Physiological roles of LOTUS, an endogenous Nogo receptor antagonist, in lateral olfactory tract formation

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Abstracts / Neuroscience Research 71S (2011) e108–e415

P4-e10 Molecular mechanism of barrel cortex development controlled by thalamocortical axon innervation Asuka Matsui , Aya Yoshida, Tomomi Shimogori Thalamus.Dev, BSI, RIKEN, Wako, Saitama, Japan In the rodent primary somatosensory cortex, layer IV cells are concentrated around barrel walls, forming cell-sparse barrel hollows and septa that delineate individual barrels. During early postnatal stage, thalamocortical axons (TCAs) from individual thalamic barrelloids are almost entirely confined to a single barrel cluster, followed by arrangement of cortical layer IV neurons into barrel hollows and septa. Addition to this, unidirectional dendrite formation of barrel neurons toward barrel hollows occurs around P7 for efficient synapse formation with TCAs. To elucidate the molecular mechanism of barrel development, we searched for genes expressed in the barrel cortex, by using Allen Brain Atlas and tested their temporal and spatial expression during early postnatal stage using in situ hybridization (ISH). As a result, we found several genes, whose expression is restricted in the barrel or septa. Among these genes, we focused on Btbd3, BTB/POZ domain containing 3, whose expression is restricted in the barrel cells. We first revealed that Btbd3 expression start around P4, when TCAs exhibit more axon branches in cortical layer IV. Next, we revealed that its barrel-like expression is disrupted in the cortex with the abnormal TCA innervation. Furthermore, we tested its function in barrel development with employing in utero electroporation and its shRNA construct. Although no major structural difference was obtained in Btbd3 knockdown barrel cortex, dendrite orientation of barrel cells are disrupted. Our results indicate that Btbd3 function to regulate dendrite patterning in TCA-dependent fashion. doi:10.1016/j.neures.2011.07.1460

P4-e11 Effects of 5-HT receptor on cytoskeletal dynamics in the dendrite formation of cortical neurons Akiko Ohtani , Fei Li, Kouji Senzaki, Takashi Shiga Grad. Sch. of Comprehensive Human Sciences, Tsukuba Univ., Tsukuba, Japan Serotonin (5-HT) has neurotrophic activity, but roles of 5-HT receptors in the development of the cerebral cortex are not well understood. We showed previously that the acute treatment of cultured cortical neurons with 5-HT2A/2C receptor agonist (DOI) increases the length of the dendritic growth cone filopodia (Ohtani et al., 2010). In the present study, we examined the dendritic growth cone filopodia in vitro in more detail. Cerebral cortex was dissected from rat embryos (Wistar/ST) at embryonic day 16 and dissociated cortical neurons were cultured for 4 days. In untreated neurons, dendritic growth cones showed various morphology including wide-shape, thin-shape and unclassified (other) shape. The time-lapse analysis revealed that single growth cone changed the shapes dynamically. Next, we examined effects of 5-HT2A/2C receptors on the dendritic growth cone filopodia with special reference to cytoskeletal proteins. Dissociated neurons were cultured for 4 days and treated with DOI for 4 h. After the culture, microtubule-associated protein 2 (MAP2), acetylated ␣-tubulin (Ace-T; stable microtubule) and tyrosinated ␣-tubulin (Tyr-T; dynamic microtubule) were immunolabeled by specific antibodies. F-actin and G-actin were labeled by rhodamine-phalloidin and fluorescent DNase I, respectively. The mean fluorescent intensity of Tyr-T increased in dendritic growth cone filopodia with other shape by DOI treatment, suggesting that DOI increases microtubule dynamics in the dendritic growth cone filopodia. The mean fluorescent intensity of G-actin decreased in growth cone filopodia with wide-shape by DOI treatment, suggesting that actin depolymerization are associated with the dendritic growth cone filopodia. Therefore, these findings suggest that 5HT2A/2C receptors regulate microtubule dynamics and actin reassembly in various shapes of dendritic growth cones. Research fund: The Long-range Research Initiative by Japan Chemical Industry Association. doi:10.1016/j.neures.2011.07.1461

