Female presence and estrous state influence mouse ultrasonic courtship vocalizations

The laboratory mouse is an emerging model for context-dependent vocal signaling and reception. Mouse ultrasonic vocalizations are robustly produced in social contexts. In adults, male vocalization during courtship has become a model of interest for signal-receiver interactions. These vocalizations can be grouped into syllable types that are consistently produced by different subspecies and strains of mice. Vocalizations are unique to individuals, vary across development, and depend on social housing conditions. The behavioral significance of different syllable types, including the contexts in which different vocalizations are made and the responses listeners have to different types of vocalizations, is not well understood. We examined the effect of female presence and estrous state on male vocalizations by exploring the use of syllable types and the parameters of syllables during courtship. We also explored correlations between vocalizations and other behaviors. These experimental manipulations produced four main findings: 1) vocalizations varied among males, 2) the production of USVs and an increase in the use of a specific syllable type were temporally related to mounting behavior, 3) the frequency (kHz), bandwidth, and duration of syllables produced by males were influenced by the estrous phase of female partners, and 4) syllable types changed when females were removed. These findings show that mouse ultrasonic courtship vocalizations are sensitive to changes in female phase and presence, further demonstrating the context-sensitivity of these calls.     

Sex differences in ultrasonic vocalizations and coordinated movement in the California mouse (Peromyscus californicus)

Geyer [Am. Zool. 21 (1981) 117] hypothesized that infant rodents increased the number of ultrasonic vocalizations when they moved in and out of the nest in order to elicit extended care from parents. We tested these hypotheses by recording ultrasonic vocalizations (USVs) and coordinated movements (locomotion and grooming) in California mouse and by recording USVs from pups before and after their parents retrieved them. In Experiment 1, USVs and coordinated movements were recorded from 2 to 30 days of age, in female and male Peromyscus californicus pups. USVs at 37, 42, 47, and 52kHz were digitized and recorded by computer and an event recorder program which was simultaneously used to record coordinated movement. Vocalizations persisted to 30 days of age. Vocalizations increased for both males and females after they spent more than 180s in coordinated movement and females vocalized more than males. Females also displayed more coordinated movement and earlier development of coordinated movement than males. There was no effect of litter size on USVs. In Experiment 2, the number of USVs emitted by pups and the latency of dams and sires to contact their pups and retrieve them was measured. There was no significant correlation between the number of USVs emitted by pups and the latency for a parent to contact or retrieve a pup. Results from Experiment 1 provided some support for Geyer's (1981) hypothesis and results from Experiment 2 did not support the hypothesis that the main function of USVs in California mouse pups was to elicit parental care.     

Development of social vocalizations in mice

Adult mice are highly vocal animals, with both males and females vocalizing in same sex and cross sex social encounters. Mouse pups are also highly vocal, producing isolation vocalizations when they are cold or removed from the nest. This study examined patterns in the development of pup isolation vocalizations, and compared these to adult vocalizations. In three litters of CBA/CaJ mice, we recorded isolation vocalizations at ages postnatal day 5 (p5), p7, p9, p11, and p13. Adult vocalizations were obtained in a variety of social situations. Altogether, 28,384 discrete vocal signals were recorded using high-frequency-sensitive equipment and analyzed for syllable type, spectral and temporal features, and the temporal sequencing within bouts. We found that pups produced all but one of the 11 syllable types recorded from adults. The proportions of syllable types changed developmentally, but even the youngest pups produced complex syllables with frequency-time variations. When all syllable types were pooled together for analysis, changes in the peak frequency or the duration of syllables were small, although significant, from p5 through p13. However, individual syllable types showed different, large patterns of change over development, requiring analysis of each syllable type separately. Most adult syllables were substantially lower in frequency and shorter in duration. As pups aged, the complexity of vocal bouts increased, with a greater tendency to switch between syllable types. Vocal bouts from older animals, p13 and adult, had significantly more sequential structure than those from younger mice. Overall, these results demonstrate substantial changes in social vocalizations with age. Future studies are required to identify whether these changes result from developmental processes affecting the vocal tract or control of vocalization, or from vocal learning. To provide a tool for further research, we developed a MATLAB program that generates bouts of vocalizations that correspond to mice of different ages.     

