神経内分泌 Neuroendocrine System
P2-1-148 コルチコトロピン放出因子産生ニューロンで蛍光タンパクを選択的に発現するマウス脳の組織学的検討 Histological characterization of the mouse brain expressing fluorescent proteins selectively in the corticotropin-releasing factor neurons 布施俊光1, 杉本直哉1, 山崎真弥3, 阿部学3, 夏目里恵3, 内田克哉1, 崎村建司3, 井樋慶一1,2 ○Ashraf H Talukder1, Toshimitsu Fuse1, Naoya Sugimoto1, Maya Yamazaki3, Manabu Abe3, Rie Natsume3, Katsuya Uchida1, Kenji Sakimura3, Keiichi Itoi1,2 東北大院 情報科学 情報生物学1, 東北大院 医学系 神経内分泌学2, 新潟大 脳研 細胞神経生物学3 Tohoku Univ Grad Sch Info Sci1, Tohoku Univ Grad Sch Med, Sendai, Japan2, Niigata Univ Brain Res Inst3 Corticotropin-releasing factor (CRF) neurons, in the paraventricular nucleus of the hypothalamus (PVN), play a pivotal role in the regulation of the hypothalamic-pituitary-adrenal axis and protect an organism from various stressors. CRF neurons are also present in other brain regions including the bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), Barrington's nucleus, and inferior olivary nucleus (IO). We developed a mouse line (CRF-Venus) in which Venus (an enhanced yellow fluorescent protein) gene was inserted to the translation initiation site of the CRF gene by homologous recombination. CRF neurons could be visualized by Venus under the fluorescent microscope. The brain of the mouse was frozen after perfusion with 4% paraformaldehyde, 24 h following injection of colchicine into the lateral ventricle, and 30 μm sections were made by cryostat. Colocalization of CRF with Venus was examined by double fluorescent immunocytochemistry throughout the brain. The sections were stained by the free-floating method and observed by a confocal fluorescent microscope. Approximately 70% of the green fluorescent protein (GFP; the antibody against GFP also recognizes Venus)-immunoreactive (ir) neurons expressed CRF-ir in the PVN. The GFP (Venus)-ir neurons never co-expressed vasopressin (AVP) or oxytocin (OXT). Almost all Venus-ir neurons expressed CRF-ir in either Barrington's nucleus or IO. We developed another mouse line (CRF-iCre) in which Cre recombinase gene was inserted to the CRF gene in the same manner as the CRF-Venus mouse. By crossing this mouse with a GFP-reporter mouse, CRF neurons could be visualized by GFP. The majority of GFP-ir neurons co-expressed CRF-ir in the PVN as well as in the Barrington's nucleus and IO. Thus, present mouse lines were demonstrated to be useful tools for studying the mechanisms of CRF-containing circuitry in the brain.	P2-1-149 C. elegansの飼育温度依存低温耐性にかかわる遺伝子の同定 Identification of genes underlying cultivation temperature-dependent cold tolerance in C. elegans ○園田悟1, 水谷仁美1, 木下ゆかり1, 長屋ひろみ1, 大久保幸恵1, 宇治澤知代1, 太田茜1, 久原篤1 ○Satoru Sonoda1, Hitomi Mizutani1, Yukari Kinoshita1, Hiromi Nagaya1, Yukie Okubo1, Tomoyo Ujisawa1, Ohta Akane1, Atsushi Kuhara1 甲南大学　理工学部　生物学科1 Dept Biol, Konan Univ, Kobe1 Responding and adapting to environmental temperature changes are important system for living and proliferation. We are utilizing a cultivation temperature-dependent cold tolerance in C. elegans, as a model for studying temperature signaling at molecular and cellular levels. After cultivation at 25°C, C. elegans were destroyed by cold stimuli. In contrast, most of animals can survive, after cultivation at 15°C. To isolate genes involved in the cultivation temperature-dependent cold tolerance, we are using four analyses, (1) DNA microarray, (2) natural variation, (3) EMS-mutagenesis and (4) artificial evolution.(1) We tested cultivation temperature-dependent cold tolerance of mutants defective in genes that have been isolated from DNA microarray analysis. Several genes such as protein protease PP1 and laminin are involved in cultivation temperature-dependent cold tolerance.(2) Natural variations of wild-type C. elegans that is isolated from various areas showed variety of cultivation temperature-dependent cold tolerance phenotypes. Responsible gene for the natural variation between Bristol N2 and California CB4854 are mapped on X-chromosome. By using deep DNA sequencer and classical three factors cross, responsible gene is narrowed down to 54 genes. We are now using detailed SNPs analysis. (3) Through 2000 genomes screen by using ethylmethane sulfonate (EMS), we isolated 10 mutants defective in the cold tolerance. We are utilizing snip-SNPs analysis to identify the responsible genes. (4) Since life cycle of C. elegans is only 3 days and strains can be permanently preserved at -80°C, C. elegans has strong advantage for artificial evolution analysis. We are maintaining C. elegans at 15 or 23°C for gradual accumulation of mutations in the genome. So far, 87th generations are frozen, and we found that cultivation temperature-dependent cold tolerance was notably changed at 61th generation. We are planning to decode whole genome by using deep DNA sequencer.
