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Characterization of cells newly generated after granule cell loss in the hippocampal dentate gyrus of mice
e368
Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-e16 Ryanodine receptor regulates proliferative activity in neural stem/progenitor cells derived from subventricular zone of adult mice Tatsuo Shiba , Masanori Yoneyama, Kiyokazu Ogita Department Pharmacol., Setsunan University Subventricular zone (SVZ) of adult brain has neural stem/progenitor cells (NPCs) that generate new neurons throughout life. In the NPCs, intracellular Ca2+ is known to play a critical role in regulating different stages of early brain development and neurogenesis. To elucidate participation of Ca2+ signaling pathway in proliferation of the NPCs, we evaluated the effect of ryanodine receptor (RyR) inhibitor dantrolene on proliferative activity in the NPCs derived from the SVZ of adult mice. Cells were prepared from the SVZ of 5-week-old Std-ddY male mice and then primarily cultured in DMEM/F12 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 8 days in vitro (DIV). After replating, the cells were secondarily cultured for 5 DIV under the same conditions in the absence or presence of dantrolene for a period of 8–13 DIV. In the presence of both EGF and bFGF, marked round spheres were formed, continued to grow, and proliferate to form large neurospheres. In addition, most cells obtained from the neurospheres were immunoreactive to nestin. To determine the expression of RyR subunits in the NPCs, we performed RT-PCR analysis using total RNA prepared from the NPCs. Although there exist 3 subunits of RyR in the NPCs, RyR3 was the highest level of 3 subunits of RyR. ELISA for 5 -bromo2 -deoxyuridine revealed that a marked decrease in the proliferative activity was seen by treatment with dantrolene in a dose-dependent manner (2, 10, 20 or 50 M). However, dantrolene was ineffective in releasing lactate dehydrogenase into the culture medium. Moreover, treatment with dantrolene resulted in a marked reduction in STAT3 and Pax6 mRNA in NPCs. These results suggest that Ca2+ release from the endoplasmic reticulum via the RyR would positively regulate proliferative activity in NPCs of adult mouse SVZ. doi:10.1016/j.neures.2010.07.1629
P3-e17 Characterization of cells newly generated after granule cell loss in the hippocampal dentate gyrus of mice Tetsuya Tanaka , Tatsuo Shiba, Masanori Yoneyama, Kiyokazu Ogita Department of Pharmacol., Setsunan University Various neurological injuries are widely recognized as promoting endogenous neurogenesis in hippocampal dentate gyrus of adulthood. Our previous studies demonstrated that the granule cells in the hippocampal dentate gyrus are injured and disappeared by treatment with trimethyltin chloride (TMT), with being regenerated in the dentate granule cell layer (GCL) after neuronal loss. To date, it has been unclear what kind of cells contributes to regeneration of the GCL. In this study, we determined characteristics of newly generated cells in the dentate gyrus after TMT treatment. Male ddY mice at 5-week-old age were given TMT (2.8 mg/kg, i.p.) to prepare slices for immunohistochemical analyses using antibody against glial fibrillary acidic protein (GFAP, astrocyte and neural stem cell marker), brain lipid binding protein (BLBP, radial glial marker), Iba1 (microglia marker) or Ki67 (cell cycle marker). Cells positive for GFAP, BLBP, Iba1 or Ki67 markedly increased in the subgranular zone (SGZ)/GCL, molecular layer, and hilus on days 3–7 (regeneration stage) after TMT treatment. Double immunostaining revealed that the majority of cells positive for GFAP or BLBP had Ki67 immunoreactivity in the SGZ/GCL on day 3 after TMT treatment. In the molecular layer, however, cells positive for GFAP or BLBP had little Ki67 immunoreactivity during the regeneration stage after TMT treatment. In addition, most Iba1-positive cells failed to express Ki67 in the SGL/GCL and molecular layer during the regeneration stage after TMT treatment. In the hilus, cells positive for GFAP, BLBP or Iba1 had little Ki67 immunoreactivity during the regeneration stage after TMT treatment. These results suggest that enhanced proliferation of radial glial cells, but not astrocytes and microglia, contributes to regenerate the GCL after dentate granule cell loss induced by TMT treatment. doi:10.1016/j.neures.2010.07.1630
