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Diffusion tensor analysis of the development of cerebral white matter bundle in cynomolgus monkey fetuses
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Abstracts / Neuroscience Research 68S (2010) e109–e222
P1-d03 Identification and characterization of a mouse recessive lethal mutation which displays cranial neural tube closure defects
Mami Tsume 1,2 , Chiharu Kimura-Yoshida 1 , Saori Amazaki 1 , Kayo Shimokawa 1 , Kyoko Mochida 1 , Isao Matsuo 1,2
1 Department of Molecular Embryology, Osaka Medical Center and Research Institute for Maternal and Child Health 2 School of Medicine, Osaka University, Suita, Osaka, Japan
Neurulation comprises several complex morphogenetic processes; the mammalian neural tube is initially formed as a simple epithelial layer from ectoderm around headfold stage (E7.5) in the mouse embryo and subsequent cell proliferation and dynamic cell movement completes elaborate structures of the central nervous system. However, the genetic and epigenetic mechanisms underlying mammalian neurulation are still poorly understood. Here, in order to elucidate the molecular mechanism of mouse neural tube formation, we identified a novel recessive mutation which displays cranial neural tube closure defects in our transgenic mouse colony. Then, we determined the cause of gene of the transgene-inserted mutation and characterized the phenotypes of homozygous mutant embryos. With Chromosomal FISH and inverse PCR experiments, we identified that the transgene was located on Chromosome 17 and upon insertion of the transgene, the endogenous genomic region was deleted as long as 33.9kb. Further complementation tests determined the responsible gene of this mutation within the deleted locus. Histological examination and molecular marker analyses of homozygous mutant embryos revealed that mutant neuroepithelial cells did not proliferate appropriately and underwent a cell death until E10.5. We will discuss the role of proliferation and/or survival of neuroepithelial cells on mammalian neurulation processes.
ond postnatal week. The other group of molecules (‘early markers’), including Pcp2 (=L7) and Wnt7b, are expressed in multiple parasagittal stripes mainly in the embryonic stage, much earlier than the late markers. The cerebellar cortex of a transgenic mouse strain, 1NM13, in which the type 1 inositol 1,4,5-trisphosphate receptor (IP3 R1) gene promoter-driven -galactosidase (LacZ) composite gene has been introduced, shows clear parasagittal stripes of LacZ expression from embryonic day 15 (E15) to postnatal day 14 (P14). To understand the development and consistency of these stripes in the growing cerebellum, we analyzed the organization of the stripes systematically with X-gal staining in whole mount preparations and in serial sections. Multiple separate patchy stripes, in which Purkinje cells expressed LacZ, emerged in the embryonic cerebellum and were transformed to long parasagittal stripes later. The most complicated stripe pattern of LacZ expression (‘LacZ stripes’) was established around P7. This pattern was still clear on P13, when the aldolase C stripes began to be stabilized. Here, the LacZ stripes were capable of full identification regarding location since they were closely parallel to the aldolase C stripes in details. The results suggest that this mouse strain would be useful in understanding how the adult pattern of cerebellar parasagittal compartmentalization is predetermined in the early developing cerebellum. For this purpose we are comparing the expression pattern of LacZ with that of early markers in the embryonic cerebellum in 1NM13 mice. doi:10.1016/j.neures.2010.07.2146
P1-d06 Promotion of neuronal differentiation in neural progenitors exposed to static magnetism Ryouta Nakazato , Noritaka Nakamichi, Yukichi Ishioka, Takeshi Takarada, Yukio Yoneda Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Tec
doi:10.1016/j.neures.2010.07.2144
P1-d04 Draxin, an axon guidance protein, is involved in retinocollicular axon projection Iftekhar B. Naser , Giasuddin Ahmed, Mahmud Hossein, Yohei Shinmyo, Hideaki Tanaka Kumamoto University, Global COE Most axonal projections in the brain establish in their target field an orderly arrangement of connections termed a topographic map. One extensively studied example of this topographic map, retinotectal projection, is organized along the anterior-posterior and dorsal-ventral axes of optic tectum. Ventral retinal axons connect to the dorsal tectum and dorsal retinal axons find their targets at ventral tectum. Temporal retinal axons terminate at anterior tectum, and nasal retinal axons project to the posterior tectum. Studies implicated A class ephrins in establishing anterior-posterior topographic mapping via repulsive mechanism through the EphA receptors. Along the dorsal-ventral axis, an attractive interaction involving ephrinB-EphB and a repulsive effect of Wnt3-Ryk were proposed to control dorsal-ventral patterning. These axon guidance cues are all expressed in the tectum in a gradient, for example, ephrin A2 expressed in a posterior high to anterior low gradient and Wnt 3 expressed in dorsal high to ventral low gradient. Our molecule draxin is expressed in medial high to lateral low gradient in superior colliculas of mouse and retina also express draxin. Draxin inhibit neurite outgrowth from mose retinal explant. Draxin knock out mice shows mapping defect along the dorso-ventral and anterior-posterior axis. Further analyses are required to reveal the role of Draxin and underlying mechanism of the mapping defect in retinocollicular projection.
