- Email: [email protected]
Analysis of formation mechanism for excitatory and inhibitory neural circuits
Abstracts improvements in functional behaviors. These results suggest that sNgR-Fc may have potential therapeutic values for treating stroke and neurodegenerative diseases. doi:10.1016/j.neures.2009.09.787
P2-d16 Expression of cyclin E in the cerebral and cerebellar cortices Yayoi Ikeda 1 , Yuko Matsunaga 2 , Masa-Aki Ikeda 2 1 Dept Histol & Cell Biol, Yokohama City Univ School of Med, Yokohama, Japan; 2 Sect Molecular Embryol, Tokyo Med & Dent Univ, Tokyo, Japan
Cyclin E is a member of G1 cyclins which promote the progression of the cell cycle. The present study examined cyclin E expression in the developing and adult central nervous system (CNS) in mice and rats. The Western blot, in situ hybridization and immunohistochemistry demonstrated that mRNA and protein of cyclin E were expressed in both proliferating and non-proliferation regions during development of the cerebral cortex and the cerebellum. Double-label immunofluorescence for cyclin E and cyclin-dependent kinase 5 (cdk5) showed that both of the two proteins were expressed by pyramidal neurons and Purkinje cells in the cerebral and cerebellar coteces, respectively. Finally, immunoprecipitation analysis reveals that cyclin E binds to cdk5 in the cytoplasm of postnatal and adult cerebral cortex with no detectable kinase activity. The fact that cyclin E is expressed in terminally differentiated neurons predict an alternative function(s) in addition to controlling the cell cycle. doi:10.1016/j.neures.2009.09.788
P2-d17 Differential Islet-1 expression among spinal motorneurons in prenatal mouse Kengo Funakoshi, Da-Young Han, Miki Kobayashi, Masato Nakano, Yoshitoshi Atobe, Tetsuo Kadota Dept Neuroana, Yokohama City Univ, Yokohama, Japan In mouse, neurons projecting to the urethral sphincter and ischiocavernosus muscles form the dorsolateral nucleus (DL) at the caudal lumbar levels, whereas neurons projecting to the limb and hip joint muscles comprise the retrodorsolateral and ventral nucleus, as well as the DL at the rostral lumbar levels. The present study showed that the expression pattern of a LIM homeodomein protein Islet-1 was different among motoneuronal groups at the prenatal stage of mouse. The highest expression was observed in the caudal DL. The high expression was also observed in the medial part of the rostral DL, whereas there was little expression in the lateral part of the rostral DL. These findings provide evidence that the DL neurons corresponding to Onuf nucleus, are chemically distinct among the motoneuronal groups at the prenatal stages. This differential Islet-1 expression among the motoneuronal groups suggests that Islet-1 not only leads to a motoneuronal lineage, but also to the differentiation of motoneuronal subsets in the lumbosacral spinal cord. doi:10.1016/j.neures.2009.09.789
P2-d18 The effect of arachidonic acid and docosahexaenoic acid in neural stem/progenitor cells Numayama-Tsuruta 1,2 , Noriko Nobuyuki Sakayori 1 , Keiko Osumi 1,2,3 1 Div. of Devel. Neurosci., Tohoku University Grad. Sch. of Med., Sendai, Japan; 2 CREST, JST, Kawauchi, Japan; 3 Tohoku Neuroscience Global COE, Tohoku University, Sendai, Japan
Arachidonic acid (ARA) and Docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids (PUFAs) in the brain, play crucial roles in brain development and function. We have shown that ARA promotes postnatal neurogenesis in the rat hippocampus. To further elucidate functions of these PUFAs in neurogenesis, we analyzed effects of ARA and DHA on proliferation, differentiation and survival of NSPCs at various concentrations using neurosphere assays. ARA at low concentrations tended to increase the number of neurospheres and also increase the number of Tuj1-positive cells, whereas DHA at low concentrations increased the number of neurospheres. Thus, ARA has a tendency to promote maintenance and neuronal differentiation of NSPCs, while DHA promoted maintenance of NSPCs. These results are consistent with our previous report that ARA promotes postnatal neurogenesis in vivo. We are now investigating effects of ARA and DHA on glial differentiation. doi:10.1016/j.neures.2009.09.790
S155
