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Network confluence in a rat neuronal culture
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Abstracts / Neuroscience Research 71S (2011) e108–e415
P2-f14 Involvement of Olig2 in the neural circuit formation in the fetal mouse forebrain Katsuhiko Ono 1,5 , Carlos M. Parras 2 , Hirohide Takebayashi 3 , Kenji Shimamura 4 , Hitoshi Gotoh 1,5 , Kazuhiro Ikenaka 5 1
Dept. of Biol., Kyoto Pref. Univ. Medicine 2 Institute of the Brain and Spinal Cord (ICM), Inserm-UPMC, Paris, France 3 Dept. of Morphological Neural Science, Grad. Sch. of Medical Sciences, Kumamoto Univ., Kumamoto, Japan 4 Dept. of Brain Morphogenesis, Inst. Mol. Embryol. Genetics, Kumamoto Univ., Kumamoto, Japan 5 Div. of Mol. Neurobiol. Bioinfo., NIPS, Okazaki, Japan Olig2 is well-known to be a transcription factor essential for oligodendrocyte and motor neuron generation in the spinal cord. In spite of its wide distribution throughout the CNS, Olig2 function in the other regions has not been uncovered well yet. To study functional implication of Olig2 in the forebrain morphogenesis, we first performed microarray analysis of Olig2 deficient basal forebrain, which showed that several axon guidance molecules and their receptors were up- or down-regulated in the Olig2 mutant mouse. Then, spatio-temporal expression pattern of these genes was examined in the fetal mouse forebrain by in situ hybridization. Among axon guidance molecules examined, EphA3 expression was altered in the diencephalon. Axonal architecture, demonstrated by neurofilament immunohistochemistry, was also malformed in this region. The present results suggest that Olig2 is involved in neural circuit formation in the forebrain through regulation of expression of axon guidance molecules. Research fund: KAKENHI (22500295). doi:10.1016/j.neures.2011.07.574
P2-f15 Morphometric analysis of late-spiking pyramidal neurons in layer 2 of the rat retrosplenial cortex Tohru Kurotani 1,2 , Kazuhisa Sakai 3 , Noritaka Ichinohe 2,3 , Kazuo Okanoya 1 , Kathleen S. Rockland 2 1
JST ERATO Okanoya Emotional Information Project 2 Lab. for Cortical Organization and Systematics, RIKEN Brain Science Institute, Wako, Japan 3 Department of Ultrastructural Research, National Institute of Neuroscience, Kodaira, Japan Callosally projecting neurons in layer 2 (L2) of granular retrosplenial (GRS) cortex of the rat form unusually distinct apical dendritic bundles and show a distinct “late-spiking” (LS) firing pattern [i.e., the initiation of the first spike was delayed 500–1000 ms from the onset of supra-threshold step current injection]. More than 90% of GRS L2 neurons turned out to be LS neurons that show distinct morphology in the apical and basal dendrite branching pattern from that observed in L2 pyramids in sensory neocortex such as S1 barrel field. For example, the total length of apical dendrite for LS neurons was shorter than that for S1 L2 pyramids (location of cell soma < 100 m below L1/L2 border, 1130 ± 64.3 m n = 22, vs. 2200 ± 352 m n = 8, P < 0.001 by one-way ANOVA). The number of branching point of apical dendrite was also smaller in LS than in S1 pyramids (16.7 ± 1.14 n = 22, vs. 24.6 ± 3.06 n = 8, p < 0.001) The Sholl analysis further revealed that LS neurons had few branching points (intersection < 3) on the proximal (<100 m) apical dendrite. The intersection reached its maximum value (7.4 ± 3.8, n = 22) at 170 m distant from the soma. On the other hand, in the S1 L2 pyramids, the dendrite intersection showed the peak values (10–14) at the proximal (40–80 m from the soma). These findings suggest that GRS L2 pyramidal neurons may have distinct information processing properties compared with L2 neocortical pyramidal neurons. It will be important to determine how this feature relates to the modular and functional properties of the GRS. Research fund: KAKENHI (22500370). doi:10.1016/j.neures.2011.07.575
P2-f16 Network confluence in a rat neuronal culture Daisuke Ito , Takumi Komatsu, Akira Shirai, Kazutoshi Gohara Div. Appl. Phys., Fac. Eng., Hokkaido Univ., Sapporo, Japan Dissociated culture in vitro is useful tool to study the development and maturation of neuronal networks. Previous studies in vitro have mainly focused on the early development of the neuronal components such as axonal elongation and synaptic formation. However, there have been few studies about the changes of the neuronal components and the electrical activity during long-term culture period. In order to investigate the long-term development and maturation of the network, we cultured rat cortical cells for 2 months. For the measurement of the electrical activity, we used multi-electrode arrays. The network activity (firing rate and synchronized burst rate) showed an