In our previous studies, a significant increase was seen in Ca2+ influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in rat hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of exposure to static magnetism on proliferation for self-replication and differentiation into neuronal, astroglial and oligodendroglial lineages in undifferentiated neural progenitor cells. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT for different periods and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Sustained exposure to static magnetism not only led to significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also resulted in promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for glial fibrillary acidic protein irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism for 12 days, moreover, a significant increase was seen in mRNA expression of several activator type proneural genes, such as Mash1, Math1 and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of several proneural genes by progenitor cells in fetal rat brain. This study would lead to the innovative development of magnetic instruments clinically useful for the noninvasive treatment of patients suffering from a variety of neurodegenerative and/or neuropsychiatric diseases relevant to neuronal dysfunctions in terms of promoted neuronal differentiation during embryonic neurogenesis. doi:10.1016/j.neures.2010.07.2147
doi:10.1016/j.neures.2010.07.2145
P1-d05 Development of cerebellar parasagittal compartmentalization studied in a transgenic mouse strain carrying the LacZ composite gene Hirofumi Fujita 1 , Teiichi Furuichi 2 , Izumi Sugihara 1 1 Dept. of Systems Neurophysiology, Tokyo Medical and Dental University, Tokyo 2 Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako
In the cerebellar cortex, the afferent and efferent neuronal connections are organized according to the parasagittal compartmentalization, which is also reflected in the cerebellar expression of specific marker molecules in adult rodents. These molecules (‘late markers’), including aldolase C (=zebrin II), are expressed in multiple parasagittal stripes mainly in and after the late sec-
P1-d07 Diffusion tensor analysis of the development of cerebral white matter bundle in cynomolgus monkey fetuses
Kazuhiko Sawada 1 , Katsuhiro Fukunishi 2 , Masatoshi Kashima 2 , Shigeyoshi Saito 3 , Xue-Zhi Sun 4 , Hiromi Sakata-Haga 5 , Takayuki Sukamoto 2 , Yoshihiro Fukui 5 1
Fac Health Sci, Tsukuba Int Univ, Tsuchiura, Japan 2 Shin Nippon Biomed Lab, Kagoshima, Japan 3 Mol Imaging Cent, NIRS, Chiba, Japan 4 Mol Imaging Cent, NIRS, Chiba, Japan 5 Dept Anat & Dev Neurobiol, Univ of Tokushima, Tokushima, Japan Diffusion tensor imaging (DTI) was acquired from the fixed brains of cynomolgus monkey fetuses at embryonic days (EDs) 70–140. On DTI color maps, the association fiber tracts in the limbic area, the commissural tracts,
Abstracts / Neuroscience Research 68S (2010) e109–e222
and the projection fiber tracts were revealed already on ED 70. The corticocortical long association fibers began to emerge sequentially from ED 70. Interestingly, the emergences of those fibers corresponded to and/or followed the formation of early-generated primary sulci: the middle longitudinal fasciculus along the superior temporal sulcus on ED 90; the superior longitudinal fasciculus along the arcuate and central sulci on ED 100; and the inferior longitudinal fasciculus along the lunate sulcus on ED 120. These results suggest that the emergence of the long corticocortical associative fibers links spatiotemporally with the formation of primary sulci in the cerebrum of cynomolgus monkey fetuses. The present results support tension-based theory of the cortical convolution. Grant: supported by the JSPS KAKENHI (20590176). Ethics: approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories. doi:10.1016/j.neures.2010.07.2148