P2-d19 15-deoxy-12,14 -prostaglandin J2 biphasically regulates the proliferation of mouse hippocampal neural stem cells by modulating the redox state Takashi Katura, Takahiro Moriya, Norimichi Nakahata Department Cellular signaling, Grad. Sch. Pharmaceut. Sci., Tohoku University, Japan In this study, we tried to examine the effect of 15-deoxy-12,14 -prostaglandin J2 (15d-PGJ2 ) on the proliferation of the neural stem cells (NSCs) derived from mouse hippocampus. 15d-PGJ2 showed the biphasic effects on EGF-dependent proliferation of the NSCs. An inhibitor of PPAR␥, known as a receptor for 15d-PGJ2 , failed to abolish the effects of 15d-PGJ2 . We also found that the elecrophilic carbon at 9 position is critical for the action of 15d-PGJ2 on the proliferation. The treatment with 15d-PGJ2 caused an increase in the levels of reactive oxygen species (ROS) accompanied with the decrease in endogenous glutathione (GSH) levels. Furthermore, the supplement with a GSH ethyl ester (GSH-EE) (2 mM), diminished the biphasic effects of 15d-PGJ2 . Taken together that GSH plays important roles in redox cellsignaling pathway, these results suggest that 15d-PGJ2 regulates the proliferation of NSCs via its electrophilic nature, which enables the covalent binding to molecules such as glutathione in PPAR␥-independent manner. doi:10.1016/j.neures.2009.09.791
P2-d20 Adenosine suppresses the proliferation of the embryonic mouse neural stem cells via equilibrative nucleoside transporters Yuko Suzuki, Takahiro Moriya, Takashi Katura, Norimichi Nakahata Dept Cell Signal, Grad Sch Pharm Sci, Tohoku Univ, Sendai, Japan Neural stem cells (NSCs) exhibit the properties of self-renewal and multipotential. While adenosine is known to be a modulator of neuronal function, its effects on NSCs remain unclear. We, therefore, investigated the effects of adenosine on the proliferation of the NSCs derived from embryonic mouse striatum.Using WST-8 assay and BrdU-incorporation assay, we found that adenosine inhibited cell proliferation of NSCs in time- and concentration-dependent manners. Adenosine receptors were not involved in these effects. We confirmed the NSCs expressed equilibrative nucleoside transporter (ENT)-1, which incorporates extracellular adenosine into cytosol. Furthermore, inhibitors of ENTs attenuated adenosine-induced growth inhibition. Furthermore, either an adenosine kinase inhibitor or uridine supplement reversed the growth inhibition induced by adenosine. These results suggest that intracellular AMP accumulation by adenosine kinase following adenosine uptake would induce NSC growth inhibition through uridine starvation. doi:10.1016/j.neures.2009.09.792
P2-d21 Synchronization of calcium oscillation between retinal neuroepithelial cells Masayuki Yamashita Dept Physiol 1, Nara Med Univ, Kashihara, Japan In neuroepithelium spontaneous and periodic increases in intracellular Ca2+ concentration (Ca2+ oscillation) occur synchronously between the cells and independently of extracellular Ca2+ . Thus it is supposed that Ca2+ releases from intracellular Ca2+ stores, which are formed from endoplasmic reticulum and nuclear envelope, are synchronized between the cells. Recently, synchronous oscillatory changes in the membrane potential of intracellular Ca2+ sores were recorded using an organellespecific voltage-sensitive dye [DiOC5 (3)]. High-speed fluorescence measurement of store membrane potential using a photomultiplier surprisingly revealed that the synchronous oscillatory change in the store potential was periodic repeats of a burst of high-frequency voltage fluctuations. The burst coincided with a Ca2+ increase. It is suggested that the synchronization of Ca2+ releases is mediated by the highfrequency fluctuation in the store potential. Close apposition of the outer nuclear membrane and plasma membrane in a pseudostratified epithelial structure would allow capacitative electrical coupling between the cells. doi:10.1016/j.neures.2009.09.793
P2-d22 Analysis of formation mechanism for excitatory and inhibitory neural circuits Mitsunori Arai, Shoji Komai Nara Institute of Science and Technology, Japan Many researchers have studied about neural stem cell and its transplantation into several brain regions to treat neurological disorders, such as Parkinson’s disease, ischemia, and spinal cord injuries, caused by damage or loss of neurons and glial cells. These therapies indeed have been helped to recover of motor functions such as