increase in the firing and burst rate within 3 weeks, and then became saturated. This activity lasted for up to 2 months. To clarify the developmental change of the network size, immunofluorescence staining of neurons was performed using antibody against microtubule associated protein 2 (MAP2). Fluorescence observation revealed that the number of neurons decreased gradually up to 1 month, and then showed constant value until 2 months. In addition, we investigated the changes of excitatory and inhibitory synaptic densities using antibodies against vesicular glutamate transporter 1 (VGluT1) and vesicular transporter of ␥-amino-butyric acid (VGAT). Both excitatory and inhibitory synaptic densities increased gradually along culture ages up to 1 month, whereas the densities became saturated and did not increase until 2 months. These results suggest that the cortical neurons became network confluence during long-term culture after the initial construction of the network. Research fund: KAKENHI (20240023, 21650049). doi:10.1016/j.neures.2011.07.576
P2-f17 Hunting for genes that regulate remodeling and lifelong maintenance of dendritic arbors Kohei Shimono , Takafumi Nomura, Tadao Usui, Tadashi Uemura Grad. Sch. of Biostudies, Kyoto Univ., Kyoto, Japan Neurons develop distinctive dendritic morphologies to receive and process sensory or synaptic inputs. Dendritic arbors that are formed in early development are often reorganized and such arbors of many neuronal types are possibly maintained throughout animal life. To genetically investigate underlying mechanisms of this remodeling and maintenance in vivo, we have employed dendritic arborization (da) neurons, which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis, as a model system. We first identified da neurons in the adult Drosophila abdomen and then clarified developmental basis of these adult neurons by tracing origins of those cells back to the larval stage. We and others also showed that the dendritic arbor of one da neuron, v’ada, exhibited prominent radial-to-lattice transformation in one day after eclosion, and the resultant lattice-shaped arbor persisted throughout adult life. To conduct a MARCM screening efficiently, we expressed FLP recombinase in sensory organ precursors (SOPs). By using this “SOP-FLP” system, we isolated several mutants that displayed defects in the remodeling and/or the life-long maintenance of dendritic arbors. To identify causative genes for these mutants, we have started employing a next-generation sequencing technique to directly compare whole-genomic sequences of several mutants with each other, in addition to conventional mapping methods. In this poster, we will discuss our on-going screening and mapping. Research fund: 10J06191. doi:10.1016/j.neures.2011.07.577
P2-f18 High intensity single cell labeling reveals precise developmental processes of barrel neuron dendrites in somatosensory cortex of neonatal mice Hidenobu Mizuno 1,2 , Yoshikazu M. Saito 3 , Shigeyoshi Itohara 3 , Takuji Iwasato 1,2 1
Div. Neurogenetics, National Institute of Genetics, Mishima 2 Dept. Genetics, The Graduate University for Advances Studies (SOKENDAI), Mishima 3 Lab. Behavioral Genetics, RIKEN BSI, Wako Information processing in the cerebral cortex relies on precise neuronal wiring. Many lines of evidence suggest that neuronal activity is involved in the refinement of neuronal connections. In the mouse somatosensory cortex, cortical barrels are stereotypically arranged in the layer 4, corresponding to one-to-one whiskers on the snout. Each cortical barrel is composed of a cell dense barrel wall and a cell sparse barrel hollow. Thalamocortical axon (TCA) terminals cluster within the barrel hollow. Layer 4 barrel neurons are located around the edge of the barrel, extend their dendrites asymmetrically toward the barrel hollow and make synapses with TCAs. These precise neuronal connections are established during early postnatal period in a whisker-dependent manner. However, when barrel neuron dendrites acquire their orientation bias and what mechanisms regulate this process are not well understood. To address these points, we developed methodology which allows us to characterize dendritic features of individual barrel neurons in correspondence with terminal clusters of TCAs. We achieved sparse and highly intensified cell labeling of barrel neurons using modified Cre/loxP-mediated RFP expression by in vivo electroporation. We visualized TCA clusters by generating a new transgenic mouse line which expresses GFP specifically in TCAs. By combining these two systems and using confo-
