P 3f 08 Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit

s / Neuroscience Research 71S (2011) e108–e415 e235 injection of the GABAA receptor antagonist pentylenetetrazole. Immunohistochemistry and Golgi staining revealed that the expression of cAMP in GCs significantly increased about 1 week before the appearance of prominent MF sprouting, which was in accordance with the progression of recurrent seizure. These results suggest that elevation of intracellular cAMP in GCs contributes to the establishment of aberrant network formation in the epileptic brain. doi:10.1016/j.neures. 2011.07. 1023 P3-f08 Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit Keisuke Yonehara 1 , Kamill Balint 1, Masaharu Noda 2,3, Georg Nagel 4, Ernst Bamberg 5,6, Botond Roska 1 1 Neural Circuit Lab, Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland 2 Div Mol Neurobiol, Nat Inst Basic Biol (NIBB), Okazaki 3 Sch Life Sci, The Graduate Univ for Advanced Studies (SOKENDAI), Okazaki 4 Univ Wuerzburg, Botanik I, Germany 5 Max-Planck-Institut fuer Biophysik, Germany 6 Johann Wolfgang Goethe-Univ, Institut fuer Biophysikalische Chemie, Germany Spatial asymmetries in neural connectivity have an important role in creating basic building blocks of neuronal processing. A key circuit module of directionally selective (DS) retinal ganglion cells is a spatially asymmetric inhibitory input from starburst amacrine cells. It is not known how and when this circuit asymmetry is established during development. Here we photostimulate mouse starburst cells targeted with channelrhodopsin-2 while recording from a single genetically labelled type of DS cell. We follow the spatial distribution of synaptic strengths between starburst and DS cells during early postnatal development before these neurons can respond to a physiological light stimulus, and confirm connectivity by monosynaptically restricted trans-synaptic rabies viral tracing. We show that asymmetry develops rapidly over a 2-day period through an intermediate state in which random or symmetric synaptic connections have been established. The development of asymmetric inputs involves the spatially selective reorganization of inhibitory synaptic inputs. Intriguingly, the spatial distribution of excitatory synaptic inputs from starburst cells is significantly more symmetric than that of the inhibitory inputs at the end of this developmental period. Our work demonstrates a rapid developmental switch from a symmetric to asymmetric input distribution for inhibition in the neural circuit of a principal cell. Research fund: The Friedrich Miescher Institute for Biomedical Research, a US Office of Naval Research Naval International Cooperative Opportunities in Science and Technology Program grant, a Marie Curie Excellence grant, a National Centre of Competence in Research Frontiers in Genetics grant, an European Research Council as well as RETICIRC, TREATRUSH and OPTONEURO grants from the European Union to B.R. and an EMBO Long-Term Fellowship to K.Y. doi:10.1016/j.neures. 2011.07. 1024 P3-f09 Npas4 regulates neurite outgrowth and phosphoryation of synapsin I Jaesuk Yun 1 , Taku Nagai 1, Yoko Hibi 1, Keisuke Kuroda 2, Kozo Kaibuchi 2, Kiyofumi Yamada 1 1 Dep Neuropsychopharmacol and Hosp Pharm, Grad Sch of Med, Nagoya Univ, Nagoya, Japan 2 Dep of Cell Pharmacol, Grad Sch of Med, Nagoya Univ, Nagoya, Japan We have previously reported that the mRNA levels of neuronal PAS domain protein 4 (Npas4), a brain specific basic helix–loop–helix transcription factor, are decreased in the hippocampus of mice following a chronic social isolation or restriction stress. A recent study has demonstrated that Npas4 regulates the development of GABAergic inhibitory neurons. In the presents study, treatment with LiCl in Neuro2a cells resulted in an increase in Npas4 mRNA and phosphorylated-synapsin I protein expression levels, which were associated with the neurite outgrowth. The knock down of Npas4 significantly inhibited the neurite outgrowth of Neuro2a cells induced by LiCl whereas the overexpression of Npas4 gene potentiated it. Overexpression of Npas4 in Neuro2a cells led to increase in the expression level of NeuN and phosphorylated synapsin I respectively. In primary cultured hippocampal neurons, Npas4 overexpression significantly increased in the protein level of phosphorylated synapsin I. These results suggest that Npas4 plays a role in the structural and functional plasticity of neurons. doi:10.1016/j.neures. 2011.07. 