S156
Abstracts
dyskinesia. However, little is known about recovery and regeneration mechanism of optimal neural circuits developing by precise neural activities and how neural activities form the appropriate local neural circuits with transplanted cells correctly processing the information with endogenous neurons. Therefore we investigated whether Neurogenin2 and Mash-1 can differentiate transplanted neural stem cells, which are thought to regulate differentiation of excitatory and inhibitory neuron respectively. This approach will allow us to reveal the formation mechanism of local excitatory and inhibitory circuits in the cerebral cortex. Additionally this method could be helpful to care the patients suffered from neurological disorders. doi:10.1016/j.neures.2009.09.794
P2-d23 Restoration of injured spinal cord by epigenetic regulation of transplanted neural stem cells Masahiko Abematsu 1,2 , Keita Tsujimura 1 , Mariko Yamano 3 , Kohno 1 , Jun Kohyama 1 , Masakazu Michiko Saito 1 , Kenji 1 2 Namihira , Setsuro Komiya , Kinichi Nakashima 1 1
Grad. Sch. Biol. Sci., NAIST, Nara, Japan; 2 Grad. Sch. Med, Kagoshima University, Kagoshima, Japan; 3 Comprehensive Rehabilitation, Osaka Prefecture University, Osaka, Japan In the injured central nervous system (CNS), restoration of damaged neural networks is severely limited. Here, using a mouse model of spinal cord injury (SCI), we show that transplanting neural stem cells (NSCs) and administering VPA to injured mice synergistically enhances the functional recovery of their hindlimbs. Neuronal but not glial differentiation of transplanted NSCs was promoted in VPA-treated mice. Anterograde corticospinal tract tracing revealed that transplant-derived neurons partially restored the broken neuronal circuits, most likely in a “relay” manner. Ablation of the transplanted cells abolished the recovery of hindlimb motor function, indicating that transplanted cells contributed directly to the improvement of motor function. These data raise the possibility that epigenetic regulation in transplanted neural stem cells can be exploited to provide treatment for SCI. doi:10.1016/j.neures.2009.09.795
P2-d24 Temporal release of GABA from anion channel on Bergmann glia (BG) accelerates granule cell precursors (GCPs) proliferation during rat cerebellar cortex development Toshitaka Morishima 1 , Tatsuro Kumada 1 , Taku Uchida 1 , Chitoshi Takayama 3 , Sachiko Yoshida 2 , Atsuo Fukuda 1 1
Hamamatsu University Sch. Med, Japan; 2 Department Material Sci., Toyohashi University Technol, Japan; 3 Department Anat. 2, University the Ryukyu Fac. Med, Japan Immature GCPs express GABAA receptor (GABAA R) in external granule layer(EGL). However its function is unknown. Therefore we have evaluated spatio-temporal distribution of GABA by means of the GABA imaging that we have developed. Interestingly, extracellular GABA was observed in the outer EGL at P3 but disappeared at P8. This extracellular GABA was affected by VGAT and anion channel inhibitor. Patch-clamp recordings revealed that GABAA R of GCPs in outer EGL were tonically activated by the ambient GABA. Immunohistological analysis revealed that GAD, GABA and VGAT were localized on BG at P3. To elucidate the role of GABAA R in the GCPs proliferation at P3, we chronically inhibited GABAA R. The proliferation rate of GCPs and CyclinD1 expression were significantly reduced in outer EGL without cell death. Thus GABA released from anion channel on BG may accelerate the proliferation of GCPs in oEGL during cerebellar development. doi:10.1016/j.neures.2009.09.796
P2-e01 Neural-specific inactivation of ERK2 in mice causes abnormal laminar formation in the cerebral cortex Osamu Imamura 1 , Yasushi Satoh 1 , Jacques Pouyssegur 2 , Shogo Endo 3 , Kunio Takishima 1 1
Dept of Biochem., Natl. Def. Med. Coll; 2 IBDC, CNRS UMR 6543, University of Nice, France; 3 OIST, Unit for Mol. Neurobiol. of Learning & Memory
The formation of the distinct laminar structure in the developing cerebral cortex requires precisely regulated generation and migration of neurons and glia that arise from neural stem/progenitor cells (NPCs) in ventricular zone (VZ). We have previously reported the generation of neural-specific ERK2 knockout mice to address the ERK2 function in NPCs and demonstrated a diminished proliferation of NPCs in the embryonic VZ from E15.5 onwards. Here, we demonstrate that ERK2, but not ERK1, plays a role in proper cortical laminar formation. We observed that the deeper layer neurons were mostly preserved, whereas the upper layer neurons were reduced in
