研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    渡辺 雅彦(ワタナベ マサヒコ), ワタナベ マサヒコ

所属(マスター)

  • 医学研究院 生理系部門 解剖学分野

所属(マスター)

  • 医学研究院 生理系部門 解剖学分野

独自項目

syllabus

  • 2020, 大学院共通授業科目(教育プログラム):外科系臨床医学研究の新展開 臨床医学・外科解剖セミナー, Inter-Graduate School Classes:New Developments in Clinical Medical Research in Surgery - Seminar of clinical medicine and surgical anatomy, 修士課程, 大学院共通科目, 臨床医学、臨床解剖、外科教育、献体を使用した手術手技研修(Cadaver Surgical Training: CST)、医療機器開発
  • 2020, 医理工学研究概論, Introduction to Biomedical Science and Engineering Research, 修士課程, 医理工学院, 動物実験、図書館、電子ジャーナル、共同利用施設、RI実験
  • 2020, 基本医学研究概論, Introduction to Basic Medical Research, 修士課程, 医学院, 動物実験、図書館、電子ジャーナル、共同利用施設、RI実験 Animal experiments, library, electric journals, common facilities, radioisotopic experiments
  • 2020, 大学院共通授業科目(教育プログラム):脳科学研究の展開, Inter-Graduate School Classes(Educational Program):Brain Science Research, 修士課程, 大学院共通科目, 脳科学,生命科学,方法論,脳機能計測
  • 2020, 大学院共通授業科目(教育プログラム):脳科学入門, Inter-Graduate School Classes(Educational Program):Basic Brain Science, 修士課程, 大学院共通科目, ニューロン、グリア、脳、神経機能
  • 2020, 大学院共通授業科目(教育プログラム):脳科学研究の展開, Inter-Graduate School Classes(Educational Program):Brain Science Research, 修士課程, 大学院共通科目, 脳科学研究、口頭発表、研究討論、合宿研修
  • 2020, 大学院共通授業科目(教育プログラム):脳科学研究の展開, Inter-Graduate School Classes(Educational Program):Brain Science Research, 修士課程, 大学院共通科目, 脳科学、講演、セミナー
  • 2020, 大学院共通授業科目(教育プログラム):外科系臨床医学研究の新展開 臨床解剖概論, Inter-Graduate School Classes:New Developments in Clinical Medical Research in Surgery - Outline of clinical anatomy, 修士課程, 大学院共通科目, 臨床医学、臨床解剖、外科教育、献体を使用した手術手技研修(Cadaver Surgical Training: CST)、医療機器開発
  • 2020, 基本医学研究, Master's Thesis Research in Medical Sciences, 修士課程, 医学院, 解剖学 Anatomy
  • 2020, 基本医学総論, Basic Principles of Medicine, 修士課程, 医学院, 脳、シナプス、解剖学brain, synapse, anatomy
  • 2020, 大学院共通授業科目(教育プログラム):脳科学入門, Inter-Graduate School Classes(Educational Program):Basic Brain Science, 修士課程, 大学院共通科目, 脳解剖実習
  • 2020, 基本医学研究法Ⅰ, Basic Research Methods in Medical Sciences, 修士課程, 医学院, 細胞小器官、膜の構造と特性、核の構造と細胞分裂、上皮組織、結合組織、細胞外マトリックス、血球 Organelles, membrane structure and properties, nuclear structure and cell division, epithelial tissue, connective tissue, extracellular matrix, blood cells
  • 2020, 医学研究概論, Introduction to Medical Research, 博士後期課程, 医学院, 動物実験、図書館、電子ジャーナル、共同利用施設、RI実験 Animal experiments, library, electric journals, common facilities, radioisotopic experiments
  • 2020, 医学総論, Principles of Medicine, 博士後期課程, 医学院, 脳、シナプス、解剖学
  • 2020, 基盤医学研究, Dissertation Research in Medical Sciences, 博士後期課程, 医学院, 解剖学
  • 2020, 医学研究法Ⅰ, Research Methods in Medical Sciences Ⅰ, 博士後期課程, 医学院, 解剖学、組織学 Anatomy, Histoloty
  • 2020, 解剖学実習, Human Gross Dissection, 学士課程, 医学部, 人体解剖学、解剖学実習
  • 2020, 解剖学(神経解剖学), Anatomy(Neuroanatomy), 学士課程, 医学部, ヒト脳、神経解剖学、ニューロン、シナプス、グリア、伝達物質、受容体
  • 2020, 解剖学(解剖発生学), Anatomy(Anatomy & Embryology), 学士課程, 医学部, 人体解剖学

timetable

  • 修士課程, 医学院, 2020, 研究発表技法Ⅰ
  • 修士課程, 医学院, 2020, 研究発表技法Ⅱ
  • 博士後期課程, 医学研究科, 2020, 医学研究概論
  • 博士後期課程, 医学研究科, 2020, 医学研究法Ⅰ
  • 博士後期課程, 医学研究科, 2020, 医学総論
  • 博士後期課程, 医学院, 2020, 研究発表技法Ⅰ
  • 博士後期課程, 医学院, 2020, 研究発表技法Ⅱ

PositionHistory

  • 大学院医学研究院附属動物実験施設長, 2017年4月1日, 2019年3月31日
  • 大学院医学研究院附属動物実験施設長, 2019年4月1日, 2021年3月31日

researchmap

プロフィール情報

学位

  • 博士(医学)(筑波大学)

プロフィール情報

  • プロフィール

    昭和59年3月 東北大学医学部卒
    昭和63年3月 筑波大学大学院医学研究科博士課程修了、医学博士
    昭和63年4月 金沢大学医学部助手(解剖学第1講座)
    平成2年1月 東北大学医学部助手(解剖学第2講座)
    平成4年2月 北海道大学医学部助教授(解剖学第1講座)
    (平成5年10月~平成6年7月 文部省在外研究員として米国ノースカロライナ大学
    「相同組み替えによる実験モデル動物の作成と解析に関する研究」)
    平成10年11月 北海道大学大学院医学研究科教授
    平成29年4月 北海道大学大学院医学研究院教授、現在に至る

    研究専門分野:分子神経解剖学

    担当学部教育:解剖学実習(6単位)、解剖発生学(2単位)、神経解剖学(1単位)
  • 渡辺, ワタナベ
  • 雅彦, マサヒコ
  • ID各種

    200901014585072736

対象リソース

業績リスト

研究キーワード

  • 包括脳ネットワーク・研究集会委員会   包括脳ネットワーク・リソース・技術開発委員会   包括脳ネットワーク・包括支援委員会・脳神経回路機能   包括脳ネットワーク   統合脳・神経回路機能   嗅神経被覆グリア   遺伝子欠損マウス   登上繊維   錐体細胞   ラット   GTP結合蛋白   GluRδ2   可塑性   シナプス可塑性   プルキン工細胞   バーグマングリア   グルタミン酸トランスポーター   3PGDH   星状膠細胞   海馬   NMDA受容体   NMDA受容体チャネル   グルタミン酸   脳   ノックアウトマウス   平行線維   シナプス   登上線維   免疫組織化学   マウス   シナプス形成   発達   プルキンエ細胞   小脳   グルタミン酸受容体   

研究分野

  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経形態学

経歴

  • 2008年 - 現在 北海道大学 医学(系)研究科(研究院) 教授
  • 2007年 北海道大学 医学(系)研究科(研究院)・医学研究科・医学研究院・医学部 大学院・教授
  • 2006年 北海道大学 医学(系)研究科(研究院)・大学院医学研究科・医学研究科 教授
  • 2005年 北海道大学 医学(系)研究科(研究院)・医学研究科 大学院・教授
  • 2004年 北海道大学 医学(系)研究科(研究院)・大学院医学研究科・医学研究科 大学院・教授
  • 2001年 - 2003年 北海道大学 医学(系)研究科(研究院)・医学研究科 大学院・教授
  • 1999年 - 2000年 北海道大学 医学部・医学研究科 大学院・教授
  • 1998年 北海道大学 医学部・医学研究科 大学院・助教授・教授
  • 1993年 - 1997年 北海道大学 医学部 助教授

学歴

  • 1984年04月 - 1988年03月   筑波大学   大学院医学研究科
  • 1978年04月 - 1984年03月   東北大学   医学部   医学科

受賞

  • 2019年10月 伊藤医薬学術交流財 伊藤太郎学術賞
     グルタミン酸伝達系による神経回路発達の分子細胞基盤
  • 2015年07月 日本神経科学学会 時実利彦記念賞
     「神経活動依存的な 神経回路発達と回路機能発現に関する分子解剖学的研究」 
    受賞者: 渡辺 雅彦
  • 2015年03月 北海道大学 北海道大学総長研究賞(優秀賞)
     
    受賞者: 渡辺 雅彦
  • 2006年03月 北海道大学医学研究科 北海道大学医学研究科優秀研究賞
     「小脳シナプス回路網の競合的発達を制御する分子機構の解明」 
    受賞者: 渡辺 雅彦
  • 1996年04月 日本解剖学会 日本解剖学会奨励賞
     「中枢神経系の発達とNMDA型グルタミン酸受容体発現調節」 
    受賞者: 渡辺 雅彦
  • 1992年01月 東北医学会 東北医学会奨励賞
     「14-3-3蛋白の遺伝子解析とその脳内発現」 
    受賞者: 渡辺 雅彦

論文

  • Toshikazu Kakizaki, Tomokazu Ohshiro, Makoto Itakura, Kohtarou Konno, Masahiko Watanabe, Hajime Mushiake, Yuchio Yanagawa
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 35 2 e21224  2021年02月 
    GABA is synthesized by glutamate decarboxylase (GAD), which has two isoforms, namely, GAD65 and GAD67, encoded by the Gad2 and Gad1 genes, respectively. GAD65-deficient (Gad2-/- ) mice exhibit a reduction in brain GABA content after 1 month of age and show spontaneous seizures in adulthood. Approximately 25% of Gad2-/- mice died by 6 months of age. Our Western blot analysis demonstrated that the protein expression ratio of GAD65 to GAD67 in the brain was greater in rats than in mice during postnatal development, suggesting that the contribution of each GAD isoform to GABA functions differs between these two species. To evaluate whether GAD65 deficiency causes different phenotypes between rats and mice, we generated Gad2-/- rats using TALEN genome editing technology. Western blot and immunohistochemical analyses with new antibodies demonstrated that the GAD65 protein was undetectable in the Gad2-/- rat brain. Gad2-/- pups exhibited spontaneous seizures and paroxysmal discharge in EEG at postnatal weeks 3-4. More than 80% of the Gad2-/- rats died at postnatal days (PNDs) 17-23. GABA content in Gad2-/- brains was significantly lower than those in Gad2+/- and Gad2+/+ brains at PND17-19. These results suggest that the low levels of brain GABA content in Gad2-/- rats may lead to epilepsy followed by premature death, and that Gad2-/- rats are more severely affected than Gad2-/- mice. Considering that the GAD65/GAD67 ratio in human brains is more similar to that in rat brains than in mouse brains, Gad2-/- rats would be useful for further investigating the roles of GAD65 in vivo.
  • Marta Valle-León, Luis F Callado, Ester Aso, María M Cajiao-Manrique, Kristoffer Sahlholm, Marc López-Cano, Concepció Soler, Xavier Altafaj, Masahiko Watanabe, Sergi Ferré, Víctor Fernández-Dueñas, José M Menchón, Francisco Ciruela
    Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 46 3 665 - 672 2021年02月 
    According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A2A receptors (A2ARs) or their degree of functional heteromerization with dopamine D2 receptors (D2R). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A2AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A2AR-D2R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A2AR knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D2R without changes in A2AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A2AR-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A2AR-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A2AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A2AR-D2R heteromers. The degree of A2AR-D2R heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.
  • Hajime Shiotani, Muneaki Miyata, Takeshi Kameyama, Kenji Mandai, Miwako Yamasaki, Masahiko Watanabe, Kiyohito Mizutani, Yoshimi Takai
    The Journal of comparative neurology 529 2 450 - 477 2021年02月 [査読有り][通常論文]
     
    The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and β-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, β-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.
  • Shigeo Miyata, Toshikazu Kakizaki, Kazuyuki Fujihara, Hideru Obinata, Touko Hirano, Junichi Nakai, Mika Tanaka, Shigeyoshi Itohara, Masahiko Watanabe, Kenji F Tanaka, Manabu Abe, Kenji Sakimura, Yuchio Yanagawa
    Molecular brain 14 1 5 - 5 2021年01月07日 
    Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the Gad1 gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed Gad1tTA/STOP-tetO biallelic knock-in mice using Gad1STOP-tetO and Gad1tTA knock-in mice, and compared them with Gad1+/+ mice. The expression level of GAD67 protein in brains of Gad1tTA/STOP-tetO mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1tTA/STOP-tetO mice. In the open-field test, Dox-treated Gad1tTA/STOP-tetO mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1tTA/STOP-tetO mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.
  • Anussara Kamnate, Juthathip Sirisin, Yada Polsan, Surang Chomphoo, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Journal of anatomy 2021年01月04日 
    In order to understand the mechanism of the endocannabinoid (eCB) signal, which has so far been shown to work in oocyte genesis and maturation, it is critical to clarify detailed localization of the eCB synthesizing enzyme molecules as well as receptors for eCBs in oocytes in the ovary in situ. For this purpose, diacylglycerol lipase (DGL) α and β are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLα/β and the cannabinoid receptor 1 (CB1) for 2-AG were shown to be localized to the primary oocytes of postnatal mice using immuno-light and electron microscopy. It was found that two types of localization existed: first, immunoreactivities for DGLα and β were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible. Secondly, DGLβ-immunoreactivity was distinctly confined to the nuage of Balbiani bodies and small nuage-derivative structures; both amorphous materials and membranes of vesicles were responsible for their localization. On the other hand, the weak immunoreactivity for CB1 was localized in a pattern similar to the first one for DGLs, but not found in a pattern for the Balbiani nuage. Two routes of functional exertion of 2-AG synthesized by DGLs were suggested from the two types of localization: one was that the eCB synthesized at all the sites of DGLs is released from the oocytes and exerts paracrine or autocrine effects on adjacent intra-ovarian cells as well as the oocytes themselves. The other was that the eCB synthesized within the nuage was involved in the modulation of the posttranscriptional processing of oocytes. Owing to the failure in the detection of CB1 in the Balbiani nuage, however, the validity of the latter possibility remains to be elucidated.
  • Toshinori Matsuoka, Miwako Yamasaki, Manabu Abe, Yukiko Matsuda, Hiroyuki Morino, Hideshi Kawakami, Kenji Sakimura, Masahiko Watanabe, Kouichi Hashimoto
    The Journal of physiology 599 2 547 - 569 2021年01月 
    KEY POINTS: Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so-called 'resonant properties'). Here, we report that heterologous expression of mouse Kv11 voltage-dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties. Kv11.3-expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor. The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice. This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. ABSTRACT: The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor-like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+ ) channels, encoded by ether-á-go-go-related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole-cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3-, Kv11.1(Merg1b)- and Kv11.1(Merg1a)-expressing cells were 2-6 Hz, 2-4 Hz, and 0.6-0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor-inductor-capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11-dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at -30 to -60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency-dependent responses in a subtype-dependent manner from resting to depolarized membrane potentials.
  • Péter Szocsics, Péter Papp, László Havas, Masahiko Watanabe, Zsófia Maglóczky
    Brain structure & function 226 1 281 - 296 2021年01月 
    Betz cells-the gigantopyramidal neurons found in high amount in the primary motor cortex-are among of the most characteristic neuronal cells. A part of them contains the calcium-binding protein parvalbumin (PV) in primates. However, less is known about these cells in the human motor cortex despite their important role in different neurological disorders. Therefore, the aim of our study was to investigate the neurochemical features and perisomatic input properties of Betz cells in control human samples with short post-mortem interval. We used different microscopic techniques to investigate the primary motor cortex of both hemispheres. The soma size and density, and expression of PV of the Betz cells were investigated. Furthermore, we used confocal fluorescent and electron microscopy to examine their perisomatic input. The soma size and density showed moderate variability among samples and hemispheres. Post-mortem interval and hemispherical localization did not influence these features. Around 70% of Betz cells expressed PV, but in less intensity than the cortical interneurons. Betz neurons receive dense perisomatic input, which are mostly VIAAT- (vesicular inhibitory amino acid transporter) and PV immunopositive. In the electron microscope, we found PV-immunolabelled terminals with asymmetric-like synaptic structure, too. Terminals with morphologically similar synaptic specialisation were also found among vGluT2- (vesicular glutamate transporter type 2) immunostained terminals contacting Betz cells. Our data suggest that Betz cells' morphological properties showed less variability among subjects and hemispheres than the density of them. Their neurochemical and perisomatic input characteristics support their role in execution of fast and precise movements.
  • Motokazu Uchigashima, Kohtarou Konno, Emily Demchak, Amy Cheung, Takuya Watanabe, David G Keener, Manabu Abe, Timmy Le, Kenji Sakimura, Toshikuni Sasaoka, Takeshi Uemura, Yuka Imamura Kawasawa, Masahiko Watanabe, Kensuke Futai
    eLife 9 2020年12月23日 
    Synapse formation and regulation require signaling interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn signaling generates distinct functional properties at synapses.
  • Kazuyuki Fujihara, Kazuo Yamada, Yukio Ichitani, Toshikazu Kakizaki, Weiru Jiang, Shigeo Miyata, Takashi Suto, Daiki Kato, Shigeru Saito, Masahiko Watanabe, Yuki Kajita, Tomokazu Ohshiro, Hajime Mushiake, Yoshiki Miyasaka, Tomoji Mashimo, Hiroki Yasuda, Yuchio Yanagawa
    Translational psychiatry 10 1 426 - 426 2020年12月08日 
    GABAergic dysfunctions have been implicated in the pathogenesis of schizophrenia, especially the associated cognitive impairments. The GABA synthetic enzyme glutamate decarboxylase 67-kDa isoform (GAD67) encoded by the GAD1 gene is downregulated in the brains of patients with schizophrenia. Furthermore, a patient with schizophrenia harboring a homozygous mutation of GAD1 has recently been discovered. However, it remains unclear whether loss of function of GAD1 leads to the symptoms observed in schizophrenia, including cognitive impairment. One of the obstacles faced in experimental studies to address this issue is the perinatal lethality of Gad1 knockout (KO) mice, which precluded characterization at the adult stage. In the present study, we successfully generated Gad1 KO rats using CRISPR/Cas9 genome editing technology. Surprisingly, 33% of Gad1 KO rats survived to adulthood and could be subjected to further characterization. The GABA concentration in the Gad1 KO cerebrum was reduced to ~52% of the level in wild-type rats. Gad1 KO rats exhibited impairments in both spatial reference and working memory without affecting adult neurogenesis in the hippocampus. In addition, Gad1 KO rats showed a wide range of behavioral alterations, such as enhanced sensitivity to an NMDA receptor antagonist, hypoactivity in a novel environment, and decreased preference for social novelty. Taken together, the results suggest that Gad1 KO rats could provide a novel model covering not only cognitive deficits but also other aspects of the disorder. Furthermore, the present study teaches an important lesson: differences between species should be considered when developing animal models of human diseases.
  • Erika Polgár, Andrew M Bell, Maria Gutierrez-Mecinas, Allen C Dickie, Oğuz Akar, Miruna Costreie, Masahiko Watanabe, Andrew J Todd
    Neuroscience 450 113 - 125 2020年12月01日 
    The tachykinin peptide substance P (SP) is expressed by many interneurons and some projection neurons in the superficial dorsal horn of the spinal cord. We have recently shown that SP-expressing excitatory interneurons in lamina II correspond largely to a morphological class known as radial cells. However, little is known about their function, or their synaptic connectivity. Here we use a modification of the Brainbow technique to define the excitatory synaptic input to SP radial cells. We show that around half of their excitatory synapses (identified by expression of Homer) are from boutons with VGLUT2, which are likely to originate mainly from local interneurons. The remaining synapses presumably include primary afferents, which generally have very low levels of VGLUT2. Our results also suggest that the SP cells are preferentially innervated by a population of excitatory interneurons defined by expression of green fluorescent protein under control of the gene for gastrin-releasing peptide, and that they receive sparser input from other types of excitatory interneuron. We show that around 40% of lamina I projection neurons express Tac1, the gene encoding substance P. Finally, we show that silencing Tac1-expressing cells in the dorsal horn results in a significant reduction in reflex responses to cold and radiant heat, but does not affect withdrawal to von Frey hairs, or chloroquine-evoked itch.
  • Ryoji Fukabori, Yoshio Iguchi, Shigeki Kato, Kazumi Takahashi, Satoshi Eifuku, Shingo Tsuji, Akihiro Hazama, Motokazu Uchigashima, Masahiko Watanabe, Hiroshi Mizuma, Yilong Cui, Hirotaka Onoe, Keigo Hikishima, Yasunobu Yasoshima, Makoto Osanai, Ryo Inagaki, Kohji Fukunaga, Takuma Nishijo, Toshihiko Momiyama, Richard Benton, Kazuto Kobayashi
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 43 8367 - 8385 2020年10月21日 [査読無し][通常論文]
     
    The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and β-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.
  • Hiroaki Sacai, Kazuto Sakoori, Kohtarou Konno, Kenichiro Nagahama, Honoka Suzuki, Takaki Watanabe, Masahiko Watanabe, Naofumi Uesaka, Masanobu Kano
    Nature communications 11 1 5140 - 5140 2020年10月12日 
    Autism spectrum disorder (ASD) is thought to result from deviation from normal development of neural circuits and synaptic function. Many genes with mutation in ASD patients have been identified. Here we report that two molecules associated with ASD susceptibility, contactin associated protein-like 2 (CNTNAP2) and Abelson helper integration site-1 (AHI1), are required for synaptic function and ASD-related behavior in mice. Knockdown of CNTNAP2 or AHI1 in layer 2/3 pyramidal neurons of the developing mouse prefrontal cortex (PFC) reduced excitatory synaptic transmission, impaired social interaction and induced mild vocalization abnormality. Although the causes of reduced excitatory transmission were different, pharmacological enhancement of AMPA receptor function effectively restored impaired social behavior in both CNTNAP2- and AHI1-knockdown mice. We conclude that reduced excitatory synaptic transmission in layer 2/3 pyramidal neurons of the PFC leads to impaired social interaction and mild vocalization abnormality in mice.
  • Sho Kakizawa, Yasushi Kishimoto, Shinichiro Yamamoto, Kazuko Onga, Kunihiko Yasuda, Yoshiaki Miyamoto, Masahiko Watanabe, Ryuichi Sakai, Nozomu Mori
    Scientific reports 10 1 14475 - 14475 2020年09月02日 
    Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.
  • Kunimichi Suzuki, Jonathan Elegheert, Inseon Song, Hiroyuki Sasakura, Oleg Senkov, Keiko Matsuda, Wataru Kakegawa, Amber J Clayton, Veronica T Chang, Maura Ferrer-Ferrer, Eriko Miura, Rahul Kaushik, Masashi Ikeno, Yuki Morioka, Yuka Takeuchi, Tatsuya Shimada, Shintaro Otsuka, Stoyan Stoyanov, Masahiko Watanabe, Kosei Takeuchi, Alexander Dityatev, A Radu Aricescu, Michisuke Yuzaki
    Science (New York, N.Y.) 369 6507 2020年08月28日 
    Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.
  • Andrew M Bell, Maria Gutierrez-Mecinas, Anna Stevenson, Adrian Casas-Benito, Hendrik Wildner, Steven J West, Masahiko Watanabe, Andrew J Todd
    Scientific reports 10 1 13176 - 13176 2020年08月06日 
    Dorsal horn excitatory interneurons that express gastrin-releasing peptide (GRP) are part of the circuit for pruritogen-evoked itch. They have been extensively studied in a transgenic line in which enhanced green fluorescent protein (eGFP) is expressed under control of the Grp gene. The GRP-eGFP cells are separate from several other neurochemically-defined excitatory interneuron populations, and correspond to a class previously defined as transient central cells. However, mRNA for GRP is widely distributed among excitatory interneurons in superficial dorsal horn. Here we show that although Grp mRNA is present in several transcriptomically-defined populations, eGFP is restricted to a discrete subset of cells in the GRP::eGFP mouse, some of which express the neuromedin receptor 2 and likely belong to a cluster defined as Glut8. We show that these cells receive much of their excitatory synaptic input from MrgA3/MrgD-expressing nociceptive/pruritoceptive afferents and C-low threshold mechanoreceptors. Although the cells were not innervated by pruritoceptors expressing brain natriuretic peptide (BNP) most of them contained mRNA for NPR1, the receptor for BNP. In contrast, these cells received only ~ 10% of their excitatory input from other interneurons. These findings demonstrate that the GRP-eGFP cells constitute a discrete population of excitatory interneurons with a characteristic pattern of synaptic input.
  • Kazuyoshi Yamazaki, Masahito Kawabori, Toshitaka Seki, Soichiro Takamiya, Takahiro Tateno, Kotaro Konno, Masahiko Watanabe, Kiyohiro Houkin
    Journal of neurotrauma 37 15 1720 - 1728 2020年08月01日 [査読有り][通常論文]
     
    Neuropathic pain severely impairs rehabilitation and quality of life after spinal cord injury (SCI). The sphingosine-1-phosphate receptor agonist, FTY720, plays an important protective role in neuronal injury. This study aims to examine the effects of FTY720 in a rat acute SCI model, focusing on neuropathic pain. Female rats with SCI induced by 1-min clip compression were administered vehicle or 1.5 mg/kg of FTY720 24 h after the injury. Using the mechanical nociceptive threshold test, we monitored neuropathic pain and performed histological analysis of the pain pathway, including the μ opioid receptor (MOR), hydroxytryptamine transporter (HTT), and calcitonin gene-related peptide (CGRP). Motor score, SCI lesion volume, residual motor axons, inflammatory response, glial scar, and microvascular endothelial dysfunction were also compared between the two groups. FTY720 treatment resulted in significant attenuation of post-traumatic neuropathic pain. It also decreased systemic and local inflammation, thereby reducing the damaged areas and astrogliosis and resulting in motor functional recovery. Whereas there was no difference in the CGRP expression between the two groups, FTY720 significantly preserved the MOR in both the caudal and rostral areas of the spinal dorsal horn. Whereas HTT was preserved in the FTY720 group, it was significantly increased in the rostral side and decreased in the caudal side of the injury in the vehicle group. These results suggest that FTY720 ameliorates post-traumatic allodynia through regulation of neuroinflammation, maintenance of the blood-brain barrier, and inhibition of glial scar formation, thereby preserving the connectivity of the descending inhibitory pathway and reducing neuropathic pain.
  • Keitaro Yoshida, Iku Tsutsui-Kimura, Anna Kono, Akihiro Yamanaka, Kenta Kobayashi, Masahiko Watanabe, Masaru Mimura, Kenji F Tanaka
    Cell reports 31 13 107829 - 107829 2020年06月30日 
    Medium spiny neurons (MSNs) of mice show opposing activities upon the initiation of a food-seeking lever press task. Ventromedial striatal (VMS)-MSNs are inhibited but ventrolateral striatal (VLS)-MSNs are activated; these activities mediate action selection and action initiation, respectively. To understand what input shapes the opposing MSN activities, here, we monitor cortical input activities at the cell population level and artificially reverse them. We demonstrate that the ventral hippocampus (vHP) and the insular cortex (IC) are major inputs to the VMS and VLS, both projections show silencing at the trial start time, and the vHP-VMS and IC-VLS pathways form functionally coupled input-output units during the task. Of note, the upstream IC silencing is converted to the downstream VLS-MSN activation. We find biased localization of striatal parvalbumin-expressing interneurons (PV INs) and verify PV IN-dependent feedforward architecture in the VLS. Our results reveal a distinct mode of cortico-striatal signal conveyance via feedforward disinhibition in behaving animals.
  • Shin-Ichiro Horigane, Shun Hamada, Satoshi Kamijo, Hirokazu Yamada, Miwako Yamasaki, Masahiko Watanabe, Haruhiko Bito, Toshihisa Ohtsuka, Sayaka Takemoto-Kimura
    Neuroscience research 2020年06月26日 
    Increasing evidence has shown that voltage-gated L-type Ca2+ channels (LTCCs) are crucial for neurodevelopmental events, including neuronal differentiation/migration and neurite morphogenesis/extension. However, the time course of their functional maturation during the development of excitatory neurons remains unknown. Using a combination of fluorescence in situ hybridization and in utero electroporation-based labeling, we found that the transcripts of Cacna1c and Cacna1d, which encode the LTCC pore-forming subunits, were upregulated in the intermediate zone (IZ) during radial migration. Ca2+ imaging using GCaMP6s in acute brain slices showed spontaneous Ca2+ transients in migrating neurons throughout the IZ. Neurons in the IZ upper layer, especially in the multipolar-to-bipolar transition layer (TL), exhibited more frequent Ca2+ transients than adjacent layers and responded to FPL64176, a potent activator of LTCC. Consistently, nimodipine, an LTCC blocker, inhibited spontaneous Ca2+ transients in neurons in the TL. Collectively, we showed a hitherto unknown increased prevalence of LTCC-dependent Ca2+ transients in the TL of the IZ upper layer during the radial migration of excitatory neurons, which could be essential for the regulation of Ca2+-dependent neurodevelopmental processes.
  • Motokazu Uchigashima, Ming Leung, Takuya Watanabe, Amy Cheung, Timmy Le, Sabine Pallat, Alexandre Luis Marques Dinis, Masahiko Watanabe, Yuka Imamura Kawasawa, Kensuke Futai
    The Journal of biological chemistry 295 25 8589 - 8595 2020年06月19日 
    Synapse formation is a dynamic process essential for the development and maturation of the neuronal circuitry in the brain. At the synaptic cleft, trans-synaptic protein-protein interactions are major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity, and dysregulation of the E-I balance has been implicated in neurodevelopmental disorders, including autism spectrum disorders. However, the molecular mechanisms underlying the E-I balance remain to be elucidated. Here, using single-cell transcriptomics, immunohistochemistry, and electrophysiology approaches to murine CA1 pyramidal neurons obtained from organotypic hippocampal slice cultures, we investigate neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that the NLGN3 protein differentially regulates inhibitory synaptic transmission in a splice isoform-dependent manner at hippocampal CA1 synapses. We also found that distinct subcellular localizations of the NLGN3 isoforms contribute to the functional differences observed among these isoforms. Finally, results from single-cell RNA-Seq analyses revealed that Nlgn1 and Nlgn3 are the major murine Nlgn genes and that the expression levels of the Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons. Our results delineate isoform-specific effects of Nlgn genes on the E-I balance in the murine hippocampus.
  • Masahiro Fukaya, Takeyuki Sugawara, Yoshinobu Hara, Makoto Itakura, Masahiko Watanabe, Hiroyuki Sakagami
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 22 4277 - 4296 2020年05月27日 
    Brefeldin A-resistant ArfGEF 2 (BRAG2) [or Iqsec1 (IQ motif and Sec7 domain-containing protein 1)] is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of AMPA receptors (AMPARs) through the direct interaction during the hippocampal long-term depression. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Herein, using mouse brains of both sexes, we demonstrated that BRAG2a, an alternative isoform with a long C-terminal insert containing a proline-rich domain and type I PDZ-binding motif, was selectively localized to the excitatory postsynaptic density (PSD). Using yeast two-hybrid screening, we identified PSD-95 and endophilin 1/3 as BRAG2a-binding partners in the brain. The interaction with PSD-95 was required for synaptic targeting of BRAG2a. In cultured hippocampal neurons, stimulation of group I metabotropic glutamate receptors (mGluRs) increased the interaction of BRAG2a with endophilin 3 and concomitant Arf6 activation in a time-dependent manner. Knockdown of BRAG2 in cultured hippocampal neurons blocked the mGluR-dependent decrease in surface AMPAR levels, which was rescued by introducing wild-type BRAG2a, but not wild-type BRAG2b or BRAG2a mutants lacking the ability to activate Arf6 or to interact with endophilin 3 or PSD-95. Further postembedding immunoelectron microscopic analysis revealed the preorganized lateral distribution of BRAG2a, Arf6, and endophilin 3 for efficient endocytosis at the postsynaptic membrane. Together, the present findings unveiled a novel molecular mechanism by which BRAG2a links AMPARs to the clathrin-dependent endocytic pathway through its interaction with PSD-95 and endophilin 3.SIGNIFICANCE STATEMENT BRAG2/Iqsec1 is a GDP/GTP exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes, and regulates Arf6-dependent endocytosis of AMPARs through direct interaction during hippocampal long-term depression, one of the mechanisms of synaptic plasticity related to learning and memory. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Here, we identified isoform-specific mechanisms of BRAG2-mediated AMPAR internalization. We demonstrated that the interaction of BRAG2a isoform with PSD-95 and endophilin 3 was required for the mGluR-dependent decrease in surface AMPARs in hippocampal neurons. These results unveiled a novel molecular mechanism by which BRAG2 links AMPARs to the clathrin-mediated endocytic machinery at postsynaptic sites.
  • Taisuke Miyazaki, Miwako Yamasaki, Kenji F Tanaka, Masahiko Watanabe
    Neuroscience 2020年05月26日 
    Purkinje cells (PCs) are principal cerebellar neurons, and several classes of interneurons modulate their activity. Lugaro cells (LCs) are one such inhibitory interneuron with distinctive cytology and location, but still most enigmatic among cerebellar neurons. Here we serendipitously produced a novel transgenic mouse line, where a half of Yellow Cameleon (YC)(+) cells in the cerebellar cortex were judged to be LCs, and YC(+) LCs were estimated to constitute one-third of the total LC populations. Neurochemically, two-thirds of YC(+) LCs were dually GABAergic/glycinergic, with the rest being GABAergic. Beneath the PC layer, they extended a sheet of somatodendritic meshwork interconnected with neighboring LCs by adherens junctions, and received various inputs from climbing fibers, mossy fibers, granule cell axons, recurrent PC axons, Golgi cell axons, LC axons, and serotonergic fibers. Intriguingly, somatodendritic elements of individual LCs preferentially extended within a given cerebellar compartment defined by aldolase C expression. In turn, YC(+) LCs projected a dense lattice of ascending and transverse axons to the molecular layer, and innervated molecular layer interneurons (basket and stellate cells) and Golgi cells, but not PCs. Of note, ascending axons profusely innervated individual targets within a cerebellar compartment, while transverse axons ran across several compartments and innervated targets sparsely. This unique circuit configuration highlights that LCs integrate various excitatory, inhibitory, and modulatory inputs coming to the belonging cerebellar compartment and that, as an interneuron-selective interneuron, LCs can effectively disinhibit cerebellar cortical activities in a compartment-dependent manner through inhibition of inhibitory interneurons selectively targeting PCs and granule cells.
  • Atthapon Pidsaya, Anussara Kamnate, Juthathip Sirisin, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Archives of oral biology 113 104689 - 104689 2020年05月 
    OBJECTIVE: The aim of this study was to clarify the mechanism of GABA (□-amino butyric acid)-signaling in the salivary glands by localization of vesicular inhibitory amino acid transporter, a key molecule in GABA-synthesis. DESIGN: Parotid, sublingual and submandibular glands of mice at various postnatal stages were examined in immuno-light and electron microscopy as well as immuno-blotting. RESULTS: Expression for vesicular inhibitory amino acid transporter was detected in parotid and sublingual glands of both sexes and female submandibular gland throughout postnatal development, while it was negligible in male submandibular glands at and after puberty. The expression in female submandibular glands attenuated after testosterone injection. The immunoreactivity was localized in striated ductal cells, but not acinar cells, in the salivary glands, and it occurred in association with intracellular and plasma membranes of the cells. It also occurred in myoepithelial and vascular smooth muscle cells. CONCLUSIONS: GABA-signaling was suggested to be a significant signaling pathway in salivary ductal cells, which was suppressed in male submandibular glands at and after puberty. The suppression in the submandibular duct was by testosterone. In addition, the participation of vesicular inhibitory amino acid transporter in GABA signaling through plasma membranes of the ductal cells was suggested. The significance of occurrence of the immunoreactivity in myoepithelial and smooth muscle cells remains to be further elucidated in terms of implication in GABA signaling.
  • Chihiro Nakamoto, Kohtarou Konno, Taisuke Miyazaki, Ena Nakatsukasa, Rie Natsume, Manabu Abe, Meiko Kawamura, Yugo Fukazawa, Ryuichi Shigemoto, Miwako Yamasaki, Kenji Sakimura, Masahiko Watanabe
    The Journal of comparative neurology 528 6 1003 - 1027 2020年04月 [査読有り][通常論文]
     
    In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.
  • Erzsébet Farkas, Edina Varga, Balázs Kovács, Anett Szilvásy-Szabó, Antonieta Cote-Vélez, Zoltán Péterfi, Magdalini Matziari, Mónika Tóth, Dóra Zelena, Zsolt Mezriczky, Andrea Kádár, Dóra Kővári, Masahiko Watanabe, Masanobu Kano, Ken Mackie, Balázs Rózsa, Yvette Ruska, Blanka Tóth, Zoltán Máté, Ferenc Erdélyi, Gábor Szabó, Balázs Gereben, Ronald M Lechan, Jean-Louis Charli, Patricia Joseph-Bravo, Csaba Fekete
    iScience 23 3 100921 - 100921 2020年03月27日 [査読有り][通常論文]
     
    Based on the type-I cannabinoid receptor (CB1) content of hypophysiotropic axons and the involvement of tanycytes in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis, we hypothesized that endocannabinoids are involved in the tanycyte-induced regulation of TRH release in the median eminence (ME). We demonstrated that CB1-immunoreactive TRH axons were associated to DAGLα-immunoreactive tanycyte processes in the external zone of ME and showed that endocannabinoids tonically inhibit the TRH release in this tissue. We showed that glutamate depolarizes the tanycytes, increases their intracellular Ca2+ level and the 2-AG level of the ME via AMPA and kainite receptors and glutamate transport. Using optogenetics, we demonstrated that glutamate released from TRH neurons influences the tanycytes in the ME. In summary, tanycytes regulate TRH secretion in the ME via endocannabinoid release, whereas TRH axons regulate tanycytes by glutamate, suggesting the existence of a reciprocal microcircuit between tanycytes and TRH terminals that controls TRH release.
  • B Anne Bannatyne, Zhao-Zhe Hao, Georgia M C Dyer, Masahiko Watanabe, David J Maxwell, Ari Berkowitz
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 13 2680 - 2694 2020年03月25日 
    The spinal cord can appropriately generate diverse movements, even without brain input and movement-related sensory feedback, using a combination of multifunctional and behaviorally specialized interneurons. The adult turtle spinal cord can generate motor patterns underlying forward swimming, three forms of scratching, and limb withdrawal (flexion reflex). We previously described turtle spinal interneurons activated during both scratching and swimming (multifunctional interneurons), interneurons activated during scratching but not swimming (scratch-specialized interneurons), and interneurons activated during flexion reflex but not scratching or swimming (flexion reflex-selective interneurons). How multifunctional and behaviorally specialized turtle spinal interneurons affect downstream neurons was unknown. Here, we recorded intracellularly from spinal interneurons activated during these motor patterns in turtles of both sexes in vivo and filled each with dyes. We labeled motoneurons using choline acetyltransferase antibodies or earlier intraperitoneal FluoroGold injection and used immunocytochemistry of interneuron axon terminals to identify their neurotransmitter(s) and putative synaptic contacts with motoneurons. We found that multifunctional interneurons are heterogeneous with respect to neurotransmitter, with some glutamatergic and others GABAergic or glycinergic, and can directly contact motoneurons. Also, scratch-specialized interneurons are heterogeneous with respect to neurotransmitter and some directly contact motoneurons. Thus, scratch-specialized interneurons might directly excite motoneurons that are more strongly activated during scratching than forward swimming, such as hip-flexor motoneurons. Finally, and surprisingly, we found that some motoneurons are behaviorally specialized, for scratching or flexion reflex. Thus, either some limb muscles are only used for a subset of limb behaviors or some limb motoneurons are only recruited during certain limb behaviors.SIGNIFICANCE STATEMENT Both multifunctional and behaviorally specialized spinal cord interneurons have been described in turtles, but their outputs are unknown. We studied responses of multifunctional interneurons (activated during swimming and scratching) and scratch-specialized interneurons, filled each with dyes, and used immunocytochemistry to determine their neurotransmitters and contacts with motoneurons. We found that both multifunctional and scratch-specialized interneurons are heterogeneous with respect to neurotransmitter, with some excitatory and others inhibitory. We found that some multifunctional and some scratch-specialized interneurons directly contact motoneurons. Scratch-specialized interneurons may excite motoneurons that are more strongly activated during scratching than swimming, such as hip-flexor motoneurons, or inhibit their antagonists, hip-extensor motoneurons. Surprisingly, we also found that some motoneurons are behaviorally specialized, for scratching or for flexion reflex.
  • Atsara Rawangwong, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Acta histochemica 122 2 151497 - 151497 2020年02月 [査読有り][通常論文]
     
