Lysophosphatidic acid (LPA) signaling plays diverse roles in the development of various vertebrates such as mammals and fish. The lamprey is a fish that retains ancestral features of vertebrates, but information regarding lamprey LPA receptor genes is limited. Here, using information from the lamprey genome database, we cloned two LPA receptor genes, Lpar1 and Lpar5, from the Japanese lamprey (Lethenteron camtschaticum). Lamprey Lpar1 had a high amino acid identity to mouse and medaka fish Lpar1, whereas Lpar5 amino acid sequences were more diverse between species. Our functional analyses using a heterologous expression system demonstrated that Lpar1 and Lpar5 responded to LPA treatment with G12/13-associated cellular responses, which are indicative of cytoskeletal actions. The existence of functional LPA receptors in the Japanese lamprey suggests that LPA receptor-dependent signals contribute to lamprey growth and development.

Di-2-ethylhexyl phthalate (DEHP) is the most commonly used phthalate for the production of flexible polyvinyl chloride. Recent studies in humans reported a widespread DEHP exposure, raising concerns in infants whose metabolic and excretory systems are immature. DEHP is a potential endocrine-disrupting chemical, but the effects of postnatal DEHP exposure on neuronal development are unclear. The dentate gyrus (DG) is critical in the consolidation of information from short- to long-term memory, as well as spatial learning. We evaluated neurodevelopmental toxicity due to neonatal DEHP exposure by assessing neurogenesis in the DG. Newborn mice were orally administered DEHP from postnatal day (PND) 12 to 25. We performed immunostaining using neuronal markers at different stages to assess whether DEHP exposure affects neurons at specific differentiation stages at PND 26 and PND 110. We found that in mice, postnatal DEHP exposure led to a decrease in the number of Type-1, -2a, -2b, and -3 neural progenitor cells, as well as granule cells in the hippocampal DG at PND 26. Further, the results showed that neural progenitor cell proliferation and differentiation were also reduced in the hippocampal DG of the DEHP-exposed mice. However, no effect on memory and learning was observed. Overall, our results suggest that neurodevelopmental toxicity due to postnatal DEHP exposure might affect postnatal DG morphogenesis.

Boldness and risk-taking behaviours in animals are important traits to obtain advantages such as habitation, food resources, reproductive success and social dominance. Risk-taking behaviour is influenced by physiological and environmental conditions; however, whether individual fish become bolder by the presence of conspecifics remains unknown. In this study, a light-dark preference test was conducted using medaka fish (Oryzias latipes) with or without a neighbouring conspecific. It was found that individual medaka male fish preferred a light environment and avoided a dark environment, whereas the display of a neighbouring conspecific enhanced the time the male spent in the dark environment (i.e., this condition encouraged risk-taking). The blood glucose level increased in fish confined to the dark condition but did not increase in light-preferring fish and risk-taking fish. Large somata expressing tyrosine hydroxylase, which is the rate-limiting enzyme in dopamine synthesis, were detected in the telencephalic and diencephalic brain regions in risk-taking medaka, whereas large somata were detected in the diencephalic region in medaka confined to the dark condition. These findings indicated that medaka is a good fish model to explore the central roles of dopaminergic neurons in the telencephalon and the diencephalon, which regulate risk-taking behaviour.

Bisphenol A (BPA) is known to cause abnormal neurogenesis in the developing neocortex. The mechanisms of BPA toxicity concerning neuroinflammatory-related endpoints are incompletely characterized. To evaluate the microglial morphology and the gene expression of pro-inflammatory cytokines in the newborn neocortex, ICR mice were exposed to BPA 200 μg/kg/d on gestational day 6 through post-partum day 21. Weanlings exposed during prenatal and postnatal period to BPA showed an increased number of amoeboid-type microglia, a microglial differentiation disruption (the M1/M2 microglial ratio), and an abnormal expression of genes encoding pro-inflammatory factors. These findings suggest that the well-known neurodevelopmental toxicity of BPA may be related to an increased microglial activation and neuroinflammation in the neocortex.

Bisphenol A (BPA), an endocrine disruptor with estrogenic effects, is widely used as a raw material for manufacturing polycarbonate plastic and epoxy resins. Prenatal and postnatal exposure to BPA affects brain morphogenesis. However, the effects of prenatal and postnatal BPA exposure on postnatal neurogenesis in mice are poorly understood. In this study, we developed a mouse model of prenatal and postnatal BPA exposure and analyzed its effects on hippocampal neurogenesis. The hippocampal dentate gyrus is vulnerable to chemical exposure, as neurogenesis continues in this region even after birth. Our results showed that in mice, prenatal and postnatal BPA exposure decreased the number of type-1, 2a, 2b, and 3 neural progenitor cells, as well as in granule cells, in the hippocampal dentate gyrus on postnatal days 16 and 70. The effect of prenatal and postnatal BPA exposure on neural progenitors were affected at all differentiation stages. In addition, prenatal and postnatal BPA exposure affects the maintenance of long-term memory on postnatal day 70. Our results suggest that neurodevelopmental toxicity due to prenatal and postnatal BPA exposure might affect postnatal morphogenesis and functional development of the hippocampal dentate gyrus.