ERK2 knockout mice. Furthermore, we found that the formation of oligodendrocytes in the ERK2 knockout cortex is compromised. Our results demonstrate that ERK2 is required for maintaining sufficient numbers of NPCs during corticogenesis and essential for proper lamination of the cerebral cortex. doi:10.1016/j.neures.2009.09.797
P2-e02 ROCK2 regulates bFGF-induced proliferation of SH-SY5Y cells through GSK3/-catenin pathway Shuken Boku, Shin Nakagawa, Katsuji Suzuki, Mai Kihara, Takeshi Inoue, Tsukasa Koyama Dept Psychiat, Hokkaido Univ, Sapporo, Japan Increased adult neurogenesis might be beneficial to the treatment of psychiatric disorders. bFGF is essential to proliferation of neural precursors and GSK3/-catenin pathway play a role in it. To elucidate its mechanism, using SH-SY5Y cells as a model of neural precursors, we investigated the role of Rho-associated coiled-coil kinases (ROCKs) in bFGF-induced cell proliferation. Y27632, an inhibitor of ROCKs, decreased proliferation. Lithium (Li), an inhibitor of GSK3, recovered proliferation and quercetin (Que), an inhibitor of -catenin pathway, reversed it. Both nuclear catenin and the expression of cyclin D1 were altered by bFGF, Li and Que in parallel with proliferation. In addition, bFGF inactivated GSK3 through increasing Ser9 phosphorylation and Y27632 activated it through increasing Tyr216 phosphorylation. Moreover, ROCK2, not ROCK1, was involved in bFGF-induced proliferation. Therefore, ROCK2 may regulate bFGF-increased proliferation of SH-SY5Y cells through GSK3/-catenin pathway. doi:10.1016/j.neures.2009.09.798
P2-e03 Role of n-3 PUFAs in the brain development Tatsuro Yamamoto, Naomichi Nishimura Dept of Nutr Sci, Faculty of Health and Welfare Sci, Nayoro City Univ, Hokkaido, Japan Fat is important to provide essential fatty acid. N−3 PUFAs is necessary to DHA biosynthesis at in vivo. DHA is an essential component for brain function. The fatty acid absorption tends to be affected by the lipid composition of food. To understand the effect of maternal intake of n−3 PUFAs on the brain development, we crossed the female rats fed an n−3 PUFAs deficient diet, and examined morphological studies in cerebral cortex. We injected BrdU into female at E17.5, and BrdU positive cells were detected at E19.5. As a result, the number of BrdU positive cells was increased in the n−3 deficient fetus compared to the control. Moreover, we studied cytoarchitecture in n−3 deficient cerebral cortex at P21. Interestingly, cytoarchitectures of n−3 deficient pups was similar to that in the control. These results suggest that disorder of cell proliferation during the embryonic period is improved during the weaning period. Altered development of the cerebral cortex would affect its refined structure. These data suggest that appropriate intake of n−3 PUFAs is important for brain development. doi:10.1016/j.neures.2009.09.799
P2-e04 Heterogeneity of neural progenitor cells during neocortical development as revealed by single cell gene expression profiles Ayano Kawaguchi 1 , Mayumi Okamoto 1 , Fumio Matsuzaki 2 1
Dep Anatomy and Cell Biol, Nagoya Univ Grad Sch of Med, Japan; for Cell Asymmetry, CDB, RIKEN, Japan
2
Lab
Mammalian neocortical development involves highly dynamic progenitor cell behavior and heterogeneous cell lineage. To explore the diversity of neural progenitor cells, we analyzed the gene expression profiles of a large number of singly isolated neocortical progenitor cells at three different developmental stages (mouse embryonic day E11, E14,E16). By using a method for single-cell cDNA amplification, we were able to perform quantitative high density oligonucleotide microarray (GeneChip, Affymetrix) analyses of the cDNA from individual cells. We systematically analyzed the obtained single-cell-cDNA profiles (N = 30 for E11, N = 70 for E14, N = 28 for E16). By cluster analysis and in situ hybridization, we have identified a set of genes that define the progenitor subclasses during cortical development. We are currently investigating the molecular mechanisms that regulate the cell lineages of these progenitor subclasses. doi:10.1016/j.neures.2009.09.800