    Our previous study demonstrated that, different from the parotid and sublingual glands, the submandibular glands of adult mice did not show an immunoblot band for PLCβ3 which is critical in the secretion mechanism by muscarinic cholinergic signaling. Therefore, the submandibular glands of mice at various stages of postnatal development were examined for this enzyme molecule in immunoblot and immunohistochemistry. In immunoblot, a weak band for PLCβ3-expression was detected only at early postnatal stages. In immunohistochemistry, PLCβ3-immunoreactivity was distinctly found in most basally located cells of immature ducts, while the immunoreactivity was weakly seen in terminal tubule cells without significant immunoreactivity in adjacent acinar cells. In contrast, the immunoreactivity was distinctly found in some basal cells of adult excretory ducts, and it was ultrastructurally localized densely in close association with bundles of tonofilaments in the cells. The present finding suggests the possibility that Ca2+ signaling governed by phospholipase Cβ3 is involved in the differentiation of ductal basal cells into apical cells through control of keratin molecule(s) in the cells.
  • Jun Yamada, Chihiro Sato, Kohtarou Konno, Masahiko Watanabe, Shozo Jinno
    The Journal of neuroscience : the official journal of the Society for Neuroscience 40 4 825 - 842 2020年01月22日 [査読有り][通常論文]
     
    The extracellular glycan polysialic acid linked to neural cell adhesion molecule (PSA-NCAM) is principally expressed in the developing brain and the adult neurogenic regions. Although colocalization of PSA-NCAM with cholecystokinin (CCK) was found in the adult brain, the role of PSA-NCAM remains unclear. In this study, we aimed to elucidate the functional significance of PSA-NCAM in the CA1 region of the male mouse hippocampus. Combined fluorescence in situ hybridization and immunohistochemistry showed that few vesicular glutamate transporter 3-negative/CCK-positive (VGluT3-/CCK+) cells were colocalized with PSA-NCAM, but most of the VGluT3+/CCK+ cells were colocalized with PSA-NCAM. The somata of PSA-NCAM+/CCK+ cells were highly innervated by serotonergic boutons than those of PSA-NCAM-/CCK+ cells. The expression ratios of 5-HT3A receptors and p11, a serotonin receptor-interacting protein, were higher in PSA-NCAM+/CCK+ cells than in PSA-NCAM-/CCK+ cells. Pharmacological digestion of PSA-NCAM impaired the efficacy of antidepressant fluoxetine (FLX), a selective serotonin reuptake inhibitor, but not the efficacy of benzodiazepine anxiolytic diazepam. A Western blot showed that restraint stress decreased the expressions of p11 and mature brain-derived neurotrophic factor (BDNF), and FLX increased them. Interestingly, the FLX-induced elevation of expression of p11, but not mature BDNF, was impaired by the digestion of PSA-NCAM. Quantitative reverse transcription-polymerase chain reaction showed that restraint stress reduced the expression of polysialyltransferase ST8Sia IV and FLX elevated it. Collectively, PSA-NCAM colocalized with VGluT3+/CCK+ cells in the CA1 region of the hippocampus may play a unique role in the regulation of antidepressant efficacy via the serotonergic pathway.SIGNIFICANCE STATEMENT Polysialic acid (PSA) is composed of eight or more α2,8-linked sialic acids. Here, we examined the functional significance of polysialic acid linked to the neural cell adhesion molecule (PSA-NCAM) in the adult mouse hippocampus. Few vesicular glutamate transporter 3-negative/cholecystokinin-positive (VGluT3-/CCK+) cells were colocalized with PSA-NCAM, but most of the VGluT3+/CCK+ cells were colocalized with PSA-NCAM. The expression ratios of 5-HT3A receptors and p11, a serotonin receptor-interacting protein, were higher in PSA-NCAM+/CCK+ cells than in PSA-NCAM-/CCK+ cells. The efficacy of antidepressants, but not anxiolytics, was impaired by the digestion of PSA-NCAM. The antidepressant-induced increase in p11 expression was inhibited following PSA-NCAM digestion. We hence hypothesize that PSA-NCAM colocalized with VGluT3+/CCK+ cells may play a unique role in regulating antidepressant efficacy.
  • Chihiro Nakamoto, Meiko Kawamura, Ena Nakatsukasa, Rie Natsume, Keizo Takao, Masahiko Watanabe, Manabu Abe, Tomonori Takeuchi, Kenji Sakimura
    PloS one 15 2 e0229288  2020年 [査読有り][通常論文]
     
    The GluD1 gene is associated with susceptibility for schizophrenia, autism, depression, and bipolar disorder. However, the function of GluD1 and how it is involved in these conditions remain elusive. In this study, we generated a Grid1 gene-knockout (GluD1-KO) mouse line with a pure C57BL/6N genetic background and performed several behavioral analyses. Compared to a control group, GluD1-KO mice showed no significant anxiety-related behavioral differences, evaluated using behavior in an open field, elevated plus maze, a light-dark transition test, the resident-intruder test of aggression and sensorimotor gating evaluated by the prepulse inhibition test. However, GluD1-KO mice showed (1) higher locomotor activity in the open field, (2) decreased sociability and social novelty preference in the three-chambered social interaction test, (3) impaired memory in contextual, but not cued fear conditioning tests, and (4) enhanced depressive-like behavior in a forced swim test. Pharmacological studies revealed that enhanced depressive-like behavior in GluD1-KO mice was restored by the serotonin reuptake inhibitors imipramine and fluoxetine, but not the norepinephrine transporter inhibitor desipramine. In addition, biochemical analysis revealed no significant difference in protein expression levels, such as other glutamate receptors in the synaptosome and postsynaptic densities prepared from the frontal cortex and the hippocampus. These results suggest that GluD1 plays critical roles in fear memory, sociability, and depressive-like behavior.
  • Takafumi Kawai, Haruhiko Miyata, Hiroki Nakanishi, Souhei Sakata, Shin Morioka, Junko Sasaki, Masahiko Watanabe, Kenji Sakimura, Toyoshi Fujimoto, Takehiko Sasaki, Masahito Ikawa, Yasushi Okamura
    Proceedings of the National Academy of Sciences of the United States of America 116 51 26020 - 26028 2019年12月17日 [査読有り][通常論文]
     
    The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.
  • Edina Varga, Erzsébet Farkas, Györgyi Zséli, Andrea Kádár, Alexandra Venczel, Dóra Kővári, Dorottya Németh, Zoltán Máté, Ferenc Erdélyi, András Horváth, Ottó Szenci, Masahiko Watanabe, Ronald M Lechan, Balázs Gereben, Csaba Fekete
    Thyroid : official journal of the American Thyroid Association 29 12 1858 - 1868 2019年12月 [査読有り][通常論文]
     
    Background: Glycine is a classical neurotransmitter that has role in both inhibitory and excitatory synapses. To understand whether glycinergic inputs are involved in the regulation of the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons, the central controllers of the hypothalamic-pituitary-thyroid axis, the glycinergic innervation of the TRH neurons was studied in the hypothalamic paraventricular nucleus (PVN). Methods: Double-labeling immunocytochemistry and patch-clamp electrophysiology were used to determine the role of glycinergic neurons in the regulation of TRH neurons in the PVN. Anterograde and retrograde tracing methods were used to determine the sources of the glycinergic input of TRH neurons. Results: Glycine transporter-2 (GLYT2), a marker of glycinergic neurons, containing axons were found to establish symmetric type of synapses on TRH neurons in the PVN. Furthermore, glycine receptor immunoreactivity was observed in these TRH neurons. The raphe magnus (RMg) and the ventrolateral periaqueductal gray (VLPAG) were found to be the exclusive sources of the glycinergic innervation of the TRH neurons within the PVN. Patch-clamp electrophysiology using sections of TRH-IRES-tdTomato mice showed that glycine hyperpolarized the TRH neurons and completely blocked the firing of these neurons. Glycine also markedly hyperpolarized the TRH neurons in the presence of tetrodotoxin demonstrating the direct effect of glycine. In more than 60% of the TRH neurons, spontaneous inhibitory postsynaptic currents (sIPSCs) were observed, even after the pharmacological inhibition of glutamatergic and GABAergic neuronal transmission. The glycine antagonist, strychnine, almost completely abolished these sIPSCs, demonstrating the inhibitory nature of the glycinergic input of TRH neurons. Conclusions: These data demonstrate that TRH neurons in the PVN receive glycinergic inputs from the RMg and the VLPAG. The symmetric type of synaptic connection and the results of the electrophysiological experiments demonstrate the inhibitory nature of these inputs.
  • Kelly M Smith, Tyler J Browne, Olivia C Davis, A Coyle, Kieran A Boyle, Masahiko Watanabe, Sally A Dickinson, Jacqueline A Iredale, Mark A Gradwell, Phillip Jobling, Robert J Callister, Christopher V Dayas, David I Hughes, Brett A Graham
    eLife 8 2019年11月12日 [査読有り][通常論文]
     
    Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.
  • Motokazu Uchigashima, Amy Cheung, Julie Suh, Masahiko Watanabe, Kensuke Futai
    The Journal of comparative neurology 527 12 1940 - 1965 2019年08月15日 [査読有り][通常論文]
     
    Synapses, highly specialized membrane junctions between neurons, connect presynaptic neurotransmitter release sites and postsynaptic ligand-gated channels. Neurexins (Nrxns), a family of presynaptic adhesion molecules, have been characterized as major regulators of synapse development and function. Via their extracellular domains, Nrxns bind to different postsynaptic proteins, generating highly diverse functional readouts through their postsynaptic binding partners. Not surprisingly given these versatile protein interactions, mutations and deletions of Nrxn genes have been identified in patients with autism spectrum disorders, intellectual disabilities, and schizophrenia. Therefore, elucidating the expression profiles of Nrxns in the brain is of high significance. Here, using chromogenic and fluorescent in situ hybridization, we characterize the expression patterns of Nrxn isoforms throughout the brain. We found that each Nrxn isoform displays a unique expression profile in a region-, cell type-, and sensory system-specific manner. Interestingly, we also found that αNrxn1 and αNrxn2 mRNAs are expressed in non-neuronal cells, including astrocytes and oligodendrocytes. Lastly, we found diverse expression patterns of genes that encode Nrxn binding proteins, such as Neuroligins (Nlgns), Leucine-rich repeat transmembrane neuronal protein (Lrrtms) and Latrophilins (Adgrls), suggesting that Nrxn proteins can mediate numerous combinations of trans-synaptic interactions. Together, our anatomical profiling of Nrxn gene expression reflects the diverse roles of Nrxn molecules.
  • Ester Aso, Víctor Fernández-Dueñas, Marc López-Cano, Jaume Taura, Masahiko Watanabe, Isidre Ferrer, Rafael Luján, Francisco Ciruela
    Molecular neurobiology 56 8 5382 - 5391 2019年08月 [査読有り][通常論文]
     
    At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.
  • Maria Gutierrez-Mecinas, Andrew M Bell, Fraser Shepherd, Erika Polgár, Masahiko Watanabe, Takahiro Furuta, Andrew J Todd
    The Journal of comparative neurology 527 11 1857 - 1871 2019年08月01日 [査読有り][通常論文]
     
    Excitatory interneurons account for the majority of dorsal horn neurons, and are required for perception of normal and pathological pain. We have identified largely non-overlapping populations in laminae I-III, based on expression of substance P, gastrin-releasing peptide, neurokinin B, and neurotensin. Cholecystokinin (CCK) is expressed by many dorsal horn neurons, particularly in the deeper laminae. Here, we have used immunocytochemistry and in situ hybridization to characterize the CCK cells. We show that they account for ~7% of excitatory neurons in laminae I-II, but between a third and a quarter of those in lamina III. They are largely separate from the neurokinin B, neurotensin, and gastrin-releasing peptide populations, but show limited overlap with the substance P cells. Laminae II-III neurons with protein kinase Cγ (PKCγ) have been implicated in mechanical allodynia following nerve injury, and we found that around 50% of CCK cells were PKCγ-immunoreactive. Neurotensin is also expressed by PKCγ cells, and among neurons with moderate to high levels of PKCγ, ~85% expressed CCK or neurotensin. A recent transcriptomic study identified mRNA for thyrotropin-releasing hormone in a specific subpopulation of CCK neurons, and we show that these account for half of the CCK/PKCγ cells. These findings indicate that the CCK cells are distinct from other excitatory interneuron populations that we have defined. They also show that PKCγ cells can be assigned to different classes based on neuropeptide expression, and it will be important to determine the differential contribution of these classes to neuropathic allodynia.
  • Víctor Fernández-Dueñas, Maricel Gómez-Soler, Marta Valle-León, Masahiko Watanabe, Isidre Ferrer, Francisco Ciruela
    International journal of molecular sciences 20 14 2019年07月23日 [査読有り][通常論文]
     
    Background: Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods: Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results: Adenosine A2A-dopamine D2 receptor (A2AR/D2R) heteromer formation was monitored in caudate from healthy and Parkinson's disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR/D2R heteromer formation in PD brains. Conclusions: The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity.
  • Kieran A Boyle, Mark A Gradwell, Toshiharu Yasaka, Allen C Dickie, Erika Polgár, Robert P Ganley, Desmond P H Orr, Masahiko Watanabe, Victoria E Abraira, Emily D Kuehn, Amanda L Zimmerman, David D Ginty, Robert J Callister, Brett A Graham, David I Hughes
    Cell reports 28 2 526 - 540 2019年07月09日 [査読有り][通常論文]
     
    Chronic pain presents a major unmet clinical problem. The development of more effective treatments is hindered by our limited understanding of the neuronal circuits underlying sensory perception. Here, we show that parvalbumin (PV)-expressing dorsal horn interneurons modulate the passage of sensory information conveyed by low-threshold mechanoreceptors (LTMRs) directly via presynaptic inhibition and also gate the polysynaptic relay of LTMR input to pain circuits by inhibiting lamina II excitatory interneurons whose axons project into lamina I. We show changes in the functional properties of these PV interneurons following peripheral nerve injury and that silencing these cells unmasks a circuit that allows innocuous touch inputs to activate pain circuits by increasing network activity in laminae I-IV. Such changes are likely to result in the development of tactile allodynia and could be targeted for more effective treatment of mechanical pain.
  • Harumi Nakao, Yasushi Kishimoto, Kouichi Hashimoto, Kazuo Kitamura, Miwako Yamasaki, Kazuki Nakao, Masahiko Watanabe, Masanobu Kano, Yutaka Kirino, Atsu Aiba
    Scientific reports 9 1 7353 - 7353 2019年05月14日 [査読有り][通常論文]
     
    Classical eyeblink conditioning is a representative associative motor learning that requires both the cerebellar cortex and the deep cerebellar nucleus (DCN). Metabotropic glutamate receptor subtype 1 (mGluR1) is richly expressed in Purkinje cells (PCs) of the cerebellar cortex. Global mGluR1 knock-out (KO) mice show a significantly lower percentage of conditioned response (CR%) than wild-type mice in eyeblink conditioning, and the impaired CR% is restored by the introduction of mGluR1 in PCs. However, the specific roles of mGluR1 in major memory processes, including formation, storage and expression have not yet been defined. We thus examined the role of mGluR1 in these processes of eyeblink conditioning, using mGluR1 conditional KO (cKO) mice harboring a selective and reversible expression of mGluR1 in PCs. We have found that eyeblink memory is not latently formed in the absence of mGluR1 in adult mouse PCs. However, once acquired, eyeblink memory is expressed even after the depletion of mGluR1 in PCs. We thus conclude that mGluR1 in PCs is indispensable for the formation of eyeblink memory, while it is not required for the expression of CR.
  • Atsara Rawangwong, Atthapon Pidsaya, Wipawee Thoungseabyoun, Apussara Tachow, Tarinee Sawatpanich, Waraporn Sakaew, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Acta histochemica 121 4 484 - 490 2019年05月 [査読有り][通常論文]
     
    Phospholipase C (PLC)β has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCβ isoforms in mouse salivary glands in situ. A single major band was detected for PLCβ3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCβ1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCβ3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCβ3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.
  • Atsara Rawangwong, Suthankamon Khrongyut, Surang Chomphoo, Kohtaro Konno, Miwako Yamasaki, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    Archives of oral biology 100 14 - 22 2019年04月 [査読有り][通常論文]
     
    We hypothesize variation in expression and localization, along the course of the glandular tubule, of muscarinic cholinergic receptor M1 which plays as a distinct contribution, though minor in comparison with M3 receptor, in saliva secretion. Localization of the M1 receptor was examined using immunohistochemistry in three major salivary glands. Although all glandular cells were more or less M1-immunoreactive, acinar cells were weakly immunoreactive, while ductal cells exhibited substantial M1-immunoreactivity. Many ductal cells exhibited clear polarity with higher immunoreactivity in their apical/supra-nuclear domain. However, some exhibited indistinct polarity because of additional higher immunoreactivity in their basal/infra-nuclear domain. A small group of cells with intense immunoreactivity was found, mostly located in the intercalated ducts or in portions of the striated ducts close to the intercalated ducts. In immuno-electron microscopy, the immunoreactive materials were mainly in the cytoplasm including various vesicles and vacuoles. Unexpectedly, distinct immunoreactivity on apical and basal plasma membranes was infrequent in most ductal cells. The heterogeneous localization of M1-immunoreactivity along the gland tubular system is discussed in view of possible modulatory roles of the M1 receptor in saliva secretion.
  • Waraporn Sakaew, Apussara Tachow, Wipawee Thoungseabyoun, Suthankamon Khrongyut, Atsara Rawangwong, Yada Polsan, Watanabe Masahiko, Hisatake Kondo, Wiphawi Hipkaeo
    Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft 222 21 - 27 2019年03月 [査読有り][通常論文]
     
    Vesicular inhibitory amino acid transporter (VIAAT) is a transmembrane transporter which is responsible for the storage of gamma-aminobutyric acid (GABA) or glycine in synaptic vesicles. According to recent studies, GABA is known to be expressed in the kidney. For clear understanding of the intra-renal GABA signaling, the localization of VIAAT was examined in the present study. Intense immunoreactivity was found largely confined to the distal tubule epithelia, especially distinct in the inner medulla, although the immunoreactivity was discerned more or less in all tubules and glomeruli. No distinct immunoreactivity was seen in capillary endothelia or interstitial fibroblasts. In immuno-DAB and immuno-gold electron microscopy, the immunoreaction was found at the basal infoldings of plasma membranes and basal portions of the lateral plasma membranes, but not in any vesicles or vacuoles within the distal tubular cells. The significance of the enigmatic finding, localization of a vesicular molecule on selected portions of the plasma membrane of distal tubular cells, was discussed in view of the possibility of paracrine or autocrine effects of GABA on some other uriniferous tubular cells or interstitial cells.
  • Dickie AC, Bell AM, Iwagaki N, Polgár E, Gutierrez-Mecinas M, Kelly R, Lyon H, Turnbull K, West SJ, Etlin A, Braz J, Watanabe M, Bennett DLH, Basbaum AI, Riddell JS, Todd AJ
    Pain 160 2 442 - 462 2019年02月 [査読有り][通常論文]
     
    Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified 2 populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here, we demonstrate that these cells show major differences in their morphological, electrophysiological, and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. By contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The 2 populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the μ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal-regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information.
  • Maria Gutierrez-Mecinas, Olivia Davis, Erika Polgár, Mahvish Shahzad, Keila Navarro-Batista, Takahiro Furuta, Masahiko Watanabe, David I Hughes, Andrew J Todd
    Neuroscience 398 171 - 181 2019年02月01日 [査読有り][通常論文]
     
    Around 75% of neurons in laminae I-II of the mouse dorsal horn are excitatory interneurons, and these are required for normal pain perception. We have shown that four largely non-overlapping excitatory interneuron populations can be defined by expression of the neuropeptides neurotensin, neurokinin B (NKB), gastrin-releasing peptide (GRP) and substance P. In addition, we recently identified a population of excitatory interneurons in glabrous skin territory that express dynorphin. The calcium-binding protein calretinin is present in many excitatory neurons in this region, but we know little about its relation to these neuropeptide markers. Here we show that calretinin is differentially expressed, being present in the majority of substance P-, GRP- and NKB-expressing cells, but not in the neurotensin or dynorphin cells. Calretinin-positive cells have been implicated in detection of noxious mechanical stimuli, but are not required for tactile allodynia after neuropathic pain. Our findings are therefore consistent with the suggestion that neuropathic allodynia involves the neurotensin and/or dynorphin excitatory interneuron populations. Around a quarter of inhibitory interneurons in lamina I-II contain calretinin, and recent transcriptomic studies suggest that these co-express substance P. We confirm this, by showing that inhibitory Cre-expressing cells in a Tac1Cre knock-in mouse are calretinin-immunoreactive. Interestingly, there is evidence that these cells express low levels of peptidylglycine alpha-amidating monooxygenase, an enzyme required for maturation of neuropeptides. This may explain our previous finding that although the substance P precursor preprotachykinin A can be detected in some inhibitory interneurons, very few inhibitory axonal boutons are immunoreactive for substance P.
  • Toshihiko Iwanaga, Kohtarou Konno, Masahiko Watanabe
    Biomedical research (Tokyo, Japan) 40 5 207 - 214 2019年 [査読有り][通常論文]
     
    Sensory circumventricular organs contain the subfornical organ, organum vasculosum of the lamina terminalis (OVLT), and area postrema. Here, immunostaining for GLUT3 in the murine brain selectively labeled the sobfornical organ and OVLT. The immunoreactive neural tract of the subfornical organ formed into thin bundles and extended ventro-rostrally over the anterior commissure. After turning over the commissure, the neural tract passed through the median preoptic nucleus (MnPO) and reached the OVLT; thus, a continuous neural tract expressing GLUT3 connected the subfornical organ, MnPO, and OVLT in the lamina terminalis. In the OVLT, GLUT3-immunoreactive fibers gathered in both the dorsal cap and lateral periventricular zone. Electron microscopically, the immunoreactive structures in the subfornical organ corresponded to nerve fibers or nerve terminals containing many small clear vesicles. The area postrema, another sensory organ, was immunonegative for GLUT3. This study not only presented a useful marker tracing the neural tract in the sensory sites of the lamina terminalis but also suggested a unique system for sensing and determining the metabolism of circulating glucose in the circumventricular organs.
  • Péterfi Z, Farkas I, Denis RGP, Farkas E, Uchigashima M, Füzesi T, Watanabe M, Lechan RM, Liposits Z, Luquet S, Fekete C
    Molecular metabolism 18 120 - 133 2018年12月 [査読有り][通常論文]
     
    OBJECTIVE: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. METHODS: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. RESULTS: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. CONCLUSION: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.
  • Li Zhou, M Ibrahim Hossain, Maya Yamazaki, Manabu Abe, Rie Natsume, Kohtaro Konno, Shun Kageyama, Masaaki Komatsu, Masahiko Watanabe, Kenji Sakimura, Hirohide Takebayashi
    Journal of neurochemistry 147 4 557 - 572 2018年11月 [査読有り][通常論文]
     
    Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and β-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
  • Tayo Katano, Keizo Takao, Manabu Abe, Maya Yamazaki, Masahiko Watanabe, Tsuyoshi Miyakawa, Kenji Sakimura, Seiji Ito
    Molecular brain 11 1 63 - 63 2018年10月25日 [査読有り][通常論文]
     
    Calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1 (Caskin1) is a direct binding partner of the synaptic adaptor protein CASK. Because Caskin1 forms homo-multimers and binds not only CASK but also other neuronal proteins in vitro, it is anticipated to have neural functions; but its exact role in mammals remains unclear. Previously, we showed that the concentration of Caskin1 in the spinal dorsal horn increases under chronic pain. To characterize this protein, we generated Caskin1-knockout (Caskin1-KO) mice and specific anti-Caskin1 antibodies. Biochemical and immunohistochemical analyses demonstrated that Caskin1 was broadly distributed in the whole brain and spinal cord, and that it primarily localized at synapses. To elucidate the neural function of Caskin1 in vivo, we subjected Caskin1-KO mice to comprehensive behavioral analysis. The mutant mice exhibited differences in gait, enhanced nociception, and anxiety-like behavior relative to their wild-type littermates. In addition, the knockouts exhibited strong freezing responses, with or without a cue tone, in contextual and cued-fear conditioning tests as well as low memory retention in the Barnes Maze test. Taken together, these results suggest that Caskin1 contributes to a wide spectrum of behavioral phenotypes, including gait, nociception, memory, and stress response, in broad regions of the central nervous system.
  • Morató X, Luján R, Gonçalves N, Watanabe M, Altafaj X, Carvalho AL, Fernández-Dueñas V, Cunha RA, Ciruela F
    Human molecular genetics 27 20 3528 - 3541 2018年10月 [査読有り][通常論文]
  • Natsumi Ageta-Ishihara, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Makoto Kinoshita
    Neurochemistry international 119 190 - 198 2018年10月 [査読有り][通常論文]
     
    Configuration of tripartite synapses, comprising the pre-, post-, and peri-synaptic components (axon terminal or bouton, dendritic spine, and astroglial terminal process), is a critical determinant of neurotransmitter kinetics and hence synaptic transmission. However, little is known about molecular basis for the regulation of tripartite synapse morphology. Previous studies showed that CDC42EP4, an effector protein of a cell morphogenesis regulator CDC42, is expressed exclusively in Bergmann glia in the cerebellar cortex, that it forms tight complex with the septin heterooligomer, and that it interacts indirectly with the glutamate transporter GLAST and MYH10/nonmuscle myosin ΙΙB. Scrutiny of Cdc42ep4-/- mice had revealed that the CDC42EP4-septins-GLAST interaction facilitates glutamate clearance, while the role for CDC42EP4-septins-MYH10 interaction has remained unsolved. Here, we find anomalous configuration of the tripartite synapses comprising the parallel fiber boutons, dendritic spines of Purkinje cells, and Bergmann glial processes in Cdc42ep4-/- mice. The complex anomalies include 1) recession of Bergmann glial membranes from the nearest active zones, and 2) extension of nonactive synaptic contact around active zone. In line with the recession of Bergmann glial membranes by the loss of CDC42EP4, overexpression of CDC42EP4 in heterologous cells promotes cell spreading and partitioning of MYH10 to insoluble (i.e., active) fraction. Paradoxically, however, Cdc42ep4-/- cerebellum contained significantly more MYH10 and N-cadherin, which is attributed to secondary neuronal response mainly in Purkinje cells. Given cooperative actions of N-cadherin and MYH10 for adhesion between neurons, we speculate that their augmentation may reflect the extension of nonactive synaptic contacts in Cdc42ep4-/- cerebellum. Transcellular mechanism that links the absence of CDC42EP4 in Bergmann glia to the augmentation of N-cadherin and MYH10 in neurons is currently unknown, but the phenotypic similarity to GLAST-null mice indicates involvement of the glutamate intolerance. Together, the unique phenotype of Cdc42ep4-/- mice provides a clue to novel molecular network underlying tripartite synapse configuration.
  • Yamagata A, Goto-Ito S, Sato Y, Shiroshima T, Maeda A, Watanabe M, Saitoh T, Maenaka K, Terada T, Yoshida T, Uemura T, Fukai S
    Nature communications 9 1 3964 - 3964 2018年09月27日 [査読有り][通常論文]
     
    Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). Here, we report the crystal structure of the Nrxn1β-LRRTM2 complex at 3.4 Å resolution. The Nrxn1β-LRRTM2 interface involves Ca2+-mediated interactions and overlaps with the Nrxn-neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn.
  • Satoshi Nonomura, Kayo Nishizawa, Yutaka Sakai, Yasuo Kawaguchi, Shigeki Kato, Motokazu Uchigashima, Masahiko Watanabe, Ko Yamanaka, Kazuki Enomoto, Satomi Chiken, Hiromi Sano, Shogo Soma, Junichi Yoshida, Kazuyuki Samejima, Masaaki Ogawa, Kazuto Kobayashi, Atsushi Nambu, Yoshikazu Isomura, Minoru Kimura
    Neuron 99 6 1302 - 1314 2018年09月19日 [査読有り][通常論文]
     
    The basal ganglia play key roles in adaptive behaviors guided by reward and punishment. However, despite accumulating knowledge, few studies have tested how heterogeneous signals in the basal ganglia are organized and coordinated for goal-directed behavior. In this study, we investigated neuronal signals of the direct and indirect pathways of the basal ganglia as rats performed a lever push/pull task for a probabilistic reward. In the dorsomedial striatum, we found that optogenetically and electrophysiologically identified direct pathway neurons encoded reward outcomes, whereas indirect pathway neurons encoded no-reward outcome and next-action selection. Outcome coding occurred in association with the chosen action. In support of pathway-specific neuronal coding, light activation induced a bias on repeat selection of the same action in the direct pathway, but on switch selection in the indirect pathway. Our data reveal the mechanisms underlying monitoring and updating of action selection for goal-directed behavior through basal ganglia circuits.
  • Notartomaso S, Nakao H, Mascio G, Scarselli P, Cannella M, Zappulla C, Madonna M, Motolese M, Gradini R, Liberatore F, Zonta M, Carmignoto G, Battaglia G, Bruno V, Watanabe M, Aiba A, Nicoletti F
    Scientific reports 8 1 13361  2018年09月 [査読有り][通常論文]
  • Kaplan E, Zubedat S, Radzishevsky I, Valenta AC, Rechnitz O, Sason H, Sajrawi C, Bodner O, Konno K, Esaki K, Derdikman D, Yoshikawa T, Watanabe M, Kennedy RT, Billard JM, Avital A, Wolosker H
    Proceedings of the National Academy of Sciences of the United States of America 115 38 9628 - 9633 2018年09月 [査読有り][通常論文]
  • Itabashi T, Arima Y, Kamimura D, Higuchi K, Bando Y, Takahashi-Iwanaga H, Murakami M, Watanabe M, Iwanaga T, Nio-Kobayashi J
    Neurochemistry international 118 176 - 184 2018年09月01日 [査読有り][通常論文]
     
    Multiple sclerosis (MS) is an autoimmune disease in which pathogenic T cells play an important role, and an experimental autoimmune encephalomyelitis (EAE) is used as an animal model of MS. Galectins are β-galactoside-binding lectins and involved in various physiological and pathological events. Among fifteen members of galectins, galectin-1, -8, and -9 play immunosuppressive roles in MS and EAE however, the role of galectin-3 (gal-3) is complex and controversial. We examined expression of gal-3 in the spinal cord and nerve roots of EAE mice. No immunohistochemical signals were detected in naïve mice, whereas gal-3 appeared at lower lumbar levels of the spinal cord and nerve roots in EAE mice. In the spinal cord, gal-3-positive cells were activated microglia and/or infiltrating macrophages, which were round in shape and intensified for the lysosomal enzyme, cathepsin D, indicating elevated phagocytic activity. Gal-3-positive cells in the spinal cord were most abundant during the peak symptomatic period. In the recovery period, they disappeared from the spinal parenchyma but remained at moderate levels in the pia mater. Interestingly, gal-3-positive cells selectively appeared in ventral, but not dorsal, nerve roots running through the spinal canal, with expression peaking during the recovery period. In ventral nerve roots, the major cell type expressing gal-3 was a specific population of Schwann cells that surround unmyelinated axons and express the biosynthetic enzyme for L-serine, a potent neurotrophic amino acid. Gal-3 was also induced in Iba1/F4/80-positive macrophages, which engulf damaged myelin and axon debris. Thus, gal-3 is induced in distinct cell types that are engaged in removal of damaged axons and cell debris and axon regeneration and remyelination, suggesting a potential neuroprotective role of gal-3 in EAE mice.
  • Zhang MD, Su J, Adori C, Cinquina V, Malenczyk K, Girach F, Peng C, Ernfors P, Löw P, Borgius L, Kiehn O, Watanabe M, Uhlén M, Mitsios N, Mulder J, Harkany T, Hökfelt T
    The Journal of clinical investigation 128 9 3757 - 3768 2018年08月 [査読有り][通常論文]
  • Kano M, Watanabe T, Uesaka N, Watanabe M
    Cerebellum (London, England) 2018年07月 [査読有り][通常論文]
  • Shiotani H, Miyata M, Itoh Y, Wang S, Kaito A, Mizoguchi A, Yamasaki M, Watanabe M, Mandai K, Mochizuki H, Takai Y
    The Journal of comparative neurology 526 9 1527 - 1549 2018年06月15日 [査読有り][通常論文]
     
    The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin-like cell adhesion molecules (CAMs) nectin-2α and nectin-2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin-2α, but not nectin-2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin-2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin-2α at this boundary was not colocalized with the nectin-2α-binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin-2α forms a cell adhesion apparatus different from the Kv4.2-associated MSs. Genetic ablation of nectin-2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin-2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.
  • Wenjie Mao, Anna C. Salzberg, Motokazu Uchigashima, Yuto Hasegawa, Hanno Hock, Masahiko Watanabe, Schahram Akbarian, Yuka Imamura Kawasawa, Kensuke Futai
    Cell Reports 23 11 3209 - 3222 2018年06月12日 [査読有り][通常論文]
     
    Homeostatic synaptic downscaling reduces neuronal excitability by modulating the number of postsynaptic receptors. Histone modifications and the subsequent chromatin remodeling play critical roles in activity-dependent gene expression. Histone modification codes are recognized by chromatin readers that affect gene expression by altering chromatin structure. We show that L3mbtl1 (lethal 3 malignant brain tumor-like 1), a polycomb chromatin reader, is downregulated by neuronal activity and is essential for synaptic response and downscaling. Genome-scale mapping of L3mbtl1 occupancies identified Ctnnb1 as a key gene downstream of L3mbtl1. Importantly, the occupancy of L3mbtl1 on the Ctnnb1 gene was regulated by neuronal activity. L3mbtl1 knockout neurons exhibited reduced Ctnnb1 expression. Partial knockdown of Ctnnb1 in wild-type neurons reduced excitatory synaptic transmission and abolished homeostatic downscaling, and transfecting Ctnnb1 in L3mbtl1 knockout neurons enhanced synaptic transmission and restored homeostatic downscaling. These results highlight a role for L3mbtl1 in regulating homeostasis of synaptic efficacy. Synaptic homeostasis is crucial for maintaining proper neuronal excitability and excitatory/inhibitory balance in the brain. Mao et al. report that an activity-dependent chromatin reader protein is required for homeostatic control of synaptic strength through the regulation of downstream target gene Ctnnb1.
  • Kikuchihara S, Sugio S, Tanaka KF, Watanabe T, Kano M, Yamazaki Y, Watanabe M, Ikenaka K
    Journal of neurochemistry 2018年06月 [査読有り][通常論文]
  • Szabolcs Takács, Zsuzsanna Bardóczi, Katalin Skrapits, Balázs Göcz, Viktória Váczi, Zsófia Maglóczky, Iván Szűcs, Gergely Rácz, András Matolcsy, Waljit S. Dhillo, Masahiko Watanabe, Andrea Kádár, Csaba Fekete, Imre Kalló, Erik Hrabovszky
    Brain Structure and Function 223 5 2143 - 2156 2018年06月01日 [査読有り][通常論文]
     
    Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.
  • Jing Huang, Erika Polgár, Hans Jürgen Solinski, Santosh K. Mishra, Pang-Yen Tseng, Noboru Iwagaki, Kieran A. Boyle, Allen C. Dickie, Mette C. Kriegbaum, Hendrik Wildner, Hanns Ulrich Zeilhofer, Masahiko Watanabe, John S. Riddell, Andrew J. Todd, Mark A. Hoon
    Nature Neuroscience 21 5 707 - 716 2018年05月01日 [査読有り][通常論文]
     
    Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide.
  • Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Masashi Watanabe, Taichi Nomura, Takahiro Kano, Masahiko Watanabe, Shigetsugu Hatakeyama
    Journal of Neurology 265 4 962 - 965 2018年04月01日 [査読有り][通常論文]
  • Rafael Luján, Carolina Aguado, Francisco Ciruela, Javier Cózar, David Kleindienst, Luis de la Ossa, Bernhard Bettler, Kevin Wickman, Masahiko Watanabe, Ryuichi Shigemoto, Yugo Fukazawa
    Brain Structure and Function 223 3 1565 - 1587 2018年04月01日 [査読有り][通常論文]
     
    Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.
  • Masanori Tachikawa, Hidetoh Toki, Masahiko Watanabe, Masatoshi Tomi, Ken-ichi Hosoya, Tetsuya Terasaki
    Anatomical Science International 93 4 1 - 8 2018年03月08日 [査読有り][通常論文]
     
    The A-subclass of ATP-binding cassette (ABC) transporters is a highly conserved superfamily of potent lipid transporters. Although the ABCA1-like subgroup of ABCA1-4, and A7 have been shown to mediate the transport of endogenous lipids, the roles of the ABCA6-like subgroup transporters, which have been identified as a unique gene cluster on human chromosome 17q24 (ABCA5, A6, A8, A9, and A10) and mouse chromosome 11 (Abca5, a6, a8a, a8b, and a9), remains largely unknown. The purpose of the present study was to clarify the spatial and temporal expression profiles of Abca6-like subgroup transporters in embryonic and postnatal mouse brains by a combination of in situ hybridization and quantitative polymerase chain reaction (PCR) using magnetically isolated brain vascular endothelial cells. In embryonic brains, the transcripts of Abca5, a8a and a8b were detected predominantly in the mantle zone, where postmitotic neurons differentiate. At the postnatal stages, they were expressed in various nuclei and neuronal layers. Abca9 mRNA was detected diffusely in the embryonic and postnatal brains and sequential and/or strong spotted signals were detected in the leptomeninges on the brain surface. PCR detected expression of Abca8a and Abca9 mRNAs in isolated vascular endothelial cells. Expression signals for Abca6 mRNA were hardly observed at any stages examined. These distinct spatio-temporal expression patterns of Abca6-like subgroup transporters may reflect their functional significance and diversity to regulate lipid transport, particularly in neurons, leptomeningeal cells, and vascular endothelial cells.
  • Naofumi Uesaka, Manabu Abe, Kohtarou Konno, Maya Yamazaki, Kazuto Sakoori, Takaki Watanabe, Tzu-Huei Kao, Takayasu Mikuni, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    Neuron 97 4 796 - 805 2018年02月21日 [査読有り][通常論文]
     
    Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination.
  • Valentin Kusch, Grit Bornschein, Desiree Loreth, Julia Bank, Johannes Jordan, David Baur, Masahiko Watanabe, Akos Kulik, Manfred Heckmann, Jens Eilers, Hartmut Schmidt
    Cell Reports 22 8 2094 - 2106 2018年02月20日 [査読有り][通常論文]
     
    Spatial relationships between Cav channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Cav2.1 and Cav2.2 channels, alters the localization of Cav2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca2+-channel pharmacology in electrophysiological and two-photon Ca2+ imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca2+ influx through Cav2.1 and Cav2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Cav2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Cav2.1 and Cav2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains. Central synapses shift from microdomain to nanodomain coupling between Ca2+ channels and release sensors during development. Kusch et al. show that Munc13-3 is a critical regulator of this process. Munc13-3 differentially regulates density and localization of Cav2.1 and Cav2.2 channels and establishes nanodomain coupling between Cav2.1 channels and release sensors.
  • Ryosuke Kaneko, Yusuke Takatsuru, Ayako Morita, Izuki Amano, Asahi Haijima, Itaru Imayoshi, Nobuaki Tamamaki, Noriyuki Koibuchi, Masahiko Watanabe, Yuchio Yanagawa
    Journal of Comparative Neurology 526 3 373 - 396 2018年02月15日 [査読有り][通常論文]
     
    Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.
  • Ogawa Y, Kakumoto K, Yoshida T, Kuwako KI, Miyazaki T, Yamaguchi J, Konno A, Hata J, Uchiyama Y, Hirai H, Watanabe M, Darnell RB, Okano H, Okano HJ
    Scientific reports 8 1 2722  2018年02月 [査読有り][通常論文]
  • Masanori Tachikawa, Masahiko Watanabe, Masahiro Fukaya, Kazuhisa Sakai, Tetsuya Terasaki, Ken-ichi Hosoya
    Neurochemical Research 43 2 500 - 510 2018年02月01日 [査読有り][通常論文]
     