P2-1-150 神経系の雄性化に対する幼若期のアンドロゲン受容体の役割 The role of androgen receptor during postnatal periods in masculinization of nervous system ○山田俊児1, 大矢未来1, 松田賢一1, 河田光博1 ○Shunji Yamada1, Miku Oya1, Ken-Ichi Matsuda1, Mitsuhiro Kawata1 京都府立医科大学大学院　医学研究科　生体構造科学1 Dept Anat and Neurobiol, Kyoto Pref Univ of Med, Kyoto1 Gonadal hormones organize the nervous system that mediates sexually dimorphic behavior in developing rodent. Many previous studies suggested that androgen secreted form immature testis is converted to estrogen by aromatase and the estrogen organizes developing brain into a masculinized phenotype via estrogen receptor in rodent. On the other hand, infusion of flutamide, which an androgen receptor (AR) antagonist, during postnatal period inhibited brain structural masculinization, suggesting that androgen signaling via AR also influence brain masculinization. In the present study, we investigated precise periods of androgen signaling involved in brain structural and behavioral masculinization in rats. We divided postnatal periods, designated postnatal day 0 to 22, into 3 stages that first (postnatal day 0 to 6: I), second (day 8 to 14: II), and third (day 16 to 22: III). Newborn male rats were received subcutaneously flutamide (100 mg/kg body weight/day) injection in each stage (I, II, and III groups) or all stages (I+II+III group) every other day. After adulthood, we examined the effect for masuculinization of the brain by analyzing male sexual behavior, size of sexually dimorphic nucleus of the preoptic area (SDN-POA), and number of cells in spinal nucleus of the bulbocavernsous (SNB). Male sexual behavior in I+II+III group was significantly reduced compared with other groups. There were no significant differences in the size of the SDN-POA and number of cells in the SNB between I+II+III group and others. These results demonstrated that continuous AR activation from birth to weaning rather than time-specific AR activation is important for brain masclinization mediating male sexual behavior.	P2-1-151 核内エストロゲン関連受容体ERRγとエストロゲン受容体ERαのラット脳における共局在 The co-localization of nuclear estrogen-related receptor (ERR) γ and estrogen receptor (ER) α in the rat brain ○谷田任司1, 松田賢一1, 山田俊児1, 高浪景子1, 河田光博1 ○Takashi Tanida1, Ken-ichi Matsuda1, Shunji Yamada1, Keiko Takanami1, Mitsuhiro Kawata1 京都府立医科大学　大学院医学研究科　解剖学・生体構造科学1 Dept Anat & Neurobiol, Kyoto Pref Univ of Med, Kyoto1 Estrogen-related receptors (ERRs) are the transcription factors that belong to nuclear receptor superfamily. They include three subtypes; &alpha, &beta, and &gamma, that have high homology to estrogen receptors (ERs). Although endogenous ligand of ERRs does not identified, they maintain constitutively active structure thereby control gene transcription after binding to estrogen responsive element (ERE) or ERR responsive element (ERRE) on the DNA. ERRs are broadly expressed in estrogen-responsive organs such as placenta, ovary, and mammary gland, and are suggested to regulate estrogen signaling. Although estrogen is essential for the reproductive function of the brain, the distributional and functional information of ERRs is few. Therefore, we investigated their localization in the adult rat brain by RT-PCR and immunohistochemistry (IHC). Since RT-PCR analysis showed that ERR&gamma was most abundantly expressed among ERRs in the brain, we focused on ERR&gamma. IHC revealed that ERR&gamma was dominantly detected in reticular thalamic nucleus (RTN), which is negative for ERs, suggesting that ERR&gamma acts independent of endogenous estrogen in this region. On the other hand, ERR&gamma was also expressed in ER-abundant regions such as ventromedial hypothalamic nucleus (VMH) and medial amygdaloid nucleus (MeA). Double-immunofluorescent analysis revealed the co-localization of ERR&gamma and ER&alpha in the MeA ventral part (MeAV), suggesting the association of ERR&gamma with estrogen function in the brain including sexual behavior. The interaction of ERR&gamma and ER&alpha has been examined by co-immunoprecipitation method and transcriptional assay.
