Beacon-based sleep-wake monitoring in dogs.

The sleep-wake cycle represents a crucial physiological process essential for maintaining homeostasis and promoting individual growth. In dogs, alterations in sleep patterns associated with age and dog's correlation with temperament factors, such as nervousness, have been reported, and there is an increasing demand for precise monitoring of sleep and physical activity in dogs. The present study aims to develop an analysis method for measuring sleep-wake patterns and physical activity in dogs by utilizing an accelerometer and a smartphone. By analyzing time series data collected from the accelerometer attached to the dog's collar, a comprehensive sleep and activity analysis model was constructed. This model classified the activity level into seven classes and effectively highlighted the variations in sleep-activity patterns. Two classes with lower activity levels were considered as sleep, while other five levels were regarded as wake based on the rate of occurrence. This protocol of data acquisition and analysis provides a methodology that enables accurate and extended evaluation of both sleep and physical activity in dogs.

Long-term monitoring of huddling behavior in mice using online image processing.

Many animal species, including mice, form societies of numerous individuals for survival. Understanding the interactions between individual animals is crucial for elucidating group behavior. One such behavior in mice is huddling, yet its analysis has been limited. In this study, we propose a cost-effective method for monitoring long-term huddling behavior in mice using online image processing with OpenCV. This method treats a single mouse or a group of mice as a cluster of pixels (a 'blob') in video images, extracting and saving only essential information such as areas, coordinates, and orientations. This approach reduces data storage needs to 1/200000th of what would be required if the video were recorded in its compressed form, thereby enabling long-term behavioral analysis. To validate the performance of our algorithm, ~2000 video frames were randomly chosen. We manually counted the number of clusters of mice in these frames and compared them with the number of blobs automatically detected by the algorithm. The results indicated a high level of consistency, exceeding 90% across the selected video frames. Initial observations of both male and female groups suggested some variations in huddling behavior among male and female groups; however, these results should be interpreted cautiously due to a small sample. Group behavior is known to be disrupted in several neuropsychiatric disorders, such as autism. Various mouse models of these disorders have been proposed. Our measurement system, when combined with drug or genetic modification screening, could provide a valuable tool for high-throughput analyses of huddling behavior.

Structural alterations in the amygdala and impaired social incentive learning in a mouse model of a genetic variant associated with neurodevelopmental disorders.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and their dimensions and changes in brain structures and behavior. However, as CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how the genes in the 22q11.2 region individually contribute to structural alterations and associated mental illnesses and their dimensions. Our previous studies have identified Tbx1, a T-box family transcription factor encoded in 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes in congenic Tbx1 heterozygous mice. Our data show that the volumes of anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were reduced in Tbx1 heterozygous mice. Moreover, we examined the behavioral consequences of an altered volume of the amygdala. Tbx1 heterozygous mice were impaired for their ability to detect the incentive value of a social partner in a task that depends on the amygdala. Our findings identify the structural basis for a specific social dimension associated with loss-of-function variants of TBX1 and 22q11.2 CNV.

Structural alterations in the amygdala and impaired social incentive learning in a mouse model of a genetic variant associated with neurodevelopmental disorders.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and their dimensions and changes in brain structures and behavior. However, as CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how the genes in the 22q11.2 region individually contribute to structural alterations and associated mental illnesses and their dimensions. Our previous studies have identified Tbx1, a T-box family transcription factor encoded in 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes in congenic Tbx1 heterozygous mice. Our data show that the volumes of anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were reduced in Tbx1 heterozygous mice. Moreover, we examined the behavioral consequences of an altered volume of the amygdala. Tbx1 heterozygous mice were impaired for their ability to detect the incentive value of a social partner in a task that depends on the amygdala. Our findings identify the structural basis for a specific social dimension associated with loss-of-function variants of TBX1 and 22q11.2 CNV.

Chronic corticosterone deteriorates latrine and nesting behaviours in mice.

Self-care behaviours are actions that help maintain good health and surroundings. For example, appropriate toileting, sleeping in the bed, and bathing and washing are among self-care behaviours in humans. Animals also perform similar self-care behaviours such as latrine, nesting and self-grooming. Studies have shown that chronic stress disrupts nesting and self-grooming behaviours. However, the effect of chronic stress on latrine behaviour, preferential, repeated defecation at specific locations, has not yet been clarified. This study aimed to investigate the influence of chronic corticosterone administration on latrine and nesting behaviours in mice. The variation in defecation location was quantified as the degree of the latrine behaviour by using Shannon entropy. The nest quality was scored based on shape. The study showed that mice exposed to chronic corticosterone had scattered defecation sites and lower nest quality compared to the control group. Furthermore, results showed that more scattered defecation behaviour was associated with lower nest quality at an individual level. Additionally, the deterioration of these self-care behaviours was associated with depression-like behaviours such as less open field activity and increased immobility time during the tail suspension test. These results suggest that chronic corticosterone deteriorates self-care behaviours such as latrine and nesting in mice.