    Creatine is synthesized by S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), and the creatine/phosphocreatine shuttle system mediated by creatine kinase (CK) is essential for storage and regeneration of high-energy phosphates in cells. Although the importance of this system in brain development is evidenced by the hereditary nature of creatine deficiency syndrome, the spatiotemporal cellular expression patterns of GAMT in developing brain remain unknown. Here we show that two waves of high GAMT expression occur in developing mouse brain. The first involves high expression in mitotic cells in the ventricular zone of the brain wall and the external granular layer of the cerebellum at the embryonic and neonatal stages. The second was initiated by striking up-regulation of GAMT in oligodendrocytes during the second and third postnatal weeks (i.e., the active myelination stage), which continued to adulthood. Distinct temporal patterns were also evident in other cell types. GAMT was highly expressed in perivascular pericytes and smooth muscle cells after birth, but not in adults. In neurons, GAMT levels were low to moderate in neuroblasts residing in the ventricular zone, increased during the second postnatal week when active dendritogenesis and synaptogenesis occur, and decreased to very low levels thereafter. Moderate levels were observed in astrocytes throughout development. The highly regulated, cell type-dependent expression of GAMT suggests that local creatine biosynthesis plays critical roles in certain phases of neural development. In accordance with this idea, we observed increased CK expression in differentiating neurons this would increase creatine/phosphocreatine shuttle system activity, which might reflect increased energy demand.
  • Sam A. Booker, Desiree Loreth, Annabelle L. Gee, Masahiko Watanabe, Peter C. Kind, David J.A. Wyllie, Ákos Kulik, Imre Vida
    Cell Reports 22 1 36 - 43 2018年01月02日 [査読有り][通常論文]
     
    Inhibition provided by local GABAergic interneurons (INs) activates ionotropic GABAA and metabotropic GABAB receptors (GABABRs). Despite GABABRs representing a major source of inhibition, little is known of their function in distinct IN subtypes. Here, we show that, while the archetypal dendritic-inhibitory somatostatin-expressing INs (SOM-INs) possess high levels of GABABR on their somato-dendritic surface, they fail to produce significant postsynaptic inhibitory currents. Instead, GABABRs selectively inhibit dendritic CaV1.2 (L-type) Ca2+ channels on SOM-IN dendrites, leading to reduced calcium influx and loss of long-term potentiation at excitatory input synapses onto these INs. These data provide a mechanism by which GABABRs can contribute to disinhibition and control the efficacy of extrinsic inputs to hippocampal networks. Booker et al. show that GABAB receptors are highly expressed on somatostatin interneuron dendrites. Rather than activating Kir3 channels, they preferentially co-cluster with, and negatively couple to, L-type calcium channels inhibiting long-term potentiation at excitatory inputs.
  • Luján R, Aguado C, Ciruela F, Arus XM, Martín-Belmonte A, Alfaro-Ruiz R, Martínez-Gómez J, de la Ossa L, Watanabe M, Adelman JP, Shigemoto R, Fukazawa Y
    Frontiers in cellular neuroscience 12 311 - 311 2018年 [査読有り][通常論文]
     
    The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.
  • Uchida H, Matsumura S, Katano T, Watanabe M, Schlossmann J, Ito S
    Molecular pain 14 1744806918796409  2018年01月 [査読有り][通常論文]
  • Haruko Kumamoto, Taku Yamaguchi, Kohtarou Konno, Takeshi Izumi, Takayuki Yoshida, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka
    Journal of Pharmacological Sciences 136 1 1 - 8 2018年01月01日 [査読有り][通常論文]
     
    Childhood maltreatment is associated with impaired adult brain function, particularly in the hippocampus, and is not only a major risk factor for some psychiatric diseases but also affects early social development and social adaptation in later life. The aims of this study were to determine whether early postnatal stress affects social behavior and whether repeated fluvoxamine treatment reverses these changes. Rat pups were exposed to footshock stress during postnatal days 21–25 (at 3 weeks old: 3wFS). During the post-adolescent period (10–14 weeks postnatal), the social interaction test and Golgi-cox staining of dorsal hippocampal pyramidal neurons were performed. Following exposure to footshock stress, 3wFS rats showed an increase in social interaction time, which might be practically synonymous with hypersociability, and a decrease in spine density in the CA3 hippocampal subregion, but not in CA1. These behavioral and morphological changes were both recovered by repeated oral administration of fluvoxamine at a dose of 10 mg/kg/day for 14 days. These findings suggest that the vulnerability of the hippocampal CA3 region is closely related to social impairments induced by physical stress during the juvenile period and shed some light on therapeutic alternatives for early postnatal stress-induced emotional dysfunction.
  • Jean-Marc Good, Michael Mahoney, Taisuke Miyazaki, Kenji F. Tanaka, Kenji Sakimura, Masahiko Watanabe, Kazuo Kitamura, Masanobu Kano
    CELL REPORTS 21 8 2066 - 2073 2017年11月 [査読有り][通常論文]
     
    Neural circuits undergo massive refinements during postnatal development. In the developing cerebellum, the climbing fiber (CF) to Purkinje cell (PC) network is drastically reshaped by eliminating early-formed redundant CF to PC synapses. To investigate the impact of CF network refinement on PC population activity during postnatal development, we monitored spontaneous CF responses in neighboring PCs and the activity of populations of nearby CF terminals using in vivo two-photon calcium imaging. Population activity is highly synchronized in newborn mice, and the degree of synchrony gradually declines during the first postnatal week in PCs and, to a lesser extent, in CF terminals. Knockout mice lacking P/Q-type voltage-gated calcium channel or glutamate receptor delta 2, in which CF network refinement is severely impaired, exhibit an abnormally high level of synchrony in PC population activity. These results suggest that CF network refinement is a structural basis for developmental desynchronization and maturation of PC population activity.
  • Kieran A. Boyle, Maria Gutierrez-Mecinas, Erika Polgar, Nicole Mooney, Emily O'Connor, Takahiro Furuta, Masahiko Watanabe, Andrew J. Todd
    NEUROSCIENCE 363 120 - 133 2017年11月 [査読有り][通常論文]
     
    Around a quarter of neurons in laminae I-II of the dorsal horn are inhibitory interneurons. These play an important role in modulating somatosensory information, including that perceived as pain or itch. Previous studies in rat identified four largely non-overlapping neurochemical populations among these cells, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin. The galanin cells were subsequently shown to coexpress dynorphin. Several recent studies have used genetically modified mice to investigate the function of different interneuron populations, and it is therefore important to determine whether the same pattern applies in mouse, and to estimate the relative sizes of these populations. We show that the neurochemical organization of inhibitory interneurons in mouse superficial dorsal horn is similar to that in the rat, although a larger proportion of these neurons (33%) express NPY. Between them, these four populations account for similar to 75% of inhibitory cells in laminae I-II. Since similar to 25% of inhibitory interneurons in this region belong to a novel calretinin-expressing type, our results suggest that virtually all inhibitory interneurons in superficial dorsal horn can be assigned to one of these five neurochemical populations. Although our main focus was inhibitory neurons, we also identified a population of excitatory dynorphin-expressing cells in laminae I-II that are largely restricted to the medial part of the mid-lumbar dorsal horn, corresponding to glabrous skin territory. These findings are important for interpretation of studies using molecular-genetic techniques to manipulate the functions of interneuron populations to investigate their roles in somatosensory processing. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of IBRO.
  • Zsuzsanna Bardoczi, Balazs Pal, Aron Koszeghy, Tamas Wilheim, Masahiko Watanabe, Laszlo Zaborszky, Zsolt Liposits, Imre Kallo
    JOURNAL OF NEUROSCIENCE 37 39 9534 - 9549 2017年09月 [査読有り][通常論文]
     
    The basal forebrain (BF) receives afferents from brainstem ascending pathways, which has been implicated first by Moruzzi and Magoun (1949) to induce forebrain activation and cortical arousal/waking behavior; however, it is very little known about how brainstem inhibitory inputs affect cholinergic functions. In the current study, glycine, a major inhibitory neurotransmitter of brainstem neurons, and gliotransmitter of local glial cells, was tested for potential interaction with BF cholinergic (BFC) neurons in male mice. In the BF, glycine receptor alpha subunit-immunoreactive (IR) sites were localized in choline acetyltransferase (ChAT)-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs (sIPSCs; 0.81 +/- 0.25 +/- 10(-1) Hz) recorded in whole-cell conditions. Potential neuronal as well as glial sources of glycine were indicated in the extracellular space of cholinergic neurons by glycine transporter type 1 (GLYT1)- and GLYT2-IR processes found in apposition to ChAT-IR cells. Ultrastructural analyses identified synapses of GLYT2-positive axon terminals on ChAT-IR neurons, as well as GLYT1-positive astroglial processes, which were localized in the vicinity of synapses ofChAT-IRneurons. The brainstem raphe magnus was determined to be a major source of glycinergic axons traced retrogradely from the BF. Our results indicate a direct effect of glycine on BFC neurons. Furthermore, the presence of high levels of plasma membrane glycine transporters in the vicinity of cholinergic neurons suggests a tight control of extracellular glycine in the BF.
  • Shioda N, Yabuki Y, Wang Y, Uchigashima M, Hikida T, Sasaoka T, Mori H, Watanabe M, Sasahara M, Fukunaga K
    Mol Psychiatry 22 8 1205 - 1222 2017年08月 [査読有り][通常論文]
     
    Aberrant dopamine D-2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/ platelet-derived growth factor receptor-beta (PDGFR beta)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFR beta are then transported to the Golgi apparatus, where those complexes trigger Gai3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFR beta is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.
  • Tomonori Aikawa, Takaki Watanabe, Taisuke Miyazaki, Takayasu Mikuni, Minoru Wakamori, Miyano Sakurai, Hidenori Aizawa, Nobutaka Ishizu, Masahiko Watanabe, Masanobu Kano, Hidehiro Mizusawa, Kei Watase
    HUMAN MOLECULAR GENETICS 26 16 3094 - 3104 2017年08月 [査読有り][通常論文]
     
    Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Ca(v)2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1a(CtmKO/CtmKO) mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Ca(v)2.1 with Ca-v beta 4 and Rimbp2 were significantly reduced while those with GABA(B2) were enhanced in the cerebellum of Cacna1a(CtmKO/CtmKO) mice. Treatment with the GABA(B) antagonist CGP35348 partially rescued the motor impairments seen in Cacna1a(CtmKO/CtmKO) mice. These results suggest that the carboxyl-terminal domain of Ca(v)2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Ca(v)2.1 with other proteins, and plays an essential role in preventing a complex neurological disease.
  • Myeongjeong Choo, Taisuke Miyazaki, Maya Yamazaki, Meiko Kawamura, Takanobu Nakazawa, Jianling Zhang, Asami Tanimura, Naofumi Uesaka, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    NATURE COMMUNICATIONS 8 1 195  2017年08月 [査読有り][通常論文]
     
    Elimination of early-formed redundant synapses during postnatal development is essential for functional neural circuit formation. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs). A single CF is strengthened whereas the other CFs are eliminated in each PC dependent on postsynaptic activity in PC, but the underlying mechanisms are largely unknown. Here, we report that brain-derived neurotrophic factor (BDNF) from PC facilitates CF synapse elimination. By PC-specific deletion of BDNF combined with knockdown of BDNF receptors in CF, we show that BDNF acts retrogradely on TrkB in CFs, and facilitates elimination of CF synapses from PC somata during the third postnatal week. We also show that BDNF shares signaling pathway with metabotropic glutamate receptor 1, a key molecule that triggers a canonical pathway for CF synapse elimination. These results indicate that unlike other synapses, BDNF mediates punishment signal for synapse elimination in the developing cerebellum.
  • Norikazu Katayama, Saori Yamamori, Masahiro Fukaya, Shizuka Kobayashi, Masahiko Watanabe, Masami Takahashi, Toshiya Manabe
    SCIENTIFIC REPORTS 7 1 7996  2017年08月 [査読有り][通常論文]
     
    Neurotransmitter release is mediated by the SNARE complex, but the role of its phosphorylation has scarcely been elucidated. Although PKC activators are known to facilitate synaptic transmission, there has been a heated debate on whether PKC mediates facilitation of neurotransmitter release through phosphorylation. One of the SNARE proteins, SNAP-25, is phosphorylated at the residue serine-187 by PKC, but its physiological significance has been unclear. To examine these issues, we analyzed mutant mice lacking the phosphorylation of SNAP-25 serine-187 and found that they exhibited reduced release probability and enhanced presynaptic short-term plasticity, suggesting that not only the release process, but also the dynamics of synaptic vesicles was regulated by the phosphorylation. Furthermore, it has been known that the release probability changes with development, but the precise mechanism has been unclear, and we found that developmental changes in release probability of neurotransmitters were regulated by the phosphorylation. These results indicate that SNAP-25 phosphorylation developmentally facilitates neurotransmitter release but strongly inhibits presynaptic short-term plasticity via modification of the dynamics of synaptic vesicles in presynaptic terminals.
  • Tomoko Yoshikawa, Natsuko F. Inagaki, Seiji Takagi, Shigeru Kuroda, Miwako Yamasaki, Masahiko Watanabe, Sato Honma, Ken-ichi Honma
    SCIENTIFIC REPORTS 7 1 8210  2017年08月 [査読有り][通常論文]
     
    The circadian pacemaker in the suprachiasmatic nucleus (SCN) yields photoperiodic response to transfer seasonal information to physiology and behavior. To identify the precise location involved in photoperiodic response in the SCN, we analyzed circadian Period1 and PERIOD2 rhythms in horizontally sectioned SCN of mice exposed to a long or short day. Statistical analyses of bioluminescence images with respective luciferase reporters on pixel level enabled us to identify the distinct localization of three oscillating regions; a large open-ring-shape region, the region at the posterior end and a sharply demarcated oval region at the center of the SCN. The first two regions are the respective sites for the so-called evening and morning oscillators, and the third region is possibly a site for mediating photic signals to the former oscillators. In these regions, there are two classes of oscillating cells in which Per1 and Per2 could play differential roles in photoperiodic responses.
  • Xavier Morato, Rafael Lujan, Marc Lopez-Cano, Jorge Gandia, Igor Stagljar, Masahiko Watanabe, Rodrigo A. Cunha, Victor Fernandez-Duenas, Francisco Ciruela
    SCIENTIFIC REPORTS 7 1 9452  2017年08月 [査読有り][通常論文]
     
    G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A(2A) receptor (A(2A)R) cell surface expression and function in vivo. In addition, we showed that GPR37 and A(2A)R do oligomerize in the striatum. Thus, a close proximity of GPR37 and A(2A)R at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal A(2A)R cell surface expression that correlated well with an increased A(2A)R agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/-mice showed enhanced A(2A)R agonist-induced catalepsy and an increased response to A(2A)R antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling A(2A)R biology in the striatum, which may be relevant for PD management.
  • Yoon-Mi Oh, Fuyuki Karube, Susumu Takahashi, Kenta Kobayashi, Masahiko Takada, Motokazu Uchigashima, Masahiko Watanabe, Kayo Nishizawa, Kazuto Kobayashi, Fumino Fujiyama
    BRAIN STRUCTURE & FUNCTION 222 5 2359 - 2378 2017年07月 [査読有り][通常論文]
     
    In the present study, we generated a novel parvalbumin (PV)-Cre rat model and conducted detailed morphological and electrophysiological investigations of axons from PV neurons in globus pallidus (GP). The GP is considered as a relay nucleus in the indirect pathway of the basal ganglia (BG). Previous studies have used molecular profiling and projection patterns to demonstrate cellular heterogeneity in the GP; for example, PV-expressing neurons are known to comprise approximately 50% of GP neurons and represent majority of prototypic neurons that project to the subthalamic nucleus and/or output nuclei of BG, entopeduncular nucleus and substantia nigra (SN). The present study aimed to identify the characteristic projection patterns of PV neurons in the GP (PV-GP neurons) and determine whether these neurons target dopaminergic or GABAergic neurons in SN pars compacta (SNc) or reticulata (SNr), respectively. We initially found that (1) 57% of PV neurons co-expressed Lim-homeobox 6, (2) the PVGP terminals were preferentially distributed in the ventral part of dorsal tier of SNc, (3) PV-GP neurons formed basket-like appositions with the somata of tyrosine hydroxylase, PV, calretinin and cholecystokinin immunoreactive neurons in the SN, and (4) in vitro wholecell recording during optogenetic photo-stimulation of PVGP terminals in SNc demonstrated that PV-GP neurons strongly inhibited dopamine neurons via GABA(A) receptors. These results suggest that dopamine neurons receive direct focal inputs from PV-GP prototypic neurons. The identification of high-contrast inhibitory systems on dopamine neurons might represent a key step toward understanding the BG function.
  • Hiromi Takahashi-Iwanaga, Shunsuke Kimura, Kohtarou Konno, Masahiko Watanabe, Toshihiko Iwanaga
    AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 313 1 F20 - F29 2017年07月 [査読有り][通常論文]
     
    The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet; 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Kazuhisa Kohda, Michisuke Yuzaki, Keiko Shimamoto, Kohichi Tanaka, Masanobu Kano, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 28 7438 - 7443 2017年07月 [査読有り][通常論文]
     
    Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the L-glutamate/L-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and non-synaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF-PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10-90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs.
  • Hozumi Y, Fujiwara H, Kaneko K, Fujii S, Topham MK, Watanabe M, Goto K
    Cell and tissue research 368 3 441 - 458 2017年06月 [査読有り][通常論文]
     
    Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGK epsilon is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGK epsilon play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGK epsilon antibody. DGK epsilon was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGK epsilon was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGK epsilon was also distributed in close apposition to DG lipase-alpha, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGK epsilon-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGK epsilon plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.
  • Takafumi Miki, Walter A. Kaufmann, Gerardo Malagon, Laura Gomez, Katsuhiko Tabuchi, Masahiko Watanabe, Ryuichi Shigemoto, Alain Marty
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 26 E5246 - E5255 2017年06月 [査読有り][通常論文]
     
    Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such "simple synapses" indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Ca(v)2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Ca(v)2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1-10) and 2.03 at 4 wk (range: 1-4), whereas the mean numbers of Ca(v)2.1 clusters were 2.84 at 2 wk (range: 1-8) and 2.37 at 4 wk (range: 1-5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 mu m(2) to 0.0234 mu m(2)), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels.
  • Nobuhiro Nakai, Masatoshi Nagano, Fumihito Saitow, Yasuhito Watanabe, Yoshinobu Kawamura, Akiko Kawamoto, Kota Tamada, Hiroshi Mizuma, Hirotaka Onoe, Yasuyoshi Watanabe, Hiromu Monai, Hajime Hirase, Jin Nakatani, Hirofumi Inagaki, Tomoyuki Kawada, Taisuke Miyazaki, Masahiko Watanabe, Yuka Sato, Shigeo Okabe, Kazuo Kitamura, Masanobu Kano, Kouichi Hashimoto, Hidenori Suzuki, Toru Takumi
    SCIENCE ADVANCES 3 6 e1603001  2017年06月 [査読有り][通常論文]
     
    Serotonin is a critical modulator of cortical function, and its metabolism is defective in autism spectrum disorder (ASD) brain. How serotonin metabolism regulates cortical physiology and contributes to the pathological and behavioral symptoms of ASD remains unknown. We show that normal serotonin levels are essential for the maintenance of neocortical excitation/inhibition balance, correct sensory stimulus tuning, and social behavior. Conversely, low serotonin levels in 15q dup mice (a model for ASD with the human 15q11-13 duplication) result in impairment of the same phenotypes. Restoration of normal serotonin levels in 15q dup mice revealed the reversibility of a subset of ASD-related symptoms in the adult. These findings suggest that serotonin may have therapeutic potential for discrete ASD symptoms.
  • Akira Ooki, Kiwamu Akagi, Toshimasa Yatsuoka, Masako Asayama, Hiroki Hara, Yoji Nishimura, Hiroshi Katoh, Keishi Yamashita, Masahiko Watanabe, Kensei Yamaguchi
    Journal of Gastrointestinal Surgery 21 5 867 - 878 2017年05月01日 [査読有り][通常論文]
     
    Background: Although several markers, including the lymph node ratio (LNR), have been proposed as a clinically prognostic tool for colorectal cancer (CRC), it remains unclear which markers have the most relevance in determining recurrence following adjuvant chemotherapy for stage III CRC. Methods: Independent risk factors for recurrence-free survival (RFS) were retrospectively determined using the Cox proportional hazard model in 360 stage III CRC patients and validated using an independent cohort comprising 172 stage III CRC patients. Results: The LNR was independently associated with RFS (HR, 1.96 95% CI, 1.11 to 3.28 P = 0.020). A higher LNR value was significantly associated with recurrence, microsatellite stable, and shorter time to recurrence. A combination of the LNR with pre-chemotherapy CEA and CA19-9, other independent risk factors, provided accurate risk stratification of RFS and conferred additional information on recurrence within each stage III CRC subgroup, which was then validated in an independent cohort. A beneficial effect in patients at risk of recurrence, and a reduced effect in patients at low risk, was exhibited by the addition of oxaliplatin to 5-fluorouracil-based adjuvant chemotherapy. Conclusion: A higher LNR is one of the most aggressive phenotypes with recurrence risk following adjuvant chemotherapy for stage III CRC.
  • Junko Kono, Kohtarou Konno, Ashraf Hossain Talukder, Toshimitsu Fuse, Manabu Abe, Katsuya Uchida, Shuhei Horio, Kenji Sakimura, Masahiko Watanabe, Keiichi Itoi
    BRAIN STRUCTURE & FUNCTION 222 4 1705 - 1732 2017年05月 [査読有り][通常論文]
     
    We examined the morphological features of corticotropin-releasing factor (CRF) neurons in a mouse line in which modified yellow fluorescent protein (Venus) was expressed under the CRF promoter. We previously generated the CRF-Venus knock-in mouse, in which Venus is inserted into the CRF gene locus by homologous recombination. In the present study, the neomycin phosphotransferase gene (Neo), driven by the pgk-1 promoter, was deleted from the CRF-Venus mouse genome, and a CRF-Venusa dagger Neo mouse was generated. Venus expression is much more prominent in the CRF-Venusa dagger Neo mouse when compared to the CRF-Venus mouse. In addition, most Venus-expressing neurons co-express CRF mRNA. Venus-expressing neurons constitute a discrete population of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) that project to the median eminence. Venus-expressing neurons were also found in brain regions outside the neuroendocrine PVH, including the olfactory bulb, the piriform cortex (Pir), the extended amygdala, the hippocampus, the neocortices, Barrington's nucleus, the midbrain/pontine dorsal tegmentum, the periaqueductal gray, and the inferior olivary nucleus (IO). Venus-expressing perikarya co-expressing CRF mRNA could be observed clearly even in regions where CRF-immunoreactive perikarya could hardly be identified. We demonstrated that the CRF neurons contain glutamate in the Pir and IO, while they contain gamma-aminobutyric acid in the neocortex, the bed nucleus of the stria terminalis, the hippocampus, and the amygdala. A population of CRF neurons was demonstrated to be cholinergic in the midbrain tegmentum. The CRF-Venusa dagger Neo mouse may be useful for studying the structural and functional properties of CRF neurons in the mouse brain.
  • Kei Hosoda, Keishi Yamashita, Hiromitsu Moriya, Hiroaki Mieno, Masahiko Watanabe
    World Journal of Gastroenterology 23 15 2723 - 2730 2017年04月21日 [査読有り][通常論文]
     
    AIM: To determine the optimal treatment strategy for Siewert type II and III adenocarcinoma of the esophagogastric junction. METHODS: We retrospectively reviewed the medical records of 83 patients with Siewert type II and III adenocarcinoma of the esophagogastric junction and calculated both an index of estimated benefit from lymph node dissection for each lymph node (LN) station and a lymph node ratio (LNR: ratio of number of positive lymph nodes to the total number of dissected lymph nodes). We used Cox proportional hazard models to clarify independent poor prognostic factors. The median duration of observation was 73 mo. RESULTS: Indices of estimated benefit from LN dissection were as follows, in descending order: lymph nodes (LN) along the lesser curvature, 26.5 right paracardial LN, 22.8 left paracardial LN, 11.6 LN along the left gastric artery, 10.6. The 5-year overall survival (OS) rate was 58%. Cox regression analysis revealed that vigorous venous invasion (v2, v3) (HR = 5.99 95%CI: 1.71-24.90) and LNR of > 0.16 (HR = 4.29, 95%CI: 1.79-10.89) were independent poor prognostic factors for OS. CONCLUSION: LN along the lesser curvature, right and left paracardial LN, and LN along the left gastric artery should be dissected in patients with Siewert type II or III adenoca rcinoma of the esophagogastric junction. Patients with vigorous venous invasion and LNR of > 0.16 should be treated with aggressive adjuvant chemotherapy to improve survival outcomes.
  • Keishi Yamashita, Akira Ema, Kei Hosoda, Hiroaki Mieno, Hiromitsu Moriya, Natsuya Katada, Masahiko Watanabe
    World Journal of Gastrointestinal Oncology 9 4 166 - 175 2017年04月01日 [査読有り][通常論文]
     
    AIM To evaluate whether a high risk macroscopic appearance (Type IV and giant Type III) is associated with a dismal prognosis after curative surgery, because its prognostic relevance remains elusive in pathological stage II/III (pStage II/III) gastric cancer. METHODS One hundred and seventy-two advanced gastric cancer (defined as pT2 or beyond) patients with pStage II /III who underwent curative surgery plus adjuvant S1 chemotherapy were evaluated, and the prognostic relevance of a high-risk macroscopic appearance was examined. RESULTS Advanced gastric cancers with a high-risk macroscopic appearance were retrospectively identified by preoperative recorded images. A high-risk macroscopic appearance showed a significantly worse relapse free survival (RFS) (35.7%) and overall survival (OS) (34%) than an average risk appearance (P = 0.0003 and P < 0.0001, respectively). A high-risk macroscopic appearance was significantly associated with the 13th Japanese Gastric Cancer Association (JGCA) pT (P = 0.01), but not with the 13th JGCA pN. On univariate analysis for RFS and OS, prognostic factors included 13th JGCA pStage (P < 0.0001) and other clinicopathological factors including macroscopic appearance. A multivariate Cox proportional hazards model for univariate prognostic factors identified highrisk macroscopic appearance (P = 0.036, HR = 2.29 for RFS and P = 0.021, HR = 2.74 for OS) as an independent prognostic indicator. CONCLUSION A high-risk macroscopic appearance was associated with a poor prognosis, and it could be a prognostic factor independent of 13th JGCA stage in pStage II/III advanced gastric cancer.
  • Noriko Kawashima-Takeda, Yoshiya Ito, Nobuyuki Nishizawa, Rei Kawashima, Kiyoshi Tanaka, Kazutake Tsujikawa, Masahiko Watanabe, Masataka Majima
    Journal of Gastroenterology and Hepatology (Australia) 32 4 809 - 818 2017年04月01日 [査読有り][通常論文]
     
    Background and Aims: Calcitonin gene-related peptide (CGRP) is thought to be involved in the modulation of intestinal motility. CGRP receptor is composed of receptor activity-modifying protein (RAMP) 1 combined with calcitonin receptor-like receptor (CRLR) for CGRP. The study investigated the role of CGRP in mice with experimentally induced colitis. Methods: The study used dextran sodium sulfate (DSS) to induce colitis in mice. The study compared the severity of colitis in wild-type (WT) mice, mice treated with a CGRP receptor antagonist (CGRP8–37), and RAMP1 knockout (−/−) mice. Pathological changes in the mucosa were assessed, and inflammatory cells and cytokine levels were measured. Results: The severity of inflammation in DSS-induced colitis increased markedly in CGRP8–37-treated mice and RAMP1−/− mice compared with WT mice. RAMP1−/− mice showed more severe damage compared with CGRP8–37-treated mice. The number of periodic acid-Schiff-positive cells decreased in CGRP8–37-treated mice compared with WT mice and was even further decreased in RAMP1−/− mice. RAMP1 was expressed by macrophages, mast cells, and T-cells. RAMP1−/− mice exhibited excessive accumulation of macrophages and mast cells into the colonic tissue with increased levels of tumor necrosis factor-α and interleukin-1β as compared with WT mice. Infiltration of T-cells into the colonic mucosa, which was associated with the expression of T helper (Th) cytokines including Th1 (interferon gamma) and Th17 (IL-17), was augmented in RAMP1−/− mice. Conclusions: The findings of this study suggest that RAMP1 exerted mucosal protection in DSS-induced colitis via attenuation of recruitment of inflammatory cells and of pro-inflammatory cytokines.
  • Keishi Yamashita, Kei Hosoda, Hiromitsu Moriya, Chikatoshi Katada, Mitsuhiro Sugawara, Hiroaki Mieno, Shoko Komori, Natsuya Katada, Masahiko Watanabe
    Oncology (Switzerland) 92 4 221 - 228 2017年04月01日 [査読有り][通常論文]
     
    Background: Neoadjuvant chemotherapy (NAC) has become prevalent in esophageal squamous cell carcinoma (ESCC), but its long-term prognostic advantages remain unclear. The latest prognostic outcomes in clinical Stage (cStage) II/III ESCC with NAC were herein elucidated. Patients and Methods: NAC prior to curative treatment was done in 115 cStage II/III ESCC patients with either cisplatin (CDDP)/5-fluorouracil (5-FU CF) (n = 41) or docetaxel/CDDP/5-FU (DCF) NAC (n = 74) between 2007 and 2013. Results: (1) Esophagectomy was finally performed in 35 of the 41 CF NAC cases and in 48 of the 74 DCF NAC cases. The preservation rate of the esophagus was higher in the DCF NAC than in the CF NAC (p = 0.018). (2) The overall survival was better in DCF NAC than in CF NAC (p = 0.071), and progression-free survivals were 58.3% with DCF and 30.5% with CF (p = 0.0060). DCF NAC was associated with fewer cases of progression than CF NAC (p = 0.0040), largely due to excellent control of the preoperative disease (p = 0.018) and postoperative lymph node recurrence (p = 0.014). Conclusion: DCF NAC in cStage II/III ESCC could have a great potential to achieve a better prognosis due to suppression of specific progression events with a higher preservation rate of the esophagus.
  • Kei Hosoda, Keishi Yamashita, Shinichi Sakuramoto, Natsuya Katada, Hiromitsu Moriya, Hiroaki Mieno, Masahiko Watanabe
    American Journal of Surgery 213 4 763 - 770 2017年04月01日 [査読有り][通常論文]
     
    Background Little is known about postgastrectomy syndrome and quality of life (QOL after laparoscopy-assisted pylorus-preserving gastrectomy (LAPPG). The aim of this study was to assess postgastrectomy syndrome and QOL after LAPPG as compared with laparoscopy-assisted distal gastrectomy with Billroth-I reconstruction (LADGBI). Methods Postgastrectomy Syndrome Assessment Scale-45 (PGSAS-45) questionnaires were sent by mail to 167 patients. To balance the characteristics of the groups, propensity score matching was performed. Results Of the 167 patients sent questionnaires, 112 (67%) responded, including 47 who underwent LAPPG and 65 who underwent LADGBI. After propensity score matching, the LAPPG group scored significantly better on the diarrhea and dumping subscales. Multiple regression analysis showed that female sex and LADGBI were independent factors predicting dumping. Evaluation of outcome measures for singular symptom showed that the LAPPG group scored significantly worse on the acid regurgitation subscale, but significantly better on the lower abdominal pain and early dumping abdominal subscales. Conclusion LAPPG is superior to LADGBI for ameliorating postgastrectomy syndrome and maintaining QOL. LAPPG is recommended for patients with cT1N0 middle third gastric cancer.
  • Sam A. Booker, Daniel Althof, Anna Gross, Desiree Loreth, Johanna Mueller, Andreas Unger, Bernd Fakler, Andrea Varro, Masahiko Watanabe, Martin Gassmann, Bernhard Bettler, Ryuichi Shigemoto, Imre Vida, Akos Kulik
    CEREBRAL CORTEX 27 3 2318 - 2334 2017年03月 [査読有り][通常論文]
     
    Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABA(B) receptors (GABA(B)Rs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABA(B)R-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABA(B)R currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABA(B)R-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABA(B)R-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABA(B)Rs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins.
  • Maria Gutierrez-Mecinas, Andrew M. Bell, Alina Marin, Rebecca Taylor, Kieran A. Boyle, Takahiro Furuta, Masahiko Watanabe, Erika Polgar, Andrew J. Todd
    PAIN 158 3 440 - 456 2017年03月 [査読有り][通常論文]
     
    The superficial dorsal horn, which is the main target for nociceptive and pruritoceptive primary afferents, contains a high density of excitatory interneurons. Our understanding of their roles in somatosensory processing has been restricted by the difficulty of distinguishing functional populations among these cells. We recently defined 3 nonoverlapping populations among the excitatory neurons, based on the expression of neurotensin, neurokinin B, and gastrin-releasing peptide. Here we identify and characterise another population: neurons that express the tachykinin peptide substance P. We show with immunocytochemistry that its precursor protein (preprotachykinin A, PPTA) can be detected in; similar to 14% of lamina I-II neurons, and these are concentrated in the outer part of lamina II. Over 80% of the PPTA-positive cells lack the transcription factor Pax2 (which determines an inhibitory phenotype), and these account for similar to 15% of the excitatory neurons in this region. They are different from the neurotensin, neurokinin B, or gastrin-releasing peptide neurons, although many of them contain somatostatin, which is widely expressed among superficial dorsal horn excitatory interneurons. We show that many of these cells respond to noxious thermal and mechanical stimuli and to intradermal injection of pruritogens. Finally, we demonstrate that these cells can also be identified in a knock-in Cre mouse line (Tac1(Cre)), although our findings suggest that there is an additional population of neurons that transiently express PPTA. This population of substance P-expressing excitatory neurons is likely to play an important role in the transmission of signals that are perceived as pain and itch.
  • Akiyo Natsubori, Iku Tsutsui-Kimura, Hiroshi Nishida, Youcef Bouchekioua, Hiroshi Sekiya, Motokazu Uchigashima, Masahiko Watanabe, Alban de Kerchove d'Exaerde, Masaru Mimura, Norio Takata, Kenji F. Tanaka
    JOURNAL OF NEUROSCIENCE 37 10 2723 - 2733 2017年03月 [査読有り][通常論文]
     
    The ventral striatum is involved in motivated behavior. Akin to the dorsal striatum, the ventral striatum contains two parallel pathways: the striatomesencephalic pathway consisting of dopamine receptor Type 1-expressing medium spiny neurons (D1-MSNs) and the striatopallidal pathway consisting of D2-MSNs. These two genetically identified pathways are thought to encode opposing functions in motivated behavior. It has also been reported that D1/D2 genetic selectivity is not attributed to the anatomical discrimination of two pathways. We wanted to determine whether D1-and D2-MSNs in the ventral striatum functioned in an opposing manner as previous observations claimed, and whether D1/D2 selectivity corresponded to a functional segregation in motivated behavior of mice. To address this question, we focused on the lateral portion of ventral striatum as a region implicated in food-incentive, goal-directed behavior, and recorded D1 or D2-MSN activity by using a gene-encoded ratiometric Ca2+ indicator and by constructing a fiberphotometry system, and manipulated their activities via optogenetic inhibition during ongoing behaviors. We observed concurrent event-related compoundCa(2+) elevations in ventrolateral D1-and D2-MSNs, especially at trial start cue-related and first lever press-related times. D1 or D2 selective optogenetic inhibition just after the trial start cue resulted in a reduction of goal-directed behavior, indicating a shared coding of motivated behavior by both populations at this time. Only D1-selective inhibition just after the first lever press resulted in the reduction of behavior, indicating D1-MSN-specific coding at that specific time. Our data did not support opposing encoding by both populations in food-incentive, goal-directed behavior.
  • Hisataka Fujimoto, Kotaro Konno, Masahiko Watanabe, Shozo Jinno
    JOURNAL OF COMPARATIVE NEUROLOGY 525 4 868 - 884 2017年03月 [査読有り][通常論文]
     
    The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non-GABAergic phenotype, while they express well-established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (-) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV+/NOS-/GAD67(+), PV+/NOS+/GAD67(+), PV+/NOS-/GAD67(-), and PV-/NOS+/GAD67(-). Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67(-) IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67(+) IC neurons remained unchanged in all subdivisions. The NDs of PV+/NOS-/GAD67(+) neurons and PV-/NOS+/GAD67(-) neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV+/NOS+/GAD67(+) neurons and PV+/NOS-/GAD67(-) neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67(+) IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868-884, 2017. (c) 2016 Wiley Periodicals, Inc.
  • Hideki Ushiku, Keishi Yamashita, Hiroshi Katoh, Akira Ema, Naoko Minatani, Mariko Kikuchi, Ken Kojo, Keigo Yokoi, Toshimichi Tanaka, Nobuyuki Nishizawa, Satoru Ishii, Kei Hosoda, Hiromitsu Moriya, Hiroaki Mieno, Natsuya Katada, Shiro Kikuchi, Masahiko Watanabe
    Diseases of the Esophagus 30 2 2017年02月01日 [査読有り][通常論文]
     
    We have demonstrated that CDO1 methylation is frequently found in various cancers, including esophageal squamous cell carcinoma (ESCC), but its clinical relevance has remained elusive. CDO1 methylation was investigated in 169 ESCC patients who underwent esophagectomy between 1996 and 2007. CDO1 methylation was assessed by Q-MSP (quantitative methylation specific PCR), and its clinical significance, including its relationship to prognosis, was analyzed. (i) The median TaqMeth value of CDO1 methylation was 9.4, ranging from 0 to 279.5. CDO1 methylation was significantly different between cStage I and cStage II/III (P=0.02). (ii) On the log-rank plot, the optimal cut-off value was determined to be 8.9 ESCC patients with high CDO1 methylation showed a significantly worse prognosis than those with low CDO1 methylation (P=0.02). (iii) A multivariate Cox proportional hazards model identified only CDO1 hypermethylation as an independent prognostic factor (HR 2.00, CI 1.09-3.78, P=0.03). (iv) CDO1 hypermethylation stratified ESCC patients' prognosis in cStage II/III for both neoadjuvant chemo(radio)therapy (NAC)-positive and NAC-negative cases.Moreover, the CDO1 methylation level was significantly lower in cases with Grade 2/3 than in those with Grade 0/1 (P=0.02) among cStage II/III ESCC patients withNAC. PromoterDNA hypermethylation of CDO1 could be an independent prognostic factor in ESCC it may also reflect NAC eradication of tumor cells in the primary tumors.
  • Iku Tsutsui-Kimura, Hiroyuki Takiue, Keitaro Yoshida, Ming Xu, Ryutaro Yano, Hiroyuki Ohta, Hiroshi Nishida, Youcef Bouchekioua, Hideyuki Okano, Motokazu Uchigashima, Masahiko Watanabe, Norio Takata, Michael R. Drew, Hiromi Sano, Masaru Mimura, Kenji F. Tanaka
    Nature Communications 8 14304  2017年02月 [査読有り][通常論文]
     
    Impaired motivation is present in a variety of neurological disorders, suggesting that decreased motivation is caused by broad dysfunction of the nervous system across a variety of circuits. Based on evidence that impaired motivation is a major symptom in the early stages of Huntington's disease, when dopamine receptor type 2-expressing striatal medium spiny neurons (D2-MSNs) are particularly affected, we hypothesize that degeneration of these neurons would be a key node regulating motivational status. Using a progressive, time-controllable, diphtheria toxin-mediated cell ablation/dysfunction technique, we find that loss-of-function of D2-MSNs within ventrolateral striatum (VLS) is sufficient to reduce goal-directed behaviours without impairing reward preference or spontaneous behaviour. Moreover, optogenetic inhibition and ablation of VLS D2-MSNs causes, respectively, transient and chronic reductions of goal-directed behaviours. Our data demonstrate that the circuitry containing VLS D2-MSNs control motivated behaviours and that VLS D2-MSN loss-of-function is a possible cause of motivation deficits in neurodegenerative diseases.
  • Jamie R. Flynn, Victoria L. Conn, Kieran A. Boyle, David I. Hughes, Masahiko Watanabe, Tomoko Velasquez, Martyn D. Goulding, Robert J. Callister, Brett A. Graham
    FRONTIERS IN NEUROANATOMY 11 5  2017年02月 [査読有り][通常論文]
     
