Novel 31-kHz calls emitted by female Lewis rats during social isolation and social inequality conditions.

Whether commonly used experimental animals show aversion toward inequality of social rewards, as humans do remains unknown. We examined whether rats emitted the 22-kHz distress calls under social reward inequality. Rats showed affiliative behavior for a specific human who repeatedly stroked and tickled them. When experimenter stroked another rat in front of them and during social isolation, these rats emitted novel calls with acoustic characteristics different from those of calls emitted under physical stress, namely air-puff. Under inequality conditions, rats emitted calls with higher frequency (∼31 kHz) and shorter duration (<0.5 s) than those emitted when receiving air-puff. However, with an affiliative human in front of them, the number of novel calls was lower and rats emitted 50-kHz calls, indicative of the appetitive state. These results indicate that rats distinguish between conditions of social reward inequality and the presence of an experimenter, and emit novel 31-kHz calls.

Higher oxytocin concentrations occur in subjects who build affiliative relationships with companion robots.

Building affiliative relationships with others is important for mental health. Recently, robots have been expected to play a role in improving mental health, but there is little scientific evidence as to whether they can build affiliative relationships with humans. To investigate that, we conducted studies combining behavior, physiology and questionnaires for companion robot Owners and Non-Owners. The results reveal that the steady-state concentration of oxytocin, a hormone related to affiliative relationships, was significantly higher in Owners than in Non-Owners. In addition, the Owners showed more behaviors indicative of intimacy than the Non-Owners. These results suggest that humans can build affiliative relationships with robots. Fifteen minutes of contact with the robot decreased the concentration of cortisol in both groups, suggesting that even a brief contact can contribute to improving mental health. Therefore, relationships between humans and robots may be one option to improve mental health and enhance well-being.

Post-weaning stroking stimuli induce affiliative behavior toward humans and influence brain activity in female rats.

Gentle touch contributes to affiliative interactions. We investigated the effects of gentle stroking in female rats on the development of affiliative behaviors toward humans and we exploratively examined brain regions in which activity was influenced by stroking. Rats that had received stroking stimuli repeatedly after weaning emitted 50-kHz calls, an index of positive emotion, and showed affiliative behaviors toward the experimenter. Hypothalamic paraventricular oxytocin neurons were activated in the rats after stroking. The septohypothalamic nucleus (SHy) in the post-weaningly stroked rats showed decreased activity in response to stroking stimuli compared with that in the non-stroked control group. There were negative correlations of neural activity in hypothalamic regions including the SHy with the number of 50-kHz calls. These findings revealed that post-weaning stroking induces an affiliative relationship between female rats and humans, possibly via activation of oxytocin neurons and suppression of the activity of hypothalamic neurons.

Gentle stroking stimuli induce affiliative responsiveness to humans in male rats.

Gentle tactile stimuli have been shown to play an important role in the establishment and maintenance of affiliative social interactions. Oxytocin has also been shown to have similar actions. We investigated the effects of gentle stroking on affiliative relationships between humans and rats and the effects of gentle stroking on activation of oxytocin neurons. Male rats received 5-min stroking stimuli from an experimenter every other day for 4 weeks between 3 and 6 weeks of age (S3-6 group), for 4 weeks between 7 and 10 weeks of age (S7-10 group), or for 8 weeks between 3 and 10 weeks of age (S3-10 group). Control rats did not receive stroking stimuli. Rats in the S7-10 and S3-10 groups emitted 50-kHz calls, an index of positive emotion, more frequently during stroking stimuli. Rats in the S3-6, S7-10, and S3-10 groups showed affiliative behaviors toward the experimenter. Oxytocin neurons in the hypothalamic paraventricular nucleus of rats in the S3-6, S7-10, and S3-10 groups were activated following stroking stimuli. These findings revealed that post-weaning repeated stroking stimuli induce an affiliative relationship between rats and humans and activation of oxytocin neurons.

USVSEG: A robust method for segmentation of ultrasonic vocalizations in rodents.

Rodents' ultrasonic vocalizations (USVs) provide useful information for assessing their social behaviors. Despite previous efforts in classifying subcategories of time-frequency patterns of USV syllables to study their functional relevance, methods for detecting vocal elements from continuously recorded data have remained sub-optimal. Here, we propose a novel procedure for detecting USV segments in continuous sound data containing background noise recorded during the observation of social behavior. The proposed procedure utilizes a stable version of the sound spectrogram and additional signal processing for better separation of vocal signals by reducing the variation of the background noise. Our procedure also provides precise time tracking of spectral peaks within each syllable. We demonstrated that this procedure can be applied to a variety of USVs obtained from several rodent species. Performance tests showed this method had greater accuracy in detecting USV syllables than conventional detection methods.

