Performance information

• Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric Müllerian duct development by eliminating asymmetric gene expression in Japanese quail embryos
  Natsuko Abe; Akari Sakiyama; Maho Suzuki; Tin-Tin Win-Shwe; Takehiro Suzuki; Takaharu Kawashima; Shinji Tsukahara
  Toxicological Sciences, Volume：199, Number：2, First page：210, Last page：226, 2024, [Reviewed], [Last, Corresponding]
  Abstract
  
  In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3β-HSD) and estrogen receptor-β, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.
  Oxford University Press (OUP), Scientific journal
  DOI：https://doi.org/10.1093/toxsci/kfae033
  DOI ID：10.1093/toxsci/kfae033, ISSN：1096-6080, eISSN：1096-0929
• In ovo o,p′‐DDT exposure induces malformation of reproductive organs and alters the expression of genes controlling sexual differentiation in Japanese quail embryo　　　　　　　　　　　　　　　
  Tin‐Tin Win‐Shwe; Natsuko Abe; Akari Sakiyama; Maho Suzuki; Kazuhiro Sano; Takaharu Kawashima; Shinji Tsukahara
  Journal of Applied Toxicology, Volume：44, Number：5, First page：699, Last page：711, 2024, [Reviewed], [Last, Corresponding]
  Abstract
  
  In ovo exposure to o,p′‐dichloro‐diphenyl‐trichloroethane (o,p′‐DDT) impairs reproduction by inducing malformation of the reproductive organs in birds, although the mechanism remains unclear. Here, we examined the effects of o,p′‐DDT on the development of the reproductive organs, the expression of genes controlling sexual differentiation, and the plasma concentrations of testosterone and estradiol in Japanese quail embryos. o,p′‐DDT‐containing sesame oil was injected into the yolk sac on Embryonic Day (E) 3 at a dose of 500, 2,000, or 8,000 μg per egg. On E15, the reproductive organs were observed; the gonads and Müllerian ducts (MDs) were sampled to measure the mRNA of steroidogenic enzymes, sex steroid receptors, anti‐Müllerian hormone (AMH), and AMH receptor 2 (AMHR2); blood samples were collected to assay plasma testosterone and estradiol levels; and the gonads were used for histological analysis. o,p′‐DDT dose‐dependently increased the prevalence of hypertrophic MDs in females and residual MDs in males. In female MDs, o,p′‐DDT dose‐dependently decreased estrogen receptor (ER) α, ERβ, and AMHR2 mRNA expression. o,p′‐DDT dose‐dependently induced left‐biased asymmetry of testis size, and ovary‐like tissue was found in the left testis after exposure to 8,000 μg per egg o,p′‐DDT, although asymmetric gene expression did not occur. o,p′‐DDT did not affect ovarian tissue but did decrease 17α‐hydroxylase/C17‐20 lyase mRNA expression and dose‐dependently increased ERβ mRNA expression. o,p′‐DDT decreased plasma testosterone concentrations in females. These findings suggest that o,p′‐DDT induces hypertrophy of the MDs and ovarian tissue formation in the left testis. Abnormal MD development may be linked to altered gene expression for sensing estrogens and AMH signals.
  Wiley, Scientific journal
  DOI：https://doi.org/10.1002/jat.4571
  DOI ID：10.1002/jat.4571, ISSN：0260-437X, eISSN：1099-1263
• Two step actions of testicular androgens in the organization of a male-specific neural pathway from the medial preoptic area to the ventral tegmental area for modulating sexually motivated behavior　　　　　　　　　　　　　　　
  Morishita M; Kobayashi K; Mitsuzuka M; Takagi R; Ono K; Monma R; Tsuneoka Y; Horio S; Tsukahara S
  Journal of Neuroscience, Volume：43, Number：44, First page：7322, Last page：7336, 2023, [Reviewed], [Last, Corresponding], ［International magazine］
  The medial preoptic area (MPOA) is a sexually dimorphic region of the brain that regulates social behaviors. The sexually dimorphic nucleus (SDN) of the MPOA has been studied to understand sexual dimorphism, although the anatomy and physiology of the SDN is not fully understood. Here, we characterized SDN neurons that contribute to sexual dimorphism and investigated the mechanisms underlying the emergence of such neurons and their roles in social behaviors. A target-specific neuroanatomical study using transgenic mice expressing Cre recombinase under the control of Calb1, a gene expressed abundantly in the SDN, revealed that SDN neurons are divided into two subpopulations: GABA neurons projecting to the ventral tegmental area (VTA), where they link to the dopamine system (CalbVTA neurons) and GABA neurons that extend axons in the MPOA or project to neighboring regions (CalbnonVTA neurons). CalbVTA neurons were abundant in males, but were scarce or absent in females. There was no difference in the number of CalbnonVTA neurons between sexes. Additionally, we found that emergence of CalbVTA neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. Chemogenetic analyses of CalbVTA neurons indicated a role in modulating sexual motivation in males. Knockdown of Calb1 in the MPOA reduced the intromission required for males to complete copulation. These findings provide strong evidence that a male-specific neural pathway from the MPOA to the VTA is organized by the two-step actions of testicular androgens for the modulation of sexually motivated behavior.Significance StatementThe medial preoptic area (MPOA) is a sexually dimorphic region of the brain that regulates social behaviors, although its sexual dimorphism is not fully understood. Here, we describe a population of MPOA neurons that contribute to the sexual dimorphism. These neurons only exist in masculinized brains, and they project their axons to the ventral tegmental area, where they link to the dopamine system. Emergence of such neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. These MPOA neurons endow masculinized brains with a neural pathway from the MPOA to the ventral tegmental area and modulate sexually motivated behavior in males.
  English, Scientific journal
  DOI：https://doi.org/10.1523/JNEUROSCI.0361-23.2023
  DOI ID：10.1523/JNEUROSCI.0361-23.2023, PubMed ID：37722849
• Sexually dimorphic expression of sexual differentiation genes in the internal genital organs of Japanese quail embryos　　　　　　　　　　　　　　　
  Shinji Tsukahara; Masahiro Morishita; Shiho Sasaki; Kanta Wakayama; Kaito Kobayashi; Koichi Ohno; Takaharu Kawashima
  General and Comparative Endocrinology, Volume：314, First page：113917, Last page：113917, Dec. 2021, [Reviewed], [Lead, Corresponding], ［International magazine］
  Japanese quail (Coturnix japonica) is an avian model used to evaluate the reproductive and developmental toxicity of chemicals. The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail, NIES-L, which may be a better model because of its highly inbred characteristics. To understand sexual differentiation of the reproductive organs and the value of using NIES-L quails for avian toxicity assessment, we profiled estradiol and androgen plasma levels by enzyme-linked immunosorbent assay; the mRNA levels of estrogen receptor-α (ERα), ERβ, and androgen receptor (AR) in the gonads, Müllerian ducts, Wolffian ducts; and the mRNA levels of steroidogenic enzymes, cholesterol side chain cleavage enzyme (P450scc), 17α-hydroxylase/C17-20 lyase (P45017α, lyase), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and aromatase (P450arom), anti-Müllerian hormone (AMH), and AMH receptor type 2 (AMHR2) in the gonads of NIES-L Japanese quails on embryonic days 9, 12, and 15 using a real-time quantitative PCR method. The plasma estradiol concentration was higher in females than males on these embryonic days, but no sex difference was found in the plasma androgens. The mRNA levels of all examined steroidogenic enzymes were significantly higher in female than male embryos. In particular, the P450arom mRNA levels showed a striking sex difference: P450arom was expressed in female but not male gonads. In contrast, the AMH and AMHR2 mRNA levels in the gonads were higher in males than females. The ERα, ERβ, and AR mRNA levels increased in the left female gonad and peaked on embryonic day 15, but not in the left and right male gonads; therefore, there was a female-biased sex difference. The ERα, ERβ, and AR mRNA levels in the left Müllerian duct, but not in the right Müllerian duct, of females increased and peaked on embryonic day 15, which resulted in asymmetric mRNA levels. The Wolffian ducts expressed ERα, ERβ, and AR in both sexes, and no sex difference or asymmetry of mRNA levels was found. The information obtained from this study helps elucidate the molecular endocrinological basis of sexual dimorphism formation of reproductive organs and clarify the value of NIES-L quails for toxicity assessment.
  Elsevier BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.ygcen.2021.113917
  DOI ID：10.1016/j.ygcen.2021.113917, ISSN：0016-6480, PubMed ID：34555414
• Neuronal activation of the sexually dimorphic nucleus of the preoptic area in female and male rats during copulation and its sex differences　　　　　　　　　　　　　　　
  Masahiro Morishita; Arisa Kamada; Shinji Tsukahara
  Neuroscience Letters, Volume：755, First page：135915, Last page：135915, Jun. 2021, [Reviewed], [Last, Corresponding], ［International magazine］
  The medial preoptic area, which plays an essential role in the control of sexual behavior in rats, contains a sexually dimorphic nucleus that consists of neurons expressing calbindin-D28 K (Calb) that is referred to as the CALB-SDN. The CALB-SDN is larger and contains more Calb neurons in males than in females. The physiological functions of the CALB-SDN are not fully understood; however, CALB-SDN neurons are activated during sexual behavior in males, suggesting that the male CALB-SDN is involved in regulation of sexual behavior. However, no information exists about the physiological functions of the female CALB-SDN. In the present study, we performed an immunohistochemical analysis of c-Fos, a neuronal activity marker, in the CALB-SDN of female and male rats that had copulated with conspecifics of the opposite sex to determine whether neurons of the female CALB-SDN are activated during copulation and whether the neuronal activity of the CALB-SDN differs between sexes. The numbers of c-Fos-immunoreactive cells with or without Calb-immunoreactivity (c-Fos+/Calb+ and c-Fos+/Calb- cells) were greater in the CALB-SDN of rats that had copulated than in rats that had not copulated in each sex. Although the number of Calb+ cells in the CALB-SDN was smaller in females than in males, the increase in the number of c-Fos+/Calb+ cells in the female CALB-SDN with copulation was comparable to that in the male CALB-SDN with copulation. The increase in the number of c-Fos+/Calb- cells in the CALB-SDN with copulation was more prominent in males than in females. These results suggest that CALB-SDN neurons are activated during copulation in both sexes. The patterns of neuronal activation in the CALB-SDN during copulation may differ between sexes.
  Elsevier BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2021.135915
  DOI ID：10.1016/j.neulet.2021.135915, ISSN：0304-3940, PubMed ID：33905774
• The role of central corticotrophin‐releasing factor receptor signalling in plasma glucose maintenance through ghrelin secretion in calorie‐restricted mice　　　　　　　　　　　　　　　
  Risa Kimura; Daisuke Kondo; Shota Takemi; Miyuki Fujishiro; Shinji Tsukahara; Takafumi Sakai; Ichiro Sakata
  Journal of Neuroendocrinology, Volume：33, Number：3, First page：e12961, Mar. 2021, [Reviewed], ［International magazine］
  Under severe calorie restriction (CR), the ghrelin-growth hormone axis in mice is involved in the maintenance of plasma glucose levels. Ghrelin, a stomach-derived acylated peptide, is up-regulated by the sympathetic nerve in the negative energy status. Central corticotrophin-releasing factor receptor (CRF-R) signalling stimulates the sympathetic tone. The present study aimed to examine the effect of central CRF-R signalling on the maintenance of plasma glucose concentrations in severe calorie-restricted mice with the involvement of ghrelin. Intracerebroventricular injections of urocorin-1 and urocorin-2, which are natural ligands for CRF-R1 and CRF-R2, elevated plasma ghrelin concentrations and ghrelin elevation with an i.c.v. injection of urocorin-1 was cancelled by atenolol (β1 adrenergic receptor antagonist) administration. We then established a mice model of 60% CR and found that the administration of [d-Lys3]-GHRP-6 (a ghrelin receptor antagonist) in mice under 60% CR reduced the plasma glucose concentration more compared to the vehicle mice. Similarly, the atenolol injection in mice under 60% CR significantly reduced the plasma glucose concentration, which was rescued by the co-administration of ghrelin. An i.c.v. injection of the alpha helical CRH, a non-selective corticotrophin-releasing factor receptor antagonist, in mice under 60% CR significantly reduced the plasma glucose concentration, although the co-administration of α-helical CRH with ghrelin maintained plasma glucose levels. These results suggest that central CRF-R signalling is involved in the maintenance of plasma glucose levels in mice under severe CR via the sympathetic-ghrelin pathway.
  Wiley, English, Scientific journal
  DOI：https://doi.org/10.1111/jne.12961
  DOI ID：10.1111/jne.12961, ISSN：0953-8194, eISSN：1365-2826, PubMed ID：33675127
• Perinatal Exposure to Diesel Exhaust-Origin Secondary Organic Aerosol Induces Autism-Like Behavior in Rats
  Tin-Tin Win-Shwe; Chaw Kyi-Tha-Thu; Yuji Fujitani; Shinji Tsukahara; Seishiro Hirano
  International Journal of Molecular Sciences, Volume：22, Number：2, First page：538, Last page：538, Jan. 2021, [Reviewed]
  Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social communication, poor social interactions, and repetitive behaviors. We aimed to examine autism-like behaviors and related gene expressions in rats exposed to diesel exhaust (DE)-origin secondary organic aerosol (DE-SOA) perinatally. Sprague–Dawley pregnant rats were exposed to clean air (control), DE, and DE-SOA in the exposure chamber from gestational day 14 to postnatal day 21. Behavioral phenotypes of ASD were investigated in 10~13-week-old offspring using a three-chambered social behavior test, social dominance tube test, and marble burying test. Prefrontal cortex was collected to examine molecular analyses including neurological and immunological markers and glutamate concentration, using RT-PCR and ELISA methods. DE-SOA-exposed male and female rats showed poor sociability and social novelty preference, socially dominant behavior, and increased repetitive behavior. Serotonin receptor (5-HT(5B)) and brain-derived neurotrophic factor (BDNF) mRNAs were downregulated whereas interleukin 1 β (IL-β) and heme oxygenase 1 (HO-1) mRNAs were upregulated in the prefrontal cortex of male and female rats exposed to DE-SOA. Glutamate concentration was also increased significantly in DE-SOA-exposed male and female rats. Our results indicate that perinatal exposure to DE-SOA may induce autism-like behavior by modulating molecules such as neurological and immunological markers in rats.
  MDPI AG, Scientific journal
  DOI：https://doi.org/10.3390/ijms22020538
  DOI ID：10.3390/ijms22020538, eISSN：1422-0067
• The glutamatergic system in the preoptic area is involved in the retention of maternal behavior in maternally experienced female rats　　　　　　　　　　　　　　　
  Eri Okino; Sayaka Morita; Yumi Hoshikawa; Shinji Tsukahara
  Psychoneuroendocrinology, Volume：120, First page：104792, Last page：104792, Oct. 2020, [Reviewed], [Last, Corresponding]
  Elsevier BV, Scientific journal
  DOI：https://doi.org/10.1016/j.psyneuen.2020.104792
  DOI ID：10.1016/j.psyneuen.2020.104792, ISSN：0306-4530
• Sexually Dimorphic Formation of the Preoptic Area and the Bed Nucleus of the Stria Terminalis by Neuroestrogens　　　　　　　　　　　　　　　
  Shinji Tsukahara; Masahiro Morishita
  Frontiers in Neuroscience, Volume：14, First page：797, Last page：797, Jul. 2020, [Reviewed], [Lead, Corresponding], ［International magazine］
  Testicular androgens during the perinatal period play an important role in the sexual differentiation of the brain of rodents. Testicular androgens transported into the brain act via androgen receptors or are the substrate of aromatase, which synthesizes neuroestrogens that act via estrogen receptors. The latter that occurs in the perinatal period significantly contributes to the sexual differentiation of the brain. The preoptic area (POA) and the bed nucleus of the stria terminalis (BNST) are sexually dimorphic brain regions that are involved in the regulation of sex-specific social behaviors and the reproductive neuroendocrine system. Here, we discuss how neuroestrogens of testicular origin act in the perinatal period to organize the sexually dimorphic structures of the POA and BNST. Accumulating data from rodent studies suggest that neuroestrogens induce the sex differences in glial and immune cells, which play an important role in the sexually dimorphic formation of the dendritic synapse patterning in the POA, and induce the sex differences in the cell number of specific neuronal cell groups in the POA and BNST, which may be established by controlling the number of cells dying by apoptosis or the phenotypic organization of living cells. Testicular androgens in the peripubertal period also contribute to the sexual differentiation of the POA and BNST, and thus their aromatization to estrogens may be unnecessary. Additionally, we discuss the notion that testicular androgens that do not aromatize to estrogens can also induce significant effects on the sexually dimorphic formation of the POA and BNST.
  Frontiers Media SA, English, Scientific journal
  DOI：https://doi.org/10.3389/fnins.2020.00797
  DOI ID：10.3389/fnins.2020.00797, eISSN：1662-453X, PubMed ID：32848568, PubMed Central ID：PMC7403479
• Detection and Characterization of Estrogen Receptor Beta Expression in the Brain with Newly Developed Transgenic Mice　　　　　　　　　　　　　　　
  Shoko Sagoshi; Sho Maejima; Masahiro Morishita; Satoshi Takenawa; Akito Otubo; Keiko Takanami; Tatsuya Sakamoto; Hirotaka Sakamoto; Shinji Tsukahara; Sonoko Ogawa
  Neuroscience, Volume：438, First page：182, Last page：197, Jul. 2020, [Reviewed], ［International magazine］
  Two types of nuclear estrogen receptors, ERα and ERβ, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERβ expression, detailed anatomical distribution and neurochemical characteristics of ERβ expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERβ-RFPtg, in which RFP was inserted downstream of ERβ BAC promotor. We verified RFP signals as ERβ by confirming: (1) high ERβ mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERβ mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERβ-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERβ, and those expressing exclusively either ERα or ERβ. The majority of PVN and DRN cells expressed only ERβ-RFP. Further, ERβ-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERβ-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERβ-RFPtg mice can be a powerful tool for future studies on ERβ function in the estrogenic regulation of social behaviors.
  Elsevier BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuroscience.2020.04.047
  DOI ID：10.1016/j.neuroscience.2020.04.047, ISSN：0306-4522, PubMed ID：32387645
• Actions of Peripubertal Gonadal Steroids in the Formation of Sexually Dimorphic Brain Regions in Mice　　　　　　　　　　　　　　　
  Masahiro Morishita; Ryoma Koiso; Shinji Tsukahara
  Endocrinology, Volume：161, Number：6, Jun. 2020, [Reviewed], [Last, Corresponding], ［International magazine］
  
  The calbindin-sexually dimorphic nucleus (CALB-SDN) and calbindin-principal nucleus of the bed nucleus of the stria terminalis (CALB-BNSTp) show male-biased sex differences in calbindin neuron number. The ventral part of the BNSTp (BNSTpv) exhibits female-biased sex differences in noncalbindin neuron number. We previously reported that prepubertal gonadectomy disrupts the masculinization of the CALB-SDN and CALB-BNSTp and the feminization of the BNSTpv. This study aimed to determine the action mechanisms of testicular androgens on the masculinization of the CALB-SDN and CALB-BNSTp and whether ovarian estrogens are the hormones that have significant actions in the feminization of the BNSTpv. We performed immunohistochemical analyses of calbindin and NeuN, a neuron marker, in male mice orchidectomized on postnatal day 20 (PD20) and treated with cholesterol, testosterone, estradiol, or dihydrotestosterone during PD20-70, female mice ovariectomized on PD20 and treated with cholesterol or estradiol during PD20-70, and PD70 mice gonadectomized on PD56. Calbindin neurons number in the CALB-SDN and CALB-BNSTp in males treated with testosterone or dihydrotestosterone, but not estradiol, was significantly larger than that in cholesterol-treated males. Noncalbindin neuron number in the BNSTpv in estradiol-treated females was significantly larger than that in cholesterol-treated females. Gonadectomy on PD56 had no significant effect on neuron numbers. Additionally, an immunohistochemical analysis revealed the expression of androgen receptors in the CALB-SDN and CALB-BNSTp of PD30 males and estrogen receptors-α in the BNSTpv of PD30 females. These results suggest that peripubertal testicular androgens act to masculinize the CALB-SDN and CALB-BNSTp without aromatization, and peripubertal ovarian estrogens act to feminize the BNSTpv.
  The Endocrine Society, English, Scientific journal
  DOI：https://doi.org/10.1210/endocr/bqaa063
  DOI ID：10.1210/endocr/bqaa063, ISSN：0013-7227, eISSN：1945-7170, PubMed ID：32303738
• Estrogenic action by tris(2,6-dimethylphenyl) phosphate impairs the development of female reproductive functions.　　　　　　　　　　　　　　　
  Kazuhiro Sano; Hidenori Matsukami; Go Suzuki; Nang Thinn Thinn Htike; Masahiro Morishita; Tin-Tin Win-Shwe; Shunji Hashimoto; Takaharu Kawashima; Tomohiko Isobe; Shoji F Nakayama; Shinji Tsukahara; Fumihiko Maekawa
  Environment international, Volume：138, First page：105662, Last page：105662, May 2020, [Reviewed], ［International magazine］
  Developmental exposure to environmental chemicals with estrogen-like activity is suspected to permanently impair women's health. In this study, a mouse model was used to evaluate whether tris(2,6-dimethylphenyl) phosphate (TDMPP), a chemical with a putative estrogen-like action, impairs sexual differentiation of the brain. Either TDMPP and 17β-estradiol (E2) as positive controls or sesame oil as a negative control were administered subcutaneously to dams from gestational day (GD) 14 to parturition, and to pups from postnatal day (PND) 0 to 9. Precocious puberty, irregular estrous cycles, and a lowered lordosis response were found in the TDMPP- and E2-treated groups. A certain amount of TDMPP and its metabolites in the perinatal brain and the masculinization of sexual dimorphic nuclei in the hypothalamus of female mice after treatment were also detected. The experimental evidence demonstrates that TDMPP directly enters the fetal and neonatal brain, thereby inducing changes of sex-related brain structures and impairing female reproductive functions.
  English, Scientific journal
  DOI：https://doi.org/10.1016/j.envint.2020.105662
  DOI ID：10.1016/j.envint.2020.105662, PubMed ID：32203809
• Estrogenic regulation of social behavior and sexually dimorphic brain formation.
  Ogawa S; Tsukahara S; Choleris E; Vasudevan N
  Volume：110, First page：46, Last page：59, 2020, [Reviewed]
  English
  DOI：https://doi.org/10.1016/j.neubiorev.2018.10.012
  DOI ID：10.1016/j.neubiorev.2018.10.012
• Region-specific effects of copulation on dendritic spine morphology and gene expression related to spinogenesis in the medial preoptic nucleus of male rats.　　　　　　　　　　　　　　　
