Abstract Oxytocin is a key regulator of social behavior, yet how it shapes the neural circuits underlying these behaviors remains unclear. Here, we show that oxytocin signaling is required for the proper maturation of terminal nerve gonadotropin-releasing hormone 3 (TN-GnRH3) circuits that regulate female mate preference in medaka. Disruption of oxytocin signaling reduced expression of the complement component C1q, altered microglial properties, and increased TN-GnRH3 innervation in the optic tectum and dorsolateral telencephalon, suggesting impaired synaptic refinement. In addition, TN-GnRH3 neurons in mutants exhibited disrupted clustering and failed to increase firing frequency in response to visual stimulation, indicating functional deficits. Furthermore, c1qb mutants phenocopied the behavioral and neuronal abnormalities observed in oxytocin signaling–deficient mutants. Together, our findings suggest that oxytocin signaling links microglia-associated synaptic refinement to neural circuit maturation, thereby revealing a previously unrecognized role for oxytocin in shaping social behavior.

Phenotypic plasticity allows organisms to adapt traits in response to environmental changes, yet the molecular basis by which such plastic traits become genetically fixed remains unclear. Here, we investigated gut-length plasticity in medaka fish (Oryzias latipes) through genome-wide methylation profiling, CRISPR/Cas9-mediated deletion, and population genomic analyses. We found that seasonal methylation of CpG sites upstream of the Plxnb3 is correlated with gut-length plasticity, and deletion of this region abolishes plasticity. Additionally, standing variation in Ppp3r1 is associated with genetically fixed longer gut length in populations lacking plasticity. These results suggest that loss of epigenetic regulation via CpG site reduction triggers the genetic fixation of novel traits. Our findings provide molecular evidence linking epigenetic plasticity and genetic assimilation, advancing understanding of plasticity-led evolution in natural populations.

GCaMP-based calcium imaging is a powerful tool for investigating neural function in specific neurons. We generated transgenic (Tg) medaka strains expressing jGCaMP7s across extensive brain regions under the control of the gap43 promoter. Using these Tg larvae, calcium imaging successfully detected a tricaine-induced suppression of spontaneous neural activity and topographical visual responses in the optic tectum elicited by moving paramecia or optical fiber stimulation. These results indicate that our Tg medaka strains provide a versatile platform for investigating neural dynamics and their responses to various stimuli.

Social familiarity within groups promotes behavioural synchrony and facilitates information transfer. Whether it shapes collective decision-making under predator threat is unknown. Here groups of six medaka (Oryzias latipes) familiarised for one month were used to test whether familiarisation promotes instantaneous collective decision-making in response to a looming stimulus (LS) mimicking a predator attack. First, we analysed behavioural transitions, defined as changes among three behavioural states: high-speed, normal and freezing-like before, during, and after LS in groups of six individuals. Individuals showing high-speed state in response to LS typically tended to shift to freezing-like state afterwards, whereas non-responders were more likely to maintain normal state. Group-level analysis revealed a bimodal distribution in the number of individuals exhibiting freezing-like state, with peaks at zero and six individuals, corresponding to ‘all non-freezing’ and ‘all-freezing’. Clustering analysis further identified three consistent group profiles: ‘freezing-dominant’, ‘non-freezing-dominant’, and ‘mixed-type’ based on behavioural tendencies across 10 trials. In contrast, in unfamiliar groups assembled immediately before testing, the ‘freezing-dominant’ profile was absent, and the distribution in the number of individuals exhibiting freezing-like state shifted to unimodal. In these groups, even at the individual level, responses more often showed a transition from high-speed state to normal state rather than freezing-like state. The results indicate that social familiarity promotes synchronous freezing-like state and consensus decisions under looming threat. Our study presents a behavioural assay for predator-evoked collective decision-making in a genetic model fish, providing a framework for future efforts to link behavioural ethology with neuroscience.

Spatial and temporal control of transgene expression is a powerful approach to understand gene functions in specific cells and tissues. The Tet-On system is a robust tool for controlling transgene expression spatially and temporally; however, few studies have examined whether this system can be applied to postembryonic stages of Medaka (Oryzias latipes) or other fishes. Here, we first improved a basal promoter sequence on the donor vector for a nonhomologous end joining (NHEJ)-based knock-in (KI) system. Next, using transgenic Medaka for establishing the Tet-On system by KI, we demonstrated that doxycycline administration for four or more days by feeding can be a stable and efficient method to achieve expression of the transduced reporter gene in adult fish. From these analyses, we propose an optimized approach for a spatio-temporal gene-expression system in the adult stage of Medaka and other small fishes.

