- Email: [email protected]
The protocadherin-α family is required for normal serotonergic projections
Abstracts / Neuroscience Research 71S (2011) e108–e415
cal microscope, we analyzed morphological characteristic of barrel neuron dendrites at various ages during the early postnatal period. Moreover, to elucidate the role of neuronal activity in the dendrite refinement, we also analyzed dendritic morphology of developing barrel neurons which genetically lack NMDA receptor subunit 1. We will discuss precise refinement processes of barrel neuron dendrites of newborn mice and roles of neuronal activity in these processes. Research fund: KAKENHI (2270034, HM) (22115009, TI), The Naito foundation (TI). doi:10.1016/j.neures.2011.07.578
P2-f19 Vascular endothelial cells promote cortical neurite elongation by a mechanism dependent on integrin  3 Sumimoto 1,2
Satoko , Rieko Toshihide Yamashita 1,2 1
Muramatsu 1,2 ,
Sakiko
Hujii 1,2 ,
2
Dept. Mol. Neurosci., Osaka Univ., Osaka Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo, Japan
During development and regeneration, neurons change their morphology that is occurred by restructuring of cytoskeleton. Interaction of neuron with the non-neuronal milieu plays a crucial role in the growth and guidance of neurite. A wide variety of cells which express the guidance molecules have been identified. In this study, we added a new member, vascular endothelial cell, which promotes neurite elongation though integrin  3 dependent manner. We employed co-culture of cortical neurons and cortical vascular endothelial cells, and found that adhesive molecules derived from vascular endothelial cells elongated cortical neurites in vitro. A large number of adhesive molecules present arginine-glycine-aspartic acid (RGD) sequence as their cell recognition sites. To confirm the involvement of RGD sequence, we treated the co-culture with an RGD peptide or control peptide. Treatment with RGD peptide inhibited vascular endothelial cell-induced neurite elongation. Then we next identified the key molecule containing RGD sequences that involve of neurite elongation. We focused on the role of integrin  3, which is confirmed the expression on vascular endothelial cells by immunocytochemical analysis. Pharmacological blockade of integrin  3 abolished the neurite elongation induced by endothelial cells. These data suggested that integrin  3 expressed on vascular endothelial cells promoted cortical neurite elongation. This molecular mechanism may contribute to formation and restoration of the neuronal network. doi:10.1016/j.neures.2011.07.579
P2-f20 Balanced bilateral sensory input is essential for target selection of callosal axons Ying Huang , Ning-Ning Song, Lei Zhang, Ze-Lan Hu, Jia-Yin Chen, Yu-Qiang Ding
e135
pus and serotonergic neurons, and that serotonergic terminal axons of Pcdh␣ mutant mice are abnormally clumped in several regions of the brain including the hippocampus and cortex (Katori et al., J. Neurosci. 2009). To identify which cells regulate these serotonergic projections, we generated telencephalon-specific Pcdh␣ conditional knockout mice (Pcdh␣flox/flox ; Emx1-Cre) and serotonergic neuron-specific Pcdh␣ conditional knockout mice (Pcdh␣flox/flox ; ePet-Cre). Though the former showed normal serotonergic projections, the latter showed abnormal ones. Our findings therefore suggest that Pcdh␣ proteins expressed in serotonergic neurons have an important function in serotonergic projections whereas those expressed in the target regions do not. Research fund: KAKENHI22700369. doi:10.1016/j.neures.2011.07.581
P2-g01 The F-BAR protein Rapostlin regulates endocytosis and dendritic spine formation in hippocampal neurons Yohei Wakita Negishi
, Tetsuhiro Kakimoto, Hironori Katoh, Manabu
Lab. of Mol. Neurobiol., Grad. Sch. of Biostudies, Kyoto Univ Prombe Cdc15 homology (PCH) proteins, characterized by F-BAR/EFC domains with membrane invaginating property, play critical roles in a variety of membrane reorganization. Among them, Rapostlin has attracted increasing attention as a critical coordinator of endocytosis. We previously reported that Rapostlin was strongly expressed in hippocampus and cerebral cortex in late developmental stages when accelerated spine formation and maturation occur, and that knockdown of Rapostlin in primary cultured rat hippocampal neurons resulted in a decrease in spine density. Here we used Rapostlin K51Q + K52Q mutant (Rapostlin-QQ), which has no ability to associate with membrane and to induce membrane invagination, to investigate whether Rapostlin regulates dendritic spine formation through the regulation of membrane trafficking. Overexpression of Rapostlin-QQ inhibited spine formation in primary cultured rat hippocampal neurons, whereas overexpression of wild-type Rapostlin had no effect. Knockdown of Rapostlin or overexpression of Rapostlin-QQ significantly reduced the uptake of fluorescently-labeled transferrin in cultured hippocampal neurons compared with that in the control cells. On the other hand, expression of shRNA-resistant wild-type Rapostlin restored spine density in Rapostlin knockdown neurons, whereas expression of Rapostlin deletion mutants lacking the HR1 or SH3 domain did not. Knockdown of Rnd2, which binds to the HR1 domain of Rapostlin and enhanced the Rapostlin-induced tubular membrane invagination, also reduced spine density and the transferrin uptake. These results suggest that the F-BAR protein Rapostlin, whose activity is regulated by Rnd2, plays a key role in spine development through the regulation of membrane dynamics. Research fund: KAKENHI 21770202.