P1-d08 Activation of 5 -AMP-activated protein kinase by metformin promotes proliferation of the neural stem/progenitor cells via ryanodine receptor-mediated Ca2+ signaling Natsuki Tanaka , Shohei Sano, Nobuyuki Kuramoto, Masanori Yoneyama, Kiyokazu Ogita Dept. Pharmcol., Setsunan Univ Neural stem/progenitor cells (NPCs) are the ability to self-renew and differentiate into 3 major cell types, such as neurons, astrocytes, and oligodendrocytes. 5 -AMP-activated protein kinase (AMPK) acts as an efficient sensor for cellular energy state by sensing cytosolic AMP. Although previous studies suggest that metformin increases the amount of cytosolic AMP, the exact mechanisms underlying acitivation of AMPK by metformin are still unclear. Evidence for activation of AMPK by Ca2+ signaling comes from the observations that calmodulin-dependent protein kinases phosphorylate and thereby activate AMPK. Recent our studies indicated that metformin enhanced proliferation of the NPCs derived from embryonic neocortex of mice. In this study, we examined if Ca2+ signaling is involved in metformin-induced enhancement of proliferation of the NPCs. Cells were prepared from the neocortex of 15-day-old embryonic mice and primarily cultured in DMEM/F12 medium with EGF and bFGF for 9 days in vitro. After re-plating, the cells were kept under the same medium in the absence or presence of metformin and Ca2+ channel blockers including ifenprodil, MK801, nifedipine, and dantrolene. Incorporation of 5 -bromo-2 -deoxyuridine was increased by metformin and decreased by any of Ca2+ channel blockers. Metformin elevated the level of phospho-AMPK (p-AMPK), phospho-ERK (pERK), and phospho-Akt (p-Akt). Of Ca2+ channel blockers used, dantrolene abolished metformin-induced elevation of p-AMPK level. However, dantrolene was ineffective in metformin elevating the level of p-ERK and p-Akt. These results suggest that metformin promotes proliferation of the NPCs through activation of AMPK by Ca2+ released by activation of ryanodine receptor from the endoplasmic reticulam in the neocortex of embryonic mouse. doi:10.1016/j.neures.2010.07.2149
P1-d10 Acceleration of neural stem cell differentiation into astrocyte by X-irradiation Ayumi Ozeki 1 , Masatoshi Suzuki 1 , Hiroki Ozawa 2 , Keiji Suzuki 1 , Shunichi Yamashita 1 1
Dept Mol Med, Nagasaki Univ Grad Sch Biomed Sci, Nagasaki 2 Dept Neuropsy, Nagasaki Univ Grad Sch Biomed Sci, Nagasaki Radiation exposure to brain causes dysfunctions of the normal central nervous system (CNS), however, the mechanism is not fully understood. Since the CNS is composed of several types of differentiated cells from neural stem cells (NSCs), one possibility mechanism could be that ionizing radiation affects differentiation process of NSCs. Here, we investigated the differentiation patterns of NSCs, isolated from rat cortex, into neuron, astrocyte and oligodendrocyte after 5 Gy of X-irradiation. Nestin-positive NSCs were cultured in the proliferation-permissive medium after X-irradiation for 7 days. They were also cultured in the differentiation-permissive medium without radiation, and then each differentiation markers were detected. We found no induction of -III tubulin-positive neurons nor O4-positive oligodendrocytes. On the other hand, X-irradiation specifically resulted in a 17.3 fold-induction of glial fibrillary acidic protein (GFAP)-positive astrocytes. Interestingly, the fraction of GFAP positive cells induced by X-irradiation was
e131
quite similar to that detected in cells cultured in differentiation-permissive medium without radiation. The combination of X-irradiation and cultivation in a differentiation-permissive medium did not cause synergistic effects. These results suggest that X-irradiation accelerates astrocyte-specific differentiation through activating the common pathway stimulated when NSCs are differentiated into astrocytes in vivo. doi:10.1016/j.neures.2010.07.2150
P1-d11 Valproic acid stimulates differentiation of CG4-16 cells via HDAC inhibitory activity Yuta Inagawa , Kaoru Nagai Department of Epigenetic Medicine, Interdisciplinary Graduate School of Medicine and Engineering, The University of Yamanashi Epigenetic regulation, such as histone modification and DNA methylation, is essential in the central nervous system (CNS). Although importance of epigenetics on neuronal or astroglial differentiation from neural precursor cells has been reported, contribution of epigenetics on the differentiation from oligodendrocyte precursor cells (OPCs) has not been studied, yet. In this study, we investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the