  Shizuka Nakashima; Masahiro Morishita; Kanna Ueno; Shinji Tsukahara
  Psychoneuroendocrinology, Volume：108, First page：1, Last page：13, 2019, [Reviewed], ［International magazine］
  The medial preoptic nucleus (MPN) plays an essential role in the control of male sexual behavior. In rats, the central part of the MPN (MPNc) contains a sexually dimorphic nucleus exhibiting male-biased morphological sex differences. Although it has been suggested that the MPNc of male rats functions to induce sexual arousal, the mechanisms by which male rats are sexually aroused to successfully achieve copulation are poorly understood. We recently showed that increased neuronal activity in the MPNc of male rats during copulation is higher at their first copulation compared with later copulations, indicating that a plastic change in excitatory synaptic transmission occurs with copulatory experience. In this study, we tested the hypothesis that changes to dendritic spines at structural and molecular levels occur following copulatory experience. First, we examined the effects of at least two copulations on the morphology of dendrites and spines in the MPNc and in the lateral and medial parts of the MPN (MPNlm) of male rats. In the MPNc, the total number of dendrites and their branches, and the surface area of dendrites were not significantly affected by copulation. However, the copulatory experience, specifically experience of ejaculation, significantly reduced the density of mushroom spines but not of filopodia, thin or stubby spines in the MPNc. In the MPNlm, the copulatory experience, specifically experience of ejaculation, significantly increased the surface area of dendrites, although there was no significant effect of copulation on spine density. Next, we measured the mRNA levels of genes encoding actin-binding proteins related to spinogenesis after male rats had copulated for their first and second times. Copulatory stimuli, especially stimuli from ejaculation, significantly reduced the mRNA levels of drebrin A and spinophilin in the MPNc but not in the MPNlm. These results indicate that copulatory experiences, especially experience of ejaculation, reduce spine density in the MPNc of male rats, which may result, in part, from downregulation of genes encoding actin-binding proteins.
  English, Scientific journal
  DOI：https://doi.org/10.1016/j.psyneuen.2019.05.025
  DOI ID：10.1016/j.psyneuen.2019.05.025, PubMed ID：31174081
• Correction to: Female-biased sexual dimorphism of corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis.　　　　　　　　　　　　　　　
  Katsuya Uchida; Hiroko Otsuka; Masahiro Morishita; Shinji Tsukahara; Tatsuya Sato; Kenji Sakimura; Keiichi Itoi
  Biology of sex differences, Volume：10, Number：6, First page：10, Last page：10, 2019, [Reviewed], ［International magazine］
  Following publication of the original article [1], we noticed a number of errors.
  English, Scientific journal
  DOI：https://doi.org/10.1186/s13293-019-0221-2
  DOI ID：10.1186/s13293-019-0221-2, PubMed ID：30782210, PubMed Central ID：PMC6381707
• Neonatal septal lesions perevent behavioral defeminization caused by neonatal treatment with estradiol in female rats.
  Kanaya M; Tsukahara S; Yamanouchi K
  Volume：694, First page：80, Last page：85, 2019, [Reviewed]
  English
  DOI：https://doi.org/10.1016/j.neulet.2018.11.032
  DOI ID：10.1016/j.neulet.2018.11.032
• Temporal expression patterns of genes related to sex steroid action in sexually dimorphic nuclei during puberty
  Moeko Kanaya; Masahiro Morishita; Shinji Tsukahara
  Frontiers in Endocrinology, Volume：9, Number：MAY, First page：213, May 2018, [Reviewed]
  Sex steroids play a major role in sexually dimorphic brain development during not only the perinatal period but also the pubertal period. We previously showed that, in male mice, the estrogen receptor-a (Esr1) and aromatase (Cyp19α1) genes are essential to the sexually dimorphic formation of the anteroventral periventricular nucleus (AVPV) and the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), but the estrogen receptor-β (Esr2) gene is not necessary. We also showed that the androgen receptor (Ar) gene is essential to the sexually dimorphic formation of the BNSTp. These genes are expressed in the AVPV and BNSTp of perinatal mice. However, it remains unknown whether these genes are expressed in the AVPV and BNSTp during puberty, and whether the expression, if any, differs by sex, age, and brain region. Here, we dissected the AVPV and BNSTp from Nissl-stained brain sections of male and female mice on postnatal day (PD) 20 (prepuberty), PD30 (puberty onset in females), PD40 (puberty onset in males), and PD60 (young adult) using a laser microdissection system. We then examined the mRNA levels of Esr1, Esr2, Cyp19α1, and Ar in these brain regions. In the AVPV, Esr1 mRNA levels were greater in females than males during PD20-60. Esr2 and Ar mRNA expressions did not differ between sexes. Ar mRNA levels were higher at PD30 than PD20. Cyp19α1 mRNA was not detected in the AVPV at PD20-60. In the BNSTp, Esr1 and Esr2 mRNA levels were higher in females than in males during PD20-60, although the mRNA levels of Cyp19α1 and Ar did not differ between sexes. Additionally, we revealed that orchiectomy at PD20 induced a failure of normal formation of the male BNSTp and testosterone replacement in the prepubertal period rescued the effect of orchiectomy at PD20. Taken together, it is suggested that pubertal testosterone transported to the AVPV is not converted to estradiol there and does not act via ESR1 and ESR2. By contrast, the formation of the male BNSTp may be affected by testicular testosterone during puberty via AR and/or via ESR1 after conversion to estradiol by CYP19A1.
  Frontiers Media S.A., English, Scientific journal
  DOI：https://doi.org/10.3389/fendo.2018.00213
  Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85046672261&origin=inward
  Scopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=85046672261&origin=inward
  DOI ID：10.3389/fendo.2018.00213, ISSN：1664-2392, eISSN：1664-2392, SCOPUS ID：85046672261
• Neuronal activity in the sagittalis nucleus of the hypothalamus after ovarian steroid hormone manipulation and sexual behavior in female rat
  Ivaylo Evgueniev Balabanov; Ken Ichi Matsuda; Hiroko Mori; Shunji Yamada; Keito Kitagawa; Yukina Yamamoto; Shinji Tsukahara; Masaki Tanaka
  Neuroscience Letters, Volume：671, First page：25, Last page：28, Apr. 2018, [Reviewed]
  During extended observation of estrogen receptor (ER) α-immunoreactive neurons in the hypothalamus, we previously identified a novel nucleus, the Sagittalis Nucleus of the Hypothalamus (SGN), in the interstitial area between the arcuate nucleus and the ventromedial hypothalamic nucleus. The SGN exhibits sexual dimorphism in its volume and cell count, and estrous cycle related variations in ERα-immunoreactivity. These characteristics of the SGN implicate the nucleus in sex-biased brain functions and behaviors. In this study, we examined involvement of the SGN in sexual arousal in female rats. Immunohistochemical staining of c-Fos, a marker of neuronal activity was performed after administration of an estrus-inducing dose of estrogen and progesterone in ovariectomized female rats. Analysis of microscopic images showed a significant increase in the number of c-Fos-expressing neurons in the SGN following hormonal manipulation. Moreover, neuronal activity in the region exhibited a further increase after each animal was coupled with a male and allowed to mate. These results suggest that the SGN plays an important role in sexual activity in female rat.
  Elsevier Ireland Ltd, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2018.02.008
  DOI ID：10.1016/j.neulet.2018.02.008, ISSN：1872-7972, SCOPUS ID：85041652129
• VGF in the Medial Preoptic Nucleus Increases Sexual Activity Following Sexual Arousal Induction in Male Rats.　　　　　　　　　　　　　　　
  Sho Maejima; Yuta Abe; Shohei Yamaguchi; Sergei Musatov; Sonoko Ogawa; Yasuhiko Kondo; Shinji Tsukahara
  Endocrinology, Volume：159, Number：12, First page：3993, Last page：4005, 2018, [Reviewed], ［International magazine］
  The central part of the medial preoptic nucleus (MPNc) is associated with sexual arousal induction in male rats. However, it is largely unclear how males are sexually aroused and achieve their first copulation. We previously reported that more MPNc neurons activate during the first copulation than the second copulation. In this study, to explore the molecules responsible for sexual arousal induction, we performed DNA microarray of the MPNc in sexually naive males and males after they copulated for their first and second times. We then performed quantitative PCR analyses to validate the results of the DNA microarray. Six genes were identified. Their expression increased following copulation and was higher in males after they copulated for the first time than after the second time. The genes encode transcription factors (Fos, Nfil3, and Nr4a3), a serine/threonine kinase (Sik1), an antioxidant protein (Srxn1), and a neuropeptide precursor VGF (Vgf), which may be the candidate genes responsible for sexual arousal induction. We examined the effects of Vgf knockdown in the MPNc on sexual partner preference and sexual behavior in sexually inexperienced and experienced males to determine the role of VGF in sexual arousal induction. A preference for estrous female rats was reinforced, and the latency of mount and intromission became short after sexually inexperienced males copulated for the first time. However, Vgf knockdown disrupted these phenomena. Vgf knockdown did not have any significant effect in sexually experienced males. VGF-derived neuropeptides presumably serve as an effector molecule to increase sexual activity following sexual arousal induction.
  English, Scientific journal
  DOI：https://doi.org/10.1210/en.2018-00804
  DOI ID：10.1210/en.2018-00804, PubMed ID：30371765
• Sexual experience reduces neuronal activity in the central part of the medial preoptic nucleus in male rats during sexual behavior.　　　　　　　　　　　　　　　
  Shohei Yamaguchi; Yuta Abe; Sho Maejima; Shinji Tsukahara
  Neuroscience letters, Volume：685, First page：155, Last page：159, 2018, [Reviewed], ［International magazine］
  The medial preoptic area (MPN) plays an important role in the control of male sexual behavior. In rats, the central part of the MPN (MPNc) is sexually dimorphic and contains a sexually dimorphic nucleus composed of neurons expressing calbindin-D28 K (CALB-SDN). Although the functions of the MPNc are not well understood, surgical destruction of the MPNc adversely affects the performance of sexual behavior in sexually naive males, but not in sexually experienced males, supporting the notion that the MPNc changes functionally with sexual experience. In this study, we aimed to determine the effects of sexual experience on the neuronal activity of the MPNc and CALB-SDN. Sexual behavior in sexually inexperienced males that had no experience of ejaculation, and experienced males that had ejaculated once was observed. After they displayed sexual behavior, the brains were sampled, and immunohistochemical analysis of c-Fos, a neuronal activity marker, in the MPNc and CALB-SDN was performed. The numbers of c-Fos-immunopositive cells with or without calbindin-D28K-immunoreactivity increased significantly in the MPNc and CALB-SDN following ejaculation in both sexually inexperienced and experienced males, although the numbers did not change significantly with exposure to estrous female odors, the first mount, and the first intromission before and after the first ejaculation. We further found that the number of c-Fos-immunopositive and calbindin-D28K-immunonegative cells in the MPNc, but not in the CALB-SDN, was significantly smaller in sexually experienced males than in sexually inexperienced males. These results suggest that a population of MPNc neurons, which is located outside the CALB-SDN and do not express calbindin-D28 K, are activated during the first copulation and then silent after acquisition of sexual experience.
  English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2018.08.037
  DOI ID：10.1016/j.neulet.2018.08.037, PubMed ID：30170041
• Strain differences in intermale aggression and possible factors regulating increased aggression in Japanese quail　　　　　　　　　　　　　　　
  Fumihiko Maekawa; Koki Nagino; Jiaxin Yang; Nang T. T. Htike; Shinji Tsukahara; Takayoshi Ubuka; Kazuyoshi Tsutsui; Takaharu Kawashima
  GENERAL AND COMPARATIVE ENDOCRINOLOGY, Volume：256, First page：63, Last page：70, 2018, [Reviewed]
  The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail (Coturnix japonica) known as NIES-L by rotation breeding in a closed colony for over 35 years; accordingly, the strain has highly inbred-like characteristics. Another strain called NIES-Brn has been maintained by randomized breeding in a closed colony to produce outbred-like characteristics. The current study aimed to characterize intermale aggressive behaviors in both strains and to identify possible factors regulating higher aggression in the hypothalamus, such as sex hormone and neuropeptide expression. Both strains displayed a common set of intermale aggressive behaviors that included pecking, grabbing, mounting, and cloacal contact behavior, although NIES-Brn quail showed significantly more grabbing, mounting, and cloacal contact behavior than did NIES-L quail. We examined sex hormone levels in the blood and diencephalon in both strains. Testosterone concentrations were significantly higher in the blood and diencephalon of NIES-Brn quail compared to NIES-L quail. We next examined gene expression in the hypothalamus of both strains using an Agilent gene expression microarray and real-time RT-PCR and found that gene expression of mesotocin (an oxytocin homologue) was significantly higher in the hypothalamus of NIES-Brn quail compared to NIES-L quail. Immunohistochemistry of the hypothalamus revealed that numbers of large cells (cell area > 500 mu m(2)) expressing mesotocin were significantly higher in the NIES-Brn strain compared to the NIES-L strain. Taken together, our findings suggest that higher testosterone and mesotocin levels in the hypothalamus may be responsible for higher aggression in the NIES-Brn quail strain. (C) 2017 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1016/j.ygcen.2017.07.025
  DOI ID：10.1016/j.ygcen.2017.07.025, ISSN：0016-6480, eISSN：1095-6840, Web of Science ID：WOS:000423013200011
• Sexual behavior-associated c-Fos induction in the sagittalis nucleus of the hypothalamus in male rat　　　　　　　　　　　　　　　
  Ken Ichi Matsuda; Kei Uchiyama; Hiroko Mori; Sho Maejima; Shohei Yamaguchi; Masaki Tanaka; Shinji Tsukahara
  NEUROSCIENCE LETTERS, Volume：661, First page：104, Last page：107, Nov. 2017, [Reviewed]
  The sagittalis nucleus of the hypothalamus (SGN) is a small nucleus located in the interstitial area between the arcuate and ventromedial nuclei of the hypothalamus in rats. The SGN exhibits male-biased sexual dimorphism and expresses estrogen receptor a and calbindin-D28 K. This suggests a contribution of the SGN to sexually differentiated brain function, but its functional role is unknown. In this study, neuronal activation in the SGN during sexual behavior in male rats was examined by c-Fos immunohistochemistry. The number of c-Fos-immunoreactive (c-Fos-ir) cells was elevated with only exposure to chemosensory cues of estrous females and significantly increased after the first mount. The first intromission and ejaculation did not induce further increases in the number of c-Fos-ir cells in the SGN. These findings suggest that the SGN is involved in regulation of the early phase of male sexual behavior, including motivation.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2017.09.053
  DOI ID：10.1016/j.neulet.2017.09.053, ISSN：0304-3940, eISSN：1872-7972, Web of Science ID：WOS:000415912800018
• Gonadal Hormone-Dependent Sexual Differentiation of a Female-Biased Sexually Dimorphic Cell Group in the Principal Nucleus of the Bed Nucleus of the Stria Terminalis in Mice　　　　　　　　　　　　　　　
  Masahiro Morishita; Sho Maejima; Shinji Tsukahara
  ENDOCRINOLOGY, Volume：158, Number：10, First page：3512, Last page：3525, Oct. 2017, [Reviewed]
  We recently reported a female-biased sexually dimorphic area in the mouse brain in the boundary region between the preoptic area and the bed nucleus of the stria terminalis (BNST). We reexamined this area and found that it is a ventral part of the principal nucleus of the BNST (BNSTp). The BNSTp is a male-biased sexually dimorphic nucleus, but the ventral part of the BNSTp (BNSTpv) exhibits female-biased sex differences in volume and neuron number. The volume and neuron number of the BNSTpv were increased in males by neonatal orchiectomy and decreased in females by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. Sex differences in the volume and neuron number of the BNSTpv emerged before puberty. These sex differences became prominent in adulthood with increasing volume in females and loss of neurons in males during the pubertal/adolescent period. Prepubertal orchiectomy did not affect the BNSTpv, although prepubertal ovariectomy reduced the volume increase and induced loss of neurons in the female BNSTpv. In contrast, the volume and neuron number of male-biased sexually dimorphic nuclei that are composed of mainly calbindin neurons and are located in the preoptic area and BNST were decreased by prepubertal orchiectomy but not affected by prepubertal ovariectomy. Testicular testosterone during the postnatal period may defeminize the BNSTpv via binding directly to the androgen receptor and indirectly to the estrogen receptor after aromatization, although defeminization may proceed independently of testicular hormones in the pubertal/adolescent period. Ovarian hormones may act to feminize the BNSTpv during the pubertal/adolescent period. A male-biased sexually dimorphic nucleus contains a cell group showing female-biased sex differences. This cell group is sexually differentiated by perinatal and pubertal gonadal hormones.
  OXFORD UNIV PRESS INC, English, Scientific journal
  DOI：https://doi.org/10.1210/en.2017-00240
  DOI ID：10.1210/en.2017-00240, ISSN：0013-7227, eISSN：1945-7170, Web of Science ID：WOS:000412298700039
• Moxd1 Is a Marker for Sexual Dimorphism in the Medial Preoptic Area, Bed Nucleus of the Stria Terminalis and Medial Amygdala　　　　　　　　　　　　　　　
  Yousuke Tsuneoka; Shinji Tsukahara; Sachine Yoshida; Kenkichi Takase; Satoko Oda; Masaru Kuroda; Hiromasa Funato
  FRONTIERS IN NEUROANATOMY, Volume：11, First page：26, Mar. 2017, [Reviewed]
  The brain shows various sex differences in its structures. Various mammalian species exhibit sex differences in the sexually dimorphic nucleus of the preoptic area (SDNPOA) and parts of the extended amygdala such as the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr) and posterodorsal part of the medial amygdala (MePD). The SDN-POA and BNSTpr are male-biased sexually dimorphic nuclei, and characterized by the expression of calbindin D-28K (calbindin 1). However, calbindinimmunoreactive cells are not restricted to the SDN-POA, but widely distributed outside of the SDN-POA. To find genes that are more specific to sexually dimorphic nuclei, we selected candidate genes by searching the Allen brain atlas and examined the detailed expressions of the candidate genes using in situ hybridization. We found that the strong expression of monooxygenase DBH-like 1 (Moxd1) was restricted to the SDN-POA, BNSTpr and MePD. The numbers of Moxd1-positive cells in the SDN-POA, BNSTpr and MePD in male mice were larger than those in female mice. Most of the Moxd1positive cells in the SDN-POA and BNSTpr expressed calbindin. Neonatal castration of male mice reduced the number of Moxd1-positive cells in the SDN-POA, whereas gonadectomy in adulthood did not change the expression of the Moxd1 gene in the SDN-POA in both sexes. These results suggest that the Moxd1 gene is a suitable marker for sexual dimorphic nuclei in the POA, BNST and amygdala, which enables us to manipulate sexually dimorphic neurons to examine their roles in sex-biased physiology and behaviors.
  FRONTIERS MEDIA SA, English, Scientific journal
  DOI：https://doi.org/10.3389/fnana.2017.00026
  DOI ID：10.3389/fnana.2017.00026, ISSN：1662-5129, Web of Science ID：WOS:000397397100003
• A Sexually Dimorphic Area of the Dorsal Hypothalamus in Mice and Common Marmosets　　　　　　　　　　　　　　　
  Yadanar Moe; Chaw Kyi-Tha-Thu; Tomoko Tanaka; Hiroto Ito; Satowa Yahashi; Ken-Ichi Matsuda; Mitsuhiro Kawata; Goro Katsuura; Fumihiro Iwashige; Ichiro Sakata; Atsushi Akune; Akio Inui; Takafumi Sakai; Sonoko Ogawa; Shinji Tsukahara
  ENDOCRINOLOGY, Volume：157, Number：12, First page：4817, Last page：4828, Dec. 2016, [Reviewed]
  We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.
  ENDOCRINE SOC, English, Scientific journal
  DOI：https://doi.org/10.1210/en.2016-1428
  DOI ID：10.1210/en.2016-1428, ISSN：0013-7227, eISSN：1945-7170, Web of Science ID：WOS:000392840400032
• Molecular Cloning of Ghrelin and Characteristics of Ghrelin-Producing Cells in the Gastrointestinal Tract of the Common Marmoset (Callithrix jacchus)　　　　　　　　　　　　　　　
  Shota Takemi; Ichiro Sakata; Auvijit Saha Apu; Shinji Tsukahara; Satowa Yahashi; Goro Katsuura; Fumihiro Iwashige; Atsushi Akune; Akio Inui; Takafumi Sakai
  ZOOLOGICAL SCIENCE, Volume：33, Number：5, First page：497, Last page：504, Oct. 2016, [Reviewed]
  Ghrelin was first isolated from human and rat as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In the present study, we determined the ghrelin cDNA sequence of the common marmoset (Callithrix jacchus), a small-bodied New World monkey, and investigated the distribution of ghrelin-producing cells in the gastrointestinal tract and localization profiles with somatostatin-producing cells. The marmoset ghrelin cDNA coding region was 354 base pairs, and showed high homology to that in human, rhesus monkey, and mouse. Marmoset ghrelin consists of 28 amino acids, and the N-terminal region is highly conserved as found in other mammalian species. Marmoset preproghrelin and mature ghrelin have 86.3% and 92.9% homology, respectively, to their human counterparts. Quantitative RT-PCR analysis showed that marmoset ghrelin mRNA is highly expressed in the stomach, but it is not detected in other tissues of the gastrointestinal tract. In addition, a large number of ghrelin mRNA-expressing cells and ghrelin-immunopositive cells were detected in the mucosal layer of the stomach, but not in the myenteric plexus. Moreover, all the ghrelin cells examined in the stomach were observed to be closed-type. Double staining showed that somatostatin-immunopositive cells were not co-localized with ghrelin-producing cells; however, a subset of somatostatin-immunopositive cells is directly adjacent to ghrelin-immunopositive cells. These findings suggest that the distribution of ghrelin cells in marmoset differs from that in rodents, and thus the marmoset may be a more useful model for the translational study of ghrelin in primates. In conclusion, we have clarified the expression and cell distribution of ghrelin in marmoset, which may represent a useful model in translational study.
  ZOOLOGICAL SOC JAPAN, English, Scientific journal
  DOI：https://doi.org/10.2108/zs160020
  DOI ID：10.2108/zs160020, ISSN：0289-0003, Web of Science ID：WOS:000385345800007
• A comparative study of sex difference in calbindin neurons among mice, musk shrews, and Japanese quails　　　　　　　　　　　　　　　
  Yadanar Moe; Tomoko Tanaka; Masahiro Morishita; Ryoko Ohata; Chihiro Nakahara; Takaharu Kawashima; Fumihiko Maekawa; Ichiro Sakata; Takafumi Sakai; Shinji Tsukahara
  NEUROSCIENCE LETTERS, Volume：631, First page：63, Last page：69, Sep. 2016, [Reviewed]
  The medial preoptic nucleus (MPN) and the bed nucleus of the stria terminalis (BNST) of mice contain sexually dimorphic nuclei (SDNs) that are larger and have more neurons expressing calbindin D-28K (CB), a calcium-binding protein, in males than females. However, it is largely unknown whether such SDNs exist in species other than rodents. In this study, we performed an immunohistochemical study of CB in the MPN and BNST of musk shrews and Japanese quails to examine the existence of homologs of SDNs in mice. Like mice, musk shrews had a SDN exhibiting male-biased sex differences in volume and CB-immunoreactive (ir) cell number in the MPN. The BNST of musk shrews also contained a male-biased SDN, but consisted of non-CB neurons. The paratenial thalamic nucleus of musk shrews, but not mice, had more CB-ir cells in males than females. In Japanese quails of both sexes, CB-ir cells in the MPN and BNST were extremely small in number and did not cluster. These results suggest that the distribution of CB neurons differs among these species. Musk shrews may have a homolog of the SDN composed of CB neurons in the MPN of mice. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2016.08.018
  DOI ID：10.1016/j.neulet.2016.08.018, ISSN：0304-3940, eISSN：1872-7972, Web of Science ID：WOS:000384786600011
• Pubertal activation of estrogen receptor a in the medial amygdala is essential for the full expression of male social behavior in mice　　　　　　　　　　　　　　　
  Kazuhiro Sano; Mariko Nakata; Sergei Musatov; Masahiro Morishita; Toshiro Sakamoto; Shinji Tsukahara; Sonoko Ogawa
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Volume：113, Number：27, First page：7632, Last page：7637, Jul. 2016, [Reviewed]
  Testosterone plays a central role in the facilitation of male-type social behaviors, such as sexual and aggressive behaviors, and the development of their neural bases in male mice. The action of testosterone via estrogen receptor (ER) alpha, after being aromatized to estradiol, has been suggested to be crucial for the full expression of these behaviors. We previously reported that silencing of ER alpha in adult male mice with the use of a virally mediated RNAi method in the medial preoptic area (MPOA) greatly reduced sexual behaviors without affecting aggressive behaviors whereas that in the medial amygdala (MeA) had no effect on either behavior. It is well accepted that testosterone stimulation during the pubertal period is necessary for the full expression of male-type social behaviors. However, it is still not known whether, and in which brain region, ER alpha is involved in this developmental effect of testosterone. In this study, we knocked down ER alpha in the MeA or MPOA in gonadally intact male mice at the age of 21 d and examined its effects on the sexual and aggressive behaviors later in adulthood. We found that the prepubertal knockdown of ER alpha in the MeA reduced both sexual and aggressive behaviors whereas that in the MPOA reduced only sexual, but not aggressive, behavior. Furthermore, the number of MeA neurons was reduced by prepubertal knockdown of ER alpha. These results indicate that ER alpha activation in the MeA during the pubertal period is crucial for male mice to fully express their male-type social behaviors in adulthood.