Acetamiprid (ACE) and imidacloprid (IMI) are widely used neonicotinoid pesticides. They bind selectively to insect nicotinic acetylcholine receptors (nAChRs) and are considered non-hazardous to mammals. Few studies have assessed the activation of vertebrate nAChRs and the neurodevelopmental toxicity following in utero or neonatal exposure to neonicotinoids; therefore, we evaluated the effects of ACE or IMI exposure on neurogenesis and microglial profiles in the developing hippocampal dentate gyrus (DG) of mouse neonates. Mice were exposed to ACE, IMI (both 5 mg/kg/day) or nicotine (0.5 mg/kg/day) from postnatal day (P)12 to P26 by oral gavage. On P27, brains were removed, and neurogenesis and microglial activation in the hippocampal DG were examined via immunohistochemistry. A reduction in neurogenesis in the hippocampal DG of neonates following ACE, IMI and nicotine treatment was found. Additionally, neonicotinoid-exposed newborns showed an increase in the number of amoeboid-type and activated M1-type microglia. These results suggest that exposure to ACE and IMI impairs neurogenesis and alters microglial profiles in the developing hippocampal DG following oral dosing in an early postnatal period. A better understanding of the potential effects of these pesticides on human infant health is an important goal of our research.

Serotonin (i.e., 5-hydroxytryptamine [5-HT]) plays a key role in stress responses in vertebrates. In mammals and teleosts, tryptophan hydroxylase (Tph), a rate-limiting enzyme in the biosynthesis of 5-HT, includes two paralogs: Tph 1 and Tph 2. The response of the Tphs to stress has been reported in mammals, but less is known about the responses of these enzymes to stress in fish. In the present study, we examined whether heat stress affects the mRNA expression of these Tphs in the brain of medaka fish (Oryzias latipes). We also determined the concentration of 5-HT in the brain, the mRNA expression of heat shock protein 90 alpha (Hsp90α) in the liver, plasma cortisol concentration, and blood glucose concentration in medaka. Whole-body exposure to repeated heat stress significantly decreased the mRNA expression of Tph1 and Tph2 in male and female medaka, whereas single heat stress did not affect the expression of either of the mRNAs. The 5-HT concentration also decreased significantly after repeated heat stress sessions in both sexes, but did not decrease after a single heat stress session. After single and repeated heat stress sessions, Hsp90α mRNA expression increased in both sexes; however, increments in the concentrations of plasma cortisol and blood glucose occurred in male, but not in female, medaka. These results suggest that both types of Tphs are involved in reducing 5-HT in the brain and are reliable indicators of chronic stress response in both sexes. However, stress responses in plasma cortisol and blood glucose concentrations differ between male and female medaka.

Social interactions regulate the expression of several neuropeptides that have a central role in the reproductive system of mammals. Nonmammalian vertebrates also have these neuropeptides or paralogs, however, studies on the social regulation of reproductive physiology in nonmammalian species are limited. In this study, we examined whether the expression of gonadotropin-releasing hormones (GnRHs) and kisspeptin (Kiss1) is affected by social hierarchy resulting from the outcomes of male-male competition in medaka fish (Oryzias latipes). Four males were introduced to each other in an experimental tank, and classified as the most aggressive dominant or the most submissive subordinate fish, based on the frequency of their aggressive acts during a short-term competition. Dominant and subordinate males maintained their social rank during a long-term competition. Immediately after short-term competition, gonadotropin-releasing hormone-3 (GnRH3) level in the olfactory bulb was significantly higher in subordinate males than in dominant males. After long-term competition, dominant males had high level of gonadotropin-releasing hormone-1 (GnRH1) in the preoptic area, whereas subordinate males had lower Kissl level in the nucleus ventral tuberis. On the other hand, the levels of gonadotropin-releasing hormone-2 (GnRH2) in the nucleus lateralis valvulae and Kissl in the nucleus posterioris periventricularis, and plasma 11-ketotestosterone (11-KT) concentration did not differ between subordinate and dominant males after the short- and long-term competitions. These results suggest that social hierarchy regulates the expression of GnRH1, GnRH3, and Kissl without affecting 11-KT level in male medaka. (C) 2017 Elsevier Inc. All rights reserved.

Fetal alcohol spectrum disorders (FASD) constitute a wide range of disorders that arise from prenatal exposure to ethanol (EtOH). However, detailed reports regarding the adverse effects of prenatal EtOH exposure on neocortical morphology and its underlying pathogenic mechanisms are limited. In the present study, we aimed to characterize the anatomical abnormalities of neocortical development and their correlation with microglial properties and neuro-inflammation in a mouse model of FASD. We evaluated the development and maturation of the neocortex in ICR mice prenatally exposed to 25% (w/v) EtOH using histological and molecular analyses. Reduced proliferation and excessive cell death were observed in the dorsal telencephalon. Abnormal neuronal distribution, layer formation, and dopaminergic neuronal projections were observed in the neocortex. Disruption of microglial differentiation (M-1/M-2 microglial ratio) and abnormal expression of pro-inflammatory and neurotrophic factors were induced, and these abnormalities were ameliorated by co-treatment with an antiinflammatory drug (pioglitazone). FASD model mice displayed histological abnormalities, microglial abnormalities, and neuro-inflammation in both the embryonic and newborn stages. Thus, antiinflammatory therapeutics may provide a novel preventive approach for the treatment of FASD.

Di(2-ethylhexyl) phthalate (DEHP) is currently the most commonly used phthalate for the production of flexible polyvinyl chloride. Phthalates including DEHP have been labeled as potential endocrine disruptors. The effect on the development of the neocortex, however, is unknown. To evaluate the neurodevelopmental effects of prenatal DEHP exposure at 1 and 100 mg/kg/day or 100 and 500 mg/kg/day in fetal and newborn mice, we performed a detailed histologic analysis of the developing dorsal telencephalon and neocortex. The observation of fetuses exposed to DEHP revealed reductions of proliferation and neurogenesis (1 and 100 mg/kg) and an increase in cell death (500 mg/kg). In addition, the newborns prenatally exposed to DEHP showed an abnormal neuronal distribution and a decrease in neurons. These findings suggest that prenatal DEHP exposure induces neurodevelopmental toxicity associated with the neural stem cell niche and corticogenesis.