Pup exposure facilitates retrieving behavior via the oxytocin neural system in female mice.

Parental behavior in mammals is innate, but it is also facilitated by social experience, specifically social interactions between the parent and infant. Social interactions with infants also induce the alloparental behavior of virgin animals. Oxytocin (OT) plays an important role in mediating alloparental behavior. Although parental behavior is modulated by the medial preoptic area (MPOA) and adjacent regions, it is unclear how OT acts in these regions as a control mechanism of alloparental behavior promoted by adult-pup interaction. The aim of this study was to investigate the role of OT for facilitating effects of adult-pup interactions on alloparental behavior via neural activity of preoptic area (POA), including MPOA and adjacent area. For this purpose, we conducted behavioral tests and examined the neural activity of the OT system in POA. Virgin female mice that were repeatedly exposed to pups showed shorter retrieving latencies and higher number of c-Fos expressing neurons in POA, particular in lateral preoptic area (LPO) compared to control animals that were exposed to pups only one time. In addition, repeated pup exposure increased the proportion of OT neurons and OTR neurons expressing c-Fos in POA. The concentration of OT also significantly increased in the POA. Finally, infusion of an OT antagonist into the POA area blocked the facilitating effects of repeated pup exposure on retrieving behavior. These results demonstrated that the facilitating effects of repeated pup exposure on alloparental behavior occurred via an organizational role of the OT system.

Mutual mother-infant recognition in mice: The role of pup ultrasonic vocalizations.

The importance of the mother-infant bond for the development of offspring health and sociality has been studied not only in primate species but also in rodent species. A social bond is defined as affiliative behaviors toward a specific partner. However, controversy remains concerning whether mouse pups can distinguish between their own mother and an alien mother, and whether mothers can differentiate their own pups from alien pups. In this study, we investigated whether mutual recognition exists between mother and infant in ICR mice. Furthermore, we studied pup ultrasonic vocalizations (USVs), which are emitted by pups when isolated from their mothers, to determine whether they constituted an individual signature used by the mother for pup recognition. We conducted a variety of two-choice tests and selective-retrieving tests. In a two-choice test for mother recognition by the pup, pups between the ages of 17 and 21days preferred their own mothers to alien mothers. In a two-choice test for pup recognition by its mother, the mothers located their own pups faster than alien pups at the beginning of the test, yet displayed similar retrieving activity for both their own and alien pups in the subsequent selective-retrieving test. Furthermore, after recording USVs from pups from subject and alien mothers, then playing them simultaneously, subject mothers displayed a preference for pup USVs emitted by their own pups. Overall, our findings support the existence of mother-infant bonding in mice and suggest that pup USVs contribute to pup recognition by mothers.

Activation of hypothalamic oxytocin neurons following tactile stimuli in rats.

Gentle touching or stroking has anxiolytic actions and contributes to the establishment of an intimate relationship between individuals. Oxytocin administration also has anxiolytic actions and facilitates social behaviors. In this study, we examined effects of stroking stimuli on activation of oxytocin neurons and emission of 50-kHz ultrasonic vocalizations, an index of positive emotion, in rats. The number of oxytocin neurons expressing Fos protein was increased in the hypothalamus, especially in the dorsal zone of the medial parvicellular part of the paraventricular nucleus. The number of 50-kHz ultrasonic vocalizations was also increased. These findings suggest that pleasant sensory stimuli activate hypothalamic oxytocin neurons.

Developmental social environment imprints female preference for male song in mice.

BACKGROUND: Sexual imprinting is important for kin recognition and for promoting outbreeding, and has been a driving force for evolution; however, little is known about sexual imprinting by auditory cues in mammals. Male mice emit song-like ultrasonic vocalizations that possess strain-specific characteristics. OBJECTIVES: In this study, we asked whether female mice imprint and prefer specific characteristics in male songs. METHODS AND FINDINGS: We used the two-choice test to determine the song preference of female C57BL/6 and BALB/c mice. By assessing the time engaged in searching behavior towards songs played back to females, we found that female mice displayed an innate preference for the songs of males from different strains. Moreover, this song preference was regulated by female reproductive status and by male sexual cues such as the pheromone ESP1. Finally, we revealed that this preference was reversed by cross-fostering and disappeared under fatherless conditions, indicating that the behavior was learned by exposure to the father's song. CONCLUSIONS: Our results suggest that female mice can discriminate among male song characteristics and prefer songs of mice from strains that are different from their parents, and that these preferences are based on their early social experiences. This is the first study in mammals to demonstrate that male songs contribute to kin recognition and mate choice by females, thus helping to avoid inbreeding and to facilitate offspring heterozygosity.

Transport Response is a filial-specific behavioral response to maternal carrying in C57BL/6 mice.

