Roles of serotonin receptors in the dendrite formation of the rat cerebral cortical neurons

Abstracts plasmid into the ventricular progenitor cells by in utero electroporation (EP). Previously, we reported that most of the labeled cells by EP at embryonic day (E) 11.5 or 12.5 were Purkinje cells and few deep cerebellar neurons and Golgi cells were labeled. Other interneurons and granule cells were not observed. We hypothesized that this was because plasmids were diluted out due to cell proliferation. To circumvent this issue, we utilized a transposon-mediated gene transfer, which enables us to express electroporated DNAs persistently by genomic integration. As a result, almost every type of the cerebellar neurons and glias was labeled by EP at E12.5. This suggests that these cells are derived from VZ or RL. doi:10.1016/j.neures.2009.09.814

P2-e19 Control of dendrite formation by Meis2-mediated transcription in the developing cerebral cortical pyramidal neurons Emi Nishiwaki, Fujio Murakami, Yasuto Tanabe Grad. Sch. Front. Biosci., Osaka University, Suita, Japan Previous studies provided evidence that transcription factors play crucial roles in the acquisition of neuronal specificity in the mammalian nervous system. However, it remains still largely unknown how the spatially- and temporally-controlled acquisition of neuronal connectivity is globally coordinated by the activity of transcription factors. Meis/Pbx belong to the TALE homeodomain-containing transcription factors. Our study showed that Meis/Pbx were expressed in most of the cortical pyramidal neurons during the period of dendrogenesis. To examine the potential roles of Meis/Pbx in the control of dendrogenesis, we established a single celllevel inducible gene expression system where we can analyze the morphology of dendrites derived from distinct cortical pyramidal neurons. We found that Meis2mediated transcriptional repression enhanced whereas its activation suppressed the dendrite development, suggesting that Meis2-mediated transcriptional activities behave as a binary switch for the control of dendrogenesis in cortical pyramidal neurons. doi:10.1016/j.neures.2009.09.815

P2-e20 Identification of a thalamus-specific molecule which affects development of cortical cells Haruka Sato, Eiichi Tatara, Yuji Yamamoto, Nobuhiko Yamamoto Grad Sch of Frontier Biosci, Osaka Univ, Osaka Not only intrinsic factors but also extrinsic factors are suggested to play a role in cortical development. Here, we investigated the influence of extrinsic molecules derived from thalamic afferent on cortical cell development. First, thalamus-specific genes were searched by constructing a cDNA library in which cortical cDNAs were subtracted from thalamic cDNAs. A systematic screening with sequence analysis and in situ hybridization showed that Cpg15, which encodes a cell surface molecule, is expressed in the sensory thalamic nuclei but not in the cortex during development. Overexpression of this gene in cultured thalamic cells demonstrated that CPG15 was transported to axon terminals. The effect of CPG15 on cortical development was then studied using cortical cell culture. Application of CPG15 was found to promote cell survival and dendritic growth. These results suggest that CPG15, which is specifically expressed in thalamic cells, may be transported to the cortex, and promote cortical cell development. doi:10.1016/j.neures.2009.09.816

P2-e21 Activity-dependent Netrin-4 expression regulates thalamocortical axon branching Yasufumi Hayano 1 , Makoto Takemoto 2 , Nobuhiko Yamamoto 1 1

Grad Sch of Frontier Biosci, Osaka Univ, Suita, Japan; Kumamoto Univ, Japan

2

Grad Sch Med Sci,

Thalamocortical (TC) axon branching is regulated by not only layer-specific cues but also neural activities. To reveal the activity-dependent mechanism, we investigated activity-dependent expression of layer-specific molecules and their roles for TC axon branching. We found that netrin-4, one of the guidance molecules, was strongly expressed in cortical layer 4, which is the primary target layer of TC axons. A quantitative PCR analysis showed that netrin-4, expression in cortical slices was reduced by blockade of firing activity. On the other hand, the candidate receptors were expressed in thalamic nuclei including dLGN and VB. A morphological study using organotypic cocultures of the thalamus and cortex further showed that TC axon branching in cortical explants was increased by Netrin-4 application, but was markedly decreased by removing endogenous ligands with Unc5h2-Fc. These results suggest that Netrin-4 contributes to TC axon branching by being expressed in a lamina-specific and activity-dependent fashion. doi:10.1016/j.neures.2009.09.817