The neurohypophysial peptide arginine vasotocin (AVT) and its mammalian ortholog arginine vasopressin function in a wide range of physiological and behavioral events. Here, we generated a new line of trans genic medaka (Oryzias latipes), which allowed us to monitor AVT neurons by enhanced green fluorescent protein (EGFP) and demonstrate AVT neuronal development in the embryo and the projection of AVT neurons in the adult brain of avt-egfp transgenic medaka. The onset of AVT expression manifested at 2 days postfertilization (dpf) as a pair of signals in the telencephalon of the brain. The telencephalic AVT neurons migrated and converged on the preoptic area (POA) by 4 dpf. At the same stage, another onset of AVT expression manifested in the central optic tectum (OT), and they migrated to the ventral part of the hypothalamus (VH) by 6 dpf. In the adult brain, the AVT somata with EGFP signals existed in the gigantocellular POA (gPOA), magnocellular POA (mPOA), and parvocellular POA (pPOA) and in the VH. Whereas the major projection of AVT fibers was found from the pPOA and VH to the posterior pituitary, it was also found that AVT neurons in the three POAs send their fibers into wide regions of the brain such as the telencephalon, mesencephalon and diencephalon. This study suggests that the avt-egfp transgenic medaka is a useful model to explore AVT neuronal development and function. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

The neurohypophysial hormones, arginine vasotocin and isotocin, regulate both hydromineral balance and social behaviors in fish. In the amphibious mudskipper, Periophthalmus modestus, we previously found arginine-vasotocin-specific regulation of aggressive behavior, including migration of the submissive subordinate into water. This migration also implies the need for adaptation to dehydration. Here, we examined the effects of arginine vasotocin and isotocin administration on the amphibious behavior of individual mudskippers in vivo. The mudskippers remained in the water for an increased period of time after 1-8 h of intracerebroventricular (ICV) injection with 500 pg/g arginine vasotocin or isotocin. The 'frequency of migration' was decreased after ICV injection of arginine vasotocin or isotocin, reflecting a tendency to remain in the water. ICV injections of isotocin receptor antagonist with arginine vasotocin or isotocin inhibited all of these hormonal effects. In animals kept out of water, mRNA expression of brain arginine vasotocin and isotocin precursors increased 3- and 1.5-fold, respectively. Given the relatively wide distribution of arginine vasotocin fibres throughout the mudskipper brain, induction of arginine vasotocin and isotocin under terrestrial conditions may be involved also in the preference for an aquatic habitat as ligands for brain isotocin receptors.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish.

Studies on the low-dose effects of xenoestrogens have yielded conflicting results that may have resulted from differences in estrogen sensitivity between the mouse strains used. We developed a mouse newborn behavioral testing method for evaluating the risk of neurotoxicity of environmental chemicals, by means of determining a newborn's motor activity through applying the tare function of an analytical balance. Motor activities including crawling, pivoting, and tremors of C57BL/6J and ICR mouse newborns exposed to bisphenol A (BPA) at 200 µg/kg/day on embryonic days 6 through 18 were evaluated for 5 min on postnatal day 1 by the testing method. Motor activities of mature male offspring exposed prenatally to BPA were also evaluated in wheel cage and open field tests. Maternal BPA oral dosing increased the motor activity in newborns of both strains and mature offspring of the C57BL/6J strain. The findings indicate that both mouse strains provide adequate models for the newborn neurobehavioral study of prenatal exposure to environmentally relevant levels of estrogen-mimicking chemicals.

Medaka fish (Oryzias latipes) were whole-bodily treated with various doses of mitomycin C (MMC), ethylmethanesulfonate (EMS), cyclophosphamide (CP), diethylnitrosamine (DEN), or colchicine (COL) for 24 h, and the frequency of micronucleated cells (MNCs) was measured in the gills at 24 and 48 h after treatment. In the present experiments, MMC, CP, and DEN were recorded as efficient inducers of micronuclei at both sampling times, and none of the MNC frequencies recorded with these agents at 24 h significantly exceeded the corresponding frequency at 48 h. For EMS and COL, positive responses were recorded only 48 h after treatment. By comparison with the time-course data reported for radiation-induced MNCs in the same MN assay system, the clear responses observed at the 48-h time point for all the chemicals used were regarded as evidence of their delayed effects on micronucleus (MN) formation. The mean sizes of micronuclei induced after exposure to COL was significantly larger by a factor 2 as compared with that induced by X-irradiation, whereas those determined for the other four chemicals were almost equal to that induced by X-irradiation. These results demonstrate that the medaka gill-cell MN assay can detect chemically-induced chromosome damage, either directly or after metabolic activation, and spindle malfunction, and provide a basis for further development of the present assay system for testing cytogenetic activities of chemical agents.

Lysophosphatidic acid (LPA) signaling is known to play biological and pathophysiological roles in many types of animals. Medaka (Oryzias latipes) is an experimental fish that can be easily maintained, propagated, and analyzed, and whose genome has been completely sequenced. However, there is limited information available regarding medaka LPA receptors. Here, using information from the medaka genome database, we examine the genomic structures, expression, and functions of six LPA receptor genes, Lpar1-Lpar6. Our analyses reveal that the genomic structures of Lpar1 and Lpar4 are different from those deduced from the database. Functional analyses using a heterologous expression system demonstrate that all medaka LPA receptors except for LPA(5b) respond to LPA treatment with cytoskeletal changes. These findings provide useful information on the structure and function of medaka LPA receptor genes, and identify medaka as a useful experimental model for exploration of the biological significance of LPA signaling. (C) 2014 Elsevier B.V. All rights reserved.

