Chronic exposure of female mice to selective serotonin reuptake inhibitors during lactation induces vocal behavior deficits in pre-weaned offspring.

Developmental factors for autism spectrum disorders (ASDs) have been an ongoing debate despite an increasing number of reports on genetic factors. Recent studies have suggested maternal intake of selective serotonin reuptake inhibitors (SSRIs) as a possible developmental factor elevating the risk for ASD in offspring. Here, we show that maternal exposure of mice to an SSRI, Fluoxetine (FLX), induces abnormal ultrasonic vocalizations (USVs), an indicator of ASD-related behavior. We tested the effect of FLX intake during pregnancy, lactation, or both. We found that the lactation and both conditions decreased the number of USVs emitted by offspring pups. An index for assessing the syllables' frequency modulation revealed that highly modulated syllables appeared to be inhibited only in both conditions. Furthermore, we found that the number of serotonergic neurons at adulthood was reduced in the progeny of mice treated with FLX in all conditions. In addition, maternal exposure to FLX through pregnancy and lactation induced a high death rate of early post-natal pups. These suggest that the maternal exposure to SSRIs affects early development of offsprings as well as the serotonergic system. Focusing on vocal communication, our results indicate that intake of an SSRI during lactation increases the risk of abnormal USVs in pups, and provides potential insights into the development of ASD.

Acoustic camera system for measuring ultrasound communication in mice.

To investigate biological mechanisms underlying social behaviors and their deficits, social communication via ultrasonic vocalizations (USVs) in mice has received considerable attention as a powerful experimental model. The advances in sound localization technology have facilitated the analysis of vocal interactions between multiple mice. However, existing sound localization systems are built around distributed-microphone arrays, which require a special recording arena and long processing time. Here, we report a novel acoustic camera system, USVCAM, which enables simpler and faster USV localization and assignment. The system comprises recently developed USV segmentation algorithms with a modification for overlapping vocalizations that results in high accuracy. Using USVCAM, we analyzed USV communications in a conventional home cage, and demonstrated novel vocal interactions in female ICR mice under a resident-intruder paradigm. The extended applicability and usability of USVCAM may facilitate future studies investigating typical and atypical vocal communication and social behaviors, as well as the underlying mechanisms.

Advanced paternal age diversifies individual trajectories of vocalization patterns in neonatal mice.

Infant crying is a communicative behavior impaired in neurodevelopmental disorders (NDDs). Because advanced paternal age is a risk factor for NDDs, we performed computational approaches to evaluate how paternal age affected vocal communication and body weight development in C57BL/6 mouse offspring from young and aged fathers. Analyses of ultrasonic vocalization (USV) consisting of syllables showed that advanced paternal age reduced the number and duration of syllables, altered the syllable composition, and caused lower body weight gain in pups. Pups born to young fathers had convergent vocal characteristics with a rich repertoire, whereas those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Additional analyses revealed that some pups from aged fathers displayed atypical USV trajectories. Thus, our study indicates that advanced paternal age has a significant effect on offspring's vocal development. Our computational analyses are effective in characterizing altered individual diversity.

Sexual excitation induces courtship ultrasonic vocalizations and cataplexy-like behavior in orexin neuron-ablated male mice.

Cataplexy is triggered by laughter in humans and palatable food in mice. To further evaluate mice's cataplexy, we examined courtship behavior in orexin neuron-ablated mice (ORX-AB), one of the animal models of narcolepsy/cataplexy. Wild-type female mice were placed into the home cage of male ORX-AB and cataplexy-like behavior was observed along with ultrasonic vocalizations (USVs), also known as the "love song". ORX-AB with a female encounter showed cataplexy-like behavior both during the dark and light periods, whereas ORX-AB with chocolate predominantly showed it during the dark period. During the light period observation, more than 85% of cataplexy-like bouts were preceded by USVs. A strong positive correlation was observed between the number of USVs and cataplexy-like bouts. Cataplexy-like behavior in narcoleptic mice is a good behavioral measure to study the brain mechanisms behind positive emotion because they can be induced by different kinds of positive stimuli, including chocolate and female courtship.

AUTS2 Regulation of Synapses for Proper Synaptic Inputs and Social Communication.

Impairments in synapse development are thought to cause numerous psychiatric disorders. Autism susceptibility candidate 2 (AUTS2) gene has been associated with various psychiatric disorders, such as autism and intellectual disabilities. Although roles for AUTS2 in neuronal migration and neuritogenesis have been reported, its involvement in synapse regulation remains unclear. In this study, we found that excitatory synapses were specifically increased in the Auts2-deficient primary cultured neurons as well as Auts2 mutant forebrains. Electrophysiological recordings and immunostaining showed increases in excitatory synaptic inputs as well as c-fos expression in Auts2 mutant brains, suggesting that an altered balance of excitatory and inhibitory inputs enhances brain excitability. Auts2 mutant mice exhibited autistic-like behaviors including impairments in social interaction and altered vocal communication. Together, these findings suggest that AUTS2 regulates excitatory synapse number to coordinate E/I balance in the brain, whose impairment may underlie the pathology of psychiatric disorders in individuals with AUTS2 mutations.