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P2-e22 Analysis of protocadherin-␣ isoforms in serotonergic neurons Shota Katori 1 , Yukiko Noguchi 1,2 , Takahiro Hirabayashi 1 , Yoshimi Kawamura 3 , Takeshi Yagi 1 1

Grad Sch of Frontier Biosciences, Osaka Univ, Osaka, Japan; Res Inst, AIST, Japan; 3 Dept Physiology, Keio Univ, Japan

2

Neurosci

Protocadherin-␣ (Pcdha) family are expressed in vertebrate brains, and belong to the cadherin superfamily, cell adhesion/recognition transmembrane proteins. The Pcdha proteins have 14 isoforms. The first exons of Pcdha genes, encoding the extracellular and transmembrane regions and a part of the cytoplasmic region, are tandemly clustered, and their second to fourth exons, encoding a part of the cytoplasmic region, are common to Pcdha genes. Recently, we reported that Pcdha genes were intensely expressed in serotonergic neurons, and that common exons-deficient mice revealed abnormal serotonergic innervation. In this study, to elucidate the roles of the individual isoforms, we examined the expression of individual Pcdha genes in serotonergic neurons. In addition, to know which isoforms are essential for normal serotonergic innervation, we generated several isoforms-deficient mice, and examined these mutant mice. doi:10.1016/j.neures.2009.09.818

P2-e23 A role of proper mitochondrial distribution for dendrite development in the cortex Toshiya Kimura, Fujio Murakami Grad Sch Frontier Biosci, Osaka Univ, Osaka, Japan Proper subcellular distribution of mitochondria is proposed to be critical for the development of fly neuromuscular junctions as well as spine formation of cultured hippocampal neurons. However, it remains unknown whether mitochondrial subcellular distribution is also important for neuronal development in mammals in vivo. Here, we examined mitochondrial distribution in embryonic mice cortical cells by labeling mitochondria with electroporation and the effect of mitochondrial distribution disruption on neuronal development. Mitochondrial distribution dramatically changed during normal development. Promotion of mitochondrial fusion by dominant negative Drp1 or Mfn1 disrupted the subcellular distribution and caused abnormal development of dendritic trees. This manipulation did not appear to reduce mitochondrial membrane potential or to cause appreciable cell death. Together, these results suggest that mitochondrial subcellular distribution is developmentally regulated and that the proper distribution is critical for dendrite development of cortical neurons. doi:10.1016/j.neures.2009.09.819

P2-e24 Two photon real-time imaging of cortical interneurons in living mouse embryos Mitsutoshi Yanagida, Ryota Miyoshi, Ryuhei Toyokuni, Fujio Murakami Graduate School of Frontier Bioscience, Osaka University, Japan During development, neurons migrate from the site of their birth to distant destinations. Although migratory behaviors of neurons have been studied extensively, most studies have utilized in vitro preparations. Previously, we developed an imaging system by which populations of migrating cortical interneurons in embryos could be stably recorded. Here, we improved the resolution of the system, which allowed us to analyze migratory behaviors of individual neurons in detail. Cortical interneurons were labeled by electroporating GFP into E12 mice medial ganglionic eminence. Four days later, labeled cells reaching the cortical surface were analyzed by two photon confocal microscopy. A subset of migrating interneurons extended and retracted multiple neurites and sometimes changed the direction of migration by choosing the direction of a neurite. We will report further details of migrating behaviors of cortical interneurons in vivo. doi:10.1016/j.neures.2009.09.820

P2-e25 Roles of serotonin receptors in the dendrite formation of the rat cerebral cortical neurons Akiko Ohtani 1 , Hidetoshi Nishiyama 2 , Mitsuo Suga 2 , Chikara Sato 3 , Kouji Senzaki 1 , Takashi Shiga 1 1 Grad Sch of Comprehensive Human Sciences, Univ of Tsukuba, Tsukuba, Japan; 2 JEOL Ltd., Japan; 3 AIST, Neuroscience Research Institute, Japan

Serotonin (5-HT) is shown to be a regulatory factor in the development of central nervous system. Among the 14 receptors, 5-HT1A and 5-HT2A receptors appear early in the developing cerebral cortex, but the roles of these receptors in the cortical devel-

