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Isolated primary culture of the terminal nerve (TN)-GnRH neurons derived from the brain of a tropical teleost
S222
Abstracts / Neuroscience Research 58S (2007) S1–S244
P3-gØ5 Sexually dimorphic metastin neurons in medaka brain Shinji Matsumoto 1 , Yasuhisa Akazome 1 , Naoyuki Yamamoto 2 , Kataaki Okubo 3 , Shunji Yamada 4 , Hiroko Tsukamura 4 , Kei-Ichiro Maeda 4 , Yoshitaka Oka 1 1 Grad Sch Sci, Univ of Tokyo, Tokyo, Japan; 2 Nippon Med Sch, Tokyo, Japan; 3 NIBB, Okazaki, Japan; 4 Grad Sch Bioagr Sci, Nagoya Univ, Nagoya, Japan Novel peptidergic neurons, metastin neurons, are thought to facilitate sexual maturation and ovulation by stimulating gonadotropin releasing hormone (GnRH) neurons, but their physiological functions are still largely unknown. Here we focused on the relationship between the metastin and hypothalamic/extrahypothalamic GnRH neurons. Taking advantage of well-developed teleost GnRH systems, we used medaka, which is a useful teleost for the application of various molecular genetic tools. First, we cloned and sequenced the metastin gene (Kiss1) in medaka for the first time in non-mammalian species. Then, Kiss1 expression in the brain was analyzed by in situ hybridization. We found two hypothalamic nuclei with Kiss1 expression and discovered prominent sexual difference in cell number (male female) in one of the two nuclei. On the other hand, we found no circadian variation in expression of Kiss1 by real-time PCR, although medaka shows daily ovulatory cyclicity.
P3-gØ8 Isolated primary culture of the terminal nerve (TN)GnRH neurons derived from the brain of a tropical teleost Hideki Abe, Yoshitaka Oka Dept. of Biol. Sci., Grad. School of Sci., Univ. of Tokyo, Tokyo, Japan The TN-GnRH neurons release GnRH peptides from widely branched neural processes, depending on their firing activity, and the released GnRH is suggested to exert their neuromodulatory action via GnRH receptors expressed in nearby as well as distant target neurons. To address questions on how electrical activities of the TN-GnRH neuronal cell bodies affect GnRH release from the widely branched neural process, we tried to establish the primary culture of TN-GnRH neurons. The whole brain of the adult dwarf gourami (Colisa lalia, a tropical freshwater fish) was dissected out and was briefly treated by protease. The TN-GnRH neurons were sucked out from the cluster of TN-GnRH neurons with the aid of a suction pipette and IR-DIC optics and cultured on coverslips. The isolated TN-GnRH neurons were kept at room temperature in a darkened humidity chamber and could survive up to around two weeks. Both axonand dendrite-like processes grew out from the cultured TN-GnRH neurons, and characteristic growth cone and varicosity-like structures were observed. Research funds: KAKENHI (17770059, 18370029, 18021009).
Research funds: KAKENHI 18370029 18021009.
P3-gØ6 Synaptic estrogen receptors in principal neurons of hippocampus Mukai 1,2 ,
Murakami 1,2 ,
Kominami 3 ,
Hideo Gen Shiro Shimpei Higo 1,2 , Yasushi Hojo 1,2 , John H. Morrison 4 , Suguru Kawato 1,2 1 Department of Biophysics and Life Sciences, The University of Tokyo, Tokyo, Japan; 2 Bioinformatics Project, JST, Japan; 3 Graduate School of Integrated Arts and Sciences, Hiroshima University, Japan; 4 Kastor Neurobiology of Aging Laboratories, Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, USA Estrogens elicit rapid non-genomic effects on the synaptic transmission and spinogenesis in the hippocampus. However, the existence of estrogen receptor alpha (ERa) still remains elusive. With highly purified antibody RC-19, mass spectrometric analysis identified ERa in the hippocampus and immunohistochemistry showed ERa localization in principal neurons of CA1, CA3, and granule cells in dentate gyrus. Further we found the synaptic distribution of ERa by immunoelectron microscopy. We also examined the distribution of putative G-protein coupled estrogen receptor, GPR30. These results imply that synaptic estrogen receptors mediate the effects of estrogen in hippocampal neurons.