BACKGROUND: A mother carries her young in many altricial mammals, such as cats, lions, rats and mice. During maternal carrying, the transported young assume a compact posture. We have recently shown that, in both humans and mice, the carried infants immediately calmed down and showed reductions in heart rate, distress vocalizations, and voluntary movement. The loss of the calming response in mouse pups hindered maternal retrieval efficacy. These findings suggested that the infant calming response functioned to reduce the maternal burden of carrying and was therefore conserved in a variety of mammalian species. However, it remains unclear how and when each component of this calming response develops and whether it is a filial-specific behavior. RESULTS: We dissected various components of the carrying-induced responses in mouse pups, collectively called the "Transport Response" herein. We showed that during the second postnatal week, pups exhibited characteristic compact posture with limb ventroflexion. The body trunk remained paradoxically pliable, suggesting complex neural regulation throughout the body. Pups also showed an increased pain tolerance to a tail pinch during the Transport Response. Analyses of the developmental courses of distinct components of the Transport Response revealed the independent regulation of each component: in the first postnatal week, the cessation of ultrasonic vocalizations was exhibited prominently; in the second postnatal week, immobilization reached its peak; and toward the third postnatal week, the postural component became fully matured. At the end of the third postnatal week, when the pups are able to transport by themselves, the pups no longer exhibited the Transport Response. CONCLUSIONS: This study has revealed the mouse Transport Response as a complex set of behavioral and physiological components, each of which has a specific postnatal time window but is orchestrated in a well-matched manner with the maturation of ambulatory ability in the pups. These findings collectively indicate that the Transport Response is a filial-specific, innate behavioral reaction and is distinct from a simple reflex or defensive freezing response. The Transport Response could be a novel index of primitive filial attachment behaviors, acting to smooth mother-infant interaction.

Testosterone inhibits facilitating effects of parenting experience on parental behavior and the oxytocin neural system in mice.

Parental behavior in mammals is facilitated by sensory experiences from infant, and by endocrine hormones. However, the interactions between these factors in the parental behavior of nonreproductive adults are not understood. We examined the interactive effects of gonadal hormones and the experience of repeated pup exposure on parental behavior in sexually naive mice. We also compared oxytocin (OT) expression levels in the paraventricular nucleus of the hypothalamus to behavioral outcomes. Clear sex differences were observed in retrieving tests; initial retrieving latency was shorter in females than in males, and 5-time pup exposure shortened retrieving latency in females only. Gonadectomy influenced neither initial retrieving latency nor pup sensitization in females. In contrast, gonadectomy shortened initial retrieving latency and caused pup sensitization in males. Estrogen implants given simultaneously with gonadectomy further shortened the initial retrieving latency in males, but pup sensitization was not affected and occurred in both sexes. In contrast, simultaneous testosterone implants impaired pup sensitization in both sexes. Similar to the results for responsiveness to pups, the number of OT neurons was increased by gonadectomy in males only. In comparison to gonadectomy only, OT neurons were decreased by simultaneous testosterone implants, but were not influenced by estrogen in either sex. Considering the parallel inhibitory effects of testosterone on both pup sensitization and number of OT neurons, we postulate that sex differences in parental responsiveness facilitated by repeated pup exposure were caused by an inhibitory effect of testosterone via the OT neural system in mice.

Infant calming responses during maternal carrying in humans and mice.

BACKGROUND: Mother-infant bonding is the earliest and most critical social relationship of mammalian infants. To promote this bond, infants have innate behaviors to seek maternal proximity and protest upon separation via communication with the mother vocally and through body movement. However, the physiological mechanisms regulating these infant behaviors remain largely undefined. RESULTS: Here we show a novel set of infant cooperative responses during maternal carrying. Infants under 6 months of age carried by a walking mother immediately stopped voluntary movement and crying and exhibited a rapid heart rate decrease, compared with holding by a sitting mother. Furthermore, we identified strikingly similar responses in mouse pups as defined by immobility and diminished ultrasonic vocalizations and heart rate. Using pharmacologic and genetic interventions in mouse pups, we identified the upstream and downstream neural systems regulating the calming response. Somatosensory and proprioceptive input signaling are required for induction, and parasympathetic and cerebellar functions mediate cardiac and motor output, respectively. The loss of the calming response hindered maternal rescue of the pups, suggesting a functional significance for the identified calming response. CONCLUSIONS: Our study has demonstrated for the first time that the infant calming response to maternal carrying is a coordinated set of central, motor, and cardiac regulations and is a conserved component of mammalian mother-infant interactions. Our findings provide evidence for and have the potential to impact current parenting theory and practice, since unsoothable crying is the major risk factor for child abuse.