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.

Sex differences in vocalizations to familiar or unfamiliar females in mice.

Mice, both wild and laboratory strains, emit ultrasound to communicate. The sex differences between male to female (male-female) and female to female (female-female) ultrasonic vocalizations (USVs) have been discussed for decades. In the present study, we compared the number of USVs emitted to familiar and unfamiliar females by both males (male-female USVs) and females (female-female USVs). We found that females vocalized more to unfamiliar than to familiar females. By contrast, males exhibited more USVs to familiar partners. This sexually dimorphic behaviour suggests that mice change their vocal behaviour in response to the social context, and their perception of the context is based on social cognition and memory. In addition, because males vocalized more to familiar females, USVs appear to be not only a response to novel objects or individuals, but also a social response.

Effect of Sociosexual Experience and Aging on Number of Courtship Ultrasonic Vocalizations in Male Mice.

Sexual behaviors are instinctually exhibited without prior training, but they are modulated by experience. One of the precopulatory behaviors in adult male mice, courtship ultrasonic vocalizations (USVs), has attracted considerable academic attention recently. Male mice emit ultrasounds as courtship behavior when encountering females. However, the modulatory effects of experience on USVs remain unclear. In the present study, we aimed to clarify the effects of sociosexual experience and aging on adult male vocalizations. First, we examined the effect of aging. The number of USVs decreased in an age-dependent manner. Following this, young adult male mice were co-housed for two weeks with normal female mice or ovariectomized (OVX) female mice, or housed without female mice, and the number of courtship USVs before and after co-housing were compared. In males housed with normal or OVX females, USVs increased significantly after co-housing. In contrast, males housed without females did not exhibit a significant increase of USVs. A facilitative effect of co-housing with female mice on vocalizations was also observed in aged males. In addition, females used as co-housing partners became pregnant, and the reproductive rate may be related to the vocal activity observed in the partnered males. These results indicate that sociosexual experience and aging affect vocalization activity, which may be related to courtship and/or reproductive function.

Impairment of interstrain social recognition during territorial aggressive behavior in oxytocin receptor-null mice.

In humans, oxytocin has been shown to be involved in in-group cooperative behaviors and out-group aggression. Studies have also demonstrated that oxytocin plays a pivotal role in social recognition. However, no empirical research has investigated the effect of oxytocin on in-group and out-group aggressiveness. We employed a resident-intruder paradigm to assess the ability of resident male mice to discriminate intruder male strain differences. We found that resident male mice exhibited higher frequencies of attack bites against intruders of different strains than against intruders of their own strain. Subsequently, we examined whether the interstrain recognition was regulated by the oxytocin system using oxytocin receptor (OTR)-null mice. OTR wild-type or heterozygous residents displayed higher aggression toward intruders of a strain different from their own (C57BL/6J). On the other hand, OTR-null residents exhibited greater aggression toward intruders of the same strain compared to OTR wild-type or heterozygous residents, and aggression levels were not different compared to those exhibited toward other strains. Our findings demonstrated that the oxytocin system contributes to interstrain social recognition in territorial aggression in male mice, implying that one function of oxytocin is to promote an in-group "tend-and-defend" response, such as in-group favoritism, which could be evolutionarily conserved in mammals.

IL1RAPL1 knockout mice show spine density decrease, learning deficiency, hyperactivity and reduced anxiety-like behaviours.

IL-1 receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic intellectual disability and is associated with autism. IL1RAPL1 mediates excitatory synapse formation through trans-synaptic interaction with PTPδ. Here, we showed that the spine density of cortical neurons was significantly reduced in IL1RAPL1 knockout mice. The spatial reference and working memories and remote fear memory were mildly impaired in IL1RAPL1 knockout mice. Furthermore, the behavioural flexibility was slightly reduced in the T-maze test. Interestingly, the performance of IL1RAPL1 knockout mice in the rotarod test was significantly better than that of wild-type mice. Moreover, IL1RAPL1 knockout mice consistently exhibited high locomotor activity in all the tasks examined. In addition, open-space and height anxiety-like behaviours were decreased in IL1RAPL1 knockout mice. These results suggest that IL1RAPL1 ablation resulted in spine density decrease and affected not only learning but also behavioural flexibility, locomotor activity and anxiety.

Enhanced prepulse inhibition and low sensitivity to a dopamine agonist in HESR1 knockout mice.