Department of Anatomy and Neurobiology, Tongji University School of Medicine, Shanghai, China
doi:10.1016/j.neures.2011.07.582
Prior to initial contact formation, intrinsic developmental programs and external guidance cues are responsible for guiding growing axons into their target field. Sensory input is generally thought to be necessary for refining and consolidating neuronal connections in developing neuronal networks. We here report that cortical callosal axons in the somatosensory cortex absolutely require sensory input for their target selection in the contralateral cortex. Eliminating sensory input to either hemisphere by unilateral transection of the infraorbital nerve (ION) prevents target selection of callosal axons in the contralateral cortex. Strikingly, blocking sensory input bilaterally, by simultaneously transecting both IONs, callosal projections are largely restored. In contrast, non-simultaneous bilateral ION transection has the same effect as unilateral transection, regardless of which side is lesioned first. Our data demonstrate the critical role of somatosensory input in guiding callosal axons into their target cortical territory.
P2-g02 Synaptogenesis in the embryonic chick ciliary ganglion – a brainbow study
doi:10.1016/j.neures.2011.07.580
P2-f21 The protocadherin-␣ family is required for normal serotonergic projections Shota Katori 1 , Atsushi Okayama 1 , Evan Deneris 2 , Takeshi Yagi 1 1
FBS, Osaka Univ., Osaka, Japan 2 Dept. of Neurosci., Case Western Reserve University, OH, USA The protocadherin-␣ (Pcdh␣) family, which consists of 14 kinds of transmembrane proteins, is expressed specifically in neurons. We previously showed that Pcdh␣ transcripts are abundantly expressed in the hippocam-
Ryo Egawa 1,2 , Shoko Hososhima 1,3,5 , Toru Harukazu Nakamura 1,3,4 , Hiromu Yawo 1,3,5
Ishizuka 1,5 ,
1
Tohoku Univ. Grad. Sch. of Life Sci., Sendai 2 Tohoku University International Advanced Research and Education Organization, Sendai 3 Tohoku University Basic and Translational Research Center for Global Brain Science, Sendai 4 Tohoku University Institute of Development, Aging and Cancer, Sendai 5 CREST, JST Although the morphology and physiology of synapses are dramatically changed during embryonic development, the precise mechanisms remain unrevealed. As one of the model systems of developing synapses, the calyx-type synapses in chick ciliary ganglion (CG) have been intensively studied for decades. Here, we transfected the plasmids containing CAGGS-BRAINBOW1.1 construct into the midbrain preganglionic neurons at embryonic day 2 (E2) using in ovo electroporation technique, and studied the development of presynaptic terminal in the CG between E6 and E14 under confocal microscopy. Preganglionic neurons extended their axons to CG by E6, developed highly-ramified axonal projections in CG by E8. The calyx-type presynaptic terminals were first generated at E9. Some of axonal branches were eliminated between E11 and E14 with the differentiation of calyx-type synapses. These results are consistent with the previous electromicroscopic studies carried out by Landmeser and Pilar in 1970s. With the combination of genetic manipulations this system would enable one to reveal the molecular mechanisms underlying embryonic synaptogenesis.