Comparison of ultrasonic vocalizations emitted by rodent pups.

Ultrasonic vocalizations (USVs) emitted by rodent pups, mouse, rat, Syrian hamster, vole, and Mongolian gerbil, were compared as a basic study for a screening test of anti-panic drugs. USVs of rodent pups, separated from their mother under a low temperature condition, were collected by Real-Time Spectrogram (RTS) apparatus, and transformed into spectrograms and power spectra by SIGNAL software. Waveforms of USVs emitted by the rodent pups showed several characteristic features, and species specificity of USVs was shown. We think that the species specificity might be due to differences of the anatomical structures in the respiratory tract and respiratory patterns in rodent pups.     

Dopamine receptor D2 deficiency reduces mouse pup ultrasonic vocalizations and maternal responsiveness

Dopamine signalling facilitates motivated behaviours, and the D2 dopamine receptor (D2R) is important in mother–infant interactions. D2R antagonists disrupt maternal behaviour and, in isolated rat pups, reduce ultrasonic vocalizations (USVs) that promote maternal interaction. Here, we examined the effects of genetic D2R signalling deficiency on pup‐dam interaction with Drd2 knockout (D2R KO) mice. Using heterozygous (HET) cross littermates, the effect of pup genotype on isolation‐induced USVs was quantified. Independent of parental genotype, D2R‐deficient pups emitted fewer USVs than wild type (WT) littermates in a gene dose‐dependent manner. Using reciprocal D2R KO‐WT crosses, we examined how parental genotype affects pup USVs. Heterozygous pups from D2R KO dams produced fewer USVs than HET pups from WT dams. Also, exposure to USV‐emitting pups increased plasma prolactin levels in WT dams but not in D2R KO dams, and KO dams showed delayed pup retrieval and nest building. These findings indicate the importance of the interaction between pup and dam genotypes on behaviour and further support the role of D2R signalling in maternal care.     

Vocal communication in adult greater horseshoe bats, Rhinolophus ferrumequinum

Whereas echolocation in horseshoe bats is well studied, virtually nothing is known about characteristics and function of their communication calls. Therefore, the communication calls produced by a group of captive adult greater horseshoe bats were recorded during various social interactions in a free-flight facility. Analysis revealed that this species exhibited an amazingly rich repertoire of vocalizations varying in numerous spectro-temporal aspects. Calls were classified into 17 syllable types (ten simple syllables and seven composites). Syllables were combined into six types of simple phrases and four combination phrases. The majority of syllables had durations of more than 100 ms with multiple harmonics and fundamental frequencies usually above 20 kHz, although some of them were also audible to humans. Preliminary behavioral observations indicated that many calls were emitted during direct interaction with and in response to social calls from conspecifics without requiring physical contact. Some echolocation-like vocalizations also appeared to clearly serve a communication role. These results not only shed light upon a so far widely neglected aspect of horseshoe bat vocalizations, but also provide the basis for future studies on the neural control of the production of communicative vocalizations in contrast to the production of echolocation pulse sequences.     

Comprehensive Analysis of Ultrasonic Vocalizations in a Mouse Model of Fragile X Syndrome Reveals Limited, Call Type Specific Deficits

Fragile X syndrome (FXS) is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP). Absence of FMRP in fragile X patients as well as in Fmr1 knockout (KO) mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs) when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT) littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.     

Male mice ultrasonic vocalizations enhance female sexual approach and hypothalamic kisspeptin neuron activity