The central nervous system is especially susceptible to toxic insults during development. Prenatal administration of bisphenol A (BPA) induces histologic anomalies in the dorsal telencephalon of the embryo. Whether these anomalies affect the morphogenesis and maturation of neuronal function of the newborn neocortex, however, is unknown. To evaluate the neurodevelopmental and behavioral effects of prenatal BPA exposure at 20 and 200 mu g/kg/day in newborn mice, we performed a detailed histologic analysis of the neocortex and tested for the presence of behavioral abnormalities in newborn mice prenatally exposed to BPA using our newly developed behavioral test. Observations of newborn mice prenatally exposed to BPA revealed abnormal neuronal distribution and layer formation, hypoplasia of layer 6b, and abnormal dopaminergic neuronal projections in the neocortex. Further, the newborn mice exhibited hyperactivity. These findings suggest that prenatal BPA exposure induces neurobehavioral toxicity associated with abnormal dopaminergic neuronal projections, and abnormal corticogenesis and lamination. Histologic and behavioral analyses of newborn mice are considered useful for assessing the neurodevelopmental and behavioral toxicity of chemicals. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

This study was performed to clarify the involvement of impaired labyrinth zone (LZ) of the placenta in the developmental toxicity of diethylstilbestrol (DES). DES at 10g/kg per day was administered orally to mice on days 4 through 8 of gestation. Histological observation of the LZ and determination of blood glucose levels in dam and fetus were performed on day 13. A high frequency of embryonic death was observed in the DES group. DES induced the underdevelopment of the plexus vasculosus, extensive maternal blood space and the decreased expression of glucose transporters in the LZ, and a reduction of the glucose level in embryos. These findings suggest that impaired LZ development may be related to the embryolethality of DES.

This study was performed to clarify the involvement of impaired labyrinth zone (LZ) of the placenta in the developmental toxicity of diethylstilbestrol (DES). DES at 10g/kg per day was administered orally to mice on days 4 through 8 of gestation. Histological observation of the LZ and determination of blood glucose levels in dam and fetus were performed on day 13. A high frequency of embryonic death was observed in the DES group. DES induced the underdevelopment of the plexus vasculosus, extensive maternal blood space and the decreased expression of glucose transporters in the LZ, and a reduction of the glucose level in embryos. These findings suggest that impaired LZ development may be related to the embryolethality of DES.

There have been few neurobehavioral toxicology studies on newborn animals. Thus, we developed a mouse newborn behavioral testing method for evaluating the risk of neurotoxicity of environmental toxicants, by means of determining the newborn's motor activity applying the tare function of an analytical balance. Motor activities including crawling, pivoting, righting or tremors of mouse newborns were evaluated. Tremors of newborns of dams exposed to bisphenol A at 2, 20 or 200 mu g/kg/day on days 5 through 18 of gestation were significantly increased when evaluated on postnatal day 1, as well as those of newborns exposed prenatally to diethylstilbestrol at 0.5 mu g/kg/day. We suggest that our developed testing method may provide a useful addition to neurobehavioral assessment in very young rodents exposed to environmental hormone mimics.

This study compared aggressiveness between two distinct wild populations of Japanese medaka: a Northern population, Oryzias sakaizumii, and a Southern population, O. latipes. When four males competed in intra-population contests, the social hierarchy was determined based on aggressive acts in both populations. Dominants of the Southern population showed higher aggressive acts than did dominants of the Northern population. Increased aggressiveness of Southern males compared with Northern males was also observed when a pair of Northern and Southern males were compared in inter-population contests. High expression of arginine vasotocin (AVT) in distinct preoptic regions were found in dominants and subordinates of the Southern population, but not in those of the Northern population. In contrast, neither 11-ketotestosterone nor cortisol levels in plasma differed between dominants and subordinates of either population, nor between pairs of the Northern and the Southern males. Taken together, these findings indicate that the two wild populations of medaka represent intriguing models for the study of neuroendocrinological correlates in behavioral traits underlying congeners of medaka fish.

Although there have been a vast number of behavioral toxicology studies carried out on adult mice and rats, there have been few neurobehavioral studies utilizing their newborn animals. Thus, we developed a mouse newborn behavioral testing method for evaluating the risk of neurotoxicity of chemicals, by means of determining the newborn's activity using the tare function of an analytical balance. The unstable weighing values resulting from movement of the newborn on the balance recorded by a personal computer every 0.1s, and the total activities of a newborn from the start time of weighing to individual times of evaluation were calculated. In addition, we confirmed the usefulness of our method by determining the activity of mouse newborns with microcephaly induced by prenatal exposure to a neurotoxicant, methylnitrosourea. Copyright (c) 2012 John Wiley & Sons, Ltd.

The hypothalamic hormones, arginine-vasotocin (VT) and isotocin (IT), play central roles in osmoregulation and in the regulation of social behaviors including aggressive behavior in many vertebrates including fish. Here, we examined whether these hormones are associated with aggressive behavior in the mudskipper (Periophthalmus modestus). The mudskipper is an amphibious fish, which lives in the brackish water of river mouths and displays unique aggressive behavior. Upon introduction to each other in an experimental tank with aquatic and terrestrial areas, a pair of males can be classified as aggressive dominant or submissive subordinate based on the frequency of their aggressive acts, which is significantly higher in dominant male. Additionally, the length of stay in terrestrial area of dominant was longer than that of the subordinate. The latter remained in aquatic area almost throughout the period of behavioral observation. The expression of brain VT mRNA was significantly higher in subordinate than in dominant, whereas neither IT mRNA expression nor plasma cortisol level differed between subordinate and dominant male. On the other hand, an intracerebroventricular injection of VT increased aggressive behaviors in mudskippers. In addition to known roles of VT in mediation of aggressive behavior, these results may shed light on the role of endogenous VT toward water migration in submissive mudskippers. The amphibious fish is a valuable experimental model to observe the relationship between effects of central VT on the osmoregulation and social behavioral regulation in vertebrates. © 2013.