    Long descending propriospinal neurons (LDPNs) are interneurons that form direct connections between cervical and lumbar spinal circuits. LDPNs are involved in interlimb coordination and are important mediators of functional recovery after spinal cord injury (SCI). Much of what we know about LDPNs comes from a range of species, however, the increased use of transgenic mouse lines to better define neuronal populations calls for a more complete characterisation of LDPNs in mice. In this study, we examined the cell body location, inhibitory neurotransmitter phenotype, developmental provenance, morphology and synaptic inputs of mouse LDPNs throughout the cervical and upper thoracic spinal cord. LDPNs were retrogradely labelled from the lumbar spinal cord to map cell body locations throughout the cervical and upper thoracic segments. Ipsilateral LDPNs were distributed throughout the dorsal, intermediate and ventral grey matter as well as the lateral spinal nucleus and lateral cervical nucleus. In contrast, contralateral LDPNs were more densely concentrated in the ventromedial grey matter. Retrograde labelling in GlyT2(GFP) and GAD67(GFP) mice showed the majority of inhibitory LDPNs project either ipsilaterally or adjacent to the midline. Additionally, we used several transgenic mouse lines to define the developmental provenance of LDPNs and found that V2b positive neurons form a subset of ipsilaterally projecting LDPNs. Finally, a population of Neurobiotin (NB) labelled LDPNs were assessed in detail to examine morphology and plot the spatial distribution of contacts from a variety of neurochemically distinct axon terminals. These results provide important baseline data in mice for future work on their role in locomotion and recovery from SCI.
  • Hiroki Harada, Keishi Yamashita, Kei Hosoda, Hiromitsu Moriya, Hiroaki Mieno, Akira Ema, Marie Washio, Chikatoshi Katada, Shoko Komori, Masahiko Watanabe
    Japanese Journal of Gastroenterological Surgery 50 12 941 - 955 2017年 [査読有り][通常論文]
     
    Purpose: Unique treatments of esophageal squamous cell carcinoma (ESCC) in our hospital are neoadjuvant chemotherapy using docetaxel/CDDP/5-FU in cStage II/III (excluding cT4) cases, and chemoradiotherapy using docetaxel/CDDP/5-FU in cT4 or cM1 lymph node metastasis cases. We herein report prognostic analysis of ESCC in a prospective database determined by treatment strategy based on a cancer board. Materials and Methods: Between January 2009 and March 2016, prognostic analyses were performed based on a prospective database of 504 ESCC debated on an esophageal cancer board. Result: (1) Five-year overall survival rate for each cStage (I/IIA/IIB/III/IV) in the surgical treatment cases were 88.3/85.2/83.8/55.8/66.7%, respectively. (2) In the cases of surgical treatment in cStage I/IIB, five-year disease specific survival rate was very good: 95.3/90.2%, respectively. In cStage IIB, neoadjuvant chemotherapy was administered to 90.5% of cases. (3) Surgical treatment outcomes in cStage IIA/III cases were significantly better than nonsurgical treatments except for cT4 cases with poor prognosis (P=0.0229). (4) Long-term survival cases in cStage IV was those with conversion surgery for positive para aortic lymph node metastasis cases. Conclusion: Prognosis was deemed to be optimized by multidisciplinary treatment decision based on esophageal cancer board. Especially, the prognosis of surgical treatment cases was the best and it was considered to be the most recommended treatment at present.
  • Satoru Ishii, Keishi Yamashita, Hiroki Harada, Hideki Ushiku, Toshimichi Tanaka, Nobuyuki Nishizawa, Keigo Yokoi, Marie Washio, Akira Ema, Hiroaki Mieno, Hiromitsu Moriya, Kei Hosoda, Mina Waraya, Hiroshi Katoh, Masahiko Watanabe
    Oncotarget 8 43 74567 - 74581 2017年 [査読有り][通常論文]
     
    We previously demonstrated that the lymph node ratio (LNR) is a prognostic factor associated with EGFR expression, among first priority genes amplified or overexpressed in cancer. Here, we investigated the associations between high LNR and second, third, and fourth priority genes. We performed mRNA expression microarray analysis of tumor tissue from patients with stage III gastric cancer and high or low LNRs. Candidate high LNR-associated genes were further evaluated in 39 patients with stage III gastric cancer. The functional relevance of these genes was evaluated in gastric cancer cell lines. We focused on five genes: H19,PEG10, IGF2BP3, CD177, and PGA3. H19 and PEG10 were confirmed as high LNR-associated genes. H19, PEG10, and IGF2BP3 were found to promote each other's expression. Knocking down H19 or PEG10 using RNAi decreased cell proliferation, invasion, anchorageindependent growth, and chemoresistance. These genes had a mutual relationship in MKN7 cells. H19 knockdown decreased expression of epithelial-mesenchymal transition-associated genes in MKN74 cells to suppress transformation. Thus, H19 promotes epithelial-mesenchymal transition in gastric cancer and is a potential therapeutic target.
  • Hiromi Takahashi-Iwanaga, Shunsuke Kimura, Kohtarou Konno, Masahiko Watanabe, Toshihiko Iwanaga
    American Journal of Physiology - Renal Physiology 313 1 F20 - F29 2017年 [査読有り][通常論文]
     
    The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.
  • Keigo Yokoi, Kei Hosoda, Natsuya Katada, Keishi Yamashita, Hiromitsu Moriya, Hiroaki Mieno, Makoto Ohbu, Masahiko Watanabe
    Japanese Journal of Gastroenterological Surgery 50 6 419 - 428 2017年 [査読有り][通常論文]
     
    A 29-year-old man was given a diagnosis as having primary mediastinal large B-cell lymphoma and received RCHOP chemotherapy followed by radiation therapy with a total dose of 30 Gy (to the upper mediastinum, bilateral irradiation of 28 Gy and oblique irradiation of 2 Gy) after the chemotherapy. He suffered dysphasia 2 months after the irradiation. Upper gastrointestinal endoscopy showed total obstruction of the upper thoracic esophagus, therefore, endoscopic dilation was impossible. Because the patient's nutritional status was very poor, and he was unable to eat anything by mouth, we first performed tube enterostomy to improve his nutritional status. After enteral feeding for 1.5 months, we then performed esophagectomy. Taking into account the pathological findings and the clinical course, we thought the cause of the esophageal obstruction was radiation esophagitis. The postoperative course was uneventful, and he was discharged 20 days after the esophagectomy. We often experience radiation esophagitis, however, only a few cases result in esophageal obstruction. In the case of esophageal obstruction due to radiation esophagitis, for which it is thought impossible to perform endoscopic dilation, and in which the possibility of malignant disease cannot be ruled out, we need to perform esophagectomy, which can also serve as accurate diagnosis.
  • Mariko Kikuchi, Hiroshi Katoh, Mina Waraya, Yoko Tanaka, Satoru Ishii, Toshimichi Tanaka, Nobuyuki Nishizawa, Keigo Yokoi, Naoko Minatani, Akira Ema, Yoshimasa Kosaka, Hirokazu Tanino, Keishi Yamashita, Masahiko Watanabe
    Cancer Letters 384 70 - 78 2017年01月01日 [査読有り][通常論文]
     
    Epigenetic silencing of HOPX has been shown to be frequent and specific in human cancers. HOPX is thought as a tumor suppressor gene and its promoter methylation is the main mechanism of down-regulation. In non-hereditary breast cancer, since roles of epigenetic modifications are more critical than in other cancers, the aim of this study is to seek into the roles and clinical relevance of epigenetic silencing of HOPX. Down-regulation of HOPX was observed in all human breast cancer cell lines tested. The promoter methylation was found in six of seven cell lines, and demethylating agents restored HOPX expression. The promoter methylation was cancer-specific in human breast tissues. Forced expression of HOPX attenuated anchorage-independent growth in vitro. HOPX promoter methylation independently predicted worse prognosis of breast cancer patients. Of note, HOPX promoter methylation was significantly associated with HER2 positivity as well as advanced lymph node metastasis. HOPX promoter methylation is not only frequent and cancer-specific but also associated with aggressive phenotype in breast cancer. Epigenetic silencing of HOPX may have clinical potential as a biomarker in the treatment strategy of breast cancer patients.
  • Fukudome Y, Ohno-Shosaku T, Matsui M, Omori Y, Watanabe M, Kano M
    Eur J Nuerosci. 19 10 2682 - 2692 2017年 [査読有り][通常論文]
  • Masanobu Kano, Takaki Watanabe
    F1000Research 6 416  2017年 [査読有り][通常論文]
     
    The cerebellum is a brain structure involved in coordination, control, and learning of movements, as well as certain aspects of cognitive function. Purkinje cells are the sole output neurons from the cerebellar cortex and therefore play crucial roles in the overall function of the cerebellum. The type-1 metabotropic glutamate receptor (mGluR1) is a key "hub" molecule that is critically involved in the regulation of synaptic wiring, excitability, synaptic response, and synaptic plasticity of Purkinje cells. In this review, we aim to highlight how mGluR1 controls these events in Purkinje cells. We also describe emerging evidence that altered mGluR1 signaling in Purkinje cells underlies cerebellar dysfunctions in several clinically relevant mouse models of human ataxias.
  • Victoria E. Abraira, Emily D. Kuehn, Anda M. Chirila, Mark W. Springel, Alexis A. Toliver, Amanda L. Zimmerman, Lauren L. Orefice, Kieran A. Boyle, Ling Bai, Bryan J. Song, Karleena A. Bashista, Thomas G. O'Neill, Justin Zhuo, Connie Tsan, Jessica Hoynoski, Michael Rutlin, Laura Kus, Vera Niederkofler, Masahiko Watanabe, Susan M. Dymecki, Sacha B. Nelson, Nathaniel Heintz, David I. Hughes, David D. Ginty
    CELL 168 1-2 295 - + 2017年01月 [査読有り][通常論文]
     
    The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.
  • Eiichi Tokuda, Itsuki Anzai, Takao Nomura, Keisuke Toichi, Masahiko Watanabe, Shinji Ohara, Seiji Watanabe, Koji Yamanaka, Yuta Morisaki, Hidemi Misawa, Yoshiaki Furukawa
    MOLECULAR NEURODEGENERATION 12 1 2  2017年01月 [査読有り][通常論文]
     
    Background: Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS) with accumulation of misfolded SOD1 proteins as intracellular inclusions in spinal motor neurons. Oligomerization of SOD1 via abnormal disulfide crosslinks has been proposed as one of the misfolding pathways occurring in mutant SOD1; however, the pathological relevance of such oligomerization in the SOD1-ALS cases still remains obscure. Methods: We prepared antibodies exclusively recognizing the SOD1 oligomers cross-linked via disulfide bonds in vitro. By using those antibodies, immunohistochemical examination and ELISA were mainly performed on the tissue samples of transgenic mice expressing mutant SOD1 proteins and also of human SOD1-ALS cases. Results: We showed the recognition specificity of our antibodies exclusively toward the disulfide-crosslinked SOD1 oligomers by ELISA using various forms of purified SOD1 proteins in conformationally distinct states in vitro. Furthermore, the epitope of those antibodies was buried and inaccessible in the natively folded structure of SOD1. The antibodies were then found to specifically detect the pathological SOD1 species in the spinal motor neurons of the SOD1-ALS patients as well as the transgenic model mice. Conclusions: Our findings here suggest that the SOD1 oligomerization through the disulfide-crosslinking associates with exposure of the SOD1 structural interior and is a pathological process occurring in the SOD1-ALS cases.
  • Hirokazu Tanino, Yoshimasa Kosaka, Hiroshi Nishimiya, Youko Tanaka, Naoko Minatani, Mariko Kikuchi, Akiko Shida, Mina Waraya, Hiroshi Katoh, Takumo Enomoto, Norihiko Sengoku, Sabine Kajita, Robert M. Hoffman, Masahiko Watanabe
    PLoS ONE 11 12 2016年12月01日 [査読有り][通常論文]
     
    BRCAness is defined as the set of traits in which BRCA1 dysfunction, arising from gene mutation, methylation or deletion, results in DNA repair deficiency. In the present study, we addressed BRCAness, therapeutic efficacy, recurrence, and survival in patients with triple negative breast cancer (TNBC) who were treated with neoadjuvant chemotherapy at Kitasato University Hospital, Japan, between April 2006 and October 2012. BRCAness was determined by preoperative core needle biopsy (CNB) specimens and surgical specimens. Assay was performed using Multiplex Ligation-dependent Probe Amplification (MLPA) with P376-B2 BRCA1ness probemix (MRC-Holland, Amsterdam, The Netherlands). The relative copy number ratio of each sample was compared to Human Genomic DNA (Promega, Madison, WI, USA) as reference samples was calculated with Coffalyser. NET default settings. The BRCAness score was calculated with the relative copy number ratio of various DNA sequences. Values of 0.5 or more were determined as the BRCA1-like Type (BRCAness) and those of less than 0.5 as the Sporadic Type to analyze pathological complete response (pCR) rate, recurrence, and survival. pCR (ypT0/Tis/N0) was observed in 15 patients (pCR rate: 37.5%). These patients had no recurrence. Twelve patients recurred, 8 died from breast cancer. The BRCA1-like Type were 22 and Sporadic Type were 18 in CNB specimens. No major differences were observed between the BRCA1-like Type and Sporadic Type with pCR rate, recurrence rate and survival. Twenty four surgical specimens of non-pCR patients were available and 9 were BRCA1-like Type, who had more recurrences (7/9 vs. 5/15), and their relapse-free survival was also lower (p< 0.05) than that of Sporadic Type. Seven BRCA1-like Type patients remained BRCA1-like Type in surgical specimens, were worse in recurrence (p< 0.01) and survival (p< 0.05) compared with 6 patients whose BRCA status in surgical specimens turned to Sporadic Type. New clinical trials assessing the true recurrence (TR) rate of BRCA-type patients are expected since neither platinum-containing drugs nor poly (ADP-ribose) polymerase (PARP) inhibitors are effective against tumors with nonfunctional BRCA genes.
  • Hasegawa S, Kumagai M, Hagihara M, Nishimaru H, Hirano K, Kaneko R, Okayama A, Hirayama T, Sanbo M, Hirabayashi M, Watanabe M, Hirabayashi T, Yagi T
    Frontiers in molecular neuroscience 9 155  2016年12月 [査読有り][通常論文]
  • Toshimichi Tanaka, Takeo Sato, Keishi Yamashita, Kei Hosoda, Takatoshi Nakamura, Masahiko Watanabe
    Digestive Surgery 34 1 68 - 77 2016年11月01日 [査読有り][通常論文]
     
    Background/Aims: Postoperative complications of colorectal cancer (CRC) can sometimes be life threatening. Prevention of morbidity is therefore the most important issue, and among such complications, surgical site infections (SSIs) are the most critical. Predictive factors for SSI were evaluated. Patients and Methods: This was a retrospective study of 432 patients with CRC. SSIs were classified into incisional SSIs (iSSIs) and organ/space SSIs (o/sSSIs). Results: Preoperative serum albumin (relative risk (RR) 2.51, p = 0.01) and body mass index (BMI: RR 2.36, p = 0.02) were the independent risk factors for iSSIs, while rectal cancer (RR 6.34, p < 0.0001), preoperative serum albumin (RR 7.03, p = 0.004), age (RR 2.71, p = 0.02), and male sex (RR 2.41, p = 0.05) were the independent risk factors for o/sSSIs. Patients with both low serum albumin and high BMI had the highest incidence of iSSIs (34.2%), and the group with rectal cancer and low serum albumin had the highest incidence of o/sSSIs (57.1%). Conclusions: Preoperative nutritional status is a risk factor for SSIs in CRC, and its proper preoperative management may reduce the risk of SSIs.
  • Wataru Mizushima, Hidehisa Takahashi, Masashi Watanabe, Shintaro Kinugawa, Shouji Matsushima, Shingo Takada, Takashi Yokota, Takaaki Furihata, Junichi Matsumoto, Masaya Tsuda, Ikuru Chiba, Shun Nagashima, Shigeru Yanagi, Masaki Matsumoto, Keiichi I. Nakayama, Hiroyuki Tsutsui, Shigetsugu Hatakeyama
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 100 43 - 53 2016年11月 [査読有り][通常論文]
     
    A failing heart shows severe energy insufficiency, and it is presumed that this energy shortage plays a critical role in the development of cardiac dysfunction. However, little is known about the mechanisms that cause energy metabolic alterations in the failing heart. Here, we show that the novel RING-finger protein 207 (RNF207), which is specifically expressed in the heart, plays a role in cardiac energy metabolism. Depletion of RNF207 in neonatal rat cardiomyocytes (NRCs) leads to a reduced cellular concentration of adenosine triphosphate (ATP) and mitochondrial dysfunction. Consistent with this result, we observed here that the expression of RNF207 was significantly reduced in mice with common cardiac diseases including heart failure. Intriguingly, proteomic approaches revealed that RNF207 interacts with the voltage-dependent anion channel (VDAC), which is considered to be a key regulator of mitochondria function, as an RNF207-interacting protein. Our findings indicate that RNF207 is involved in ATP production by cardiomyocytes, suggesting that RNF207 plays an important role in the development of heart failure. (C) 2016 Elsevier Ltd. All rights reserved.
  • Armania Nurdin, Yutaro Hoshi, Toshihiro Yoneyama, Eisuke Miyauchi, Masanori Tachikawa, Michitoshi Watanabe, Tetsuya Terasaki
    JOURNAL OF PHARMACEUTICAL SCIENCES 105 11 3440 - 3452 2016年11月 [査読有り][通常論文]
     
    Prostate-specific antigen is currently the only protein biomarker routinely used as a diagnostic tool for early detection and treatment monitoring of prostate cancer. However, it remains questionable whether prostate-specific antigen-based screening can sensitively and selectively identify the presence and progression status of primary and metastatic prostate cancers. Hence, the purpose of this study was to identify potential biomarker candidates in the secretome of primary and metastatic prostate cancer cells by using a combination of global and targeted proteomics. Quantitative comparisons among secretome proteins derived from androgen-responsive primary cancer cells (P-22Rv1), androgen-irresponsive bone metastatic cancer cells (M-PC-3), and noncancerous prostate cells (N-PNT2) were performed using 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry followed by in silico selection selected reaction monitoring analysis. Mediator of RNA polymerase II transcription subunit 13-like, insulin-like growth factor-binding protein 2, and hepatocyte growth factor were identified as highly secreted proteins from P-22Rv1 cells compared with N-PNT2 cells. Prostate-associated microseminoprotein, proactivator polypeptide, collagen-alpha-1 (VI) chain, and neuropilin-1 were identified as predominantly secreted proteins in M-PC-3 cells compared with N-PNT2 cells. These proteins in biological fluids are considered to be candidate biomarkers of primary and/or metastatic prostate cancer. (C) 2016 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.
  • Shintaro Otsuka, Kohtarou Konno, Manabu Abe, Junko Motohashi, Kazuhisa Kohda, Kenji Sakimura, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 36 46 11801 - 11816 2016年11月 [査読有り][通常論文]
     
    The cerebellum is thought to be involved in cognitive functions in addition to its well established role in motor coordination and motor learning in humans. Cerebellin 1 (Cbln1) is predominantly expressed in cerebellar granule cells and plays a crucial role in the formation and function of parallel fiber-Purkinje cell synapses. Although genes encoding Cbln1 and its postsynaptic receptor, the delta2 glutamate receptor (GluD2), are suggested to be associated with autistic-like traits and many psychiatric disorders, whether such cognitive impairments are caused by cerebellar dysfunction remains unclear. In the present study, we investigated whether and how Cbln1 signaling is involved in non-motor functions in adult mice. We show that acquisition and retention/retrieval of cued and contextual fear memory were impaired in Cbln1-null mice. In situ hybridization and immunohistochemical analyses revealed that Cbln1 is expressed in various extracerebellar regions, including the retrosplenial granular cortex and the hippocampus. In the hippocampus, Cbln1 immunoreactivity was present at the molecular layer of the dentate gyrus and the stratum lacunosum-moleculare without overt mRNA expression, suggesting that Cbln1 is provided by perforant path fibers. Retention/retrieval, but not acquisition, of cued and contextual fear memory was impaired in forebrain-predominant Cbln1-null mice. Spatial learning in the radial arm water maze was also abrogated. In contrast, acquisition of fear memory was affected in cerebellum-predominant Cbln1-null mice. These results indicate that Cbln1 in the forebrain and cerebellum mediates specific aspects of fear conditioning and spatial memory differentially and that Cbln1 signaling likely regulates motor and non-motor functions in multiple brain regions.
  • Kitaro Okamoto, Miwako Yamasaki, Keizo Takao, Shingo Soya, Monica Iwasaki, Koh Sasaki, Kenta Magoori, Iori Sakakibara, Tsuyoshi Miyakawa, Michihiro Mieda, Masahiko Watanabe, Juro Sakai, Masashi Yanagisawa, Takeshi Sakurai
    PLOS ONE 11 11 e0164716  2016年11月 [査読有り][通常論文]
     
    How the hypothalamus transmits hunger information to other brain regions to govern whole brain function to orchestrate feeding behavior has remained largely unknown. Our present study suggests the importance of a recently found lateral hypothalamic neuropeptide, QRFP, in this signaling. Qrfp(-/-) mice were hypophagic and lean, and exhibited increased anxiety-like behavior, and were hypoactive in novel circumstances as compared with wild type littermates. They also showed decreased wakefulness time in the early hours of the dark period. Histological studies suggested that QRFP neurons receive rich innervations from neurons in the arcuate nucleus which is a primary region for sensing the body's metabolic state by detecting levels of leptin, ghrelin and glucose. These observations suggest that QRFP is an important mediator that acts as a downstream mediator of the arcuate nucleus and regulates feeding behavior, mood, wakefulness and activity.
  • Rie Kusano, Kousuke Fujita, Yasuharu Shinoda, Yuko Nagaura, Hiroshi Kiyonari, Takaya Abe, Toshio Watanabe, Yasuhisa Matsui, Masahiro Fukaya, Hiroyuki Sakagami, Tatsuya Sato, Jun-ichi Funahashi, Motoko Ohnishi, Shinri Tamura, Takayasu Kobayashi
    FEBS LETTERS 590 20 3606 - 3615 2016年10月 [査読有り][通常論文]
     
    PPM1L, a member of the metal-dependent protein phosphatase (PPM) family, is involved in regulating the stress-activated protein kinase pathway and ceramide trafficking. However, the physiological function of PPM1L in the brain is unclear. In this study, we generated and analyzed ppm1l-deficient mice in order to investigate PPM1L functions in the brain. Our results indicate that ppm1l is highly expressed in the central nervous system during mouse development and that ppm1l(Delta/Delta) mice display impaired motor performance and morphological abnormalities in the forebrain. Electron microscopic and immunohistochemical analyses suggest that these abnormalities are due to impaired axonal tract formation. Our novel findings suggest an important role for PPM1L in brain development.
  • Ming-Dong Zhang, Swapnali Barde, Edit Szodorai, Anna Josephson, Nicholas Mitsios, Masahiko Watanabe, Johannes Attems, Gert Lubec, Gábor G. Kovács, Mathias Uhlén, Jan Mulder, Tibor Harkany, Tomas Hökfelt
    Brain Structure and Function 221 7 3843  2016年09月01日 [査読有り][通常論文]
     
    The original article was published with an error in the author name. The author name Gábor M. Kovács should be changed to Gábor G. Kovács. The original article has been updated accordingly.
  • Madoka Narushima, Motokazu Uchigashima, Yuki Yagasaki, Takeshi Harada, Yasuyuki Nagumo, Naofumi Uesaka, Kouichi Hashimoto, Atsu Aiba, Masahiko Watanabe, Mariko Miyata, Masanobu Kano
    NEURON 91 5 1097 - 1109 2016年09月 [査読有り][通常論文]
     
    Neural circuits formed during postnatal development have to be maintained stably thereafter, but their mechanisms remain largely unknown. Here we report that the metabotropic glutamate receptor subtype 1 (mGluR1) is essential for the maintenance of mature synaptic connectivity in the dorsal lateral geniculate nucleus (dLGN). In mGluR1 knockout (mGluR1-KO) mice, strengthening and elimination at retinogeniculate synapses occurred normally until around postnatal day 20 (P20). However, during the subsequent visual-experience-dependent maintenance phase, weak retinogeniculate synapses were newly recruited. These changes were similar to those of wild-type (WT) mice that underwent visual deprivation or inactivation of mGluR1 in the dLGN from P21. Importantly, visual deprivation was ineffective in mGluR1-KO mice, and the changes induced by visual deprivation in WT mice were rescued by pharmacological activation of mGluR1 in the dLGN. These results demonstrate that mGluR1 is crucial for the visual-experience-dependent maintenance of mature synaptic connectivity in the dLGN.
  • Tomonori Takeuchi, Adrian J. Duszkiewicz, Alex Sonneborn, Patrick A. Spooner, Miwako Yamasaki, Masahiko Watanabe, Caroline C. Smith, Guillen Fernandez, Karl Deisseroth, Robert W. Greene, Richard G. M. Morris
    NATURE 537 7620 357 - + 2016年09月 [査読有り][通常論文]
     
    The retention of episodic-like memory is enhanced, in humans and animals, when something novel happens shortly before or after encoding. Using an everyday memory task in mice, we sought the neurons mediating this dopamine-dependent novelty effect, previously thought to originate exclusively from the tyrosine-hydroxylase-expressing (TH+) neurons in the ventral tegmental area. Here we report that neuronal firing in the locus coeruleus is especially sensitive to environmental novelty, locus coeruleus TH+ neurons project more profusely than ventral tegmental area TH+ neurons to the hippocampus, optogenetic activation of locus coeruleus TH+ neurons mimics the novelty effect, and this novelty-associated memory enhancement is unaffected by ventral tegmental area inactivation. Surprisingly, two effects of locus coeruleus TH+ photoactivation are sensitive to hippocampal D-1/D-5 receptor blockade and resistant to adrenoceptor blockade: memory enhancement and long-lasting potentiation of synaptic transmission in CA1 ex vivo. Thus, locus coeruleus TH+ neurons can mediate post-encoding memory enhancement in a manner consistent with possible co-release of dopamine in the hippocampus.
  • Tayo Katano, Masafumi Fukuda, Hidemasa Furue, Maya Yamazaki, Manabu Abe, Masahiko Watanabe, Kazuhiko Nishida, Ikuko Yao, Akihiro Yamada, Yutaka Hata, Nobuaki Okumura, Takanobu Nakazawa, Tadashi Yamamoto, Kenji Sakimura, Toshifumi Takao, Seiji Ito
    eNeuro 3 5 2016年09月01日 [査読有り][通常論文]
     
    Maintenance of neuropathic pain caused by peripheral nerve injury crucially depends on the phosphorylation of GluN2B, a subunit of the N-methyl-d-aspartate (NMDA) receptor, at Tyr1472 (Y1472) and subsequent formation of a postsynaptic density (PSD) complex of superficial spinal dorsal horn neurons. Here we took advantage of comparative proteomic analysis based on isobaric stable isotope tags (iTRAQ) between wild-type and knock-in mice with a mutation of Y1472 to Phe of GluN2B (Y1472F-KI) to search for PSD proteins in the spinal dorsal horn that mediate the signaling downstream of phosphorylated Y1472 GluN2B. Among several candidate proteins, we focused on brain-enriched guanylate kinase-associated protein (BEGAIN), which was specifically up-regulated in wild-type mice after spared nerve injury (SNI). Immunohistochemical analysis using the generated antibody demonstrated that BEGAIN was highly localized at the synapse of inner lamina II in the spinal dorsal horn and that its expression was up-regulated after SNI in wild-type, but not in Y1472F-KI, mice. In addition, alteration of the kinetics of evoked excitatory postsynaptic currents for NMDA but not those for _-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in spinal lamina II was demonstrated by BEGAIN deletion. We demonstrated that mechanical allodynia, a condition of abnormal pain induced by innocuous stimuli, in the SNI model was significantly attenuated in BEGAIN-deficient mice. However, there was no significant difference between naive wild-type and BEGAIN-knockout mice in terms of physiological threshold for mechanical stimuli. These results suggest that BEGAIN was involved in pathological pain transmission through NMDA receptor activation by the phosphorylation of GluN2B at Y1472 in spinal inner lamina II.
  • Shizuka Kobayashi, Yamato Hida, Hiroyoshi Ishizaki, Eiji Inoue, Miki Tanaka-Okamoto, Miwako Yamasaki, Taisuke Miyazaki, Masahiro Fukaya, Isao Kitajima, Yoshimi Takai, Masahiko Watanabe, Toshihisa Ohtsuka, Toshiya Manabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 44 5 2272 - 2284 2016年09月 [査読有り][通常論文]
     
    Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.
  • Maria Gutierrez-Mecinas, Emily D. Kuehn, Victoria E. Abraira, Erika Polgar, Masahiko Watanabe, Andrew J. Todd
    NEUROSCIENCE 329 171 - 181 2016年08月 [査読有り][通常論文]
     
    The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I-III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of IBRO.
  • Yuki Sugaya, Maya Yamazaki, Motokazu Uchigashima, Kenta Kobayashi, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    CELL REPORTS 16 5 1405 - 1415 2016年08月 [査読有り][通常論文]
     
    Endocannabinoid signaling is considered to suppress excessive excitability of neural circuits and to protect the brain from seizures. However, the precise mechanisms of this effect are poorly understood. Here, we report that 2-arachidonoylglycerol (2-AG), one of the two major endocannabinoids, is crucial for suppressing seizures. We found that kainate-induced seizures in mice lacking the 2-AG synthesizing enzyme, diacylglycerol lipase alpha, were much more severe compared with those in cannabinoid CB1 receptor knockout mice and were comparable to those in mice lacking both CB1- and CB2-receptor-mediated signaling. In the dentate gyrus, 2-AG suppressed excitatory input around the inner and middle molecular layers through CB1 and presumably CB2 receptors, respectively. This 2-AG-mediated suppression contributed to decreased granule cell excitability and the dampening of seizures. Furthermore, lack of 2-AG signaling enhanced kindling epilepto-genesis and spontaneous seizures after kainate-induced status epilepticus. These results highlight critical roles of 2-AG signaling in the suppression of epileptic seizures.
  • Ken Kojo, Yoshiya Ito, Koji Eshima, Nobuyuki Nishizawa, Hirotoki Ohkubo, Takehiko Yokomizo, Takao Shimizu, Masahiko Watanabe, Masataka Majima
    Scientific Reports 6 2016年07月11日 [査読有り][通常論文]
     
    Leukotriene B 4 (LTB 4) is a potent chemoattractant for neutrophils. Signalling of LTB 4 receptor type 1 (BLT 1) has pro-inflammatory functions through neutrophil recruitment. In this study, we investigated whether BLT 1 signalling plays a role in acetaminophen (APAP)-induced liver injury by affecting inflammatory responses including the accumulation of hepatic neutrophils. BLT 1 -knockout (BLT 1-/-) mice and their wild-type (WT) counterparts were subjected to a single APAP overdose (300 mg/kg), and various parameters compared within 24 h after treatment. Compared with WT mice, BLT 1-/- mice exhibited exacerbation of APAP-induced liver injury as evidenced by enhancement of alanine aminotransferase level, necrotic area, hepatic neutrophil accumulation, and expression of cytokines and chemokines. WT mice co-treated with APAP and ONO-0457, a specific antagonist for BLT 1, displayed amplification of the injury, and similar results to those observed in BLT 1-/- mice. Hepatic neutrophils in BLT 1-/- mice during APAP hepatotoxicity showed increases in the production of reactive oxygen species and matrix metalloproteinase-9. Administration of isolated BLT 1 -deficient neutrophils into WT mice aggravated the liver injury elicited by APAP. These results demonstrate that BLT 1 signalling dampens the progression of APAP hepatotoxicity through inhibiting an excessive accumulation of activated neutrophils. The development of a specific agonist for BLT 1 could be useful for the prevention of APAP hepatotoxicity.
  • Christoph Straub, Yoav Noam, Toshihiro Nomura, Miwako Yamasaki, Dan Yan, Herman B. Fernandes, Ping Zhang, James R. Howe, Masahiko Watanabe, Anis Contractor, Susumu Tomita
    CELL REPORTS 16 2 531 - 544 2016年07月 [査読有り][通常論文]
     
    Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.
  • Yoshiko Matsumoto-Makidono, Hisako Nakayama, Miwako Yamasaki, Taisuke Miyazaki, Kazuto Kobayashi, Masahiko Watanabe, Masanobu Kano, Kenji Sakimura, Kouichi Hashimoto
    CELL REPORTS 16 4 994 - 1004 2016年07月 [査読有り][通常論文]
     
    Some neurons have the ability to enhance output voltage to input current with a preferred frequency, which is called resonance. Resonance is thought to be a basis for membrane potential oscillation. Although ion channels responsible for resonance have been reported, the precise mechanisms by which these channels work remain poorly understood. We have found that resonance is reduced but clearly present in the inferior olivary neurons of Cav3.1 T-type voltage-dependent Ca2+ channel knockout (KO) mice. The activation of Cav3.1 channels is strongly membrane potential dependent, but less frequency dependent. Residual resonance in Cav3.1 KO mice is abolished by a hyper-polarization-activated cyclic nucleotide-gated (HCN) channel blocker, ZD7288, and is partially suppressed by voltage-dependent K+ channel blockers. Resonance is inhibited by ZD7288 in wild-type mice and impaired in HCN1 KO mice, suggesting that the HCN1 channel is essential for resonance. The ZD7288-sensitive current is nearly sinusoidal and strongly frequency dependent. These results suggest that Cav3.1 and HCN1 channels act as amplifying and resonating conductances, respectively.
  • Keishi Yamashita, Shinichi Sakuramoto, Shiro Kikuchi, Nobue Futawatari, Natsuya Katada, Kei Hosoda, Hiromitsu Moriya, Hiroaki Mieno, Masahiko Watanabe
    SURGERY TODAY 46 6 741 - 749 2016年06月 [査読有り][通常論文]
     
    We report the long-term clinical outcomes of a randomized clinical trial comparing laparoscopy-assisted distal gastrectomy (LADG) with open DG (ODG). Between 2005 and 2008, 63 patients with clinical T1 (cT1) gastric cancer were randomly assigned to undergo either LADG or ODG. Long-term clinical outcomes included prospective questionnaire-based symptoms and survival. Based on the responses to the prospective questionnaires, patients who underwent LADG reported greater satisfaction and were more likely to favor the procedure than those who underwent ODG. The most notable difference in symptoms was related to wound pain and diarrhea. After ODG, wound pain reduced in intensity but persisted throughout the follow-up. Surprisingly, diarrhea was more frequent after LADG than after ODG, possibly due to overeating, because symptoms elicited by overeating, such as vomiting after a meal or heartburn, were also more frequent after LADG. In terms of long-term survival, there were no cases of recurrence in either group. LADG was associated with less wound pain during long-term follow-up after surgery, whereas symptoms related to overeating were common. Based on our findings and the patients' reported satisfaction, we recommend LADG for cT1 gastric cancer as an effective procedure with excellent long-term survival.
  • Chiaki Itami, Jui-Yen Huang, Miwako Yamasaki, Masahiko Watanabe, Hui-Chen Lu, Fumitaka Kimura
    JOURNAL OF NEUROSCIENCE 36 26 7039 - 7054 2016年06月 [査読有り][通常論文]
     
    The formation and refinement of thalamocortical axons (TCAs) is an activity-dependent process (Katz and Shatz, 1996), but its mechanism and nature of activity are elusive. We studied the role of spike timing-dependent plasticity (STDP) in TCA formation and refinement in mice. At birth (postnatal day 0, P0), TCAs invade the cortical plate, from which layers 4 (L4) and L2/3 differentiate at P3-P4. A portion of TCAs transiently reach toward the pia surface around P2-P4 (Senft and Woolsey, 1991; Rebsam et al., 2002) but are eventually confined below the border between L2/3 and L4. We previously showed that L4-L2/3 synapses exhibit STDP with only potentiation (timing-dependent long-term potentiation [t-LTP]) during synapse formation, then switch to a Hebbian form of STDP. Here we show that TCA-cortical plate synapses exhibit robust t-LTP in neonates, whose magnitude decreased gradually after P4-P5. After L2/3 is differentiated, TCA-L2/3 gradually switched to STDP with only depression (t-LTD) after P7-P8, whereas TCA-L4 lost STDP. t-LTP was dependent on NMDA receptor and PKA, whereas t-LTD was mediated by Type 1 cannabinoid receptors (CB1Rs) probably located at TCA terminals, revealed by global and cortical excitatory cell-specific knock-out of CB1R. Moreover, we found that administration of CB1R agonists, including Delta(9)-tetrahydrocannabinol, caused substantial retraction of TCAs. Consistent with this, individual thalamocortical axons exuberantly innervated L2/3 at P12 in CB1R knock-outs, indicating that endogenous cannabinoid signaling shapes TCA projection. These results suggest that the developmental switch in STDP and associated appearance of CB1R play important roles in the formation and refinement of TCAs.
  • Izumi Kaji, Yasutada Akiba, Kohtarou Konno, Masahiko Watanabe, Shunsuke Kimura, Toshihiko Iwanaga, Ayaka Kuri, Ken-ichi Iwamoto, Atsukazu Kuwahara, Jonathan D. Kaunitz
    JOURNAL OF PHYSIOLOGY-LONDON 594 12 3339 - 3352 2016年06月 [査読有り][通常論文]
     
    The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl- secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13m. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the G(i/o) pathway. Luminal propionate induced Cl- secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.
  • Yu Ohmura, Takayuki Yoshida, Kohtarou Konno, Masabumi Minami, Masahiko Watanabe, Mitsuhiro Yoshioka
    INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY 19 6 2016年06月 [査読有り][通常論文]
     
    Background: Patients with posttraumatic stress disorder or panic disorder are often troubled by inappropriate retrieval of fear memory. Moreover, these disorders are often comorbid with irritable bowel syndrome. The main aim of the present study is to elucidate the involvement of hippocampal serotonergic systems in fear memory retrieval and stress-induced defecation. Methods and Results: Microinjection of serotonin 7 receptor antagonist, but not other serotonin receptor antagonists (serotonin (1A), (2A), (2C), (3), (4), and (6)), into the rat ventral hippocampus significantly suppressed the expression of freezing behavior, an index of fear memory retrieval, and decreased the amount of feces, an index of stress-induced defecation, in the contextual fear conditioning test. Electrophysiological data indicated that the serotonin 7 receptor agonist increased the frequency of action potentials in the ventral hippocampal CA3 pyramidal neuron via the activation of the hyperpolarization-activated nonselective cation current I-h. Moreover, in situ hybridization demonstrated that Htr7 mRNA was abundantly expressed in the CA3 compared with other subregions of the hippocampus and that these Htr7 mRNA-positive cells coexpressed hyperpolarization-activated cyclic nucleotide-gated channel 2 and 4 mRNAs, which are components of the I-h channel. Conclusions: These results indicated that the released serotonin activates the serotonin 7 receptor in the CA3 ventral hippocampus subregion, enhances the sensitivity to inputs via hyperpolarization-activated cyclic nucleotide 2 and 4 channels, and thereby facilitates fear memory retrieval. The serotonin 7 receptor might be a target of drug development for the treatment of mental disorders involving fear memory and gastrointestinal problems.
  • Gireesh Gangadharan, Jonghan Shin, Seong-Wook Kim, Angela Kim, Afshin Paydar, Duk-Soo Kim, Taisuke Miyazaki, Masahiko Watanabe, Yuchio Yanagawa, Jinhyun Kim, Yeon-Soo Kim, Daesoo Kim, Hee-Sup Shin
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 23 6550 - 6555 2016年06月 [査読有り][通常論文]
     
    Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Ca(v)3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca2+ channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca2+ channels in the medial septal GABAergic projection neurons in this behavior.
  • Keiko Matsuda, Timotheus Budisantoso, Nikolaos Mitakidis, Yuki Sugaya, Eriko Miura, Wataru Kakegawa, Miwako Yamasaki, Kohtarou Konno, Motokazu Uchigashima, Manabu Abe, Izumi Watanabe, Masanobu Kano, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, Michisuke Yuzaki
    NEURON 90 4 752 - 767 2016年05月 [査読有り][通常論文]
     
    Postsynaptic kainate-type glutamate receptors (KARs) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. Here, we show that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.
  • Takanobu Nakazawa, Ryota Hashimoto, Kazuto Sakoori, Yuki Sugaya, Asami Tanimura, Yuki Hashimotodani, Kazutaka Ohi, Hidenaga Yamamori, Yuka Yasuda, Satomi Umeda-Yano, Yuji Kiyama, Kohtarou Konno, Takeshi Inoue, Kazumasa Yokoyama, Takafumi Inoue, Shusuke Numata, Tohru Ohnuma, Nakao Iwata, Norio Ozaki, Hitoshi Hashimoto, Masahiko Watanabe, Toshiya Manabe, Tadashi Yamamoto, Masatoshi Takeda, Masanobu Kano
    NATURE COMMUNICATIONS 7 11466  2016年04月 [査読有り][通常論文]
  • Yamasaki M, Fukaya M, Yamazaki M, Azechi H, Natsume R, Abe M, Sakimura K, Watanabe M
    The Journal of neuroscience : the official journal of the Society for Neuroscience 36 15 4296 - 4312 2016年04月 [査読有り][通常論文]
     