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Abstracts

opment are unknown. Here, we examined roles of 5-HT1A and 5-HT2A receptors in the dendrite formation of rat cerebral cortex using a dissociation culture. Cerebral cortex was dissected from rat embryos (Wistar/ST) at embryonic day 16 and dissociated cortical neurons were cultured up to 5 days. During the culture, neurons were treated chronically or acutely with selective receptor agonists. After the culture, the neurons were double-immunostained by antibodies against microtubule-associated protein 2 (MAP2) and glutamate decarboxylase 65/67 (GAD65/67). We found that both chronic and acute treatment with 5-HT1A agonist (8-OH DPAT) decreased the dendiritic length of GAD65/67 negative non-GABAergic neurons. doi:10.1016/j.neures.2009.09.821

fication of TrkC+ TG neurons which convey the mechanoreceptive sensation from the Merkel cells to Sp5I. doi:10.1016/j.neures.2009.09.824

P2-e29 Developmental changes of signaling molecules that regulate neurite outgrowth and myelination in the rat orbitofrontal and visual cortex Satoshi Ichisaka 1 , Manami Miyahara 1 , Ayano Yamamoto 1 , Kazuhiro Kojima 2 , Yoshio Hata 1,2 1

P2-e26 Axons of medial habenular nucleus topographically sorted in the core of fasciculus retroflexus Tomoko Toyama, Satoru Takahashi, Hiroyuki Ichijo Department Anat Embryol., University Tsukuba, Ibaraki, Japan Axons of medial (MHb) and lateral nucleus (LHb) run in the core and sheath parts of fasciculus retroflexus (FR), respectively. MHb neurons topographically project to interpeduncular nucleus (IPN): medial, dorsal, and lateral MHb neurons project to ventral, lateral, and dorsal IPN, respectively. However, topographic arrangement of these axons is not known in the core of FR. We developed transgenic mice that labeled MHb neurons by fluorescent protein, Venus. In the mice, the topographic projection of medial and dorsal MHb to ventral and lateral IPN was confirmed. Moreover, we show the topographic arrangement of these axons in the core of FR. After postnatal days 5 (P5), medial MHb and ventral IPN were labeled. After P24, medial, dorsal MHb and ventral, lateral IPN were labeled. After P5, the core of FR consisted of labeled and unlabeled fasciculi; the labeled fasciculi were formed by axons of medial MHb after P5, and by axons of medial and dorsal MHb after P24. Therefore, the results indicate that these axons are topographically sorted out in the core of FR. doi:10.1016/j.neures.2009.09.822

Div Neurobiol, Fac Med, Tottori Univ, Yonago, Japan; Biosci, Tottori Univ Grad Sch Med Sci, Yonago, Japan

2

Div Integrative

Neural circuits in the visual cortex (V1) exhibit experience-dependent plasticity during the critical period (CP). Growing evidences suggest a role of structural dynamics of synapses and myelination in the plasticity. To investigate whether similar age-specific mechanism underlies development of the orbitofrontal cortex (OFC) involved in personality, we compared developmental changes of the following molecules that regulate neurite outgrowth and myelination: BDNF, p-Erk, p-CREB, Cdk5/p35, NgR1, active RhoA, p-LIMK, p-Cofilin, Drebrin A. We found significant differences between V1 and OFC in the developmental profile of the molecules involved in neurite outgrowth but not in actin stabilization. Although the circuit remodeling would be most active during CP in V1, OFC might preserve high ability to add new circuits in adulthood. On the other hand, consolidation mechanisms of neural circuits in childhood might operate similarly in the two areas. doi:10.1016/j.neures.2009.09.825

P2-e30 Morphological study of the corticoceptive spinal neurons in the early postnatal period Hitoshi Maeda 1 , Yuchio Yanagawa 2 , Yasuo Kawaguchi 3 , Masaki Sakurai 1 1

P2-e27 Different effects of SulfFP1 and SulfFP2 electroporation in rescuing axon guidance defects in SulfFP double mutant mice Takuya Okada 1 , Kazuko Keino-Masu 1 , Satoshi Nagamine 1 , Satoshi Kunita 2 , Satoru Takahashi 2 , Masayuki Masu 1 1