differentiation of OPCs model cell line CG4-16 cells. VPA reduced cell number dose dependently. And the reduction was inhibited by DNA polymerase inhibitor aphidicolin. VPA increased dendritic number, and upregulated oligodendrocyte marker, MAG on oligodendrocyte differentiating condition. While, on OPCs maintaining condition, VPA altered the cell shape to epithelial like morphology, and upregulated an astrocyte marker, GFAP. Then, we evaluated if the effect of VPA was dependent on HDAC activity. Valpromide, which is a VPA derivative without HDAC inhibitory activity, did not show any effect on CG4-16 cells. Our data suggests that VPA arrested cell growth and promoted glial differentiation from OPCs via its inhibitory activity of histone deacetylation. doi:10.1016/j.neures.2010.07.2151
P1-d12 Characterization of CD44-positive cells in the developing mouse cerebellum Masashi Kurachi , Na Cai, Koji Shibasaki, Takayuki Okano-Uchida, Yasuki Ishizaki Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, Maebashi, Japan It is generally considered that neural stem cells are committed to become either neuronal precursor cells or glial precursor cells before terminally differentiating into neurons or glial cells. Up to now, neuronal precursor cells and oligodendrocyte precursor cells have been identified in several areas in the murine CNS. Presence of astrocyte precursor cells (APCs), however, has been reported only rarely so far. In this study, we report isolation of CD44positive cells, candidates for APCs, from the developing mouse cerebellum. CD44-positive cells were purified from the early postnatal mouse cerebellum using FACS, and were characterized in vitro. The purified cells expressed neither GFAP nor S100-b, markers for mature astrocytes. In contrast, they expressed brain-lipid binding protein (BLBP), a marker for immature astrocyte lineage cells. When the cells were cultured without trophic factors, many cells died by apoptosis. The surviving cells, however, gradually expressed GFAP, indicating that differentiation into astrocytes is the default program for these cells. In the presence of basic fibroblast growth factor (bFGF), many cells incorporated BrdU. Most of the BrdU-positive cells were GFAP-negative, suggesting that bFGF is essential for their proliferation and the maintenance of their undifferentiated state. Many of these cells, however, underwent apoptosis, suggesting bFGF alone is not sufficient for their survival. In the presence of both bFGF and bone morphogenetic protein-4 (BMP4), the cells maintained their survival and proliferation, indicating BMP4 is a survival factor for these cells. When CD44-positive cells were treated with leukemia inhibitory factor (LIF), the proportion of GFAP-positive cells increased dramatically, suggesting LIF is an inducer of their astrocytic differentiation. Characterization of these cells may be useful for understanding the mechanism for generation of various astrocytes during cerebellar development. doi:10.1016/j.neures.2010.07.2152
Abstracts / Neuroscience Research 68S (2010) e109–e222
P1-d03 Identification and characterization of a mouse recessive lethal mutation which displays cranial neural tube closure defects
Mami Tsume 1,2 , Chiharu Kimura-Yoshida 1 , Saori Amazaki 1 , Kayo Shimokawa 1 , Kyoko Mochida 1 , Isao Matsuo 1,2
1 Department of Molecular Embryology, Osaka Medical Center and Research Institute for Maternal and Child Health 2 School of Medicine, Osaka University, Suita, Osaka, Japan
Neurulation comprises several complex morphogenetic processes; the mammalian neural tube is initially formed as a simple epithelial layer from ectoderm around headfold stage (E7.5) in the mouse embryo and subsequent cell proliferation and dynamic cell movement completes elaborate structures of the central nervous system. However, the genetic and epigenetic mechanisms underlying mammalian neurulation are still poorly understood. Here, in order to elucidate the molecular mechanism of mouse neural tube formation, we identified a novel recessive mutation which displays cranial neural tube closure defects in our transgenic mouse colony. Then, we determined the cause of gene of the transgene-inserted mutation and