Spatio-temporal control of targeted gene expression in combination with CRISPR/Cas and Tet-On systems in Medaka.

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 Oryzias celebensis as a social signal influenced by environmental background.

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.

Optogenetic control of medaka behavior with channelrhodopsin.

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.

Sexually dimorphic role of oxytocin in medaka mate choice.

Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are up-regulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish.

Development of a classifier for gambling disorder based on functional connections between brain regions.

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.

A modified Tet-ON system minimizing leaky expression for cell-type specific gene induction in medaka fish.

The Tet-ON system is an important molecular tool for temporally and spatially-controlled inducible gene expression. Here, we developed a Tet-ON system to induce transgene expression specifically in the rod photoreceptors of medaka fish. Our modified reverse tetracycline-controlled transcriptional transactivator (rtTAm) with 5 amino acid substitutions dramatically improved the leakiness of the transgene in medaka fish. We generated a transgenic line carrying a self-reporting vector with the rtTAm gene driven by the Xenopus rhodopsin promoter and a tetracycline response element (TRE) followed by the green fluorescent protein (GFP) gene. We demonstrated that GFP fluorescence was restricted to the rod photoreceptors in the presence of doxycycline in larval fish (9 days post-fertilization). The GFP fluorescence intensity was enhanced with longer durations of doxycycline treatment up to 72 h and in a dose-dependent manner (5-45 µg/ml). These findings demonstrate that the Tet-ON system using rtTAm allows for spatiotemporal control of transgene expression, at least in the rod photoreceptors, in medaka fish.

Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

The cooperative induction of macrophage activation by fucoidan derived from Cladosiphon okamuranus and ß-glucan derived from Saccharomyces cerevisiae.

The present study investigated immune stimulatory effects of Cladosiphon okamuranus-derived fucoidan to activate murine macrophage-like cell line RAW264, and the functional relationship with zymosan, a Saccharomyces cerevisiae-derived ß-glucan. The production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in RAW264 cells were remarkably enhanced in the presence of 10 µg/mL fucoidan, and the stimulatory effects of fucoidan were maximally augmented in combinational treatment with 500 ng/mL zymosan, whereas any TLR ligands had no those effects. Confocal microscopic analyses suggested that fucoidan bound on plasma membrane, and it was estimated that some cell surface molecules acted as receptor for fucoidan because cytochalasin D, an inhibitor of phagocytosis, did not affect the immune enhancing activities, whereas methyl-ß-cyclodextrin (MßCD), a general agent for disruption of lipid rafts, diminished that. Furthermore, it was revealed that the additive effects of zymosan on the immune activation with fucoidan was thought to be mediated by dectin-1 based on the results with dectin-1-knockdown RAW264 cells. All of results suggested that fucoidan and some kinds of ß-glucan would cooperatively reinforce the activity of innate immune cells via interactive receptor crosstalk.

Amygdala volume is associated with risky probability cognition in gambling disorder.

Gambling disorder (GD) is characterized by continual gambling despite negative consequences. Risky decision-making is a hallmark of the disorder. We applied a tool from behavioral economics for assessing probability cognition in both gain and loss domains to GD. We aimed to examine the alteration of probability cognition and its relationship with brain structure in GD. Forty-six GD patients and 52 age-matched healthy controls (HCs) conducted a risky choice task in which subjects should choose between a sure and a risky option in both loss and gain domains. The distortion and elevation parameters of the probability weighting function were estimated. We compared the parameters between GD and HC and examined their relationships with the striatum and amygdala volumes in GD. GD showed greater elevation parameter in the gain domain and smaller regional gray matter volume in the left amygdala than HC. The elevation parameter in the gain domain showed a negative correlation with the left amygdala volume in GD. Altered probability cognition in the gain domain but not in the loss domain might be more relevant to risky decision-making in GD. Our findings indicate that alteration in the amygdala might play a significant role in risky decision-making of GD. Longitudinal studies are recommended to examine the causal relationship between brain abnormalities and risky decision-making in GD.

An essential role of the arginine vasotocin system in mate-guarding behaviors in triadic relationships of medaka fish (Oryzias latipes).