Placental growth and function are of biological significance in that placental tissue promotes prenatal life and the maintenance of pregnancy. Exposure to synthetic estrogens causes embryonic mortality and placental growth restriction in mice. The aim of the present study was to examine the effects of diethylstilbestrol (DES) on placenta in mice. DES at 1, 5, 10 or 15 mu g kg(-1) day(-1), or 17-estradiol (E-2) at 50 mu g kg(-1) day(-1), was administered orally to ICR mice on days 4 through to 8 of gestation. Expression of ER, ER, ERR or ERR mRNA in the junctional or labyrinth zone of the placentas on day 13 was assessed using RT-PCR, as well as the embrynic mortality, embryonic and placental weight, histological changes of labyrinth and ultrastructural changes of the trophoblast giant cells (TGCs). Embryo mortalities in the DES 10 and 15 mu g kg(-1) day(-1) groups were markedly increased. No significant changes in embryonic and placental weight were observed in any DES- or E-2-exposed groups. Expression of ER mRNA in the junctional zone with male embryos in the 5 mu g kg(-1) day(-1) group was significantly higher than that in the control, whereas expression was not determined in the 15 mu g kg(-1) day(-1) group. Histological observation revealed that the placentas exposed to DES at 10 mu g kg(-1) day(-1) lacked the developing labyrinth. Ultrastructural observation of the TGCs showed poor rough-surfaced endoplasmic reticulum in the DES 10 mu g kg(-1) day(-1) group. The present data suggest that developmental changes induced by DES may be related to interference with the nutrition and oxygen exchange between mother and embryo or decreased protein synthesis, resulting in a high frequency of embryo mortality. Copyright (c) 2012 John Wiley & Sons, Ltd.

This study examined arginine vasotocin (AVT) expression in the brains of dominant and subordinate male medaka Oryzias latipes after short- and long-term competition. High AVT expression in distinct preoptic regions was found in dominants and subordinates within minutes of encountering each other. During long-term competition, AVT expression remained high in dominants but not in subordinates. © 2012 The Author. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

The effect of dose rate on radiation-induced mutations in two somatic tissues, the spleen and liver, was examined in transgenic gpt delta mice. These mice can be used for the detection of deletion-type mutations, and these are the major type of mutation induced by radiation. The dose rates examined were 920 mGy/min, 1 mGy/min and 12.5 microGy/min. In both tissues, the number of mutations increased with increasing dose at each of the three dose rates examined. The mutation induction rate was dependent on the dose rate. The mutation induction rate was higher in the spleen than in the liver at the medium dose rate but was similar in the two tissues at the high and low dose rates. The mutation induction rate in the liver did not show much change between the medium and low dose rates. Analysis of the molecular nature of the mutations indicated that 2- to 1,000-bp deletion mutations were specifically induced by radiation in both tissues after high- and low-dose-rate irradiation. The occurrence of deletion mutation without any sequence homology at the break point was elevated in spleen after high-dose-rate irradiation. The results indicate that the mutagenic effects of radiation in somatic tissues are dependent on dose rate and that there is some variability between tissues.

To understand the effects of low dose-rate radiation on genome structure in vivo, we examined gene mutation and chromosomal abnormality in mouse tissues. The mutation was studied on transgenes in the spleen and liver and also on the Dlb1 gene in intestinal stem cells. The mice were irradiated for 483 consecutive days at three dose-rates of 0.0323, 0.65, and 12.5 μGy/min, which resulted in total doses of 21, 414, and 8,000 mGy, respectively. Statistically significant increases were observed for all indices examined when the total dose was 8,000 mGy, whereas no significant difference was observed with 21 mGy or 414 mGy.

The frequency of micronucleated cells (MNCs) was measured in acridine orange (AO) stained kidney cells obtained from male and female medaka fish (Oryzias latipes) of known body weight before and after X-irradiation at a dose of 4Gy. The MNC frequency in the kidney cells obtained from untreated and X-ray treated fish did not vary in correlation with the body weight in either gender, and neither did it show a significant gender-related difference. In both the untreated and treated groups, the number of MNCs in kidney cells per fish appeared to follow a Poisson distribution. These results are in agreement with those previously reported for the MNC frequency in gill cells of medaka fish. The average MNC frequency in kidney cells for the untreated fish was 0.4‰ (12/30000), which was significantly lower than 0.8‰ (24/30000) reported for gill cells. Similarly, a relatively lower frequency of X-ray-induced MNCs was evident for kidney cells (2.2‰; 69/30000) vs. gill cells (6.3‰; 191/30000). These results combined with previous reports support the use of AO to detect micronucleus formation in kidney cells, and further indicate that the kidney cell micronucleus assay can be used as a reliable and coincidently applied adjunct to the gill cell assay for medaka fish.