P2-d16 Expression of cyclin E in the cerebral and cerebellar cortices Yayoi Ikeda 1 , Yuko Matsunaga 2 , Masa-Aki Ikeda 2 1 Dept Histol & Cell Biol, Yokohama City Univ School of Med, Yokohama, Japan; 2 Sect Molecular Embryol, Tokyo Med & Dent Univ, Tokyo, Japan
Cyclin E is a member of G1 cyclins which promote the progression of the cell cycle. The present study examined cyclin E expression in the developing and adult central nervous system (CNS) in mice and rats. The Western blot, in situ hybridization and immunohistochemistry demonstrated that mRNA and protein of cyclin E were expressed in both proliferating and non-proliferation regions during development of the cerebral cortex and the cerebellum. Double-label immunofluorescence for cyclin E and cyclin-dependent kinase 5 (cdk5) showed that both of the two proteins were expressed by pyramidal neurons and Purkinje cells in the cerebral and cerebellar coteces, respectively. Finally, immunoprecipitation analysis reveals that cyclin E binds to cdk5 in the cytoplasm of postnatal and adult cerebral cortex with no detectable kinase activity. The fact that cyclin E is expressed in terminally differentiated neurons predict an alternative function(s) in addition to controlling the cell cycle. doi:10.1016/j.neures.2009.09.788
P2-d17 Differential Islet-1 expression among spinal motorneurons in prenatal mouse Kengo Funakoshi, Da-Young Han, Miki Kobayashi, Masato Nakano, Yoshitoshi Atobe, Tetsuo Kadota Dept Neuroana, Yokohama City Univ, Yokohama, Japan In mouse, neurons projecting to the urethral sphincter and ischiocavernosus muscles form the dorsolateral nucleus (DL) at the caudal lumbar levels, whereas neurons projecting to the limb and hip joint muscles comprise the retrodorsolateral and ventral nucleus, as well as the DL at the rostral lumbar levels. The present study showed that the expression pattern of a LIM homeodomein protein Islet-1 was different among motoneuronal groups at the prenatal stage of mouse. The highest expression was observed in the caudal DL. The high expression was also observed in the medial part of the rostral DL, whereas there was little expression in the lateral part of the rostral DL. These findings provide evidence that the DL neurons corresponding to Onuf nucleus, are chemically distinct among the motoneuronal groups at the prenatal stages. This differential Islet-1 expression among the motoneuronal groups suggests that Islet-1 not only leads to a motoneuronal lineage, but also to the differentiation of motoneuronal subsets in the lumbosacral spinal cord. doi:10.1016/j.neures.2009.09.789
P2-d18 The effect of arachidonic acid and docosahexaenoic acid in neural stem/progenitor cells Numayama-Tsuruta 1,2 , Noriko Nobuyuki Sakayori 1 , Keiko Osumi 1,2,3 1 Div. of Devel. Neurosci., Tohoku University Grad. Sch. of Med., Sendai, Japan; 2 CREST, JST, Kawauchi, Japan; 3 Tohoku Neuroscience Global COE, Tohoku University, Sendai, Japan
Arachidonic acid (ARA) and Docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids (PUFAs) in the brain, play crucial roles in brain development and function. We have shown that ARA promotes postnatal neurogenesis in the rat hippocampus. To further elucidate functions of these PUFAs in neurogenesis, we analyzed effects of ARA and DHA on proliferation, differentiation and survival of NSPCs at various concentrations using neurosphere assays. ARA at low concentrations tended to increase the number of neurospheres and also increase the number of Tuj1-positive cells, whereas DHA at low concentrations increased the number of neurospheres. Thus, ARA has a tendency to promote maintenance and neuronal differentiation of NSPCs, while DHA promoted maintenance of NSPCs. These results are consistent with our previous report that ARA promotes postnatal neurogenesis in vivo. We are now investigating effects of ARA and DHA on glial differentiation. doi:10.1016/j.neures.2009.09.790
S155