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (avt) and its receptors (V1a1, V1a2) and revealed that two genes, avt and V1a2, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.

Molecular basis of social competence in medaka fish.

Oryzias latipes (Medaka) is an established vertebrate model for studying developmental genetics, genomics, and evolutionary biology. The physiology, embryology, and genetics of this species have been extensively investigated for centuries. Medaka fish recently attracted attention in the field of social neuroscience. This review introduces recent advances in medaka behavioral studies, focusing on female mating preferences and male mate-guarding behaviors. The medaka female has the ability to discriminate male individuals and prefers to mate with socially familiar males (female mating preference). In triadic relationships (two males and one female), the dominant male remains closer to the female and repels the other male (mate-guarding). Interestingly, mate-guarding blocks female social familiarization of the rival male, which can increase the mating success of the dominant male. Importantly, behavioral analyses using a series of medaka mutants revealed critical roles of neuropeptide neuromodulatory systems in regulating their social behaviors. The extra-hypothalamic gonadotropin-releasing hormone system has a central role in activating female mating preference. The arginine-vasotocin system is required for the emergence of mate-guarding behavior.

Mate-guarding behavior enhances male reproductive success via familiarization with mating partners in medaka fish.

BACKGROUND: Male-male competition and female mating preference are major mechanisms of sexual selection, which influences individual fitness. How male-male competition affects female preference, however, remains poorly understood. Under laboratory conditions, medaka (Oryzias latipes) males compete to position themselves between a rival male and the female (mate-guarding) in triadic relationships (male, male, and female). In addition, females prefer to mate with visually familiar males. In the present study, to examine whether mate-guarding affects female preference via visual familiarization, we established a novel behavioral test to simultaneously quantify visual familiarization of focal males with females and mate-guarding against rival males. In addition, we investigated the effect of familiarization on male reproductive success in triadic relationships. RESULTS: Three fish (female, male, male) were placed separately in a transparent three-chamber tank, which allowed the male in the center (near male) to maintain closer proximity to the female than the other male (far male). Placement of the wild-type male in the center blocked visual familiarization of the far male by the female via mate-guarding. In contrast, placement of an arginine-vasotocin receptor mutant male, which exhibits mate-guarding deficits, in the center, allowing for maintaining close proximity to the female, did not block familiarization of the far male by the female. We also demonstrated that the reproductive success of males was significantly decreased by depriving females visual familiarization with the males. CONCLUSIONS: Our findings indicated that, at least in triadic relationships, dominance in mate-guarding, not simply close proximity, allows males to gain familiarity with the female over their rivals, which may enhance female preference for the dominant male. These findings focusing on the triadic relationships of medaka may contribute to our understanding of the adaptive significance of persistent mate-guarding, as well as female preference for familiar mates.

Ontogeny and Sexual Differences in Swimming Proximity to Conspecifics in Response to Visual Cues in Medaka Fish.

Adult medaka fish (Oryzias latipes) exhibit complex social behaviors that depend mainly on visual cues from conspecifics. The ontogeny of visually-mediated social behaviors from larval/juvenile to adult medaka fish, however, is unknown. In the present study, we established a simple behavioral paradigm to evaluate the swimming proximity to conspecifics based on visual cues in an inter-individual interaction of two medaka fish throughout life. When two fish were placed separately in a cylindrical tank with a concentric transparent wall, the two fish maintained close proximity to each other. A normal fish inside the tank maintained proximity to an optic nerve-cut fish outside of the tank, while the converse was not true. This behavioral paradigm enabled us to quantify visually-induced motivation of a single fish inside the tank. The proximity was detected from larval/juvenile to adult fish. Larval fish, however, maintained close proximity not only to conspecifics, but also to heterospecifics. As the growth stage increased, the degree of proximity to heterospecifics decreased, suggesting that shoaling preferences toward conspecifics and/or visual ability to recognize conspecifics is refined and established according to the growth stage. Furthermore, the proximity of adult female fish was affected by their reproductive status and social familiarity. Only before spawning, adult females maintained closer proximity to familiar males rather than to unfamiliar males, suggesting that proximity was affected by familiarity in a female-specific manner. This simple behavioral paradigm will contribute to our understanding of the neural basis of the development of visually-mediated social behavior using medaka fish.