Prelimbic cortex responds to male ultrasonic vocalizations in the presence of a male pheromone in female mice.

Sensory signals are critical to perform adaptive social behavior. During copulation, male mice emit ultrasonic vocalizations (USVs). Our previous studies have shown that female mice exhibit approach behavior toward sound sources of male USVs and that, after being exposed to a male pheromone, exocrine gland-secreting peptide 1 (ESP1), female mice exhibited a preference toward a particular type of male USVs. These findings suggest that male USVs modulate female courtship behavior. However, it remains unclear which brain regions and what cell types of neurons are involved in neuronal processing of male USVs. To clarify this issue, immediate early gene analysis, behavioral analysis, and neurochemical analysis were performed. The in situ hybridization analysis of c-fos mRNA in multiple brain regions showed that neurons in the prelimbic cortex were responsive to presentation of male USVs in the presence of ESP1. Furthermore, this study found that activity of prelimbic cortex was correlated with the duration of female exploration behavior toward a sound source of the USVs. Finally, by using double immunohistochemistry, the present study showed that the prelimbic neurons responding to the presentation of male USVs were presumably excitatory glutamatergic neurons. These results suggest that the prelimbic cortex may facilitate female courtship behavior in response to male USVs.

Humanized substitutions of Vmat1 in mice alter amygdala-dependent behaviors associated with the evolution of anxiety.

The human vesicular monoamine transporter 1 (VMAT1) harbors unique substitutions (Asn136Thr/Ile) that affect monoamine uptake into synaptic vesicles. These substitutions are absent in all known mammals, suggesting their contributions to distinct aspects of human behavior modulated by monoaminergic transmissions, such as emotion and cognition. To directly test the impact of these human-specific mutations, we introduced the humanized residues into mouse Vmat1 via CRISPR/Cas9-mediated genome editing and examined changes at the behavioral, neurophysiological, and molecular levels. Behavioral tests revealed reduced anxiety-related traits of Vmat1 Ile mice, consistent with human studies, and electrophysiological recordings showed altered oscillatory activity in the amygdala under anxiogenic conditions. Transcriptome analyses further identified changes in gene expressions in the amygdala involved in neurodevelopment and emotional regulation, which may corroborate the observed phenotypes. This knock-in mouse model hence provides compelling evidence that the mutations affecting monoaminergic signaling and amygdala circuits have contributed to the evolution of human socio-emotional behaviors.

Microendoscopic calcium imaging of the primary visual cortex of behaving macaques.

In vivo calcium imaging with genetically encoded indicators has recently been applied to macaque brains to monitor neural activities from a large population of cells simultaneously. Microendoscopic calcium imaging combined with implantable gradient index lenses captures neural activities from deep brain areas with a compact and convenient setup; however, this has been limited to rodents and marmosets. Here, we developed miniature fluorescent microscopy to image neural activities from the primary visual cortex of behaving macaques. We found tens of clear fluorescent signals from three of the six brain hemispheres. A subset of these neurons showed clear retinotopy and orientation tuning. Moreover, we successfully decoded the stimulus orientation and tracked the cells across days. These results indicate that microendoscopic calcium imaging is feasible and reasonable for investigating neural circuits in the macaque brain by monitoring fluorescent signals from a large number of neurons.

Testosterone Increases the Emission of Ultrasonic Vocalizations With Different Acoustic Characteristics in Mice.

Testosterone masculinizes male sexual behavior through an organizational and activational effects. We previously reported that the emission of ultrasonic vocalizations (USVs) in male mice was dependent on the organizational effects of testosterone; females treated with testosterone in the perinatal and peripubertal periods, but not in adults, had increased USV emissions compared to males. Recently, it was revealed that male USVs have various acoustic characteristics and these variations were related to behavioral interactions with other mice. In this regard, the detailed acoustic characteristic changes induced by testosterone have not been fully elucidated. Here, we revealed that testosterone administered to female and male mice modulated the acoustic characteristics of USVs. There was no clear difference in acoustic characteristics between males and females. Call frequencies were higher in testosterone propionate (TP)-treated males and females compared to control males and females. When the calls were classified into nine types, there was also no distinctive difference between males and females, but TP increased the number of calls with a high frequency, and decreased the number of calls with a low frequency and short duration. The transition analysis by call type revealed that even though there was no statistically significant difference, TP-treated males and females had a similar pattern of transition to control males and females, respectively. Collectively, these results suggest that testosterone treatment can enhance the emission of USVs both in male and female, but the acoustic characteristics of TP-treated females were not the same as those of intact males.