  NATL ACAD SCIENCES, English, Scientific journal
  DOI：https://doi.org/10.1073/pnas.1524907113
  DOI ID：10.1073/pnas.1524907113, ISSN：0027-8424, Web of Science ID：WOS:000379021700086
• Arsenic Exposure Induces Unscheduled Mitotic S Phase Entry Coupled with Cell Death in Mouse Cortical Astrocytes　　　　　　　　　　　　　　　
  Nang T. T. Htike; Fumihiko Maekawa; Haruka Soutome; Kazuhiro Sano; Sho Maejima; Kyaw H. Aung; Masaaki Tokuda; Shinji Tsukahara
  FRONTIERS IN NEUROSCIENCE, Volume：10, First page：297, Jun. 2016, [Reviewed]
  There is serious concern about arsenic in the natural environment, which exhibits neurotoxicity and increases the risk of neurodevelopmental disorders. Adverse effects of arsenic have been demonstrated in neurons, but it is not fully understood how arsenic affects other cell types in the brain. In the current study, we examined whether sodium arsenite (NaAsO2) affects the cell cycle, viability, and apoptosis of in vitro-cultured astrocytes isolated from the cerebral cortex of mice. Cultured astrocytes from transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were subjected to live imaging analysis to assess the effects of NaAsO2 (0, 1, 2, and 4 mu M) on the cell cycle and number of cells. Fucci was designed to express monomeric Kusabira Orange2 (mKO(2)) fused with the ubiquitylation domain of hCdt1, a marker of G1 phase, and monomeric Azami Green (mAG) fused with the ubiquitylation domain of hGem, a marker of S, G2, and M phases. NaAsO2 concentration-dependently decreased the peak levels of the mAG/mKO2 emission ratio when the ratio had reached a peak in astrocytes without NaAsO2 exposure, which was due to attenuating the increase in the mAG-expressing cell number. In contrast, the mAG/mKO(2) emission ratio and number of mAG-expressing cells were concentration-dependently increased by NaAsO2 before their peak levels, indicating unscheduled S phase entry. We further examined the fate of cells forced to enter S phase by NaAsO2. We found that most of these cells died up to the end of live imaging. In addition, quantification of the copy number of the glial fibrillary acidic protein gene expressed specifically in astrocytes revealed a concentration dependent decrease caused by NaAsO2. However, NaAsO2 did not increase the amount of nucleosomes generated from DNA fragmentation and failed to alter the gene expression of molecules relevant to unscheduled S phase entry-coupled apoptosis (p21, p53, E2F1, E2F4, and Gm36566). These findings suggest that NaAsO2 adversely affects the cell cycle and viability of astrocytes by inducing unscheduled S phase entry coupled with cell death that may be caused by mechanisms other than apoptosis.
  FRONTIERS MEDIA SA, English, Scientific journal
  DOI：https://doi.org/10.3389/fnins.2016.00297
  DOI ID：10.3389/fnins.2016.00297, ISSN：1662-453X, Web of Science ID：WOS:000379396400001
• Prenatal Exposure to Arsenic Impairs Behavioral Flexibility and Cortical Structure in Mice　　　　　　　　　　　　　　　
  Kyaw H. Aung; Chaw Kyi-Tha-Thu; Kazuhiro Sano; Kazuaki Nakamura; Akito Tanoue; Keiko Nohara; Masaki Kakeyama; Chiharu Tohyama; Shinji Tsukahara; Fumihiko Maekawa
  FRONTIERS IN NEUROSCIENCE, Volume：10, First page：137, Mar. 2016, [Reviewed]
  Exposure to arsenic from well water in developing countries is suspected to cause developmental neurotoxicity. Although, it has been demonstrated that exposure to sodium arsenite (NaAsO2) suppresses neurite outgrowth of cortical neurons in vitro, it is largely unknown how developmental exposure to NaAsO2 impairs higher brain function and affects cortical histology. Here, we investigated the effect of prenatal NaAsO2 exposure on the behavior of mice in adulthood, and evaluated histological changes in the prelimbic cortex (PrL), which is a part of the medial prefrontal cortex that is critically involved in cognition. Drinking water with or without NaAsO2 (85 ppm) was provided to pregnant C3H mice from gestational days 8 to 18, and offspring of both sexes were subjected to cognitive behavioral analyses at 60 weeks of age. The brains of female offspring were subsequently harvested and used for morphometrical analyses. We found that both male and female mice prenatally exposed to NaAsO2 displayed an impaired adaptation to repetitive reversal tasks. In morphometrical analyses of Nissl- or Golgi-stained tissue sections, we found that NaAsO2 exposure was associated with a significant increase in the number of pyramidal neurons in layers V and VI of the PrL, but not other layers of the PrL. More strikingly, prenatal NaAsO2 exposure was associated with a significant decrease in neurite length but not dendrite spine density in all layers of the PrL. Taken together, our results indicate that prenatal exposure to NaAsO2 leads to behavioral inflexibility in adulthood and cortical disarrangement in the PrL might contribute to this behavioral impairment.
  FRONTIERS MEDIA SA, English, Scientific journal
  DOI：https://doi.org/10.3389/fnins.2016.00137
  DOI ID：10.3389/fnins.2016.00137, ISSN：1662-453X, Web of Science ID：WOS:000373037400001
• Exposure of BALB/c Mice to Diesel Engine Exhaust Origin Secondary Organic Aerosol (DE-SOA) during the Developmental Stages Impairs the Social Behavior in Adult Life of the Males　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Chew Kyi-Tha-Thu; Yadanar Moe; Yuji Fujitani; Shinji Tsukahara; Seishiro Hirano
  FRONTIERS IN NEUROSCIENCE, Volume：9, First page：524, Jan. 2016, [Reviewed]
  Secondary organic aerosol (SOA) is a component of particulate matter (PM) 2.5 and formed in the atmosphere by oxidation of volatile organic compounds. Recently, we have reported that inhalation exposure to diesel engine exhaust (DE) originated SOA (DE-SOA) affect novel object recognition ability and impair maternal behavior in adult mice. However, it is not clear whether early life exposure to SOA during the developmental stages affect social behavior in adult life or not. In the present study, to investigate the effects of early life exposure to DE-SOA during the gestational and lactation stages on the social behavior in the adult life, BALB/c mice were exposed to clean air (control), DE, DE-SOA and gas without any PM in the inhalation chambers from gestational day 14 to postnatal day 21 for 5 h a day and 5 days per week. Then adult mice were examined for changes in their social behavior at the age of 13 week by a sociability and social novelty preference, social interaction with a juvenile mouse and light-dark transition test, hypothalamic mRNA expression levels of social behavior-related genes, estrogen receptor-alpha and oxytocin receptor as well as of the oxidative stress marker gene, heme oxygenase (HO)-1 by real-time RT-PCR method. In addition, hypothalamic level of neuronal excitatory marker, glutamate was determined by ELISA method. We observed that sociability and social novelty preference as well as social interaction were remarkably impaired, expression levels of estrogen receptor-alpha, oxytocin receptor mRNAs were significantly decreased, expression levels of HO-1 mRNAs and glutamate levels were significantly increased in adult male mice exposed to DE-SOA compared to the control ones. Findings of this study indicate early life exposure of BALB/c mice to DE-SOA may affect their late-onset hypothalamic expression of social behavior related genes, trigger neurotoxicity and impair social behavior in the males.
  FRONTIERS MEDIA SA, English, Scientific journal
  DOI：https://doi.org/10.3389/fnins.2015.00524
  DOI ID：10.3389/fnins.2015.00524, ISSN：1662-453X, Web of Science ID：WOS:000368586000001
• Sex differences in cells expressing green fluorescent protein under the control of the estrogen receptor-alpha promoter in the hypothalamus of mice　　　　　　　　　　　　　　　
  Chaw Kyi-Tha-Thu; Kota Okoshi; Hiroto Ito; Ken-Ichi Matsuda; Mitsuhiro Kawata; Shinji Tsukahara
  NEUROSCIENCE RESEARCH, Volume：101, First page：44, Last page：52, Dec. 2015, [Reviewed]
  Estradiol that originates from testicular testosterone and binds to estrogen receptor-alpha (ER alpha) during developing period acts to organize the male-type brain in mice. Here, we examined transgenic mice expressing green fluorescent protein (GFP) under the control of the ER alpha promoter, in which ER alpha-expressing cells in the brain can be visualized by GFP. Fluorescence microscopy revealed the existence of many GFP-expressing cells in the medial preoptic area, medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BNST), and striohypothalamic nucleus (StHy) of adult transgenic mice. Neuronal nuclear antigen, a neuron marker, but not glial fibrillary acidic protein, an astrocyte marker, was mostly expressed by GFP-expressing cells. Analysis of GFP expression area showed that adult females had higher GFP expression in a region including the ventral part of the BNST, StHy, and dorsal part of the MPN than in adult males. Such female-biased sex difference was also found in transgenic pups on postnatal day 5 and 8. The GFP expression area of adult females was decreased by postnatal treatment with testosterone or estradiol. These results indicate that GFP visualizes a sex difference of ER alpha-expressing neurons. The transgenic mice may be useful for the analysis of the sexual differentiation of the brain. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neures.2015.07.006
  DOI ID：10.1016/j.neures.2015.07.006, ISSN：0168-0102, eISSN：1872-8111, Web of Science ID：WOS:000364954800006
• A neural connection between the central part of the medial preoptic nucleus and the bed nucleus of the stria terminalis to regulate sexual behavior in male rats　　　　　　　　　　　　　　　
  Sho Maejima; Naoya Ohishi; Shohei Yamaguchi; Shinji Tsukahara
  NEUROSCIENCE LETTERS, Volume：606, First page：66, Last page：71, Oct. 2015, [Reviewed]
  The medial preoptic nucleus (MPN) is a regulatory center for male sexual behavior. It consists of sexually dimorphic structures that are male biased, and these structures are found in the central part of the MPN (MPNc). The bed nucleus of the stria terminalis (BNST) also participates in male sexual behavior, and receives efferent neural projections from the MPNc. In this study, we examined if MPNc neurons projecting to the BNST are activated in male rats displaying sexual behavior. Fluoro-Gold (FG; a retrograde neural tracer) was injected into the BNST of male rats, which were separated into two groups: (1) those in contact with estrus female rats and displayed sexual behavior followed by ejaculation and (2) those without contact with estrous female rats. In both groups, protein expression of c-Fos (a neuronal activity marker) and calbindin (a location marker of the MPNc) were detected by fluorescent immunohistochemistry. The number of c-Fos-immunoreactive cells with or without FG labeling in the MPNc was also measured. The number of c-Fos-immunoreactive cells significantly increased following ejaculation. Approximately 10% of FG-labeled cells in ejaculation male rats were immunoreactive for c-Fos, and this percentage value was significantly higher in this group compared with control male rats. Overall, these results suggest that efferent projections from the MPNc to the BNST function to control sexual behavior in male rats. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2015.08.047
  DOI ID：10.1016/j.neulet.2015.08.047, ISSN：0304-3940, eISSN：1872-7972, Web of Science ID：WOS:000363346900012
• Nano-Sized Secondary Organic Aerosol of Diesel Engine Exhaust Origin Impairs Olfactory-Based Spatial Learning Performance in Preweaning Mice　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Chaw Kyi-Tha-Thu; Yadanar Moe; Fumihiko Maekawa; Rie Yanagisawa; Akiko Furuyama; Shinji Tsukahara; Yuji Fujitani; Seishiro Hirano
  NANOMATERIALS, Volume：5, Number：3, First page：1147, Last page：1162, Sep. 2015, [Reviewed]
  The aims of our present study were to establish a novel olfactory-based spatial learning test and to examine the effects of exposure to nano-sized diesel exhaust-origin secondary organic aerosol (SOA), a model environmental pollutant, on the learning performance in preweaning mice. Pregnant BALB/c mice were exposed to clean air, diesel exhaust (DE), or DE-origin SOA (DE-SOA) from gestational day 14 to postnatal day (PND) 10 in exposure chambers. On PND 11, the preweaning mice were examined by the olfactory-based spatial learning test. After completion of the spatial learning test, the hippocampus from each mouse was removed and examined for the expressions of neurological and immunological markers using real-time RT-PCR. In the test phase of the study, the mice exposed to DE or DE-SOA took a longer time to reach the target as compared to the control mice. The expression levels of neurological markers such as the N-methyl-d-aspartate (NMDA) receptor subunits NR1 and NR2B, and of immunological markers such as TNF-alpha, COX2, and Iba1 were significantly increased in the hippocampi of the DE-SOA-exposed preweaning mice as compared to the control mice. Our results indicate that DE-SOA exposure in utero and in the neonatal period may affect the olfactory-based spatial learning behavior in preweaning mice by modulating the expressions of memory function-related pathway genes and inflammatory markers in the hippocampus.
  MDPI AG, English, Scientific journal
  DOI：https://doi.org/10.3390/nano5031147
  DOI ID：10.3390/nano5031147, ISSN：2079-4991, Web of Science ID：WOS:000362643900002
• Role of Environmental Chemical Insult in Neuronal Cell Death and Cytoskeleton Damage　　　　　　　　　　　　　　　
  Kyaw Htet Aung; Shinji Tsukahara; Fumihiko Maekawa; Keiko Nohara; Kazuaki Nakamura; Akito Tanoue
  BIOLOGICAL & PHARMACEUTICAL BULLETIN, Volume：38, Number：8, First page：1109, Last page：1112, Aug. 2015, [Reviewed]
  Environmental influences, such as chemical exposure, have long been considered potential risk factors for neurodegenerative disorders, including neuromuscular diseases. However, no definitive links between environmental chemical exposure and a pathogenic mechanism of neurodegenerative disease has yet been established. In this study, we describe that exposure to arsenic, an environmental pollutant naturally found in drinking water, induces neuronal cell death and alteration of morphology, particularly neurite outgrowth and in the cytoskeleton of neurons. Since progressive cell loss accompanied by the alteration of neuronal structures and cytoskeleton is considered the major pathologic feature of neurodegenerative disorders, arsenic-induced neurotoxicity might contribute to an etiologic mechanism of some neurodegenerative diseases. Further, we discuss the importance of in vitro assay, particularly an embryonic toxicity test, for assessing the neurotoxicity of chemicals, because most of chemicals found in our environment remain to be evaluated regarding their neurotoxicity risk for neurodegenerative diseases.
  PHARMACEUTICAL SOC JAPAN, English, Scientific journal
  DOI：https://doi.org/10.1248/bpb.b14-00890
  DOI ID：10.1248/bpb.b14-00890, ISSN：0918-6158, Web of Science ID：WOS:000358820000005
• Neural, hormonal and experiential control of sex-typical expression of social behavior.　　　　　　　　　　　　　　　
  Ogawa S; Tsuda MC; Sano K; Tsukahara S; Musatov S
  Volume：21, Number：3, First page：181, Last page：187, 2015, [Reviewed]
  English
• Effects of Diesel Engine Exhaust Origin Secondary Organic Aerosols on Novel Object Recognition Ability and Maternal Behavior in BALB/C Mice　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Yuji Fujitani; Chaw Kyi-Tha-Thu; Akiko Furuyama; Takehiro Michikawa; Shinji Tsukahara; Hiroshi Nitta; Seishiro Hirano
  INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH, Volume：11, Number：11, First page：11286, Last page：11307, Nov. 2014, [Reviewed]
  Epidemiological studies have reported an increased risk of cardiopulmonary and lung cancer mortality associated with increasing exposure to air pollution. Ambient particulate matter consists of primary particles emitted directly from diesel engine vehicles and secondary organic aerosols (SOAs) are formed by oxidative reaction of the ultrafine particle components of diesel exhaust (DE) in the atmosphere. However, little is known about the relationship between exposure to SOA and central nervous system functions. Recently, we have reported that an acute single intranasal instillation of SOA may induce inflammatory response in lung, but not in brain of adult mice. To clarify the whole body exposure effects of SOA on central nervous system functions, we first created inhalation chambers for diesel exhaust origin secondary organic aerosols (DE-SOAs) produced by oxidation of diesel exhaust particles caused by adding ozone. Male BALB/c mice were exposed to clean air (control), DE and DE-SOA in inhalation chambers for one or three months (5 h/day, 5 days/week) and were examined for memory function using a novel object recognition test and for memory function-related gene expressions in the hippocampus by real-time RT-PCR. Moreover, female mice exposed to DE-SOA for one month were mated and maternal behaviors and the related gene expressions in the hypothalamus examined. Novel object recognition ability and N-methyl-D-aspartate (NMDA) receptor expression in the hippocampus were affected in male mice exposed to DE-SOA. Furthermore, a tendency to decrease maternal performance and significantly decreased expression levels of estrogen receptor (ER)-alpha, and oxytocin receptor were found in DE-SOA exposed dams compared with the control. This is the first study of this type and our results suggest that the constituents of DE-SOA may be associated with memory function and maternal performance based on the impaired gene expressions in the hippocampus and hypothalamus, respectively.
  MDPI AG, English, Scientific journal
  DOI：https://doi.org/10.3390/ijerph111111286
  DOI ID：10.3390/ijerph111111286, ISSN：1660-4601, Web of Science ID：WOS:000345532000016
• Regional Difference in Sex Steroid Action on Formation of Morphological Sex Differences in the Anteroventral Periventricular Nucleus and Principal Nucleus of the Bed Nucleus of the Stria Terminalis　　　　　　　　　　　　　　　
  Moeko Kanaya; Mumeko C. Tsuda; Shoko Sagoshi; Kazuyo Nagata; Chihiro Morimoto; Chaw Kyi Tha Thu; Katsumi Toda; Shigeaki Kato; Sonoko Ogawa; Shinji Tsukahara
  PLOS ONE, Volume：9, Number：11, First page：e112616, Nov. 2014, [Reviewed]
  Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-alpha (ER alpha), but not estrogen receptor-beta (ER beta). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ER alpha, or ER beta genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ER alpha genes, but not the ERb gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ER alpha, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ER alpha mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ER alpha mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ERa during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.
  PUBLIC LIBRARY SCIENCE, English, Scientific journal
  DOI：https://doi.org/10.1371/journal.pone.0112616
  DOI ID：10.1371/journal.pone.0112616, ISSN：1932-6203, Web of Science ID：WOS:000345558500066
• Neuroanatorny and sex differences of the lordosis-inhibiting system in the lateral septum　　　　　　　　　　　　　　　
  Shinji Tsukahara; Moeko Kanaya; Korehito Yamanouchi
  FRONTIERS IN NEUROSCIENCE, Volume：8, First page：299, Sep. 2014, [Reviewed]
  Female sexual behavior in rodents, termed lordosis, is controlled by facilitatory and inhibitory systems in the brain. It has been well demonstrated that a neural pathway from the ventromedial hypothalamic nucleus (VMN) to the midbrain central gray (MCG) is essential for facilitatory regulation of lordosis. The neural pathway from the arcuate nucleus to the VMN, via the medial preoptic nucleus, in female rats mediates transient suppression of lordosis, until female sexual receptivity is induced. In addition to this pathway, other regions are involved in inhibitory regulation of lordosis in female rats. The lordosis-inhibiting systems exist not only in the female brain but also in the male brain. The systems contribute to suppression of heterotypical sexual behavior in male rats, although they have the potential ability to display lordosis. The lateral septum (LS) exerts an inhibitory influence on lordosis in both female and male rats. This review focuses on the neuroanatomy and sex differences of the lordosis-inhibiting system in the LS. The LS functionally and anatomically links to the MCG to exert suppression of lordosis. Neurons of the intermediate part of the LS (LSi) serve as lordosis-inhibiting neurons and project axons to the MCG. The LSi-MCG neural connection is sexually dimorphic, and formation of the male-like LSi-MCG neural connection is affected by aromatized testosterone originating from the testes in the postnatal period. The sexually dimorphic LSi-MCG neural connection may reflect the morphological basis of sex differences in the inhibitory regulation of lordosis in rats.
  FRONTIERS RESEARCH FOUNDATION, English
  DOI：https://doi.org/10.3389/fnins.2014.00299
  DOI ID：10.3389/fnins.2014.00299, ISSN：1662-453X, Web of Science ID：WOS:000346527100001
• The mechanisms underlying sexual differentiation of behavior and physiology in mammals and birds: relative contributions of sex steroids and sex chromosomes　　　　　　　　　　　　　　　
  Fumihiko Maekawa; Shinji Tsukahara; Takaharu Kawashima; Keiko Nohara; Hiroko Ohki-Hamazaki
  FRONTIERS IN NEUROSCIENCE, Volume：8, First page：242, Aug. 2014, [Reviewed]
  From a classical viewpoint, sex-specific behavior and physiological functions as well as the brain structures of mammals such as rats and mice, have been thought to be influenced by perinatal sex steroids secreted by the gonads. Sex steroids have also been thought to affect the differentiation of the sex-typical behavior of a few members of the avian order Galliformes, including the Japanese quail and chickens, during their development in ovo. However, recent mammalian studies that focused on the artificial shuffling or knockout of the sex-determining gene, Sry, have revealed that sex chromosomal effects may be associated with particular types of sex-linked differences such as aggression levels, social interaction, and autoimmune diseases, independently of sex steroid-mediated effects. In addition, studies on naturally occurring, rare phenomena such as gynandromorphic birds and experimentally constructed chimeras in which the composition of sex chromosomes in the brain differs from that in the other parts of the body, indicated that sex chromosomes play certain direct roles in the sex-specific differentiation of the gonads and the brain. In this article, we review the relative contributions of sex steroids and sex chromosomes in the determination of brain functions related to sexual behavior and reproductive physiology in mammals and birds.