Following whole-body irradiation of ICR mice with various doses of fission neutrons or X-rays, the frequency of micronuclei (MNs) in peripheral blood reticulocytes was measured at 12 h intervals beginning immediately after irradiation and ending at 72 h after irradiation. The resulting time-course curve of MN frequency had a clear peak 36 h after irradiation, irrespective of the type of radiation applied and the dose used. The MN frequency, averaged as the unweighted mean over the experimental time course, showed a linear increase with increasing dose of either fission neutrons or X-rays. The linear response to X-rays supports reported conclusion that induction of MN formation in reticulocytes is a dose-rate independent phenomenon. The relative biological effectiveness (RBE) of fission neutrons to X-rays for MN induction was estimated to be 1.9 +/- 0.3. This value is considerably lower than the RBE value of 4.6 +/- 0.5 reported for the same fission neutrons for induction of lymphocyte apoptosis in the thymus of ICR mice that represents dose-rate independent, one-track event. Based on these results, we propose that MNs increased in reticulocytes after irradiation mostly represent acentric fragments caused by single chromosome breaks, and that some confounding factor is operating in erythroblasts for the formation of aberrations from non-rejoining DNA double-strand breaks more severely after high-LET radiation than after low-LET radiation.

Geldanamycin (GA), a specific inhibitor of the chaperoning function of heat-shock protein 90 (Hsp90), has been shown to mimic heat shock (HS) in inducing expression of Hsp90, Hsp72 and other Hsps in unstressed mammalian cells. In the present study, intra-cerebral treatment of goldfish with GA (at a dose of 0.1 microg/g-body weight) diminished basal Hsp90 level to a 30-40% level in the brain, without affecting the basal Hsp72 level, as assayed 28-48 h after treatment. Whole-body exposure to HS significantly increased Hsp90 level in GA-untreated fish but not in GA-treated fish, while it significantly increased Hsp72 level in both GA-untreated and -treated fish. In both GA-untreated and -treated fish, plasma cortisol (PC) levels increased considerably 4 h after HS and then decreased in a time-dependent manner to the control levels 24 h after HS, showing no evidence of a GA effect on the time course of PC level. These results suggest that in the brain of goldfish, Hsp90 may not be involved as a key factor either in regulating Hsp72 expression both before and after HS or in the feedback regulation of HS-increased PC level, and support the idea that GA can be used in fish brain as a tool in elucidating the role of Hsp90 in complicated, Hsp-mediated biological processes.

The frequency of micronucleated cells (MNCs) was measured in acridine-orange (AO) stained RNA-rich gill cells from male and female medaka (Oryzias latipes) fish of known body weight. Spontaneous MNC frequencies were not significantly correlated with body weight, despite the fact that the heaviest of the 30 fish used outweighed the lightest by a factor of 3. Average MNC frequencies were identical in males and females at 0.8‰. An X-ray dose of 4Gy increased the frequency of MNCs over the spontaneous level in all 30 of the fish used, reaching a level of 7.2‰ on average when assayed 24h after exposure. In X-ray treated fish, MNC frequency and body weight were not significantly correlated, nor was there any difference between the sexes. These and other results support our primary conclusion that AO-staining is suitable for the medaka micronucleus assay in gill cells, and indicate that male and female medaka fish are similarly and size-independently susceptible to both spontaneous and X-ray induced micronucleus formation in gill cells. © 2003 Elsevier B.V. All rights reserved.

The frequency of micronucleated cells (MNCs) was measured in acridine-orange (AO) stained RNA-rich gill cells from male and female medaka (Oryzias latipes) fish of known body weight. Spontaneous MNC frequencies were not significantly correlated with body weight, despite the fact that the heaviest of the 30 fish used outweighed the lightest by a factor of 3. Average MNC frequencies were identical in males and females at 0.8 per thousand. An X-ray dose of 4 Gy increased the frequency of MNCs over the spontaneous level in all 30 of the fish used, reaching a level of 7.2 per thousand on average when assayed 24 h after exposure. In X-ray treated fish, MNC frequency and body weight were not significantly correlated, nor was there any difference between the sexes. These and other results support our primary conclusion that AO-staining is suitable for the medaka micronucleus assay in gill cells, and indicate that male and female medaka fish are similarly and size-independently susceptible to both spontaneous and X-ray induced micronucleus formation in gill cells.

Medaka fish (Oryzias latipes) were exposed to various doses of X-rays or fast neutrons, and the frequency of micronucleated cells (MNCs) was measured in gills sampled at 12- or 24-hr intervals from 12 to 96 hr after exposure. The resulting time course of MNC frequency was biphasic, with a clear peak 24 hr after exposure, irrespective of the kind of radiation applied and the dose used. The half-life of MNCs induced in the gill tissues by the two exposures fluctuated around 28 hr, with no significant dose-dependent trend for either X-ray- or neutron-exposed fish. As assayed 24 hr after exposure, the MNC frequency increased linearly over the control level with increasing doses of both X-rays and fast neutrons. The relative biological effectiveness (RBE) of fast neutrons to X-rays for MNC induction was estimated to be 4.3 +/- 0.6. This value is close to the RBE value of 5.1 +/- 0.3 reported for fast neutron induction of somatic crossing-over mutations in Drosophila melanogaster that arise from recombination repair of DNA double-strand breaks. These results and other data support our conclusion that the medaka gill cell micronucleus assay is a reliable short-term test for detecting potential inducers of DNA double-strand breaks.

We previously found that when goldfish were exposed to a potential predator, bluegills, the goldfish experienced an increase in HSP70 mRNA expression in the brains and increased plasma cortisol levels. In the present study, we examined the potential causative relationship between HSP70 mRNA expression and plasma cortisol levels. Cortisol agonists (corticotropin releasing factor and cortisol) and antagonists (metyrapone and betamethasone) were used to modulate plasma cortisol levels. HSP70 mRNA expression and plasma cortisol levels were analyzed by Northern blotting and ELISA, respectively. Goldfish treated with the cortisol agonists showed marked increases in plasma cortisol levels and also in brain HSP70 mRNA expression. When goldfish were exposed to bluegills, plasma cortisol levels increased and HSP70 mRNA expression was enhanced after 6 hr. However, pre-treatment with the cortisol antagonists 24 hr prior to the exposure inhibited the enhancement as well as the increase in plasma cortisol levels. These results suggest that plasma cortisol plays a key role in the enhancement of brain HSP70 mRNA expression in goldfish stressed by exposure to bluegills.