cal microscope, we analyzed morphological characteristic of barrel neuron dendrites at various ages during the early postnatal period. Moreover, to elucidate the role of neuronal activity in the dendrite refinement, we also analyzed dendritic morphology of developing barrel neurons which genetically lack NMDA receptor subunit 1. We will discuss precise refinement processes of barrel neuron dendrites of newborn mice and roles of neuronal activity in these processes. Research fund: KAKENHI (2270034, HM) (22115009, TI), The Naito foundation (TI). doi:10.1016/j.neures.2011.07.578
P2-f19 Vascular endothelial cells promote cortical neurite elongation by a mechanism dependent on integrin  3 Sumimoto 1,2
Satoko , Rieko Toshihide Yamashita 1,2 1
Muramatsu 1,2 ,
Sakiko
Hujii 1,2 ,
2
Dept. Mol. Neurosci., Osaka Univ., Osaka Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo, Japan
During development and regeneration, neurons change their morphology that is occurred by restructuring of cytoskeleton. Interaction of neuron with the non-neuronal milieu plays a crucial role in the growth and guidance of neurite. A wide variety of cells which express the guidance molecules have been identified. In this study, we added a new member, vascular endothelial cell, which promotes neurite elongation though integrin  3 dependent manner. We employed co-culture of cortical neurons and cortical vascular endothelial cells, and found that adhesive molecules derived from vascular endothelial cells elongated cortical neurites in vitro. A large number of adhesive molecules present arginine-glycine-aspartic acid (RGD) sequence as their cell recognition sites. To confirm the involvement of RGD sequence, we treated the co-culture with an RGD peptide or control peptide. Treatment with RGD peptide inhibited vascular endothelial cell-induced neurite elongation. Then we next identified the key molecule containing RGD sequences that involve of neurite elongation. We focused on the role of integrin  3, which is confirmed the expression on vascular endothelial cells by immunocytochemical analysis. Pharmacological blockade of integrin  3 abolished the neurite elongation induced by endothelial cells. These data suggested that integrin  3 expressed on vascular endothelial cells promoted cortical neurite elongation. This molecular mechanism may contribute to formation and restoration of the neuronal network. doi:10.1016/j.neures.2011.07.579
P2-f20 Balanced bilateral sensory input is essential for target selection of callosal axons Ying Huang , Ning-Ning Song, Lei Zhang, Ze-Lan Hu, Jia-Yin Chen, Yu-Qiang Ding
e135
pus and serotonergic neurons, and that serotonergic terminal axons of Pcdh␣ mutant mice are abnormally clumped in several regions of the brain including the hippocampus and cortex (Katori et al., J. Neurosci. 2009). To identify which cells regulate these serotonergic projections, we generated telencephalon-specific Pcdh␣ conditional knockout mice (Pcdh␣flox/flox ; Emx1-Cre) and serotonergic neuron-specific Pcdh␣ conditional knockout mice (Pcdh␣flox/flox ; ePet-Cre). Though the former showed normal serotonergic projections, the latter showed abnormal ones. Our findings therefore suggest that Pcdh␣ proteins expressed in serotonergic neurons have an important function in serotonergic projections whereas those expressed in the target regions do not. Research fund: KAKENHI22700369. doi:10.1016/j.neures.2011.07.581
P2-g01 The F-BAR protein Rapostlin regulates endocytosis and dendritic spine formation in hippocampal neurons Yohei Wakita Negishi
, Tetsuhiro Kakimoto, Hironori Katoh, Manabu
Lab. of Mol. Neurobiol., Grad. Sch. of Biostudies, Kyoto Univ Prombe Cdc15 homology (PCH) proteins, characterized by F-BAR/EFC domains with membrane invaginating property, play critical roles in a variety of membrane reorganization. Among them, Rapostlin has attracted increasing attention as a critical coordinator of endocytosis. We previously reported that Rapostlin was strongly expressed in hippocampus and cerebral cortex in late developmental stages when accelerated spine formation and maturation occur, and that knockdown of Rapostlin in primary cultured rat hippocampal neurons resulted in a decrease in spine density. Here we used Rapostlin K51Q + K52Q mutant (Rapostlin-QQ), which has no ability to associate with membrane and to induce membrane invagination, to investigate whether Rapostlin regulates dendritic spine formation through the regulation of membrane trafficking. Overexpression of Rapostlin-QQ inhibited spine formation in primary cultured rat hippocampal neurons, whereas overexpression of wild-type Rapostlin had no effect. Knockdown of Rapostlin or overexpression of Rapostlin-QQ significantly reduced the uptake of fluorescently-labeled transferrin in cultured hippocampal neurons compared with that in the control cells. On the other hand, expression of shRNA-resistant wild-type Rapostlin restored spine density in Rapostlin knockdown neurons, whereas expression of Rapostlin deletion mutants lacking the HR1 or SH3 domain did not. Knockdown of Rnd2, which binds to the HR1 domain of Rapostlin and enhanced the Rapostlin-induced tubular membrane invagination, also reduced spine density and the transferrin uptake. These results suggest that the F-BAR protein Rapostlin, whose activity is regulated by Rnd2, plays a key role in spine development through the regulation of membrane dynamics. Research fund: KAKENHI 21770202.