Transcription factor Hesr family genes are important in neuronal development. We demonstrated previously that HESR1 and HESR2 modified expression of the dopamine transporter (DAT) reporter gene. HESR-family genes have been investigated in development, but their functions, especially in relation to behaviors regulated by dopamine, in adult animals remain unclear. In the present study, we investigated the effects of Hesr1 and Hesr2 on behavior. A behavioral test battery to examine spontaneous activity, anxiety-like behavior, aggressive behavior, pain sensitivity, and sensorimotor gating was conducted in Hesr1 and Hesr2 knockout (KO) mice. Enhanced prepulse inhibition (PPI), which is a form of sensorimotor gating, was observed in only Hesr1 KO mice; other behavioral traits were mostly comparable to wild-type animals in both the Hesr1 and the Hesr2 KO lines. Next, we used a dopamine agonist, apomorphine, to confirm the involvement of the dopaminergic system. Injection of apomorphine reduced the enhanced PPI in Hesr1 KO mice. Additionally, dose-dependent sensitivity to the agonist was lower in the Hesr1 KO mice than in wild-type mice, suggesting that the enhanced PPI resulted from this alteration in dopamine sensitivity. Furthermore, DAT mRNA was downregulated in Hesr1 KO mice, whereas the dopamine D1 and D2 receptors were comparable. These findings suggest Hesr1 to be a novel factor that affects dopamine sensitivity and the sensorimotor gating system.

The androgen receptor facilitates inhibition of human dopamine transporter (DAT1) reporter gene expression by HESR1 and HESR2 via the variable number of tandem repeats.

A functional genetic polymorphism in the 3'-untranslated region (UTR) within exon 15 of the human DAT gene (DAT1) has been described. This 3'-UTR contains a variable number of tandem repeats (VNTR) 40 bp in length; many association studies of psychiatric or developmental disorders with this VNTR have been conducted. We previously demonstrated that HESR1 (the Hairy/enhancer of split related transcriptional factor 1 with YRPW motif) and HESR2 reduced DAT reporter gene expression via this 3'-UTR. VNTR allele-dependent altered reporter gene expression was also observed. In the present study, we wanted to clarify the molecular characterization of HESR1 and HESR2, focusing on its cis-element and co-factor. Deletion of the VNTR domain increased reporter gene expression both with and without transfection of HESRs, suggesting that the VNTR inhibits DAT expression, and is responsive to HESRs. In the presence of transfected androgen receptor (AR), activity of the luciferase reporter with the nine-repeat allele (9r) decreased, while that with the ten-repeat allele (10r), the most frequent in the population, increased significantly. Furthermore, co-expression of HESR1 or HESR2 with AR increased the inhibitory effect of the HESRs. Our data indicate that a functional modification occurs when the HESRs are coupled with AR. This HESR-AR interaction could be the molecular basis of sexual dimorphisms in DAT expression, or other dopamine-related behavioral traits.

Differential effects of the HESR/HEY transcription factor family on dopamine transporter reporter gene expression via variable number of tandem repeats.

The 3'-untranslated region (UTR) of the human dopamine transporter (DAT1) gene contains a variable number of tandem repeats (VNTR) domain, which is thought to be associated with dopamine-related psychiatric disorders, personality, and behavior. However, the molecular and neuronal functions of polymorphisms within the VNTR domain are unknown. We previously identified the transcription factor HESR1 (HEY1) as a VNTR-binding protein. Hesr1 knockout mice exhibit DAT up-regulation in the brain and low levels of spontaneous activity. Other members of the HESR (HEY) family, including HESR2 (HEY2) and 3 (HEYL), have similar DNA-binding domains. In this study, we analyzed the effects of HESR1, -2, and -3 on DAT1 expression in human neuroblastoma SH-SY5Y cells using luciferase reporter assays. We found that the VNTR domain played an inhibitory role in DAT1 reporter gene expression and that HESR1 and -2 inhibited expression via both the core promoter and the VNTR. The inhibitory effects of HESR family members on DAT reporter gene expression differed depending on the number of repeats in the VNTR domain. We also found that each Hesr was expressed in the dopaminergic neurons in the mouse midbrain. These results suggest that the HESR family is involved in DAT expression via the VNTR domain.

Ipsilateral and contralateral serotonergic projections from dorsal and median raphe nuclei to the forebrain in rats: immunofluorescence quantitative analysis.

The objective of this study was to clarify the 5-HT projections from the right and left sides of the dorsal (DRD), ventral (DRV) and lateral (DRL) subdivisions of the middle level of the dorsal (DR) raphe nucleus and median (MR) raphe nucleus to the lateral septum (LS), preoptic area (POA) or ventromedial hypothalamus (VMH), which are important neural substrates for neuroendocrine regulation of reproduction. A retrograde neural tracer, Fluoro-Gold (FG), was infused into the right side of these regions in ovariectomized rats and the numbers of FG and/or 5-HT immunopositive cells in the right and left sides of the raphe nuclei were counted. It was found that the POA and VMH received more 5-HT projections than the LS from the DR and MR. In the subdivisions of the DR, 70% of all 5-HT projections from the DR to these 3 areas originated from the DRL. Furthermore, ipsilateral projections from the DR to the POA and VMH but not to the LS were dominant, compared to the contralateral projections. A right-left difference was not seen among the MR 5-HT projections. Thus, laterality of the projections is thought to be strong in the 5-HT clusters located far from the midline of the midbrain raphe nuclei.