回路形成2 Circuit Formation 2
O2-7-6-1 新生仔体性感覚野2光子イメージングによるNMDA受容体依存的なバレル細胞樹状突起精緻化過程の解析 In vivo imaging of NMDAR-dependent dendritic refinement of barrel cells in neonatal somatosensory cortex ○水野秀信1,2, , 斉藤芳和3, 糸原重美3, 岩里琢治1,2 ○Hidenobu Mizuno1,2, Wenshu Luo1,2, Yoshikazu M. Saito3, Shigeyoshi Itohara3, Takuji Iwasato1,2 国立遺伝研・形質遺伝1, 総合研究大学院大学　遺伝学専攻2, 理化学研究所・脳センター・行動遺伝学技術開発チーム3 Div. Neurogenetics, National Institute of Genetics, Mishima1, Dept. Genetics, The Grad. Univ. for Advanced Studies, Mishima2, Lab. Behavioral Genetics3 Activity-dependent refinement during early postnatal period underlies establishment of precise neuronal connections. The mouse somatosensory cortex has an array of barrels corresponding to the arrangement of whiskers on the face. Layer 4 spiny stellate neurons (barrel cells) are located around the barrel wall and have dendrites with preferential orientation toward the barrel center, where thalamocortical axon (TCA) terminals are clustered. This dendritic asymmetry, which makes neurons belonging to a barrel receive the information from the corresponding single whisker, is established during neonatal stages in an NMDA receptor (NMDAR)-dependent manner. However, the precise time course of dendritic refinement of barrel cells and roles of NMDAR in this refinement remain totally unknown due to technical limitations. Here we directly observed the processes of dendritic refinement of barrel cells by two-photon in vivo time-lapse imaging. First, we developed a method based on in utero electroporation to express specific genes in a sparse population of layer 4 neurons. This method allowed us to visualize dendrites of barrel cells by RFP, and also to knockout a gene in sparsely labeled neurons by expressing Cre in floxed mice. Second, we generated a transgenic mouse line which labels TCA termini with GFP in vivo. By combining these novel approaches, we simultaneously labeled the TCA termini and dendrites of barrel cells with or without NMDAR in living newborn mice. Time-lapse imaging demonstrated that barrel cell dendrites have large motility and reinforce their orientation bias towards the barrel center by concurrent elongation and retraction of their branches both inside and outside the barrels. Dendrites of barrel cells devoid of NMDAR showed much larger motility, but they failed to reinforce their asymmetry. Our results suggest critical roles of NMDAR in neonatal cortex for the stabilization of dendritic branches, which is necessary for the dendritic refinement.	O2-7-6-2 小脳辺縁系神経回路のシナプス接続を制御する分子機構の解析 Analysis of mechanism regulating circuit connectivity in cerebellar limbic system ○桑子賢一郎1, 岡野栄之1 ○Kenichiro Kuwako1, Hideyuki Okano1 慶應義塾大学　医学部　生理学教室1 Dept Physiol, Keio Univ, Sch of Med, Tokyo1 To establish functionally precise patterns of neural connectivity, growing axons navigate long and complicated routes to reach their final destinations and form synapses with specific target cells. However, little is known about molecular mechanism regulating synaptic specificity, especially in the mammalian nervous system. The synapse formation is thought to require trans-synaptic interactions through adhesion molecules. Although numerous synaptic adhesion molecules have been identified in mammals, the molecules responsible for the establishment of specific synaptic connectivity in most circuits have not been elucidated. Moreover, gene expression program governing the circuit-specific expression of the synaptic molecules that regulate synaptic specificity is totally unknown. To reveal molecular basis for synaptic specificity, we analyzed the cerebellar limbic circuits, as a model system, that are the important afferent networks conveying information to the cerebellar cortex. The cerebellar limbic circuits are composed of the axons of precerebellar neurons including pontine neurons (PN) and inferior olivary neurons (ION). Although PN and ION have similarities in the steps of circuit formation, the axons of PN and ION form synapses exclusively and selectively with granule cells and purkinje cells, respectively. Thus, highly possible candidates regulating synaptic specificity of the cerebellar limbic circuits are likely to be expressed specifically in PN or ION at the synaptogenic stage. Through the transcriptome analysis using high purity tissue samples from PN and ION at postnatal day 12, we identified six cell adhesion molecules that were specifically expressed in PN. Furthermore, we found that a knockdown of the candidate molecule in PN using in utero electroporation method resulted in the abnormal connections of PN axons to purkinje cells in vivo. We will report additional data of functional analysis for those candidate molecules.