Vocal communication in animals is important for ensuring reproductive success. Male mice emit song-like “ultrasonic vocalizations (USVs)” when they encounter female mice, and females show approach to the USVs. However, it is unclear whether USVs of male mice trigger female behavioral and endocrine responses in reproduction. In this study, we first investigated the relationship between the number of deliveries in breeding pairs for 4 months and USVs syllables emitted from those paired males during 3 min of sexual encounter with unfamiliar female mice. There was a positive correlation between these two indices, which suggests that breeding pairs in which males could emit USVs more frequently had more offspring. Further, we examined the effect of USVs of male mice on female sexual behavior. Female mice showed more approach behavior towards vocalizing males than devocalized males. Finally, to determine whether USVs of male mice could activate the neural system governing reproductive function in female mice, the activation of kisspeptin neurons, key neurons to drive gonadotropin-releasing hormone neurons in the hypothalamus, was examined using dual-label immunocytochemistry with cAMP response element-binding protein phosphorylation (pCREB). In the arcuate nucleus (Arc), the number of kisspeptin neurons expressing pCREB significantly increased after exposure to USVs of male as compared with noise exposure group. In conclusion, our results suggest that USVs of male mice promote fertility in female mice by activating both their approaching behavior and central kisspeptin neurons.     

Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism

BTBR T+ tf/J (BTBR) is an inbred mouse strain that displays social abnormalities and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. Here we investigate ultrasonic vocalizations in BTBR, to address the second diagnostic symptom of autism, communication deficits. As compared to the commonly used C57BL/6J (B6) strain, BTBR pups called more loudly and more frequently when separated from their mothers and siblings. Detailed analysis of ten categories of calls revealed an unusual pattern in BTBR as compared to B6. BTBR emitted high levels of harmonics, two-syllable, and composite calls, but minimal numbers of chevron-shaped syllables, upward, downward, and short calls. Because body weights were higher in BTBR than B6 pups, one possible explanation was that larger thoracic size was responsible for the louder calls and different distribution of syllable categories. To test this possibility, we recorded separation calls from FVB/NJ, a strain with body weights similar to BTBR, and 129X1/SvJ, a strain with body weights similar to B6. BTBR remained the outlier on number of calls, displaying low numbers of complex, upward, chevron, short, and frequency steps calls, along with high harmonics and composites. Further, developmental milestones and growth rates were accelerated in BTBR, indicating an unusual neurodevelopmental trajectory. Overall, our findings demonstrate strain-specific patterns of ultrasonic calls that may represent different lexicons, or innate variations in complex vocal repertoires, in genetically distinct strains of mice. Particularly intriguing is the unusual pattern of vocalizations and the more frequent, loud harmonics evident in the BTBR mouse model of autism that may resemble the atypical vocalizations seen in some autistic infants.     

Sexual dimorphism and gonadal control of ultrasonic vocalizations in adult pine voles, Microtus pinetorum

We investigated the vocalizations produced by adult pine voles during various social interactions by presenting an experimental animal with either an anesthetized or awake (unanesthetized) conspecific. Ultrasonic vocalizations (USVs) occurred frequently during tests in which an awake male was present, but were rarely detected in tests involving only awake females, or when a female was presented with an anesthetized conspecific. Higher rates of USVs were produced when males were tested with a familiar female than when tested with an unfamiliar male or female. Equivalent rates were produced when males were presented with either anesthetized or awake animals, but female-soiled bedding failed to elicit USVs from males. Sonic vocalizations (SVs) were produced by both sexes and were associated with aggressive behavior, but occurred only in tests between awake, unfamiliar animals. Castration greatly reduced and testosterone therapy restored USVs emitted by males in response to anesthetized conspecifics. Our results suggest that (i) USVs are emitted predominantly by males; (ii) familiarity enhances USV response; (iii) SVs are produced during aggressive interactions; and (iv) androgens regulate the production of USVs by males. Possible roles for pine vole vocalizations are discussed.     

Ultrasonic Vocalizations of Male Mice Differ among Species and Females Show Assortative Preferences for Male Calls