Abstracts / Neuroscience Research 71S (2011) e108–e415
P2-f14 Involvement of Olig2 in the neural circuit formation in the fetal mouse forebrain Katsuhiko Ono 1,5 , Carlos M. Parras 2 , Hirohide Takebayashi 3 , Kenji Shimamura 4 , Hitoshi Gotoh 1,5 , Kazuhiro Ikenaka 5 1
Dept. of Biol., Kyoto Pref. Univ. Medicine 2 Institute of the Brain and Spinal Cord (ICM), Inserm-UPMC, Paris, France 3 Dept. of Morphological Neural Science, Grad. Sch. of Medical Sciences, Kumamoto Univ., Kumamoto, Japan 4 Dept. of Brain Morphogenesis, Inst. Mol. Embryol. Genetics, Kumamoto Univ., Kumamoto, Japan 5 Div. of Mol. Neurobiol. Bioinfo., NIPS, Okazaki, Japan Olig2 is well-known to be a transcription factor essential for oligodendrocyte and motor neuron generation in the spinal cord. In spite of its wide distribution throughout the CNS, Olig2 function in the other regions has not been uncovered well yet. To study functional implication of Olig2 in the forebrain morphogenesis, we first performed microarray analysis of Olig2 deficient basal forebrain, which showed that several axon guidance molecules and their receptors were up- or down-regulated in the Olig2 mutant mouse. Then, spatio-temporal expression pattern of these genes was examined in the fetal mouse forebrain by in situ hybridization. Among axon guidance molecules examined, EphA3 expression was altered in the diencephalon. Axonal architecture, demonstrated by neurofilament immunohistochemistry, was also malformed in this region. The present results suggest that Olig2 is involved in neural circuit formation in the forebrain through regulation of expression of axon guidance molecules. Research fund: KAKENHI (22500295). doi:10.1016/j.neures.2011.07.574
P2-f15 Morphometric analysis of late-spiking pyramidal neurons in layer 2 of the rat retrosplenial cortex Tohru Kurotani 1,2 , Kazuhisa Sakai 3 , Noritaka Ichinohe 2,3 , Kazuo Okanoya 1 , Kathleen S. Rockland 2 1
JST ERATO Okanoya Emotional Information Project 2 Lab. for Cortical Organization and Systematics, RIKEN Brain Science Institute, Wako, Japan 3 Department of Ultrastructural Research, National Institute of Neuroscience, Kodaira, Japan Callosally projecting neurons in layer 2 (L2) of granular retrosplenial (GRS) cortex of the rat form unusually distinct apical dendritic bundles and show a distinct “late-spiking” (LS) firing pattern [i.e., the initiation of the first spike was delayed 500–1000 ms from the onset of supra-threshold step current injection]. More than 90% of GRS L2 neurons turned out to be LS neurons that show distinct morphology in the apical and basal dendrite branching pattern from that observed in L2 pyramids in sensory neocortex such as S1 barrel field. For example, the total length of apical dendrite for LS neurons was shorter than that for S1 L2 pyramids (location of cell soma < 100 m below L1/L2 border, 1130 ± 64.3 m n = 22, vs. 2200 ± 352 m n = 8, P < 0.001 by one-way ANOVA). The number of branching point of apical dendrite was also smaller in LS than in S1 pyramids (16.7 ± 1.14 n = 22, vs. 24.6 ± 3.06 n = 8, p < 0.001) The Sholl analysis further revealed that LS neurons had few branching points (intersection < 3) on the proximal (<100 m) apical dendrite. The intersection reached its maximum value (7.4 ± 3.8, n = 22) at 170 m distant from the soma. On the other hand, in the S1 L2 pyramids, the dendrite intersection showed the peak values (10–14) at the proximal (40–80 m from the soma). These findings suggest that GRS L2 pyramidal neurons may have distinct information processing properties compared with L2 neocortical pyramidal neurons. It will be important to determine how this feature relates to the modular and functional properties of the GRS. Research fund: KAKENHI (22500370). doi:10.1016/j.neures.2011.07.575
P2-f16 Network confluence in a rat neuronal culture Daisuke Ito , Takumi Komatsu, Akira Shirai, Kazutoshi Gohara Div. Appl. Phys., Fac. Eng., Hokkaido Univ., Sapporo, Japan Dissociated culture in vitro is useful tool to study the development and maturation of neuronal networks. Previous studies in vitro have mainly focused on the early development of the neuronal components such as axonal elongation and synaptic formation. However, there have been few studies about the changes of the neuronal components and the electrical activity during long-term culture period. In order to investigate the long-term development and maturation of the network, we cultured rat cortical cells for 2 months. For the measurement of the electrical activity, we used multi-electrode arrays. The network activity (firing rate and synchronized burst rate) showed an