    The number of AMPA-type glutamate receptors (AMPARs) at synapses is the major determinant of synaptic strength and varies from synapse to synapse. To clarify the underlying molecular mechanisms, the density of AMPARs, PSD-95, and transmembrane AMPAR regulatory proteins (TARPs) were compared at Schaffer collateral/commissural (SCC) synapses in the adult mouse hippocampal CA1 by quantitative immunogold electron microscopy using serial sections. We examined four types of SCC synapses: perforated and nonperforated synapses on pyramidal cells and axodendritic synapses on parvalbumin-positive (PV synapse) and pravalbumin-negative interneurons (non-PV synapse). SCC synapses were categorized into those expressing high-density (perforated and PV synapses) or low-density (nonperforated and non-PV synapses) AMPARs. Although the density of PSD-95 labeling was fairly constant, the density and composition of TARP isoforms was highly variable depending on the synapse type. Of the three TARPs expressed in hippocampal neurons, the disparity inTARP gamma-2 labeling was closely related to that of AMPAR labeling. Importantly, AMPAR density was significantly reduced at perforated and PV synapses in TARP gamma-2-knock-out (KO) mice, resulting in a virtual loss of AMPAR disparity among SCC synapses. In comparison, TARP gamma-8 was the only TARP expressed at nonperforated synapses, where AMPAR labeling further decreased to a background level in TARP gamma-8-KO mice. These results show that synaptic inclusion of TARP gamma-2 potently increases AMPAR expression and transforms low-density synapses into high-density ones, whereas TARP gamma-8 is essential for low-density or basal expression of AMPARs at nonperforated synapses. Therefore, these TARPs are critically involved in AMPAR density control at SCC synapses.
  • Ryoichi Ichikawa, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 36 17 4846 - 4858 2016年04月 [査読有り][通常論文]
     
    Although injured axons usually do not regenerate in the adult CNS, parallel fibers (PFs) regenerate synaptic connections onto cerebellar Purkinje cells (PCs). In this study, we investigated the role of GluD2 in this regenerative process after PF transection using GluD2-knock-out (KO) mice. All dendritic spines on distal dendrites were innervated by PFs in sham-operated wild-type controls, whereas one-third were devoid of innervation in GluD2-KO mice. In both genotypes, a steep drop in the number of PF synapses occurred with a reciprocal surge in the number of free spines on postlesion day 1, when the PF territory aberrantly expanded toward the proximal dendrites. In wild-type mice, the territory and number of PF synapses were nearly fully restored to normal on postlesion day 7, although PF density remained low. Moreover, presynaptic and postsynaptic elements were markedly enlarged, and the PF terminal-to-PC spine contact ratio increased from 1:1 to 1:2 at most synapses. On postlesion day 30, the size and contact ratio of PF synapses returned to sham-operated control values and PF density recovered through the sprouting and elongation of PF collaterals. However, GluD2-KO mice showed neither a hypertrophic response nor territorial restoration 7 d postlesion, nor the recovery of PF axons or synapses on postlesion day 30. This suggests that PF wiring regenerates initially by inducing hypertrophic responses in surviving synaptic elements (hypertrophic phase), followed by collateral formation by PF axons and retraction of PF synapses (remodeling phase). Without GluD2, no transition to these regenerative phases occurs.
  • Motokazu Uchigashima, Toshihisa Ohtsuka, Kazuto Kobayashi, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 15 4206 - 4211 2016年04月 [査読有り][通常論文]
     
    Midbrain dopamine neurons project densely to the striatum and form so-called dopamine synapses on medium spiny neurons (MSNs), principal neurons in the striatum. Because dopamine receptors are widely expressed away from dopamine synapses, it remains unclear how dopamine synapses are involved in dopaminergic transmission. Here we demonstrate that dopamine synapses are contacts formed between dopaminergic presynaptic and GABAergic postsynaptic structures. The presynaptic structure expressed tyrosine hydroxylase, vesicular monoamine transporter-2, and plasmalemmal dopamine transporter, which are essential for dopamine synthesis, vesicular filling, and recycling, but was below the detection threshold for molecules involving GABA synthesis and vesicular filling or for GABA itself. In contrast, the postsynaptic structure of dopamine synapses expressed GABAergic molecules, including postsynaptic adhesion molecule neuroligin-2, postsynaptic scaffolding molecule gephyrin, and GABA(A) receptor alpha 1, without any specific clustering of dopamine receptors. Of these, neuroligin-2 promoted presynaptic differentiation in axons of midbrain dopamine neurons and striatal GABAergic neurons in culture. After neuroligin-2 knockdown in the striatum, a significant decrease of dopamine synapses coupled with a reciprocal increase of GABAergic synapses was observed on MSN dendrites. This finding suggests that neuroligin-2 controls striatal synapse formation by giving competitive advantage to heterologous dopamine synapses over conventional GABAergic synapses. Considering that MSN dendrites are preferential targets of dopamine synapses and express high levels of dopamine receptors, dopamine synapse formation may serve to increase the specificity and potency of dopaminergic modulation of striatal outputs by anchoring dopamine release sites to dopamine-sensing targets.
  • Noboru Iwagaki, Robert P. Ganley, Allen C. Dickie, Erika Polgar, David I. Hughes, Patricia Del Rio, Yulia Revina, Masahiko Watanabe, Andrew J. Todd, John S. Riddell
    PAIN 157 3 598 - 612 2016年03月 [査読有り][通常論文]
     
    The spinal dorsal horn contains numerous inhibitory interneurons that control transmission of somatosensory information. Although these cells have important roles in modulating pain, we still have limited information about how they are incorporated into neuronal circuits, and this is partly due to difficulty in assigning them to functional populations. Around 15% of inhibitory interneurons in laminae I-III express neuropeptide Y (NPY), but little is known about this population. We therefore used a combined electrophysiological/morphological approach to investigate these cells in mice that express green fluorescent protein (GFP) under control of the NPY promoter. We show that GFP is largely restricted to NPY-immunoreactive cells, although it is only expressed by a third of those in lamina I-II. Reconstructions of recorded neurons revealed that they were morphologically heterogeneous, but never islet cells. Many NPY-GFP cells (including cells in lamina III) appeared to be innervated by C fibres that lack transient receptor potential vanilloid-1, and consistent with this, we found that some lamina III NPY-immunoreactive cells were activated by mechanical noxious stimuli. Projection neurons in lamina III are densely innervated by NPY-containing axons. Our results suggest that this input originates from a small subset of NPY-expressing interneurons, with the projection cells representing only a minority of their output. Taken together with results of previous studies, our findings indicate that somatodendritic morphology is of limited value in classifying functional populations among inhibitory interneurons in the dorsal horn. Because many NPY-expressing cells respond to noxious stimuli, these are likely to have a role in attenuating pain and limiting its spread.
  • Andras Szonyi, Marton I. Mayer, Csaba Cserep, Virag T. Takacs, Masahiko Watanabe, Tamas F. Freund, Gabor Nyiri
    BRAIN STRUCTURE & FUNCTION 221 2 735 - 751 2016年03月 [査読有り][通常論文]
     
    The median raphe region (MRR) is thought to be serotonergic and plays an important role in the regulation of many cognitive functions. In the hippocampus (HIPP), the MRR exerts a fast excitatory control, partially through glutamatergic transmission, on a subpopulation of GABAergic interneurons that are key regulators of local network activity. However, not all receptors of this connection in the HIPP and in synapses established by MRR in other brain areas are known. Using combined anterograde tracing and immunogold methods, we show that the GluN2A subunit of the NMDA receptor is present in the synapses established by MRR not only in the HIPP, but also in the medial septum (MS) and in the medial prefrontal cortex (mPFC) of the mouse. We estimated similar amounts of NMDA receptors in these synapses established by the MRR and in local adjacent excitatory synapses. Using retrograde tracing and confocal laser scanning microscopy, we found that the majority of the projecting cells of the mouse MRR contain the vesicular glutamate transporter type 3 (vGluT3). Furthermore, using double retrograde tracing, we found that single cells of the MRR can innervate the HIPP and mPFC or the MS and mPFC simultaneously, and these double-projecting cells are also predominantly vGluT3-positive. Our results indicate that the majority of the output of the MRR is glutamatergic and acts through NMDA receptor-containing synapses. This suggests that key forebrain areas receive precisely targeted excitatory input from the MRR, which is able to synchronously modify activity in those regions via individual MRR cells with dual projections.
  • Maria Gutierrez-Mecinas, Takahiro Furuta, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 12 2016年03月 [査読有り][通常論文]
     
    Background: Excitatory interneurons account for the majority of neurons in laminae I-III, but their functions are poorly understood. Several neurochemical markers are largely restricted to excitatory interneuron populations, but we have limited knowledge about the size of these populations or their overlap. The present study was designed to investigate this issue by quantifying the neuronal populations that express somatostatin (SST), neurokinin B (NKB), neurotensin, gastrin-releasing peptide (GRP) and the gamma isoform of protein kinase C (PKC gamma), and assessing the extent to which they overlapped. Since it has been reported that calretinin- and SST-expressing cells have different functions, we also looked for co-localisation of calretinin and SST. Results: SST, preprotachykinin B (PPTB, the precursor of NKB), neurotensin, PKC gamma or calretinin were detected with antibodies, while cells expressing GRP were identified in a mouse line (GRP-EGFP) in which enhanced green fluorescent protein (EGFP) was expressed under control of the GRP promoter. We found that SST-, neurotensin-, PPTB-and PKC gamma-expressing cells accounted for 44%, 7%, 12% and 21% of the neurons in laminae I-II, and 16%, 8%, 4% and 14% of those in lamina III, respectively. GRP-EGFP cells made up 11% of the neuronal population in laminae I-II. The neurotensin, PPTB and GRP-EGFP populations showed very limited overlap, and we estimate that between them they account for similar to 40% of the excitatory interneurons in laminae I-II. SST which is expressed by similar to 60% of excitatory interneurons in this region, was found in each of these populations, as well as in cells that did not express any of the other peptides. Neurotensin and PPTB were often found in cells with PKC gamma, and between them, constituted around 60% of the PKC gamma cells. Surprisingly, we found extensive co-localisation of SST and calretinin. Conclusions: These results suggest that cells expressing neurotensin, NKB or GRP form largely non-overlapping sets that are likely to correspond to functional populations. In contrast, SST is widely expressed by excitatory interneurons that are likely to be functionally heterogeneous.
  • Suzuki M, Watanabe M, Nakamaru Y, Takagi D, Takahashi H, Fukuda S, Hatakeyama S
    Cellular and molecular life sciences : CMLS 73 5 1085 - 1101 2016年03月 [査読有り][通常論文]
     
    NF kappa B is one of the central regulators of cell survival, immunity, inflammation, carcinogenesis and organogenesis. The activation of NF kappa B is strictly regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. Several types of ubiquitination play important roles in multi-step regulations of the NF kappa B pathway. Some of the tripartite motif-containing (TRIM) proteins functioning as E3 ubiquitin ligases are known to regulate various biological processes such as inflammatory signaling pathways. One of the TRIM family proteins, TRIM39, for which the gene has single nucleotide polymorphisms, has been identified as one of the genetic factors in Behcet's disease. However, the role of TRIM39 in inflammatory signaling had not been fully elucidated. In this study, to elucidate the function of TRIM39 in inflammatory signaling, we performed yeast two-hybrid screening using TRIM39 as a bait and identified Cactin, which has been reported to inhibit NF kappa B- and TLR-mediated transcriptions. We show that TRIM39 stabilizes Cactin protein and that Cactin is upregulated after TNF alpha stimulation. TRIM39 knockdown also causes activation of the NF kappa B signal. These findings suggest that TRIM39 negatively regulates the NF kappa B signal in collaboration with Cactin induced by inflammatory stimulants such as TNF alpha.
  • Gukhan Kim, Rafael Lujan, Jochen Schwenk, Melissa H. Kelley, Carolina Aguado, Masahiko Watanabe, Bernd Fakler, James Maylie, John P. Adelman
    ELIFE 5 2016年02月 [査読有り][通常論文]
     
    Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca2+ influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
  • Hiroshi Ueda, Shingo Nakamura, Taro Nakamura, Kaoru Inada, Takashi Okubo, Naohiro Furukawa, Reiichi Murakami, Shigeo Tsuchida, Yonathan Zohar, Kotaro Konno, Masahiko Watanabe
    SCIENTIFIC REPORTS 6 2016年02月 [査読有り][通常論文]
     
    The olfactory hypothesis for salmon imprinting and homing to their natal stream is well known, but the endocrine hormonal control mechanisms of olfactory memory formation in juveniles and retrieval in adults remain unclear. In brains of hatchery-reared underyearling juvenile chum salmon (Oncorhynchus keta), thyrotropin-releasing hormone gene expression increased immediately after release from a hatchery into the natal stream, and the expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor increased during downstream migration. Gene expression of salmon gonadotropin-releasing hormone (sGnRH) and NR1 increased in the adult chum salmon brain during homing from the Bering Sea to the natal hatchery. Thyroid hormone treatment in juveniles enhanced NR1 gene activation, and GnRHa treatment in adults improved stream odour discrimination. Olfactory memory formation during juvenile downstream migration and retrieval during adult homing migration of chum salmon might be controlled by endocrine hormones and could be clarified using NR1 as a molecular marker.
  • Ryoichi Ichikawa, Kouichi Hashimoto, Taisuke Miyazaki, Motokazu Uchigashima, Miwako Yamasaki, Atsu Aiba, Masanobu Kano, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 8 2282 - 2287 2016年02月 [査読有り][通常論文]
     
    In Purkinje cells (PCs) of the cerebellum, a single "winner" climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9-15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase C gamma (PKC gamma), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1 alpha into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKC gamma signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.
  • Takanobu Nakazawa, Ryota Hashimoto, Kazuto Sakoori, Yuki Sugaya, Asami Tanimura, Yuki Hashimotodani, Kazutaka Ohi, Hidenaga Yamamori, Yuka Yasuda, Satomi Umeda-Yano, Yuji Kiyama, Kohtarou Konno, Takeshi Inoue, Kazumasa Yokoyama, Takafumi Inoue, Shusuke Numata, Tohru Ohnuma, Nakao Iwata, Norio Ozaki, Hitoshi Hashimoto, Masahiko Watanabe, Toshiya Manabe, Tadashi Yamamoto, Masatoshi Takeda, Masanobu Kano
    NATURE COMMUNICATIONS 7 2016年02月 [査読有り][通常論文]
     
    Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders.
  • Taku Nagai, Shinichi Nakamuta, Keisuke Kuroda, Sakura Nakauchi, Tomoki Nishioka, Tetsuya Takano, Xinjian Zhang, Daisuke Tsuboi, Yasuhiro Funahashi, Takashi Nakano, Junichiro Yoshimoto, Kenta Kobayashi, Motokazu Uchigashima, Masahiko Watanabe, Masami Miura, Akinori Nishi, Kazuto Kobayashi, Kiyofumi Yamada, Mutsuki Amano, Kozo Kaibuchi
    NEURON 89 3 550 - 565 2016年02月 [査読有り][通常論文]
     
    Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.
  • Izumi Watanabe-Iida, Kohtarou Konno, Kaori Akashi, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
    JOURNAL OF NEUROCHEMISTRY 136 2 295 - 305 2016年01月 [査読有り][通常論文]
     
    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KARsubunits are predominant in the brain, with distinct subunitcombinations between the hippocampus and cerebellum.
  • Noemi Holderith, Andrea Lorincz, Gergely Katona, Balazs Rozsa, Akos Kulik, Masahiko Watanabe, Zoltan Nusser
    NATURE NEUROSCIENCE 19 1 172 - 172 2016年01月 [査読有り][通常論文]
  • Natsumi Ageta-Ishihara, Maya Yamazaki, Kohtarou Konno, Hisako Nakayama, Manabu Abe, Kenji Hashimoto, Tomoki Nishioka, Kozo Kaibuchi, Satoko Hattori, Tsuyoshi Miyakawa, Kohichi Tanaka, Fathul Huda, Hirokazu Hirai, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Makoto Kinoshita
    NATURE COMMUNICATIONS 6 10090  2015年12月 [査読有り][通常論文]
     
    The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (gamma DGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance.
  • Yasukazu Hozumi, Ryo Akimoto, Akihito Suzuki, Koichi Otani, Masahiko Watanabe, Kaoru Goto
    CELL AND TISSUE RESEARCH 362 2 295 - 305 2015年11月 [査読有り][通常論文]
     
    Adrenal glands play a central role in the secretion of steroid hormones and catecholamines. Previous studies have revealed that molecules engaged in phosphoinositide (PI) turnover are expressed in the adrenal gland, suggesting the importance of PI signaling in adrenal signal transduction. Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DG), a major second messenger in the PI signaling cascade. The DGK family is expressed in distinct patterns in endocrine organs at the mRNA and protein levels. Nevertheless, little is known about the characteristics and morphological aspects of DGKs in the adrenal gland. We have performed immunohistochemical analyses to investigate the expression and localization of DGK isozymes, together with PI signaling molecules, in the adrenal gland at the protein level. Our results show that the DGK family and a set of PI signaling molecules are expressed intensely in zona glomerulosa cells and medullary chromaffin cells in the adrenal gland. In adrenal cells, DGK gamma localizes to the Golgi complex, DGK epsilon to the plasma membrane, and DGK zeta to the nucleus. These findings show the distinct expression and subcellular localization of DGK isozymes and PI signaling molecules in the adrenal gland, suggesting that each DGK isozyme has a role in signal transduction in adrenal cells, especially in the zona glomerulosa and medulla.
  • Darren Cameron, Erika Polgar, Maria Gutierrez-Mecinas, Maria Gomez-Lima, Masahiko Watanabe, Andrew J. Todd
    PAIN 156 10 2061 - 2071 2015年10月 [査読有り][通常論文]
     
    The anterolateral tract (ALT), which originates from neurons in lamina I and the deep dorsal horn, represents a major ascending output through which nociceptive information is transmitted to brain areas involved in pain perception. Although there is detailed quantitative information concerning the ALT in the rat, much less is known about this system in the mouse, which is increasingly being used for studies of spinal pain mechanisms because of the availability of genetically modified lines. The aim of this study was therefore to determine the extent to which information about the ALT in the rat can be extrapolated to the mouse. Our results suggest that as in the rat, most lamina I ALT projection neurons in the lumbar enlargement can be retrogradely labelled from the lateral parabrachial area, that the majority of these cells (similar to 90%) express the neurokinin 1 receptor (NK1r), and that these are larger than other NK1r-expressing neurons in this lamina. This means that many lamina I spinoparabrachial cells can be identified in NK1r-immunostained sections from animals that have not received retrograde tracer injections. However, we also observed certain species differences, in particular we found that many spinoparabrachial cells in laminae III and IV lack the NK1r, meaning that they cannot be identified based solely on the expression of this receptor. We also provide evidence that the majority of spinoparabrachial cells are glutamatergic and that some express substance P. These findings will be important for studies designed to unravel the complex neuronal circuitry that underlies spinal pain processing.
  • Mina Waraya, Keishi Yamashita, Akira Ema, Natsuya Katada, Shiro Kikuchi, Masahiko Watanabe
    PLOS ONE 10 10 2015年10月 [査読有り][通常論文]
     
    Background A comprehensive search for DNA methylated genes identified candidate tumor suppressor genes that have been proven to be involved in the apoptotic process of the p53 pathway. In this study, we investigated p53 mutation in relation to such epigenetic alteration in primary gastric cancer. Methods The methylation profiles of the 3 genes: PGP9.5, NMDAR2B, and CCNA1, which are involved in the p53 tumor suppressor pathway in combination with p53 mutation were examined in 163 primary gastric cancers. The effect of epigenetic reversion in combination with chemotherapeutic drugs on apoptosis was also assessed according to the tumor p53 mutation status. Results p53 gene mutations were found in 44 primary gastric tumors (27%), and super-high methylation of any of the 3 genes was only found in cases with wild type p53. Higher p53 pathway aberration was found in cases with male gender (p = 0.003), intestinal type (p = 0.005), and non-infiltrating type (p = 0.001). The p53 pathway aberration group exhibited less recurrence in lymph nodes, distant organs, and peritoneum than the p53 non-aberration group. In the NUGC4 gastric cancer cell line (p53 wild type), epigenetic treatment augmented apoptosis by chemotherapeutic drugs, partially through p53 transcription activity. On the other hand, in the KATO III cancer cell line (p53 mutant), epigenetic treatment alone induced robust apoptosis, with no trans-activation of p53. Conclusion In gastric cancer, p53 relevant and non-relevant pathways exist, and tumors with either pathway type exhibited unique clinical features. Epigenetic treatments can induce apoptosis partially through p53 activation, however their apoptotic effects may be explained largely by mechanism other than through p53 pathways.
  • Takashi Watanabe, Mai Kakeno, Toshinori Matsui, Ikuko Sugiyama, Nariko Arimura, Kenji Matsuzawa, Aya Shirahige, Fumiyoshi Ishidate, Tomoki Nishioka, Shinichiro Taya, Mikio Hoshino, Kozo Kaibuchi
    JOURNAL OF CELL BIOLOGY 210 5 737 - 751 2015年08月 [査読有り][通常論文]
     
    Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.
  • Yoshinori Shirai, Masahiko Watanabe, Hiroyuki Sakagami, Tatsuo Suzuki
    JOURNAL OF NEUROCHEMISTRY 134 3 578 - 589 2015年08月 [査読有り][通常論文]
     
    General transcription factor II-I (Gtf2i) is a transcription factor and one of the genes implicated in Willams-Beuren syndrome, an autism spectrum disorder. In this study, we investigated splice variants of the Gtf2i gene in both the 5'untranslated region (5'UTR) and the coding region. To search for novel 5'UTRs of Gtf2i, we utilized the cap analysis gene expression database of the mouse. We identified seven novel Gtf2i transcripts with alternatively spliced 5'UTRs in the rat brain. We also identified four novel splice variants in the coding sequence of Gtf2i. Furthermore, we identified a selective usage of certain types of 5'UTR by coding variants. In situ hybridization demonstrated a differential pattern of expression of Gtf2i mRNAs with alternatively spliced 5'UTRs among neuronal cells, and the localization of one of the variants in neuronal dendrites in the rat brain. Immunohistochemistry also demonstrated a distribution of Gtf2i-immunoreactivity in the dendrites. These results suggest multiple pathways of expression of Gtf2i gene in the brain. The expression patterns may be under the control of alternative promoters coupled to the alternative splicing in the coding region. Differential localization of mRNA to neuronal dendrites suggests spatiotemporal-specific translation at the post-synaptic sites that is involved in transfer of synaptic activity to expression of specific sets of genes in the nucleus.
  • Kerise Lyttle, Yu Ohmura, Kohtarou Konno, Takayuki Yoshida, Takeshi Izumi, Masahiko Watanabe, Mitsuhiro Yoshioka
    BRAIN RESEARCH 1616 88 - 100 2015年08月 [査読有り][通常論文]
     
    Human studies have suggested that early life stress such as child abuse could enhance susceptibility to depressive disorders. Moreover, the abnormalities of the prefrontal cortex have been associated with depression. Although clinical studies have implied the negative effects of early life stress on brain development, the causality and the detailed morphogenetic changes has not been clearly elucidated. In the present study, we determined the effect of juvenile stress exposure on the presentation of depressive-like behavior and the neural mechanisms involved using a rodent model. Rat pups were exposed to footshock stress during postnatal days 21-25 followed by repeated oral administration of fluvoxamine (0 or 10 mg/kg/d x 14 days), which is a selective serotonin reuptake inhibitor. At the postadolescent stage forced swim test assessment of depressive-like behavior and Golgi Cox staining of medial prefrontal cortex pyramidal neurons followed by morphological analyses were carried out. Post-adolescent behavioral and morphological studies identified the presentation of increased depressive-like behaviors and reduced spine densities and dendritic lengths of layer II/III pyramidal neuron in the infralimbic cortex, but not in the prelimbic cortex of rats exposed to juvenile stress. Repeated fluvoxamine treatment recovered the increased depressive-like behavior and reduced spine densities/dendritic lengths observed in rats exposed to footshock stress. Cortical thicknesses in the infralimbic cortex and prelimbic cortex were also reduced by juvenile stress, but these reductions were not recovered by fluvoxamine treatment. The results demonstrate cortical sensitivities to stress exposures during the juvenile stage which mediate behavioral impairments, and provide a clue to find therapeutics for early life stress-induced emotional dysfunctions. (C) 2015 Elsevier B.V. All rights reserved.
  • Wiphawi Hipkaeo, Masahiko Watanabe, Hisatake Kondo
    INTERNATIONAL JOURNAL OF MORPHOLOGY 33 2 695 - 700 2015年06月 [査読有り][通常論文]
     
    In view of the fact that human marijuana users often show dry mouth symptom, the present study was attempted to examine the localization of CB1, which was originally identified in brain, in the submandibular and sublingual salivary glands of postnatal developing male mice by immunohistochemistry. In submandibular gland, CB1-immunoreactivity was positive in a majority of acinar cells in forms of granular appearance in their apical cytoplasm, while it was negative in the ducts at newborn stage. The immunoreactivity decreased in the acinar cells at P1W and no immunoreactivity was detected in the acinar cells at P3W and thereafter. The immunoreactivity was positive in ductal cells at P3W and it remained positive thereafter until P8W stage. The immunoreaction was distinct on the apical plasmalemma of the intercalated ductal cells, while it was distinct on the basal plasmalemma of the granular convoluted ductal cells. The enhanced immunostaining on the lateral plasmalemma of the granular ductal cells was discerned only on P6W. In sublingual gland, CB1-immunoreactivity was detected in the demilune acinar cells and ductal cells only on P4W. Furthermore, CB1-immunoreactivity was shown to occur in the salivary ganglionic neurons, suggesting the CB1-inhibitory action in the saliva secretion through the parasympathetic nervous transmission.
  • Kazuto Masamoto, Miyuki Unekawa, Tatsushi Watanabe, Haruki Toriumi, Hiroyuki Takuwa, Hiroshi Kawaguchi, Iwao Kanno, Ko Matsui, Kenji F. Tanaka, Yutaka Tomita, Norihiro Suzuki
    SCIENTIFIC REPORTS 5 11455  2015年06月 [査読有り][通常論文]
     
    Cortical neural activities lead to changes in the cerebral blood flow (CBF), which involves astrocytic control of cerebrovascular tone. However, the manner in which astrocytic activity specifically leads to vasodilation or vasoconstriction is difficult to determine. Here, cortical astrocytes genetically expressing a light-sensitive cation channel, channelrhodopsin-2 (ChR2), were transcranially activated with a blue laser while the spatiotemporal changes in CBF were noninvasively monitored with laser speckle flowgraphy in the anesthetised mouse cortex. A brief photostimulation induced a fast transient increase in CBF. The average response onset time was 0.7 +/- 0.7 sec at the activation foci, and this CBF increase spread widely from the irradiation spot with an apparent propagation speed of 0.8-1.1 mm/sec. The broad increase in the CBF could be due to a propagation of diffusible vasoactive signals derived from the stimulated astrocytes. Pharmacological manipulation showed that topical administration of a K+ channel inhibitor (BaCl2; 0.1-0.5 mM) significantly reduced the photostimulation-induced CBF responses, which indicates that the ChR2-evoked astrocytic activity involves K+ signalling to the vascular smooth muscle cells. These findings demonstrate a unique model for exploring the role of the astrocytes in gliovascular coupling using non-invasive, time-controlled, cell-type specific perturbations.
  • Hiroyuki Takeda, Tomio Ogasawara, Tatsuhiko Ozawa, Atsushi Muraguchi, Pei-Ju Jih, Ryo Morishita, Motokazu Uchigashima, Masahiko Watanabe, Toyoshi Fujimoto, Takahiro Iwasaki, Yaeta Endo, Tatsuya Sawasaki
    SCIENTIFIC REPORTS 5 11333  2015年06月 [査読有り][通常論文]
     
    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.
  • Ivan Soltesz, Bradley E. Alger, Masanobu Kano, Sang-Hun Lee, David M. Lovinger, Takako Ohno-Shosaku, Masahiko Watanabe
    NATURE REVIEWS NEUROSCIENCE 16 6 264 - 277 2015年06月 [査読無し][通常論文]
  • Robert P. Ganley, Noboru Iwagaki, Patricia del Rio, Najma Baseer, Allen C. Dickie, Kieran A. Boyle, Erika Polgar, Masahiko Watanabe, Victoria E. Abraira, Amanda Zimmerman, John S. Riddell, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 35 19 7626 - 7642 2015年05月 [査読有り][通常論文]
     
    The superficial dorsal horn of the spinal cord contains numerous inhibitory interneurons, which regulate the transmission of information perceived as touch, pain, or itch. Despite the importance of these cells, our understanding of their roles in the neuronal circuitry is limited by the difficulty in identifying functional populations. One group that has been identified and characterized consists of cells in the mouse that express green fluorescent protein (GFP) under control of the prion protein (PrP) promoter. Previous reports suggested that PrP-GFP cells belonged to a single morphological class (central cells), received inputs exclusively from unmyelinated primary afferents, and had axons that remained in lamina II. However, we recently reported that the PrP-GFP cells expressed neuronal nitric oxide synthase (nNOS) and/or galanin, and it has been shown that nNOS-expressing cells are more diverse in their morphology and synaptic connections. We therefore used a combined electrophysiological, pharmacological, and anatomical approach to reexamine the PrP-GFP cells. We provide evidence that they are morphologically diverse (corresponding to "unclassified" cells) and receive synaptic input from a variety of primary afferents, with convergence onto individual cells. We also show that their axons project into adjacent laminae and that they target putative projection neurons in lamina I. This indicates that the neuronal circuitry involving PrP-GFP cells is more complex than previously recognized, and suggests that they are likely to have several distinct roles in regulating the flow of somatosensory information through the dorsal horn.
  • Watanabe M, Takahashi H, Saeki Y, Ozaki T, Itoh S, Suzuki M, Mizushima W, Tanaka K, Hatakeyama S
    eLife 4 e05615  2015年04月 [査読有り][通常論文]
     
    Adipocyte differentiation is a strictly controlled process regulated by a series of transcriptional activators. Adipogenic signals activate early adipogenic activators and facilitate the transient formation of early enhanceosomes at target genes. These enhancer regions are subsequently inherited by late enhanceosomes. PPAR gamma is one of the late adipogenic activators and is known as a master regulator of adipogenesis. However, the factors that regulate PPAR gamma expression remain to be elucidated. Here, we show that a novel ubiquitin E3 ligase, tripartite motif protein 23 (TRIM23), stabilizes PPAR gamma protein and mediates atypical polyubiquitin conjugation. TRIM23 knockdown caused a marked decrease in PPAR gamma protein abundance during preadipocyte differentiation, resulting in a severe defect in late adipogenic differentiation, whereas it did not affect the formation of early enhanceosomes. Our results suggest that TRIM23 plays a critical role in the switching from early to late adipogenic enhanceosomes by stabilizing PPAR gamma protein possibly via atypical polyubiquitin conjugation.
  • Yanyong Toomsan, Surang Chomphoo, Sawetree Pakkarato, Masahiko Watanabe, Hisatake Kondo, Wiphawi Hipkaeo
    INTERNATIONAL JOURNAL OF MORPHOLOGY 33 1 113 - 118 2015年03月 [査読有り][通常論文]
     
    According to recent studies, it is highly possible that the occurrence of vesicular inhibitory amino acid transporter (VIAAT) is a good marker of GABA-signaling not only in the brain, but also in extra-brain tissue cells containing GABA and GAD. In view of this, the present study was attempted to localize VIAAT-immunoreactivity in the submandibular gland of mice. In the present study, the submandibular glands of male mice at various postnatal developmental stages were examined for detailed localization of VIAAT-immunoreactivity in immunohistochemistry at light microscopic level. The immunoreactivity for VIAAT was localized in epithelial cells of proximal and distal excretory ducts with the striated portion more intensely immunopositive at young postnatal stages. No significant immunoreactivity was seen in the acinar cells throughout the postnatal development. In addition, the immunoreactivity for VIAAT was detected in the salivary parasympathetic ganglionic neurons, but not in any nerve fibers surrounding the glandular cells. Furthermore, VIAAT-immunoreactivity was found in smooth muscle cells forming the outermost layer of intralobular arterioles. From the present findings, it is possible that GABA plays roles as paracrine and autocrine regulators in the saliva secretion as well as the gland development.
  • Kohtarou Konno, Hiromi Takahashi-Iwanaga, Motokazu Uchigashima, Kyoko Miyasaka, Akihiro Funakoshi, Masahiko Watanabe, Toshihiko Iwanaga
    HISTOCHEMISTRY AND CELL BIOLOGY 143 3 301 - 312 2015年03月 [査読有り][通常論文]
     
    Information concerning the cellular localization of cholecystokinin (CCK)-1 receptors has been discrepant and remained scanty at ultrastructural levels. The present immunohistochemical study at light and electron microscopic levels revealed the distinct localization of CCK1 receptors in visceral organs. Immunohistochemistry by use of a purified antibody against mouse CCK1 receptor was applied to fixed tissue sections of the pancreas, gallbladder, stomach, and intestine of mice. A silver-intensified immunogold method revealed the subcellular localization under electron microscope. The immunoreactivity for CCK1 receptors was selectively found in the basolateral membrane of pancreatic acinar cells and gastric chief cells but was absent in pancreatic islets and gastric D cells. Another intense expression in the gut was seen in the myenteric nerve plexus of the antro-duodenal region and some populations of c-Kit-expressing pacemaker cells in the duodenal musculature. The gallbladder contained smooth muscle fibers with an intense immunoreactivity of CCK1 receptors on cell surfaces. The restricted localization of CCK1 receptors on the basolateral membrane of pancreatic acinar cells and gastric chief cells, along with their absence in the islets of Langerhans and gastric D cells, provides definitive information concerning the regulatory mechanism by circulating CCK. Especially, the subcellular localization in the acinar cells completes the investigation for the detection of circulating CCK by the basolateral membrane.
  • Yuki Omiya, Motokazu Uchigashima, Kohtarou Konno, Miwako Yamasaki, Taisuke Miyazaki, Takayuki Yoshida, Ichiro Kusumi, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 35 10 4215 - 4228 2015年03月 [査読有り][通常論文]
     
    Invaginating synapses in the basal amygdala are a unique type of GABAergic synapses equipped with molecular-anatomical organization specialized for 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling. Cholecystokinin (CCK)-positive basket cell terminals protrude into pyramidal cell somata and form invaginating synapses, where apposing presynaptic and postsynaptic elements are highly loaded with cannabinoid receptor CB1 or 2-AG synthetic enzyme diacylglycerol lipase-alpha (DGL alpha), respectively. The present study scrutinized their neurochemical and neuroanatomical phenotypes in adult mouse telencephalon. In the basal amygdala, vesicular glutamate transporter-3 (VGluT3) was transcribed in one-fourth of CB1-expressing GABAergic interneurons. The majority of VGluT3-positive CB1-expressing basket cell terminals apposed DGL alpha clusters, whereas the majority of VGluT3-negative ones did not. Importantly, VGluT3-positive basket cell terminals selectively constructed invaginating synapses. GABA(A) receptors accumulated on the postsynaptic membrane of invaginating synapses, whereas metabotropic glutamate receptor-5 (mGluR5) was widely distributed on the somatodendritic surface of pyramidal cells. Moreover, CCK2 receptor (CCK2R) was highly transcribed in pyramidal cells. In cortical regions, pyramidal cells equipped with such VGluT3/CB1/DGL alpha-accumulated invaginating synapses were found at variable frequencies depending on the subregions. Therefore, in addition to extreme proximity of CB1- and DGL alpha-loaded presynaptic and postsynaptic elements, tripartite transmitter phenotype of GABA/glutamate/CCK is the common neurochemical feature of invaginating synapses, suggesting that glutamate, CCK, or both can promote 2-AG synthesis through activating G alpha(q/11) protein-coupled mGluR5 and CCK2R. These molecular configurations led us to hypothesize that invaginating synapses might be evolved to provide some specific mechanisms of induction, regulation, and cooperativity for 2-AG-mediated retrograde signaling in particular cortical and cortex-like amygdaloid regions.
  • Taisuke Miyazaki, Masahiko Watanabe
    Immunocytochemistry and Related Techniques 299 - 311 2015年02月24日 [査読有り][通常論文]
     
    Neuronal tracing ( neurotracing ) using anterograde and retrograde tracers is widely used to study the projections between different brain regions and the wiring between individual neurons . Neurotracing is a technique essential not only for examining the connectivity of complex neuronal networks but also for providing the neuroanatomical basis for electrophysiological, pharmacological and behavioral experiments. If neurotracing is combined with immunocytochemical labeling, the combined technique can characterize the neurochemical properties, postsynaptic targets and innervation modes of neurons. The utility and versatility of this approach can be further extended by adopting appropriate cellular and subcellular markers for immunocytochemistry, by applying the approach to animal models generated by advanced genemanipulation technology, and by using single-cell labeling techniques, e.g., after viral transfection of fluorescent proteins or in utero / in vivo electroporation . In this chapter, we introduce the methods for combined immunocytochemistry and neurotracing at both light and electron microscopic levels. We have developed and employed these combined approaches to study the mechanisms underlying the development and refinement of climbing fiber mono-innervation in cerebellar Purkinje cells . Therefore, we present some examples of the images obtained in this experimental context.
  • Yoshio Bando, Taichi Nomura, Hiroki Bochimoto, Koichi Murakami, Tatsuhide Tanaka, Tsuyoshi Watanabe, Shigetaka Yoshida
    NEUROCHEMISTRY INTERNATIONAL 81 16 - 27 2015年02月 [査読有り][通常論文]
     
    Demyelination and axonal damage are responsible for neurological deficits in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. However, the pathology of axonal damage in MS is not fully understood. In this study, histological analysis of morphological changes of axonal organelles during demyelination in murine models was investigated by scanning electron microscopy (SEM) using an osmium-maceration method. In cuprizone-induced demyelination, SEM showed typical morphology of demyelination in the corpus callosum of mouse brain. In contrast, SEM displayed variations in ultrastructural abnormalities of myelin structures and axonal organelles in spinal cord white matter of experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS. Myelin detachment and excessive myelin formation were observed as typical morphological myelin abnormalities in EAE. In addition, well-developed axoplasmic reticulum-like structures and accumulated mitochondria were observed in tortuous degenerating/degenerated axons and the length of mitochondria in axons of EAE spinal cord was shorter compared with naive spinal cord. Immunohistochemistry also revealed dysfunction of mitochondrial fusion/fission machinery in EAE spinal cord axons. Moreover, the number of Y-shaped mitochondria was significantly increased in axons of the EAE spinal cord. Axonal morphologies in myelin basic proteindeficient shiverer mice were similar to those in EAE. However, shiverer mice had "tortuous" (S-curve shaped mitochondria) and larger mitochondria compared with wild-type and EAE mice. Lastly, analysis of human MS patient autopsied brains also demonstrated abnormal myelin structures in demyelinating lesions. These results indicate that morphological abnormalities of myelin and axonal organelles play important role on the pathogenesis of axonal injury in demyelinating diseases. (C) 2015 Elsevier Ltd. All rights reserved.
  • Naofumi Uesaka, Motokazu Uchigashima, Takayasu Mikuni, Hirokazu Hirai, Masahiko Watanabe, Masanobu Kano
    CEREBELLUM 14 1 4 - 7 2015年02月 [査読有り][通常論文]
     
    Neurons form exuberant synapses with target cells early in development. Then, necessary synapses are selectively strengthened whereas unnecessary connections are weakened and eventually eliminated during postnatal development. This process is known as synapse elimination and is a crucial step for shaping immature neural circuits into functionally mature versions. Accumulating evidence suggests that retrograde signaling from postsynaptic cells regulates synapse elimination, but the underlying mechanisms remain unknown. Here, we show that semaphorin3A (Sema3A) and semaphorin7A (Sema7A) mediate retrograde signals for elimination of redundant climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum, a representative model of synapse elimination in the central nervous system. We picked up candidate retrograde signaling molecules that are expressed in PCs during the period of CF synapse elimination and the receptors of these candidate molecules that are present in CFs. We then assessed the effects of lentivirus-mediated RNAi-knockdown of these molecules on CF synapse elimination. By this systematic screening, we found that knockdown of Sema3A in PCs or its co-receptor, plexinA4 (PlxnA4), in CFs accelerated CF synapse elimination and decreased CF-mediated synaptic inputs. Conversely, knockdown of Sema7A in PCs or either of the two receptors for Sema7A, plexinC1 (PlxnC1) and integrinB1 (ItgB1), in CFs impaired CF synapse elimination. Importantly, the effect of Sema7A involves signaling by type 1 metabotropic glutamate receptor (mGluR1), a canonical pathway in PCs for the final stage of CF synapse elimination. These results demonstrate that specific semaphorins act as retrograde signaling molecules and regulate distinct processes of CF synapse elimination during postnatal cerebellar development.
  • Izumi Kaji, Toshihiko Iwanaga, Masahiko Watanabe, Paul H. Guth, Eli Engel, Jonathan D. Kaunitz, Yasutada Akiba
    AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY 308 3 G188 - G197 2015年02月 [査読有り][通常論文]
     