1025 P3-f10 Control of rotational neuronal motility by engineering of molecular motors Atsushi Tamada 1,2 , Hiroyuki Kamiguchi 1 1 Lab. for Neuronal Growth Mechanisms, RIKEN BSI, Japan 2 Decoding and Controlling Brain Information, PRESTO, JST, Japan In the developing nervous system, neurons can extend long processes called neurites toward their final targets. It is well established that the direction of neurite elongation is controlled by various guidance cues in the environment. Even in the absence of extrinsic directional signals, however, neurites turn rightward or clockwise on two-dimensional substrates (Heacock and Agranoff, Science 198, 64, 1977). Inspired by this asymmetric phenomenon, we discovered a novel neuronal motility where growth cone filopodia at the tip of neurites autonomously rotate in the direction of a right-handed screw (Tamada et al., J. Cell Biol. 188, 429, 2010). We further provided evidence that the right-screw filopodial rotation causes the rightward neurite turning and that it is driven by myosin V, a left-handed spiral molecular motor along a right-handed double-helical actin filament. These findings reveal the hierarchical structure of the rotational motility from molecular interactions to neuronal behavior. Here we hypothesize that such asymmetry at the molecular and cellular levels affect brain organization, including the formation of precise neural circuits and the emergence of structural and functional leftright brain asymmetry. To test this, we have been developing the methods for manipulating the filopodial rotation. We first overexpressed myosin II, an opposite right-handed spiral motor, but it did not change the filopodial rotation. Next, we generated myosin V motors with different length of the neck domain, which could be the determinant of the step size and the spiral motility. Overexpression of these mutants accelerated the filopodial rotation. We are currently testing the effect of insertion of rigid peptides in the neck domain. Generation of engineered myosin motors with diminished, accelerated or reversed spiral motility would be an effective tool for assessing the functional roles of the rotational neuronal motility. Research fund: JST PRESTO, KAKENHI (18500255, 23111532). doi:10.1016/j.neures. 2011.07. 1026 P3-f11 Excitatory cortical neurons with multipolar shape establish neuronal polarity by forming a tangentially oriented axon in the intermediate zone Yumiko Hatanaka 1,2 , Kenta Yamauchi 3 1 Div Cerebral Circurity, NIPS, Okazaki, Japan 2 CREST, Japan 3 Grad Sch of Frontier Biosci, Osaka Univ, Osaka, Japan Although the processes of neuronal polarity formation have been extensively studied using neurons in dissociated culture, the corresponding developmental processes in vivo are still unclear. In particular, whether neuronal polarization relates to the apico-basal polarity of the neuroepithelial progenitor remains an open question. Here we illuminate the initial steps of morphological polarization of excitatory cortical neurons in situ, by sparsely labeling their neuroepithelial progenitors using in utero electroporation and then examining their neuronal progeny in brain sections and in slice cultures. Morphological analysis showed that an axon-like long tangential process formed in progeny cells in the intermediate zone (IZ). Time-lapse imaging analysis using slice culture revealed that progeny cells with multipolar shape, after alternately extending and retracting their processes for several hours, suddenly elongated a long process tangentially. These cells then transformed into a bipolar shape, extending a pia-directed leading process, and migrated radially leaving a tangential process behind, which gave rise to an “L-shaped” axon. These findings suggest that neuronal polarity in these cells is established de novo from a non-polarized stage in vivo, and indicate that excitatory cortical neurons with multipolar shape in the IZ initiate axon outgrowth before radial migration into the cortical plate. Research fund: KAKENHI 22500310. doi:10.1016/j.neures. 2011.07. 1027 P3-f12 Analyses of nervous system in knockout mice deficient in SNAREs Akihiro Harada Dept. Cell Biol., Grad. Sch. Med., Osaka Univ., Osaka, Japan SNAREs are known to mediate fusion between vesicles and target membranes. Though some SNAREs are known to be involved in axonal elongation, their roles in vivo has been largely elusive. We produced conventional and conditional knockout mice for various SNAREs believed to be involved in axonal elongation. The results of knockout mice of syntaxin3, VAMP7 and