P2-1-152 下垂体における浸透圧感受性TRPチャネルの解析 Functional analysis of osomosensitive TRP channels in the pituitary gland ○植田高史1, 熊本奈緒子1, 柴田泰宏1, 鵜川眞也1 ○Takashi Ueda1, Naoko Kumamoto1, Yasuhiro Shibata1, Shinya Ugawa1 名古屋市立大学大学院　医学研究科　機能組織学1 Dept Neurobiol Anat, Nagoya City Univ, Nagoya1 Transient receptor potential vanilloid (TRPV) family comprises calcium-permeable channels involved in sensing chemical and physical changes inside and outside of cells. Out of mammalian TRP channels, TRPV1, TRPV2, TRPV4 and TRPM7 were demonstrated to function in transduction of osmotic stimuli. Previously we've showed the transcripts of TRPV1, TRPV2, TRPV4 and TRPM7 channels in mouse pituitary glands by RT-PCR and that the functional expression of TRPV2 was apparently observed in the neurohypophysis. However, many things about the other TRP channels are still unknown . To examine the functional expression of TRPV1, TRPV2, TRPV4 and TRPM7 in the pituitary glands, we performed RT-PCR, immunohistochemistry, and calcium imaging analysis of the primary pituitary cells subjected to the ligands (capsaicin, 2-APB, probenecid, 4alpha-PDD, acid and hypoosmotic stress). The involvement of the osmosensitive TRP channels in the pituitary functions was further investigated and discussed.	P2-1-153 授乳期ラットの視索上核オキシトシン細胞は対側視索上核および室傍核から興奮性シナプス入力を受ける Oxytocin neurones in the supraoptic nucleus receive excitatory synaptic inputs from the contra-lateral supraoptic and paraventricular nuclei in lactating rats ○本田和正1, 須藤彩子1, 池田健太郎1 ○Kazumasa Honda1, Ayako Sudou1, Kentarou Ikeda1 福井県立大学 看護福祉学部 看護学科 解剖生理学1 Dept Nursing Science, Faculty of Nursing and Welfare Science, Fukui Pref Univ, Fukui, Japan1 Present experiments were undertaken to examine whether oxytocin neurones in the supraoptic nucleus receive synaptic inputs from the contra-lateral supraoptic nucleus or paraventricular nucleus. Using urethane-anesthetized lactating rats, extracellular action potentials were recorded from single oxytocin or vasopressin neurones in the supraoptic nucleus. Electrical stimulation was applied to the contra-lateral supraoptic nucleus or paraventricular nucleus and responses of oxytocin or vasopressin neurones were analyzed by peri-stimulus time histogram or by change of firing rate of oxytocin or vasopressin neurones. Electrical stimulation of contra-lateral supraoptic nucleus or paraventricular nucleus did not cause antidromic excitation neither in oxytocin nor vasopressin neurones, but caused orthodromic responses. Although analysis by peri-stimulus time histogram showed that electrical stimulation of the contra-lateral supraoptic nucleus or paraventricular nucleus caused orthodromic excitation in both oxytocin and vasopressin neurones, the effect was remarkable in oxytocin neurones. Train stimulation applied to the contra-lateral supraoptic nucleus or paraventricular nucleus at 10Hz increased firing rates of oxytocin neurones and decreased those of vasopressin neurones. The results of the present experiments suggest that oxytocin neurones in the supraoptic nucleus receive mainly excitatory synaptic inputs from the contra-lateral supraoptic nucleus and paraventricular nucleus. These synaptic inputs to oxytocin neurone may contribute to the synchronized activation of oxytocin neurones during milk-ejection reflex.
P2-1-154 Species and sex differences in androgen receptor expression in the preoptic areas and anterior hypothalamic areas of the adult rats and mice ○Mir R. Jahan1, Keiji Kokubu1, Ryutaro Fujinaga1, Islam Md N.1, Akie Yanai1, Chikahisa Matsuo1, Nagisa Takemoto1, Hiromi Yoshidome1, Naoto Hayasaka1, Koh Shinoda1 Division of Neuroanatomy, Yamaguchi Uni. Grad. Sch. of Medicine1 Species difference has been suggested on androgen-induced functions of the preoptic and anterior hypothalamic areas (PO/AH). Using paraformaldehyde-fixed completely serial frozen sections, expression of androgen receptor (AR) was immunohistochemically compared in the PO/AH between adult rats (Wistar, SD) and mice (C57BL/6, DBA/2j, Balb/c) of both sexes. In general, AR expression was stronger in mice than rats, particularly in the medial preoptic area (MPO), posterodorsal preoptic nucleus and suprachiasmatic nucleus (SCN). Exceptionally, more AR-immunoreactive (AR-ir) cells were seen in the sexually dimorphic nucleus (SDN) of the MPO and periventricular zone of the AH in male rats than male mice. Interestingly, we discovered three new species specific AR-ir cells cluster in the peAH during the observation of AR immunostained serial sections of the rat and mouse hypothalamus, which have never been reported. We found two distinct rat-specific very small clusters as the "rostral nebular island" and "caudal nebular island" and theses clusters exhibits significant sex differences in its volume and cell numbers, as defined by Nissl staining. We found a distinct mouse-specific AR-ir neuronal cluster in the AH as the "tear drop nucleus" (TDN) which exhibits significant sex differences in its volume and cell numbers as defined by AR immunoreactivity of gonadectomized mice after single injection of dihydrotestosterone. The present data might explain different androgen-induced psychotic, behavioral and endocrinergic responses between the two rodents, warning that data cannot directly be applied each other.

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