Dept Mol Neurobiol, Inst Basic Medical Sci, Grad Sch of Comprehensive Human Sci, Univ of Tsukuba, Tsukuba, Japan; 2 Laboratory Animal Resource Center, Univ of Tsukuba, Tsukuba, Japan Heparan sulfate (HS) plays a crucial role in the neural development through interactions with extracellular molecules. The specificity and affinity of the interactions are determined by the sulfation state of HS. HS 6-O-endosulfatases, SulfFP1 and SulfFP2, are extracellular enzymes that control the sulfation patterns of HS, thereby regulating cellular signaling in vivo. We show that the double mutant mice show axon guidance defects, which can be normalized by introducing exogenous SulfFP1 or SulfFP2 genes using electroporation. However, the potency in rescuing the phenotype is slightly different between two subtypes. In addition, the distribution and expression levels of SulfFP1 and SulfFP2 proteins are different in vivo. Our findings suggest that the regulation of localization and stability of SulfFP proteins is important in determining their functions. doi:10.1016/j.neures.2009.09.823

Department Physiol., Teikyo University Sch. Med., Tokyo, Japan; Department Genet. Behavi. Neurosci, Gunma University Grad. Sch. Med., Maebashi, Japan; 3 Division of Cerebral Circuitry, NIPS, Japan 2

We investigated the morphological and electrophysiological properties of the neurons that receive CS synapses in the acute spinal cord slices of P6–P15 rats. In addition, using the VGAT-Venus transgenic rats, we selectively recorded from the inhibitory interneurons in the spinal cord. The postsynaptic neurons identified by the CS-EPSC were whole-cell patch clamped and labeled. They were later reconstructed three dimensionally. Those neurons were widely distributed from the laminae III–IX, showing a large variety of morphologies. However at least a part of them located in the same lamina can be grouped into several distinctive morphological types chiefly according to their pattern of dendritic arborization. Furthermore, we recorded CSEPSCs from the commissural interneurons in the lamina VIII projecting their axons to contralateral side; and putative motoneurons in the lamina IX extending their axons to the ventral root. doi:10.1016/j.neures.2009.09.826

P2-e31 Retrograde labeling of corticospinal neurons innervating the spinal gray of C7 using fluorescent beads (RetroBeadsTM ) Tsutomu Kamiyama, Naoyuki Murabe, Masaki Sakurai Dept Physiol, Teikyo Univ Sch Med, Tokyo, Japan

P2-e28 Runx3 is required for the specification of TrkC-positive mechanoreceptive neurons in the trigeminal ganglion Kouji Senzaki 1 , Shigeru Ozaki 1 , Masaaki Yoshikawa 1 , Yoshiaki Ito 2 , Takashi Shiga 1 1 Graduate School of Comprehensive Human Sciences, Univ of Tsukuba, Japan; 2 Institute of Molecular and Cell Biology, Singapore

Sensory neurons specifically project axons to peripheral and central targets according to their sensory modality. Transcription factor Runx3 plays crucial roles in the development of proprioceptive dorsal root ganglion neurons. Runx3 is also expressed in trigeminal ganglion (TG) neurons. However, the roles of Runx3 in TG are largely unknown, because TG does not contain proprioceptive neurons. In Runx3−/− mice, TrkB-expressing (TrkB+ ) TG neurons were increased, whereas TrkC+ TG neurons decreased. In addition, in Runx3−/− mice, peripheral projections of TrkC+ TG neruons to the Merkel cells in the outer root sheath of whisker vibrissae were lost, and central projections to the spinal trigeminal nucleus pars interpolaris (Sp5I) were also lost. These findings suggest that Runx3 is required for the speci-

To study the developmental change of distribution of corticospinal (CS) axon in rat’s cervical segment 7 (C7), we produced the animals in which CS neurons (CSNs) are labeled with EYFP by way of electroporation at an embryonic stage. To confirm the labeled area covers the distribution of the CSNs projecting to C7, we labeled the CSNs retrogradely. In this study, the fluorescent beads (RetroBeadsTM , Lumafluor Inc.) which is taken up selectively from nerve terminals, was injected as a retrograde tracer in rat’s C7 at postnatal day 6 (P6) and the animals were fixed at P18. Serial cortical sections were prepared and analyzed by an intelligent microscope. In the rostro-caudal direction, the CSNs distributed from 1 mm caudal to frontal tip until the level where the hippocampus disappeared. In the medio-lateral direction, they were observed in medial half or one-third of the cortex from cerebral fissure to rhinal fissure. The CNSs distributed to unexpectedly large cortical area, which was too large to be labeled with classical anterograde tracers. doi:10.1016/j.neures.2009.09.827