  FRONTIERS RESEARCH FOUNDATION, English
  DOI：https://doi.org/10.3389/fnins.2014.00242
  DOI ID：10.3389/fnins.2014.00242, ISSN：1662-453X, Web of Science ID：WOS:000346509300001
• Involvement of hemeoxygenase-1 in di(2-ethylhexyl) phthalate (DEHP)-induced apoptosis of Neuro-2a cells　　　　　　　　　　　　　　　
  Kyaw Htet Aung; Tin-Tin Win-Shwe; Moeko Kanaya; Hirohisa Takano; Shinji Tsukahara
  JOURNAL OF TOXICOLOGICAL SCIENCES, Volume：39, Number：2, First page：217, Last page：229, Apr. 2014, [Reviewed]
  A widely-used plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to induce apoptosis in neurons, although the mechanisms responsible for DEHP-induced apoptosis is not well explored yet. We recently showed that exposure to DEHP increases the expression of hemeoxygenase (HO)-1, an oxidative stress related enzyme, in the mice brain. In this study, we investigated whether HO-1 is involved in DEHP-induced apoptosis using a mouse neuroblastoma cell line Neuro-2a, which forcibly express SCAT3, a fluorescent indicator of caspase-3 activity. The doses of DEHP at 1, 10 or 100 mu M were used in the present study to mimic the level of human exposure to DEHP. Live image analysis of SCAT3-expressing Neuro-2a cells revealed that caspase-3 activity in the cells was significantly increased by DEHP at 100 mu M but not 1 or 10 mu M. We measured HO-1 mRNA level in Neuro-2a cells exposed to DEHP and found significant increase in HO-1 mRNA level by DEHP at 100 mu M but not 1 or 10 mu M. Live image analysis of SCAT3-expresisng Neuro-2a cells was further performed to determine the effects of HO-1 siRNA in DEHP-induced apoptosis via caspase-3 activation. We found that knockdown of HO-1 gene nullifies the effects of DEBT to activate caspase-3. These results suggest that HO-1 is involved in DEHP-induced apoptosis. Moreover, this study demonstrates that high-dose DEHP exposure induces caspase-3-dependent apoptosis, which is at least partially mediated by the up-regulation of HO-1 gene, in Neuro-2a cells.
  JAPANESE SOC TOXICOLOGICAL SCIENCES, English, Scientific journal
  DOI：https://doi.org/10.2131/jts.39.217
  DOI ID：10.2131/jts.39.217, ISSN：0388-1350, eISSN：1880-3989, Web of Science ID：WOS:000334013300005
• Neuroanatomy and sex differences of the lordosis-inhibiting system in the lateral septum　　　　　　　　　　　　　　　
  Shinji Tsukahara; Moeko Kanaya; Korehito Yamanouchi
  Frontiers in Neuroscience, Volume：8, First page：299, 2014, [Reviewed]
  Female sexual behavior in rodents, termed lordosis, is controlled by facilitatory and inhibitory systems in the brain. It has been well demonstrated that a neural pathway from the ventromedial hypothalamic nucleus (VMN) to the midbrain central gray (MCG) is essential for facilitatory regulation of lordosis. The neural pathway from the arcuate nucleus to the VMN, via the medial preoptic nucleus, in female rats mediates transient suppression of lordosis, until female sexual receptivity is induced. In addition to this pathway, other regions are involved in inhibitory regulation of lordosis in female rats. The lordosis-inhibiting systems exist not only in the female brain but also in the male brain. The systems contribute to suppression of heterotypical sexual behavior in male rats, although they have the potential ability to display lordosis. The lateral septum (LS) exerts an inhibitory influence on lordosis in both female and male rats. This review focuses on the neuroanatomy and sex differences of the lordosis-inhibiting system in the LS. The LS functionally and anatomically links to the MCG to exert suppression of lordosis. Neurons of the intermediate part of the LS (LSi) serve as lordosis-inhibiting neurons and project axons to the MCG. The LSi-MCG neural connection is sexually dimorphic, and formation of the male-like LSi-MCG neural connection is affected by aromatized testosterone originating from the testes in the postnatal period. The sexually dimorphic LSi-MCG neural connection may reflect the morphological basis of sex differences in the inhibitory regulation of lordosis in rats.
  Frontiers Research Foundation, English
  DOI：https://doi.org/10.3389/fnins.2014.00299
  DOI ID：10.3389/fnins.2014.00299, ISSN：1662-453X, PubMed ID：25278832, SCOPUS ID：84907174430
• The mechanisms underlying sexual differentiation of behavior and physiology in mammals and birds: relative contributions of sex steroids and sex chromosomes.　　　　　　　　　　　　　　　
  Maekawa F; Tsukahara S; Kawashima T; Nohara K; Ohki-Hamazaki H
  Frontiers in neuroscience, Volume：8, First page：242, 2014, [Reviewed]
  DOI：https://doi.org/10.3389/fnins.2014.00242
  DOI ID：10.3389/fnins.2014.00242, ISSN：1662-4548, PubMed ID：25177264
• Effects of sodium arsenite on neurite outgrowth and glutamate AMPA receptor expression in mouse cortical neurons　　　　　　　　　　　　　　　
  Fumihiko Maekawa; Takashi Tsuboi; Manami Oya; Kyaw Htet Aung; Shinji Tsukahara; Luc Pellerin; Keiko Nohara
  NEUROTOXICOLOGY, Volume：37, First page：197, Last page：206, Jul. 2013, [Reviewed]
  There has been broad concern that arsenic in the environment exerts neurotoxicity. To determine the mechanism by which arsenic disrupts neuronal development, primary cultured neurons obtained from the cerebral cortex of mouse embryos were exposed to sodium arsenite (NaAsO2) at concentrations between 0 and 2 mu M from days 2 to 4 in vitro and cell survival, neurite outgrowth and expression of glutamate AMPA receptor subunits were assessed at day 4 in vitro. Cell survival was significantly decreased by exposure to 2 mu M NaAsO2, whereas 0.5 mu M NaAsO2 increased cell survival instead. The assessment of neurite outgrowth showed that total neurite length was significantly suppressed by 1 mu M and 2 mu M NaAsO2, indicating that the lower concentration of NaAsO2 impairs neuritogenesis before inducing cell death. Immunoblot analysis of AMPA receptor subunit expression showed that the protein level of GluA1, a specific subunit of the AMPA receptor, was significantly decreased by 1 mu M and 2 mu M NaAsO2. When immunocytochemistry was used to confirm this effect by staining for GluA1 expression in neuropeptide Y neurons, most of which contain GluA1, GluA1 expression in neuropeptide Y neurons was found to be significantly suppressed by 1 mu M and 2 mu M NaAsO2 but to be increased at the concentration of 0.5 mu M. Finally, to determine whether neurons could be rescued from the NaAsO2-induced impairment of neuritogenesis by compensatory overexpression of GluA1, we used primary cultures of neurons transfected with a plasmid vector to overexpress either GluA1 or GluA2, and the results showed that GluA1/2 overexpression protected against the deleterious effects of NaAsO2 on neurite outgrowth. These results suggest that the NaAsO2 concentration inducing neurite suppression is lower than the concentration that induces cell death and is the same as the concentration that suppresses GluA1 expression. Consequently, the suppression of GluA1 expression by NaAsO2 seems at least partly responsible for neurite suppression induced by NaAsO2. (C) 2013 Elsevier Inc. All rights reserved.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuro.2013.05.006
  DOI ID：10.1016/j.neuro.2013.05.006, ISSN：0161-813X, Web of Science ID：WOS:000321413400024
• Visualisation and characterisation of oestrogen receptor alpha-positive neurons expressing green fluorescent protein under the control of the oestrogen receptor alpha promoter　　　　　　　　　　　　　　　
  Ken Ichi Matsuda; Miho Yanagisawa; Kazuhiro Sano; Ikuo Ochiai; Sergei Musatov; Kota Okoshi; Shinji Tsukahara; Sonoko Ogawa; Mitsuhiro Kawata
  EUROPEAN JOURNAL OF NEUROSCIENCE, Volume：38, Number：2, First page：2242, Last page：2249, Jul. 2013, [Reviewed]
  Oestrogen receptor (ER)alpha plays important roles in the development and function of various neuronal systems through activation by its ligands, oestrogens. To visualise ER alpha-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ER alpha promoter. In three independent tg lines, GFP-positive neurons were observed in areas previously reported to express ER alpha mRNA, including the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus (MPO), hypothalamus, and amygdala. In these areas, GFP signals mostly overlapped with ER alpha immunoreactivity. GFP fluorescence was seen in neurites and cell bodies of neurons. In addition, the network and detailed structure of neurites were visible in dissociated and slice cultures of hypothalamic neurons. We examined the effect of oestrogen deprivation by ovariectomy on the structure of the GFP-positive neurons. The area of ER alpha-positive cell bodies in the bed nucleus of the stria terminalis and MPO was measured by capturing the GFP signal and was found to be significantly smaller in ovariectomy mice than in control mice. When neurons in the MPO were infected with an adeno-associated virus that expressed small hairpin RNA targeting the ER alpha gene, an apparent induction of GFP was observed in this area, suggesting a negative feedback mechanism in which ER alpha controls expression of the ER alpha gene itself. Thus, the ER alpha promoter-GFP tg mice will be useful to analyse the development and plastic changes of the structure of ER alpha-expressing neurons and oestrogen and its receptor-mediated neuronal responses.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1111/ejn.12227
  DOI ID：10.1111/ejn.12227, ISSN：0953-816X, Web of Science ID：WOS:000321704800005
• Collapsin response mediator protein 4 affects the number of tyrosine hydroxylase-immunoreactive neurons in the sexually dimorphic nucleus in female mice　　　　　　　　　　　　　　　
  Takashi Iwakura; Miyuki Sakoh; Atsuhiro Tsutiya; Naoya Yamashita; Akiko Ohtani; Mumeko C. Tsuda; Sonoko Ogawa; Shinji Tsukahara; Masugi Nishihara; Takashi Shiga; Yoshio Goshima; Tomohiro Kato; Ritsuko Ohtani-Kaneko
  DEVELOPMENTAL NEUROBIOLOGY, Volume：73, Number：7, First page：502, Last page：517, Jul. 2013, [Reviewed]
  In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV. (c) 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 502-517, 2013
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1002/dneu.22076
  DOI ID：10.1002/dneu.22076, ISSN：1932-8451, Web of Science ID：WOS:000320190800002
• Inhibition of neurite outgrowth and alteration of cytoskeletal gene expression by sodium arsenite　　　　　　　　　　　　　　　
  Kyaw Htet Aung; Ryohei Kurihara; Shizuka Nakashima; Fumihiko Maekawa; Keiko Nohara; Tetsuya Kobayashi; Shinji Tsukahara
  NEUROTOXICOLOGY, Volume：34, First page：226, Last page：235, Jan. 2013, [Reviewed]
  Arsenic compounds that are often found in drinking water increase the risk of developmental brain disorders. In this study, we performed live imaging analyses of Neuro-2a cells expressing SCAT3, a caspase-3 cleavage peptide sequence linking two fluorescent proteins; enhanced cyan fluorescence protein (ECFP) and Venus, to determine whether sodium arsenite (NaAsO2; 0, 1, 5, or 10 mu M) affects both neurite outgrowth and/or induces apoptosis with the same doses and in the same cell cultures. We observed that the area ratio of neurite to cell body in SCAT3-expressing cells was significantly reduced by 5 and 10 mu M NaAsO2, but not by 1 mu M, although the emission ratio of ECFP to Venus, an endpoint of caspase-3 activity, was not changed. However, cytological assay using apoptotic and necrotic markers resulted in that apoptosis, but not necrosis, was significantly induced in Neuro-2a cells when NaAsO2 exposure continued after the significant effects of NaAsO2 on neurite outgrowth were found by live imaging. These results suggested that neurite outgrowth was suppressed by NaAsO2 prior to NaAsO2-induced apoptosis. Next, we examined the effects of NaAsO2 on cytoskeletal gene expression in Neuro-2a cells. NaAsO2 increased the mRNA levels of the light and medium subunits of neurofilament and decreased the mRNA levels of tau and tubulin in a dose-dependent manner; no significant effect was found in the mRNA levels of the heavy subunit of neurofilament, microtubule-associated protein 2, or actin. The changes in cytoskeletal gene expression are likely responsible for the inhibitory effects of NaAsO2 on neurite outgrowth. (C) 2012 Elsevier Inc. All rights reserved.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuro.2012.09.008
  DOI ID：10.1016/j.neuro.2012.09.008, ISSN：0161-813X, Web of Science ID：WOS:000314626100024
• Involvement of postnatal apoptosis on sex difference in number of cells generated during late fetal period in the sexually dimorphic nucleus of the preoptic area in rats　　　　　　　　　　　　　　　
  Yukinori Kato; Shizuka Nakashima; Fumihiko Maekawa; Shinji Tsukahara
  NEUROSCIENCE LETTERS, Volume：516, Number：2, First page：290, Last page：295, May 2012, [Reviewed]
  Postnatal apoptosis is involved in formation of the sex difference in neuron number of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in rats. In this study, we examined the origin of neurons that die with apoptosis on the postnatal period to exhibit the sex difference in neuron number of the SDN-POA. First, we measured the number of cells that were labeled with 5-bromo-2'-deoxyuridine (BrdU) on embryonic day (ED) 17, ED18, and ED19 in the SDN-POA of rats on postnatal day (PD) 4 and PD8. The SDN-POA had many more cells labeled with BrdU on ED17 and ED18 than those on ED19. Significantly fewer cells labeled with BrdU on ED18 in the female SDN-POA from PD4 to PD8 resulted in a significant sex difference in the number at PD8. Next, combination analyses of BrdU-labeling and immunohistochemistry for single-stranded DNA (ssDNA), an apoptotic marker, were succeeded to investigate whether SDN-POA neurons generated during ED17-18 were removed by apoptosis. Many more ssDNA-immunoreactive cells that had been labeled with BrdU during ED17-18 were found in the SDN-POA of PD8 females, but few in the SDN-POA of PD8 males and PD4 females and males. These results suggest that the sex difference in the number of SDN-POA neurons generated during the late fetal period was caused by postnatal apoptosis. (c) 2012 Elsevier Ireland Ltd. All rights reserved.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2012.04.017
  DOI ID：10.1016/j.neulet.2012.04.017, ISSN：0304-3940, Web of Science ID：WOS:000304520300025
• Differential mRNA expression of neuroimmune markers in the hippocampus of infant mice following toluene exposure during brain developmental period　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Naoki Kunugita; Yasuhiro Yoshida; Daisuke Nakajima; Shinji Tsukahara; Hidekazu Fujimaki
  JOURNAL OF APPLIED TOXICOLOGY, Volume：32, Number：2, First page：126, Last page：134, Feb. 2012, [Reviewed]
  Toluene, a volatile organic compound with a wide range of industrial applications, can exert neurotoxic and immunotoxic effects. However, the effects of toluene exposure on developmental immunotoxicity in the brain have not yet been characterized. To investigate the susceptible window to toluene exposure during development and the effects of fetal and neonatal toluene exposure on the neuroimmune markers, gestational day (GD) 14 pregnant mice, postnatal day (PND) 2 and PND 8 male offspring were exposed to filtered air (control; 0?ppm), or 5 or 50 ppm toluene for 6?h per day for five consecutive days. The neuroimmune markers in the hippocampus of PND 21 were examined using a real-time RT-PCR and immunohistochemical analysis. Mice exposed to 50 ppm toluene on PND 26 showed significantly increased levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)-a mRNAs. In contrast, NGF and brain-derived neurotrophic factor (BDNF) and proinflammatory cytokines TNF-a, CCL3, NF-B, toll-like receptor (TLR)-4, astrocyte marker glial fibrillary acidic protein (GFAP), and microglia marker ionized calcium binding adapter molecule (Iba)-1 mRNAs were increased significantly in mice exposed to 5ppm toluene on PND 812. These results indicate that low-level toluene exposure during the late postnatal period (PND 812) might induce neuroinflammatory mediators via TLR4-dependent NF-B pathway in the hippocampus of PND 21 male mice. Among the three developmental phases, PND 812 seems to be most sensitive to toluene exposure. This is the first study to show developmental phase- and dose-specific changes in neuroimmune markers in infant mice following toluene exposure. Copyright (C) 2011 John Wiley & Sons, Ltd.
  WILEY-BLACKWELL, English, Scientific journal
  DOI：https://doi.org/10.1002/jat.1643
  DOI ID：10.1002/jat.1643, ISSN：0260-437X, Web of Science ID：WOS:000298597900006
• Effects of Aromatase or Estrogen Receptor Gene Deletion on Masculinization of the Principal Nucleus of the Bed Nucleus of the Stria Terminalis of Mice　　　　　　　　　　　　　　　
  Shinji Tsukahara; Mumeko C. Tsuda; Ryohei Kurihara; Yukinori Kato; Yoshiko Kuroda; Mariko Nakata; Kai Xiao; Kazuyo Nagata; Katsumi Toda; Sonoko Ogawa
  NEUROENDOCRINOLOGY, Volume：94, Number：2, First page：137, Last page：147, 2011, [Reviewed]
  The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is a sexually dimorphic nucleus, and the male BNSTp is larger and has more neurons than the female BNSTp. To assess the roles of neuroestrogen synthesized from testicular androgen by brain aromatase in masculinization of the BNSTp, we performed morphometrical analyses of the adult BNSTp in aromatase knockout (ArKO), estrogen receptor-alpha knockout (alpha ERKO), and estrogen receptor-beta knockout (beta ERKO) mice and their respective wild-type littermates. In wild-type littermates, the BNSTp of males had a larger volume and greater numbers of neuronal and glial cells than did that of females. The volume and neuron number of the BNSTp in ArKO and alpha ERKO males and glial cell number of the BNSTp in alpha ERKO males were significantly smaller than those of wild-type male littermates, and they were not significantly different from those in female mice with either gene knockout. In contrast, there was no significant morphological difference in the BNSTp between beta ERKO and wild-type mice. Next, we examined the BNSTp of ArKO males subcutaneously injected with estradiol benzoate (EB) on postnatal days 1, 2, and 3 (1.5 mu g/day). EB-treated ArKO males had a significantly greater number of BNSTp neurons than did oil-treated ArKO males. The number of BNSTp neurons in EB-treated ArKO males was comparable to that in wild-type males. These findings suggested that masculinization of the BNSTp in mice involves the actions of neuroestrogen that was synthesized by aromatase and that this estrogen mostly binds to ER alpha during the postnatal period. Copyright (C) 2011 S. Karger AG, Basel
  KARGER, English, Scientific journal
  DOI：https://doi.org/10.1159/000327541
  DOI ID：10.1159/000327541, ISSN：0028-3835, Web of Science ID：WOS:000295158800005
• Intrauterine Position Affects Estrogen Receptor alpha Expression in the Ventromedial Nucleus of the Hypothalamus via Promoter DNA Methylation　　　　　　　　　　　　　　　
  Hiroko Mori; Ken Ichi Matsuda; Shinji Tsukahara; Mitsuhiro Kawata
  ENDOCRINOLOGY, Volume：151, Number：12, First page：5775, Last page：5781, Dec. 2010, [Reviewed]
  There is well-established evidence in many mammalian species for effects of the intrauterine position (IUP) (the sex-specific positioning of the embryo) on postnatal brain function and behavior. We found that the IUP affects estrogen receptor (ER)alpha expression in adult female rats in the ventrolateral region of the hypothalamic ventromedial nucleus (vlVMH), which is associated with sexual behavior. The ER alpha expression level in the vlVMH was higher in females that developed in utero between two male siblings (2M females) than in those that developed between female siblings (2F females). We also found that the cytosine methylation status across the ER alpha promoter in the vlVMH was affected by the IUP, with greater methylation in 2F females. These findings showed a negative correlation between ER alpha expression levels in the vlVMH and methylation frequency in the ER alpha promoter. This suggests that genomic methylation sustains the effect of the fetal IUP on ER alpha expression in the vlVMH. (Endocrinology 151: 5775-5781, 2010)
  ENDOCRINE SOC, English, Scientific journal
  DOI：https://doi.org/10.1210/en.2010-0646
  DOI ID：10.1210/en.2010-0646, ISSN：0013-7227, Web of Science ID：WOS:000284489800022
• Does early life toluene exposure alter the expression of NMDA receptor subunits and signal transduction pathway in infant mouse hippocampus?　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Yasuhiro Yoshida; Naoki Kunugita; Shinji Tsukahara; Hidekazu Fujimaki
  NEUROTOXICOLOGY, Volume：31, Number：6, First page：647, Last page：653, Dec. 2010, [Reviewed]
  We aim to investigate the critical window of susceptibility to toluene exposure during brain development and the effects of fetal and neonatal toluene exposure on the expression of N-methyl-D-aspartate (NMDA) receptor subunits and related transduction pathway in infant mice hippocampus. Pregnant mice (GD 14), male offspring (postnatal day; PND 2) or PND 8 were exposed to either a filtered air control (0 ppm), or 5, or 50 ppm of toluene for 6 h per day for 5 consecutive days. On PND 21, the expression levels of NMDA receptor subunits, cyclic AMP responsive element binding protein (CREB)-1, calcium/calmodulin-dependent protein kinase (CaMK)-IV, and apoptotic related genes (Bax, Bcl) mRNAs in the hippocampus were estimated using quantitative real-time RT-PCR and immunohistochemical analyses. NR2B, CaMKIV and CREB1 mRNAs increased significantly in the hippocampus of mice exposed to 50 ppm toluene on PND 2-6. In contrast, almost all memory function-related gene mRNAs and proapoptotic and anti-apoptotic ratio increased significantly in mice exposed to 5 or 50 ppm toluene on PND 8-12. However, mice exposed to toluene on GD 14-18 showed no significant change. Increased active caspase-3 immunoreactive cells were found in hippocampal CA1 area of PND 21 male mice exposed to 5 ppm toluene during PND 8-12. Our results suggest that late postnatal period may be a vulnerable and critical period to toluene exposure. Then, we have also examined the effect of toluene exposure in brain development on learning ability in young adult mice and found that poor spatial learning performance in PND 49 male mice exposed to 5 ppm toluene during critical period. This is the first study to show that the early toluene exposure induces persistent of the alteration of memory function-related genes in infant mice and memory deficit in later life via modulating the synaptic morphology and function. (C) 2010 Elsevier Inc. All rights reserved.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuro.2010.08.006
  DOI ID：10.1016/j.neuro.2010.08.006, ISSN：0161-813X, Web of Science ID：WOS:000284199800003
• Use of live imaging analysis for evaluation of cytotoxic chemicals that induce apoptotic cell death　　　　　　　　　　　　　　　
  Yoshiko Koike-Kuroda; Masaki Kakeyama; Hidekazu Fujimaki; Shinji Tsukahara
  TOXICOLOGY IN VITRO, Volume：24, Number：7, First page：2012, Last page：2020, Oct. 2010, [Reviewed]
  We carried out live imaging of PC12 cells expressing SCAT3, a caspase-3 cleavage peptide sequence linking two fluorescent proteins, ECFP and Venus, which function respectively as the donor and acceptor for FRET. Live imaging of SCAT3-expressing cells was performed from 60 to 300 min after exposure to sodium arsenite (NaAsO(2): 0, 1, 5, or 10 mu M) was initiated. We then measured the emission ratio of ECFP to Venus to monitor the activity of caspase-3 and found that the ratio was temporally and dose-dependently increased by NaAsO(2). The mean ECFP/Venus emission ratio between 200 and 300 min after exposure to NaAsO(2) at a dose of 5 or 10 mu M, but not at 1 mu M, was significantly higher than that in the control group. We showed by other methods that NaAsO(2) significantly increased the amount and activity of mature caspase-3 and the amount of nucleosomes generated from DNA fragmentation, and decreased cell viability. However, methods other than live imaging required a longer time and higher doses of NaAsO(2) than did live imaging to detect significant effects. This result suggests that live imaging using SCAT3 is a useful method for the screening of chemical toxicities and for improving the efficiency of toxicity evaluation. (C) 2010 Elsevier Ltd. All rights reserved.
