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Early tangential axonal extension from migrating excitatory neurons in the cerebral cortex
Abstracts P2-e32 Early tangential axonal extension from migrating excitatory neurons in the cerebral cortex Yumiko Hatanaka 1 , Tomohiro Namikawa 1 , Kenta Yamauchi 2 , Sadao Shiosaka 1 1
Grad Sch of Biol Sci, NAIST, Japan; Univ, Japan
2
Grad Sch of Frontier Biosci, Osaka
S161
P2-e35 Single-cell level multilayered substructure of neocortical layer V Rumi Kurokawa, Kazumasa Kubota, Hisato Maruoka, Shun Tsuruno, Toshihiko Hosoya RIKEN, BSI, Hosoya Unit, Japan
Cortical excitatory neurons extend axons radially toward the white matter, and subsequently make a right-angled turn to run tangentially through the white matter. Previous our in vitro study indicated that radial axonal extension seemed to occur as a result of trailing process extension during radial migration. However, the precise timing of tangential axonal extension remains obscure. To address this issue, we labeled a limited number of mouse ventricular zone (VZ) cells by in vivo electroporation and examined morphology of their progeny in detail. When we labeled VZ cells at E12.5 or E15.5 and analyzed two days later, almost all labeled cells in the cortical plate, as well as many in the intermediate zone (IZ), exhibited a long process extending tangentially within the IZ. Moreover, the process showed directionality: those derived from E12.5 labeled cells extended laterally, while those from E15.5 extended medially. These results suggest that cortical excitatory neurons initiate tangential axonal growth when they migrate through the IZ.
The neocortex contains a large number of neuronal types. Understanding their organization is important for the analysis of neocortical circuit. We previously showed that in layer V of the mouse neocortex a major type of pyramidal neurons, i.e. subcerebral projection neurons (SCPNs), form microcolumns that are periodically repeated in the tangential direction. We further found that single SCPNs expressed different combination of marker genes. Interestingly, this difference in gene expression was not random, but was regulated and dependent on the radial position in the microcolumn. Typically one to four cells expressing a specific combination of marker genes were located in a specific radial position in microcolumns. Using combinations of expressed genes, single microcolumns can be divided into at least three parts located at different radial positions. Therefore, microcolumns have subparts each of which is composed of one to four neurons, and this structure makes at least three sublayers of SCPNs in layer V. This sublayer structure may underlie a precise network structure in the neocortical local circuit.
doi:10.1016/j.neures.2009.09.828
doi:10.1016/j.neures.2009.09.831
P2-e33 Analysis of relative roles of the individual phosphotyrosines of Disabled-1 in Reelin signaling Toshifumi Morimura 2 , Masaharu Ogawa 1
P2-e36 Mediolateral compartments determined by the birthdate of Purkinje cells correlates closely with Zebrin II-pattern in adult cerebellum Mitsuhiro Hashimoto 1 , Kazunori Namba 1 , Izumi Sugihara 2
1
Brain Science Institute, RIKEN, Japan; 2 Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Japan Reelin is secreted by Cajal-Retzius cells and regulates radial migration of projection neurons in the developing cerebral cortex by inducing tyrosine phosphorylation of an intracellular adaptor protein, Disabled-1 (Dab1), through activation of Src family tyrosine kinases (SFKs). Dab1 contains five potential tyrosine residues phosphorylated by SFKs, and phosphorylation of three of them (Y198, Y220 and Y232) has been demonstrated after Reelin stimulation. Reelin may also induce phosphorylation of an additional tyrosine residue, Y185, but its phosphorylation has not been confirmed in vivo. Here, we established a reconstitution system for Dab1-deficient cerebral cortex with the wild-type Dab1 gene by in utero electroporation. Using this reconstitution system combined with site-directed mutations of the Dab1 gene, we clarified that Reelin also induces phosphorylation of Y185 of Dab1, and identified the relative roles of the individual phosphotyrosines of Dab1 to trasmit Reelin signaling.
1
RIKEN BSI, Hashimoto Res Unit, Saitama, Japan; physiol, Tokyo Med Dental University Tokyo, Japan
2
Depa Systems Neuro-
Using an adenoviral vector system, we reveal that the birthdate of Purkinje cells (PCs) determines the M-L compartmentalization of the cerebellum. However, the process of M-L compartmentalization and the biological significance of M-L compartments determined by the birthdate of PCs (PC-birthdate compartments) have been not elucidated. To examine this issue, we carefully compared PC-birthdate compartments with the striped pattern of Zebrin II-immunoreactivity (Zebrin II-bands) in adult cerebella. Our observation indicated that PC-birthdate compartments were correlated strikingly with Zebrin II-bands, and moreover, with the olivocerebellar projections. It suggested that PC-birthdate compartments (early-onset) play an important role in establishing the M-L compartments revealed by Zebrin II (late-onset pattern) and the olivocerebellar projection in mature cerebellum.