Hypothalamic perifornical Urocortin-3 neurons modulate defensive responses to a potential threat stimulus.

Defensive behaviors are evolved responses to threat stimuli, and a potential threat elicits risk assessment (RA) behavior. However, neural mechanisms underlying RA behavior are hardly understood. Urocortin-3 (Ucn3) is a member of corticotropin-releasing factor peptide family and here, we report that Ucn3 neurons in the hypothalamic perifornical area (PeFA) are involved in RA of a novel object, a potential threat stimulus, in mice. Histological and in vivo fiber photometry studies revealed that the activity of PeFA Ucn3 neurons was associated with novel object investigation involving the stretch-attend posture, a behavioral marker for RA. Chemogenetic activation of these neurons increased RA and burying behaviors toward a novel object without affecting anxiety and corticosterone levels. Ablation of these neurons caused the abnormal behaviors of gnawing and direct contacts with novel objects, especially in a home-cage. These results suggest that PeFA Ucn3 neurons modulate defensive responses to a potential threat stimulus.

Testosterone regulates the emission of ultrasonic vocalizations and mounting behavior during different developmental periods in mice.

Testosterone masculinizes male sexual behavior by providing organizational and activational effects during the perinatal and peripubertal periods and during adulthood, respectively. We revealed that the emission of ultrasonic vocalizations (USVs) and mounting behavior was regulated by different neural circuits. However, the detailed testosterone effects on these two behaviors have not been fully elucidated. Here, we evaluated the time-dependent effects of testosterone on USVs and mounting behavior in mice using a testosterone treatment model, in which females were treated with testosterone to assess the "gain-of-function" and a "loss-of-function" model. In the loss-of-function model, we used Ad4BP/SF-1ΔFLC/- male mice, in which testosterone production was abolished in prenatal and postnatal stages, and Ad4BP/SF-1ΔFLC/ΔFLC mice, in which testosterone production was markedly reduced only in prenatal stages. When testosterone was administered to female mice during the neonatal and peripubertal periods, but not during adulthood, USV emissions increased. Conversely, testosterone treatment in adult female mice increased the mounting behavior, but not USVs. In Ad4BP/SF-1ΔFLC/- mice, USVs and mounting behavior was completely absent. Ad4BP/SF-1ΔFLC/ΔFLC male mice displayed equivalent levels of USVs but less mounting behavior. Collectively, these results suggest that testosterone has dual regulatory roles in USV emissions and mounting behavior.

Maternal approach behaviors toward neonatal calls are impaired by mother's experiences of raising pups with a risk gene variant for autism.

How the intrinsic sequence structure of neonatal mouse pup ultrasonic vocalization (USV) and maternal experiences determine maternal behaviors in mice is poorly understood. Our previous work showed that pups with a Tbx1 heterozygous (HT) mutation, a genetic risk for autism spectrum disorder (ASD), emit altered call sequences that do not induce maternal approach behaviors in C57BL6/J mothers. Here, we tested how maternal approach behaviors induced by wild-type and HT USVs are influenced by the mother's experience in raising pups of these two genotypes. The results showed that wild-type USVs were effective in inducing maternal approach behaviors when mothers raised wild-type but not HT pups. The USVs of HT pups were ineffective regardless of whether mothers raised HT or wild-type pups. However, the sequence structure of pup USVs had no effect on the general, non-directional incentive motivation of maternal behaviors. Our data show how the mother's experience with a pup with a genetic risk for ASD alters the intrinsic incentive values of USV sequences in maternal approach behaviors.

Differential Encoding of Predator Fear in the Ventromedial Hypothalamus and Periaqueductal Grey.