  PERGAMON-ELSEVIER SCIENCE LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.tiv.2010.07.022
  DOI ID：10.1016/j.tiv.2010.07.022, ISSN：0887-2333, Web of Science ID：WOS:000283567500017
• Up-regulation of neurotrophin-related gene expression in mouse hippocampus following low-level toluene exposure　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Shinji Tsukahara; Shoji Yamamoto; Atsushi Fukushima; Naoki Kunugita; Keiichi Arashidani; Hidekazu Fujimaki
  NEUROTOXICOLOGY, Volume：31, Number：1, First page：85, Last page：93, Jan. 2010, [Reviewed]
  To investigate the role of strain differences in sensitivity to low-level toluene exposure on neurotrophins and their receptor levels in the mouse hippocampus, 8-week-old male C3H/HeN, BALB/c and C57BL/10 mice were exposed to 0, 5, 50, or 500 ppm toluene for 6 h per day, 5 days per week for 6 weeks in an inhalation chamber. We examined the expressions of neurotrophin-related genes and receptors in the mouse hippocampus using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression of nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), tyrosine kinase (Trk) A, and TrkB mRNAs in the C3H/HeN mice hippocampus was significantly higher in the mice exposed to 500 ppm toluene. Among the three strains of mice, the C3H/HeN mice seemed to be sensitive to toluene exposure. To examine the combined effect of toluene exposure and allergic challenge, the C3H/HeN mice stimulated with ovalbumin were exposed to toluene. The allergy group of C3H/HeN mice showed significantly elevated level of NGF mRNA in the hippocampus following exposure to 50 ppm toluene. Then, we also examined the expression of transcription factor, dopamine markets and oxidative stress marker in the hippocampus of sensitive strain C3H/HeN mice and found that the expression of CREB1 mRNA was significantly increased at 50 ppm toluene. In immunohistochemical analysis, the density of the NGF-immunoreactive signal was significantly stronger in the hippocampal CA3 region of the C3H/HeN mice exposed to 500 ppm toluene in non-allergy group and 50 ppm in allergy group. Our results indicate that low-level toluene exposure may induce up-regulation of neurotrophin-related gene expression in the mouse hippocampus depending on the mouse strain and an allergic stimulation in sensitive strain may decrease the threshold for sensitivity at lower exposure level. (C) 2009 Elsevier Inc. All rights reserved.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuro.2009.11.004
  DOI ID：10.1016/j.neuro.2009.11.004, ISSN：0161-813X, Web of Science ID：WOS:000274611000009
• The effect of D-cycloserine on spatial learning performance and memory function-related gene expression in mice following toluene exposure　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Shiho Kageyama; Shinji Tsukahara; Daisuke Nakajima; Hidekazu Fujimaki
  Journal of UOEH, Volume：32, Number：2, First page：127, Last page：140, 2010, [Reviewed]
  D-cycloserine (DCS), a partial N-methyl-D-aspartate (NMDA) receptor agonist, is a well-known cognitive enhancer. To investigate the effect of DCS in cognitive function following toluene exposure, eight-week-old male C3H/HeN mice were exposed to filtered air (0 ppm) or 50 ppm toluene for 6 h a day on 5 consecutive days a week for 6 weeks. The day after the day of final exposure, a spatial learning task was performed using a Morris water maze apparatus. During the learning task, some mice were treated with DCS intraperitoneally (20 mg/kg) 30 min before the first trial of the acquisition phase and probe trial. After completion of the spatial learning task, the hippocampus was collected from each mouse to examine memory function-related gene expression using the real-time RT-PCR method. During the acquisition phase, on day 3 and 4, toluene-exposed mice with DCS treatment showed significantly better learning performance than corresponding saline treated groups. Moreover, the toluene-exposed mice with DCS treatment also showed significantly improved memory retention during the probe trial and up-regulation of hippocampal NMDA receptor subunit 2B mRNA expression compared with the saline treated groups. Our findings indicate that a subunit-specific modulation of hippocampal NMDA receptor mRNA expression by DCS contributes to improvement of spatial learning performance in mice following toluene exposure.
  University of Occupational and Environmental Health, English, Scientific journal
  DOI：https://doi.org/10.7888/juoeh.32.127
  DOI ID：10.7888/juoeh.32.127, ISSN：0387-821X, PubMed ID：20549902, SCOPUS ID：77955898534
• Sex Differences and the Roles of Sex Steroids in Apoptosis of Sexually Dimorphic Nuclei of the Preoptic Area in Postnatal Rats　　　　　　　　　　　　　　　
  S. Tsukahara
  JOURNAL OF NEUROENDOCRINOLOGY, Volume：21, Number：4, First page：370, Last page：376, Apr. 2009, [Reviewed]
  The brain contains several sexually dimorphic nuclei that exhibit sex differences with respect to cell number. It is likely that the control of cell number by apoptotic cell death in the developing brain contributes to creating sex differences in cell number in sexually dimorphic nuclei, although the mechanisms responsible for this have not been determined completely. The milieu of sex steroids in the developing brain affects sexual differentiation in the brain. The preoptic region of rats has two sexually dimorphic nuclei. The sexually dimorphic nucleus of the preoptic area (SDN-POA) has more neurones in males, whereas the anteroventral periventricular nucleus (AVPV) has a higher cell density in females. Sex differences in apoptotic cell number arise in the SDN-POA and AVPV of rats in the early postnatal period, and an inverse correlation exists between sex differences in apoptotic cell number and the number of living cells in the mature period. The SDN-POA of postnatal male rats exhibits a higher expression of anti-apoptotic Bcl-2 and lower expression of pro-apoptotic Bax compared to that in females and, as a potential result, apoptotic cell death via caspase-3 activation more frequently occurs in the SDN-POA of females. The patterns of expression of Bcl-2 and Bax in the SDN-POA of postnatal female rats are changed to male-typical ones by treatment with oestrogen, which is normally synthesised from testicular androgen and affects the developing brain in males. In the AVPV of postnatal rats, apoptotic regulation also differs between the sexes, although Bcl-2 expression is increased and Bax expression and caspase-3 activity are decreased in females. The mechanisms of apoptosis possibly contributing to the creation of sex differences in cell number and the roles of sex steroids in apoptosis are discussed.
  WILEY-BLACKWELL PUBLISHING, INC, English
  DOI：https://doi.org/10.1111/j.1365-2826.2009.01855.x
  DOI ID：10.1111/j.1365-2826.2009.01855.x, ISSN：0953-8194, Web of Science ID：WOS:000264635700024
• Effects of maternal toluene exposure on testosterone levels in fetal rats　　　　　　　　　　　　　　　
  Shinji Tsukahara; Daisuke Nakajima; Yoshiko Kuroda; Rieko Hojo; Shiho Kageyama; Hidekazu Fujimaki
  TOXICOLOGY LETTERS, Volume：185, Number：2, First page：79, Last page：84, Mar. 2009, [Reviewed]
  The goal of our study was to determine if toluene affected the synthesis and secretion of testosterone in fetal rats. Dams were exposed to atmospheres that contained 0.09 ppm, 0.9 ppm or 9 ppm of toluene for 90 min/day from gestational days (GDs) 14.5 to 18.5 via nasal inhalation. Fetal plasma testosterone concentrations determined by enzyme immunoassay were significantly reduced on GD18.5 after exposure to 0.9 and 9 ppm, but not to 0.09 ppm, of toluene in male, but not in female, fetuses. We measured, using real-time PCR methods, mRNA levels in fetal testes for several steroidogenic enzymes involved in testosterone synthesis and insulin-like 3 (Insl3), a maker of Leydig cell differentiation. The mRNA levels of 3p-hydroxysteroid dehydrogenase (30-HSD) were significantly reduced after exposure to 0.9-ppm toluene. However, the mRNA levels of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/c17-20 lyase, 17 beta-hydroxysteroid dehydrogenase, and Insl3 were not significantly altered by exposure to 0.9-ppm toluene. In addition, immunohistochemical analysis showed reduced 3 beta-HSD-immunoreactive areas in the interstitial region of fetal testes after exposure to 0.9 and 9 ppm, but not 0.09 ppm, toluene. These findings indicate that toluene reduced the synthesis and secretion of testosterone in fetal testes from rats possibly as a consequence of reduced 30-HSD expression. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.toxlet.2008.12.001
  DOI ID：10.1016/j.toxlet.2008.12.001, ISSN：0378-4274, Web of Science ID：WOS:000263946400002
• Measurement of Toluene Concentrations in the Blood of Fetuses of Pregnant Rats Exposed to Low Concentration Toluene Using Headspace-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry　　　　　　　　　　　　　　　
  Daisuke Nakajima; Shinji Tsukahara; Rieko Hojo; Shiho Kageyama; Sumio Goto; Hiroaki Shiraishi; Fujio Shiraishi; Hidekazu Fujimaki
  JOURNAL OF HEALTH SCIENCE, Volume：55, Number：1, First page：50, Last page：55, Feb. 2009, [Reviewed]
  In order to measure the level of toluene in the blood of fetuses of pregnant rats exposed to toluene, application of headspace-solid phase micro extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was studied. Pregnant rats from gestational day 15 (GD15) to GD19 were subjected to inhalational exposure to toluene for 90 min per day. They were obtained by Cesarean section on the fifth day of exposure (on GD 19), and the level of toluene in the blood of mother rats and in fetuses were quantified using 5 mu l blood. The levels of toluene in the blood of the fetuses in the groups exposed to toluene at 9 ppm and 90 ppm were 0.07 +/- 0.03 pg/ml (n = 17) and 2.0 +/- 0.51 mu g/ml (n = 16) respectively, which were significantly higher than the blood level of the fetuses in the control group (0.02 +/- 0.02 mu g/ml, n = 12). Meanwhile, the blood concentrations of mother rats were 0.06 +/- 0.06 mu g/ml (n = 3 control group), 0.14 +/- 0.03 mu g/ml (n = 3, 9 ppm exposure group), and 3.5 +/- 1.5 mu g/ml (n = 4, 90 ppm exposure group), respectively, which were higher than those of the fetuses per unit blood volume.
  PHARMACEUTICAL SOC JAPAN, English, Scientific journal
  DOI：https://doi.org/10.1248/jhs.55.50
  DOI ID：10.1248/jhs.55.50, ISSN：1344-9702, eISSN：1347-5207, Web of Science ID：WOS:000262843400006
• Estrogen modulates Bcl-2 family protein expression in the sexually dimorphic nucleus of the preoptic area of postnatal rats　　　　　　　　　　　　　　　
  Shinji Tsukahara; Rieko Hojo; Yoshiko Kuroda; Hidekazu Fujimaki
  NEUROSCIENCE LETTERS, Volume：432, Number：1, First page：58, Last page：63, Feb. 2008, [Reviewed]
  In the sexually dimorphic nucleus of the preoptic area (SDN-POA) of postnatal rats, apoptotic cells are detected more frequently in females than males. This sex difference is under the influence of aromatized androgen. We have reported that there are sex differences in the levels of Bcl-2 (female male) and Bax (female male) in the central division of the media] preoptic nucleus (MPNc), a significant component of the SDN-POA, followed by a sex difference in induction of apoptosis via caspase-3 activation (female> male). In the present study, we examined effects of estradiol benzoate (EB) on expression of Bcl-2 and Bax in the MPNc. Female rats were subcutaneously injected with EB (25 or 50 mu g per head) on postnatal day 5. MPNc and caudate putamen (CP) tissues were obtained from EB-treated female and male rats on postnatal day 6. Protein levels of Bcl-2 and Bax were determined by Western blotting. In the MPNc of female rats, EB at a dose of 50 mu g/head but not 25 mu g/head significantly increased Bcl-2 protein level and decreased Bax protein level. The levels of Bcl-2 and Bax of female rats treated with 50 mu g of EB were comparable to those of male rats. However, the protein levels of Bcl-2 and Bax in the CP did not change with EB treatment. These results suggest that estrogen up-regulates Bcl-2 expression and down-regulates Bax expression in the MPNc of postnatal rats. Effects of estrogen on the Bcl-2 family are presumably responsible for sex difference in postnatal apoptosis of the SDN-POA. (c) 2007 Elsevier Ireland Ltd. All rights reserved.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neulet.2007.12.006
  DOI ID：10.1016/j.neulet.2007.12.006, ISSN：0304-3940, Web of Science ID：WOS:000253807600012
• Evaluation of olfactory detection threshold of odor in mice using operant learning procedure　　　　　　　　　　　　　　　
  HOJO Rieko; KUROKAWA Yoshika; TSUKAHARA Shinji; NAKAJIMA Daisuke; FUJIMAKI Hidekazu
  Journal of Japan Association on Odor Environment, Volume：39, Number：3, First page：186, Last page：191, 2008
  Japanese
  DOI ID：10.2171/jao.39.186, ISSN：1348-2904, CiNii Articles ID：10021104019, CiNii Books ID：AA11827805
• Effects of toluene exposure on estradiol level in the fetal brain and postnatal apoptosis in the sexually dimorphic nucleus of the preoptic area [SDN-POA] in rats　　　　　　　　　　　　　　　
  Shinji Tsukahara; Daisuke Nakajima; Yoshiko Kuroda; Shiho Kageyama; Hidekazu Fujimaki
  NEUROSCIENCE RESEARCH, Volume：61, First page：S108, Last page：S108, 2008, [Reviewed]
  ELSEVIER IRELAND LTD, English
  ISSN：0168-0102, Web of Science ID：WOS:000261548100644
• Athymic nude mice are insensitive to low-level toluene-induced up-regulation of memory-related gene expressions in the hippocampus　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; Shinji Tsukahara; Sohel Ahmed; Atsushi Fukushima; Shoji Yamamoto; Masaki Kakeyama; Daisuke Nakajima; Surnio Goto; Takahiro Kobayashi; Hidekazu Fujimaki
  NEUROTOXICOLOGY, Volume：28, Number：5, First page：957, Last page：964, Sep. 2007, [Reviewed]
  The function of the N-methyl-D-aspartate (NMDA) subtype of glutamatergic receptors is known to be antagonized by toluene, a well-characterized neurotoxic chemical known to impair memory functions. Recently, peripheral T cells have been clearly shown to play an important role in cognitive and behavioral functions. In the present study, we investigated the role of peripheral T cells in the hippocampal mRNA expression of memory-related genes induced by low levels of toluene exposure in mice. BALB/c wild-type (WT) and nude mice were exposed to 9 ppm of toluene or filtered air (0 ppm toluene; control groups) in a nose-only exposure chamber for 30 min on 3 consecutive days followed by weekly sessions for 4 weeks. Twenty-four hours after the last exposure, the hippocampi were collected and the inducibility of memory-related genes was examined using a real-time quantitative PCR method. NMDA NR2A, calcium/calmodulin-dependent protein kinase IV (CaMKIV), cyclic AMP-responsive element binding protein I (CREB 1), and BDNF were significantly up-regulated in the hippocampi of WT mice exposed to 9 ppm of toluene, compared to the expressions observed in WT mice exposed to filtered air, but similar results were not observed in nude mice. To investigate the possible involvement of peripheral T cells in the toluene-induced up-regulation of memory-related genes in WT mice, we examined the mRNA expression of Thy-1 (a pan T cell-specific marker) and quantified the number of cells that were immunoreactive to a T cell antigen receptor, CD3 (CD3-ir). Both the expression of Thy-1 mRNA and the number of CD3-ir cells were significantly higher in the hippocampi of the WT mice exposed to 9 ppm of toluene, compared with that in WT mice exposed to filtered air; similar results were not observed in nude mice. We also examined the expression of chemokine genes like CCL2 and CCL3. The expression of CCL3 mRNA was significantly up-regulated only in the toluene-exposed WT mice. Although other differences unrelated to immune function may exist between WT and nude mice from the same background, the findings of the present study strongly suggest that the recruitment of peripheral T cells in the hippocampi of BALB/c WT mice exposed to low levels of toluene may be involved in the toluene-induced up-regulation of memory-related genes at the mRNA level. (C) 2007 Elsevier Inc. All rights reserved.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuro.2007.08.002
  DOI ID：10.1016/j.neuro.2007.08.002, ISSN：0161-813X, Web of Science ID：WOS:000250905100008
• Activation of cholecystokinin neurons in the dorsal pallium of the telencephalon is indispensable for the acquisition of chick imprinting behavior　　　　　　　　　　　　　　　
  Fumihiko Maekawa; Tomoharu Nakamori; Motoaki Uchimura; Ken Fujiwara; Toshihiko Yada; Shinji Tsukahara; Tomoyuki Kanamatsu; Kohichi Tanaka; Hiroko Ohki-Hamazaki
  JOURNAL OF NEUROCHEMISTRY, Volume：102, Number：5, First page：1645, Last page：1657, Sep. 2007, [Reviewed]
  Chick imprinting behavior is a good model for the study of learning and memory. Imprinting object is recognized and processed in the visual wulst, and the memory is stored in the intermediate medial mesopallium in the dorsal pallium of the telencephalon. We identified chicken cholecystokinin (CCK)expressing cells localized in these area. The number of CCK mRNA-positive cells increased in chicks underwent imprinting training, and these cells expressed nuclear Fos immunoreactivity at high frequency in these regions. Most of these CCK-positive cells were glutamatergic and negative for parvalbumin immunoreactivity. Semi-quantitative PCR analysis revealed that the CCK mRNA levels were significantly increased in the trained chicks compared with untrained chicks. In contrast, the increase in CCK- and c- Fos-double-positive cells associated with the training was not observed after closure of the critical period. These results indicate that CCK cells in the dorsal pallium are activated acutely by visual training that can elicit imprinting. In addition, the CCK receptor antagonist significantly suppressed the acquisition of memory. These results suggest that the activation of CCK cells in the visual wulst as well as in the intermediate medial mesopallium by visual stimuli is indispensable for the acquisition of visual imprinting.
  BLACKWELL PUBLISHING, English, Scientific journal
  DOI：https://doi.org/10.1111/j.1471-4159.2007.04733.x
  DOI ID：10.1111/j.1471-4159.2007.04733.x, ISSN：0022-3042, CiNii Articles ID：80018283044, PubMed ID：17697050, Web of Science ID：WOS:000249267300021
• Effects of low-level formaldehyde exposure on synaptic plasticity-related gene expression in the hippocampus of immunized mice　　　　　　　　　　　　　　　
  Sohel Ahmed; Shinji Tsukahara; Tin-Tin-Win-Shwe; Shoji Yamamoto; Naoki Kunugita; Keiichi Arashidani; Hidekazu Fujimaki
  JOURNAL OF NEUROIMMUNOLOGY, Volume：186, Number：1-2, First page：104, Last page：111, May 2007, [Reviewed]
  We examined the effects of inhalative exposure to formaldehyde (FA, 400 ppb) on N-methyl-D-aspartate (NMDA) receptor subunits (NR2A and NR2B), dopamine receptor subtypes (D1 and D2), cyclic AMP responsive element-binding protein (CREB)-1, CREB-2, FosB/Delta FosB, and transient receptor potential vanilloid receptor (TRPV1) in the hippocampus of ovalbumin-immunized mice using quantitative real-time PCR. Western blot analyses for pCREB were performed. The mRNA levels of NR2A, D1 and D2 receptors, and CREB-1 were significantly increased by FA, but NR2B, CREB-2, FosB/Delta FosB, and TRPV 1 mRNA levels remained unchanged. Treatment with MK-801 normalized the mRNA levels induced by FA. There was no significant effect of FA exposure and MK-801 treatment on the protein level of pCREB. These results indicate that FA exposure selectively up-regulates hippocampal gene expression in immunologically sensitized mice. The FA effects are presumably mediated by glutamatergic neurotransmission through NMDA receptors. (c) 2007 Elsevier B.V. All rights reserved.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.jneuroim.2007.03.010
  DOI ID：10.1016/j.jneuroim.2007.03.010, ISSN：0165-5728, Web of Science ID：WOS:000247452700011
• Toluene induces rapid and reversible rise of hippocampal glutamate and taurine neurotransmitter levels in mice　　　　　　　　　　　　　　　
  Tin-Tin Win-Shwe; D. Mitsushima; D. Nakajima; S. Ahmed; S. Yamamoto; S. Tsukahara; M. Kakeyama; S. Goto; H. Fujimaki
  TOXICOLOGY LETTERS, Volume：168, Number：1, First page：75, Last page：82, Jan. 2007, [Reviewed]
  Toluene. a widely used aromatic organic solvent, has been well characterized as a neurotoxic chemical. Although the neurobehavioral effects of toluene have been studied substantially, the mechanisms involved are not clearly understood. Hippocampus, which is one of the limbic areas of brain associated with neuronal plasticity, and learning and memory functions, may be a principal target of toluene. In the present study, to establish a mouse model for investigating the effects of acute toluene exposure on the amino acid neurotransmitter levels in the hippocampus, in vivo microdialysis study was performed in freely moving mice after a single intraperitoneal administration of toluene (150 and 300 mg/kg,). Amino acid neurotransmitters in microdialysates were measured by a high performance liquid chromatography system. The extracellular levels of glutamate and taurine were rapidly and reversibly increased within 30 min after the toluene administration in a dose-dependent manner and returned to the basal level by 1 h. Conversely, the extracellular level of glycine and GABA were stable, and no significant change was observed after the toluene administration. To further investigate the brain toluene level in the hippocampus of toluene-administered mice, we used a solid-phase microextraction (SPME) method and examined the time course changes of toluene in the hippocampus of living mice. The brain toluene level reached the peak at 30 min after injection and returned to the basal level after 2 h. In the present study, we observed the relationship between brain toluene levels and amino acid neurotransmitter glutamate and taurine levels in the hippocampus. Therefore, we suggest that toluene may mediate its action through the glutamatergic and taurinergic neurotransmission in the hippocampus of freely moving mice. (c) 2006 Published by Elsevier Ireland Ltd.
  ELSEVIER IRELAND LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.toxlet.2006.10.017
  DOI ID：10.1016/j.toxlet.2006.10.017, ISSN：0378-4274, Web of Science ID：WOS:000243886400009
• Increased hippocampal mRNA expression of neuronal synaptic plasticity related genes in mice chronically exposed to toluene at a low-level human occupational-exposure　　　　　　　　　　　　　　　
  Sohel Ahmed; Tin-Tin-Win-Shwe; Shoji Yamamoto; Shinji Tsukahara; Naoki Kunugita; Keiichi Arashidani; Hidekazu Fujimaki
  NEUROTOXICOLOGY, Volume：28, Number：1, First page：168, Last page：174, Jan. 2007, [Reviewed]
  Although neurological symptoms in individuals exposed to toluene both inside and outside the homes have been reported well, the chronic effects of low-level toluene-exposure on the hippocampal expression of neuronal synaptic plasticity related genes have not been studied in vivo. In the present study, to understand the possible adult hippocampal neurobiological responses of mice chronic exposure to toluene at a low-level human occupational-exposure, we exposed 10-week-old C3H/HeN female mice to 50 ppm toluene or filtered air for 6 h a day, on 5-consecutive days of a week for 6 and 12 weeks, in a whole-body exposure chamber. Then, by a quantitative real-time PCR method, we investigated the hippocampal mRNA-expression of several genes, functions of which are necessary to maintain the homeostasis of neuronal synaptic plasticity. We observed that chronic exposure of mice to 50 ppm toluene for a longer period (12 weeks) caused a significant up-regulation of NMDA receptor subunit 2B (NMDA NR2B) expression associated with a simultaneous induction of CaMKIV, CREB-1, and FosB/Delta FosB in the same hippocampal tissues. Our data indicate that the in vivo transcriptional up-regulation of these genes in the adult hippocampus of our experimental mouse model following the chronic exposure to toluene may be an NMDA-receptor related neuroprotective mechanism of gene expression. (c) 2006 Elsevier Inc. All rights reserved.