characterized the phenotypes of homozygous mutant embryos. With Chromosomal FISH and inverse PCR experiments, we identified that the transgene was located on Chromosome 17 and upon insertion of the transgene, the endogenous genomic region was deleted as long as 33.9kb. Further complementation tests determined the responsible gene of this mutation within the deleted locus. Histological examination and molecular marker analyses of homozygous mutant embryos revealed that mutant neuroepithelial cells did not proliferate appropriately and underwent a cell death until E10.5. We will discuss the role of proliferation and/or survival of neuroepithelial cells on mammalian neurulation processes.
ond postnatal week. The other group of molecules (‘early markers’), including Pcp2 (=L7) and Wnt7b, are expressed in multiple parasagittal stripes mainly in the embryonic stage, much earlier than the late markers. The cerebellar cortex of a transgenic mouse strain, 1NM13, in which the type 1 inositol 1,4,5-trisphosphate receptor (IP3 R1) gene promoter-driven -galactosidase (LacZ) composite gene has been introduced, shows clear parasagittal stripes of LacZ expression from embryonic day 15 (E15) to postnatal day 14 (P14). To understand the development and consistency of these stripes in the growing cerebellum, we analyzed the organization of the stripes systematically with X-gal staining in whole mount preparations and in serial sections. Multiple separate patchy stripes, in which Purkinje cells expressed LacZ, emerged in the embryonic cerebellum and were transformed to long parasagittal stripes later. The most complicated stripe pattern of LacZ expression (‘LacZ stripes’) was established around P7. This pattern was still clear on P13, when the aldolase C stripes began to be stabilized. Here, the LacZ stripes were capable of full identification regarding location since they were closely parallel to the aldolase C stripes in details. The results suggest that this mouse strain would be useful in understanding how the adult pattern of cerebellar parasagittal compartmentalization is predetermined in the early developing cerebellum. For this purpose we are comparing the expression pattern of LacZ with that of early markers in the embryonic cerebellum in 1NM13 mice. doi:10.1016/j.neures.2010.07.2146
P1-d06 Promotion of neuronal differentiation in neural progenitors exposed to static magnetism Ryouta Nakazato , Noritaka Nakamichi, Yukichi Ishioka, Takeshi Takarada, Yukio Yoneda Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Tec
doi:10.1016/j.neures.2010.07.2144
P1-d04 Draxin, an axon guidance protein, is involved in retinocollicular axon projection Iftekhar B. Naser , Giasuddin Ahmed, Mahmud Hossein, Yohei Shinmyo, Hideaki Tanaka Kumamoto University, Global COE Most axonal projections in the brain establish in their target field an orderly arrangement of connections termed a topographic map. One extensively studied example of this topographic map, retinotectal projection, is organized along the anterior-posterior and dorsal-ventral axes of optic tectum. Ventral retinal axons connect to the dorsal tectum and dorsal retinal axons find their targets at ventral tectum. Temporal retinal axons terminate at anterior tectum, and nasal retinal axons project to the posterior tectum. Studies implicated A class ephrins in establishing anterior-posterior topographic mapping via repulsive mechanism through the EphA receptors. Along the dorsal-ventral axis, an attractive interaction involving ephrinB-EphB and a repulsive effect of Wnt3-Ryk were proposed to control dorsal-ventral patterning. These axon guidance cues are all expressed in the tectum in a gradient, for example, ephrin A2 expressed in a posterior high to anterior low gradient and Wnt 3 expressed in dorsal high to ventral low gradient. Our molecule draxin is expressed in medial high to lateral low gradient in superior colliculas of mouse and retina also express draxin. Draxin inhibit neurite outgrowth from mose retinal explant. Draxin knock out mice shows mapping defect along the dorso-ventral and anterior-posterior axis. Further analyses are required to reveal the role of Draxin and underlying mechanism of the mapping defect in retinocollicular projection.