Rapid body colouration changes in some animals, such as chameleons and octopuses, serve dual functions: camouflage and intraspecific communication. It has been hypothesized that these colouration changes originally evolved to provide camouflage and subsequently were co-opted as social signals; however, experimental model systems that are suitable for studying such evolutionary processes are limited. Here, we investigated the relationship between rapid colouration changes of the blackened markings and aggressive behaviours in male Oryzias celebensis , an Indonesian medaka fish, under triadic relationships (two males and one female) or three males conditions with two different environmental backgrounds. In an algae-covered tank, mimicking the common laboratory rearing conditions, males with blackened markings exhibited more frequent attacks towards different conspecific individuals compared with non-blackened males and females. The blackened males were seldom attacked by non-blackened males and females. By contrast, neither aggressive behaviours nor black colouration changes were observed in the transparent background condition with a brighter environment. These indicated that the blackened markings in O. celebensis serve as a social signal depending on the environmental backgrounds. Considering that such colouration changes for camouflage are widely conserved among teleost fishes, the traits are likely to be co-opted for displaying social signals in O. celebensis .

Previously, the integration of comparative biological and neuroscientific approaches has led to significant advancements in social neuroscience. This review highlights the potential and future directions of evolutionary social neuroscience research utilizing medaka fishes (the family Adrianichthyidae) including Japanese medaka (Oryzias latipes). We focus on medaka social cognitive capabilities and mate choice behavior, particularly emphasizing mate preference using visual cues. Medaka fishes are also advantageous due to their abundant genetic resources, extensive genomic information, and the relative ease of laboratory breeding and genetic manipulation. Here we present some research examples of both the conventional neuroscience approach and evolutionary approach involving medaka fishes and other species. We also discuss the prospects of uncovering the molecular and cellular mechanisms underlying the diversity of visual mate preference among species. Especially, we introduce that the single-cell transcriptome technology, particularly in conjunction with 'Adaptive Circuitry Census', is an innovative tool that bridges comparative biological methods and neuroscientific approaches. Evolutionary social neuroscience research using medaka has the potential to unveil fundamental principles in neuroscience and elucidate the mechanisms responsible for generating diversity in mating strategies.

Honey bees are social insects, and each colony member has unique morphological and physiological traits associated with their social tasks. Previously, we identified a long non-coding RNA from honey bees, termed Nb-1, whose expression in the brain decreases associated with the age-polyethism of workers and is detected in some neurosecretory cells and octopaminergic neurons, suggesting its role in the regulation of worker labor transition. Herein, we investigated its spatially and temporary-regulated/sex-specific expression. Nb-1 was expressed as an abundant maternal RNA during oogenesis and embryogenesis in both sexes. In addition, Nb-1 was expressed preferentially in the proliferating neuroblasts of the mushroom bodies (a higher-order center of the insect brain) in the pupal brains, suggesting its role in embryogenesis and mushroom body development. On the contrary, Nb-1 was expressed in a drone-specific manner in the pupal and adult retina, suggesting its role in the drone visual development and/or sense. Subcellular localization of Nb-1 in the brain during development differed depending on the cell type. Considering that Nb-1 is conserved only in Apidae, our findings suggest that Nb-1 potentially has pleiotropic functions in the expression of multiple developmental, behavioral, and physiological traits, which are closely associated with the honey bee lifecycle.

Aggressive behavior, exhibiting a unidirectional behavioral event across various animal species, is instrumental in fortifying the existing hierarchical order and maintaining social stability within the group. In the present study, we investigated the types of aggressive behavior exhibiting unidirectional properties that function as dominance signals among three male medaka fish (Oryzias latipes). We first examined the directionality of attack/bite and chase behaviors, revealing that males often exhibited bidirectional physical attacks, while demonstrating predominantly unidirectional chasing behavior. This directionality was maintained throughout the hierarchy formation. Upon observing that male medaka fish exhibit courtship-like displays during male-male interactions, we subsequently investigated the timing and directionality of attack/bite, chasing, and courtship-like displays. Males exhibited unidirectional courtship-like displays, with the directionality matching that of chasing behavior. In addition, the number of attack/bite behaviors significantly decreased after the first chasing as well as the first courtship-like displays. Taken together, our findings strongly suggest that chasing and courtship-like displays may function as dominance signals, reinforcing established dominance-subordinate relationships. Furthermore, when the three males were separated for half a day and then reunited the following day, the number of physical attacks decreased significantly, while the directionality of chasing behavior and courtship-like displays remained unchanged. This finding suggests that once a dominance-subordinate relationship is established, it can be maintained through chasing behavior and courtship-like displays. These observations provide insight into how unilateral pseudo-sexual behavior between males can influence hierarchy formation in social animals.