Department of Anatomy and Neurobiology, Tongji University School of Medicine, Shanghai, China
doi:10.1016/j.neures.2011.07.582
Prior to initial contact formation, intrinsic developmental programs and external guidance cues are responsible for guiding growing axons into their target field. Sensory input is generally thought to be necessary for refining and consolidating neuronal connections in developing neuronal networks. We here report that cortical callosal axons in the somatosensory cortex absolutely require sensory input for their target selection in the contralateral cortex. Eliminating sensory input to either hemisphere by unilateral transection of the infraorbital nerve (ION) prevents target selection of callosal axons in the contralateral cortex. Strikingly, blocking sensory input bilaterally, by simultaneously transecting both IONs, callosal projections are largely restored. In contrast, non-simultaneous bilateral ION transection has the same effect as unilateral transection, regardless of which side is lesioned first. Our data demonstrate the critical role of somatosensory input in guiding callosal axons into their target cortical territory.
P2-g02 Synaptogenesis in the embryonic chick ciliary ganglion – a brainbow study
doi:10.1016/j.neures.2011.07.580
P2-f21 The protocadherin-␣ family is required for normal serotonergic projections Shota Katori 1 , Atsushi Okayama 1 , Evan Deneris 2 , Takeshi Yagi 1 1
FBS, Osaka Univ., Osaka, Japan 2 Dept. of Neurosci., Case Western Reserve University, OH, USA The protocadherin-␣ (Pcdh␣) family, which consists of 14 kinds of transmembrane proteins, is expressed specifically in neurons. We previously showed that Pcdh␣ transcripts are abundantly expressed in the hippocam-
Ryo Egawa 1,2 , Shoko Hososhima 1,3,5 , Toru Harukazu Nakamura 1,3,4 , Hiromu Yawo 1,3,5
Ishizuka 1,5 ,
1
Tohoku Univ. Grad. Sch. of Life Sci., Sendai 2 Tohoku University International Advanced Research and Education Organization, Sendai 3 Tohoku University Basic and Translational Research Center for Global Brain Science, Sendai 4 Tohoku University Institute of Development, Aging and Cancer, Sendai 5 CREST, JST Although the morphology and physiology of synapses are dramatically changed during embryonic development, the precise mechanisms remain unrevealed. As one of the model systems of developing synapses, the calyx-type synapses in chick ciliary ganglion (CG) have been intensively studied for decades. Here, we transfected the plasmids containing CAGGS-BRAINBOW1.1 construct into the midbrain preganglionic neurons at embryonic day 2 (E2) using in ovo electroporation technique, and studied the development of presynaptic terminal in the CG between E6 and E14 under confocal microscopy. Preganglionic neurons extended their axons to CG by E6, developed highly-ramified axonal projections in CG by E8. The calyx-type presynaptic terminals were first generated at E9. Some of axonal branches were eliminated between E11 and E14 with the differentiation of calyx-type synapses. These results are consistent with the previous electromicroscopic studies carried out by Landmeser and Pilar in 1970s. With the combination of genetic manipulations this system would enable one to reveal the molecular mechanisms underlying embryonic synaptogenesis.