    Bacterial or ingested food-derived short-chain fatty acids (SCFAs) are present in the duodenal lumen. Acetate, the most abundant SCFA in the foregut lumen, is absorbed immediately after ingestion, although the mechanism by which this absorption occurs is not fully understood. We investigated the distribution and function of candidate SCFA transporters in rat duodenum. The Na+ -coupled monocarboxylate transporter-1 (SMCT1) was localized to the brush border, whereas the pH-dependent monocarboxylate transporter (MCT) 1 and MCT4 were localized to the duodenocyte basolateral membrane. In Ussing chambered duodenal mucosa, luminal acetate dose-dependently increased short-circuit current (I-sc) in the presence of serosal bumetanide and indomethacin by a luminal Na+ -dependent, ouabain-sensitive mechanism. The Isc response was inhibited dose-dependently by the SMCT1 nonsubstrate inhibitor ibuprofen, consistent with net electrogenic absorption of acetate via SMCT1. Other SCFAs and lactate also increased Isc. Furthermore, duodenal loop perfusion of acetate increased portal venous acetate concentration, inhibited by coperfusion of ibuprofen or a MCT inhibitor. Luminal acetate perfusion increased duodenal HCO3 secretion via capsaicin-sensitive afferent nerve activation and cyclooxygenase activity, consistent with absorption-mediated HCO3- secretion. These results suggest that absorption of luminal SCFA via SMCT1 and MCTs increases duodenal HCO3- secretion. In addition to SCFA sensing via free fatty acid receptors, the presence of rapid duodenal SCFA absorption may be important for the suppression of luminal bacterial colonization and implicated in the generation of functional dyspepsia due to bacterial overgrowth.
  • Barna Dudok, László Barna, Marco Ledri, Szilárd I. Szabó, Eszter Szabadits, Balázs Pintér, Stephen G. Woodhams, Christopher M. Henstridge, Gyula Y. Balla, Rita Nyilas, Csaba Varga, Sang-Hun Lee, Máté Matolcsi, Judit Cervenak, Imre Kacskovics, Masahiko Watanabe, Claudia Sagheddu, Miriam Melis, Marco Pistis, Ivan Soltesz, István Katona
    Nature Neuroscience 18 1 75 - 86 2015年01月01日 [査読有り][通常論文]
     
    A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type-and subcellular compartment-specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB 1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ9-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB 1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of Δ9-tetrahydrocannabinol treatment. These findings indicate that cell type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.
  • Kei Hosoda, Keishi Yamashita, Natsuya Katada, Hiromitsu Moriya, Hiroaki Mieno, Shinichi Sakuramoto, Shiro Kikuchi, Masahiko Watanabe
    SURGERY TODAY 45 1 68 - 77 2015年01月 [査読有り][通常論文]
     
    This study was designed to clarify whether preoperative tumor size is an independent prognostic factor (IPF) for patients with Borrmann type III gastric cancer. The study group comprised 350 patients with Borrmann type III gastric cancer. We performed a log-rank plot analysis to establish the threshold value of preoperative tumor size for the prediction of overall survival (OS). Factors with P < 0.10 on univariate prognostic analyses for OS were put into a Cox's proportional hazards model to identify the IPFs. Peritoneal lavage cytology (CY) was the strongest IPF for patients with Borrmann type III gastric cancer (P < 0.0001). We were able to measure the tumor size preoperatively in 135 patients with negative CY results (CY0). The cutoff tumor size for the prediction of OS was 5.3 cm. A Cox's proportional hazards model showed that pathological lymph-node metastasis (P = 0.007) and preoperative tumor size (P = 0.018) were significant IPFs in the CY0 patients. Patients with a preoperative tumor size of < 5.3 cm had satisfactory outcomes, with a 5-year OS rate of > 80 %. Preoperative tumor size is an IPF for patients with Borrmann type III gastric cancer and CY0. Thus, preoperative tumor size may be a useful factor for deciding on whether neoadjuvant chemotherapy is indicated.
  • Claire Piochon, Alexander D. Kloth, Giorgio Grasselli, Heather K. Titley, Hisako Nakayama, Kouichi Hashimoto, Vivian Wan, Dana H. Simmons, Tahra Eissa, Jin Nakatani, Adriana Cherskov, Taisuke Miyazaki, Masahiko Watanabe, Toru Takumi, Masanobu Kano, Samuel S. -H. Wang, Christian Hansel
    NATURE COMMUNICATIONS 6 6014  2015年01月 [査読有り][通常論文]
  • Hidehisa Takahashi, Ichigaku Takigawa, Masashi Watanabe, Delnur Anwar, Mio Shibata, Chieri Tomomori-Sato, Shigeo Sato, Amol Ranjan, Chris W. Seidel, Tadasuke Tsukiyama, Wataru Mizushima, Masayasu Hayashi, Yasuyuki Ohkawa, Joan W. Conaway, Ronald C. Conaway, Shigetsugu Hatakeyama
    NATURE COMMUNICATIONS 6 5941  2015年01月 [査読有り][通常論文]
     
    Regulation of transcription elongation by RNA polymerase II (Pol II) is a key regulatory step in gene transcription. Recently, the little elongation complex (LEC)-which contains the transcription elongation factor ELL/EAF-was found to be required for the transcription of Pol II-dependent small nuclear RNA (snRNA) genes. Here we show that the human Mediator subunit MED26 plays a role in the recruitment of LEC to a subset of snRNA genes through direct interaction of EAF and the N-terminal domain (NTD) of MED26. Loss of MED26 in cells decreases the occupancy of LEC at a subset of snRNA genes and results in a reduction in their transcription. Our results suggest that the MED26-NTD functions as a molecular switch in the exchange of TBP-associated factor 7 (TAF7) for LEC to facilitate the transition from initiation to elongation during transcription of a subset of snRNA genes.
  • Mahnaz Mansouri, Yu Kasugai, Yugo Fukazawa, Federica Bertaso, Fabrice Raynaud, Julie Perroy, Laurent Fagni, Walter A. Kaufmann, Masahiko Watanabe, Ryuichi Shigemoto, Francesco Ferraguti
    EUROPEAN JOURNAL OF NEUROSCIENCE 41 2 157 - 167 2015年01月 [査読有り][通常論文]
     
    Type 1 metabotropic glutamate (mGlu1) receptors play a pivotal role in different forms of synaptic plasticity in the cerebellar cortex, e.g. long-term depression at glutamatergic synapses and rebound potentiation at GABAergic synapses. These various forms of plasticity might depend on the subsynaptic arrangement of the receptor in Purkinje cells that can be regulated by protein-protein interactions. This study investigated, by means of the freeze-fracture replica immunogold labelling method, the subcellular localization of mGlu1 receptors in the rodent cerebellum and whether Homer proteins regulate their subsynaptic distribution. We observed a widespread extrasynaptic localization of mGlu1 receptors and confirmed their peri-synaptic enrichment at glutamatergic synapses. Conversely, we detected mGlu1 receptors within the main body of GABAergic synapses onto Purkinje cell dendrites. Although Homer proteins are known to interact with the mGlu1 receptor C-terminus, we could not detect Homer3, the most abundant Homer protein in the cerebellar cortex, at GABAergic synapses by pre-embedding and post-embedding immunoelectron microscopy. We then hypothesized a critical role for Homer proteins in the peri-junctional localization of mGlu1 receptors at glutamatergic synapses. To disrupt Homer-associated protein complexes, mice were tail-vein injected with the membrane-permeable dominant-negative TAT-Homer1a. Freeze-fracture replica immunogold labelling analysis showed no significant alteration in the mGlu1 receptor distribution pattern at parallel fibre-Purkinje cell synapses, suggesting that other scaffolding proteins are involved in the peri-synaptic confinement. The identification of interactors that regulate the subsynaptic localization of the mGlu1 receptor at neurochemically distinct synapses may offer new insight into its trafficking and intracellular signalling.
  • Norihiko Yokoi, Yuko Fukata, Daisuke Kase, Taisuke Miyazaki, Martine Jaegle, Toshika Ohkawa, Naoki Takahashi, Hiroko Iwanari, Yasuhiro Mochizuki, Takao Hamakubo, Keiji Imoto, Dies Meijer, Masahiko Watanabe, Masaki Fukata
    NATURE MEDICINE 21 1 19 - 26 2015年01月 [査読有り][通常論文]
     
    Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI 1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.
  • Sasaki K, Sumiyoshi A, Nonaka H, Kasahara Y, Ikeda K, Hall FS, Uhl GR, Watanabe M, Kawashima R, Sora I
    Br J Pharmacol 172 2 654 - 667 2015年01月 [査読有り][通常論文]
     
    Background and Purpose Opioid receptor knockout (MOP-KO) mice display several behavioural differences from wild-type (WT) littermates including differential responses to nociceptive stimuli. Brain structural changes have been tied to behavioural alterations noted in transgenic mice with targeting of different genes. Hence, we assess the brain structure of MOP-KO mice. Experimental ApproachMagnetic resonance imaging (MRI) voxel-based morphometry (VBM) and histological methods were used to identify structural differences between extensively backcrossed MOP-KO mice and WT mice. Key ResultsMOP-KO mice displayed robust increases in regional grey matter volume in olfactory bulb, several hypothalamic nuclei, periaqueductal grey (PAG) and several cerebellar areas, most confirmed by VBM analysis. The largest increases in grey matter volume were detected in the glomerular layer of the olfactory bulb, arcuate nucleus of hypothalamus, ventrolateral PAG (VLPAG) and cerebellar regions including paramedian and cerebellar lobules. Histological analyses confirm several of these results, with increased VLPAG cell numbers and increased thickness of the olfactory bulb granule cell layer and cerebellar molecular and granular cell layers. Conclusions and ImplicationsMOP deletion causes previously undescribed structural changes in specific brain regions, but not in all regions with high MOP receptor densities (e.g. thalamus, nucleus accumbens) or that exhibit adult neurogenesis (e.g. hippocampus). Volume differences in hypothalamus and PAG may reflect behavioural changes including hyperalgesia. Although the precise relationship between volume change and MOP receptor deletion was not determined from this study alone, these findings suggest that levels of MOP receptor expression may influence a broader range of neural structure and function in humans than previously supposed. Linked ArticlesThis article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit
  • Ayumi Tanaka-Hayashi, Shuuhei Hayashi, Ran Inoue, Tomokazu Ito, Kohtarou Konno, Tomoyuki Yoshida, Masahiko Watanabe, Tohru Yoshimura, Hisashi Mori
    AMINO ACIDS 47 1 79 - 86 2015年01月 [査読有り][通常論文]
     
    d-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, d-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, d-aspartate strongly enhances N-methyl-d-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for d-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially d-aspartate from l-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where d-aspartate is abundant. The d-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed l-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown d-aspartate-synthesizing enzyme.
  • Wataru Kakegawa, Nikolaos Mitakidis, Eriko Miura, Manabu Abe, Keiko Matsuda, Yukari H. Takeo, Kazuhisa Kohda, Junko Motohashi, Akiyo Takahashi, Soichi Nagao, Shin-ichi Muramatsu, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, Michisuke Yuzaki
    NEURON 85 2 316 - 329 2015年01月 [査読有り][通常論文]
     
    Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.
  • Kishimoto A, Ishiguro-Oonuma T, Takahashi R, Maekawa M, Toshimori K, Watanabe M, Iwanaga T
    Biomedical research (Tokyo, Japan) 36 4 225 - 234 2015年 [査読有り][通常論文]
  • Hozumi Y, Kakefuda K, Yamasaki M, Watanabe M, Hara H, Goto K
    Brain Research 1594 36 - 45 2015年01月 [査読有り][通常論文]
     
    Spine formation, a salient feature underlying neuronal plasticity to adapt to a changing environment, is regulated by complex machinery involving membrane signal transduction. The diacylglycerol kinase (DGK) family, which is involved in membrane lipid metabolism, catalyzes the phosphorylation of a lipid second messenger, diacylglycerol (DG). Of the DGKs, DGK beta is characterized by predominant expression in a specific brain region: the striatum. We previously demonstrated that DGK beta is expressed selectively in medium spiny neurons (MSNs) and that it is highly enriched in the perisynaptic membrane on dendritic spines contacted with excitatory terminals. Moreover, DGK beta regulates spinogenesis through actin-based remodeling in an activity-dependent manner. However, the detailed mechanisms of spinogenesis regulation and its functional significance remain unclear. To address these issues, we performed Golgi-Cox staining to examine morphological aspects of MSNs in the striatum of DGK beta-knockout (KO) mice. Results show that striatal MSNs of DGK beta-KO mice exhibited lower dendritic spine density at distal dendrites than wild-type mice did. We also sought protein targets that interact with DGK beta and identified the GluA2 AMPA receptor subunit as a novel DGK beta binding partner. In addition, DGK beta-deficient brain exhibits significant reduction of TARP gamma-8, which represents a transmembrane AMPA receptor regulatory protein. These findings suggest that DGK beta regulates the spine formation at distal dendrites in MSNs. (C) 2014 Elsevier B.V. All rights reserved.
  • Natsuya Katada, Jiro Shibuya, Takafumi Ichikawa, Kei Hosoda, Keishi Yamashita, Masahiko Watanabe
    ESOPHAGUS 12 1 65 - 72 2015年01月 [査読有り][通常論文]
     
    The objectives of this study were to delineate differences in the morphologic characteristics of reflux esophagitis between a rat gastroesophageal reflux (GER) model and a duodenoesophageal reflux (DER) model and to evaluate the effects of H2-receptor antagonists on morphologic characteristics of reflux esophagitis in DER model. Wistar rats were divided into 3 groups: GER model group (Group G), DER model group (Group D), and control group (Group C). Rats in each group were sacrificed 1 or 12 weeks after surgery. Intraesophageal pH was measured, and the excised esophagus was examined macroscopically and histologically. Subgroups of rats in Group D were given famotidine (10 mg/kg) or lafutidine (30 mg/kg) orally once daily for 1 week after surgery. The rats were then sacrificed, and histological findings were compared. Intraesophageal pH was significantly lower in Group G than in Group C. At 12 weeks, the epithelium of the lower esophagus in Groups G and D was significantly thicker than that in Group C and showed remarkable hyperplastic changes in Group D. The thickness of the epithelium in Group D + famotidine did not differ significantly from that in Group D. In contrast, the epithelium was significantly thinner in Group D + lafutidine than in Group D. As a rat model of reflux esophagitis, DER causes severer damage to the esophageal epithelium, including hyperplastic changes, than does GER. Famotidine had no apparent effect on esophageal epithelial damage caused by DER, whereas lafutidine was suggested to attenuate such damage.
  • Shinichiro Tsutsumi, Maya Yamazaki, Taisuke Miyazaki, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano, Kazuo Kitamura
    JOURNAL OF NEUROSCIENCE 35 2 843 - 852 2015年01月 [査読有り][通常論文]
     
    Simple and regular anatomical structure is a hallmark of the cerebellar cortex. Parasagittally arrayed alternate expression of aldolase C/zebrin II in Purkinje cells (PCs) has been extensively studied, but surprisingly little is known about its functional significance. Here we found a precise structure-function relationship between aldolase C expression and synchrony of PC complex spike activities that reflect climbing fiber inputs to PCs. We performed two-photon calcium imaging in transgenic mice in which aldolase C compartments can be visualized in vivo, and identified highly synchronous complex spike activities among aldolase C-positive or aldolase C-negative PCs, but not across these populations. The boundary of aldolase C compartments corresponded to that of complex spike synchrony at single-cell resolution. Sensory stimulation evoked aldolase C compartment-specific complex spike responses and synchrony. This result further revealed the structure-function segregation. In awake animals, complex spike synchrony both within and between PC populations across the aldolase C boundary were enhanced in response to sensory stimuli, in a way that two functionally distinct PC ensembles are coactivated. These results suggest that PC populations characterized by aldolase C expression precisely represent distinct functional units of the cerebellar cortex, and these functional units can cooperate to process sensory information in awake animals.
  • Tomoaki Sobajima, Shin-ichiro Yoshimura, Tomohiko Iwano, Masataka Kunii, Masahiko Watanabe, Nur Atik, Sotaro Mushiake, Eiichi Morii, Yoshihisa Koyama, Eiji Miyoshi, Akihiro Harada
    BIOLOGY OPEN 4 1 86 - 94 2015年01月 [査読有り][通常論文]
     
    The small GTPase Rab11 plays an important role in the recycling of proteins to the plasma membrane as well as in polarised transport in epithelial cells and neurons. We generated conditional knockout mice deficient in Rab11a. Rab11a-deficient mice are embryonic lethal, and brain-specific Rab11a knockout mice show no overt abnormalities in brain architecture. In contrast, intestine-specific Rab11a knockout mice begin dying approximately 1 week after birth. Apical proteins in the intestines of knockout mice accumulate in the cytoplasm and mislocalise to the basolateral plasma membrane, whereas the localisation of basolateral proteins is unaffected. Shorter microvilli and microvillus inclusion bodies are also observed in the knockout mice. Elevation of a serum starvation marker was also observed, likely caused by the mislocalisation of apical proteins and reduced nutrient uptake. In addition, Rab8a is mislocalised in Rab11a knockout mice. Conversely, Rab11a is mislocalised in Rab8a knockout mice and in a microvillus atrophy patient, which has a mutation in the myosin Vb gene. Our data show an essential role for Rab11a in the localisation of apical proteins in the intestine and demonstrate functional relationships between Rab11a, Rab8a and myosin Vb in vivo.
  • Maria Gutierrez-Mecinas, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 10 2014年12月 [査読有り][通常論文]
     
    Background: Gastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter. Results: GRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP(+) cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase C gamma, which is expressed by a subset of the excitatory interneurons in this region. Conclusions: These results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A.
  • Carmen Ballesteros-Merino, Jose Martinez-Hernandez, Carolina Aguado, Masahiko Watanabe, John P. Adelman, Rafael Lujan
    FRONTIERS IN NEUROANATOMY 8 2014年12月 [査読有り][通常論文]
     
    Small-conductance, Ca2+-activated K+ (SK) channels regulate neuronal excitability in a variety of ways. To understand their roles in different neuronal subtypes it is important to determine their precise subcellular distribution. Here, we used biochemical, light microscopy immunohistochemical and immunoelectron microscopy techniques, combined with quantitative approaches, to reveal the expression and subcellular localization patterns of SK2 in the developing cerebellum. Using western blots, the SK2 protein showed a progressive increase during postnatal development. At the light microscopic level, SK2 immunoreactivity was very prominent in the developing Purkinje cells (PC), particularly in the molecular layer (ML). Electron microscopy revealed that throughout development SK2 was mostly detected at the extrasynaptic and perisynaptic plasma membrane of dendritic shafts and dendritic spines of PCs. However, there was some localization at axon terminals as well. Quantitative analyses and 3D reconstructions further revealed a progressive developmental change of SK2 on the surface of PCs from dendritic shafts to dendritic spines. Together, these results indicate that SK2 channels undergo dynamic spatial regulation during cerebellar development, and this process is associated with the formation and maturation of excitatory synaptic contacts to PCs.
  • Víctor Fernández-Dueñas, Maricel Gómez-Soler, Marc López-Cano, Jaume J. Taura, Catherine Ledent, Masahiko Watanabe, Kenneth A. Jacobson, Jean-Pierre Vilardaga, Francisco Ciruela
    ACS Chemical Biology 9 11 2496 - 2501 2014年11月21日 [査読有り][通常論文]
     
    Caffeine, the most consumed psychoactive substance worldwide, may have bene fi cial effects on Parkinson's disease (PD) therapy. The mechanism by which caffeine contributes to its antiparkinsonian effects by acting as either an adenosine A< inf> 2A< /inf> receptor (A< inf> 2A< /inf> R) neutral antagonist or an inverse agonist is unresolved. Here we show that caffeine is an A< inf> 2A< /inf> R inverse agonist in cell-based functional studies and in experimental parkinsonism. Thus, we observed that caffeine triggers a distinct mode, opposite to A< inf> 2A< /inf> R agonist, of the receptor's activation switch leading to suppression of its spontaneous activity. These inverse agonist-related effects were also determined in the striatum of a mouse model of PD, correlating well with increased caffeine-mediated motor effects. Overall, caffeine A< inf> 2A< /inf> R inverse agonism may be behind some of the well-known physiological effects of this substance both in health and disease. This information might have a critical mechanistic impact for PD pharmacotherapeutic design. (Chemical Equation Presented).
  • Najma Baseer, Abdullah S. Al-Baloushi, Masahiko Watanabe, Safa A. S. Shehab, Andrew J. Todd
    PAIN 155 11 2291 - 2300 2014年11月 [査読有り][通常論文]
     
    Fine myelinated (A delta) nociceptors are responsible for fast, well-localised pain, but relatively little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. Here we show that transganglionically transported cholera toxin B subunit (CTb) labels a distinct set of afferents in lamina I that are likely to correspond to A delta nociceptors, and that most of these lack neuropeptides. The vast majority of lamina I projection neurons can be retrogradely labelled from the lateral parabrachial area, and these can be divided into 2 major groups based on expression of the neurokinin 1 receptor (NK1r). We show that CTb-labelled afferents form contacts on 43% of the spinoparabrachial lamina I neurons that lack the NK1r, but on a significantly smaller proportion (26%) of those that express the receptor. We also confirm with electron microscopy that these contacts are associated with synapses. Among the spinoparabrachial neurons that received contacts from CTb-labelled axons, contact density was considerably higher on NK1r-lacking cells than on those with the NK1r. By comparing the density of CTb contacts with those from other types of glutamatergic bouton, we estimate that nonpeptidergic A delta nociceptors may provide over half of the excitatory synapses on some NK1r-lacking spinoparabrachial cells. These results provide further evidence that synaptic inputs to dorsal horn projection neurons are organised in a specific way. Taken together with previous studies, they suggest that both NK1r(+) and NK1r-lacking lamina I projection neurons are directly innervated by A delta nociceptive afferents. (C) 2014 The Authors. Published by Elsevier B.V. on behalf of International Association for the Study of Pain. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
  • Claire Piochon, Alexander D. Kloth, Giorgio Grasselli, Heather K. Titley, Hisako Nakayama, Kouichi Hashimoto, Vivian Wan, Dana H. Simmons, Tahra Eissa, Jin Nakatani, Adriana Cherskov, Taisuke Miyazaki, Masahiko Watanabe, Toru Takumi, Masanobu Kano, Samuel S. -H. Wang, Christian Hansel
    NATURE COMMUNICATIONS 5 2014年11月 [査読有り][通常論文]
     
    A common feature of autism spectrum disorder (ASD) is the impairment of motor control and learning, occurring in a majority of children with autism, consistent with perturbation in cerebellar function. Here we report alterations in motor behaviour and cerebellar synaptic plasticity in a mouse model (patDp/+) for the human 15q11-13 duplication, one of the most frequently observed genetic aberrations in autism. These mice show ASD-resembling social behaviour deficits. We find that in patDp/+ mice delay eyeblink conditioning-a form of cerebellum-dependent motor learning-is impaired, and observe deregulation of a putative cellular mechanism for motor learning, long-term depression (LTD) at parallel fibre-Purkinje cell synapses. Moreover, developmental elimination of surplus climbing fibres-a model for activity-dependent synaptic pruning-is impaired. These findings point to deficits in synaptic plasticity and pruning as potential causes for motor problems and abnormal circuit development in autism.
  • Gloria Garcia-Negredo, David Soto, Javier Llorente, Xavier Morato, Koen M. O. Galenkamp, Maricel Gomez-Soler, Victor Fernandez-Duenas, Masahiko Watanabe, John P. Adelman, Ryuichi Shigemoto, Yugo Fukazawa, Rafael Lujan, Francisco Ciruela
    JOURNAL OF NEUROSCIENCE 34 44 14793 - 14802 2014年10月 [査読無し][通常論文]
     
    Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca2+ wave-dependent activation of small-conductance Ca2+-activated K+ (SK) channels. Here, we show that mGlu(5) receptors and SK2 channels coassemble in heterologous coexpression systems and in rat brain. Further, in cotransfected cells or rat primary hippocampal neurons, mGlu(5) receptor stimulation activated apamin-sensitive SK2-mediated K+ currents. In addition, coexpression of mGlu(5) receptors and SK2 channels promoted plasma membrane targeting of both proteins and correlated with increased mGlu(5) receptor function that was unexpectedly blocked by apamin. These results demonstrate a reciprocal functional interaction between mGlu(5) receptors and SK2 channels that reflects their molecular coassembly.
  • Shun Hamada, Itone Ogawa, Miwako Yamasaki, Yuji Kiyama, Hidetoshi Kassai, Ayako M. Watabe, Kazuki Nakao, Atsu Aiba, Masahiko Watanabe, Toshiya Manabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 40 8 3136 - 3146 2014年10月 [査読有り][通常論文]
     
    The N-methyl-d-aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B-containing receptors, which are gradually replaced with GluN2A-containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock-in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A-containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A-containing NMDARs could not be substituted for GluN2B-containing NMDARs. Importantly, the synaptic -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR-mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B-GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.
  • Xiaohong Song, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Motokazu Uchigashima, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 40 5 2797 - 2810 2014年09月 [査読有り][通常論文]
     
    Slc4a10 was originally identified as a Na+-driven Cl-/HCO3- exchanger NCBE that transports extracellular Na+ and HCO3- in exchange for intracellular Cl-, whereas other studies argue against a Cl--dependence for Na+-HCO3- transport, and thus named it the electroneutral Na+/HCO3- cotransporter NBCn2. Here we investigated Slc4a10 expression in adult mouse brains by in situ hybridization and immunohistochemistry. Slc4a10 mRNA was widely expressed, with higher levels in pyramidal cells in the hippocampus and cerebral cortex, parvalbumin-positive interneurons in the hippocampus, and Purkinje cells (PCs) in the cerebellum. Immunohistochemistry revealed an uneven distribution of Slc4a10 within the somatodendritic compartment of cerebellar neurons. In the cerebellar molecular layer, stellate cells and their innervation targets (i.e. PC dendrites in the superficial molecular layer) showed significantly higher labeling than basket cells and their targets (PC dendrites in the basal molecular layer and PC somata). Moreover, the distal dendritic trees of PCs (i.e. parallel fiber-targeted dendrites) had significantly greater labeling than the proximal dendrites (climbing fiber-targeted dendrites). These observations suggest that Slc4a10 expression is regulated in neuron type- and input pathway-dependent manners. Because such an elaborate regulation is also found for K+-Cl- cotransporter KCC2, a major neuronal Cl- extruder, we compared their expression. Slc4a10 and KCC2 overlapped in most somatodendritic elements. However, relative abundance was largely complementary in the cerebellar cortex, with particular enrichments of Slc4a10 in PC dendrites and KCC2 in molecular layer interneurons, granule cells and PC somata. These properties might reflect functional redundancy and distinction of these transporters, and their differential requirements by individual neurons and respective input domains.
  • Yuka Miyoshi, Yoshichika Yoshioka, Kinuko Suzuki, Taisuke Miyazaki, Minako Koura, Kazumasa Saigoh, Naoko Kajimura, Yoko Monobe, Susumu Kusunoki, Junichiro Matsuda, Masahiko Watanabe, Naoto Hayasaka
    PLOS ONE 9 9 e107867 - e107867 2014年09月 [査読有り][通常論文]
     
    Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.
  • Miwako Yamasaki, Rieko Okada, Chihiro Takasaki, Shima Toki, Masahiro Fukaya, Rie Natsume, Kenji Sakimura, Masayoshi Mishina, Tetsuo Shirakawa, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 34 35 11534 - 11548 2014年08月 [査読有り][通常論文]
     
    Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluR epsilon 2 or NR2B) and GluN2D (GluR epsilon 4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B(+/-) mice but advanced by nearly a day in GluN2D(-/-) mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B(+/-) and GluN2D(-/-) mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.
  • Shinya Kawata, Taisuke Miyazaki, Maya Yamazaki, Takayasu Mikuni, Miwako Yamasaki, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    CELL REPORTS 8 4 1119 - 1129 2014年08月 [査読有り][通常論文]
     
    Synapse elimination is crucial for precise neural circuit formation during postnatal development. We examined how relative differences in synaptic strengths among competing inputs and/or absolute synaptic strengths contribute to climbing fiber (CF) to Purkinje cell (PC) synapse elimination in the cerebellum. We generated mice with PC-selective deletion of stargazin (TARP gamma-2), the major AMPA receptor auxiliary subunit in PCs (gamma-2 PC-KO mice). Whereas relative differences between "strong'' and "weak'' CF-mediated postsynaptic response are preserved, absolute strengths of CF inputs are scaled down globally in PCs of gamma-2 PC-KO mice. Although the early phase of CF elimination is normal, dendritic translocation of the strongest CF and the late phase of CF elimination that requires Ca2+-dependent activation of Arc/Arg3.1 in PCs are both impaired in gamma-2 PC-KO mice. We conclude that, although relative differences in CF synaptic inputs are initially essential, proper synaptic scaling is crucial for accomplishing CF synapse elimination.
  • Keishi Yamashita, Natsuya Katada, Hiromitsu Moriya, Kei Hosoda, Shinichi Sakuramoto, Shiroh Kikuchi, Masahiko Watanabe
    HEPATO-GASTROENTEROLOGY 61 132 1042 - 1048 2014年06月 [査読有り][通常論文]
     
    Background/Aims: Multimodality treatment improves esophageal squamous cell carcinoma (ESCC) prognosis, however the most optimal treatment still remains controversial. Methodology: In 213 ESCC patients who had operation at Kitasato University Hospital between 1996 and 2007, prognosis was evaluated in terms of cStage and adjuvant therapy. Results: Survival outcome of with the 62 cStage I ESCC were excellent (2 recurrences) with infrequent postoperative treatment (Adj). Of 129 ESCC patients with cStage II/III, 45 patients including suspicious cT4 (cT3.5) were given neoadjuvant therapy (Neo), and pStage 0/I patients showed excellent prognosis. Of 84 cStage II/III patients not given Neo, 67 (79.8%) underwent R0 esophagectomy, among whom 20 cases had pN0 (pStage I/IIa) with 3 recurrences (15%) despite infrequent Adj. On the other hand, among the 45 cStage II/III patients with Neo, 8 pStage ha patients exhibited poor prognosis with 5 recurrences (62.5%) as well as pStage III/IV. In pStage Ilb ESCC, cStage ha showed poorer prognosis than cStage I/lIb (P = 0.0072). Among the 21 cStage IV ESCC patients, 5 long term survivors were experienced by multimodality treatments. Conclusions: Both cStage and Neo greatly affect ESCC prognosis irrespective of identical pStage, which must be allowed for in order to determine therapeutic strategy against ESCC.
  • Adam P. Kardon, Erika Polgar, Junichi Hachisuka, Lindsey M. Snyder, Darren Cameron, Sinead Savage, Xiaoyun Cai, Sergei Karnup, Christopher R. Fan, Gregory M. Hemenway, Carcha S. Bernard, Erica S. Schwartz, Hiroshi Nagase, Christoph Schwarzer, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, H. Richard Koerber, Andrew J. Todd, Sarah E. Ross
    NEURON 82 3 573 - 586 2014年05月 [査読有り][通常論文]
     
    Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5(-/-) mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch.
  • Kohtarou Konno, Keiko Matsuda, Chihiro Nakamoto, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Kenji Sakimura, Michisuke Yuzaki, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 34 22 7412 - 7424 2014年05月 [査読有り][通常論文]
     
    Of the two members of the delta subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.
  • Naofumi Uesaka, Motokazu Uchigashima, Takayasu Mikuni, Takanobu Nakazawa, Harumi Nakao, Hirokazu Hirai, Atsu Aiba, Masahiko Watanabe, Masanobu Kano
    SCIENCE 344 6187 1020 - 1023 2014年05月 [査読有り][通常論文]
     
    Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination.
  • Ming-Dong Zhang, Giuseppe Tortoriello, Brian Hsueh, Raju Tomer, Li Ye, Nicholas Mitsios, Lotta Borgius, Gunnar Grant, Ole Kiehn, Masahiko Watanabe, Mathias Uhlen, Jan Mulder, Karl Deisseroth, Tibor Harkany, Tomas G. M. Hokfelt
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 111 12 E1149 - E1158 2014年03月 [査読有り][通常論文]
     
    Neuronal calcium (Ca2+)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca2+-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small-and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca2+-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca2+-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.
  • Keishi Yamashita, Shinichi Sakuramoto, Natsuya Katada, Shiro Kikuchi, Masahiko Watanabe
    HEPATO-GASTROENTEROLOGY 61 130 512 - 517 2014年03月 [査読有り][通常論文]
     
    Background/Aim: Macroscopic features and age may be important prognostic factors that discriminate survival among clinical conditions requiring different therapeutic strategies of advanced gastric cancer (AGC), and this study aimed to identify their clinical relevance. Methodology: A total of 232 AGC patients who had Surgical T2b or beyond was enrolled to identify clinical indicators, including macroscopic features in combination with age. Results: Macroscopic features were divided into 3 categories (types I/II/V, III, and IV), which included stage IV in 24%, 53%, and 72% (P < 0.0001), respectively. Macroscopic features (P < 0.0001), histological features (P = 0.025), and pathological infiltration type (P = 0.0003) were all univariate prognostic factors, as well as stage (P < 0.0001) and age (P = 0.009). However, the multivariate proportional hazards model found that macroscopic features (P = 0.0013) and age (P = 0.0091) were the only factors independent of stage (P < 0.0001). Both factors clearly classified the patients into 4 groups (young type I/II/V (group 1), elderly type I/II/V (group 2), type III and young type IV (group 3), and elderly type IV (group 4) with different prognoses. Conclusions: Macroscopic features and age were simple indicators of prognosis in AGC. Both factors may have great potential to develop prognostic categories that effectively classify AGC into categories requiring different therapeutic strategies.
  • Watanabe T, Shimazaki T, Mishiro A, Suzuki T, Hirata H, Tanimoto M, Oda Y
    Journal of neurophysiology 111 6 1153 - 1164 2014年03月 [査読有り][通常論文]
     
    Each neuron possesses a unique firing property, which is largely attributed to heterogeneity in the composition of voltage-gated ion channel complexes. Zebrafish Mauthner (M) cells, which are bilaterally paired giant reticulospinal neurons (RSNs) in the hindbrain and induce rapid escape behavior, generate only a single spike at the onset of depolarization. This single spiking is in contrast with the repetitive firing of the M cell's morphologically homologous RSNs, MiD2cm and MiD3cm, which are also involved in escapes. However, how the unique firing property of M cells is established and the underlying molecular mechanisms remain unclear. In the present study, we first demonstrated that the single-spiking property of M cells was acquired at 4 days postfertilization (dpf), accompanied by an increase in dendrotoxin I (DTX)-sensitive low-threshold K+ currents, prior to which the M cell repetitively fires as its homologs. Second, in situ hybridization showed that among DTX-sensitive K(v)1 channel alpha-subunits, zKv1.1a was unexpectedly expressed even in the homologs and the bursting M cells at 2 dpf. In contrast, zKv beta 2b, an auxiliary beta-subunit of K(v)1 channels, was expressed only in the single-spiking M cells. Third, zKv1.1a expressed in Xenopus oocytes functioned as a low-threshold K+ channel, and its currents were enhanced by coexpression of zKv beta 2b subunits. Finally, knockdown of zKv beta 2b expression in zebrafish larvae resulted in repetitive firing of M cells at 4 dpf. Taken together, these results suggest that associative expression of K-v beta 2 subunits with K(v)1.1 channels is crucial for developmental acquisition of the unique firing properties of the M cells among homologous neurons.
  • Carmen Ballesteros-Merino, Masahiko Watanabe, Ryuichi Shigemoto, Yugo Fukazawa, John P. Adelman, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 39 6 883 - 892 2014年03月 [査読有り][通常論文]
     
    Small-conductance, Ca2+-activated K+ (SK) channels are expressed in the hippocampus where they regulate synaptic responses, plasticity, and learning and memory. To investigate the expression of SK3 (KCNN3) subunits, we determined the developmental profile and subcellular distribution of SK3 in the developing mouse hippocampus using western blots, immunohistochemistry and high-resolution immunoelectron microscopy. The results showed that SK3 expression increased during postnatal development, and that the localization of SK3 changed from being mainly associated with the endoplasmic reticulum and intracellular sites during the first postnatal week to being progressively concentrated in dendritic spines during later stages. In the adult, SK3 was localized mainly in postsynaptic compartments, both at extrasynaptic sites and along the postsynaptic density of excitatory synapses. Double labelling showed that SK3 co-localized with SK2 (KCNN2) and with N-methyl-D-aspartate receptors. Finally, quantitative analysis of SK3 density revealed two subcellular distribution patterns in different hippocampal layers, with SK3 being unevenly distributed in CA1 region of the hippocampus pyramidal cells and homogeneously distributed in dentate gyrus granule cells. Our results revealed a complex cell surface distribution of SK3-containing channels and a distinct developmental program that may influence different hippocampal functions.
  • Eszter Horvath, Stephen G. Woodhams, Rita Nyilas, Christopher M. Henstridge, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe, Istvan Katona
    EUROPEAN JOURNAL OF NEUROSCIENCE 39 3 419 - 434 2014年02月 [査読有り][通常論文]
     
    Monoacylglycerol lipase (MGL) is a multifunctional serine hydrolase, which terminates anti-nociceptive endocannabinoid signaling and promotes pro-nociceptive prostaglandin signaling. Accordingly, both acute nociception and its sensitization in chronic pain models are prevented by systemic or focal spinal inhibition of MGL activity. Despite its analgesic potential, the neurobiological substrates of beneficial MGL blockade have remained unexplored. Therefore, we examined the regional, cellular and subcellular distribution of MGL in spinal circuits involved in nociceptive processing. All immunohistochemical findings obtained with light, confocal or electron microscopy were validated in MGL-knockout mice. Immunoperoxidase staining revealed a highly concentrated accumulation of MGL in the dorsal horn, especially in superficial layers. Further electron microscopic analysis uncovered that the majority of MGL-immunolabeling is found in axon terminals forming either asymmetric glutamatergic or symmetric -aminobutyric acid/glycinergic synapses in laminae I/IIo. In line with this presynaptic localization, analysis of double-immunofluorescence staining by confocal microscopy showed that MGL colocalizes with neurochemical markers of peptidergic and non-peptidergic nociceptive terminals, and also with markers of local excitatory or inhibitory interneurons. Interestingly, the ratio of MGL-immunolabeling was highest in calcitonin gene-related peptide-positive peptidergic primary afferents, and the staining intensity of nociceptive terminals was significantly reduced in MGL-knockout mice. These observations highlight the spinal nociceptor synapse as a potential anatomical site for the analgesic effects of MGL blockade. Moreover, the presence of MGL in additional terminal types raises the possibility that MGL may play distinct regulatory roles in synaptic endocannabinoid or prostaglandin signaling according to its different cellular locations in the dorsal horn pain circuitry.
  • Yoshiaki Ohtani, Mariko Miyata, Kouichi Hashimoto, Toshihide Tabata, Yasushi Kishimoto, Masahiro Fukaya, Daisuke Kase, Hidetoshi Kassai, Kazuki Nakao, Tatsumi Hirata, Masahiko Watanabe, Masanobu Kano, Atsu Aiba
    JOURNAL OF NEUROSCIENCE 34 7 2702 - 2712 2014年02月 [査読有り][通常論文]
     
    The metabotropic glutamate receptor subtype 1(mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical(TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca2+ release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.
  • Xiaojing Dai, Hirohide Iwasaki, Masahiko Watanabe, Shigeo Okabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 39 4 531 - 547 2014年02月 [査読有り][通常論文]
  • Kimio Watanabe, Kana Okada, Ryoji Fukabori, Yoshimitsu Hayashi, Koichi Asahi, Hiroyuki Terawaki, Kazuto Kobayashi, Tsuyoshi Watanabe, Masaaki Nakayama
    TOXINS 6 1 254 - 269 2014年01月 [査読有り][通常論文]
     