In our previous studies, a significant increase was seen in Ca2+ influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in rat hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of exposure to static magnetism on proliferation for self-replication and differentiation into neuronal, astroglial and oligodendroglial lineages in undifferentiated neural progenitor cells. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT for different periods and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Sustained exposure to static magnetism not only led to significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also resulted in promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for glial fibrillary acidic protein irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism for 12 days, moreover, a significant increase was seen in mRNA expression of several activator type proneural genes, such as Mash1, Math1 and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of several proneural genes by progenitor cells in fetal rat brain. This study would lead to the innovative development of magnetic instruments clinically useful for the noninvasive treatment of patients suffering from a variety of neurodegenerative and/or neuropsychiatric diseases relevant to neuronal dysfunctions in terms of promoted neuronal differentiation during embryonic neurogenesis. doi:10.1016/j.neures.2010.07.2147
doi:10.1016/j.neures.2010.07.2145
P1-d05 Development of cerebellar parasagittal compartmentalization studied in a transgenic mouse strain carrying the LacZ composite gene Hirofumi Fujita 1 , Teiichi Furuichi 2 , Izumi Sugihara 1 1 Dept. of Systems Neurophysiology, Tokyo Medical and Dental University, Tokyo 2 Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako
In the cerebellar cortex, the afferent and efferent neuronal connections are organized according to the parasagittal compartmentalization, which is also reflected in the cerebellar expression of specific marker molecules in adult rodents. These molecules (‘late markers’), including aldolase C (=zebrin II), are expressed in multiple parasagittal stripes mainly in and after the late sec-
P1-d07 Diffusion tensor analysis of the development of cerebral white matter bundle in cynomolgus monkey fetuses
Kazuhiko Sawada 1 , Katsuhiro Fukunishi 2 , Masatoshi Kashima 2 , Shigeyoshi Saito 3 , Xue-Zhi Sun 4 , Hiromi Sakata-Haga 5 , Takayuki Sukamoto 2 , Yoshihiro Fukui 5 1
Fac Health Sci, Tsukuba Int Univ, Tsuchiura, Japan 2 Shin Nippon Biomed Lab, Kagoshima, Japan 3 Mol Imaging Cent, NIRS, Chiba, Japan 4 Mol Imaging Cent, NIRS, Chiba, Japan 5 Dept Anat & Dev Neurobiol, Univ of Tokushima, Tokushima, Japan Diffusion tensor imaging (DTI) was acquired from the fixed brains of cynomolgus monkey fetuses at embryonic days (EDs) 70–140. On DTI color maps, the association fiber tracts in the limbic area, the commissural tracts,
Abstracts / Neuroscience Research 68S (2010) e109–e222
and the projection fiber tracts were revealed already on ED 70. The corticocortical long association fibers began to emerge sequentially from ED 70. Interestingly, the emergences of those fibers corresponded to and/or followed the formation of early-generated primary sulci: the middle longitudinal fasciculus along the superior temporal sulcus on ED 90; the superior longitudinal fasciculus along the arcuate and central sulci on ED 100; and the inferior longitudinal fasciculus along the lunate sulcus on ED 120. These results suggest that the emergence of the long corticocortical associative fibers links spatiotemporally with the formation of primary sulci in the cerebrum of cynomolgus monkey fetuses. The present results support tension-based theory of the cortical convolution. Grant: supported by the JSPS KAKENHI (20590176). Ethics: approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories. doi:10.1016/j.neures.2010.07.2148