doi:10.1016/j.neures.2009.09.829
doi:10.1016/j.neures.2009.09.832
P2-e34 Involvement of asymmetric endocytosis in repulsive growth cone guidance Takuro Tojima, Rurika Itofusa, Hiroyuki Kamiguchi
P2-f01 Localization of BDNF in CNS neurons Tomoya Matsumoto 1,2 , Sandra Dieni 3 , Stefanie Michael Frotscher 3 , Yves-Alain Barde 2
Lab for Neuronal Growth Mechanisms, RIKEN BSI, Wako, Japan
1
Rauskolb 2 ,
Dept Psychiatry and Neurosci, Hiroshima Univ, Hiroshima, Japan; Biozentrum, Univ Basel, Switzerland; 3 Inst Anatomy and Cell Biol, Univ Freiburg, Germany
2
Asymmetric Ca2+ signals across the growth cone can mediate both attractive and repulsive axon guidance. We previously reported that attractive, but not repulsive, Ca2+ signals facilitate asymmetric membrane transport and exocytosis in the growth cone. Here we report the involvement of asymmetric endocytosis in Ca2+ -induced growth cone repulsion. Using total internal reflection fluorescence microscopy, we detect repulsive, but not attractive, (Tojima et al., 2007) Ca2+ signals induce asymmetry of clathrin-dependent endocytosis across the growth cone. Inhibition of clathrin or dynamin-dependent endocytosis abolishes Ca2+ -induced growth cone repulsion, but not attraction. Furthermore, pharmacological perturbation of endocytosis on one side of the growth cone triggers its turning to the side with lower endocytic activity. These results suggest that preferential addition and removal of plasmalemmal components of the growth cone on the side with Ca2+ elevation provides a driving force for growth cone attraction and repulsion, respectively. Reference
Brain-derived neurotrophic factor (BDNF) is initially synthesized as a precursor protein (pro-BDNF) that is post-translationally processed to mature BDNF. We previously used pulse-chase experiments with hippocampal neurons to show that this processing occurs rapidly in an intracellular compartment (Matsumoto et al., NN 2008). Here, we biochemically detected the cleaved pro-domain BDNF in hippocampal extracts, with a pro-BDNF:mature BDNF:pro-domain BDNF ratio of 1:9:9 suggesting that the cleaved pro-domain and mature peptides are both transported to the axon terminals. In support of this idea, confocal analysis of hippocampal sections double-labelled with anti-pro-domain and anti-mature BDNF antibodies reveals co-localization not only in dentate granule cell bodies, but also in mossy fiber terminals. Immunogold-EM further shows that each peptide is presynaptically localized to large secretory vesicles. It is thus conceivable that both peptides may be secreted, possibly from the same vesicles as a protein complex. doi:10.1016/j.neures.2009.09.833
Tojima, et al., 2007. Nat. Neurosci. 10, 58–66. doi:10.1016/j.neures.2009.09.830
Grad Sch of Biol Sci, NAIST, Japan; Univ, Japan
2
Grad Sch of Frontier Biosci, Osaka
S161
P2-e35 Single-cell level multilayered substructure of neocortical layer V Rumi Kurokawa, Kazumasa Kubota, Hisato Maruoka, Shun Tsuruno, Toshihiko Hosoya RIKEN, BSI, Hosoya Unit, Japan
Cortical excitatory neurons extend axons radially toward the white matter, and subsequently make a right-angled turn to run tangentially through the white matter. Previous our in vitro study indicated that radial axonal extension seemed to occur as a result of trailing process extension during radial migration. However, the precise timing of tangential axonal extension remains obscure. To address this issue, we labeled a limited number of mouse ventricular zone (VZ) cells by in vivo electroporation and examined morphology of their progeny in detail. When we labeled VZ cells at E12.5 or E15.5 and analyzed two days later, almost all labeled cells in the cortical plate, as well as many in the intermediate zone (IZ), exhibited a long process extending tangentially within the IZ. Moreover, the process showed directionality: those derived from E12.5 labeled cells extended laterally, while those from E15.5 extended medially. These results suggest that cortical excitatory neurons initiate tangential axonal growth when they migrate through the IZ.
The neocortex contains a large number of neuronal types. Understanding their organization is important for the analysis of neocortical circuit. We previously showed that in layer V of the mouse neocortex a major type of pyramidal neurons, i.e. subcerebral projection neurons (SCPNs), form microcolumns that are periodically repeated in the tangential direction. We further found that single SCPNs expressed different combination of marker genes. Interestingly, this difference in gene expression was not random, but was regulated and dependent on the radial position in the microcolumn. Typically one to four cells expressing a specific combination of marker genes were located in a specific radial position in microcolumns. Using combinations of expressed genes, single microcolumns can be divided into at least three parts located at different radial positions. Therefore, microcolumns have subparts each of which is composed of one to four neurons, and this structure makes at least three sublayers of SCPNs in layer V. This sublayer structure may underlie a precise network structure in the neocortical local circuit.
