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Cell-cycle analysis of neural stem cells in the adult mouse dentate gyrus
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Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-e07 Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus Koji Ohira 1 , Hideo Hagihara 1,2 , Keiko Toyama 1,2 , Keizo Kanai 4 , Hiroshi Funakoshi 4 , Toshikazu Takao 2,3 , Masaaki Nakamura 4 , Tsuyoshi Miyakawa 1,2,3 1
Division of Systems Medical Science, Fujita Health University 2 CREST, Saitama 3 Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi 4 Division of Molecular Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO2), whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO2 was co-expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO2 immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a bromodeoxyuridine labeling experiment revealed that new neurons began to express TDO2 between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO2 might be required at a late-stage of granule cell development, such as during axonal and dendritic growth and synaptogenesis, and for the maintenance of mature cells. doi:10.1016/j.neures.2010.07.1620
P3-e08 Cell-cycle analysis of neural stem cells in the adult mouse dentate gyrus Jun Kaneko , Tatsuhiro Hisatsune Department of Integrated Biosci, The University of Tokyo, Tokyo, Japan Newborn neurons are continuously generated from neural stem cells through neuronal progenitor cells. Neural stem cells divide at the subventricular zone of the dentate gyrus, and generate progenitor cells. In order to further elucidate this phenomenon, it has become necessary to develop a video-imaging system through which this process could be observed. For this purpose, we utilized the Fucci system, in which a protein, Geminin, is accumulated in S/G2/M phases of the cell-cycle, in combination with neural stem cells labeling with Nestin-GFP. In this method, neural stem cells approaching the M phase can be distinguished from G0/G1 phases of neural stem cells. The Geminin lentiviral vector was inoculated into both sides of the dentate gyrus. 4 days after the injection, we carried out immunostaining to confirm whether dividing neural stem cells from Nestin-GFP mice were labeled with Fucci system. As a result, we found that Geminin tagged by fluorescent protein transduced by lentiviral vector was expressed in neural stem cells in adult mice. We are developing an organotypic slice culture from mouse brain inoculated with the Geminin viral vector. doi:10.1016/j.neures.2010.07.1621
P3-e09 Exercise-induced adult hippocampal neurogenesis is inhibited by Flutamide (androgen receptor antagonist) Masahiro Okamoto , Koshiro Inoue, Takashi Matsui, Minchul Lee, Hideaki Soya Laboratory of Exercise Biochemistry, Graduate School of Comprehensive Human Sciences, The University of Tsukuba Androgens play a significant role not only in muscular development, but also in numerous brain actions such as neurotrophic effects. However, it is unclear how they mediate exercise-induced adult hippocampal neurogenesis (AHN). Our current findings, which show that up-regulation of gene expression of androgen receptors occurs in the rat hippocampus with two weeks of treadmill running, allows us to postulate that neurogenesis induced by treadmill running could mediate androgen. To address this issue, we conducted the following studies with adult male Wistar rats (11 weeks, n=60). In experiment 1, males were either gonadectomized (GDX) or sham-castrated (Sham). In experiment 2, rats were injected Flutamide (androgen receptor antagonist) or sesame oil (vehicle) subcutaneously. Each group was divided into a control group (0 min/m) and an exercise group (13.5 m/min). During
running training for two weeks, non-runners were placed on a stationary treadmill while runners exercised for 30 min/day at indicated speeds. All the animals received injection of the DNA synthesis marker BrdU (50 mg/kg B.W, i.p.), a short cell survival marker, on the day before training. Two days after the last bout of training, the rats were anesthetized and perfused transcardially with saline and fixative. BrdU-labeled cells, Ki67 (proliferation marker) and DCX (immature marker) positive cells were immunohistochemically examined from the brain sections. The GDX group showed significantly decreased testosterone levels compared to the sham group. Treadmill running for two weeks significantly increased the number of cells in the GDX group as well as the Sham group (experiment (1). However, the effect of exercise was completely blocked by Flutamide (experiment (2). Here we found that exercise-induced AHN would occur irrespective of circulating androgens. These results, together with our previous findings, provide an alternative hypothesis that exercise may cause AHN via locally synthesized androgen. doi:10.1016/j.neures.2010.07.1622
P3-e10 Embryonic neural stem cells of the dentate granule cells express GFAP Tatsunori Shioda 4
Seki 1,2
, Noriko
Osumi 2 , Tetsuya
Imura 3 , Sekiji
1