Optogenetics enables the manipulation of neural activity with high spatiotemporal resolution in genetically defined neurons. The method is widely used in various model animals in the neuroscience and physiology fields. Channelrhodopsins are robust tools for optogenetic manipulation, but they have not yet been used for studies in medaka. In the present study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knock-in approach to establish a transgenic medaka strain expressing the Chloromonas oogama channelrhodopsin (CoChR) in the ISL LIM homeobox 1 (isl1) locus. We demonstrated that light stimuli elicited specific behavioral responses, such as bending or turning locomotion in the embryos and pectoral fin movements in the larvae and adults. The response probabilities and intensities of these movements could be controlled by adjusting the intensity, duration, or wavelength of each light stimulus. Furthermore, we demonstrated that the pectoral fin movements in the adult stage could be elicited using a laser pointer to irradiate region including the caudal hind brain and the rostral spinal cord. Our results indicate that CoChR allows for manipulation of medaka behaviors by activating targeted neurons, which will further our understanding of the detailed neural mechanisms of motor control or social behaviors in medaka.

In some species, such as chameleon and cichlid fish, rapid body colouration changes (within seconds or minutes) serve as visual social signals in male-male competition. This study investigated the relationship between aggressive behaviour and body colouration changes in Oryzias celebensis , an Indonesian medaka fish. We analysed aggressive behaviours and corresponding body colouration changes during attack events in a controlled laboratory setting using groups of 3 adult fish in a small tank. In a triadic relationship consisting of 2 males and a female, males with blackened markings attacked more frequently than males without blackened markings and females. Additionally, we observed that the males with blackened markings were seldom attacked by males without blackened markings and females. These tendencies persisted even in groups consisting of 3 males. Our results suggest that the blackened markings in male O. celebensis not only indicate the level of aggression but also serve as a social signal to suppress attacks by other individuals. ### Competing Interest Statement The authors have declared no competing interest.

AbstractIn some species, such as chameleon and cichlid fish, rapid body colouration changes (within seconds or minutes) serve as visual social signals in male-male competition. This study investigated the relationship between aggressive behaviour and body colouration changes inOryzias celebensis, an Indonesian medaka fish. We analysed aggressive behaviours and corresponding body colouration changes during attack events in a controlled laboratory setting using groups of 3 adult fish in a small tank. In a triadic relationship consisting of 2 males and a female, males with blackened markings attacked more frequently than males without blackened markings and females. Additionally, we observed that the males with blackened markings were seldom attacked by males without blackened markings and females. These tendencies persisted even in groups consisting of 3 males. Our results suggest that the blackened markings in maleO. celebensisnot only indicate the level of aggression but also serve as a social signal to suppress attacks by other individuals.

The dorsal telencephalon (i.e. the pallium) exhibits high anatomical diversity across vertebrate classes. The non-mammalian dorsal pallium accommodates various compartmentalized structures among species. The developmental, functional, and evolutional diversity of the dorsal pallium remain unillustrated. Here, we analyzed the structure and epigenetic landscapes of cell lineages in the telencephalon of medaka fish ( Oryzias latipes ) that possesses a clearly delineated dorsal pallium (Dd2). We found that pallial anatomical regions, including Dd2, are formed by mutually exclusive clonal units, and that each pallium compartment exhibits a distinct epigenetic landscape. In particular, Dd2 possesses a unique open chromatin pattern that preferentially targets synaptic genes. Indeed, Dd2 shows a high density of synapses. Finally, we identified several transcription factors as candidate regulators. Taken together, we suggest that cell lineages are the basic components for the functional regionalization in the pallial anatomical compartments and that their changes have been the driving force for evolutionary diversity.

Zebrafish (Danio rerio) and medaka fish (Oryzias latipes) are popular vertebrate models for various biology fields. Medaka fish have recently attracted attention in the field of social neuroscience due to some of their unique advantages. First, medaka fish females have an extremely short ovarian cycle (24 h), which allows one to obtain a large number of female individuals of a similar physiological status according to their synchronized reproductive rhythms, facilitating quantitative analysis of female behaviors that are strongly influenced by the ovarian cycle; other experimental models such as mice and zebrafish have a longer ovarian cycle and/or unpredictable ovarian maturation. Second, medaka fish possess the visual ability to recognize conspecifics and make appropriate behavioral choices based on their social relationship. Third, the medaka fish are an established model in the field of molecular genetics (as are zebrafish) and state-of-the-art molecular-genetic methods are available. Here we describe several experimental procedures for behavioral paradigms to quantify medaka social behaviors, which are primarily mediated by visual cues.