P2-d19 15-deoxy-12,14 -prostaglandin J2 biphasically regulates the proliferation of mouse hippocampal neural stem cells by modulating the redox state Takashi Katura, Takahiro Moriya, Norimichi Nakahata Department Cellular signaling, Grad. Sch. Pharmaceut. Sci., Tohoku University, Japan In this study, we tried to examine the effect of 15-deoxy-12,14 -prostaglandin J2 (15d-PGJ2 ) on the proliferation of the neural stem cells (NSCs) derived from mouse hippocampus. 15d-PGJ2 showed the biphasic effects on EGF-dependent proliferation of the NSCs. An inhibitor of PPAR␥, known as a receptor for 15d-PGJ2 , failed to abolish the effects of 15d-PGJ2 . We also found that the elecrophilic carbon at 9 position is critical for the action of 15d-PGJ2 on the proliferation. The treatment with 15d-PGJ2 caused an increase in the levels of reactive oxygen species (ROS) accompanied with the decrease in endogenous glutathione (GSH) levels. Furthermore, the supplement with a GSH ethyl ester (GSH-EE) (2 mM), diminished the biphasic effects of 15d-PGJ2 . Taken together that GSH plays important roles in redox cellsignaling pathway, these results suggest that 15d-PGJ2 regulates the proliferation of NSCs via its electrophilic nature, which enables the covalent binding to molecules such as glutathione in PPAR␥-independent manner. doi:10.1016/j.neures.2009.09.791
P2-d20 Adenosine suppresses the proliferation of the embryonic mouse neural stem cells via equilibrative nucleoside transporters Yuko Suzuki, Takahiro Moriya, Takashi Katura, Norimichi Nakahata Dept Cell Signal, Grad Sch Pharm Sci, Tohoku Univ, Sendai, Japan Neural stem cells (NSCs) exhibit the properties of self-renewal and multipotential. While adenosine is known to be a modulator of neuronal function, its effects on NSCs remain unclear. We, therefore, investigated the effects of adenosine on the proliferation of the NSCs derived from embryonic mouse striatum.Using WST-8 assay and BrdU-incorporation assay, we found that adenosine inhibited cell proliferation of NSCs in time- and concentration-dependent manners. Adenosine receptors were not involved in these effects. We confirmed the NSCs expressed equilibrative nucleoside transporter (ENT)-1, which incorporates extracellular adenosine into cytosol. Furthermore, inhibitors of ENTs attenuated adenosine-induced growth inhibition. Furthermore, either an adenosine kinase inhibitor or uridine supplement reversed the growth inhibition induced by adenosine. These results suggest that intracellular AMP accumulation by adenosine kinase following adenosine uptake would induce NSC growth inhibition through uridine starvation. doi:10.1016/j.neures.2009.09.792
P2-d21 Synchronization of calcium oscillation between retinal neuroepithelial cells Masayuki Yamashita Dept Physiol 1, Nara Med Univ, Kashihara, Japan In neuroepithelium spontaneous and periodic increases in intracellular Ca2+ concentration (Ca2+ oscillation) occur synchronously between the cells and independently of extracellular Ca2+ . Thus it is supposed that Ca2+ releases from intracellular Ca2+ stores, which are formed from endoplasmic reticulum and nuclear envelope, are synchronized between the cells. Recently, synchronous oscillatory changes in the membrane potential of intracellular Ca2+ sores were recorded using an organellespecific voltage-sensitive dye [DiOC5 (3)]. High-speed fluorescence measurement of store membrane potential using a photomultiplier surprisingly revealed that the synchronous oscillatory change in the store potential was periodic repeats of a burst of high-frequency voltage fluctuations. The burst coincided with a Ca2+ increase. It is suggested that the synchronization of Ca2+ releases is mediated by the highfrequency fluctuation in the store potential. Close apposition of the outer nuclear membrane and plasma membrane in a pseudostratified epithelial structure would allow capacitative electrical coupling between the cells. doi:10.1016/j.neures.2009.09.793
P2-d22 Analysis of formation mechanism for excitatory and inhibitory neural circuits Mitsunori Arai, Shoji Komai Nara Institute of Science and Technology, Japan Many researchers have studied about neural stem cell and its transplantation into several brain regions to treat neurological disorders, such as Parkinson’s disease, ischemia, and spinal cord injuries, caused by damage or loss of neurons and glial cells. These therapies indeed have been helped to recover of motor functions such as
S156
Abstracts