Department of Histol & Neuroanat, Tokyo Medical University 2 Dev. Dev Neurosci, Tohoku University Grad. School Med 3 Department of Pathol Appl Neurobiol, Kyoto Prefectural University, Grad. School Med 4 Dep. of Anat., Showa University School Med Stem cell-like primary progenitors in the adult dentate gyrus have features of astocytes, including glial fibrilar acidic protein (GFAP) expression. Here we explored when and where GFAP+ neural progenitors appear in embryonic and early postnatal stages using GFAP-GFP and GFAP-Cre transgenic mice. In GFAP-GFP mice, progeny or daughter cells of GFAP+ primary progenitors can be analyzed in the GFAP-EGFP mice due to the higher stability of exogenous EGFP. According to Altman and Bayer (1990), putative stem cells of the granule cells initially appear in the neuroepithelium around a ventricular indentation, or the dentate notch at the dorsal edge of the fimbria, and travel through the dorsal portion of the fimbria to the pial side where the dentate gyrus is formed. EGFP+ cells first appeared in the dentate notch on embryonic day (E) 13.5. On E15.5–17.5, the distribution of the EGFP+ cells was expanded, and a number of EGFP+ cells were present in the suprafimbrial, subpial and prospective dentate gyrus. These EGFP+ cells expressed not only GFAP, but also some neuronal markers such as neurogenin 2, neuroD and Hu. However, only a very small number of EGFP+ cells were seen in the prospective neocortical portion. After birth, EGFP+ cells were found in the granule cell layer and hilus. These results show that GFAP expressing progenitors appear when and where progenitors of the granule cells begin to generate and migrate, and suggest that progenitors producing dentate granule cells differ from those generating neocortical neurons from the beginning. doi:10.1016/j.neures.2010.07.1623
P3-e11 The spatio-temporal context for turnover of an adult-born interneuron subtype revealed by in vivo twophoton laser ablation Masato Sawada 1 , Naoko Kaneko 1 , Hiroyuki Inada 2,3 , Hiroaki Wake 2,4 , Yasuko Kato 1 , Yuchio Yanagawa 4,5 , Kazuto Kobayashi 6 , Tomomi Nemoto 4,7 , Jyunichi Nabekura 2,3,4 , Kazunobu 1 Sawamoto 1
Nagoya City University, Nagoya, Japan 2 NIPS, Okazaki, Japan 3 Sokendai, Hayama, Japan 4 CREST, Saitama, Japan 5 Gunma University, Maebashi, Japan 6 Fukushima Med. University, Fukushima, Japan 7 Hokkaido University, Sapporo, Japan Olfactory interneurons, granule cells and periglomerular cells (PGCs), are continuously replaced within the adult olfactory bulb (OB). However, very little is known about the spatio-temporal context for this neuronal turnover in the OB. We have previously developed a technique for in vivo two-photon imaging of PGCs to study their turnover in live mice. Here, we characterized the turnover of PGCs more in detail, focusing on the PGC subtypes and their location in the olfactory glomeruli. PGCs consist of various chemically nonoverlapping subtypes including dopaminergic (DAergic) PGCs. By performing in vivo imaging in TH-GFP mice, we found that DAergic PGCs were the most-intensively replaced subtype under normal physiological condi-
Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-e07 Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus Koji Ohira 1 , Hideo Hagihara 1,2 , Keiko Toyama 1,2 , Keizo Kanai 4 , Hiroshi Funakoshi 4 , Toshikazu Takao 2,3 , Masaaki Nakamura 4 , Tsuyoshi Miyakawa 1,2,3 1
Division of Systems Medical Science, Fujita Health University 2 CREST, Saitama 3 Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi 4 Division of Molecular Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO2), whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO2 was co-expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO2 immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a bromodeoxyuridine labeling experiment revealed that new neurons began to express TDO2 between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO2 might be required at a late-stage of granule cell development, such as during axonal and dendritic growth and synaptogenesis, and for the maintenance of mature cells. doi:10.1016/j.neures.2010.07.1620
P3-e08 Cell-cycle analysis of neural stem cells in the adult mouse dentate gyrus Jun Kaneko , Tatsuhiro Hisatsune Department of Integrated Biosci, The University of Tokyo, Tokyo, Japan Newborn neurons are continuously generated from neural stem cells through neuronal progenitor cells. Neural stem cells divide at the subventricular zone of the dentate gyrus, and generate progenitor cells. In order to further elucidate this phenomenon, it has become necessary to develop a video-imaging system through which this process could be observed. For this purpose, we utilized the Fucci system, in which a protein, Geminin, is accumulated in S/G2/M phases of the cell-cycle, in combination with neural stem cells labeling with Nestin-GFP. In this method, neural stem cells approaching the M phase can be distinguished from G0/G1 phases of neural stem cells. The Geminin lentiviral vector was inoculated into both sides of the dentate gyrus. 4 days after the injection, we carried out immunostaining to confirm whether dividing neural stem cells from Nestin-GFP mice were labeled with Fucci system. As a result, we found that Geminin tagged by fluorescent protein transduced by lentiviral vector was expressed in neural stem cells in adult mice. We are developing an organotypic slice culture from mouse brain inoculated with the Geminin viral vector. doi:10.1016/j.neures.2010.07.1621