Abstract The first mating (sexual) experience leads to the maturation of male mating behavior across species (insects, fish, rodents). Here, we investigated whether the first mating experience leads to maturation of male mating behavior in medaka using repeated mating tests. In “naïve” (sexually inexperienced) males after the first mating experience, the latency to mate with the same female partner was significantly decreased, whereas when the partner was swapped, the latency to mate was not affected. These findings suggest that repeated matings (3 times) enhanced male mating activity for the familiarized female, but not for an unfamiliarized female. In “experienced” (> 7 matings) males, repeated matings (3 times) with the same partner did not influence the latency to mate, suggesting that multiple matings (> 7 times) abolish the mate preference of naïve males. Furthermore, we identified 10 highly and differentially expressed genes after the mating experience in the brains of the post-naïve males, and revealed 3 genes that are required for a functional cascade of the thyroid hormone system. These findings together suggest that the first mating experience abolishes the preference to mate with a familiarized female via neural maturation triggered by thyroid hormone activation in the medaka brain.

Various social animals synchronously make behavioral choices among alternatives (i.e., collective decision-making) to efficiently migrate, forage, or escape from predators. Here, we demonstrated that medaka fish, Oryzias latipes , make collective decisions in response to visual looming stimuli (LS), a simulated predator attack. The group-level response to the LS could be classified into 2 patterns: freezing synchronously or not freezing, indicating that medaka selected their response to the LS from 2 alternative choices. Furthermore, the behavioral response to the LS persisted over multiple behavioral trials for 3 days, indicating that each group made a consistent decision to freeze or not to freeze. Finally, we showed that the group response could be predicted by the maximum swimming speed among individuals during the LS presentation. If a group included at least 1 individual that responded to the LS with high-speed movement (>7.4 cm/s), the group tended to freeze synchronously in response to the LS. If no member of the group was moving at high-speed during the LS presentation, the group tended to continue moving after the LS presentation. Our findings suggest that the presence of 1 or more individual medaka with a strong fear response in a group determines the group-level decision of how to respond to a threatening stimulus.

Aim Recently, a machine-learning (ML) technique has been used to create generalizable classifiers for psychiatric disorders based on information of functional connections (FCs) between brain regions at resting state. These classifiers predict diagnostic labels by a weighted linear sum (WLS) of the correlation values of a small number of selected FCs. We aimed to develop a generalizable classifier for gambling disorder (GD) from the information of FCs using the ML technique and examine relationships between WLS and clinical data. Methods As a training dataset for ML, data from 71 GD patients and 90 healthy controls (HCs) were obtained from two magnetic resonance imaging sites. We used an ML algorithm consisting of a cascade of an L1-regularized sparse canonical correlation analysis and a sparse logistic regression to create the classifier. The generalizability of the classifier was verified using an external dataset. This external dataset consisted of six GD patients and 14 HCs, and was collected at a different site from the sites of the training dataset. Correlations between WLS and South Oaks Gambling Screen (SOGS) and duration of illness were examined. Results The classifier distinguished between the GD patients and HCs with high accuracy in leave-one-out cross-validation (area under curve (AUC = 0.89)). This performance was confirmed in the external dataset (AUC = 0.81). There was no correlation between WLS, and SOGS and duration of illness in the GD patients. Conclusion We developed a generalizable classifier for GD based on information of functional connections between brain regions at resting state.

Mating experience shapes male mating behavior across species, from insects, fish, and birds, to rodents. Here, we investigated the effect of multiple mating experiences on male mating behavior in "naïve" (defined as sexually inexperienced) male medaka fish. The latency to mate with the same female partner significantly decreased after the second encounter, whereas when the partner was changed, the latency to mate was not decreased. These findings suggest that mating experiences enhanced the mating activity of naïve males for the familiar female, but not for an unfamiliar female. In contrast, the mating experiences of "experienced" (defined as those having mated > 7 times) males with the same partner did not influence their latency to mate. Furthermore, we identified 10 highly and differentially expressed genes in the brains of the naïve males after the mating experience and revealed 3 genes that are required for a functional cascade of the thyroid hormone system. Together, these findings suggest that the mating experience of naïve male medaka fish influences their mating behaviors, with neural changes triggered by thyroid hormone activation in the brain.