The ventromedial hypothalamus is a central node of the mammalian predator defense network. Stimulation of this structure in rodents and primates elicits abrupt defensive responses, including flight, freezing, sympathetic activation, and panic, while inhibition reduces defensive responses to predators. The major efferent target of the ventromedial hypothalamus is the dorsal periaqueductal gray (dPAG), and stimulation of this structure also elicits flight, freezing, and sympathetic activation. However, reversible inhibition experiments suggest that the ventromedial hypothalamus and periaqueductal gray play distinct roles in the control of defensive behavior, with the former proposed to encode an internal state necessary for the motivation of defensive responses, while the latter serves as a motor pattern initiator. Here, we used electrophysiological recordings of single units in behaving male mice exposed to a rat to investigate the encoding of predator fear in the dorsomedial division of the ventromedial hypothalamus (VMHdm) and the dPAG. Distinct correlates of threat intensity and motor responses were found in both structures, suggesting a distributed encoding of sensory and motor features in the medial hypothalamic-brainstem instinctive network.SIGNIFICANCE STATEMENT Although behavioral responses to predatory threat are essential for survival, the underlying neuronal circuits remain undefined. Using single unit in vivo electrophysiological recordings in mice, we have identified neuronal populations in the medial hypothalamus and brainstem that encode defensive responses to a rat predator. We found that both structures encode both sensory as well as motor aspects of the behavior although with different kinetics. Our findings provide a framework for understanding how innate sensory cues are processed to elicit adaptive behavioral responses to threat and will help to identify targets for the pharmacological modulation of related pathologic behaviors.

Sexual imprinting overrides order effects during sampling of prospective mates.

Through crossfostering experiments between two subspecies of mice, Moreira et al. show that females normally undergo sexual imprinting early in life. When fostered by individuals from another subspecies, they tend to prefer males from the sub-species they first encounter, suggesting sexual imprinting normally over-rides this inclination.

Female C57BL/6 and BALB/c mice differently use the acoustic features of male ultrasonic vocalizations for social preferences.

Male mice emit ultrasonic vocalizations (USVs) in response to the presence of female mice and their urine. Male USVs attract females, enhancing female reproductive functions, and are thus considered as the courtship song. Previous studies have shown that female mice exhibit disassortative social preferences for male USVs. However, it remains unclear what acoustic features female mice use for the development of these preferences. To address this, we examined social preferences of female C57BL/6 and BALB/c mice using the three-chamber preference test using recorded male USVs. To dissociate the peak frequencies of these USVs from their syllable structure, we digitally manipulated the peak frequencies accordingly. We found that female mice preferred USVs that were dissimilar to those of their own strain. We also observed that, while female C57BL/6 mice were sensitive to changes in the syllable structure and the peak frequency, female BALB/c mice were sensitive to differences in the syllable structure. Our results demonstrate that female C57BL/6 and BALB/c mice differently use the acoustic features such as the peak frequency and the syllable structure for exhibiting disassortative social preferences.

Endocrine Regulations in Human-Dog Coexistence through Domestication.

Endocrine system regulation is important for the maintenance of homeostasis; it controls hormonal functions in complex physiology and behavior and adaptations to social environments. Evidence indicates that for more than 35 000 years, dogs (Canis familiaris) have been domesticated through living with humans. For example, they have acquired human-like social skills, such as eye gazing and pointing gestures. These unique behaviors are, at least partially, regulated by hormones and are thought to have been genetically altered throughout domestication. Glucocorticoids affect social tolerance, while oxytocin facilitates social coordination and familiarity between individuals. We review historical and recent literature to facilitate an understanding of the roles of glucocorticoid and oxytocin functions in the human-canine coexistence dynamic established during domestication.

Emotional Contagion From Humans to Dogs Is Facilitated by Duration of Ownership.

Emotional contagion is a primitive form of empathy that does not need higher psychological functions. Recent studies reported that emotional contagion exists not only between humans but also among various animal species. The dog (Canis familiaris) is a unique animal and the oldest domesticated species. Dogs have coexisted with humans for more than 30,000 years and are woven into human society as partners bonding with humans. Dogs have acquired human-like communication skills and, likely as a result of the domestication process, the ability to read human emotions; therefore, it is feasible that there may be emotional contagion between human and dogs. However, the higher time-resolution of measurement of emotional contagion between them is yet to be conducted. We assessed the emotional reactions of dogs and humans by heart rate variability (HRV), which reflects emotion, under a psychological stress condition on the owners. The correlation coefficients of heart beat (R-R) intervals (RRI), the standard deviations of all RR intervals (SDNN), and the square root of the mean of the sum of the square of differences between adjacent RR intervals (RMSSD) between dogs and owners were positively correlated with the duration of dog ownership. Dogs' sex also influenced the correlation coefficients of the RRI, SDNN, and RMSSD in the control condition; female showed stronger values. These results suggest that emotional contagion from owner to dog can occur especially in females and the time sharing the same environment is the key factor in inducing the efficacy of emotional contagion.