P4-e12 Dynamics of presynaptic structures and axons in the development of corticospinal innervation in vitro: Analysis by live imaging Noriko Isoo , Takae Ohno, Naoyuki Murabe, Noboru Yoshioka, Masaki Sakurai Dept. of Physiol., Teikyo Univ., Sch. of Med., Tokyo, Japan We previously reconstructed the corticospinal (CS) projection with functional synapses in vitro using the slice co-culture system of the rat cerebral cortex and spinal cord. In this system, it was shown by electrophysiolog-

ical experiments, anterograde labeling and live imaging of CS axons that once CS axons innervated the ventral side of the spinal cord, both synapses and axons were mostly eliminated from the ventral side in a postsynaptic GluN2B-containing NMDA recepter-dependent manner. In this study, we studied through live imaging the developmental dynamics of CS synapses labeled with synaptophysin-EGFP along with CS axons labeled with DsRed. Expression vectors of these fluorescent proteins were transfected to embryonic cortical cells by the exo utero electroporation technique. From 5 to 7 DIV, synaptophysin-EGFP labeled puncta (putative presynaptic vesicle clusters) were formed throughout the spinal cord. From 9 to 12 DIV, most of them were eliminated from the ventral side, which was followed by the regression of CS axons. After this elimination, CS axons reprojected to the spinal cord (second wave of innervation) from 14 to 19 DIV. Following the reprojection of CS axons, puncta were newly formed at the dorsal side and extended to the entire spinal cord including the ventral side. Puncta sizes increased during the first projection period, decreased during the elimination period and increased again during the second projection period. The puncta formed during the second projection period were larger than preexisting uneliminated ones. These results suggest that (1) the ventral synapse elimination does not coincide with but precede axon regression, (2) the second wave of CS axonal reprojection follows the elimination, and (3) puncta sizes shrink with elimination and increase with maturation. Short-interval time-lapse imaging is now in progress to analyze the detailed behavior of puncta during the elimination and the second projection period. doi:10.1016/j.neures.2011.07.1462

P4-e13 Physiological roles of LOTUS, an endogenous Nogo receptor antagonist, in lateral olfactory tract formation Masumi Iketani , Yuji Kurihara, Yasufumi Sato, Hiromu Ito, Kuniyuki Nishiyama, Yoshio Goshima, Kohtaro Takei Dept. of Mol. Pharmacol. and Neurobiol., Grad. Sch. of Med., Yokohama City Univ., Yokohama, Japan We discovered a novel axon guidance molecule LOTUS that serves for lateral olfactory tract (LOT) development of mouse telencephalon, and identified Nogo-66 receptor (NgR1) as its binding partner. We have found that binding of NgR1 ligands such as Nogo-66 (Ng66) to NgR1 was inhibited by LOTUS coexpressed with NgR1 in vitro. We examined development of LOT visualized with DiI staining and found defasciculated axon tract and increased axon collaterals of LOT in lotus deficient mice, whereas these abnormal phenotypes were not observed in ngr1 deficient mice. Furthermore, the both of abnormal phenotypes seen in single mutant of lotus deficient mice was rescued in double mutant of lotus and ngr1 deficient mice. These findings suggest that NgR1 induces defasciculation and axonal branching of LOT and the antagonism of LOTUS to NgR1 may physiologically contribute to LOT development. Research fund: Research Fellow of the Japan Society for the Promotion of Science. doi:10.1016/j.neures.2011.07.1463

P4-e14 BubR1 regulates neurite outgrowth of neuroblastoma cells Hirotaka Yamagata , Shusaku Uchida, Koji Otsuki, Teruyuki Hobara, Tomohiko Shibata, Fumihiro Higuchi, Naoko Abe, Yoshifumi Watanabe Div. of Neuropsy., Department Neurosci., Grad. Sch. of Med., Yamaguchi Univ., Ube, Japan Neural plasticity has been suggested to play an important role in the treatment of mood disorders. Antidepressant treatment has been reported to increase neurogenesis and neural plasticity in hippocampus. For example, antidepressants have regulated cAMP-CREB pathway, which is known to increase neurogenesis in adult hippocampus. Antidepressants have also increased adult neurotrophic factor including BDNF, GDNF, and VEGF. Recently, chronic imipramine treatment reported to decrease hippocampal p21, which inhibits cyclin-dependent kinase and controls the cell cycle. In addition, p21 knockout mice increased neuronal proliferation in the hippocampus. These reports suggest that cell cycle protein can regulate anti-depressive effect. Some studies demonstrated that cell cycle proteins have regulated neural plasticity. The APC complex, which has the ubiquitin ligase activity, is critical for normal cell cycle transitions. APC recruits the coactivator Cdc20 and Cdh1, and degrades proteins to drive mitosis in cycling cells. In postmitotic neurons, APC has a crucial role for neuronal morphogenesis. Cdh1-APC has played a role in regulating axonal growth, and Cdc20-APC also has regulated dendrite morphogenesis. BubR1 has been known to inhibit