P3-gØ9 Oxytocin receptor-Venus knock-in mice enable direct visualization of oxytocin receptor-expressing neurons Masahide Yoshida 1 , Yuki Takayanagi 2 , Tatushi Onaka 2 , Katuhiko Nishimori 1 1 Department of Molecular and Cell Biology, Tohoku University, Sendai, Japan; 2 Department of Physiology, Jichi Medical School, Tochigi, Japan The oxytocin receptor (OXTR) regulates reproductive physiology and sociosexual behaviors. Recently, we generated Oxtr gene-deficient mice (Oxtr −/−) and reported that Oxtr −/− exhibited pervasive social deficits [Takayanagi et al., PNAS, 2005]. To further study the functions of OXTR, we newly generated knock-in mice that express Venus (variant of YFP), which was replaced with the Oxtr coding region. Distruption of Oxtr gene locus, absence of Oxtr transcripts and expression of Venus transcripts were comfirmed in OXTR-Venus knock-in mice. Interestingly, in these mice, the fluorescence was observed not only at regions previously identified to be expressing OXTR, but also at where its expression had not been described. The OXTR-Venus knock-in mice would be a powerful new tool to study the physiological role of OXTR-expressing neurons and the dynamics of OXTR signaling in the living neurons [Takayanagi et al., PNAS, 2005]. Research funds: KAKENHI (18380063) and JSPS research fellowship for young scientists.
P3-gØ7 Accumulated fatigue induces over-activation and subsequent degeneration of melanotrophs in rat pituitary gland
P3-g1Ø Taste may not affect salt appetite in zinc deficient rats
Tokiko Ogawa 1,3 , Hiroyuki Konishi 1,3 , Masaaki Tanaka 2,3 , Sumiko Kiryu-Seo 1,3 , Saya Nakagomi 1,3 , Yasuyoshi Watanabe 2,3 , Hiroshi Kiyama 1,3 1 Dept. Anatomy & Neurobiology, Grad. Sch. Med., Osaka City Univ., Osaka, Japan; 2 Dept. Physiology, Grad. Sch. Med., Osaka City Univ., Osaka, Japan; 3 The 21st Century COE program ‘Base to overcome Fatigue’, Japan
Noritaka Sako 1 , Hideo Katsukawa 1 , Kiyohito Nakashima 2 , Tadataka Sugimura 1 1 Department of Oral Physiology, Asahi University School of Dentistry, Japan; 2 Department of Chemistry, Asahi University School of Dentistry, Japan
Accumulated fatigue causes dysfunction of cellular mechanisms or homeostasis in various organs. In this study we have employed an animal fatigue model in which rat was kept in a cage filled with water height of 1.5 cm for up to 7 days. The melanotrophs in the intermediate lobe (IL) of the fatigued rat pituitary demonstrated marked morphological changes of rER and Golgi apparatus, and subsequent cell degeneration. The tyrosine hydroxylase positive nerve fiber in IL was decreased during the fatigue stimulation. The application of dopamine antagonist to normal rats mimicked the morphological changes of rER and Golgi apparatus, and dopamine agonist to fatigued rats inhibited the changes. These results suggested that prolonged fatigue suppressed dopamine release in IL, and thereby caused an over secretion of ␣-MSH from melanotrophs, leading to ER dysfunction and subsequent cell death.
Some reports demonstrated that zinc deficient (ZnX) brought about taste disturbance and that ZnX rats enhanced their preference to sodium solution. In the present study, we conducted some experiments to investigate whether or not taste affected this preference for sodium. Results were as follows: in the long-term (48 h) two-bottle preference test, the preference percents for 0.1 and 0.3 M NaCl in the ZnX rats were higher than those in the control rats. But in the short-term (10 min) test, there was no significant difference in the preference percents between ZnX and control rats. There was no significant difference in the neural responses of chorda tympani nerves to some sodium solutions between ZnX and control rats. By the assay of aldosterone in serum of the ZnX rats, a disorder of sodium metabolism was found in these rats. These results suggest that the enhancement of preference for sodium is caused by the disorder of sodium metabolism rather than taste disturbance. Research funds: KAKENHI (15390629, 15591984), Miyata Fund (A).