Effects of Al and Cd on vitellogenin (VTG) and VTG mRNA induction by estradiol-17 β (E2) were examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then E2 (2 x 10-6 M) and Al (10-6-10-4 M) or Cd (10-9-10-6 M) were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days. Media and hepatocytes were then analyzed by SDS-PAGE and Northern blotting for VTG and VTG mRNA, respectively. These metals had no appreciable effect on the viability of hepatocytes in culture. However, Al and Cd interfered with VTG production and VTG mRNA expression. Al reduced VTG production in a concentration-dependent way and a significant reduction occurred at Al concentrations greater than 5 x 10-5 M. VTG mRNA expression also decreased with a negative correlation with Al concentrations (r = -0.98). The inhibition of VTG production by Cd was not concentration-dependent. This metal markedly inhibited VTG production and VTG mRNA expression at 10-6 M. The Al-induced inhibition of VTG production was restored 7 days after Al removal, but the Cd-induced inhibition was not restored. These results suggest that Al and Cd inhibit VTG production at the transcriptional level to reduce VTG mRNA expression by different mechanisms. (C) 2000 Academic Press.

The effect of psychological stress on HSP70 mRNA in the brains and plasma cortisol levels in goldfish was examined. Stress was induced by exposure to a predator (bluegills). HSP70 mRNA and cortisol were determined by Northern blotting and ELISA, respectively. Goldfish exposed to four predators in the same tank without a partition showed marked increases in HSP70 mRNA and cortisol levels 6 hr and 12 hr after commencement of exposure. When goldfish were separated from bluegills with a net partition, HSP70 mRNA expression was enhanced after 6 hr, and returned to the control level after 12 hr. Plasma cortisol levels increased after 2 hr, and returned to the control level after 6 hr. When goldfish were placed in a transparent tank around which bluegills were swimming, HSP70 mRNA expression and cortisol levels increased after 6 hr and 12 hr. Goldfish exposed to water circulating through a tank with bluegills showed no sign of changes in HSP70 mRNA expression or cortisol levels. These results suggest that psychological stress enhanced HSP70 mRNA expression in the brains and increased plasma cortisol levels via visual perception.

Induction of stress protein (HSP70) was studied in goldfish (Carassius auratus) reared with bluegills (Lepomis macrochirus), a predator of small fish. HSP70 was identified by Western blotting and quantified by optical density after SDS-PAGE. The expression of HSP70 was significantly enhanced in the brains of goldfish reared with bluegills for 6 and 12 h in a single tank. The hepatopancreas and the kidney were not affected by the treatment. When goldfish were separated from bluegills with a partition net (1 × 1 cm mesh size), this protein also increased in the brains after 6 h but then returned to the control level after 12 h. When the goldfish and bluegills were kept in separate tanks and were not able to see each other but were connected by circulating water, HSP70 levels in the goldfish were unaffected. Immunohistochemical observations indicated that an anti-HSP70 antibody was found to react predominantly with the optic and vagal lobes of the brains. These results suggest that visual perception plays a primary role in enhancement of HSP70 expression in the goldfish reared with bluegills.

本研究は、魚類の大胆または慎重行動が仲間の有無によってどのように調節されるか、また、それらの行動調節に神経ペプチドや神経伝達物質が関与するかを解明することを目的としている。本年度は、メダカを用いてrisk-taking行動試験を行い、同種他個体の存在下で大胆行動をとる個体と、単独で慎重行動をとる個体を得た後、両者の脳内アルギニンバソトシン（AVT）の発現量および各脳領域における神経活動活性を解析、比較した。 大胆行動をとった個体では、脳視床下部の視索前核に相当するgPOA領域におけるAVT発現量が慎重行動をとった個体に比べて高い傾向がみられた。一方、AVT神経の脳内投射領域は大胆行動をとる個体および慎重行動をとる個体のいずれにおいても、同様に観察され、大きな違いは認められなかった。これらの結果から、大胆個体と慎重個体では、AVT神経の機能する脳領域に相違はなく、特定の脳領域で受容されるAVT量が増えることで大胆行動が亢進される可能性が示唆された。 大胆個体と慎重個体の各脳領域における神経活動活性をEgr1遺伝子の発現量を指標に比較したところ、AVT神経細胞が局在するgPOA領域の他にも、終脳の扁桃体に相当するVs領域を含む複数の脳領域においても、Egr1発現量に差がみられた。これらの終脳領域には、AVT神経の投射領域に加えて、ドーパミン神経の細胞体局在領域も含まれることがわかっている。さらに、Vs領域およびgPOA領域に局在するドーパミン神経の細胞体には、AVT受容体（V1A型）が発現することを確認した。これらの結果から、AVT神経およびドーパミン神経の活動や相互の神経連絡が、メダカにおける大胆または慎重行動の選択に影響する可能性が示唆された。

Here, we determined when medaka juveniles display schooling behavior during the developmental stages after hatching. We also studied whether social isolated juvenile changes schooling behavior and whether social isolation affects the expression of neuropeptide hormone (AVT). Juveniles exhibited schooling behavior at 2 weeks post-hatch (wph). After then, schooling behavior was increased until 8 wph. AVT expression was increased at 2 wph, and then, an increase of AVT expression was found until 12 wph. Schooling behavior in a juvenile isolated from 1 wph to 2 wph was decreased when isolated fish was introduced to grouped conspecifics. In isolated fish, AVT expression was slightly decreased. In the Avt gene knock-out medaka, the juveniles did not exhibit schooling behavior at 2 wph. This study indicates that social interaction among juveniles is necessary for behavioral development in medaka fish. It also suggests that AVT are associated with behavioral development.