dyskinesia. However, little is known about recovery and regeneration mechanism of optimal neural circuits developing by precise neural activities and how neural activities form the appropriate local neural circuits with transplanted cells correctly processing the information with endogenous neurons. Therefore we investigated whether Neurogenin2 and Mash-1 can differentiate transplanted neural stem cells, which are thought to regulate differentiation of excitatory and inhibitory neuron respectively. This approach will allow us to reveal the formation mechanism of local excitatory and inhibitory circuits in the cerebral cortex. Additionally this method could be helpful to care the patients suffered from neurological disorders. doi:10.1016/j.neures.2009.09.794
P2-d23 Restoration of injured spinal cord by epigenetic regulation of transplanted neural stem cells Masahiko Abematsu 1,2 , Keita Tsujimura 1 , Mariko Yamano 3 , Kohno 1 , Jun Kohyama 1 , Masakazu Michiko Saito 1 , Kenji 1 2 Namihira , Setsuro Komiya , Kinichi Nakashima 1 1
Grad. Sch. Biol. Sci., NAIST, Nara, Japan; 2 Grad. Sch. Med, Kagoshima University, Kagoshima, Japan; 3 Comprehensive Rehabilitation, Osaka Prefecture University, Osaka, Japan In the injured central nervous system (CNS), restoration of damaged neural networks is severely limited. Here, using a mouse model of spinal cord injury (SCI), we show that transplanting neural stem cells (NSCs) and administering VPA to injured mice synergistically enhances the functional recovery of their hindlimbs. Neuronal but not glial differentiation of transplanted NSCs was promoted in VPA-treated mice. Anterograde corticospinal tract tracing revealed that transplant-derived neurons partially restored the broken neuronal circuits, most likely in a “relay” manner. Ablation of the transplanted cells abolished the recovery of hindlimb motor function, indicating that transplanted cells contributed directly to the improvement of motor function. These data raise the possibility that epigenetic regulation in transplanted neural stem cells can be exploited to provide treatment for SCI. doi:10.1016/j.neures.2009.09.795
P2-d24 Temporal release of GABA from anion channel on Bergmann glia (BG) accelerates granule cell precursors (GCPs) proliferation during rat cerebellar cortex development Toshitaka Morishima 1 , Tatsuro Kumada 1 , Taku Uchida 1 , Chitoshi Takayama 3 , Sachiko Yoshida 2 , Atsuo Fukuda 1 1
Hamamatsu University Sch. Med, Japan; 2 Department Material Sci., Toyohashi University Technol, Japan; 3 Department Anat. 2, University the Ryukyu Fac. Med, Japan Immature GCPs express GABAA receptor (GABAA R) in external granule layer(EGL). However its function is unknown. Therefore we have evaluated spatio-temporal distribution of GABA by means of the GABA imaging that we have developed. Interestingly, extracellular GABA was observed in the outer EGL at P3 but disappeared at P8. This extracellular GABA was affected by VGAT and anion channel inhibitor. Patch-clamp recordings revealed that GABAA R of GCPs in outer EGL were tonically activated by the ambient GABA. Immunohistological analysis revealed that GAD, GABA and VGAT were localized on BG at P3. To elucidate the role of GABAA R in the GCPs proliferation at P3, we chronically inhibited GABAA R. The proliferation rate of GCPs and CyclinD1 expression were significantly reduced in outer EGL without cell death. Thus GABA released from anion channel on BG may accelerate the proliferation of GCPs in oEGL during cerebellar development. doi:10.1016/j.neures.2009.09.796
P2-e01 Neural-specific inactivation of ERK2 in mice causes abnormal laminar formation in the cerebral cortex Osamu Imamura 1 , Yasushi Satoh 1 , Jacques Pouyssegur 2 , Shogo Endo 3 , Kunio Takishima 1 1
Dept of Biochem., Natl. Def. Med. Coll; 2 IBDC, CNRS UMR 6543, University of Nice, France; 3 OIST, Unit for Mol. Neurobiol. of Learning & Memory
The formation of the distinct laminar structure in the developing cerebral cortex requires precisely regulated generation and migration of neurons and glia that arise from neural stem/progenitor cells (NPCs) in ventricular zone (VZ). We have previously reported the generation of neural-specific ERK2 knockout mice to address the ERK2 function in NPCs and demonstrated a diminished proliferation of NPCs in the embryonic VZ from E15.5 onwards. Here, we demonstrate that ERK2, but not ERK1, plays a role in proper cortical laminar formation. We observed that the deeper layer neurons were mostly preserved, whereas the upper layer neurons were reduced in