P3-e18 Activity-dependent regulation of adult hippocampal neurogenesis Tomomasa Kato , Rokuya Nochi, Hiroshi Kuribayashi, Kentaro Ishinabe, Yoshie Itou, Satoshi Fukuda, Tatsuhiro Hisatsune Department of Integrated Bioscience, Graduate School of Frontier Sciences, The University of Tokyo, Japan Neurogenesis occurs in the dentte gyrus of adult hippocampus. Regulation of adult hippocampal neurogenesis is one of key questions in functional and structural plasticity of the adult brain. Simultaneous enhancement of neural circuit activity and neural stem cell (NSC) proliferation have been suggested after ischemic stroke, epileptic seizure, and the administration of NMDA receptor antagonist. In a previous study, we found that elevated proliferation of NSCs is inbibited by blocking the elevation of neural circuit activity by the administration of a GABAergic allosteric modulator, diazepam. These observations may imply the causal relationship between the increase on the proliferation of NSCs and the enhancement of neural circuit activity. In this study, we tried to elucidate the molecular mechanism underlying this putative causal relationship. By means of brain slice calcium imaging for NSC (Itou et al., Hippocampus, online); we detected the robust calcium response in NSCs after the electric stimulation of perforant pathway by a theta-burst protocol. This calcium response of NSC was significantly blocked by ATPreceptor antagonist, suramin (100 M), and was often suppressed by mGluR5 antagonist, MPEP (10 M). The puffing application of ATP evoked the calcium response in all NSCs, but the application of mGluR5 agonist (CHPG or tADA) often induced the response of NSCs. These results indicate the pivotal role of purinergic system in the activity-dependent stimulation of NSCs in the adult dentate gyrus. doi:10.1016/j.neures.2010.07.1631
P3-e19 Regulation of survival and death of adult-born neurons in the local area of the olfactory bulb by local sensory input Daisuke Mochimaru 1 , Ko Kobayakawa 3 , Reiko Kobayakawa 3 , Hitoshi Sakano 2 , Kensaku Mori 1 , Masahiro Yamaguchi 1 1
Dept. of Physiology, the University of Tokyo, Tokyo, Japan 2 Dept. of Biophysics and Biochemistry, the University of Tokyo, Tokyo, Japan 3 Dept. of Functional Neuroscience, Osaka Bioscience Institute, Osaka, Japan In the olfactory bulb (OB) of adult mammalian brain, new granule cells (GCs) are continually generated. Among new GCs, nearly a half are integrated into OB circuitry while the other half are eliminated by apoptotic cell death. Although olfactory sensory deprivation in the entire OB by nostril occlusion reduces the survival rate of new GCs in the entire OB, it is not well understood whether the survival of new GCs is regulated locally by local olfactory sensory input. In this study, we examined the fate of new GCs in mice depleted with olfactory sensory neurons in the dorsal zone (D-zone) of the olfactory epithelium (D mouse), in which sensory input to the D-zone of the OB was selectively deprived while sensory input to the ventral zone (V-zone) remained intact. A larger number of apoptotic GCs were observed in the D-zone compared to V-zone, and long-term survival of new GCs was considerably reduced in the D-zone compared to V-zone. These results indicated that enhanced cell death of new GCs in the local sensory input-deprived area leads to the reduction in their survival in the local area. Morphological analysis of virus-labeled new GCs revealed that new GCs in the D-zone had decreased spine density in the external plexiform layer compared to those in the V-zone, suggesting that local olfactory sensory inputs also regulate the morphological maturation of new GCs in the local area doi:10.1016/j.neures.2010.07.1632
P3-e20 Analysis of newly generated neurons in the accessory olfactory bulb Masayuki Sakamoto 1,2,3 Yamaguchi 5 , Kensaku Mori 5
, Itaru
Imayoshi 1,3,4 , Masahiro
1
Growth Regulation, Institute for Virus Research, Kyoto University Graduate School of Biostudies, Kyoto University, Japan 3 Japan Society of Technology Agency (CREST) 4 Japan Society of Technology Agency (PRESTO) 5 Graduate School of Medicine, University of Tokyo, Japan 2
Neurogenesis occurs continuously in two regions of adult mammals, the subventricular zone (SVZ) of the lateral ventricles (LV) and of subgranular zone (SGZ) of hippocampal dentate gyrus (DG). New neurons born in the SVZ pro-
Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-e16 Ryanodine receptor regulates proliferative activity in neural stem/progenitor cells derived from subventricular zone of adult mice Tatsuo Shiba , Masanori Yoneyama, Kiyokazu Ogita Department Pharmacol., Setsunan University Subventricular zone (SVZ) of adult brain has neural stem/progenitor cells (NPCs) that generate new neurons throughout life. In the NPCs, intracellular Ca2+ is known to play a critical role in regulating different stages of early brain development and neurogenesis. To elucidate participation of Ca2+ signaling pathway in proliferation of the NPCs, we evaluated the effect of ryanodine receptor (RyR) inhibitor dantrolene on proliferative activity in the NPCs derived from the SVZ of adult mice. Cells were prepared from the SVZ of 5-week-old Std-ddY male mice and then primarily cultured in DMEM/F12 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 8 days in vitro (DIV). After replating, the cells were secondarily cultured for 5 DIV under the same conditions in the absence or presence of dantrolene for a period of 8–13 DIV. In the presence of both EGF and bFGF, marked round spheres were formed, continued to grow, and proliferate to form large neurospheres. In addition, most cells obtained from the neurospheres were immunoreactive to nestin. To determine the expression of RyR subunits in the NPCs, we performed RT-PCR analysis using total RNA prepared from the NPCs. Although there exist 3 subunits of RyR in the NPCs, RyR3 was the highest level of 3 subunits of RyR. ELISA for 5 -bromo2 -deoxyuridine revealed that a marked decrease in the proliferative activity was seen by treatment with dantrolene in a dose-dependent manner (2, 10, 20 or 50 M). However, dantrolene was ineffective in releasing lactate dehydrogenase into the culture medium. Moreover, treatment with dantrolene resulted in a marked reduction in STAT3 and Pax6 mRNA in NPCs. These results suggest that Ca2+ release from the endoplasmic reticulum via the RyR would positively regulate proliferative activity in NPCs of adult mouse SVZ. doi:10.1016/j.neures.2010.07.1629
P3-e17 Characterization of cells newly generated after granule cell loss in the hippocampal dentate gyrus of mice Tetsuya Tanaka , Tatsuo Shiba, Masanori Yoneyama, Kiyokazu Ogita Department of Pharmacol., Setsunan University Various neurological injuries are widely recognized as promoting endogenous neurogenesis in hippocampal dentate gyrus of adulthood. Our previous studies demonstrated that the granule cells in the hippocampal dentate gyrus are injured and disappeared by treatment with trimethyltin chloride (TMT), with being regenerated in the dentate granule cell layer (GCL) after neuronal loss. To date, it has been unclear what kind of cells contributes to regeneration of the GCL. In this study, we determined characteristics of newly generated cells in the dentate gyrus after TMT treatment. Male ddY mice at 5-week-old age were given TMT (2.8 mg/kg, i.p.) to prepare slices for immunohistochemical analyses using antibody against glial fibrillary acidic protein (GFAP, astrocyte and neural stem cell marker), brain lipid binding protein (BLBP, radial glial marker), Iba1 (microglia marker) or Ki67 (cell cycle marker). Cells positive for GFAP, BLBP, Iba1 or Ki67 markedly increased in the subgranular zone (SGZ)/GCL, molecular layer, and hilus on days 3–7 (regeneration stage) after TMT treatment. Double immunostaining revealed that the majority of cells positive for GFAP or BLBP had Ki67 immunoreactivity in the SGZ/GCL on day 3 after TMT treatment. In the molecular layer, however, cells positive for GFAP or BLBP had little Ki67 immunoreactivity during the regeneration stage after TMT treatment. In addition, most Iba1-positive cells failed to express Ki67 in the SGL/GCL and molecular layer during the regeneration stage after TMT treatment. In the hilus, cells positive for GFAP, BLBP or Iba1 had little Ki67 immunoreactivity during the regeneration stage after TMT treatment. These results suggest that enhanced proliferation of radial glial cells, but not astrocytes and microglia, contributes to regenerate the GCL after dentate granule cell loss induced by TMT treatment. doi:10.1016/j.neures.2010.07.1630