  ELSEVIER SCIENCE BV, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuro.2006.03.011
  DOI ID：10.1016/j.neuro.2006.03.011, ISSN：0161-813X, eISSN：1872-9711, Web of Science ID：WOS:000244844100021
• Progression of the dose-related effects of estrogenic endocrine disruptors, an important factor in declining fertility, differs between the hypothalamo-pituitary axis and reproductive organs of male mice　　　　　　　　　　　　　　　
  Katsuhiko Warita; Teruo Sugawara; Zhan-Peng Yue; Shinji Tsukahara; Ken-ichiro Mutoh; Yoshihisa Hasegawa; Hiroshi Kitagawa; Chisato Mori; Nobuhiko Hoshi
  JOURNAL OF VETERINARY MEDICAL SCIENCE, Volume：68, Number：12, First page：1257, Last page：1267, Dec. 2006, [Reviewed]
  For the purpose of investigation of working mechanisms in endocrine disruptors, we evaluated the dose-related effects of fetal and/or neonatal exposure to an estrogenic compound on the male reproductive organs in adult mice, particularly with respect to gene expression of steroidogenic acute regulatory protein (StAR). The pregnant ICR mice were given subcutaneous injections of 10 mu g/day/animal of diethylstilbestrol (DES) to subject the fetal mice to in utero exposure (IUE). Subsequently, the newborn male mice were subjected to neonatal exposure (NE) by treatment with vehicle or 0.1-10 mu g/day/animal of DES. Fertility rates of each group were as follows: control, 100%; IUE only, 60%; IUE+NE 0.1 mu g, 25%; IUE+NE 1 mu g, 0%; IUE+NE 10 mu g, 0%. In general histology, germ cell layers in the seminiferous tubules were thinned in the group of IUE+NE 10 mu g. Hypoplasia of the Leydig cells, in which the staining intensity of eosin was diminished, was also observed in the groups of IUE+NE 0.1-10 mu g. The androgen receptor (AR) and estrogen receptor alpha (ERA) immunoexpression in the Leydig cells of IUE+NE 1-10 mu g was slightly lower than that in the controls. Longterm dysfunction of the hypothalamo-pituitary-testicular axis, including sustained hypoproduction of gonadotropin and testosterone, and altered expressions of steroid hormone receptors and StAR genes were observed. The hypothalamo-pituitary control of gonadotropin secretion may be affected by the smaller doses of estrogenic agents than the reproductive organs. Furthermore, the fertility rate in the male mice exposed to this estrogenic agent was closely correlated with the testosterone levels, and even more so with the rate-limiting factor of steroidogenesis, StAR. This finding suggests that endocrine disruptors have an important pronounced effect on StAR gene expression.
  JAPAN SOC VET SCI, English, Scientific journal
  DOI：https://doi.org/10.1292/jvms.68.1257
  DOI ID：10.1292/jvms.68.1257, ISSN：0916-7250, Web of Science ID：WOS:000243327100002
• Sex differences in the level of Bcl-2 family proteins and caspase-3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal rats　　　　　　　　　　　　　　　
  Shinji Tsukahara; Masaki Kakeyama; Yuki Toyofuku
  JOURNAL OF NEUROBIOLOGY, Volume：66, Number：13, First page：1411, Last page：1419, Nov. 2006, [Reviewed]
  In developing rats, sex differences in the number of apoptotic cells are found in the central division of the medial preoptic nucleus (MPNc), which is a significant component of the sexually dimorphic nucleus of the preoptic area, and in the anteroventral periventricular nucleus (AVPV). Specifically, male rats have more apoptotic cells in the developing AVPV, whereas females have more apoptotic cells in the developing MPNc. To determine the mechanisms for the sex differences in apoptosis in these nuclei, we compared the expression of the Bcl-2 family members and active caspase-3 in postnatal female and male rats. Western blot analyses for the Bcl-2 family proteins were performed using preoptic tissues isolated from the brain on postnatal day (PD) 1 (day of birth) or on PD8. In the AVPV-containing tissues of PD1 rats, there were significant sex differences in the level of Bcl-2 (female > male) and Bax (female < male) proteins, but not of Bcl-xL or Bad proteins. In the MPNc-containing tissues of PD8 rats, there were significant sex differences in the protein levels for Bcl-2 (female < male), Bax (female > male), and Bad (female < male), but not for Bcl-xL. Immunohistochemical analyses showed significant sex differences in the number of active caspase-3-immunoreactive cells in the AVPV on PD1 (female < male) and in the MPNc on PD8 (female > male). We further found that active caspase-3-immunoreactive cells of the AVPV and MPNc were immunoreactive for NeuN, a neuronal marker. These results suggest that there are sex differences in the induction of apoptosis via the mitochondrial pathway during development of the AVPV and MPNc. (c) 2006 Wiley Periodicals, Inc.
  JOHN WILEY & SONS INC, English, Scientific journal
  DOI：https://doi.org/10.1002/neu.20276
  DOI ID：10.1002/neu.20276, ISSN：0022-3034, Web of Science ID：WOS:000241903700002
• Modulation of pro-inflammatory and neural activity-related gene expression in the olfactory bulb of mice by nasal inhalation of low-level toluene　　　　　　　　　　　　　　　
  Sohel Ahmed; Hidekazu Fujimaki; Tin-Tin-Win-Shwe; Shoji Yamamoto; Shinji Tsukahara; Yoshika Kurokawa; Daisuke Nakajima; Sumio Goto
  JOURNAL OF NEUROIMMUNOLOGY, Volume：178, First page：182, Last page：182, Sep. 2006, [Reviewed]
  ELSEVIER SCIENCE BV, English
  ISSN：0165-5728, Web of Science ID：WOS:000241633101152
• Pituitary adenylate cyclase-activating polypeptide neurons of the ventromedial hypothalamus project to the midbrain central gray　　　　　　　　　　　　　　　
  F Maekawa; K Fujiwara; S Tsukahara; T Yada
  NEUROREPORT, Volume：17, Number：2, First page：221, Last page：224, Feb. 2006, [Reviewed]
  The pituitary adenylate cyclase-activating polypeptide (PACAP) and brain-derived neurotrophic factor (BDNF) neurons have recently been established as markers of the ventromedial hypothalamic nucleus (VMH). However, their neural projections from the VMH remained unknown. We examined whether PACAP and BDNF neurons in the VMH connected to the mesencephalic central gray (MCG), using the combination of in-situ hybridization and immunohistochemical tracing of Fluorogold (FG) injected into the MCG. Approximately 49% of PACAP neurons and 19% of BDNF neurons in the VMH contained the retrograde-transported FG, and 52% of FG-positive cells were PACAP neurons. These results indicate that a large number of PACAP neurons and a small number of BDNF neurons of the VMH project to the MCG.
  LIPPINCOTT WILLIAMS & WILKINS, English, Scientific journal
  DOI：https://doi.org/10.1097/01.wnr.0000198945.62326.ba
  DOI ID：10.1097/01.wnr.0000198945.62326.ba, ISSN：0959-4965, Web of Science ID：WOS:000235349800022
• Increased Fos Immunoreactivity in Suprachiasmatic Nucleus before Luteinizing Hormone Surge in Estrogen-Treated Ovariectomized Female Rats　　　　　　　　　　　　　　　
  Shinji Tsukahara
  NEUROENDOCRINOLOGY, Volume：83, Number：5-6, First page：303, Last page：312, 2006, [Reviewed]
  Background/Aims: The suprachiasmatic nucleus (SCN) is thought to control the timing of luteinizing hormone (LH) surges. The present study was designed to examine temporal patterns of Fos expression in the dorsomedial and ventrolateral parts of the SCN (SCNdm and SCNvl) of female rats during an LH surge. It also included examination of temporal changes in plasma LH levels and temporal changes in Fos levels in the anteroventral periventricular nucleus (AVPV) and gonadotropin-releasing hormone (GnRH) neurons. Methods: Ovariectomized rats injected with 20 mu g estradiol benzoate (EB) or vehicle were sacrificed at various times from Zeitgeber time (ZT) 8:00 to 16:30 h (ZT8-16.5; ZT0 = lights on; ZT12 = lights off) on the 2nd day after the injection. Immunohistochemical analyses for Fos and GnRH and enzyme-linked immunosorbent assays for LH were then performed. Results: In both the SCNdm and SCNvl of EB rats, the number of Fos-immunoreactive cells significantly increased between ZT9.5-10.5 and ZT11-12. On the other hand, in EB rats there were significant peaks of LH levels and Fos levels in GnRH neurons and the AVPV between ZT11-12 and ZT13-14. There was no significant difference in the number of Fos-immunoreactive cells between EB and control rats in either the SCNdm or SCNvl at ZT9.5-10.5, or in the SCNdm at ZT11-12, whereas the SCNvl of EB rats contained more Fos-immunoreactive cells than that of control rats at ZT11-12. Conclusion: These results suggest that in female rats during an LH surge, a peak in the Fos level in the SCN precedes peaks in Fos levels in the AVPV and GnRH neurons. Copyright (C) 2006 S. Karger AG, Basel
  KARGER, English, Scientific journal
  DOI：https://doi.org/10.1159/000095341
  DOI ID：10.1159/000095341, ISSN：0028-3835, Web of Science ID：WOS:000208027900003
• Inhalation of low-level formaldehyde increases the Bcl-2/Bax expression ratio in the hippocampus of immunologically sensitized mice　　　　　　　　　　　　　　　
  Shinji Tsukahara; Shoji Yamamoto; Tin-Tin-Win-Shwe; Sohel Ahmed; Naoki Kunugita; Keiichi Arashidani; Hidekazu Fujimaki
  NEUROIMMUNOMODULATION, Volume：13, Number：2, First page：63, Last page：68, 2006, [Reviewed]
  Objective: A recent study from our research group showed that repeated exposure to low-level formaldehyde (FA) increases the production of nerve growth factor, involving the survival and maintenance of neurons, in the hippocampus of immunized mice. In the present study, we examined the effects of FA on apoptotic mechanisms regulating survival and death of cells and on N-methyl-D-aspartate (NMDA) receptors related to hippocampal functions in the mouse hippocampus. Methods: Western blot analyses were performed for Bcl-2, Bax and NMDA receptor subtypes 2A and 2B of the hippocampus taken from C3H mice exposed to 0 or 400 ppb of FA with or without ovalbumin (OVA) immunization. Immunohistochemical analysis for active caspase-3 was also carried out for these mice. Results: The ratio of Bcl-2 to Bax expression levels significantly increased with 400-ppb FA exposure in OVA-immunized mice but not in mice without OVA immunization, although differences in each protein level were not significant among groups. Active caspase-3-immunoreactive cells were found in the hippocampus. However, the number was only a few and not significantly affected by FA exposure and OVA immunization. NMDA receptor type 2A and 2B expression levels of FA-exposed mice were sustained at comparative levels with those for the control mice with or without OVA immunization. Conclusions: These results indicate that changes in the Bcl-2/Bax expression ratio, which occurs with low-level FA exposure and immunization and may follow enhancement of nerve growth factor production, exerts a protective effect against cell death by apoptosis. Copyright (c) 2006 S. Karger AG, Basel.
  KARGER, English, Scientific journal
  DOI：https://doi.org/10.1159/000094829
  DOI ID：10.1159/000094829, ISSN：1021-7401, Web of Science ID：WOS:000241944000001
• Age-related change and its sex differences in histoarchitecture of the hypothalamic suprachiasmatic nucleus of F344/N rats　　　　　　　　　　　　　　　
  S Tsukahara; S Tanaka; K Ishida; N Hoshi; H Kitagawa
  EXPERIMENTAL GERONTOLOGY, Volume：40, Number：3, First page：147, Last page：155, Mar. 2005, [Reviewed]
  The present study examined the effects of aging and sex differences on the suprachiasmatic nucleus (SCN) of F344/N rats. In juveniles (1.6-1.9 months of age), adults (11.7-16.3 months of age), and old (29.2-34 months of age) rats, the volume, size of neuronal nucleus and neuronal cell number of the SCN were determined with cresyl fast violet-stained sections. In addition, immunohistochemical analysis was performed for glial fibrillary acidic protein (GFAP). There was no significant effect of aging and sex differences on the SCN volume. The number of neurons in the SCN gradually decreased from juvenile to old age in females. However, in males, the number was significantly decreased in adult and old age rats. The size of neuronal nuclei in the SCN was significantly decreased by increasing age in both sexes, except for the ventrolateral part of the SCN of males. In the dorsomedial part of the SCN of females, the density of GFAP-immunoreactive components was significantly higher in adult age rats than in rats of other ages. However, there was no significant increase in the density of the SCN in adult males. These results suggest that morphological changes in neuronal and astroglial cells occur in the SCN with aging in a sex-specific manner. (c) 2004 Elsevier Inc. All rights reserved.
  PERGAMON-ELSEVIER SCIENCE LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.exger.2004.10.003
  DOI ID：10.1016/j.exger.2004.10.003, ISSN：0531-5565, Web of Science ID：WOS:000228158500004
• Postnatal apoptosis, development, and sex difference in the lateral septum of rats　　　　　　　　　　　　　　　
  S Tsukahara; K Inami; F Maekawa; M Kakeyama; T Yokoyama; M Yuji; H Kitagawa; Y Kannan; K Yamanouchi
  JOURNAL OF COMPARATIVE NEUROLOGY, Volume：475, Number：2, First page：177, Last page：187, Jul. 2004, [Reviewed]
  To determine whether apoptosis is involved in the formation of the structure and morphological sex difference of the lateral septum (LS), the postnatal developmental changes in the number of apoptotic cells were examined in the LS on postnatal day 1 (PD1 = birth day), 4, 6, 8, 11, 16, and 31 in male and female rats. Apoptotic cells were imimmohistochemically detected by antibody against single-stranded DNA (ssDNA) or active caspase-3. The volume of the LS was also measured and was found to increase with age. The number of apoptotic cells detected by anti-ssDNA in the LS increased from PD1 to PD8 but decreased after PD11. Also, the LS was divided into dorsal, intermediate, and ventral parts (LSd, LSi, and LSv), and the volume and number of ssDNA-immunoreactive cells in each part were measured on PD6, 8, 11, 16, and 31. In both sexes, a large number of ssDNA-immunoreactive cells was found in the LSd and LSi on PD8 (but not on PD6) and in the LSv on PD6 and PD8. On PD6, the number of active caspase-3-immunoreactive cells was significantly greater in the LSv than in the LSd or LSi, in both sexes. Only the LSi of males had a high number of ssDNA-immunoreacitve cells on PD16; the number was significantly greater than that of females of the same age. However, there was no significant sex difference in the number of active caspase-3-immunoreacitve cells in the LSi on PD16. On PD31, the volume of the LSi was significantly greater in females than in males. There was no sex difference in volume or number of apoptotic cells in the LSd or LSv. These findings indicate that loss of cells due to apoptosis, which is partially caused by activation of caspase-3, occurs in the LS during postnatal development, with regional differences. They also indicate that sex difference in caspase-3-independent apoptosis contributes to morphological sexual differentiation of the LSi. (C) 2004 Wiley-Liss, Tue.
  WILEY-LISS, English, Scientific journal
  DOI：https://doi.org/10.1002/cne.20184
  DOI ID：10.1002/cne.20184, ISSN：0021-9967, Web of Science ID：WOS:000222414500003
• Distributions of two chicken bombesin receptors, bombesin receptor subtype-3.5 (chBRS-3.5) and gastrin-releasing peptide receptor (chGRP-R) mRNAS in the chicken telencephalon　　　　　　　　　　　　　　　
  F Maekawa; S Tsukahara; K Tanaka; H Ohki-Hamazaki
  NEUROSCIENCE, Volume：125, Number：3, First page：569, Last page：582, 2004, [Reviewed]
  Bombesin (BN)-like peptide receptors are known to be essential to the regulation of not only homeostasis, including feeding behavior, but also of emotional systems in mammal. Recently, two novel BN receptors, chicken BN-like peptide receptor subtype-3.5 (chBRS-3.5) and gastrin-releas-ing peptide receptor (chGRP-R), have been identified. Here, we report the localizations of these receptors' mRNAs in the chick brain through development using in situ hybridization. First, chBRS-3.5 mRNA signals were found in the dorsal ventricular ridge at embryonic day (ED) 9. Strong signals were observed in the hyperpallium accessorium, nidopallium and nucleus basorostralis pallii, and moderate signals were found in the hippocampus, cortex piriformis, hyperpallium intercalatum, area temporo-parieto-occipitalis, nucleus striae terminalis lateralis, nucleus olfactorius anterior and organum septi lateralis at ED16. This wide expression in the palliurn persisted during posthatch periods. Abundant expressions in the hyperpallium, nidopallium, considered to be similar to the mammalian cortex, as well as in the hippocampus, indicate participation of these molecules in the processing of sensory information, motor function, learning and memory. Telencephalic areas devoid of chBRS-3.5 signals were the entopallium, arcopallium anterius, globus pallidus, nucleus intrapeduncularis, tuberculum olfactorius, nucleus septalis lateralis, hypothalamic and thalamic areas. In contrast to chBRS-3.5, chGRP-R mRNA signals were found in the pallidum at ED5 and 9. At ED16, chGRP-R mRNA signals were localized in the medial striatum and hypothalamus. GRP-R expression in the hypothalamic region was phylogenically conserved.
  Thus, chBRS-3.5 mRNA signals were distributed in a broader region and were more intense than chGRP-R mRNA. Taken together, chGRP-R and chBRS-3.5 mRNA occurred in similar regions of mammals that express GRP-R.
  BN/GRP-immunoreactive neurons and varicosities were found mainly in the pallium, especially in the hyperpallium accessorium and nidopallium, and this distribution coincided with that of chBRS-3.5 mRNA. This result suggests that the endogenous ligands for chBRS-3.5 were likely BN-like peptides produced in the pallium. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.
  PERGAMON-ELSEVIER SCIENCE LTD, English, Scientific journal
  DOI：https://doi.org/10.1016/j.neuroscience.2004.01.057
  DOI ID：10.1016/j.neuroscience.2004.01.057, ISSN：0306-4522, Web of Science ID：WOS:000221278000006
• Neonatal estrogen decreases neural density of the septum-midbrain central gray connection underlying the lordosis-inhibiting system in female rats　　　　　　　　　　　　　　　
  S Tsukahara; N Ezawa; K Yamanouchi
  NEUROENDOCRINOLOGY, Volume：78, Number：4, First page：226, Last page：233, Oct. 2003, [Reviewed]
  Neurons in the lateral septum (LS) with projecting axons to the midbrain central gray (MCG) exert an inhibitory influence on lordosis. The number of such neurons is greater in female than in male rats. In this experiment, effects of neonatal estrogen on the density of the LS-MCG connections and on lordosis behavior were examined in female rats. On postnatal day 4 (day 0 = day of birth), females were injected subcutaneously with 50 or 100 mug estradiol benzoate (EB) or oil. On postnatal day 60, females and control males were gonadectomized. Behavioral tests were carried out after the implantation of silicone tubes containing estradiol. Lordotic activities in both males and EB-treated females were lower than in oil-treated females. After completing the behavioral tests, the animals were injected with Fluoro-Gold (FG), a retrograde tracer, into the right-side MCG and the number of FG-labeled neurons in the LS was measured. In all groups, the right-side LS ipsilateral to the FG injection had more FG-labeled neurons than the left-side LS. The number of FG-labeled neurons in the LS of oil-treated females was larger than that of males on both right and left sides. In the females treated with 100 mug EB (EB100), the number of FG-labeled neurons was comparable with that of males and lower than that of oil-treated females. The number of FG-labeled neurons in the EB50 females was also lower than that in oil-treated females, but tended to be larger than that observed in the EB100 group. These results indicate that neonatal estrogen decreases both lordotic activity and the density of the LS-MCG neural connections in female rats. Copyright (C) 2003 S. Karger AG, Basel.
  KARGER, English, Scientific journal
  DOI：https://doi.org/10.1159/000073706
  DOI ID：10.1159/000073706, ISSN：0028-3835, Web of Science ID：WOS:000186459200005
• Migration of erythroblastic islands toward the sinusoid as erythroid maturation proceeds in rat bone marrow　　　　　　　　　　　　　　　
  T Yokoyama; T Etoh; H Kitagawa; S Tsukahara; Y Kannan
  JOURNAL OF VETERINARY MEDICAL SCIENCE, Volume：65, Number：4, First page：449, Last page：452, Apr. 2003, [Reviewed]
  It has been hitherto considered that mature erythroblasts migrate toward the sinusoid along the cytoplasmic processes of macrophages of erythroblastic islands in bone marrow. Our previous report, however, has demonstrated the morphological features of a mature erythroblastic island passing through the sinusoidal endothelium. In this study, the possibility of migration of erythroblastic islands toward the sinusoid was examined in rat bone marrow by light microscopical histoplanimetry. As a result, the more mature erythroblasts were not regularly arranged in the peripheral direction of the erythroblastic islands. Immature erythroblasts were populated more in the erythroblastic islands away from the sinusoid than in those islands neighboring the sinusoid, whereas mature erythroblasts were more in erythroblastic islands neighboring the sinusoid. These findings suggest that the formation of erythroblastic islands occurs in a region away from the sinusoid, and that erythroblastic islands migrate towards the sinusoids as erythroid maturation proceeds.
  JAPAN SOC VET SCI, English, Scientific journal
  DOI：https://doi.org/10.1292/jvms.65.449
  DOI ID：10.1292/jvms.65.449, ISSN：0916-7250, Web of Science ID：WOS:000182670800002
• Distribution of glutamic acid decarboxylase, neurotensin, enkephalin, neuropeptide Y, and cholecystokinin neurons in the septo-preoptic region of male rats　　　　　　　　　　　　　　　
  S Tsukahara; K Yamanouchi
  JOURNAL OF REPRODUCTION AND DEVELOPMENT, Volume：49, Number：1, First page：67, Last page：77, Feb. 2003, [Reviewed]
  Neurons in the lateral septum (LS) and preoptic area (POA) are known to play an inhibitory role in feminine sexual behavior regulation in male rats. In this study, the distribution of neurons containing glutamic acid decarboxylase (GAD) and of the peptidergic neurotransmitters neurotensin (NT), enkephalin (ENK), neuropeptide Y (NPY), and cholecystokinin (CCK), was examined immunohistochemically in the LS and POA of castrated male rats subcutaneously implanted with estrogen-containing Silastic tubes. Colchicine was injected into the lateral ventricle of the animals. The forebrain sections were immunostained for each substance. A large number of GAD-immunoreactive (ir) cells were found in the LS. Many NT-ir cells were seen in the intermediate and ventral parts of the LS at the rostral and middle levels. A considerable number of ENK-ir cells were scattered over the LS at the rostral and middle levels and were observed in the ventral part of the caudal LS. There were only a few NPY-ir cells in the LS. No CCK-ir cells were observed in the LS. In the POA, GAD-ir cells were observed in abundance. Many NT-ir cells were seen, especially in the medial preoptic nucleus. Some ENK-ir cells and a few NPY-ir cells were found in the medial POA. CCK-ir cells of the POA were restricted to the periventricular and paraventricular hypothalamic nuclei.