ERK2 knockout mice. Furthermore, we found that the formation of oligodendrocytes in the ERK2 knockout cortex is compromised. Our results demonstrate that ERK2 is required for maintaining sufficient numbers of NPCs during corticogenesis and essential for proper lamination of the cerebral cortex. doi:10.1016/j.neures.2009.09.797
P2-e02 ROCK2 regulates bFGF-induced proliferation of SH-SY5Y cells through GSK3/-catenin pathway Shuken Boku, Shin Nakagawa, Katsuji Suzuki, Mai Kihara, Takeshi Inoue, Tsukasa Koyama Dept Psychiat, Hokkaido Univ, Sapporo, Japan Increased adult neurogenesis might be beneficial to the treatment of psychiatric disorders. bFGF is essential to proliferation of neural precursors and GSK3/-catenin pathway play a role in it. To elucidate its mechanism, using SH-SY5Y cells as a model of neural precursors, we investigated the role of Rho-associated coiled-coil kinases (ROCKs) in bFGF-induced cell proliferation. Y27632, an inhibitor of ROCKs, decreased proliferation. Lithium (Li), an inhibitor of GSK3, recovered proliferation and quercetin (Que), an inhibitor of -catenin pathway, reversed it. Both nuclear catenin and the expression of cyclin D1 were altered by bFGF, Li and Que in parallel with proliferation. In addition, bFGF inactivated GSK3 through increasing Ser9 phosphorylation and Y27632 activated it through increasing Tyr216 phosphorylation. Moreover, ROCK2, not ROCK1, was involved in bFGF-induced proliferation. Therefore, ROCK2 may regulate bFGF-increased proliferation of SH-SY5Y cells through GSK3/-catenin pathway. doi:10.1016/j.neures.2009.09.798
P2-e03 Role of n-3 PUFAs in the brain development Tatsuro Yamamoto, Naomichi Nishimura Dept of Nutr Sci, Faculty of Health and Welfare Sci, Nayoro City Univ, Hokkaido, Japan Fat is important to provide essential fatty acid. N−3 PUFAs is necessary to DHA biosynthesis at in vivo. DHA is an essential component for brain function. The fatty acid absorption tends to be affected by the lipid composition of food. To understand the effect of maternal intake of n−3 PUFAs on the brain development, we crossed the female rats fed an n−3 PUFAs deficient diet, and examined morphological studies in cerebral cortex. We injected BrdU into female at E17.5, and BrdU positive cells were detected at E19.5. As a result, the number of BrdU positive cells was increased in the n−3 deficient fetus compared to the control. Moreover, we studied cytoarchitecture in n−3 deficient cerebral cortex at P21. Interestingly, cytoarchitectures of n−3 deficient pups was similar to that in the control. These results suggest that disorder of cell proliferation during the embryonic period is improved during the weaning period. Altered development of the cerebral cortex would affect its refined structure. These data suggest that appropriate intake of n−3 PUFAs is important for brain development. doi:10.1016/j.neures.2009.09.799
P2-e04 Heterogeneity of neural progenitor cells during neocortical development as revealed by single cell gene expression profiles Ayano Kawaguchi 1 , Mayumi Okamoto 1 , Fumio Matsuzaki 2 1
Dep Anatomy and Cell Biol, Nagoya Univ Grad Sch of Med, Japan; for Cell Asymmetry, CDB, RIKEN, Japan
2
Lab
Mammalian neocortical development involves highly dynamic progenitor cell behavior and heterogeneous cell lineage. To explore the diversity of neural progenitor cells, we analyzed the gene expression profiles of a large number of singly isolated neocortical progenitor cells at three different developmental stages (mouse embryonic day E11, E14,E16). By using a method for single-cell cDNA amplification, we were able to perform quantitative high density oligonucleotide microarray (GeneChip, Affymetrix) analyses of the cDNA from individual cells. We systematically analyzed the obtained single-cell-cDNA profiles (N = 30 for E11, N = 70 for E14, N = 28 for E16). By cluster analysis and in situ hybridization, we have identified a set of genes that define the progenitor subclasses during cortical development. We are currently investigating the molecular mechanisms that regulate the cell lineages of these progenitor subclasses. doi:10.1016/j.neures.2009.09.800