Male house mice (Mus musculus) emit ultrasonic vocalizations (USVs) during courtship, which attract females, and we aimed to test whether females use these vocalizations for species or subspecies recognition of potential mates. We recorded courtship USVs of males from different Mus species, Mus musculus subspecies, and populations (F1 offspring of wild-caught Mus musculus musculus, Mus musculus domesticus (and F1 hybrid crosses), and Mus spicilegus), and we conducted playback experiments to measure female preferences for male USVs. Male vocalizations contained at least seven distinct syllable types, whose frequency of occurrence varied among species, subspecies, and populations. Detailed analyses of multiple common syllable types indicated that Mus musculus and Mus spicilegus could be discriminated based on spectral and temporal characteristics of their vocalizations, and populations of Mus musculus were also distinctive regardless of the classification model used. Females were able to discriminate USVs from different species, and showed assortative preferences for conspecific males. We found no evidence that females discriminate USVs of males from a different subspecies or separate populations of the same species, even though our spectral analyses identified acoustic features that differ between species, subspecies, and populations of the same species. Our results provide the first comparison of USVs between Mus species or between Mus musculus subspecies, and the first evidence that male USVs potentially facilitate species recognition.     

Comparative study on isolation calls emitted from hamster pups.

Waveforms of isolation calls emitted from hamster pups, which were Syrian hamsters, Djungarian hamsters, and Chinese hamsters, were compared in a basic study on improving reproduction by decrease of cannibalism, because it was reported that maternal behavior was induced by isolation calls in rodents. Isolation calls of hamster pups, isolated from their mother and receiving cold stress, were collected by Real-Time Spectrogram (RTS), and calculated to spectrograms and power spectra by SIGNAL. Isolation calls consisted of ultrasonic vocalizations (USVs) and audible vocalizations (ADVs) in each species. Waveforms of isolation calls emitted by the hamster pups, were shown to have several characteristic features. In this study, the species specificity of isolation calls was shown in hamster pups. It would seem that the species specificity originates in the differences of sensitivity to cold stress via the autonomic nerve in hamsters.     

Phase-Specific Vocalizations of Male Mice at the Initial Encounter during the Courtship Sequence

Mice produce ultrasonic vocalizations featuring a variety of syllables. Vocalizations are observed during social interactions. In particular, males produce numerous syllables during courtship. Previous studies have shown that vocalizations change according to sexual behavior, suggesting that males vary their vocalizations depending on the phase of the courtship sequence. To examine this process, we recorded large sets of mouse vocalizations during male–female interactions and acoustically categorized these sounds into 12 vocal types. We found that males emitted predominantly short syllables during the first minute of interaction, more long syllables in the later phases, and mainly harmonic sounds during mounting. These context- and time-dependent changes in vocalization indicate that vocal communication during courtship in mice consists of at least three stages and imply that each vocalization type has a specific role in a phase of the courtship sequence. Our findings suggest that recording for a sufficiently long time and taking the phase of courtship into consideration could provide more insights into the role of vocalization in mouse courtship behavior in future study.     

Ultrasonic courtship vocalizations in wild house mice: spectrographic analyses

House mice emit ultrasonic vocalizations (USVs) during courtship, which are sexually dimorphic and function to attract mates. Spectrographic analyses of laboratory mice show that USVs are surprisingly complex and have features of song. In this study, we conducted the first spectral and temporal analyses of recordings from wild house mice (F1 from wild-caught Mus musculus musculus). Inspection of the spectral shape of syllables shows that the USVs from wild mice can be classified by both frequency and duration, and the most apparent distinction is between low- versus high-frequency calls. High-frequency calls of wild mice seem to be emitted at a much higher frequency range than previously found in some laboratory mice. Interestingly, we found that 20% of males do not vocalize at all, though the reason for their behaviour is unclear. Future studies are needed to determine what kind of information is conveyed in these complex vocalizations, and why some males appear to be non-vocalizers.     

Reduction in Ultrasonic Vocalizations in Pups Born to Rapid Eye Movement Sleep Restricted Mothers in Rat Model