P3-e09 Exercise-induced adult hippocampal neurogenesis is inhibited by Flutamide (androgen receptor antagonist) Masahiro Okamoto , Koshiro Inoue, Takashi Matsui, Minchul Lee, Hideaki Soya Laboratory of Exercise Biochemistry, Graduate School of Comprehensive Human Sciences, The University of Tsukuba Androgens play a significant role not only in muscular development, but also in numerous brain actions such as neurotrophic effects. However, it is unclear how they mediate exercise-induced adult hippocampal neurogenesis (AHN). Our current findings, which show that up-regulation of gene expression of androgen receptors occurs in the rat hippocampus with two weeks of treadmill running, allows us to postulate that neurogenesis induced by treadmill running could mediate androgen. To address this issue, we conducted the following studies with adult male Wistar rats (11 weeks, n=60). In experiment 1, males were either gonadectomized (GDX) or sham-castrated (Sham). In experiment 2, rats were injected Flutamide (androgen receptor antagonist) or sesame oil (vehicle) subcutaneously. Each group was divided into a control group (0 min/m) and an exercise group (13.5 m/min). During
running training for two weeks, non-runners were placed on a stationary treadmill while runners exercised for 30 min/day at indicated speeds. All the animals received injection of the DNA synthesis marker BrdU (50 mg/kg B.W, i.p.), a short cell survival marker, on the day before training. Two days after the last bout of training, the rats were anesthetized and perfused transcardially with saline and fixative. BrdU-labeled cells, Ki67 (proliferation marker) and DCX (immature marker) positive cells were immunohistochemically examined from the brain sections. The GDX group showed significantly decreased testosterone levels compared to the sham group. Treadmill running for two weeks significantly increased the number of cells in the GDX group as well as the Sham group (experiment (1). However, the effect of exercise was completely blocked by Flutamide (experiment (2). Here we found that exercise-induced AHN would occur irrespective of circulating androgens. These results, together with our previous findings, provide an alternative hypothesis that exercise may cause AHN via locally synthesized androgen. doi:10.1016/j.neures.2010.07.1622
P3-e10 Embryonic neural stem cells of the dentate granule cells express GFAP Tatsunori Shioda 4
Seki 1,2
, Noriko
Osumi 2 , Tetsuya
Imura 3 , Sekiji
1
Department of Histol & Neuroanat, Tokyo Medical University 2 Dev. Dev Neurosci, Tohoku University Grad. School Med 3 Department of Pathol Appl Neurobiol, Kyoto Prefectural University, Grad. School Med 4 Dep. of Anat., Showa University School Med Stem cell-like primary progenitors in the adult dentate gyrus have features of astocytes, including glial fibrilar acidic protein (GFAP) expression. Here we explored when and where GFAP+ neural progenitors appear in embryonic and early postnatal stages using GFAP-GFP and GFAP-Cre transgenic mice. In GFAP-GFP mice, progeny or daughter cells of GFAP+ primary progenitors can be analyzed in the GFAP-EGFP mice due to the higher stability of exogenous EGFP. According to Altman and Bayer (1990), putative stem cells of the granule cells initially appear in the neuroepithelium around a ventricular indentation, or the dentate notch at the dorsal edge of the fimbria, and travel through the dorsal portion of the fimbria to the pial side where the dentate gyrus is formed. EGFP+ cells first appeared in the dentate notch on embryonic day (E) 13.5. On E15.5–17.5, the distribution of the EGFP+ cells was expanded, and a number of EGFP+ cells were present in the suprafimbrial, subpial and prospective dentate gyrus. These EGFP+ cells expressed not only GFAP, but also some neuronal markers such as neurogenin 2, neuroD and Hu. However, only a very small number of EGFP+ cells were seen in the prospective neocortical portion. After birth, EGFP+ cells were found in the granule cell layer and hilus. These results show that GFAP expressing progenitors appear when and where progenitors of the granule cells begin to generate and migrate, and suggest that progenitors producing dentate granule cells differ from those generating neocortical neurons from the beginning. doi:10.1016/j.neures.2010.07.1623
P3-e11 The spatio-temporal context for turnover of an adult-born interneuron subtype revealed by in vivo twophoton laser ablation Masato Sawada 1 , Naoko Kaneko 1 , Hiroyuki Inada 2,3 , Hiroaki Wake 2,4 , Yasuko Kato 1 , Yuchio Yanagawa 4,5 , Kazuto Kobayashi 6 , Tomomi Nemoto 4,7 , Jyunichi Nabekura 2,3,4 , Kazunobu 1 Sawamoto 1
Nagoya City University, Nagoya, Japan 2 NIPS, Okazaki, Japan 3 Sokendai, Hayama, Japan 4 CREST, Saitama, Japan 5 Gunma University, Maebashi, Japan 6 Fukushima Med. University, Fukushima, Japan 7 Hokkaido University, Sapporo, Japan Olfactory interneurons, granule cells and periglomerular cells (PGCs), are continuously replaced within the adult olfactory bulb (OB). However, very little is known about the spatio-temporal context for this neuronal turnover in the OB. We have previously developed a technique for in vivo two-photon imaging of PGCs to study their turnover in live mice. Here, we characterized the turnover of PGCs more in detail, focusing on the PGC subtypes and their location in the olfactory glomeruli. PGCs consist of various chemically nonoverlapping subtypes including dopaminergic (DAergic) PGCs. By performing in vivo imaging in TH-GFP mice, we found that DAergic PGCs were the most-intensively replaced subtype under normal physiological condi-