P1-d08 Activation of 5 -AMP-activated protein kinase by metformin promotes proliferation of the neural stem/progenitor cells via ryanodine receptor-mediated Ca2+ signaling Natsuki Tanaka , Shohei Sano, Nobuyuki Kuramoto, Masanori Yoneyama, Kiyokazu Ogita Dept. Pharmcol., Setsunan Univ Neural stem/progenitor cells (NPCs) are the ability to self-renew and differentiate into 3 major cell types, such as neurons, astrocytes, and oligodendrocytes. 5 -AMP-activated protein kinase (AMPK) acts as an efficient sensor for cellular energy state by sensing cytosolic AMP. Although previous studies suggest that metformin increases the amount of cytosolic AMP, the exact mechanisms underlying acitivation of AMPK by metformin are still unclear. Evidence for activation of AMPK by Ca2+ signaling comes from the observations that calmodulin-dependent protein kinases phosphorylate and thereby activate AMPK. Recent our studies indicated that metformin enhanced proliferation of the NPCs derived from embryonic neocortex of mice. In this study, we examined if Ca2+ signaling is involved in metformin-induced enhancement of proliferation of the NPCs. Cells were prepared from the neocortex of 15-day-old embryonic mice and primarily cultured in DMEM/F12 medium with EGF and bFGF for 9 days in vitro. After re-plating, the cells were kept under the same medium in the absence or presence of metformin and Ca2+ channel blockers including ifenprodil, MK801, nifedipine, and dantrolene. Incorporation of 5 -bromo-2 -deoxyuridine was increased by metformin and decreased by any of Ca2+ channel blockers. Metformin elevated the level of phospho-AMPK (p-AMPK), phospho-ERK (pERK), and phospho-Akt (p-Akt). Of Ca2+ channel blockers used, dantrolene abolished metformin-induced elevation of p-AMPK level. However, dantrolene was ineffective in metformin elevating the level of p-ERK and p-Akt. These results suggest that metformin promotes proliferation of the NPCs through activation of AMPK by Ca2+ released by activation of ryanodine receptor from the endoplasmic reticulam in the neocortex of embryonic mouse. doi:10.1016/j.neures.2010.07.2149
P1-d10 Acceleration of neural stem cell differentiation into astrocyte by X-irradiation Ayumi Ozeki 1 , Masatoshi Suzuki 1 , Hiroki Ozawa 2 , Keiji Suzuki 1 , Shunichi Yamashita 1 1
Dept Mol Med, Nagasaki Univ Grad Sch Biomed Sci, Nagasaki 2 Dept Neuropsy, Nagasaki Univ Grad Sch Biomed Sci, Nagasaki Radiation exposure to brain causes dysfunctions of the normal central nervous system (CNS), however, the mechanism is not fully understood. Since the CNS is composed of several types of differentiated cells from neural stem cells (NSCs), one possibility mechanism could be that ionizing radiation affects differentiation process of NSCs. Here, we investigated the differentiation patterns of NSCs, isolated from rat cortex, into neuron, astrocyte and oligodendrocyte after 5 Gy of X-irradiation. Nestin-positive NSCs were cultured in the proliferation-permissive medium after X-irradiation for 7 days. They were also cultured in the differentiation-permissive medium without radiation, and then each differentiation markers were detected. We found no induction of -III tubulin-positive neurons nor O4-positive oligodendrocytes. On the other hand, X-irradiation specifically resulted in a 17.3 fold-induction of glial fibrillary acidic protein (GFAP)-positive astrocytes. Interestingly, the fraction of GFAP positive cells induced by X-irradiation was
e131
quite similar to that detected in cells cultured in differentiation-permissive medium without radiation. The combination of X-irradiation and cultivation in a differentiation-permissive medium did not cause synergistic effects. These results suggest that X-irradiation accelerates astrocyte-specific differentiation through activating the common pathway stimulated when NSCs are differentiated into astrocytes in vivo. doi:10.1016/j.neures.2010.07.2150