Production of Knockout Mutants by CRISPR/Cas9 in the European Honeybee, Apis mellifera L.

The European honeybee (Apis mellifera L.) is used as a model organism in studies of the molecular and neural mechanisms underlying social behaviors and/or advanced brain functions. The entire honeybee genome has been sequenced, which has further advanced molecular biologic studies of the honeybee. Functions of genes of interest, however, remain largely to be elucidated in the honeybee due to the lack of effective reverse genetic methods. Moreover, genetically modified honeybees must be maintained under restricted laboratory conditions due to legal restrictions, further complicating the application of reverse genetics to this species. Here we applied CRISPR/Cas9 to the honeybee to develop an effective reverse genetic method. We targeted major royal jelly protein 1 (mrjp1) for genome editing, because this gene is predominantly expressed in adult workers and its mutation is not expected to affect normal development. By injecting sgRNA and Cas9 mRNA into 57 fertilized embryos collected within 3 h after oviposition, we successfully created six queens, one of which produced genome-edited male offspring. Of the 161 males produced, genotyping demonstrated that the genome was edited in 20 males. All of the processes necessary for producing these genome-edited queens and males were performed in the laboratory. Therefore, we developed essential techniques to create knockout honeybees by CRISPR/Cas9. Our findings also suggested that mrjp1 is dispensable for normal male development, at least till the pupal stage. This new technology could pave the way for future functional analyses of candidate genes involved in honeybee social behaviors.

Heterogeneity of Loss Aversion in Pathological Gambling.

Pathological gambling (PG) is characterized by continual repeated gambling behavior despite negative consequences. PG is considered to be a disorder of altered decision-making under risk, and behavioral economics tools were utilized by studies on decision-making under risk. At the same time, PG was suggested to be a heterogeneous disorder in terms of personality traits as well as risk attitude. We aimed to examine the heterogeneity of PG in terms of loss aversion, which means that a loss is subjectively felt to be larger than the same amount of gain. Thirty-one male PG subjects and 26 male healthy control (HC) subjects underwent a behavioral economics task for estimation of loss aversion and personality traits assessment. Although loss aversion in PG subjects was not significantly different from that in HC subjects, distributions of loss aversion differed between PG and HC subjects. HC subjects were uniformly classified into three levels (low, middle, high) of loss aversion, whereas PG subjects were mostly classified into the two extremes, and few PG subjects were classified into the middle range. PG subjects with low and high loss aversion showed a significant difference in anxiety, excitement-seeking and craving intensity. Our study suggested that PG was a heterogeneous disorder in terms of loss aversion. This result might be useful for understanding cognitive and neurobiological mechanisms and the establishment of treatment strategies for PG.

Unique spatially and temporary-regulated/sex-specific expression of a long ncRNA, Nb-1, suggesting its pleiotropic functions associated with honey bee lifecycle.

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.

Epigenetically distinct synaptic architecture in clonal compartments in the teleostean dorsal pallium.

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.

p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes).

Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.

Mating experiences with the same partner enhanced mating activities of naïve male medaka fish.

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.

Induction of c-fos transcription in the medaka brain (Oryzias latipes) in response to mating stimuli.

Immediate-early genes (IEGs) are useful for mapping active brain regions in various vertebrates. Here we identified a c-fos homologue gene in medaka and demonstrated that the amounts of c-fos transcripts and proteins in the medaka brain increased in relation to an artificially evoked seizure, suggesting that the homologue gene has the characteristics of IEGs, which are used as markers of neural activity. Next, quantitative reverse-transcription-polymerase chain reaction revealed that female mating behaviors upregulated c-fos transcription in some brain regions including the telencephalon, optic tectum, and cerebellum. In addition, we performed in situ hybridization with a c-fos intron probe to detect the de novo synthesis of c-fos transcripts and confirmed induction of c-fos transcription in these brain regions after mating. This is the first report of IEG induction in response to mating stimuli in teleost fish. Our results indicated that c-fos expression was induced in response to behavioral stimuli in the medaka brain and that medaka c-fos could be a useful marker of neural activity.