(1-1) Melanin-concentrating hormone (MCH) had a slight suppressing effect on feeding behavior of Rhabdoblennius nitidus males, but there was no significant difference in MCH levels between males cannibalizing eggs and males consuming food. (1-2) Administration of prolactin-releasing peptide (PrRP) showed a suppression effect on feeding during the breeding season and food provisioning experiments induced increase in parental care behaviors. These results are consistent with the prolactin hypothesis. (2) In the experiments inducing total-filial cannibalism, males cannibalizing all eggs showed significantly lower plasma 11-KT levels than males who did not, suggesting that males cannibalize eggs when they become incapable of courting due to the low levels of 11-KT. (3) The egg-water exposure experiments induced non care-giving males to parental care behaviors. This suggests the possibility that some kind of chemicals released from eggs stimulate the expression of parental care of males.

In this study, we examined the neural mechanisms underlying the regulation of aggressive and submissive behaviors by neuropeptide hormone (arginine vasotocin; AVT) in male medaka fish. We demonstrated that the neurons with high expression of AVT mainly send their fibers into the distinct brain regions between the aggressive (dominant) male and the submissive (subordinate) male after their competition. The different subtypes of AVT-receptors (AVT-R) was shown at a high level in each brain region of the dominants and the subordinates. In addition, we conducted a preliminary contest between two males of the AVT-R gene knock-out medaka. These males have similar high aggression throughout the contest.

Little is still known about mutagenic effect in fetal animals of fission neutrons. The present study was conducted in which fetal mice heterozygous at the Dlb-1 locus were irradiated at the organogenic stage with fission neutrons, X-rays at a high dose rate or gamma-rays at a low dose rate. Teratogenic and clastogenic effects of these radiations in fetal ICR mice were also investigated. Irrespective of the kind of endpoints used, fission neutrons were the most effective, followed by X-rays and gamma-rays in this sequence. Induced rates of mutations by fission neutrons determined at two time points during the organogenic stage do not differ significantly from each other. The same was true for the induction by other radiations. These results suggest fairly uniform sensitivity of fetal somatic cells to ionizing radiation for mutation induction during the organogenic stage in contrast to a marked shift in X-ray sensitivity seen for the induction of malformation.

(1) The male sexual hormone testosterone, which controls brood cycling of Rhabdoblennius nitidus males, has no effect on the reduction of their immune function. The base sequence of a pituitary hormone prolactin (PRL), which is expected to promote parental care behavior in animals, was determined. (2) The nest egg-removal and -adding experiments strongly suggested that the presence of eggs in the nests is a key stimulus for the switch between reproductive phases by regulating plasma androgen levels of the males. (3) There was no significant relationship between male body condition and the number of cannibalized eggs. These results suggested that males cannibalize all eggs not to take nutritional resource but to delete eggs from the nests. (4) The effects of peptide hormones neuropeptide-Y (NPY) and orexin (ORX) on male feeding activity were examined to discriminate total-filial cannibalism from feeding behavior.

We demonstrated the increased incidence of congenital defects in the offspring of male mice exposed in utero to synthetic estrogens and that the induction of malformations by the estrogens showed a clear threshold effect. Since estrogens have been reported to be non-genotoxic, epigenetic mechanisms may be involved in the transgenerational teratogenesis by estrogen. The expression patterns of Dnmts mRNA, global DNA methylation levels in testicular cells of embryos exposed to estrogen drugs or in sperm of mature male mice exposed prenatally to estrogen drugs were different from those in the controls. These results support that, when evaluating the toxicities of environmental chemicals, epigenetic effects such as DNA methylation should be taken into account.

本研究は、異なる種族の野生型メダカ(北日本集団および南日本集団)を用いて、魚類のなわばり行動すなわち他個体への攻撃行動の誘発に関連する新たな生体内因子を探索することを目的として実施した。材料には、従来から研究代表者が飼育維持してきた北日本集団(新潟産)および南日本集団(奈良産)のクローズドコロニーを用いた。昨年度までに、雌雄共に奈良産の方が新潟産に比べて高い攻撃性を有することを明らかにした。また、安静な条件下で単独飼育した奈良産および新潟産の脳視索前野におけるアルギニンバソトシン(AVT)含有ニューロン数が奈良産の方で新潟産に比べて多いことを明らかにした。視索前野におけるAVT発現は、鳥類や両生類において攻撃行動に深く関与することが報告されている。そこで本年度は、両種族の攻撃行動に際する視索前野AVT含有ニューロンの動態について精査した。実験では、雄の奈良産または新潟産をそれぞれ個別に3日間馴化後、共通の標的であるヒメダカ雄と混合飼育し、ヒメダカに対する攻撃行動を観察するとともに、混合飼育直後の脳視索前野におけるAVT含有ニューロン数および細胞体サイズを計測した。その結果、奈良産では、攻撃行動回数およびAVT含有ニューロンの数ならびに細胞体サイズのいずれにおいても新潟産に比べて約2倍高い値を示した。この際、AVT含有ニューロン数は安静時とほぼ同値であったが、安静時には認められなかった顕著に大きい細胞体サイズを有するAVT含有ニューロンが確認された。以上の結果は、ニホンメダカ種族間における攻撃性の違いに、脳視索前野のAVTニューロン細胞体サイズの違いが深く関連することを示唆している。