenic mouse lines, contribute to provide new guidelines for assessing ultrasonic communication in male and female adult mice.

Long-term behavioral effects of prenatal stress in the Fmr1-knock-out mouse model for fragile X syndrome

“…The estrous cycle of the stimulus female (estrous versus diestrous, n = 3–6 for each condition) did not affect the social and ultrasonic behaviors of mice at both ages ( Table 2 ). This lack of effect could be due to the small sample size, although this was similar to the one used in previous studies on estrous cycle and ultrasonic communication in laboratory mice ( Premoli et al, 2022 ).…”

Beyond the three-chamber test: toward a multimodal and objective assessment of social behavior in rodents

“…As for female mice, earlier studies showed that interactions between devocalized males and intact females abolished detected USVs, while interactions between intact males and devocalized females had little effect on the number of detected USVs, thus leading to the conclusion that USVs in male–female interactions are mainly emitted by the male [ 181 ]. However, later research found that female mice do vocalize during interactions with males, although to a lesser extent of 15–18% of the total USVs recorded [ 159 , 160 , 182 , 183 ] and with USVs of different acoustic features than those of males [ 159 , 162 , 183 ]. Females also emit USVs in female-female interactions [ 184 , 185 ] and in response to an awake or anesthetized female intruder in the resident-intruder test [ 165 , 186 ] (F i g. 3 I–K).…”

“…It should be pointed out, however, that some experimental designs include isolating subjects prior to the experiment so as to induce emission of a greater number of USVs [ 159 , 167 , 184 , 187 ], which may compromise the generality of the results by introducing the confounding effects of isolation on USV emission and social behavior in both males [ 162 , 170 , 174 ] and females [ 165 ]. To overcome such limitations, de Chaumont et al [ 37 ] recorded same-sex pairs of mice over three days in a home-like environment to examine differences in spontaneously emitted USVs without the contribution of prior isolation or constrained interaction in limited recording sessions.…”

“…However, mice predominantly emit USVs in social contexts with frequencies above 20 kHz. Mice USVs present many structurally and temporally complex acoustic features that can vary across developmental stage [10,90,105,156], genetic strains [157,158], gender [159][160][161][162], and social context [163][164][165] (Fig. 3C).…”

Mouse and rat ultrasonic vocalizations in neuroscience and neuropharmacology: State of the art and future applications

“…Both male and female mice emit USVs at a higher rate and higher sound frequencies during opposite‐sex compared to same‐sex interactions; furthermore, call emission is modulated based on the sex of a potential receiver, since male mice emit USVs with higher amplitude in presence of a male mouse compared to a female mouse (Zala et al, 2017). Experiments on C57BL/6 J mice, in a resident‐intruder context, demonstrated that USVs emitted during male–female versus female–female social interaction are qualitatively different, with females producing longer and more complex calls compared to male mice; these sex‐dependent qualitative differences of USVs were stable across multiple social encounters (Premoli et al, 2022). Beside the social context, other stimuli that induce the emission of USVs, albeit to a much lesser extent, include the exposure to a novel environment or to restrain stress, that is, situations both associated with anxiety‐like behaviour and emission of low‐frequency USVs (≤60 kHz) (Chabout et al, 2012; Lefebvre et al, 2020).…”