    Methylglyoxal (MG), one of the uremic toxins, is a highly reactive alpha-dicarbonyl compound. Recent clinical studies have demonstrated the close associations of cognitive impairment (CI) with plasma MG levels and presence of kidney dysfunction. Therefore, the present study aims to examine whether MG is a direct causative substance for CI development. Eight-week-old male Sprague-Dawley (SD) rats were divided into two groups: control (n = 9) and MG group (n = 10; 0.5% MG in drinking water), and fed a normal diet for 12 months. Cognitive function was evaluated by two behavioral tests (object exploration test and radial-arm maze test) in early (4-6 months of age) and late phase (7-12 months of age). Serum MG was significantly elevated in the MG group (495.8 +/- 38.1 vs. 244.8 +/- 28.2 nM; p < 0.001) at the end of study. The groups did not differ in cognitive function during the course of study. No time-course differences were found in oxidative stress markers between the two groups, while, antioxidants such as glutathione peroxidase and superoxide dismutase activities were significantly increased in the MG group compared to the control. Long-term MG administration to rats with normal kidney function did not cause CI. A counter-balanced activation of the systemic anti-oxidant system may offset the toxicity of MG in this model. Pathogenetic significance of MG for CI requires further investigation.
  • Toshiharu Yasaka, Sheena Y. X. Tiong, Erika Polgar, Masahiko Watanabe, Eiichi Kumamoto, John S. Riddell, Andrew J. Todd
    MOLECULAR PAIN 10 2014年01月 [査読有り][通常論文]
     
    Background: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. Results: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. Conclusions: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia.
  • Erika Polgar, Thomas C. P. Sardella, Sheena Y. X. Tiong, Samantha Locke, Masahiko Watanabe, Andrew J. Todd
    PAIN 154 12 2606 - 2615 2013年12月 [査読有り][通常論文]
     
    In order to understand how nociceptive information is processed in the spinal dorsal horn we need to unravel the complex synaptic circuits involving interneurons, which constitute the vast majority of the neurons in laminae I-III. The main limitation has been the difficulty in defining functional populations among these cells. We have recently identified 4 non-overlapping classes of inhibitory interneuron, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) and parvalbumin, in the rat spinal cord. In this study we demonstrate that these form distinct functional populations that differ in terms of sst(2A) receptor expression and in their responses to painful stimulation. The sst(2A) receptor was expressed by nearly all of the nNOS-and galanin-containing inhibitory interneurons but by few of those with NPY and none of the parvalbumin cells. Many galanin-and NPY-containing cells exhibited phosphorylated extracellular signal-regulated kinases (pERK) after mechanical, thermal or chemical noxious stimuli, but very few nNOS-containing cells expressed pERK after any of these stimuli. However, many nNOS-positive inhibitory interneurons up-regulated Fos after noxious thermal stimulation or injection of formalin, but not after capsaicin injection. Parvalbumin cells did not express either activity-dependent marker following any of these stimuli. These results suggest that interneurons belonging to the NPY, nNOS and galanin populations are involved in attenuating pain, and for NPY and nNOS cells this is likely to result from direct inhibition of nociceptive projection neurons. They also suggest that the nociceptive inputs to the nNOS cells differ from those to the galanin and NPY populations. (C) 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
  • Ana Fajardo-Serrano, Nicole Wydeven, Daniele Young, Masahiko Watanabe, Ryuichi Shigemoto, Kirill A. Martemyanov, Kevin Wickman, Rafael Lujan
    HIPPOCAMPUS 23 12 1231 - 1245 2013年12月 [査読有り][通常論文]
     
    In the hippocampus, signaling through G protein-coupled receptors is modulated by Regulators of G protein signaling (Rgs) proteins, which act to stimulate the rate of GTP hydrolysis, and consequently, G protein inactivation. The R7-Rgs subfamily selectively deactivates the G(i/o)-class of G subunits that mediate the action of several GPCRs. Here, we used co-immunoprecipitation, electrophysiology and immunoelectron microscopy techniques to investigate the formation of macromolecular complexes and spatial relationship of Rgs7/G5 complexes and its prototypical signaling partners, the GABA(B) receptor and Girk channel. Co-expression of recombinant GABA(B) receptors and Girk channels in combination with co-immunoprecipitation experiments established that the Rgs7/G5 forms complexes with GABA(B) receptors or Girk channels. Using electrophysiological experiments, we found that GABA(B)-Girk current deactivation kinetics was markedly faster in cells coexpressing Rgs7/G5. At the electron microscopic level, immunolabeling for Rgs7 and G5 proteins was found primarily in the dendritic layers of the hippocampus and showed similar distribution patterns. Immunoreactivity was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of pyramidal cells and, to a lesser extent, to that of presynaptic terminals. Quantitative analysis of immunogold particles for Rgs7 and G5 revealed an enrichment of the two proteins around excitatory synapses on dendritic spines, virtually identical to that of Girk2 and GABA(B1). These data support the existence of macromolecular complexes composed of GABA(B) receptor-G protein-Rgs7-Girk channels in which Rgs7 and G5 proteins may preferentialy modulate GABA(B) receptor signaling through the deactivation of Girk channels on dendritic spines. In contrast, Rgs7 and Girk2 were associated but mainly segregated from GABA(B1) in dendritic shafts, where Rgs7/G5 signaling complexes might modulate Girk-dependent signaling via a different metabotropic receptor(s). (c) 2013 Wiley Periodicals, Inc.
  • Yumi Watanabe, Norikazu Katayama, Kosei Takeuchi, Tetsuya Togano, Rieko Itoh, Michiko Sato, Maya Yamazaki, Manabu Abe, Toshiya Sato, Kanako Oda, Minesuke Yokoyama, Keizo Takao, Masahiro Fukaya, Tsuyoshi Miyakawa, Masahiko Watanabe, Kenji Sakimura, Toshiya Manabe, Michihiro Igarashi
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 48 34906 - 34919 2013年11月 [査読有り][通常論文]
     
    Background: Roles of the syntaxin-1ACaMKII interaction are not physiologically understood in vivo.Results: A point mutation in syntaxin-1A caused abnormal plasticity, recycling, and behaviors in mice. Conclusion: The CaMKII/syntaxin-1A interaction is essential for maintenance of neuronal plasticity. Significance: Syntaxin-1A is involved in regulatory pathways in higher brain functions. Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.
  • L. Benjamin Hills, Amira Masri, Kotaro Konno, Wataru Kakegawa, Anh-Thu N. Lam, Elizabeth Lim-Melia, Nandini Chandy, R. Sean Hill, Jennifer N. Partlow, Muna Al-Saffar, Ramzi Nasir, Joan M. Stoler, A. James Barkovich, Masahiko Watanabe, Michisuke Yuzaki, Ganeshwaran H. Mochida
    NEUROLOGY 81 16 1378 - 1386 2013年10月 [査読無し][通常論文]
     
    Objective: To identify the genetic cause of a syndrome causing cerebellar ataxia and eye movement abnormalities. Methods: We identified 2 families with cerebellar ataxia, eye movement abnormalities, and global developmental delay. We performed genetic analyses including single nucleotide polymorphism genotyping, linkage analysis, array comparative genomic hybridization, quantitative PCR, and Sanger sequencing. We obtained eye movement recordings of mutant mice deficient for the ortholog of the identified candidate gene, and performed immunohistochemistry using human and mouse brain specimens. Results: All affected individuals had ataxia, eye movement abnormalities, most notably tonic upgaze, and delayed speech and cognitive development. Homozygosity mapping identified the disease locus on chromosome 4q. Within this region, a homozygous deletion of GRID2 exon 4 in the index family and compound heterozygous deletions involving GRID2 exon 2 in the second family were identified. Grid2-deficient mice showed larger spontaneous and random eye movements compared to wild-type mice. In developing mouse and human cerebella, GRID2 localized to the Purkinje cell dendritic spines. Brain MRI in 2 affected children showed progressive cerebellar atrophy, which was more severe than that of Grid2-deficient mice. Conclusions: Biallelic deletions of GRID2 lead to a syndrome of cerebellar ataxia and tonic upgaze in humans. The phenotypic resemblance and similarity in protein expression pattern between humans and mice suggest a conserved role for GRID2 in the synapse organization between parallel fibers and Purkinje cells. However, the progressive and severe cerebellar atrophy seen in the affected individuals could indicate an evolutionarily unique role for GRID2 in the human cerebellum.
  • Sonia Marco, Albert Giralt, Milos M. Petrovic, Mahmoud A. Pouladi, Rebeca Martínez-Turrillas, José Martínez-Hernández, Linda S. Kaltenbach, Jesús Torres-Peraza, Rona K. Graham, Masahiko Watanabe, Rafael Luján, Nobuki Nakanishi, Stuart A. Lipton, Donald C. Lo, Michael R. Hayden, Jordi Alberch, John F. Wesseling, Isabel Pérez-Otaño
    Nature Medicine 19 8 1030 - 1038 2013年08月 [査読有り][通常論文]
     
    Huntington's disease is caused by an expanded polyglutamine repeat in the huntingtin protein (HTT), but the pathophysiological sequence of events that trigger synaptic failure and neuronal loss are not fully understood. Alterations in N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) have been implicated. Yet, it remains unclear how the HTT mutation affects NMDAR function, and direct evidence for a causative role is missing. Here we show that mutant HTT redirects an intracellular store of juvenile NMDARs containing GluN3A subunits to the surface of striatal neurons by sequestering and disrupting the subcellular localization of the endocytic adaptor PACSIN1, which is specific for GluN3A. Overexpressing GluN3A in wild-type mouse striatum mimicked the synapse loss observed in Huntington's disease mouse models, whereas genetic deletion of GluN3A prevented synapse degeneration, ameliorated motor and cognitive decline and reduced striatal atrophy and neuronal loss in the YAC128 Huntington's disease mouse model. Furthermore, GluN3A deletion corrected the abnormally enhanced NMDAR currents, which have been linked to cell death in Huntington's disease and other neurodegenerative conditions. Our findings reveal an early pathogenic role of GluN3A dysregulation in Huntington's disease and suggest that therapies targeting GluN3A or pathogenic HTT-PACSIN1 interactions might prevent or delay disease progression. © 2013 Nature America, Inc. All rights reserved.
  • Hashizume M, Miyazaki T, Sakimura K, Watanabe M, Kitamura K, Kano M
    Frontiers in neural circuits 7 130  2013年08月 [査読有り][通常論文]
     
    Cerebellar cortex has an elaborate rostrocaudal organization comprised of numerous microzones. Purkinje cells (PCs) in the same microzones how synchronous activity of complex spikes (CSs) evoked by excitatory inputs from climbing fibers (CFs) that arise from neurons in the inferior olive (IO). The synchronous CS activity is considered to depend on electrical coupling among IO neurons and anatomical organization of the olivo-cerebellar projection. To determine how the CF-PC wiring contributes to the formation of microzone, we examined the synchronous CS activities between neighboring PCs in the glutamate receptor delta 2 knockout (GluD2 KO) mouse in which exuberant surplus CFs make ectopic innervations onto distal dendrites of PCs. We performed in vivo two-photon calcium imaging for PC populations to detect CF inputs. Neighboring PCs in GluD2 KO mice showed higher synchrony of calcium transients than those in wild-type(control) mice. Moreover, the synchrony in GluD2 KO mice hardly declined with mediolateral separation between PCs up to similar to 200 mu m, which was in marked contrast to the fall off of the synchrony in control mice. The enhanced synchrony was only partially affected by the blockade of gap junctional coupling. On the other hand, transverse CF collaterals in GluD2 KO mice extended beyond the border of microzone and formed locally clustered ectopic synapses onto dendrites of neighboring PCs. Furthermore, PCs in GluD2 KO mice exhibited clustered firing (Cf), the characteristic CF response that was not found in PCs of wild type mice. Importantly, Cf was often associated with localized calcium transients in distal dendrites of PCs, which are likely to contribute to the enhanced synchrony of calcium signals in GluD2 KO mice. Thus, our results indicate that CF signals in GluD2 KO mice propagate across multiple microzones, and that proper formation of longitudinal olivo-cerebellar projection is essential for the spatio temporal organization of CS activity in the cerebellum.
  • Masanobu Kano, Hisako Nakayama, Kouichi Hashimoto, Kazuo Kitamura, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF PHYSIOLOGY-LONDON 591 13 3151 - 3158 2013年07月 [査読有り][通常論文]
     
    Functional neural circuit formation during postnatal development involves massive elimination of early-formed redundant synapses and strengthening of necessary synaptic connections. In the cerebellum, one-to-one connection from a climbing fibre (CF) to a Purkinje cell (PC) is established through four distinct phases: (1) strengthening of a single CF among multiple CFs in each PC at postnatal age P3-P7 days, (2) translocation of a single strengthened CF to PC dendrites from around P9, (3) early-phase (P7 to around P11) and (4) late-phase (around P12-P17) elimination of weak CF synapses from PC somata. Mice with PC-selective deletion of the P/Q-type voltage-dependent Ca2+ channel (VDCC) exhibit severe defects in strengthening of single CFs, dendritic translocation of single CFs and CF elimination from P7. In contrast, mice with a mutation of a single allele for the GABA synthesizing enzyme GAD67 show selective impairment of CF elimination from P10. Electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto PC somata from putative basket cells influences CF-induced Ca2+ transients and regulates elimination of redundant CF synapses from PC somata at P10-P16. Thus, regulation of Ca2+ influx to PCs through VDCCs is crucial for the four phases of CF synapse elimination during postnatal development.
  • Natsuko Tsujino, Tomomi Tsunematsu, Motokazu Uchigashima, Kohtarou Konno, Akihiro Yamanaka, Kazuto Kobayashi, Masahiko Watanabe, Yoshimasa Koyama, Takeshi Sakurai
    PLOS ONE 8 7 2013年07月 [査読有り][通常論文]
     
    Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.
  • Keimpema E, Alpár A, Howell F, Malenczyk K, Hobbs C, Hurd YL, Watanabe M, Sakimura K, Kano M, Doherty P, Harkany T
    Scientific reports 3 2093  2013年06月28日 [査読有り][通常論文]
     
    Endocannabinoids are small signaling lipids, with 2-Arachidonoylglycerol (2-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic 2-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing 2-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB1 cannabinoid receptors (CB1Rs) remains poorly understood. Here, we first show that cholinergic innervation of CA1 pyramidal cells of the hippocampus is sensitive to the genetic disruption of 2-AG signaling in DAGLa null mice. Next, we exploit a hybrid COS-7-cholinergic neuron co-culture system to demonstrate that heterologous DAGLa overexpression spherically excludes cholinergic growth cones from 2-AG-rich extracellular environments, and minimizes cell-cell contact in vitro. CB1R-mediated exclusion responses lasted 3 days, indicating sustained spherical 2-AG availability. Overall, these data suggest that extracellular 2-AG concentrations can be sufficient to activate CB1Rs along discrete spherical boundaries to modulate neuronal responsiveness.
  • Yasukazu Hozumi, Hirooki Matsui, Fumio Sakane, Masahiko Watanabe, Kaoru Goto
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 61 6 462 - 476 2013年06月 [査読有り][通常論文]
     
    Recent studies have revealed that phosphoinositide (PI) signaling molecules are expressed in mammalian retinas, suggesting their importance in its signal transduction. We previously showed that diacylglycerol kinase (DGK) isozymes are expressed in distinct patterns in rat retina at the mRNA level. However, little is known about the nature and morphological aspects of DGKs in the retina. For this study, we performed immunohistochemical analyses to investigate in the retina the expression and localization of DGK isozymes at the protein level. Here, we show that both DGK beta and DGK iota localize in the outer plexiform layer, within which photoreceptor cells make contact with bipolar and horizontal cells. These isozymes exhibit distinct subcellular localization patterns: DGK iota localizes to the synaptic area of bipolar cells in a punctate manner, whereas DGK beta distributes diffusely in the subsynaptic and dendritic regions of bipolar and horizontal cells. However, punctate labeling for DGK epsilon is evident in the outer limiting membrane. DGK zeta and DGK alpha localize predominantly to the nucleus of ganglion cells. These findings show distinct expression and localization of DGK isozymes in the retina, suggesting a different role of each isozyme.
  • Naosuke Hoshina, Asami Tanimura, Miwako Yamasaki, Takeshi Inoue, Ryoji Fukabori, Teiko Kuroda, Kazumasa Yokoyama, Tohru Tezuka, Hiroshi Sagara, Shinji Hirano, Hiroshi Kiyonari, Masahiko Takada, Kazuto Kobayashi, Masahiko Watanabe, Masanobu Kano, Takanobu Nakazawa, Tadashi Yamamoto
    NEURON 78 5 839 - 854 2013年06月 [査読有り][通常論文]
     
    Highly topographic organization of neural circuits exists for the regulation of various brain functions in corticobasal ganglia circuits. Although neural circuit-specific refinement during synapse development is essential for the execution of particular neural functions, the molecular and cellular mechanisms for synapse refinement are largely unknown. Here, we show that protocadherin 17 (PCDH17), one of the nonclustered delta 2-protocadherin family members, is enriched along corticobasal ganglia synapses in a zone-specific manner during synaptogenesis and regulates presynaptic assembly in these synapses. PCDH17 deficiency in mice causes facilitated presynaptic vesicle accumulation and enhanced synaptic transmission efficacy in corticobasal ganglia circuits. Furthermore, PCDH17(-/-) mice exhibit antidepressant-like phenotypes that are known to be regulated by corticobasal ganglia circuits. Our findings demonstrate a critical role for PCDH17 in the synaptic development of specific corticobasal ganglia circuits and suggest the involvement of PCDH17 in such circuits in depressive behaviors.
  • Pablo R. Brumovsky, Rebecca P. Seal, Kerstin H. Lundgren, Kim B. Seroogy, Masahiko Watanabe, G. F. Gebhart
    JOURNAL OF UROLOGY 189 6 2342 - 2349 2013年06月 [査読有り][通常論文]
     
    Purpose: VGLUTs, which are essential for loading glutamate into synaptic vesicles, are present in various neuronal systems. However, to our knowledge the expression of VGLUTs in neurons innervating the bladder has not yet been analyzed. We studied VGLUT(1), VGLUT(2) and VGLUT(3) in mouse bladder neurons. Materials and Methods: We analyzed the expression of VGLUT(1), VGLUT(2) and calcitonin gene-related peptide by immunohistochemistry in the retrograde labeled primary afferent and autonomic neurons of BALB/c mice after injecting fast blue in the bladder wall. To study VGLUT(3) we traced the bladder of transgenic mice, in which VGLUT(3) is identified by enhanced green fluorescent protein detection. Results: Most bladder dorsal root ganglion neurons expressed VGLUT(2). A smaller percentage of neurons also expressed VGLUT(1) or VGLUT(3). Co-expression with calcitonin gene-related peptide was only observed for VGLUT(2). Occasional VGLUT(2) immunoreactive neurons were seen in the major pelvic ganglia. Abundant VGLUT(2) immunoreactive nerves were detected in the bladder dome and trigone, and the urethra. VGLUT(1) immunoreactive nerves were discretely present. Conclusions: We present what are to our knowledge novel data on VGLUT expression in sensory and autonomic neurons innervating the mouse bladder. The frequent association of VGLUT(2) and calcitonin gene-related peptide in sensory neurons suggests interactions between glutamatergic and peptidergic neurotransmissions, potentially influencing commonly perceived sensations in the bladder, such as discomfort and pain.
  • Yoshiaki Shinohara, Hajime Hirase, Masahiko Watanabe, Makoto Itakura, Masami Takahashi, Ryuichi Shigemoto
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 24 10045 - 10046 2013年06月 [査読有り][通常論文]
  • Takayuki Yoshida, Masanobu Kano, Masahiko Watanabe, Mitsuhiro Yoshiok
    Japanese Journal of Neuropsychopharmacology 33 3 95 - 99 2013年06月 [査読有り][通常論文]
     
    In the amygdala, it has been demonstrated that cannabinoid CB1 receptors are particularly enriched in GABAergic axon terminals and moderately expressed on glutamatergic fibers, and involved in the fear coping strategies. In this study, we found predominant neuronal projections of serotonergic, dopaminergic and cholinergic fibers in the basolateral amygdala (BA), and CB1 receptors were frequently localized on serotonergic axons but not dopaminergic, noradrenergic or cholinergic fibers. Furthermore, extracellular release of serotonin was significantly reduced by WIN55212-2, a CB agonist, whereas dopamine concentration was not altered, indicating presynaptic serotonin release is modulated by CB1 receptors. On the other hand, α-methyl-5-HT, 5-HT2 receptor agonist decreased the evoked IPSC and EPSC amplitude accompanied with enhancement of paired pulse ratio and induced inward currents from the patch-clamp recording BA pyramidal neurons these are parameters of presynaptic effect and postsynaptic localization of 5-HT2 receptors, respectively. This suppression of IPSC amplitude was completely blocked by MDL100907, a 5-HT2A antagonist, or AM251, a CB1 antagonist. These findings suggest that endocannabinoid is synthesized via activation of postsynaptic 5-HT2A receptors, and regulates not only inhibitory presynaptic GABA release but also local serotonergic transmission in the BA.
  • Dan Yan, Miwako Yamasaki, Christoph Straub, Masahiko Watanabe, Susumu Tomita
    NEURON 78 4 687 - 699 2013年05月 [査読有り][通常論文]
     
    Glutamate is the most abundant excitatory neurotransmitter in the brain, and distinct classes of glutamate receptors coordinate synaptic transmission and spike generation upon various levels of neuronal activity. However, the mechanisms remain unclear. Here, we found that loss of synaptic AMPA receptors increased kainate receptor activity in cerebellar granule cells without changing NMDA receptors. The augmentation of kainate receptor-mediated currents in the absence of AMPA receptor activity is required for spike generation and is mediated by the increased expression of the GluK5 high-affinity kainate receptor subunit. Increase in GluK5 expression is sufficient to enhance kainate receptor activity by modulating receptor channel properties, but not localization. Furthermore, we demonstrate that the combined loss of the AMPA receptor auxiliary TARP gamma-2 subunit and the GluK5 subunit leads to early mouse lethality. Our findings reveal mechanisms mediated by distinct classes of postsynaptic glutamate receptors for the homeostatic maintenance of the neuronal activity.
  • Yuto Sugawara, Ryousuke Echigo, Kousuke Kashima, Hanae Minami, Megumi Watanabe, Yuiko Nishikawa, Miho Muranishi, Mitsugu Yoneda, Takako Ohno-Shosaku
    BRAIN RESEARCH 1512 9 - 21 2013年05月 [査読無し][通常論文]
     
    Signaling pathways involving phospholipase C (PLC) are involved in various neural functions. Understanding how these pathways are regulated will lead to a better understanding of their roles in neural functions. Previous studies demonstrated that receptor-driven PLC beta activation depends on intracellular Ca2+ concentration ([Ca2+](i)), suggesting the possibility that PLC beta-dependent cellular responses are basically Ca2+ dependent. To test this possibility, we examined whether modulations of ion channels driven by PLC-coupled metabotropic receptors are sensitive to [Ca2+](i) using cultured hippocampal neurons. Muscarinic activation triggered an inward current at -100 mV (the equilibrium potential for K+) in a subpopulation of neurons. This current response was suppressed by pirenzepine (an M-1-preferring antagonist), PLC inhibitor, non-selective cation channel blocker, and lowering [Ca2+](i). Using the neurons showing no response at -100 mV, effects of muscarinic activation on K+ channels were examined at -40 mV. Muscarinic activation induced a transient decrease of the holding outward current. This current response was mimicked and occluded by XE991, an M-current K+ channel blocker, suppressed by pirenzepine, PLC inhibitor and lowering [Ca2+](i), and enhanced by elevating [Ca2+](i). Similar results were obtained when group I metabotropic. glutamate receptors were activated instead of muscarinic receptors. These results clearly show that ion channel modulations driven by PLC-coupled metabotropic receptors are dependent on [Ca2+](i), supporting the hypothesis that cellular responses induced by receptor-driven PLC beta activation are basically Ca2+ dependent. (C) 2013 Elsevier B.V. All rights reserved.
  • Hiroyuki Sakagami, Osamu Katsumata, Yoshinobu Hara, Hideaki Tamaki, Masahiko Watanabe, Robert J. Harvey, Masahiro Fukaya
    Journal of Comparative Neurology 521 4 860 - 876 2013年03月01日 [査読有り][通常論文]
     
    The BRAG/IQSEC is a family of guanine nucleotide exchange factors for ADP ribosylation factors, small GTPases that regulate membrane trafficking and actin cytoskeleton, and comprises three structurally related members (BRAG1-3) generated from different genes. In the mouse retina, BRAG1 (also known as IQSEC2) was previously shown to localize at synaptic ribbons of photoreceptor terminals and to form a protein complex with RIBEYE. In this study, we examined the immunohistochemical localization of BRAG2 (IQSEC1) and BRAG3 (IQSEC3) in the adult mouse retina at the light and electron microscopic levels. In the outer plexiform layer, BRAG2 showed a punctate distribution in intimate association with dystrophin and β-dystroglycan. Immunoelectron microscopic analysis revealed that BRAG2 localized at specific subcompartments of photoreceptor terminals in both rod spherules and cone pedicles. In the inner plexiform layer, immunolabeling for both BRAG2 and BRAG3 had a punctate appearance, suggestive of synaptic labeling. Double immunostaining demonstrated that BRAG2 colocalized preferentially with PSD-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors (AMPARs). By contrast, BRAG3 colocalized with gephyrin and a subpopulation of inhibitory synapses expressing glycine receptors or γ-aminobutyric acid type A receptors (GABAARs). Immunoelectron microscopic analysis revealed that BRAG2 localized to postsynaptic processes at bipolar dyads, while BRAG3 localized to postsynaptic components at conventional synapses. These findings suggest that BRAG/IQSEC family members have key roles in the function and organization of distinct excitatory and inhibitory synapses in the retina. © 2012 Wiley Periodicals, Inc.
  • K. Mizukoshi, M. Sasaki, Y. Izumi, M. Miura, M. Watanabe, F. Amaya
    NEUROSCIENCE 234 77 - 87 2013年03月 [査読有り][通常論文]
     
    Background The phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglion (DRG) promotes primary afferent sensitization. The role of p38MAPK signaling in the DRG in the pathogenesis of plantar incision hyperalgesia has not been investigated. Results: Levels of phosphorylated p38MAPK (p-p38MAPK) obviously increased in the DRG after plantar incision. Unmyelinated and myelinated DRG neurons that express p-p38MAPK contained small to medium cell bodies, suggesting that p-p38MAPK expression is induced in neurons with C- and A delta-fibers. The p-p38MAPK inhibitors FR167653 or SB203580 inhibited incision-induced mechanical hypersensitivity and spontaneous pain behavior. The systemic administration of tumor necrosis factor-alpha (TNF-alpha) inhibitor prevented subsequent incision-induced activation of p38MAPK in the DRG and alleviated mechanical hypersensitivity after the incision. Conclusions: p38MAPK signaling in the DRG plays a crucial role in the development of primary afferent sensitization and pain behavior caused by plantar incision. (C) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Taichi Nomura, Yoshio Bando, Hiroki Bochimoto, Daisuke Koga, Tsuyoshi Watanabe, Shigetaka Yoshida
    Neuroscience Research 75 3 190 - 197 2013年03月 [査読有り][通常論文]
     
    Axonal injury and demyelination are observed in demyelinating diseases such as multiple sclerosis. However, pathological changes that underlie these morphologies are not fully understood. We examined in vivo morphological changes using a new histological technique, scanning electron microscopy (SEM) with osmium maceration method to observe three-dimensional structures such as myelin and axons in the spinal cord. Myelin basic protein-deficient shiverer mice and mice with experimental autoimmune encephalomyelitis (EAE) were used to visualize how morphological changes in myelin and axons are induced by dysmyelination and demyelination.SEM revealed following morphological changes during dysmyelination of shiverer mice. First, enriched mitochondria and well-developed sER in axons were observed in shiverer, but not in wild-type mice. Second, the processes from some perinodal glial cells ran parallel to internodes of axons in addition to the process that covered the nodal region of the axon in shiverer mice. Last, this technique left myelin and axonal structures undisturbed. Moreover, SEM images showed clear variations in the ultrastructural abnormalities of myelin and axons in the white matter of the EAE spinal cord. This technique will be a powerful tool for identifying the mechanisms underlying the pathogenesis in demyelination. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Tomoko Sayano, Yuriko Kawakami, Wataru Kusada, Takeshi Suzuki, Yuki Kawano, Akihiro Watanabe, Kana Takashima, Yashiho Arimoto, Kayoko Esaki, Akira Wada, Fumiaki Yoshizawa, Masahiko Watanabe, Masahiro Okamoto, Yoshio Hirabayashi, Shigeki Furuya
    FEBS Journal 280 6 1502 - 1517 2013年03月 [査読有り][通常論文]
     
    Targeted disruption in mice of the gene encoding d-3-phosphoglycerate dehydrogenase (Phgdh) results in embryonic lethality associated with a striking reduction in free l-serine and growth retardation including severe brain malformation. We previously observed a severe impairment in neurogenesis of the central nervous system of Phgdh knockout (KO) embryos and a reduction in the protein content of their brains. Although these findings suggest that l-serine deficiency links attenuation of mRNA translation to severe developmental malformation of the central nervous system, the underlying key molecular event remains unexplored. Here we demonstrate that mRNA of Eif4ebp1 encoding eukaryotic initiation factor 4 binding protein 1 and its protein, 4E-BP1, are markedly induced in the central nervous system of Phgdh KO embryos, whereas a modest induction is observed in the liver. The increase in 4E-BP1 was associated with a decrease in the cap initiation complex in the brain, as shown by lower levels of eukaryotic translation initiation factor 4G bound to eukaryotic translation initiation factor 4E (eIF4E) and increased eIF4E interaction with 4E-BP1 based on 7-methyl-GTP chromatography. eIF4E protein and polysomes were also diminished in Phgdh KO embryos. Induction of Eif4ebp1 mRNA and of 4E-BP1 was reproduced in mouse embryonic fibroblasts established from Phgdh KO embryos under the condition of l-serine deprivation. Induction of Eif4ebp1 mRNA was suppressed only when l-serine was supplemented in the culture medium, indicating that reduced l-serine availability regulates the induction of Eif4ebp1/4E-BP1. These data suggest that elevated levels of 4E-BP1 may be involved in a mechanism to arrest brain development in Phgdh KO embryos. © 2013 FEBS.
  • Mika Kato Kaneko, Shunpei Morita, Yuta Tsujimoto, Ryo Yanagiya, Kana Nasu, Hiroko Sasaki, Yasukazu Hozumi, Kaoru Goto, Atsushi Natsume, Mika Watanabe, Toshihiro Kumabe, Shingo Takano, Yukinari Kato
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 432 1 40 - 45 2013年03月 [査読有り][通常論文]
     
    Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in gliomas, cartilaginous tumors, and leukemias. IDH1/2 mutations are early and frequent genetic alterations, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1 and Arginine 172 (R172) in IDH2. We previously established several monoclonal antibodies (mAbs), which are specific for IDH1 mutations: clones IMab-1 or HMab-1 against IDH1-R132H or clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH2 mutations have not been reported. To establish IDH2-mutation-specific mAbs, we immunized mice or rats with each mutation-containing IDH2 peptides including IDH2-R172K and IDH2-R172M. After cell fusion, IDH2 mutation-specific mAbs were screened in Enzyme-Linked Immunosorbent Assay (ELISA). Established mAbs KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M peptides, respectively, but not with IDH2-wild type (WT) in ELISA. Western-blot analysis also showed that KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M recombinant proteins, respectively, not with IDH2-WT or other IDH2 mutants, indicating that KMab-1 and MMab-1 are IDH2-mutation-specific. Furthermore, MMab-1 specifically stained the IDH2-R172M-expressing cells in immunocytochemistry, but did not stain IDH2-WT and other IDH2-mutation-containing cells. In immunohistochemical analysis, MMab-1 specifically stained IDH2-R172M-expressing glioma. This is the first report to establish anti-IDH2-mutation-specific mAbs, which could be useful in diagnosis of mutation-bearing tumors. (C) 2013 Elsevier Inc. All rights reserved.
  • Issei Kawakita, Motokazu Uchigashima, Kohtarou Konno, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 37 4 532 - 543 2013年02月 [査読有り][通常論文]
     
    Postnatal expression of the type 2K+Cl cotransporter (KCC2) in neurons lowers the Cl equilibrium potential to values that are more negative than the resting potential, thereby converting the action of Cl-permeable GABAA and glycine receptors from excitatory to inhibitory. In the present study, we investigated the spatiotemporal expression of KCC2 in mouse cerebella, particularly focusing on Purkinje cells (PCs). First, we confirmed the fundamental expression profiles of KCC2 in the cerebellum, i.e. neuron-specific expression, somatodendritic distribution, and postnatal upregulation. We also found preferential recruitment to climbing fiber (CF) synapses during the second and third postnatal weeks, when perisomatic innervation in PCs switches from CFs to basket cell axons (BAs) and also when single winner CFs translocate from somata to dendrites. In parallel with this synaptic recruitment, the intracellular distribution shifted from a diffuse cytoplasmic to a predominantly cell surface pattern. In adult PCs, CF synapse-associated accumulation was obscured. Instead, significantly high expression was noted on the surface of PC dendrites in the superficial two-thirds of the molecular layer, in which stellate cells reside and project axons to innervate PC dendrites. Thus, the somatodendritic distribution in PCs is regulated in relation to particular inputs or input zones. During development, timed recruitment of KCC2 to CF synapses will augment inhibitory GABAergic actions by incoming BAs, promoting the CF-to-BA switchover in perisomatic PC innervation. In adulthood, enriched KCC2 expression at the stellate cell-targeting territory of PC dendrites might help in maintaining intracellular Cl homeostasis and the polarity of GABAA receptor-mediated responses upon sustained activity of this interneuron.
  • Dwi Wahyu Indriati, Naomi Kamasawa, Ko Matsui, Andrea L. Meredith, Masahiko Watanabe, Ryuichi Shigemoto
    JOURNAL OF NEUROSCIENCE 33 8 3668 - + 2013年02月 [査読有り][通常論文]
     
    P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca(v)2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Ca(v)2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Ca(v)2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Ca(v)2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Ca(v)2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of similar to 40 nm. Calcium nanodomain created by the opening of Ca(v)2.1 channels likely activates the two channels that limit the extent of depolarization.
  • Taisuke Yoneda, Katsuro Kameyama, Kazusa Esumi, Yohei Daimyo, Masahiko Watanabe, Yoshio Hata
    PLoS ONE 8 1 2013年01月08日 [査読有り][通常論文]
     
    The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience. © 2013 Yoneda et al.
  • Tie-Jun Sten Shi, Ming-Dong Zhang, Hugo Zeberg, Johanna Nilsson, Jacob Grunler, Su-Xing Liu, Qiong Xiang, Jonas Persson, Kaj J. Fried, Sergiu Bogdan Catrina, Masahiko Watanabe, Peter Arhem, Kerstin Brismar, Tomas G. M. Hokfelt
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 2 690 - 695 2013年01月 [査読有り][通常論文]
     
    Diabetic peripheral neuropathy (DPN) is the most common complication in both type 1 and type 2 diabetes. Here we studied some phenotypic features of a well-established animal model of type 2 diabetes, the leptin receptor-deficient db(-)/db(-) mouse, and also the effect of long-term (6 mo) treatment with coenzyme Q10 (CoQ10), an endogenous antioxidant. Diabetic mice at 8 mo of age exhibited loss of sensation, hypoalgesia (an increase in mechanical threshold), and decreases in mechanical hyperalgesia, cold allodynia, and sciatic nerve conduction velocity. All these changes were virtually completely absent after the 6-mo, daily CoQ10 treatment in db(-)/db(-) mice when started at 7 wk of age. There was a 33% neuronal loss in the lumbar 5 dorsal root ganglia (DRGs) of the db(-)/db(-) mouse versus controls at 8 mo of age, which was significantly attenuated by CoQ10. There was no difference in neuron number in 5/6-wk-old mice between diabetic and control mice. We observed a strong down-regulation of phospholipase C (PLC) beta 3 in the DRGs of diabetic mice at 8 mo of age, a key molecule in pain signaling, and this effect was also blocked by the 6-mo CoQ10 treatment. Many of the phenotypic, neurochemical regulations encountered in lumbar DRGs in standard models of peripheral nerve injury were not observed in diabetic mice at 8 mo of age. These results suggest that reactive oxygen species and reduced PLC beta 3 expression may contribute to the sensory deficits in the late-stage diabetic db(-)/db(-) mouse, and that early long-term administration of the antioxidant CoQ10 may represent a promising therapeutic strategy for type 2 diabetes neuropathy.
  • Sam A. Booker, Anna Gross, Daniel Althof, Ryuichi Shigemoto, Bernhard Bettler, Michael Frotscher, Matthew Hearing, Kevin Wickman, Masahiko Watanabe, Ákos Kulik, Imre Vida
    Journal of Neuroscience 33 18 7961 - 7974 2013年 [査読有り][通常論文]
     
    Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gammafrequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABAB receptors (GABABRs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABABRs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity forGABABRs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABABR-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABABR activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABAB IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABABRs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABABRs may shift the balance between perisomatic and dendritic inhibition. © 2013 the authors.
  • Shinya Kawata, Kouichi Hashimoto, Maya Yamazaki, Taisuke Miyazaki, Miwako Yamasaki, Takayasu Mikuni, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    JOURNAL OF PHYSIOLOGICAL SCIENCES 63 S188 - S188 2013年 [査読有り][通常論文]
  • Hiroki Shikanai, Takayuki Yoshida, Kohtarou Konno, Miwako Yamasaki, Takeshi Izumi, Yu Ohmura, Keiichi Shimamura, Masahiko Watanabe, Mitsuhiro Yoshioka
    JOURNAL OF PHARMACOLOGICAL SCIENCES 121 65P - 65P 2013年 [査読有り][通常論文]
  • Toshika Ohkawa, Yuko Fukata, Miwako Yamasaki, Taisuke Miyazaki, Norihiko Yokoi, Hiroshi Takashima, Masahiko Watanabe, Osamu Watanabe, Masaki Fukata
    Journal of Neuroscience 33 46 18161 - 18174 2013年 [査読有り][通常論文]
     
    More than 30 mutations in LGI1, a secreted neuronal protein, have been reported with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although LGI1 haploinsufficiency is thought to cause ADLTE, the underlying molecular mechanism that results in abnormal brain excitability remains mysterious. Here, we focused on a mode of action of LGI1 autoantibodies associated with limbic encephalitis (LE), which is one of acquired epileptic disorders characterized by subacute onset of amnesia and seizures.Wecomprehensively screened human sera from patients with immune-mediated neurological disorders for LGI1 autoantibodies, which also uncovered novel autoantibodies against six cell surface antigens including DCC, DPP10, and ADAM23. Our developed ELISA arrays revealed a specific role for LGI1 antibodies in LE and concomitant involvement of multiple antibodies, including LGI1 antibodies in neuromyotonia, a peripheral nerve disorder. LGI1 antibodies associated with LE specifically inhibited the ligand-receptor interaction between LGI1 and ADAM22/23 by targeting the EPTP repeat domain of LGI1 and reversibly reduced synaptic AMPA receptor clusters in rat hippocampal neurons. Furthermore, we found that disruption of LGI1-ADAM22 interaction by soluble extracellular domain ofADAM22was sufficient to reduce synapticAMPAreceptors in rat hippocampal neurons and that levels ofAMPAreceptor were greatly reduced in the hippocampal dentate gyrus in the epileptic LGI1 knock-out mouse. Therefore, either genetic or acquired loss of the LGI1-ADAM22 interaction reduces theAMPAreceptor function, causing epileptic disorders. These results suggest that by finely regulating the synapticAMPAreceptors, the LGI1-ADAM22 interaction maintains physiological brain excitability throughout life. © 2013 the authors.
  • Rebeca Martinez-Turrillas, Elena Puerta, Dhrubajyoti Chowdhury, Sonia Marco, Masahiko Watanabe, Norberto Aguirre, Isabel Perez-Otano
    NEUROBIOLOGY OF DISEASE 48 3 290 - 298 2012年12月 [査読有り][通常論文]
     