Abstracts / Neuroscience Research 58S (2007) S1–S244
P3-gØ5 Sexually dimorphic metastin neurons in medaka brain Shinji Matsumoto 1 , Yasuhisa Akazome 1 , Naoyuki Yamamoto 2 , Kataaki Okubo 3 , Shunji Yamada 4 , Hiroko Tsukamura 4 , Kei-Ichiro Maeda 4 , Yoshitaka Oka 1 1 Grad Sch Sci, Univ of Tokyo, Tokyo, Japan; 2 Nippon Med Sch, Tokyo, Japan; 3 NIBB, Okazaki, Japan; 4 Grad Sch Bioagr Sci, Nagoya Univ, Nagoya, Japan Novel peptidergic neurons, metastin neurons, are thought to facilitate sexual maturation and ovulation by stimulating gonadotropin releasing hormone (GnRH) neurons, but their physiological functions are still largely unknown. Here we focused on the relationship between the metastin and hypothalamic/extrahypothalamic GnRH neurons. Taking advantage of well-developed teleost GnRH systems, we used medaka, which is a useful teleost for the application of various molecular genetic tools. First, we cloned and sequenced the metastin gene (Kiss1) in medaka for the first time in non-mammalian species. Then, Kiss1 expression in the brain was analyzed by in situ hybridization. We found two hypothalamic nuclei with Kiss1 expression and discovered prominent sexual difference in cell number (male female) in one of the two nuclei. On the other hand, we found no circadian variation in expression of Kiss1 by real-time PCR, although medaka shows daily ovulatory cyclicity.
P3-gØ8 Isolated primary culture of the terminal nerve (TN)GnRH neurons derived from the brain of a tropical teleost Hideki Abe, Yoshitaka Oka Dept. of Biol. Sci., Grad. School of Sci., Univ. of Tokyo, Tokyo, Japan The TN-GnRH neurons release GnRH peptides from widely branched neural processes, depending on their firing activity, and the released GnRH is suggested to exert their neuromodulatory action via GnRH receptors expressed in nearby as well as distant target neurons. To address questions on how electrical activities of the TN-GnRH neuronal cell bodies affect GnRH release from the widely branched neural process, we tried to establish the primary culture of TN-GnRH neurons. The whole brain of the adult dwarf gourami (Colisa lalia, a tropical freshwater fish) was dissected out and was briefly treated by protease. The TN-GnRH neurons were sucked out from the cluster of TN-GnRH neurons with the aid of a suction pipette and IR-DIC optics and cultured on coverslips. The isolated TN-GnRH neurons were kept at room temperature in a darkened humidity chamber and could survive up to around two weeks. Both axonand dendrite-like processes grew out from the cultured TN-GnRH neurons, and characteristic growth cone and varicosity-like structures were observed. Research funds: KAKENHI (17770059, 18370029, 18021009).
Research funds: KAKENHI 18370029 18021009.
P3-gØ6 Synaptic estrogen receptors in principal neurons of hippocampus Mukai 1,2 ,
Murakami 1,2 ,
Kominami 3 ,
Hideo Gen Shiro Shimpei Higo 1,2 , Yasushi Hojo 1,2 , John H. Morrison 4 , Suguru Kawato 1,2 1 Department of Biophysics and Life Sciences, The University of Tokyo, Tokyo, Japan; 2 Bioinformatics Project, JST, Japan; 3 Graduate School of Integrated Arts and Sciences, Hiroshima University, Japan; 4 Kastor Neurobiology of Aging Laboratories, Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, USA Estrogens elicit rapid non-genomic effects on the synaptic transmission and spinogenesis in the hippocampus. However, the existence of estrogen receptor alpha (ERa) still remains elusive. With highly purified antibody RC-19, mass spectrometric analysis identified ERa in the hippocampus and immunohistochemistry showed ERa localization in principal neurons of CA1, CA3, and granule cells in dentate gyrus. Further we found the synaptic distribution of ERa by immunoelectron microscopy. We also examined the distribution of putative G-protein coupled estrogen receptor, GPR30. These results imply that synaptic estrogen receptors mediate the effects of estrogen in hippocampal neurons.