O2-7-6-3 転写因子Npas4によるマウス嗅球新生介在ニューロンの感覚入力依存的なスパイン形成機構 Npas4 transcription factor regulates the sensory experience-dependent dendritic spine development of newborn interneurons in the mouse olfactory bulb ○吉原誠一1, 高橋弘雄1, 木下雅仁1, 西村信城1, 永井拓2, 山田清文2, 坪井昭夫1 ○Sei-ichi Yoshihara1, Hiroo Takahashi1, Masahito Kinoshita1, Nobushiro Nishimura1, Taku Nagai2, Kiyofumi Yamada2, Akio Tsuboi1 奈良県立医科大学　先端医学研究機構　脳神経システム医科学1, 名古屋大学大学院医学系研究科医療薬学2 Lab for Mol Biol of Neural System, Nara Med Univ, Kashihara1, Dept Neuropsychopharm, Nagoya Univ Grad Sch Med, Nagoya2 Sensory experience has been shown to regulate development in a variety of species and in various structures, including the retina, cortex and olfactory bulb (OB). Within the mammalian OB specifically, the development of dendrites in mitral/tufted cells is well known to be odor experience-dependent. However, little is known about the developmental role of odor experience in the other major OB population of the GABAgenic interneurons, such as granule cells. Here we identified, by in situ hybridization screening, a transcription factor gene Npas4, whose expression in the OB interneurons is dependent on the sensory experience. Npas4 is an abbreviation for neuronal Per-Arnt-Sim (PAS) domain protein 4, whose N-terminal region contains a basic helix-loop-helix domain for DNA binding and two PAS domains involving in the adaptation of environmental factors such as odors. Overexpression of Npas4 restored a reduction of the dendritic spine-density and -head size in newborn OB granule cells under sensory deprivation, while either its knockdown or knockout in these cells showed a significant reduction. Further, overexpression of Npas4 in its knockout OB granule cells restored the reduction of the dendritic spine-density and -head size. We also present data about factors downstream of Npas4, identified by ChIP-Seq on the OB. These results demonstrate that Npas4 contributes to regulate the experience-dependent dendritic spine development of newborn interneurons and the formation of functional neural circuitry in the OB.	O2-7-6-4 皮質下I型アデニル酸シクラーゼは体性感覚マップにおける軸索末端の適切な分離に必須である Roles of subcortical adenylyl cyclase 1 in formation of whisker-related axonal patterns in mouse somatosensory system ○鈴木亜友美1,2, 糸原重美3, 岩里琢治1,2 ○Ayumi Suzuki1,2, Shigeyoshi Itohara3, Takuji Iwasato1,2 遺伝研・形質遺伝1, 総研大・遺伝学2, 理研・行動遺伝3 Div Neurogenetics, NIG, Mishima1, Dept Genetics, SOKENDAI, Mishima2, Lab Behavioral Genetics, RIKEN BSI, Wako3 Formation of functional neural circuits is thought to require refinements in the developmental stage. The mouse somatosensory system is an excellent model to investigate mechanisms underlying refinement. In mice, whisker-related patterns are recapitulated in the cortex (as barrels) and subcortical regions, the thalamus (barreloids) and brainstems (barrelettes). These patterns are consolidated during the first postnatal week through refinements. Type 1 adenylyl cyclase (AC1) is a neuron-specific enzyme that generates the major second messenger cAMP from ATP by Ca2+/CaM stimulation. AC1 is expressed throughout the somatosensory system in newborn brain. Previous studies reported that global AC1 knockout (AC1KO) mice have impaired barrels and barreloids, however cortex-specific AC1KO mice have grossly normal barrels and barreloids. Thus, subcortical AC1 may play important roles in the barrel formation. To examine this possibility, we here generated thalamus-specific AC1KO mice (Th-AC1KO). The segregation of thalamocortical axons (TCAs) was clearly reduced in Th-AC1KO mice. On the other hand, patterns in subcortical regions were grossly normal. We also generated cortex and thalamus-specific AC1KO mice (Cx;Th-AC1DKO). Phenotypes of these mice were similar to those of Th-AC1KO mice. These results demonstrate that AC1 in the thalamus is important for axonal patterning at the cortex. Our results show significance of subcortical mechanisms for neuronal circuit refinement in the somatosensory system.

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