The effects of rapid eye movement sleep restriction (REMSR) in rats during late pregnancy were studied on the ultrasonic vocalizations (USVs) made by the pups. USVs are distress calls inaudible to human ears. Rapid eye movement (REM) sleep was restricted in one group of pregnant rats for 22 hours, starting from gestational day 14 to 20, using standard single platform method. The USVs of male pups were recorded after a brief isolation from their mother for two minutes on alternate post-natal days, from day one till weaning. The USVs were recorded using microphones and were analysed qualitatively and quantitatively using SASPro software. Control pups produced maximum vocalization on post-natal days 9 to 11. In comparison, the pups born to REMSR mothers showed not only a reduction in vocalization but also a delay in peak call making days. The experimental group showed variations in the types and characteristics of call types, and alteration in temporal profile. The blunting of distress call making response in these pups indicates that maternal sleep plays a role in regulating the neural development involved in vocalizations and possibly in shaping the emotional behaviour in neonates. It is suggested that the reduced ultrasonic vocalizations can be utilized as a reliable early marker for affective state in rat pups. Such impaired vocalization responses could provide an important lead in understanding mother-child bonding for an optimal cognitive development during post-partum life. This is the first report showing a potential link between maternal REM sleep deprivation and the vocalization in neonates and infants.     

Social vocalizations of big brown bats vary with behavioral context

Bats are among the most gregarious and vocal mammals, with some species demonstrating a diverse repertoire of syllables under a variety of behavioral contexts. Despite extensive characterization of big brown bat (Eptesicus fuscus) biosonar signals, there have been no detailed studies of adult social vocalizations. We recorded and analyzed social vocalizations and associated behaviors of captive big brown bats under four behavioral contexts: low aggression, medium aggression, high aggression, and appeasement. Even limited to these contexts, big brown bats possess a rich repertoire of social vocalizations, with 18 distinct syllable types automatically classified using a spectrogram cross-correlation procedure. For each behavioral context, we describe vocalizations in terms of syllable acoustics, temporal emission patterns, and typical syllable sequences. Emotion-related acoustic cues are evident within the call structure by context-specific syllable types or variations in the temporal emission pattern. We designed a paradigm that could evoke aggressive vocalizations while monitoring heart rate as an objective measure of internal physiological state. Changes in the magnitude and duration of elevated heart rate scaled to the level of evoked aggression, confirming the behavioral state classifications assessed by vocalizations and behavioral displays. These results reveal a complex acoustic communication system among big brown bats in which acoustic cues and call structure signal the emotional state of a caller.     

The effects of repeated early deprivation on ultrasonic vocalizations and ontogenetic development in mandarin vole pups

Early deprivation is popularly used in rodent models as an early life social stress to investigate and determine the factors that affect the development of the brain and behavior. Ultrasonic calls made by pups play an important role in parental–pup interactions during the neonatal period. However, whether repeated early deprivation affects the properties of ultrasonic vocalizations (USVs) produced by mandarin vole (Microtus mandarinus) pups, and whether ontogenetic development is subsequently affected, remains unclear. Here we measured USVs and developmental parameters in mandarin vole pups deprived of their parents and littermates for 3 h per day (ED, which is significantly different from 5 min isolation used to induce USVs) and another pup group developed under normal nest conditions (PC). Repeated measures analysis indicated that the number of USVs from ED pups was significantly lower than those from PC pups during the postnatal period (p < 0.05). The pulse durations of ED pups were longer than those of PC pups at two (p < 0.001) and five days of age (p < 0.05), but shorter at 14 days of age (p < 0.001). Compared with PC pups, the frequency range of the ED pups was wider at 18–45 kHZ, variable during the first week, smaller and narrower at 18–30 kHZ at eight and 11 days of age, and became stable similar to PC pups at 25 kHZ after 14 days of age. ED also reduced pup body weight significantly and resulted in earlier eye opening compared with PC pups (p < 0.001). A positive relationship was also found between USV emissions and levels of parental care received by pups. It appears that pup USVs are an important age-dependent behavioral phenotype and an effective communicative method between parents and offspring. Prolonged parental and littermate deprivation (ED) may alter USVs emitted by pups and then ontogenetic development and parental care. Mandarin voles show USV properties similar to socially monogamous rodents and this add further support to the hypothesis that species with different social systems produce different patterns of ultrasonic vocalizations. USVs, ontogenetic development and parental care are closely associated.     