  SOCIETY REPRODUCTION & DEVELOPMENT-SRD, English, Scientific journal
  DOI：https://doi.org/10.1262/jrd.49.67
  DOI ID：10.1262/jrd.49.67, ISSN：0916-8818, Web of Science ID：WOS:000181142400008
• No apoptotic cell death of erythroid cells of erythroblastic islands in bone marrow of healthy rats　　　　　　　　　　　　　　　
  T Yokoyama; H Kitagawa; T Takeuchi; S Tsukahara; Y Kannan
  JOURNAL OF VETERINARY MEDICAL SCIENCE, Volume：64, Number：10, First page：913, Last page：919, Oct. 2002, [Reviewed]
  A possibility of apoptotic cell death in erythropoietic regulation was examined by means of detailed light microscopical histoplanimetry, electron microscopy, the in situ nick-end labeling method, and an immunohistological method in the rat bone marrow. Serum erythropoietin concentrations were shown at normal levels. The erythroid series on a mature process presented several morphological features of apoptosis, i.e. the shrinkage of both nuclei and cytoplasm and the chromatin condensation. In the light microscopical histoplanimetry, however, morphological signs of final apoptotic cell death were never found in any erythroid cell within the erythroblastic islands. This finding was also supported by detailed ultrastructural observation: No erythroid cell bodies were trapped and degraded by the central macrophages of the erythroblastic islands, while the denucleated nuclei with small amount of cytoplasm of late erythroblasts were often trapped and degraded in the macrophages. Nuclear DNA fragmentation was not detected in any erythroblasts, but was detected in the lysosomes of the central macrophages. These findings suggest that erythropoiesis is regulated by other regulatory mechanisms than apoptotic cell death. An additional ultrastructural finding shows that the reticulocytes anchored to the central macrophages are transported into the peripheral blood circulation.
  JAPAN SOC VET SCI, English, Scientific journal
  DOI：https://doi.org/10.1292/jvms.64.913
  DOI ID：10.1292/jvms.64.913, ISSN：0916-7250, Web of Science ID：WOS:000179072500008
• The arcuate nucleus mediates facilitating effect of estrogen on glutamate-induced in vitro GnRH release from nerve terminals of female rats　　　　　　　　　　　　　　　
  K Murahashi; S Tsukahara; H Tsukamura
  JOURNAL OF REPRODUCTION AND DEVELOPMENT, Volume：48, Number：2, First page：183, Last page：188, Apr. 2002, [Reviewed]
  It is well accepted that glutamate stimulates in vivo and in vitro gonadotropin-releasing hormone (GnRH) and/or luteinizing hormone (LH) release. The present study aimed to determine whether the arcuate nucleus (ARC) has a role in exerting the modulating effect of estrogen on GnRH response to glutamate. The effects of estrogen on glutamate-induced in vitro GnRH release from either the ARC-median eminence (ME) or ME fragment were examined in female rats. Both high and low doses of estradiol-17beta treatment in ovariectomized OVX animals enhanced glutamate-induced GnRH release from the ARC-ME fragment but not from the ME fragment. This suggests that the ARC mediates the facilitating effect of estrogen on glutamate-induced GnRH release from the ARC-ME. The facilitating effect of estrogen was also observed on GnRH release from the ARC-ME treated with selective excitatory amino acid agonists, i.e., N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. These results suggest that the ARC plays an important role in facilitating estrogen actions on GnRH response to glutamate via both NMDA and non-NMDA receptors. The estrogen-induced enhancement of GnRH secretory response to glutamate in the ARC-ME region could partly contribute to the induction of preovulatory GnRH/LH surge.
  SOCIETY REPRODUCTION & DEVELOPMENT-SRD, English, Scientific journal
  ISSN：0916-8818, Web of Science ID：WOS:000179039800011
• Sex difference in septal neurons projecting axons to midbrain central gray in rats: A combined double retrograde tracing and ER-immunohistochemical study　　　　　　　　　　　　　　　
  Shinji Tsukahara; Korehito Yamanouchi
  Endocrinology, Volume：143, Number：1, First page：285, Last page：294, 2002, [Reviewed]
  Sex difference in the number of neurons projecting axons from the lateral septum (LS) to the midbrain central gray (MCG) that are concerned with the lordosis-inhibiting system was investigated by injection of Fluoro-Gold (FG), a retrograde tracer, into the rostral MCG on the right side in male and female rats. Immunohistochemistry for ER-α and -β was also performed with or without combination with FG immunostaining. All animals were gonadectomized. Lordosis was observed after treatment with E2 in some animals. In the results, lordosis was rare in males, compared with females. FG-immunoreactive (ir) cells were concentrated in the intermediate LS on the right side, and its number in the females was significantly higher than that in the males. There was no sex difference in the distribution and number of ERα-ir and ERβ-ir cells in the LS. Furthermore, the number of ERs-ir cells was not influenced by E2 in either males or females. Double FG-ERβ-ir cells were less than 20% of total FG-ir cells in the LS in both males and females. These data suggest that the LS-MCG connection is sexually dimorphic but that there is no sex difference in the expression of ERs in the LS.
  Endocrine Society, English, Scientific journal
  DOI：https://doi.org/10.1210/endo.143.1.8588
  DOI ID：10.1210/endo.143.1.8588, ISSN：0013-7227, PubMed ID：11751620, SCOPUS ID：0036141055
• Inhibitory effect of postpartum lesions or cuts in median raphe nucleus on maternal behavior in female rats.　　　　　　　　　　　　　　　
  H Yurino; S Tsukahara; L Koranyi; K Yamanouchi
  ZOOLOGICAL SCIENCE, Volume：18, Number：9, First page：1225, Last page：1230, Dec. 2001, [Reviewed]
  In order to clarify the role of the median (MRN) and dorsal (DRN) raphe nuclei in regulating maternal care (retrieving and licking behavior), radiofrequency lesions or microknife cuts were made in postpartum rats on the day after delivery. Animals were housed individually without pups after the operation. One week after the surgery, maternal behavioral test was carried out daily for 3 days using pups of 2-6 days age. The results demonstrated that rats with MRN lesions or ventral horizontal cuts of the MRN showed extremely low incidence of the maternal behavior, as compared to those in control and sham-operated groups. DRN-lesions or dorsal cuts of the MRN had no effect. In locomotor activities measured by the infrared sensor system, there was no difference between the groups. This suggest that the MRN but not DRN plays a critical role in regulating retrieving and licking behaviors and ventral outputs are involved in this function in postpartum rats.
  ZOOLOGICAL SOC JAPAN, English, Scientific journal
  DOI：https://doi.org/10.2108/zsj.18.1225
  DOI ID：10.2108/zsj.18.1225, ISSN：0289-0003, Web of Science ID：WOS:000174124200006
• Neurohistological and behavioral evidence for lordosis-inhibiting tract from lateral septum to periaqueductal gray in male rats
  S Tsukahara; K Yamanouchi
  JOURNAL OF COMPARATIVE NEUROLOGY, Volume：431, Number：3, First page：293, Last page：310, Mar. 2001, [Reviewed]
  To verify the anatomical and functional connection of the lateral septum (LS) and periaqueductal gray (PAG) in inhibiting female sexual behavior, lordosis, in male rats, retrograde (Fluoro-Gold, FG) or anterograde (Phaseolus vulgaris-leucoagglutinin, PHA-L) tracer was injected into the PAG or LS on the right side, respectively, and FG-labeled cells or PHA-L-labeled axons in the forebrain and mesencephalon were determined in estrogen-treated castrated male rats. A ventral cut (VC) of the septum and a behavioral test were also conducted in some FG-injected rats. Furthermore, lordosis behavior was observed after chemical destruction of the septum by ibotenate. As a result, the lordosis quotient (LQ) in VC males was higher than that in control males without VC. FG-labeled neuronal cell bodies were found in the ipsilateral intermediate part of the LS in the control males but not in this area of the VC males. When neuronal cells in the intermediate part of the bilateral LS were completely destroyed by ibotenate, the LQ was higher than that in sham-lesioned male rats. These results suggest that a direct neural connection of the intermediate LS to the PAG has an inhibitory role in regulating lordosis in male rats. In addition, neuronal cell bodies in the intermediate LS exert an inhibitory influence. In the PHA-L experiment, labeled axons were seen in the ventral part of the LS, the medial forebrain bundle at the chiasmatic level, the lateral hypothalamus, the median region of the mesencephalon, and the rostral PAG in the side ipsilateral to the tracer injection site of the LS. Thus, these areas are thought to be involved in the pathway for lordosis-inhibition from the intermediate part of the LS to the PAG in male rats.
  WILEY-LISS, English, Scientific journal
  ISSN：0021-9967, Web of Science ID：WOS:000166761200004
• Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat　　　　　　　　　　　　　　　
  H Tsukamura; RC Thompson; S Tsukahara; S Ohkura; F Maekawa; R Moriyama; Y Niwa; DL Foster; KI Maeda
  JOURNAL OF NEUROENDOCRINOLOGY, Volume：12, Number：6, First page：529, Last page：534, Jun. 2000, [Reviewed]
  Melanin-concentrating hormone (MCH) has been reported to be involved in the regulation of feeding behaviour in rats and mice. Because many neuropeptides that influence ingestive behaviour also regulate reproductive function, the present study was designed to determine if central administration of MCH changes pulsatile secretion of luteinizing hormone (LH) in the rats. Wistar-Imamichi strain female rats were ovariectomized and implanted with oestradiol to produce a moderate inhibitory feedback effect on LH release. The effects of i.c.v. injections of MCH on LH release were examined in freely moving animals. Blood samples were collected every 6 min for 3 h through an indwelling cannula. After 1 h of sampling, MCH (0.1, 1 or 10 mu g/animal) or vehicle (saline) was injected into the third cerebroventricle. Because MCH is also reported to affect the hypothalamo-pituitary-adrenal (HPA) axis, which in turn, can influence reproductive function, plasma corticosterone concentrations were determined in the same animals at 30-min intervals during the first and last hours and every 12 min during the second hour of the 3-h sampling period. When expressed as per cent changes, mean plasma LH concentrations after MCH administration were significantly tower in the animals injected with all doses of the peptide compared with vehicle-treated animals; LH pulse frequency was significantly lowered by 1 mu g of MCH. Per cent changes in mean plasma corticosterone levels were not significantly affected by MCH administration. These results in oestradiol-treated ovariectomized rats indicate that central MCH is capable of inhibiting pulsatile LH secretion. We have previously shown that 48-h fasting suppresses pulsatile LH release in the presence of oestrogen, Take together, these results raise the possibility that MCH could play a role in mediating the suppression of LH secretion during periods of reduced nutrition.
  BLACKWELL SCIENCE LTD, English, Scientific journal
  DOI：https://doi.org/10.1046/j.1365-2826.2000.00482.x
  DOI ID：10.1046/j.1365-2826.2000.00482.x, ISSN：0953-8194, Web of Science ID：WOS:000087420000009
• Peripheral or central administration of motilin suppresses LH release in female rats: A novel role for motilin　　　　　　　　　　　　　　　
  H Tsukamura; S Tsukahara; F Maekawa; R Moriyama; BAS Reyes; T Sakai; Y Niwa; DL Foster; KI Maeda
  JOURNAL OF NEUROENDOCRINOLOGY, Volume：12, Number：5, First page：403, Last page：408, May 2000, [Reviewed]
  Motilin is secreted in a clear episodic pattern during fasting or during the interdigestive phase, but feeding promptly stops this secretory pattern, and plasma concentrations of motilin decrease. We have previously determined that fasting markedly suppresses pulsatile luteinizing hormone (LH) secretion in female rats in the presence of oestrogen. In the present study, we wished to learn if motilin may mediate the fasting-induced suppression of LH secretion by determining the effects of motilin administration on LH release and on food intake. Intravenous (i.v.) injection of motilin (37 nmol/rat) suppressed LH release and significantly decreased mean LH concentrations both in ovariectomized (OVX) and oestradiol-implanted ovariectomized (OVX + E-2) rats. Food intake was significantly increased by i.v. motilin injection in OVX rats, but not in OVX + E-2 rats. It is likely that motilin inhibits LH release via inhibition of the gonadotrophin-releasing hormone (GnRH)-releasing mechanism at the hypothalamic level, because motilin (3.7 nmol/rat) also suppressed LH secretion when centrally administered, and because LH release in i.v. motilin-treated rats increased in response to exogenous GnRH. These results suggest that motilin may be a peripheral signal for the suppression of LH secretion through central sensors.
  BLACKWELL SCIENCE LTD, English, Scientific journal
  DOI：https://doi.org/10.1046/j.1365-2826.2000.00467.x
  DOI ID：10.1046/j.1365-2826.2000.00467.x, ISSN：0953-8194, Web of Science ID：WOS:000086702400005
• Localization of glucokinase-like immunoreactivity in the rat lower brain stem: For possible location of brain glucose-sensing mechanisms　　　　　　　　　　　　　　　
  F Maekawa; Y Toyoda; N Torii; Miwa, I; RC Thompson; DL Foster; S Tsukahara; H Tsukamura; K Maeda
  ENDOCRINOLOGY, Volume：141, Number：1, First page：375, Last page：384, Jan. 2000, [Reviewed]
  Pancreatic glucokinase (GK) is considered an important element of the glucose-sensing unit in pancreatic P-cells. It is possible that the brain uses similar glucose-sensing units, and we employed GK immunohistochemistry and confocal microscopy to examine the anatomical distribution of GK-like immunoreactivities in the rat brain. We found strong GK-like immunoreactivities in the ependymocytes, endothelial cells, and many serotonergic neurons. In the ependymocytes, the GK-like immunoreactivity was located in the cytoplasmic area, but not in the nucleus. The GK-positive ependymocytes were found to have glucose transporter-2 (GLUT2)-like immunoreactivities on the cilia. In addition, the ependymocytes had GLUT1-like immunoreactivity on the cilia and GLUT4-like immunoreactivity densely in the cytoplasmic area and slightly in the plasma membrane. In serotonergic neurons, GK-like immunoreactivity was found in the cytoplasm and their processes. The present results raise the possibility that these GK-like immunopositive cells comprise a part of a vast glucose-sensing mechanism in the brain.
  ENDOCRINE SOC, English, Scientific journal
  DOI：https://doi.org/10.1210/endo.141.1.7234
  DOI ID：10.1210/endo.141.1.7234, ISSN：0013-7227, eISSN：1945-7170, Web of Science ID：WOS:000084332200045
• Prevention of inhibitory effect of dorsal raphe nucleus lesions on ovulation and LH surge by 5-HT 2A/2C receptor agonists in female rats　　　　　　　　　　　　　　　
  F Maekawa; S Tsukahara; H Tsukamura; K Maeda; K Yamanouchi
  NEUROSCIENCE RESEARCH, Volume：35, Number：4, First page：291, Last page：298, Dec. 1999, [Reviewed]
  In order to investigate the role of the dorsal raphe nucleus and the serotonergic system in the regulation of ovulation, the number of ova and plasma luteinizing hormone (LH) concentrations were measured in female rats after making lesions in this nucleus (DRL and/or treatment with 5-hydroxytryptamine (5-HT) receptor agonists or antagonists. DRL or sham lesion was made on the afternoon of proestrous (12:00-14:00 h) under ether anesthesia and the number of ova in the oviduct was counted on the next estrous and diestrous morning. In some animals, blood samples were taken via the atrial cannula during the proestrous evening for the radioimmunoassay of LH. All intact control and sham-operated females ovulated and plasma LH increased between 19:00 and 21:00 h. In contrast, ovulation was seen in only 36% of DRL rats. LH surge did not occur in this group. However, 80% of DRL rats ovulated after treatment with (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride hCRF-(21-41), or vehicle was injected into the third cerebral ventricle through the implanted cannula before 2DG administration through the indwelling atrial cannula, Pulsatile LH secretion was suppressed by 2DG (200 mg/kg b.w.) in the vehicle-treated rats bearing OE2 implants. The CRH antagonist (5.65 nmol) blocked the suppressive effect of 2DG on pulsatile LH secretion in the OE2-treated OVX animals. On the other hand, in the absence of oestrogen, the effect of a twice greater dose of 2DG (400 mg/kg b.w.) was not blocked by five times greater amount of CRH antagonist (28.3 nmol). These results suggest the mechanisms mediating glucoprivic suppression of LH secretion involve two components: one is oestrogen-dependent and the other oestrogenin-dependent. CRH may be involved in the oestrogen-dependent component of glucoprivic suppression of LH secretion but not the oestrogen-independent one.
  BLACKWELL SCIENCE LTD, English, Scientific journal
  DOI：https://doi.org/10.1046/j.1365-2826.1999.00312.x
  DOI ID：10.1046/j.1365-2826.1999.00312.x, ISSN：0953-8194, Web of Science ID：WOS:000078761400003
• Inhibitory effect of neural transections of dorsal raphe nucleus on induction of nocturnal prolactin surge by vaginal stimulation in ovariectomized rats.　　　　　　　　　　　　　　　
  MAEKAWA F.
  Brain Res., Volume：813, Number：1, First page：195, Last page：199, 1998, [Reviewed]
  English
  DOI ID：10.1016/s0006-8993(98)01019-1, CiNii Articles ID：80010697066, CiNii Books ID：AA0057324X
• Estrogen modulates effects of glutamate on in vitro gonadotropin-releasing hormone release by altering nitric oxide action in female rats.　　　　　　　　　　　　　　　
  Tsukahara S; Tsukamura H; Maeda K
  Volume：44, Number：4, First page：399, Last page：405, 1998, [Reviewed]
  English
• Vagus nerve mediates the increase in estrogen receptors in the hypothalamic paraventricular nucleus and nucleus of the solitary tract during fasting in ovariectomized rats　　　　　　　　　　　　　　　
  Maria Amelita C. Estacio; Hiroko Tsukamura; Sakiko Yamada; Shinji Tsukahara; Kanjun Hirunagi; Kei-Ichiro Maeda
  Neuroscience Letters, Volume：208, Number：1, First page：25, Last page：28, Apr. 1996, [Reviewed]
  The effect of total subdiaphragmatic vagotomy on estrogen-receptor immunoreactivity (ERIR) in the paraventricular nucleus (PVN) and nucleus of the solitary tract (NTS) was examined in fasted ovariectomized rats to clarify the peripheral inputs mediating fasting-induced increase in ERIR in these two nuclei. Vagotomy abolished the effect of 48-h fasting on the expression of ER in these two areas. The result indicates that the neural signal(s) that increase the expression of ER in the PVN and A2 region of the NTS following 48-h fasting is transmitted through the vagus. The involvement of the vagus in the fasting-induced increase in ER in the PVN and A2 region may also be the same neural pathway involved in the suppression of pulsatile luteinizing hormone secretion in fasted female rats.
  Elsevier Ireland Ltd, English, Scientific journal
  DOI：https://doi.org/10.1016/0304-3940(96)12534-9
  Scopus：https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=0029917573&origin=inward
  Scopus Citedby：https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=0029917573&origin=inward
  DOI ID：10.1016/0304-3940(96)12534-9, ISSN：0304-3940, PubMed ID：8731166, SCOPUS ID：0029917573
• Suppression of luteinizing hormone pulses by restriction of glucose availability is mediated by sensors in the brain stem　　　　　　　　　　　　　　　
  K Murahashi; DC Bucholtz; S Nagatani; S Tsukahara; H Tsukamura; DL Foster; KI Maeda
  ENDOCRINOLOGY, Volume：137, Number：4, First page：1171, Last page：1176, Apr. 1996, [Reviewed]
  The availability of metabolic fuels such as glucose is known to influence reproductive function. Peripheral administration of 2-deoxyglucose (2DG), a competitive inhibitor of glycolysis, inhibits pulsatile LH secretion in the rat and growth-retarded lamb. We hypothesized that such glucoprivic suppression of LH secretion is mediated by the lower brain stem, because studies of both ingestive and reproductive behavior implicate lower brain stem structures, such as the area postrema, as a site that is sensitive to glucose availability. In the present study, the effect of a 2DG infusion, targeted to the fourth ventricle, on pulsatile LH secretion was examined in male rats. The males were castrated or castrated and immediately implanted with testosterone. Blood samples were collected through an indwelling atrial cannula every 6 min for 4 h for LH determination. After the first hour of blood sampling, 2DG (4 or 40 mg/kg) was infused into the fourth ventricle at a now rate of 0.2 mu l/min through a cannula that had been stereotaxically implanted 1 week before sampling. The high dose of 2DG (40 mg/kg), but not the low dose (4 mg/kg), suppressed pulsatile LH secretion and increased food intake in both castrated and testosterone-treated castrated rats. LH secretion and food intake were not affected by the infusion of xylose (40 mg/kg) as an isoosmotic control. The site specificity of the 2DG treatment was confirmed by histological examination after an isovolumetric infusion of dye (0.2 mu l/min). These results suggest that glucose availability could influence LH secretion as well as feeding through a central sensor in the lower brain stem and are consistent with the idea that the area postrema might be an important glucosensor involved in the modulation of LH secretion.
  ENDOCRINE SOC, English, Scientific journal
  DOI：https://doi.org/10.1210/endo.137.4.8625886
  DOI ID：10.1210/endo.137.4.8625886, ISSN：0013-7227, eISSN：1945-7170, Web of Science ID：WOS:A1996UB89100005

• マウスの内側視索前野に存在するカルビンディン-D28Kニューロンの神経投射の性差(Sexually dimorphic neural projections of calbindin-D28K neurons in the medial preoptic area of mice)　　　　　　　　　　　　　　　
  Morishita Masahiro; Ono Kota; Tsuneoka Yousuke; Horio Shuhei; Kato Shigeki; Kobayashi Kazuto; Tsukahara Shinji
  The Journal of Physiological Sciences, Volume：70, Number：Suppl.1, First page：S132, Last page：S132, Mar. 2020
  (一社)日本生理学会, English
  ISSN：1880-6546, eISSN：1880-6562, 医中誌Web ID：2021042274
• マウスの脳内で新たに見つかった性的二型核　　　　　　　　　　　　　　　
  塚原伸治
  Volume：44, Number：163, First page：7, Last page：11, 2018, [Invited]
  Japanese, Introduction scientific journal
• 培養細胞を用いた発達神経毒性評価試験法の近年の動向　　　　　　　　　　　　　　　
  前川文彦; チョウ テッ アウン; 中村和昭; 佐野一広; ティン ティン; ウィン シュイ; 田上昭人; 野原恵子; 塚原 伸治
  Volume：65, Number：7, First page：538, Last page：547, 2014, [Invited]
  Japanese
• ライブイメージングを活用したin vitro神経毒性試験法の開発　　　　　　　　　　　　　　　
  チョウ テッ アウン; 塚原 伸治
  Volume：64, Number：4, First page：281, Last page：286, 2013, [Invited]
  Japanese
• Effects of developmental exposure to toluene on the sexual differentiation of the brain and action mechanisms of toluene　　　　　　　　　　　　　　　
  TSUKAHARA Shinji; NAKAJIMA Daisuke; FUJIMAKI Hidekazu
  Indoor Environment : Journal of Society of Indoor Environment, Japan, Volume：13, Number：1, First page：1, Last page：8, 2010, [Reviewed], [Invited]
  Immature individuals under the development are considered to have high sensitivity and vulnerability to chemical exposure. The brain is sexually differentiated under the influence of testosterone secreted from the testes during developing period. Inhalation exposure to toluene reduces blood testosterone levels in men and male rats in adulthood. We reported that developmental exposure to toluene via maternal inhalation decreases blood testosterone levels and the expression of 3β-HSD, a steroidogenic enzyme involved in testosterone synthesis, in fetal male rats. Our findings suggest that decreased expression of 3β-HSD in the fetal testes is responsible for the reduction of blood testosterone level. The brain has nuclei exhibiting the morphological sex differences, sexually dimorphic nuclei. The SDN-POA, a sexually dimorphic nucleus of the rat brain, is larger and has more neurons in males than in females. We recently found that the volume of the SDN-POA became smaller in male rats exposed to toluene during the perinatal period. We here discuss the effects of developmental exposure to toluene on the sexual differentiation of the brain in rats.