P3-e18 Activity-dependent regulation of adult hippocampal neurogenesis Tomomasa Kato , Rokuya Nochi, Hiroshi Kuribayashi, Kentaro Ishinabe, Yoshie Itou, Satoshi Fukuda, Tatsuhiro Hisatsune Department of Integrated Bioscience, Graduate School of Frontier Sciences, The University of Tokyo, Japan Neurogenesis occurs in the dentte gyrus of adult hippocampus. Regulation of adult hippocampal neurogenesis is one of key questions in functional and structural plasticity of the adult brain. Simultaneous enhancement of neural circuit activity and neural stem cell (NSC) proliferation have been suggested after ischemic stroke, epileptic seizure, and the administration of NMDA receptor antagonist. In a previous study, we found that elevated proliferation of NSCs is inbibited by blocking the elevation of neural circuit activity by the administration of a GABAergic allosteric modulator, diazepam. These observations may imply the causal relationship between the increase on the proliferation of NSCs and the enhancement of neural circuit activity. In this study, we tried to elucidate the molecular mechanism underlying this putative causal relationship. By means of brain slice calcium imaging for NSC (Itou et al., Hippocampus, online); we detected the robust calcium response in NSCs after the electric stimulation of perforant pathway by a theta-burst protocol. This calcium response of NSC was significantly blocked by ATPreceptor antagonist, suramin (100 M), and was often suppressed by mGluR5 antagonist, MPEP (10 M). The puffing application of ATP evoked the calcium response in all NSCs, but the application of mGluR5 agonist (CHPG or tADA) often induced the response of NSCs. These results indicate the pivotal role of purinergic system in the activity-dependent stimulation of NSCs in the adult dentate gyrus. doi:10.1016/j.neures.2010.07.1631
P3-e19 Regulation of survival and death of adult-born neurons in the local area of the olfactory bulb by local sensory input Daisuke Mochimaru 1 , Ko Kobayakawa 3 , Reiko Kobayakawa 3 , Hitoshi Sakano 2 , Kensaku Mori 1 , Masahiro Yamaguchi 1 1
Dept. of Physiology, the University of Tokyo, Tokyo, Japan 2 Dept. of Biophysics and Biochemistry, the University of Tokyo, Tokyo, Japan 3 Dept. of Functional Neuroscience, Osaka Bioscience Institute, Osaka, Japan In the olfactory bulb (OB) of adult mammalian brain, new granule cells (GCs) are continually generated. Among new GCs, nearly a half are integrated into OB circuitry while the other half are eliminated by apoptotic cell death. Although olfactory sensory deprivation in the entire OB by nostril occlusion reduces the survival rate of new GCs in the entire OB, it is not well understood whether the survival of new GCs is regulated locally by local olfactory sensory input. In this study, we examined the fate of new GCs in mice depleted with olfactory sensory neurons in the dorsal zone (D-zone) of the olfactory epithelium (D mouse), in which sensory input to the D-zone of the OB was selectively deprived while sensory input to the ventral zone (V-zone) remained intact. A larger number of apoptotic GCs were observed in the D-zone compared to V-zone, and long-term survival of new GCs was considerably reduced in the D-zone compared to V-zone. These results indicated that enhanced cell death of new GCs in the local sensory input-deprived area leads to the reduction in their survival in the local area. Morphological analysis of virus-labeled new GCs revealed that new GCs in the D-zone had decreased spine density in the external plexiform layer compared to those in the V-zone, suggesting that local olfactory sensory inputs also regulate the morphological maturation of new GCs in the local area doi:10.1016/j.neures.2010.07.1632
P3-e20 Analysis of newly generated neurons in the accessory olfactory bulb Masayuki Sakamoto 1,2,3 Yamaguchi 5 , Kensaku Mori 5
, Itaru
Imayoshi 1,3,4 , Masahiro
1
Growth Regulation, Institute for Virus Research, Kyoto University Graduate School of Biostudies, Kyoto University, Japan 3 Japan Society of Technology Agency (CREST) 4 Japan Society of Technology Agency (PRESTO) 5 Graduate School of Medicine, University of Tokyo, Japan 2
Neurogenesis occurs continuously in two regions of adult mammals, the subventricular zone (SVZ) of the lateral ventricles (LV) and of subgranular zone (SGZ) of hippocampal dentate gyrus (DG). New neurons born in the SVZ pro-