P3-gØ9 Oxytocin receptor-Venus knock-in mice enable direct visualization of oxytocin receptor-expressing neurons Masahide Yoshida 1 , Yuki Takayanagi 2 , Tatushi Onaka 2 , Katuhiko Nishimori 1 1 Department of Molecular and Cell Biology, Tohoku University, Sendai, Japan; 2 Department of Physiology, Jichi Medical School, Tochigi, Japan The oxytocin receptor (OXTR) regulates reproductive physiology and sociosexual behaviors. Recently, we generated Oxtr gene-deficient mice (Oxtr −/−) and reported that Oxtr −/− exhibited pervasive social deficits [Takayanagi et al., PNAS, 2005]. To further study the functions of OXTR, we newly generated knock-in mice that express Venus (variant of YFP), which was replaced with the Oxtr coding region. Distruption of Oxtr gene locus, absence of Oxtr transcripts and expression of Venus transcripts were comfirmed in OXTR-Venus knock-in mice. Interestingly, in these mice, the fluorescence was observed not only at regions previously identified to be expressing OXTR, but also at where its expression had not been described. The OXTR-Venus knock-in mice would be a powerful new tool to study the physiological role of OXTR-expressing neurons and the dynamics of OXTR signaling in the living neurons [Takayanagi et al., PNAS, 2005]. Research funds: KAKENHI (18380063) and JSPS research fellowship for young scientists.
P3-gØ7 Accumulated fatigue induces over-activation and subsequent degeneration of melanotrophs in rat pituitary gland
P3-g1Ø Taste may not affect salt appetite in zinc deficient rats
Tokiko Ogawa 1,3 , Hiroyuki Konishi 1,3 , Masaaki Tanaka 2,3 , Sumiko Kiryu-Seo 1,3 , Saya Nakagomi 1,3 , Yasuyoshi Watanabe 2,3 , Hiroshi Kiyama 1,3 1 Dept. Anatomy & Neurobiology, Grad. Sch. Med., Osaka City Univ., Osaka, Japan; 2 Dept. Physiology, Grad. Sch. Med., Osaka City Univ., Osaka, Japan; 3 The 21st Century COE program ‘Base to overcome Fatigue’, Japan
Noritaka Sako 1 , Hideo Katsukawa 1 , Kiyohito Nakashima 2 , Tadataka Sugimura 1 1 Department of Oral Physiology, Asahi University School of Dentistry, Japan; 2 Department of Chemistry, Asahi University School of Dentistry, Japan
Accumulated fatigue causes dysfunction of cellular mechanisms or homeostasis in various organs. In this study we have employed an animal fatigue model in which rat was kept in a cage filled with water height of 1.5 cm for up to 7 days. The melanotrophs in the intermediate lobe (IL) of the fatigued rat pituitary demonstrated marked morphological changes of rER and Golgi apparatus, and subsequent cell degeneration. The tyrosine hydroxylase positive nerve fiber in IL was decreased during the fatigue stimulation. The application of dopamine antagonist to normal rats mimicked the morphological changes of rER and Golgi apparatus, and dopamine agonist to fatigued rats inhibited the changes. These results suggested that prolonged fatigue suppressed dopamine release in IL, and thereby caused an over secretion of ␣-MSH from melanotrophs, leading to ER dysfunction and subsequent cell death.
Some reports demonstrated that zinc deficient (ZnX) brought about taste disturbance and that ZnX rats enhanced their preference to sodium solution. In the present study, we conducted some experiments to investigate whether or not taste affected this preference for sodium. Results were as follows: in the long-term (48 h) two-bottle preference test, the preference percents for 0.1 and 0.3 M NaCl in the ZnX rats were higher than those in the control rats. But in the short-term (10 min) test, there was no significant difference in the preference percents between ZnX and control rats. There was no significant difference in the neural responses of chorda tympani nerves to some sodium solutions between ZnX and control rats. By the assay of aldosterone in serum of the ZnX rats, a disorder of sodium metabolism was found in these rats. These results suggest that the enhancement of preference for sodium is caused by the disorder of sodium metabolism rather than taste disturbance. Research funds: KAKENHI (15390629, 15591984), Miyata Fund (A).