Female mice exhibit both sexual and social partner preferences for vocalizing males.

Acoustic signals are widely used as courtship signals in the animal kingdom. It has long been known that male mice emit ultrasonic vocalizations (USVs) in the presence of female mice or in response to female secretions. This observation led to the hypothesis that male USVs play a role in courtship behavior. Although previous studies showed that female mice have a social partner preference for vocalizing males, it is not known if they exhibit a sexual partner preference when given a choice. To address this issue, we examined the copulatory behaviors of female mice with either devocalized males (with or without the playback of the USVs) or sham-operated males in 2 different behavioral paradigms: the no-choice paradigm in the home cage of a male mouse (without choice of mating partners) or the mate-choice paradigm in a 3-chambered apparatus (with choice of mating partners). In the no-choice paradigm, female mice exhibited comparable sexual receptivity with sham-operated and devocalized males. In addition, we found that female mice showed more approach behavior towards devocalized males when male USVs were played back. In the mate-choice paradigm, female mice visited more frequently and stayed longer with sham-operated than devocalized males. Furthermore, we showed that female mice received more intromissions from sham-operated males than devocalized males. In summary, our results suggested that, although female mice can copulate equally with both devocalized and vocalizing males when given no choice of mating partner, female mice exhibit both sexual and social partner preferences for vocalizing males in the mate-choice paradigm.

Midbrain Dopamine Neurons Signal Belief in Choice Accuracy during a Perceptual Decision.

Central to the organization of behavior is the ability to predict the values of outcomes to guide choices. The accuracy of such predictions is honed by a teaching signal that indicates how incorrect a prediction was ("reward prediction error," RPE). In several reinforcement learning contexts, such as Pavlovian conditioning and decisions guided by reward history, this RPE signal is provided by midbrain dopamine neurons. In many situations, however, the stimuli predictive of outcomes are perceptually ambiguous. Perceptual uncertainty is known to influence choices, but it has been unclear whether or how dopamine neurons factor it into their teaching signal. To cope with uncertainty, we extended a reinforcement learning model with a belief state about the perceptually ambiguous stimulus; this model generates an estimate of the probability of choice correctness, termed decision confidence. We show that dopamine responses in monkeys performing a perceptually ambiguous decision task comply with the model's predictions. Consequently, dopamine responses did not simply reflect a stimulus' average expected reward value but were predictive of the trial-to-trial fluctuations in perceptual accuracy. These confidence-dependent dopamine responses emerged prior to monkeys' choice initiation, raising the possibility that dopamine impacts impending decisions, in addition to encoding a post-decision teaching signal. Finally, by manipulating reward size, we found that dopamine neurons reflect both the upcoming reward size and the confidence in achieving it. Together, our results show that dopamine responses convey teaching signals that are also appropriate for perceptual decisions.

Enhanced male-evoked responses in the ventromedial hypothalamus of sexually receptive female mice.

Social encounters often start with routine investigatory behaviors before developing into distinct outcomes, such as affiliative or aggressive actions. For example, a female mouse will initially engage in investigatory behavior with a male but will then show copulation or rejection, depending on her reproductive state. To promote adaptive social behavior, her brain must combine internal ovarian signals and external social stimuli, but little is known about how socially evoked neural activity is modulated across the reproductive cycle [1]. To investigate this, we performed single-unit recordings in the ventrolateral region of the ventromedial hypothalamus (VMHvl) in freely behaving, naturally cycling, female mice interacting with conspecifics of both genders. The VMHvl has been implicated in rodent sociosexual behavior [2, 3]: it has access to social sensory stimuli [4-8] and is involved in aggression and mating [9-11]. Furthermore, many VMHvl neurons express ovarian hormone receptors [12, 13], which play a central role in female sociosexual behavior [14-16]. We found that a large fraction of VMHvl neurons was activated in the presence of conspecifics with preference to male stimuli and that the activity of most VMHvl neurons was modulated throughout social interactions rather than in response to specific social events. Furthermore, neuronal responses to male, but not female, conspecifics in the VMHvl were enhanced during the sexually receptive state. Thus, male-evoked VMHvl responses are modulated by the reproductive state, and VMHvl neural activity could drive gender-specific and reproductive state-dependent sociosexual behavior.