The effects of social experience and gonadal hormones on retrieving behavior of mice and their responses to pup ultrasonic vocalizations

Pup ultrasonic vocalizations (USVs) are emitted from maternally separated pups and are thought to be a trigger for eliciting maternal behavior in mice. We investigated the effects of social experience and gonadectomy on the retrieving behavior of mice and their responses to pup USVs produced by a nanocrystalline silicon thermo-acoustic emitter. In each experiment, virgin, gonadectomized, sham-operated, sexually experienced, and parenting mice of both sexes were used, and the effects of these manipulations were compared in each sex. The retrieving behavior of both sexes increased with social experience or gonadectomy. In particular, mothers showed the highest retrieving activity among female groups, while castrated male mice showed the highest retrieving activity among male groups. All groups of female mice responded to pup USVs, with the responsiveness of sexually experienced female mice being the most enhanced. Unlike the females, virgin male mice did not respond to pup USVs, although socially experienced or castrated males showed this response; fathers exhibited the highest responsiveness. These results suggest that not only parenting experience, but also mating experience, may enhance retrieving activity and response to pup USVs in mice of both sexes. Nevertheless, the degree to which parenting experience contributed to the enhancement of both activities differed between the sexes. Furthermore, gonadectomy enhanced both activities in both sexes, although its effect was more prominent in males. Overall, our findings suggest that alteration in responsiveness of mice to pup USVs might be one of the changes in parental behavior caused by social experiences or gonadal hormones.     

Ultrasonic vocalizations induced by sex and amphetamine in M2, M4, M5 muscarinic and D2 dopamine receptor knockout mice

Adult mice communicate by emitting ultrasonic vocalizations (USVs) during the appetitive phases of sexual behavior. However, little is known about the genes important in controlling call production. Here, we study the induction and regulation of USVs in muscarinic and dopaminergic receptor knockout (KO) mice as well as wild-type controls during sexual behavior. Female mouse urine, but not female rat or human urine, induced USVs in male mice, whereas male urine did not induce USVs in females. Direct contact of males with females is required for eliciting high level of USVs in males. USVs (25 to120 kHz) were emitted only by males, suggesting positive state; however human-audible squeaks were produced only by females, implying negative state during male-female pairing. USVs were divided into flat and frequency-modulated calls. Male USVs often changed from continuous to broken frequency-modulated calls after initiation of mounting. In M2 KO mice, USVs were lost in about 70-80% of the mice, correlating with a loss of sexual interaction. In M5 KO mice, mean USVs were reduced by almost 80% even though sexual interaction was vigorous. In D2 KOs, the duration of USVs was extended by 20%. In M4 KOs, no significant differences were observed. Amphetamine dose-dependently induced USVs in wild-type males (most at 0.5 mg/kg i.p.), but did not elicit USVs in M5 KO or female mice. These studies suggest that M2 and M5 muscarinic receptors are needed for male USV production during male-female interactions, likely via their roles in dopamine activation. These findings are important for the understanding of the neural substrates for positive affect.     

Development of echolocation and communication vocalizations in the big brown bat, <Emphasis Type="Italic">Eptesicus fuscus</Emphasis>

Big brown bats form large maternity colonies of up to 200 mothers and their pups. If pups are separated from their mothers, they can locate each other using vocalizations. The goal of this study was to systematically characterize the development of echolocation and communication calls from birth through adulthood to determine whether they develop from a common precursor at the same or different rates, or whether both types are present initially. Three females and their six pups were isolated from our captive breeding colony. We recorded vocal activity from postnatal day 1 to 35, both when the pups were isolated and when they were reunited with their mothers. At birth, pups exclusively emitted isolation calls, with a fundamental frequency range <20 kHz, and duration >30 ms. By the middle of week 1, different types of vocalizations began to emerge. Starting in week 2, pups in the presence of their mothers emitted sounds that resembled adult communication vocalizations, with a lower frequency range and longer durations than isolation calls or echolocation signals. During weeks 2 and 3, these vocalizations were extremely heterogeneous, suggesting that the pups went through a babbling stage before establishing a repertoire of stereotyped adult vocalizations around week 4. By week 4, vocalizations emitted when pups were alone were identical to adult echolocation signals. Echolocation and communication signals both appear to develop from the isolation call, diverging during week 2 and continuing to develop at different rates for several weeks until the adult vocal repertoire is established.