increase in the firing and burst rate within 3 weeks, and then became saturated. This activity lasted for up to 2 months. To clarify the developmental change of the network size, immunofluorescence staining of neurons was performed using antibody against microtubule associated protein 2 (MAP2). Fluorescence observation revealed that the number of neurons decreased gradually up to 1 month, and then showed constant value until 2 months. In addition, we investigated the changes of excitatory and inhibitory synaptic densities using antibodies against vesicular glutamate transporter 1 (VGluT1) and vesicular transporter of ␥-amino-butyric acid (VGAT). Both excitatory and inhibitory synaptic densities increased gradually along culture ages up to 1 month, whereas the densities became saturated and did not increase until 2 months. These results suggest that the cortical neurons became network confluence during long-term culture after the initial construction of the network. Research fund: KAKENHI (20240023, 21650049). doi:10.1016/j.neures.2011.07.576
P2-f17 Hunting for genes that regulate remodeling and lifelong maintenance of dendritic arbors Kohei Shimono , Takafumi Nomura, Tadao Usui, Tadashi Uemura Grad. Sch. of Biostudies, Kyoto Univ., Kyoto, Japan Neurons develop distinctive dendritic morphologies to receive and process sensory or synaptic inputs. Dendritic arbors that are formed in early development are often reorganized and such arbors of many neuronal types are possibly maintained throughout animal life. To genetically investigate underlying mechanisms of this remodeling and maintenance in vivo, we have employed dendritic arborization (da) neurons, which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis, as a model system. We first identified da neurons in the adult Drosophila abdomen and then clarified developmental basis of these adult neurons by tracing origins of those cells back to the larval stage. We and others also showed that the dendritic arbor of one da neuron, v’ada, exhibited prominent radial-to-lattice transformation in one day after eclosion, and the resultant lattice-shaped arbor persisted throughout adult life. To conduct a MARCM screening efficiently, we expressed FLP recombinase in sensory organ precursors (SOPs). By using this “SOP-FLP” system, we isolated several mutants that displayed defects in the remodeling and/or the life-long maintenance of dendritic arbors. To identify causative genes for these mutants, we have started employing a next-generation sequencing technique to directly compare whole-genomic sequences of several mutants with each other, in addition to conventional mapping methods. In this poster, we will discuss our on-going screening and mapping. Research fund: 10J06191. doi:10.1016/j.neures.2011.07.577
P2-f18 High intensity single cell labeling reveals precise developmental processes of barrel neuron dendrites in somatosensory cortex of neonatal mice Hidenobu Mizuno 1,2 , Yoshikazu M. Saito 3 , Shigeyoshi Itohara 3 , Takuji Iwasato 1,2 1
Div. Neurogenetics, National Institute of Genetics, Mishima 2 Dept. Genetics, The Graduate University for Advances Studies (SOKENDAI), Mishima 3 Lab. Behavioral Genetics, RIKEN BSI, Wako Information processing in the cerebral cortex relies on precise neuronal wiring. Many lines of evidence suggest that neuronal activity is involved in the refinement of neuronal connections. In the mouse somatosensory cortex, cortical barrels are stereotypically arranged in the layer 4, corresponding to one-to-one whiskers on the snout. Each cortical barrel is composed of a cell dense barrel wall and a cell sparse barrel hollow. Thalamocortical axon (TCA) terminals cluster within the barrel hollow. Layer 4 barrel neurons are located around the edge of the barrel, extend their dendrites asymmetrically toward the barrel hollow and make synapses with TCAs. These precise neuronal connections are established during early postnatal period in a whisker-dependent manner. However, when barrel neuron dendrites acquire their orientation bias and what mechanisms regulate this process are not well understood. To address these points, we developed methodology which allows us to characterize dendritic features of individual barrel neurons in correspondence with terminal clusters of TCAs. We achieved sparse and highly intensified cell labeling of barrel neurons using modified Cre/loxP-mediated RFP expression by in vivo electroporation. We visualized TCA clusters by generating a new transgenic mouse line which expresses GFP specifically in TCAs. By combining these two systems and using confo-