doi:10.1016/j.neures.2009.09.828
doi:10.1016/j.neures.2009.09.831
P2-e33 Analysis of relative roles of the individual phosphotyrosines of Disabled-1 in Reelin signaling Toshifumi Morimura 2 , Masaharu Ogawa 1
P2-e36 Mediolateral compartments determined by the birthdate of Purkinje cells correlates closely with Zebrin II-pattern in adult cerebellum Mitsuhiro Hashimoto 1 , Kazunori Namba 1 , Izumi Sugihara 2
1
Brain Science Institute, RIKEN, Japan; 2 Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Japan Reelin is secreted by Cajal-Retzius cells and regulates radial migration of projection neurons in the developing cerebral cortex by inducing tyrosine phosphorylation of an intracellular adaptor protein, Disabled-1 (Dab1), through activation of Src family tyrosine kinases (SFKs). Dab1 contains five potential tyrosine residues phosphorylated by SFKs, and phosphorylation of three of them (Y198, Y220 and Y232) has been demonstrated after Reelin stimulation. Reelin may also induce phosphorylation of an additional tyrosine residue, Y185, but its phosphorylation has not been confirmed in vivo. Here, we established a reconstitution system for Dab1-deficient cerebral cortex with the wild-type Dab1 gene by in utero electroporation. Using this reconstitution system combined with site-directed mutations of the Dab1 gene, we clarified that Reelin also induces phosphorylation of Y185 of Dab1, and identified the relative roles of the individual phosphotyrosines of Dab1 to trasmit Reelin signaling.
1
RIKEN BSI, Hashimoto Res Unit, Saitama, Japan; physiol, Tokyo Med Dental University Tokyo, Japan
2
Depa Systems Neuro-
Using an adenoviral vector system, we reveal that the birthdate of Purkinje cells (PCs) determines the M-L compartmentalization of the cerebellum. However, the process of M-L compartmentalization and the biological significance of M-L compartments determined by the birthdate of PCs (PC-birthdate compartments) have been not elucidated. To examine this issue, we carefully compared PC-birthdate compartments with the striped pattern of Zebrin II-immunoreactivity (Zebrin II-bands) in adult cerebella. Our observation indicated that PC-birthdate compartments were correlated strikingly with Zebrin II-bands, and moreover, with the olivocerebellar projections. It suggested that PC-birthdate compartments (early-onset) play an important role in establishing the M-L compartments revealed by Zebrin II (late-onset pattern) and the olivocerebellar projection in mature cerebellum.
doi:10.1016/j.neures.2009.09.829
doi:10.1016/j.neures.2009.09.832
P2-e34 Involvement of asymmetric endocytosis in repulsive growth cone guidance Takuro Tojima, Rurika Itofusa, Hiroyuki Kamiguchi
P2-f01 Localization of BDNF in CNS neurons Tomoya Matsumoto 1,2 , Sandra Dieni 3 , Stefanie Michael Frotscher 3 , Yves-Alain Barde 2
Lab for Neuronal Growth Mechanisms, RIKEN BSI, Wako, Japan
1
Rauskolb 2 ,
Dept Psychiatry and Neurosci, Hiroshima Univ, Hiroshima, Japan; Biozentrum, Univ Basel, Switzerland; 3 Inst Anatomy and Cell Biol, Univ Freiburg, Germany
2
Asymmetric Ca2+ signals across the growth cone can mediate both attractive and repulsive axon guidance. We previously reported that attractive, but not repulsive, Ca2+ signals facilitate asymmetric membrane transport and exocytosis in the growth cone. Here we report the involvement of asymmetric endocytosis in Ca2+ -induced growth cone repulsion. Using total internal reflection fluorescence microscopy, we detect repulsive, but not attractive, (Tojima et al., 2007) Ca2+ signals induce asymmetry of clathrin-dependent endocytosis across the growth cone. Inhibition of clathrin or dynamin-dependent endocytosis abolishes Ca2+ -induced growth cone repulsion, but not attraction. Furthermore, pharmacological perturbation of endocytosis on one side of the growth cone triggers its turning to the side with lower endocytic activity. These results suggest that preferential addition and removal of plasmalemmal components of the growth cone on the side with Ca2+ elevation provides a driving force for growth cone attraction and repulsion, respectively. Reference
Brain-derived neurotrophic factor (BDNF) is initially synthesized as a precursor protein (pro-BDNF) that is post-translationally processed to mature BDNF. We previously used pulse-chase experiments with hippocampal neurons to show that this processing occurs rapidly in an intracellular compartment (Matsumoto et al., NN 2008). Here, we biochemically detected the cleaved pro-domain BDNF in hippocampal extracts, with a pro-BDNF:mature BDNF:pro-domain BDNF ratio of 1:9:9 suggesting that the cleaved pro-domain and mature peptides are both transported to the axon terminals. In support of this idea, confocal analysis of hippocampal sections double-labelled with anti-pro-domain and anti-mature BDNF antibodies reveals co-localization not only in dentate granule cell bodies, but also in mossy fiber terminals. Immunogold-EM further shows that each peptide is presynaptically localized to large secretory vesicles. It is thus conceivable that both peptides may be secreted, possibly from the same vesicles as a protein complex. doi:10.1016/j.neures.2009.09.833
Tojima, et al., 2007. Nat. Neurosci. 10, 58–66. doi:10.1016/j.neures.2009.09.830