P1-d11 Valproic acid stimulates differentiation of CG4-16 cells via HDAC inhibitory activity Yuta Inagawa , Kaoru Nagai Department of Epigenetic Medicine, Interdisciplinary Graduate School of Medicine and Engineering, The University of Yamanashi Epigenetic regulation, such as histone modification and DNA methylation, is essential in the central nervous system (CNS). Although importance of epigenetics on neuronal or astroglial differentiation from neural precursor cells has been reported, contribution of epigenetics on the differentiation from oligodendrocyte precursor cells (OPCs) has not been studied, yet. In this study, we investigated the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the differentiation of OPCs model cell line CG4-16 cells. VPA reduced cell number dose dependently. And the reduction was inhibited by DNA polymerase inhibitor aphidicolin. VPA increased dendritic number, and upregulated oligodendrocyte marker, MAG on oligodendrocyte differentiating condition. While, on OPCs maintaining condition, VPA altered the cell shape to epithelial like morphology, and upregulated an astrocyte marker, GFAP. Then, we evaluated if the effect of VPA was dependent on HDAC activity. Valpromide, which is a VPA derivative without HDAC inhibitory activity, did not show any effect on CG4-16 cells. Our data suggests that VPA arrested cell growth and promoted glial differentiation from OPCs via its inhibitory activity of histone deacetylation. doi:10.1016/j.neures.2010.07.2151
P1-d12 Characterization of CD44-positive cells in the developing mouse cerebellum Masashi Kurachi , Na Cai, Koji Shibasaki, Takayuki Okano-Uchida, Yasuki Ishizaki Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, Maebashi, Japan It is generally considered that neural stem cells are committed to become either neuronal precursor cells or glial precursor cells before terminally differentiating into neurons or glial cells. Up to now, neuronal precursor cells and oligodendrocyte precursor cells have been identified in several areas in the murine CNS. Presence of astrocyte precursor cells (APCs), however, has been reported only rarely so far. In this study, we report isolation of CD44positive cells, candidates for APCs, from the developing mouse cerebellum. CD44-positive cells were purified from the early postnatal mouse cerebellum using FACS, and were characterized in vitro. The purified cells expressed neither GFAP nor S100-b, markers for mature astrocytes. In contrast, they expressed brain-lipid binding protein (BLBP), a marker for immature astrocyte lineage cells. When the cells were cultured without trophic factors, many cells died by apoptosis. The surviving cells, however, gradually expressed GFAP, indicating that differentiation into astrocytes is the default program for these cells. In the presence of basic fibroblast growth factor (bFGF), many cells incorporated BrdU. Most of the BrdU-positive cells were GFAP-negative, suggesting that bFGF is essential for their proliferation and the maintenance of their undifferentiated state. Many of these cells, however, underwent apoptosis, suggesting bFGF alone is not sufficient for their survival. In the presence of both bFGF and bone morphogenetic protein-4 (BMP4), the cells maintained their survival and proliferation, indicating BMP4 is a survival factor for these cells. When CD44-positive cells were treated with leukemia inhibitory factor (LIF), the proportion of GFAP-positive cells increased dramatically, suggesting LIF is an inducer of their astrocytic differentiation. Characterization of these cells may be useful for understanding the mechanism for generation of various astrocytes during cerebellar development. doi:10.1016/j.neures.2010.07.2152