Pup odor and ultrasonic vocalizations synergistically stimulate maternal attention in mice.

Pup ultrasonic vocalizations (USVs), which are emitted by hypothermic pups, and pup odor are thought to be triggers of maternal behavior in mice. We investigated whether pup odor stimulated maternal responses to pup USVs in mother C57BL/6 mice. Two-choice tests were conducted by introducing mothers into a test cage in which a tube was attached on each long wall, and the duration spent in each tube was compared. Pup USVs were reproduced by an ultrasonic speaker at the tube end. In some cases, cotton with pup odor was also presented at the end of the tube. Compared to no stimuli, mothers did not specifically approach the sole presentation of either reproduced pup USVs or pup odor. However, compared to the sole presentation of pup odor, the simultaneous presentation of pup USVs and odor induced a specific approach response. These results suggested that pup USVs and odor synergistically stimulated maternal behavior. In addition, it was confirmed that mothers approached hypothermic pups emitting pup USVs for longer than anesthetized silent pups. To investigate the underlying neural mechanisms, we observed neural responses to various stimuli with the immunohistochemistry of c-fos expression. In the bed nucleus of the stria terminalis, the medial preoptic area, the central nucleus of the amygdala, and the basolateral amygdala, the numbers of c-fos-positive cells were significantly increased following the simultaneous presentation of pup USVs and odor compared to the presentation of each alone, suggesting that these nuclei were involved in multimodal processing related to maternal behavior.

Importance of mother-infant communication for social bond formation in mammals.

Mother-infant bonding is a universal relationship of all mammalian species. Here, we describe the role of reciprocal communication between mother and infant in the formation of bonding for several mammalian species. Mother-infant bond formation is reinforced by various social cues or stimuli, including communicative signals, such as odor and vocalizations, or tactile stimuli. The mother also develops cross-modal sensory recognition of the infant, during bond formation. Many studies have indicated that the oxytocin neural system plays a pivotal role in bond formation by the mother; however, the underlying neural mechanisms for infants have not yet been clarified. The comparative understanding of cognitive functions of mother and infants may help us understand the biological significance of mother-infant communication in mammalian species.

Oxytocin and mutual communication in mother-infant bonding.

Mother-infant bonding is universal to all mammalian species. In this review, we describe the manner in which reciprocal communication between the mother and infant leads to mother-infant bonding in rodents. In rats and mice, mother-infant bond formation is reinforced by various social stimuli, such as tactile stimuli and ultrasonic vocalizations (USVs) from the pups to the mother, and feeding and tactile stimulation from the mother to the pups. Some evidence suggests that mother and infant can develop a cross-modal sensory recognition of their counterpart during this bonding process. Neurochemically, oxytocin in the neural system plays a pivotal role in each side of the mother-infant bonding process, although the mechanisms underlying bond formation in the brains of infants has not yet been clarified. Impairment of mother-infant bonding, that is, deprivation of social stimuli from the mother, strongly influences offspring sociality, including maternal behavior toward their own offspring in their adulthood, implying a "non-genomic transmission of maternal environment," even in rodents. The comparative understanding of cognitive functions between mother and infants, and the biological mechanisms involved in mother-infant bonding may help us understand psychiatric disorders associated with mother-infant relationships.

A role for strain differences in waveforms of ultrasonic vocalizations during male-female interaction.

Male mice emit ultrasonic vocalizations (USVs) towards females during male-female interaction. It has been reported that USVs of adult male mice have the capability of attracting females. Although the waveform pattern of USVs is affected by genetic background, differences among strains with respect to USV and the effects of these differences on courtship behavior have not been analyzed fully. We analyzed USV patterns, as well as actual social behavior during USV recording, in 13 inbred mouse strains, which included laboratory and wild-derived strains. Significant effects of strain were observed for the frequency of USV emission, duration, and frequency of the waveform category. Principal component (PC) analysis showed that PC1 was related to frequency and duration, and PC2-4 were related to each waveform. In the comparison of USV patterns and behaviors among strains, wild-derived KJR mice displayed the highest scores for PC2-4, and female mice paired with KJR males did not emit rejection-related click sounds. It is assumed that the waveforms emitted by KJR males have a positive effect in male-female interaction. Therefore, we extracted waveforms in PC2-4 from the USV recordings of KJR mice to produce a sound file, "HIGH2-4". As a negative control, another sound file ("LOW2-4") was created by extracting waveforms in PC2-4 from strains with low scores for these components. In the playback experiments using these sound files, female mice were attracted to the speaker that played HIGH2-4 but not the speaker that played LOW2-4. These results highlight the role of strain differences in the waveforms of male USVs during male-female interaction. The results indicated that female mice use male USVs as information when selecting a suitable mate.

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.