  Society of Indoor Environment, Japan, Japanese, Introduction scientific journal
  DOI：https://doi.org/10.7879/siej.13.1
  DOI ID：10.7879/siej.13.1, ISSN：1882-0395, CiNii Articles ID：130002151350, CiNii Books ID：AA11443020
• ロードーシス神経制御機構の性差　　　　　　　　　　　　　　　
  山内兄人; 塚原伸治
  Volume：27, Number：10, First page：1120, Last page：1123, 2009
• Current status and progress of research on the effects of volatile organic compounds (VOCs) on fetuses and infants　　　　　　　　　　　　　　　
  NAKAJIMA Daisuke; TSUKAHARA Shinji; KAGEYAMA Shiho; SHIRAISHI Fujio; FUJIMAKI Hidekazu
  Indoor Environment, Volume：11, Number：2, First page：103, Last page：109, 2008
  In response to heightened awareness of protecting the health of infants and fetuses, as shown by the Miami Declaration adopted in 1997 and the Dubai Declaration in 2006, various actions have been taken. The effect of exposure to toluene, which is a typical indoor air contaminant, on the cerebral nervous system of humans during development is now being studied, in addition to its well-known toxicity. When investigating the effects of volatile organic compounds such as toluene, it is essential to examine their dynamic behavior in the human body as well as their toxicity. However, such research on fetuses and newborns requires a simple method applicable to micro-samples. The solid phase micro extraction (SPME) method is one such technique and is effective. Detailed susceptibility studies on the vulnerability of immature individuals must be conducted in the future.
  Society of Indoor Environment, Japan, Japanese
  DOI：https://doi.org/10.7879/siej2007.11.103
  DOI ID：10.7879/siej2007.11.103, ISSN：1882-0395, CiNii Articles ID：130004989128, CiNii Books ID：AA11443020
• Survey on the article reporting the effects of developmental exposure to volatile organic compounds and pesticides on the central nervous system　　　　　　　　　　　　　　　
  Tsukahara Shinji; Ishido Masami; Kurokawa Yoshika; Fujimaki Hidekazu
  Journal of Japan Society of Air Pollution, Volume：43, Number：3, First page：180, Last page：190, 2008, [Reviewed]
  Immature individuals under development are considered to have high sensitivity and vulnerability to chemicals. It therefore pays attention to the risk assessment for environmental chemical exposure in fetuses, infants and children. We here focused on the developmental neurotoxicities of VOCs and pesticides and reviewed articles in the studies reporting the effects of developmental exposure to these chemicals on the central nervous system. Our reviews could be contributed to the hazard assessment of VOCs and pesticides in fetuses, infants and children.
  Japan Society for Atmospheric Environment, Japanese, Introduction scientific journal
  DOI：https://doi.org/10.11298/taiki1995.43.180
  DOI ID：10.11298/taiki1995.43.180, ISSN：1341-4178, CiNii Articles ID：130004377533
• 雄ラット中枢神経系における雌型性行動制御機構: 中隔外側核の抑制機構を中心として
  塚原伸治; 山内兄人
  Volume：15, Number：3, First page：343, Last page：352, 2000, [Invited]
  Japanese, Introduction scientific journal
  CiNii Articles ID：50001324786
• 雌性行動神経制御におけるエストロゲンの作用と性分化　　　　　　　　　　　　　　　
  山内兄人; 塚原伸治
  Volume：37, Number：2, First page：221, Last page：228, 2000, [Invited]
  Japanese, Introduction scientific journal
• 視床下部におけるエストロゲンによる性腺刺激ホルモン放出ホルモン分泌制御　　　　　　　　　　　　　　　
  塚原伸治
  Volume：12, Number：2, First page：38, Last page：47, 2000, [Invited]
  Japanese, Introduction research institution

• 脳とホルモンの行動学 : わかりやすい行動神経内分泌学　　　　　　　　　　　　　　　
  近藤, 保彦; 小川, 園子; 菊水, 健史; 山田,一夫; 富原, 一哉; 塚原, 伸治, [Joint editor]
  Mar. 2023
  Japanese, Total pages：xi, 353p
  CiNii Books：http://ci.nii.ac.jp/ncid/BD00980265
  ISBN：9784867060438, CiNii Books ID：BD00980265
• ホルモンから見た生命現象と進化シリーズIV：求愛・性行動と脳の性分化　　　　　　　　　　　　　　　
  塚原伸治, [Contributor]
  2016
• 脳とホルモンの行動学—行動神経内分泌学への招待−　　　　　　　　　　　　　　　
  塚原伸治, [Contributor]
  2010
• 脳の性分化　　　　　　　　　　　　　　　
  塚原伸治; 掛山正心, [Contributor]
  2006
• 塚原伸治、掛山正心 神経核形成のメカニズム（アポトーシス） 脳の性分化　　　　　　　　　　　　　　　
  裳華房, 2006
• 塚原伸治 下位脳幹の性差 脳の性分化　　　　　　　　　　　　　　　
  裳華房, 2006

• 脳科学から紐解くこころの性　　　　　　　　　　　　　　　
  塚原伸治
  Jun. 2024, [Invited]
• 脳の性について、最新の脳科学から ーこころの性とその多様性の理解のためにー　　　　　　　　　　　　　　　
  塚原伸治
  Nov. 2023, [Invited]
• 細胞数の正確な見積のためのステレオロジー解析　　　　　　　　　　　　　　　
  塚原伸治
  Sep. 2023, [Invited]
• 性ホルモンによって構築される脳の性差 ―性のグラデーションと多様性が生じる仕組みの理解を目指して―　　　　　　　　　　　　　　　
  塚原伸治
  Sep. 2023, [Invited]
• 最新の脳科学から ヒトの脳はグラデーション　　　　　　　　　　　　　　　
  塚原伸治
  Dec. 2022, [Invited]
• Sexual differentiation of calbindin neuron in the preoptic area.　　　　　　　　　　　　　　　
  Tsukahara S
  Jul. 2019, [Invited], [Domestic conference]
  English, Public symposium
• A neuronal mechanism of sexual arousal in male rats.　　　　　　　　　　　　　　　
  Tsukahara S
  Mar. 2019, [Invited], [International conference]
  English, Nominated symposium
• 生命科学の視点から「こころの性」を考える　　　　　　　　　　　　　　　
  塚原伸治
  Jan. 2019, [Domestic conference]
  Japanese, Public discourse
• 雄性機能を覚醒する神経機構　　　　　　　　　　　　　　　
  塚原伸治
  Nov. 2018, [Domestic conference]
  Japanese, Public discourse
• 性機能を覚醒する神経メカニズム　　　　　　　　　　　　　　　
  塚原伸治
  Oct. 2018, [Invited], [Domestic conference]
  Japanese, Nominated symposium
• Formation and function of female-biased sexually dimorphic cell group in male-biased sexually dimorphic nucleus.　　　　　　　　　　　　　　　
  Tsukahara S
  Mar. 2018, [Domestic conference]
  English, Public symposium
• 新たに見つかった性的二型核の性差形成機構と生理機能　　　　　　　　　　　　　　　
  塚原伸治
  Aug. 2017, [Invited], [Domestic conference]
  Japanese, Nominated symposium
• Sexual differentiation of novel sexually dimorphic nucleus of dorsal hypothalamus in mice.　　　　　　　　　　　　　　　
  Tsukahara S
  Jun. 2017, [Invited], [International conference]
  English, Public symposium
• 性行動の神経制御と性ホルモンの働き　　　　　　　　　　　　　　　
  塚原伸治
  Jun. 2017, [Invited], [Domestic conference]
  Japanese, Invited oral presentation
• 哺乳類における性的二型核の比較と性差形成機構　　　　　　　　　　　　　　　
  塚原伸治
  Mar. 2017, [Invited], [Domestic conference]
  Japanese, Invited oral presentation
• 脳の性差を構築する性ホルモンの作用機構　　　　　　　　　　　　　　　
  塚原伸治
  Nov. 2016, [Invited], [Domestic conference]
  Japanese, Public symposium
• Sex and species differences in brain structure.　　　　　　　　　　　　　　　
  Tsukahara S
  Nov. 2016, [Invited], [Domestic conference]
  English, Public discourse
• Difference and homology of sexually dimorphic brain structures among quails, mice, musk shrews, and common marmosets.　　　　　　　　　　　　　　　
  Tsukahara S
  Nov. 2016, [International conference]
  English, Public symposium
• Gonadal steroid action on sex-specific formation of the brain.　　　　　　　　　　　　　　　
  Tsukahara S
  Jul. 2016, [Domestic conference]
  English, Public symposium
• Region-specific actions of sex steroids on the formation of morphological sex difference in the brain.　　　　　　　　　　　　　　　
  Tsukahara S; Kanaya M
  Mar. 2016, [Domestic conference]
  English, Public symposium
• こころの性を考える　　　　　　　　　　　　　　　
  塚原伸治
  Dec. 2015, [Invited], [Domestic conference]
  Japanese, Public discourse
• 脳の性差：新たに見つかった視床下部の性的二型領域　　　　　　　　　　　　　　　
  塚原伸治
  Sep. 2015, [Domestic conference]
  Japanese, Nominated symposium
• 性経験によって促進する雄性行動の神経制御機構　　　　　　　　　　　　　　　
  塚原伸治
  Dec. 2014, [Invited], [Domestic conference]
  Japanese, Public discourse
• The Year（基礎編）神経内分泌学の基礎研究における最近の動向　　　　　　　　　　　　　　　
  塚原伸治
  Nov. 2014, [Domestic conference]
  Japanese, Others
• Sex steroid actions on the formation of morphological sex difference in the rodent brain.　　　　　　　　　　　　　　　
  Tsukahara S
  Aug. 2014, [Invited], [International conference]
  English, Invited oral presentation
• 交尾経験によって引き起こされる雄性行動発現促進のメカニズム：視索前野の性的二型核（SDN-POA）の生理機能の解明をめざして　　　　　　　　　　　　　　　
  塚原伸治
  Mar. 2014, [Invited], [Domestic conference]
  Japanese, Invited oral presentation
• 行動の性差を生み出す脳の性分化機構　　　　　　　　　　　　　　　
  塚原伸治
  Mar. 2014, [Invited], [Domestic conference]
  Japanese, Public symposium
• Mechanism of brain sexual differentiation and development of neurotoxicology method using live imaging.　　　　　　　　　　　　　　　
  Tsukahara S
  Nov. 2013, [Invited], [International conference]
  English, Public discourse
• 雄ラットの性的覚醒を引き起こす神経機構　　　　　　　　　　　　　　　
  塚原伸治
  Sep. 2013, [Domestic conference]
  Japanese, Public symposium
• 雄の性行動を調節する脳機能の解析と少子化対策に向けて　　　　　　　　　　　　　　　
  塚原伸治
  Jun. 2013, [Invited], [Domestic conference]
  Japanese, Public discourse
• 脳の性差形成と性機能:視索前野における性的二型核の性差形成機構と雄性機能における関与　　　　　　　　　　　　　　　
  塚原伸治
  May 2013, [Invited], [Domestic conference]
  Japanese, Public discourse
• 脳構造の性差形成とエストロゲンの働き　　　　　　　　　　　　　　　
  塚原伸治
  Dec. 2011, [Invited], [Domestic conference]
  Japanese, Public discourse
• Formation and function of tissue structure in the sexually dimorphic nucleus of the preoptic area in rats.　　　　　　　　　　　　　　　
  Tsukahara S
  Jul. 2011, [Invited], [International conference]
  English, Nominated symposium
• 脳の性分化機構：雌雄で異なる脳の構造と機能について　　　　　　　　　　　　　　　
  塚原伸治
  Mar. 2011, [Invited], [Domestic conference]
  Japanese, Public discourse
• げっ歯類における性的二型核の形成と性ステロイドの役割　　　　　　　　　　　　　　　
  塚原伸治
  Sep. 2010, [Invited], [Domestic conference]
  Japanese, Public symposium
• Involvement of postnatal apoptosis and effects of developmental chemical exposure on SDN-POA formation in rats.　　　　　　　　　　　　　　　
  Tsukahara S
  Sep. 2010, [Invited], [Domestic conference]
  English, Public symposium
• 脳の性分化に及ぼすトルエンの発達期曝露の影響　　　　　　　　　　　　　　　
  塚原伸治; 中島大介
  Feb. 2009, [Domestic conference]
  Japanese, Nominated symposium
• Effects of developmental exposure to toluene on the sexual differentiation of the brain　　　　　　　　　　　　　　　
  Tsukahara S
  Program and Abstracts, Oct. 2008, [Invited], [International conference]
  English, Nominated symposium
• Sex difference in apoptosis and role of estrogen in the sexually dimorphic nucleus of the preoptic area (SDN-POA) in postnatal rats　　　　　　　　　　　　　　　
  Tsukahara S
  Program and Abstracts, Sep. 2008, [Invited], [International conference]
  English, Nominated symposium
• 発達期の性的二型核におけるアポトーシスに関する研究（日本神経内分泌学会川上賞受賞講演）　　　　　　　　　　　　　　　
  塚原伸治
  Aug. 2008, [Invited], [Domestic conference]
  Japanese, Invited oral presentation
• 発達期の脳の性分化機構におよぼす化学物質の影響　　　　　　　　　　　　　　　
  塚原伸治
  Mar. 2008, [Invited], [Domestic conference]
  Japanese, Invited oral presentation
• VOCの脳神経系におよぼす影響　　　　　　　　　　　　　　　
  塚原伸治
  Sep. 2007, [Invited], [Domestic conference]
  Japanese, Invited oral presentation
• 生殖機能制御を司る視床下部の性差と形成機構　　　　　　　　　　　　　　　
  塚原伸治
  Jan. 2006, [Invited], [Domestic conference]
  Japanese, Public discourse
• ラットの雌性行動抑制に関与する外側中隔の性分化　　　　　　　　　　　　　　　
  塚原伸治
  Jun. 2004, [Invited], [Domestic conference]
  Japanese, Public symposium
• ラットの雌性行動を抑制する外側中隔の性差と性分化　　　　　　　　　　　　　　　
  塚原伸治
  Nov. 2003, [Invited], [Domestic conference]
  Japanese, Public discourse
• Lordosis-inhibiting tract from septum to central gray in male rats.　　　　　　　　　　　　　　　
  Tsukahara S; Yamanouchi K
  Dec. 2000, [International conference]
  English, Nominated symposium

• Society for Neuroscience
• THE ZOOLOGICAL SOCIETY OF JAPAN
• THE PHYSIOLOGICAL SOCIETY OF JAPAN

• こころの性とその多様性を生み出す神経基盤の解明を目指した性的二型核の解析　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Apr. 2020 - Mar. 2023
  Saitama University
  Grant amount(Total)：4290000, Direct funding：3300000, Indirect funding：990000
  Grant number：20K07257
• Neuroendocrinology of Social Behavior　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (S), 29 May 2015 - 31 Mar. 2020
  OGAWA Sonoko, University of Tsukuba
  Grant amount(Total)：196690000, Direct funding：151300000, Indirect funding：45390000
  Estrogen receptors alpha (ERa) and beta (ERb) play a critical role in the regulation and modulation of social behavior by sex steroid hormones. In the present study, we first defined distribution of each of ERa and ERb at cellular levels in various brain areas in the limbic system, hypothalamus, and midbrain, which constitute the social behavioral network. We then identified their functions and mechanisms of action using a number of cutting-edge techniques in behavioral neuroscience. We concluded that harmonious action of ERa and ERb which are different in distribution and downstream effects is essential for adaptive expression of social behavior.
  Grant number：15H05724
• 性的二型核の生理機能と性差構築機構の解明　　　　　　　　　　　　　　　
  2017 - 2019
  Principal investigator
  Competitive research funding
• 心の性とその多様性の理解を目指した脳の性差解析　　　　　　　　　　　　　　　
  2018
  Principal investigator
  Competitive research funding
• Sexual differentiation of the sexually dimorphic nucleus of the preoptic area and the bed nucleus of the stria terminalis responsible for the gender identity.　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 01 Apr. 2013 - 31 Mar. 2017
  Hamada Tomohiro, Nippon Medical School
  Grant amount(Total)：4940000, Direct funding：3800000, Indirect funding：1140000
  Sex differences of the brain would be important for the gender identity, however, the mechanism of the sexual differentiation of the brain remains largely unknown. The sexually dimorphic nucleus of the preoptic area (SDN-POA) is thought to contribute to the gender identity. In this study, we examined the sexual differentiation of the SDN-POA using the organotypic brain slice cultures and time-lapse imaging in vitro to test the hypothesis that neural migration regulated by estrogen is critical for the sexual differentiation of the SDN-POA. The scattering neural migration by estrogen was observed in the masculinization of the SDN-POA. This estrogen induced scattering migration was mediated through the estrogen receptor alpha/Rac1/cofilin/actin pathway. These results suggest that scattering neural migration by estrogen is critical role for the masculinization of the SDN-POA.
  Grant number：25460320
• 性ステロイドホルモンによる社会行動神経ネットワーク機能の制御メカニズム　　　　　　　　　　　　　　　
  01 Apr. 2015 - 31 Mar. 2016
  Grant amount(Total)：13780000, Direct funding：10600000, Indirect funding：3180000
  Grant number：15H01844
• 性的二型核に着目した脳への系統進化的アプローチ　　　　　　　　　　　　　　　
  2015 - 2016
  Principal investigator
  Competitive research funding
• Neural Mechanism of the Regulation of Social Behavior by Estrogen　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (A), 01 Apr. 2011 - 31 Mar. 2015
  OGAWA Sonoko; TSUKAHARA Shinji; SAKAMOTO Hirotaka; NISHIMORI Katsuhiko; SAKAMOTO Toshiro, University of Tsukuba
  Grant amount(Total)：48880000, Direct funding：37600000, Indirect funding：11280000
  Estrogen plays a central role in the regulation of social behavior. We aimed to elucidate neurobiological basis of social behavior by focusing on neural mechanisms of 1) sex-specific, 2) time-dependent, and 3) brain site-specific action of estrogen. For this purpose, we performed behavioral, neuroanatomical and molecular biological analyses in global knockout and brain site-specific knockdown mice of two types of estrogen receptors. We have found that estrogen receptor alpha and beta in the medial amygdala, medial preoptic area, hypothalamic ventromedial nucleus, and dorsal raphe nucleus might have differential roles in the regulation of sexual, aggressive behavior and social anxiety levels in male and female mice.
  Grant number：23240057
• 新規な性的二型核の探索と探索した性的二型核の性差形成機構および生理機能の解明　　　　　　　　　　　　　　　
  2013 - 2014
  Principal investigator
  Competitive research funding
• ライブイメージングを利用した新規試験法による発達神経毒性評価と毒性発現機序の解明　　　　　　　　　　　　　　　
  2011 - 2013
  Principal investigator
  Competitive research funding
• ヒトの発達神経毒性評価を念頭に置いた新規in vitro試験法開発　　　　　　　　　　　　　　　
  2012
  Principal investigator
  Competitive research funding
• ニューロンの微細構造に着目した発達神経毒性試験法の開発　　　　　　　　　　　　　　　
  2010
  Principal investigator
  Competitive research funding
• 化学物質の有害性評価の効率化を目指した新たな神経毒性試験法の開発に関する研究　　　　　　　　　　　　　　　
  2008 - 2009
  Principal investigator
  Competitive research funding
• 脳の発達・性分化に及ぼす揮発性有機化合物の影響と作用機序の解明　　　　　　　　　　　　　　　
  2007 - 2009
  Principal investigator
  Competitive research funding
• Mechanisms underlying sexual dimorphism of the brain　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research on Priority Areas, 2007 - 2008
  OHTANI-KANEKO Ritsuko; OGAWA Sonoko; KATO Tomohiro; TSUKAHARA Shinji, Toyo University
  Grant amount(Total)：6900000, Direct funding：6900000
  Grant number：19040025
• 視床下部における生殖中枢の性差と性分化機構の解明　　　　　　　　　　　　　　　
  2005 - 2006
  Principal investigator
  Competitive research funding
• 生殖のライフサイクルにおける脳の機能構造の変化と性特異性に関する研究　　　　　　　　　　　　　　　
  2005 - 2006
  Principal investigator
  Competitive research funding
• Studies on the colonization mechanism of indigenous bacteria in alimentary tract　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 2004 - 2006
  KITAGAWA Hiroshi; SHIMIZU Akira, Kobe University
  Grant amount(Total)：3700000, Direct funding：3700000
  This research clarified the colonization mechanisms of indigenous bacteria in rat alimentary tracts. The results obtained in this research are as follows:
  1. Fundamental colonization site of indigenous bacteria is the epithelial cells with late apoptotic stage in intestinal villi and Peyer's patches.
  2. Differentiation of M cells in the follicle-associated epithelium (FAE) is co-related with the colonization of indigenous bacteria on FAE and neighboring mucosa. The ordinary columnar epithelial cells engulf the degenerated bacteria, whereas M cells engulf the intact bacteria in FAE of Peyer's patches. This engulfment of bacteria might be mediated with membrane receptor in M cells form ultrastructural findings.
  3. Specific sugar expression was histochemically determined on the epithelial cells with late apoptotic stage, whereas M cells expressed no specific sugar.
  4. Fundamental colonization site of indigenous bacteria is the epithelial cells with late apoptotic stage in entire alimentary tract. From these sites, indigenous bacteria proliferate deeply into mucous membrane. This proliferation is monitored by neighboring FAE of mucous lymphatic follicles.
  5. At bacterial attachment sites, indigenous bacteria are eliminated by both the physical defense of rearrangement of epithelial cytoskeletons and the chemical defense of secretion of antibactericidal substances.
  6. The detailed differentiation and re differentiation processes were clarified in FAE of Peyer's patches under transmission electron microscope.
  Grant number：16580239
• 雌雄で異なる生殖現象を支配調節する脳の構造と機能　　　　　　　　　　　　　　　
  2003 - 2004
  Principal investigator
  Competitive research funding
• 雄ラットにおける雌性行動抑制神経機構に関する研究　　　　　　　　　　　　　　　
  2000 - 2001
  Principal investigator
  Competitive research funding
• -　　　　　　　　　　　　　　　
  Competitive research funding