    Excitotoxicity due to excessive activation of glutamate receptors is a primary mediator of cell death in acute and chronic neurological disorders, and NMDA-type glutamate receptors (NMDARs) are thought to be involved. NMDARs assemble from heteromeric combinations of GluNl, GluN2 and GluN3 subunits, yielding a variety of receptor subtypes that differ in biophysical properties, signaling, and synaptic targeting. Inclusion of inhibitory GluN3 subunits reduces Ca2+ influx via NMDAR channels and alters their synaptic targeting, thus modifying the two hallmarks of NMDARs that are critical for their roles on neuronal death and survival. Here we evaluated the neuroprotective potential of GluN3A subunits by analyzing the susceptibility to striatal excitotoxic damage of transgenic mice overexpressing GluN3A. We found that mild GluN3A overexpression protected susceptible striatal neurons from lesions induced by the neurotoxin 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex 11/succinate dehydrogenase. GluN3A-mediated neuroprotection was dose-dependent and correlated with the levels of transgenic GluN3A expressed by two different mice strains. Neuroprotection was associated with a potent reduction of the activation of calpain, a Ca2+-dependent protease, which was measured as a decrease in 3-NP-induced fodrin and STEP cleavage in GluN3A transgenic mice relative to controls. We further show that transgenic GluN3A subunits incorporate into extrasynaptic compartments in mouse striatum, suggesting that reductions of toxic calpain activation might be linked to inhibition by GluN3A of pathological extrasynaptic NMDAR activity. (c) 2012 Elsevier Inc. All rights reserved.
  • Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 50 18035 - 18046 2012年12月 [査読有り][通常論文]
     
    Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis ( BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-)neurons constituted the majority (similar to 90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor alpha 1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter ( VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.
  • Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Kohtarou Konno, Miwako Yamasaki, Yuchio Yanagawa, Masabumi Minami, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 50 18035 - 18046 2012年12月 [査読有り][通常論文]
     
    Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis ( BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-)neurons constituted the majority (similar to 90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor alpha 1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter ( VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.
  • Aya Ito-Ishida, Taisuke Miyazaki, Eriko Miura, Keiko Matsuda, Masahiko Watanabe, Michisuke Yuzaki, Shigeo Okabe
    NEURON 76 3 549 - 564 2012年11月 [査読有り][通常論文]
     
    Differentiation of pre- and postsynaptic sites is coordinated by reciprocal interaction across synaptic clefts. At parallel fiber (PF)-Purkinje cell (PC) synapses, dendritic spines are autonomously formed without PF influence. However, little is known about how presynaptic structural changes are induced and how they lead to differentiation of mature synapses. Here, we show that Cbln1 released from PFs induces dynamic structural changes in PFs by a mechanism that depends on postsynaptic glutamate receptor delta2 (GluD2) and presynaptic neurexin (Nrx). Time-lapse imaging in organotypic culture and ultrastructural analyses in vivo revealed that Nrx-Cbln1-GluD2 signaling induces PF protrusions that often formed circular structures and encapsulated PC spines. Such structural changes in PFs were associated with the accumulation of synaptic vesicles and GluD2, leading to formation of mature synapses. Thus, PF protrusions triggered by Nrx-Cbln1-GluD2 signaling may promote bidirectional maturation of PF-PC synapses by a positive feedback mechanism.
  • N. M. Derks, O. Pinter, W. Zuure, C. Ledent, M. Watanabe, C. S. Molnar, Y. Wei, E. W. Roubos, S. Wu, E. Hrabovszky, D. Zelena, T. Kozicz
    JOURNAL OF NEUROENDOCRINOLOGY 24 11 1447 - 1461 2012年11月 [査読有り][通常論文]
     
    Neurones in the centrally projecting EdingerWestphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 similar to h of restraint) and chronic (14 similar to days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour.
  • Takatoshi Nakamura, Hiroyuki Mitomi, Wataru Onozato, Takeo Sato, Atsushi Ikeda, Masanori Naito, Naoto Ogura, Hiroki Kamata, Akira Ooki, Masahiko Watanabe
    Surgery Today 42 11 1096 - 1099 2012年11月 [査読有り][通常論文]
     
    This report describes the laparoscopic resection of a rectal GIST after treatment with imatinib mesylate. A 56-year-old male presented with a submucosal tumor (longest diameter, 8 cm) arising in the lower rectum. A core needle biopsy revealed that the tumor contained bundles of spindle-like cells. Immunostaining revealed that the tumor was positive for c-kit and CD34. Analysis of the c-kit gene revealed a substitution of ACA (threonine) by GCA (alanine) at codon 574 of exon 11. Imatinib mesylate (400 mg/day) was given as preoperative adjuvant therapy for 3 months, and the tumor shrank to 5 cm in diameter. Proctectomy with transanal anastomosis could be performed laparoscopically, while preserving the anus. There was no evidence of recurrence 2 years 6 months after surgery. Preoperative adjuvant chemotherapy with imatinib mesylate may permit the use of less invasive treatment procedures, allowing anal preservation. © Springer 2012.
  • Yoshimasa Kosaka, Akemi Kataoka, Hiroshi Yamaguchi, Hiroaki Ueo, Sayuri Akiyoshi, Norihiko Sengoku, Masaru Kuranami, Shinji Ohno, Masahiko Watanabe, Koshi Mimori, Masaki Mori
    Breast Cancer Research 14 5 2012年10月31日 [査読有り][通常論文]
     
    Introduction: Identification of useful markers associated with poor prognosis in breast cancer patients is critically needed. We previously showed that expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful to predict distant metastasis in gastric cancer patients. However, expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood of breast cancer patients has not yet been studied.Methods: Real-time reverse transcriptase-PCR was used to analyze vascular endothelial growth factor receptor-1 mRNA expression status with respect to various clinical parameters in 515 patients with breast cancer and 25 controls.Results: Expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood was higher in breast cancer patients than in controls. Increased vascular endothelial growth factor receptor-1 mRNA expression was associated with large tumor size, lymph node metastasis and clinical stage. Patients with high vascular endothelial growth factor receptor-1 mRNA expression also experienced a poorer survival rate than those with low expression levels, including those patients with triple-negative type and luminal-HER2(-) type disease.Conclusions: Expression of vascular endothelial growth factor receptor-1 mRNA in peripheral blood may be useful for prediction of poor prognosis in breast cancer, especially in patients with triple-negative type and luminal-HER2(-) type disease. © 2012 Kosaka et al. licensee BioMed Central Ltd.
  • Hiroki Shikanai, Takayuki Yoshida, Kohtarou Konno, Miwako Yamasaki, Takeshi Izumi, Yu Ohmura, Masahiko Watanabe, Mitsuhiro Yoshioka
    JOURNAL OF NEUROSCIENCE 32 41 14415 - 14426 2012年10月 [査読有り][通常論文]
     
    The serotonergic (5-HTergic) system arising from the dorsal raphe nucleus (DRN) is implicated in various physiological and behavioral processes, including stress responses. The DRN is comprised of several subnuclei, serving specific functions with distinct afferent and efferent connections. Furthermore, subsets of 5-HTergic neurons are known to coexpress other transmitters, including GABA, glutamate, or neuropeptides, thereby generating further heterogeneity. However, despite the growing evidence for functional variations among DRN subnuclei, relatively little is known about how they map onto neurochemical diversity of 5-HTergic neurons. In the present study, we characterized functional properties of GAD67-expressing 5-HTergic neurons (5-HT/GAD67 neurons) in the rat DRN, and compared with those of neurons expressing 5-HTergic molecules (5-HT neurons) or GAD67 alone. While 5-HT/GAD67 neurons were absent in the dorsomedial (DRD) or ventromedial (DRV) parts of the DRN, they were selectively distributed in the lateral wing of the DRN (DRL), constituting 12% of the total DRL neurons. They expressed plasmalemmal GABA transporter 1, but lacked vesicular inhibitory amino acid transporter. By using whole-cell patch-clamp recording, we found that 5-HT/GAD67 neurons had lower input resistance and firing frequency than 5-HT neurons. As revealed by c-Fos immunohistochemistry, neurons in the DRL, particularly 5-HT/GAD67 neurons, showed higher responsiveness to exposure to an open field arena than those in the DRD and DRV. By contrast, exposure to contextual fear conditioning stress showed no such regional differences. These findings indicate that 5-HT/GAD67 neurons constitute a unique neuronal population with distinctive neurochemical and electrophysiological properties and high responsiveness to innocuous stressor.
  • Shohei Oshima, Masahiko Watanabe
    EUROPEAN JOURNAL OF ORAL SCIENCES 120 5 386 - 394 2012年10月 [査読有り][通常論文]
     
    Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase consisting of two subunits catalytic subunit A (CnA) and regulatory subunit B (CnB) and plays a critical role in transducing Ca2+ signals into cellular responses. In this study, we investigated the expression of calcineurin in the mouse developing tooth. In-situ hybridization detected mRNAs for the CnAa and CnA beta isoforms of CnA and for the CnB1 isoform of CnB in the upper molar tooth germ at embryonic day 15. Immunohistochemistry with antibodies specific for CnAa, CnA beta, and CnB1 showed strong immunoreactivity of these proteins in secretory-stage ameloblasts and in odontoblasts during dentin formation. CnA beta and CnB1 were strongly immunoreactive in ruffle-ended ameloblasts at the enamel-maturation stage. In ameloblasts and odontoblasts, we also noted different subcellular distributions of CnAa and CnA beta. From these data, temporal profiles of calcineurin expression appear to correlate with active mineralization in tooth development. Furthermore, the distinct subcellular distribution of the two CnA subunits may reflect their distinct substrates or responsive sites within single cells, thus contributing to the diversity of calcineurin-dependent cellular responses during active tooth mineralization.
  • Kayo Nishizawa, Ryoji Fukabori, Kana Okada, Nobuyuki Kai, Motokazu Uchigashima, Masahiko Watanabe, Akira Shiota, Masatsugu Ueda, Yuji Tsutsui, Kazuto Kobayashi
    JOURNAL OF NEUROSCIENCE 32 39 13421 - 13432 2012年09月 [査読有り][通常論文]
     
    The dorsal striatum, which contains the dorsolateral striatum (DLS) and dorsomedial striatum (DMS), integrates the acquisition and implementation of instrumental learning in cooperation with the nucleus accumbens (NAc). The dorsal striatum regulates the basal ganglia circuitry through direct and indirect pathways. The mechanism by which these pathways mediate the learning processes of instrumental actions remains unclear. We investigated how the striatal indirect (striatopallidal) pathway arising from the DLS contributes to the performance of conditional discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D-2 receptor in the DLS of transgenic rats resulted in selective, efficient elimination of the striatopallidal pathway. This elimination impaired the accuracy of response selection in a two-choice reaction time task dependent on different auditory stimuli. The impaired response selection was elicited early in the test sessions and was gradually restored as the sessions continued. The restoration from the deficits in auditory discrimination was prevented by excitotoxic lesion of the NAc but not by that of the DMS. In addition, lesion of the DLS mimicked the behavioral consequence of the striatopallidal removal at the early stage of test sessions of discriminative performance. Our results demonstrate that the DLS-derived striatopallidal pathway plays an essential role in the execution of conditional discrimination, showing its contribution to the control of selection accuracy of learned motor responses. The results also suggest the presence of a mechanism that compensates for the learning deficits during the repetitive sessions, at least partly, demanding accumbal function.
  • Hidetaka Matsumoto, Koji Shibasaki, Motokazu Uchigashima, Amane Koizumi, Masashi Kurachi, Yasuhiro Moriwaki, Hidemi Misawa, Koichiro Kawashima, Masahiko Watanabe, Shoji Kishi, Yasuki Ishizaki
    PLOS ONE 7 8 2012年08月 [査読有り][通常論文]
     
    It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that alpha 7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the alpha 7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through alpha 7 nAChRs in their microvilli.
  • Najma Baseer, Erika Polgar, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 32 34 11854 - 11863 2012年08月 [査読有り][通常論文]
     
    Large projection neurons in lamina III of the rat spinal cord that express the neurokinin 1 receptor are densely innervated by peptidergic primary afferent nociceptors and more sparsely by low-threshold myelinated afferents. However, we know little about their input from other glutamatergic neurons. Here we show that these cells receive numerous contacts from nonprimary boutons that express the vesicular glutamate transporter 2 (VGLUT2), and form asymmetrical synapses on their dendrites and cell bodies. These synapses are significantly smaller than those formed by peptidergic afferents, but provide a substantial proportion of the glutamatergic synapses that the cells receive (over a third of those in laminae I-II and half of those in deeper laminae). Surprisingly, although the dynorphin precursor preprodynorphin (PPD) was only present in 4-7% of VGLUT2 boutons in laminae I-IV, it was found in 58% of the VGLUT2 boutons that contacted these cells. This indicates a highly selective targeting of the lamina III projection cells by glutamatergic neurons that express PPD, and these are likely to correspond to local neurons (interneurons and possibly projection cells). Since many PPD-expressing dorsal horn neurons respond to noxious stimulation, this suggests that the lamina III projection cells receive powerful monosynaptic and polysynaptic nociceptive input. Excitatory interneurons in the dorsal horn have been shown to possess I-A currents, which limit their excitability and can underlie a form of activity-dependent intrinsic plasticity. It is therefore likely that polysynaptic inputs to the lamina III projection neurons are recruited during the development of chronic pain states.
  • Najma Baseer, Erika Polgar, Masahiko Watanabe, Takahiro Furuta, Takeshi Kaneko, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 32 34 11854 - 11863 2012年08月 [査読有り][通常論文]
     
    Large projection neurons in lamina III of the rat spinal cord that express the neurokinin 1 receptor are densely innervated by peptidergic primary afferent nociceptors and more sparsely by low-threshold myelinated afferents. However, we know little about their input from other glutamatergic neurons. Here we show that these cells receive numerous contacts from nonprimary boutons that express the vesicular glutamate transporter 2 (VGLUT2), and form asymmetrical synapses on their dendrites and cell bodies. These synapses are significantly smaller than those formed by peptidergic afferents, but provide a substantial proportion of the glutamatergic synapses that the cells receive (over a third of those in laminae I-II and half of those in deeper laminae). Surprisingly, although the dynorphin precursor preprodynorphin (PPD) was only present in 4-7% of VGLUT2 boutons in laminae I-IV, it was found in 58% of the VGLUT2 boutons that contacted these cells. This indicates a highly selective targeting of the lamina III projection cells by glutamatergic neurons that express PPD, and these are likely to correspond to local neurons (interneurons and possibly projection cells). Since many PPD-expressing dorsal horn neurons respond to noxious stimulation, this suggests that the lamina III projection cells receive powerful monosynaptic and polysynaptic nociceptive input. Excitatory interneurons in the dorsal horn have been shown to possess I-A currents, which limit their excitability and can underlie a form of activity-dependent intrinsic plasticity. It is therefore likely that polysynaptic inputs to the lamina III projection neurons are recruited during the development of chronic pain states.
  • Asami Tanimura, Motokazu Uchigashima, Maya Yamazaki, Naofumi Uesaka, Takayasu Mikuni, Manabu Abe, Kouichi Hashimoto, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 30 12195 - 12200 2012年07月 [査読有り][通常論文]
     
    The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, 2-AG-mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened 2-AG-mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of 2-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, 2-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates 2-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.
  • Atsushi Iwakura, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 27 9438 - 9448 2012年07月 [査読有り][通常論文]
     
    The axon initial segment (AIS) of cerebellar Purkinje cells (PCs) is embraced by ramified axons of GABAergic basket cells (BCs) called the pinceau formation. This unique structure has been assumed to be a device for the modulation of PC outputs through electrical and/or GABAergic inhibition. Electrical inhibition is supported by enriched potassium channels, absence of sodium channels, and developed septate-like junctions between BC axons. The neurochemical basis for GABAergic inhibition, however, has not been well investigated. Here we addressed this issue using C56BL/6 mice. First, we confirmed previous observations that typical synaptic contacts were rare and confined to proximal axonal portions, with the remaining portions being mostly covered by astrocytic processes. Then we examined the expression of molecules involved in GABAergic signaling, including GABA synthetic enzyme glutamic acid decarboxylase (GAD), vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT), cytomatrix active zone protein bassoon, GABA receptor GABA(A)R alpha 1, and cell adhesion molecule neuroligin-2. These molecules were recruited to form a functional assembly at perisomatic BC-PC synapses and along the AIS of hippocampal and neocortical pyramidal cells. GAD and VIAAT immunogold labeling was five times lower in the pinceau formation compared with perisomatic BC terminals and showed no accumulation toward the AIS. Moreover, bassoon, neuroligin-2, and GABA(A)R alpha 1 formed no detectable clusters along the ankyrin-G-positive AIS proper. These findings indicate that GABAergic signaling machinery is organized loosely and even incompletely in the pinceau formation. Together, BCs do not appear to exert GABAergic synaptic inhibition on the AIS, although the mode of action of the pinceau formation remains to be explored.
  • Noemi Holderith, Andrea Lorincz, Gergely Katona, Balazs Rozsa, Akos Kulik, Masahiko Watanabe, Zoltan Nusser
    NATURE NEUROSCIENCE 15 7 988 - 997 2012年07月 [査読有り][通常論文]
     
    Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active-zone area. Peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active-zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated calcium channel subunit Cav2.1 and the presynaptic protein Rim 1/2 are confined to the active zone and their numbers scale linearly with the active-zone area. Gold particles labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.
  • Laura C. Gregg, Kwang-Mook Jung, Jessica M. Spradley, Rita Nyilas, Richard L. Suplita, Andreas Zimmer, Masahiko Watanabe, Ken Mackie, Istvan Katona, Daniele Piomelli, Andrea G. Hohmann
    JOURNAL OF NEUROSCIENCE 32 28 9457 - 9468 2012年07月 [査読有り][通常論文]
     
    Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [( S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB1 cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)tetrahydrolipstatin (THL), which inhibit activity of DGL-alpha and DGL-beta isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-alpha colocalized with mGlu5 at postsynaptic sites of the dlPAG, whereas CB1 was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-alpha, but not DGL-alpha, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-alpha cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.
  • Yoshimasa Kosaka, Koshi Mimori, Takeo Fukagawa, Kenji Ishikawa, Tsuyoshi Etoh, Hitoshi Katai, Takeshi Sano, Masahiko Watanabe, Mitsuru Sasako, Masaki Mori
    Annals of Surgical Oncology 19 3 S430 - S437 2012年07月 [査読有り][通常論文]
     
    Purpose: Matrix metalloproteinases are responsible for proteolytic degradation of basement membrane and extracellular matrix. In tumor tissues, elevated expression of matrix metalloproteinase-1 (MMP-1) has been associated with tumor invasion and metastasis. However, little is known about the expression of MMP-1 in peripheral blood (PB) and bone marrow (BM) in gastric cancer patients. Thus, the aim of the present study is to determine MMP-1 messenger RNA (mRNA) expression levels in BM and PB of patients with gastric cancer. Methods: The study group consisted of 857 patients with gastric cancer (577 males and 280 females) ranging in age from 27 to 87 years (average 61.6 years). MMP-1 mRNA expression levels in BM and PB were evaluated quantitatively by real-time reverse-transcription polymerase chain reaction (RT-PCR). Results: Expression of MMP-1 mRNA in BM and PB of patients with gastric cancer was significantly higher than in noncancer patients. High levels of MMP-1 mRNA expression were significantly associated with differentiated histology, tumor size, tumor invasiveness, lymph node metastasis, liver metastasis, and clinical stage. Particularly importantly, MMP-1 mRNA expression in PB was an independent factor of distant metastasis. Conclusions: We disclosed that MMP-1 mRNA expression in peripheral blood and bone marrow of gastric cancer patients was very high, precisely reflecting staging of gastric cancer. MMP-1 mRNA expression in peripheral blood may be a useful marker for distant metastasis in gastric cancer. © 2011 Society of Surgical Oncology.
  • Hiroshi Katoh, Keishi Yamashita, Mina Waraya, Ofer Margalit, Akira Ooki, Hideaki Tamaki, Hiroyuki Sakagami, Kenichi Kokubo, David Sidransky, Masahiko Watanabe
    NEOPLASIA 14 7 559 - + 2012年07月 [査読有り][通常論文]
     
    Homeodomain-only protein X (HOPX)-beta promoter methylation was recently shown to be frequent in human cancers and was suggested as tumor suppressor gene in esophageal and gastric cancer. The aim of this study was to investigate the mechanistic roles of HOPX-beta promoter methylation and its clinical relevance in colorectal cancer (CRC). HOPX-beta promoter methylation was assessed in human CRC cell lines and 294 CRC tissues. HOPX mRNA and protein levels were measured in relation to HOPX-beta promoter methylation. The effects of forced HOPX expression on tumorigenesis were studied using in vitro and in vivo assays. The association between HOPX-beta promoter methylation and clinical relevance of CRC patients was determined. HOPX-beta promoter methylation is cancer-specific and frequently found in CRC cell lines and tissues, resulting in the down-regulation of HOPX mRNA and protein levels. In CRC cell lines, forced expression of HOPX suppressed proliferation, invasion, and anchorage-independent growth. DNA microarray analyses suggested critical downstream genes that are associated with cancer cell proliferation, invasion or angiogenesis. In a mouse xenograft model, HOPX inhibited tumorigenesis and angiogenesis. Finally, HOPX-beta promoter methylation was associated with worse prognosis of stage III CRC patients (hazard ratio = 1.40, P = .035) and also with poor differentiation (P = .014). In conclusion, HOPX-beta promoter methylation is a frequent and cancer-specific event in CRC progression. This epigenetic alteration may have clinical ramifications in the diagnosis and treatment of CRC patients.
  • Erik Hrabovszky, Csilla S. Molnar, Robert Nagy, Barbara Vida, Beata A. Borsay, Kalman Racz, Laszlo Herczeg, Masahiko Watanabe, Imre Kallo, Zsolt Liposits
    ENDOCRINOLOGY 153 6 2766 - 2776 2012年06月 [査読有り][通常論文]
     
    Amino acid (aa) neurotransmitters in synaptic afferents to hypothalamic GnRH-I neurons are critically involved in the neuroendocrine control of reproduction. Although in rodents the major aa neurotransmitter in these afferents is gamma-aminobutyric acid (GABA), glutamatergic axons also innervate GnRH neurons directly. Our aim with the present study was to address the relative contribution of GABAergic and glutamatergic axons to the afferent control of human GnRH neurons. Formalin-fixed hypothalamic samples were obtained from adult male individuals (n = 8) at autopsies, and their coronal sections processed for dual-label immunohistochemical studies. GABAergic axons were labeled with vesicular inhibitory aa transporter antibodies, whereas glutamatergic axons were detected with antisera against the major vesicular glutamate transporter (VGLUT) isoforms, VGLUT1 and VGLUT2. The relative incidences of GABAergic and glutamatergic axonal appositions to GnRH-immunoreactive neurons were compared quantitatively in two regions, the infundibular and paraventricular nuclei. Results showed that GABAergic axons established the most frequently encountered type of axo-somatic apposition. Glutamatergic contacts occurred in significantly lower numbers, with similar contributions by their VGLUT1 and VGLUT2 subclasses. The innervation pattern was different on GnRH dendrites where the combined incidence of glutamatergic (VGLUT1 + VGLUT2) contacts slightly exceeded that of the GABAergic appositions. We conclude that GABA represents the major aa neurotransmitter in axo-somatic afferents to human GnRH neurons, whereas glutamatergic inputs occur somewhat more frequently than GABAergic inputs on GnRH dendrites. Unlike in rats, the GnRH system of the human receives innervation from the VGLUT1, in addition to the VGLUT2, subclass of glutamatergic neurons. (Endocrinology 153: 2766-2776, 2012)
  • Carmen Ballesteros-Merino, Mike Lin, Wendy W. Wu, Clotilde Ferrandiz-Huertas, Maria J. Cabanero, Masahiko Watanabe, Yugo Fukazawa, Ryuichi Shigemoto, James Maylie, John P. Adelman, Rafael Lujan
    HIPPOCAMPUS 22 6 1467 - 1480 2012年06月 [査読有り][通常論文]
     
    We investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy, and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Consistent with this redistribution and gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. However, the effect of apamin on EPSPs was not different between synapses in proximal or distal stratum radiatum or stratum lacunosum-moleculare in adult. These results show a developmental increase and gradient in SK2-containing channel surface expression that underlie their influence on neurotransmission, and that may contribute to increased memory acquisition during early development. 2011 Wiley Periodicals, Inc.
  • Kondo T, Watanabe M, Hatakeyama S
    Biochemical and biophysical research communications 422 3 501 - 507 3 2012年06月 [査読有り][通常論文]
     
    Innate immune responses are triggered by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) and then activate intracellular signaling pathways including NF-kappa B and interferon regulatory factors. Recently, it has been reported that tripartite motif (TRIM) proteins function as crucial regulators via ubiquitin-mediated modifications for these signaling pathways. In this study, we showed that one of the TRIM family ubiquitin ligases, TRIM59, interacts with ECSIT as an adaptor protein required for the TLR-mediated transduction pathway. Luciferase reporter assays using reporter plasmids including NF-kappa B responsive element, interferon beta (IFN-beta) promoter and interferon-sensitive response element (ISRE) showed that overexpression of TRIM59 repressed their transcriptional activities, whereas knockdown of TRIM59 enhanced their transcriptional activities. Furthermore, TRIM59 inhibited phosphorylation and dimerization of IRF3 and IRF7, suggesting that TRIM59 negatively regulates upstream kinases for IRFs. These findings indicate that TRIM59 may serve as a multifunctional regulator for innate immune signaling pathways. (c) 2012 Elsevier Inc. All rights reserved.
  • Yuichi Takeuchi, Miwako Yamasaki, Yasuyuki Nagumo, Keiji Imoto, Masahiko Watanabe, Mariko Miyata
    JOURNAL OF NEUROSCIENCE 32 20 6917 - 6930 2012年05月 [査読有り][通常論文]
     
    The remodeling of neural circuitry and changes in synaptic efficacy after peripheral sensory nerve injury are considered the basis for functional reorganization in the brain, including changes in receptive fields. However, when or how the remodeling occurs is largely unknown. Here we show the rapid rewiring of afferent fibers in the mature ventral posteromedial thalamic nucleus of mice after transection of the peripheral whisker sensory nerve, using the whole-cell voltage-clamp technique. Transection induced the recruitment of afferent fibers to a thalamic relay neuron within 5-6 d of injury. The rewiring was pathway specific, but not sensory experience dependent or peripheral nerve activity dependent. The newly recruited fibers mediated small EPSCs, and postsynaptic GluA2-containing AMPA receptors were selectively upregulated at the new synapses. This rapid and pathway-specific remodeling of thalamic circuitry may be an initial step in the massive axonal reorganization at supraspinal levels, which occurs months or years after peripheral sensory nerve injury.
  • Csaba Cserep, Eszter Szabadits, Andras Szonyi, Masahiko Watanabe, Tamas F. Freund, Gabor Nyiri
    PLOS ONE 7 5 2012年05月 [査読有り][通常論文]
     
    GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6-7 and we observed a direct colocalization of GABA(A)- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of preembedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABA(A)R-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABA(A)R-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca2+-influx through the NMDA receptors.
  • Hiroaki Yaguchi, Fumihiko Okumura, Hidehisa Takahashi, Takahiro Kano, Hiroyuki Kameda, Motokazu Uchigashima, Shinya Tanaka, Masahiko Watanabe, Hidenao Sasaki, Shigetsugu Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 15 12050 - 12059 2012年04月 [査読有り][通常論文]
     
    Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.
  • Hisako Nakayama, Taisuke Miyazaki, Kazuo Kitamura, Kouichi Hashimoto, Yuchio Yanagawa, Kunihiko Obata, Kenji Sakimura, Masahiko Watanabe, Masanobu Kano
    NEURON 74 2 384 - 396 2012年04月 [査読有り][通常論文]
     
    Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABAA receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.
  • Okada M, Hozumi Y, Iwazaki K, Misaki K, Yanagida M, Araki Y, Watanabe T, Yagisawa H, Topham MK, Kaibuchi K, Goto K
    Biochemical Biophysical Research Communications 420 2 479 - 484 2012年04月 [査読有り][通常論文]
     
    Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGK zeta-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGK zeta or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGK zeta(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGK zeta is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages. (C) 2012 Elsevier Inc. All rights reserved.
  • Daiki D. Horikawa, Ayami Yamaguchi, Tetsuya Sakashita, Daisuke Tanaka, Nobuyuki Hamada, Fumiko Yukuhiro, Hirokazu Kuwahara, Takekazu Kunieda, Masahiko Watanabe, Yuichi Nakahara, Seiichi Wada, Tomoo Funayama, Chihiro Katagiri, Seigo Higashi, Shin-Ichi Yokobori, Mikinori Kuwabara, Lynn J. Rothschild, Takashi Okuda, Hirofumi Hashimoto, Yasuhiko Kobayashi
    ASTROBIOLOGY 12 4 283 - 289 2012年04月 [査読有り][通常論文]
     
    Tardigrades are tiny (less than 1 mm in length) invertebrate animals that have the potential to survive travel to other planets because of their tolerance to extreme environmental conditions by means of a dry ametabolic state called anhydrobiosis. While the tolerance of adult tardigrades to extreme environments has been reported, there are few reports on the tolerance of their eggs. We examined the ability of hydrated and anhydrobiotic eggs of the tardigrade Ramazzottius varieornatus to hatch after exposure to ionizing irradiation (helium ions), extremely low and high temperatures, and high vacuum. We previously reported that there was a similar pattern of tolerance against ionizing radiation between hydrated and anhydrobiotic adults. In contrast, anhydrobiotic eggs (50% lethal dose; 1690 Gy) were substantially more radioresistant than hydrated ones (50% lethal dose; 509 Gy). Anhydrobiotic eggs also have a broader temperature resistance compared with hydrated ones. Over 70% of the anhydrobiotic eggs treated at either -196 degrees C or +50 degrees C hatched successfully, but all the hydrated eggs failed to hatch. After exposure to high-vacuum conditions (5.3 x 10(-4) Pa to 6.2 x 10(-5) Pa), the hatchability of the anhydrobiotic eggs was comparable to that of untreated control eggs.
  • Claire L. Padgett, Arnaud L. Lalive, Kelly R. Tan, Miho Terunuma, Michaelanne B. Munoz, Menelas N. Pangalos, Jose Martinez-Hernandez, Masahiko Watanabe, Stephen J. Moss, Rafael Lujan, Christian Luescher, Paul A. Slesinger
    NEURON 73 5 978 - 989 2012年03月 [査読有り][通常論文]
     
    Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABABR-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.
  • Ryoji Fukabori, Kana Okada, Kayo Nishizawa, Nobuyuki Kai, Kenta Kobayashi, Motokazu Uchigashima, Masahiko Watanabe, Yuji Tsutsui, Kazuto Kobayashi
    EUROPEAN JOURNAL OF NEUROSCIENCE 35 5 784 - 797 2012年03月 [査読有り][通常論文]
     
    The dorsal striatum in the basal ganglia circuitry is a principal structure that mediates the acquisition and performance of instrumental learning. The projections from the dorsal striatum are composed of two subpopulations of medium spiny neurons that constitute the direct and indirect pathways. The mechanism by which these striatal projections control the learning processes of instrumental actions remains unknown. We addressed the behavioral role of the striatal direct (striatonigral) pathway in the performance of visual discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D1 receptor in mice resulted in a moderate level of elimination of the striatonigral pathway. Targeting of the neural pathway from the whole region of the dorsal striatum lengthened the response time but did not affect the accuracy of response selection in a two-choice reaction time task dependent on light stimulus. This lengthened motor response was induced early in the test sessions and was gradually restored to normal levels during repetitive sessions. In addition, subregion-specific pathway targeting revealed that the delay in learned motor response was generated by the elimination of the striatonigral pathway arising from the dorsomedial striatum but not from the dorsolateral striatum. Our findings indicate that the striatonigral pathway, in particular from the dorsomedial striatum, contributes to the regulation of response time in the execution of visual discrimination. The restoration of motor response deficits during repetitive sessions suggests the presence of a mechanism by which the response facilitation is acquired through continuation of learning despite the removal of the striatonigral pathway.
  • Valentina Chiodi, Motokazu Uchigashima, Sarah Beggiato, Antonella Ferrante, Monica Armida, Alberto Martire, Rosa Luisa Potenza, Luca Ferraro, Sergio Tanganelli, Masahiko Watanabe, Maria Rosaria Domenici, Patrizia Popoli
    NEUROBIOLOGY OF DISEASE 45 3 983 - 991 2012年03月 [査読有り][通常論文]
     
    Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice. (C) 2011 Elsevier Inc. All rights reserved.
  • The interaction of the cannabinoidergic and serotonergic system modulating synaptic transmission in the basal amygdala
    Yoshida, T, Izumi, T, Ohmura, Y, Watanabe, M, Yoshioka, M
    Journal of Pharmacological Sciences 118 179P - 179P 2012年 [査読有り][通常論文]
  • Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia
    Okubo, Y, Mashimo, M, Yamazawa, T, Yamasaki, M, Watanabe, M, Murayama, T, Lino, M
    Journal of Pharmacological Sciences 118 102P - 102P 2012年 [査読有り][通常論文]
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Maya Yamazaki, Manabu Abe, Hiroshi Usui, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 32 4 1311 - 1328 2012年01月 [査読有り][通常論文]
     
    In the adult cerebellum, each Purkinje cell (PC) is innervated by a single climbing fiber (CF) in proximal dendrites and 10(5)-10(6) parallel fibers (PFs) in distal dendrites. This organized wiring is established postnatally through heterosynaptic competition between PFs and CFs and homosynaptic competition among multiple CFs. Using PC-specific Ca(v)2.1 knock-out mice (PC-Ca(v)2.1 KO mice), we have demonstrated recently that postsynaptic Ca(v)2.1 plays a key role in the homosynaptic competition by promoting functional strengthening and dendritic translocation of single "winner" CFs. Here, we report that Ca(v)2.1 in PCs, but not in granule cells, is also essential for the heterosynaptic competition. In PC-Ca(v)2.1 KO mice, the extent of CF territory was limited to the soma and basal dendrites, whereas PF territory was expanded reciprocally. Consequently, the proximal somatodendritic domain of PCs displayed hyperspiny transformation and fell into chaotic innervation by multiple CFs and numerous PFs. PC-Ca(v)2.1 KO mice also displayed patterned degeneration of PCs, which occurred preferentially in aldolase C/zebrin II-negative cerebellar compartments. Furthermore, the mutually complementary expression of phospholipase C beta 3 (PLC beta 3) and PLC beta 4 was altered such that their normally sharp boundary was blurred in the PCs of PC-Cav2.1 KO mice. This blurring was caused by an impaired posttranscriptional downregulation of PLC beta 3 in PLC beta 4-dominant PCs during the early postnatal period. A similar alteration was noted in the banded expression of the glutamate transporter EAAT4 in PC-Cav2.1 KO mice. Therefore, Cav2.1 in PCs is essential for competitive synaptic wiring, cell survival, and the establishment of precise boundaries and reciprocity of biochemical compartments in PCs.
  • Sho Kakizawa, Toshiko Yamazawa, Yili Chen, Akihiro Ito, Takashi Murayama, Hideto Oyamada, Nagomi Kurebayashi, Osamu Sato, Masahiko Watanabe, Nozomu Mori, Katsuji Oguchi, Takashi Sakurai, Hiroshi Takeshima, Nobuhito Saito, Masamitsu Iino
    EMBO JOURNAL 31 2 417 - 428 2012年01月 [査読有り][通常論文]
     
    Mobilization of intracellular Ca2+ stores regulates a multitude of cellular functions, but the role of intracellular Ca2+ release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca2+ release from intracellular stores of central neurons, and thereby promote prolonged Ca2+ signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca2+ release. NO-induced Ca2+ release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain. The EMBO Journal (2012) 31, 417-428. doi:10.1038/emboj.2011.386; Published online 28 October 2011
  • Natsuya Katada, Shinichi Sakuramoto, Keishi Yamashita, Tomotaka Shibata, Hiromitsu Moriya, Shiro Kikuchi, Masahiko Watanabe
    Annals of Thoracic and Cardiovascular Surgery 18 5 420 - 428 2012年 [査読有り][通常論文]
     
    Radical treatment for achalasia is currently unavailable. At present, most palliative procedures are designed improve the passage of food through the gastroesophageal junction and thereby alleviate symptoms. Drug therapy is of limited, transient effectiveness. Pneumatic dilation (PD) is considered superior to endoscopic botulinum toxin injection (EBTI). The mainstay of surgical treatment for achalasia is laparoscopic Heller myotomy (LHM) with fundoplication, currently considered superior to PD. Per oral endoscopic myotomy (POEM), a "state-of-the-art" procedure for minimally invasive surgery, holds great promise for the future management of achalasia. Definitive conclusions regarding the benefits and risks of currently available treatments for achalasia must await the accumulation of evidence from well-designed clinical trials. © 2012 The Editorial Committee of Annals of Thoracic and Cardiovascular Surgery. All rights reserved.
  • Yuki Nakatani, Yumi Iwamitsu, Masaru Kuranami, Shigemi Okazaki, Kenji Yamamoto, Masahiko Watanabe, Hitoshi Miyaoka
    Shinrigaku Kenkyu 83 2 126 - 134 2012年 [査読有り][通常論文]
     
    This study examined the relationship between emotional suppression and psychological distress in response to a diagnosis related to breast cancer. After their first visit, 31 patients with breast cancer and 90 with benign breast conditions completed the Courtauld Emotional Control Scale (CECS) and the Profile of Mood States (POMS) and were interviewed about their concerns after being diagnosed. Breast cancer and benign breast condition patients were divided into separate emotional suppression groups or emotional expression groups based on their median CECS score. The POMS scores of breast cancer patients were higher than those of benign breast condition patients scores in the emotional suppression groups were higher than in the emotional expression groups. Breast cancer patients in the emotional suppression group expressed more negative emotions and fewer positive emotions than benign breast condition patients. Our results suggest that patients who suppressed their emotions experienced and reported more psychological distress when diagnosed with breast cancer.
  • Duane Allen, Shin Nakayama, Masayuki Kuroiwa, Takaaki Nakano, Julie Palmateer, Yasuharu Kosaka, Carmen Ballesteros, Masahiko Watanabe, Chris T. Bond, Rafael Lujan, James Maylie, John P. Adelman, Paco S. Herson
    JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 31 12 2302 - 2312 2011年12月 [査読有り][通常論文]
     
    In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca2+-activated K+ channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg2+ block that reduces Ca2+ influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2302-2312; doi:10.1038/jcbfm.2011.90; published online 29 June 2011
  • Laura Fernandez-Alacid, Masahiko Watanabe, Elek Molnar, Kevin Wickman, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 34 11 1724 - 1736 2011年12月 [査読有り][通常論文]
     
    G protein-gated inwardly-rectifying K+ (GIRK/family 3 of inwardly-rectifying K+) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development. Together, these findings shed new light on the developmental regulation and subcellular diversity of neuronal GIRK channels, and support the contention that distinct subpopulations of GIRK channels exert separable influences on neuronal excitability. The ability to selectively target specific subpopulations of GIRK channels may prove effective in the treatment of disorders of excitability.
  • Duane Allen, Shin Nakayama, Masayuki Kuroiwa, Takaaki Nakano, Julie Palmateer, Yasuharu Kosaka, Carmen Ballesteros, Masahiko Watanabe, Chris T. Bond, Rafael Lujan, James Maylie, John P. Adelman, Paco S. Herson
    JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 31 12 2302 - 2312 2011年12月 [査読有り][通常論文]
     
    In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca2+-activated K+ channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg2+ block that reduces Ca2+ influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2302-2312; doi:10.1038/jcbfm.2011.90; published online 29 June 2011
  • Noriko Ando, Yumi Iwamitsu, Masaru Kuranami, Shigemi Okazaki, Kenji Yamamoto, Masahiko Watanabe, Hitoshi Miyaoka
    Familial Cancer 10 4 681 - 689 2011年12月 [査読有り][通常論文]
     
    The purpose of this study was to reveal characteristics that underlie patients' expressions of concern about their genetic risk of breast cancer at an initial outpatient clinic visit prior to a definitive diagnosis of cancer. A total of 233 women, at their initial outpatient clinic visits, participated in semi-structured and open-ended interviews that surveyed their anxieties, worries, thoughts, and feelings. Each patient completed a self-administered survey comprising questions about her family history of breast cancer and responded to a questionnaire that assessed psychological distress. Patients were divided into those who expressed concerns about inherited risk of breast cancer during the interview (expression group, n = 39) and those who did not (non-expression group, n = 194). Although patients in the expression group tended to have higher rates of breast cancer in their families, patients without a family history of breast cancer also expressed concerns. We used qualitative content analysis to identify the emotions and thoughts of patients expressing these concerns. Patients with a family history of breast cancer experienced anxiety/worry, risk-reducing behavior, acceptance, objective fact, and denial, whereas patients without a family history of breast cancer experienced anxiety/worry, risk-reducing behavior, surprise/shock, acceptance, objective fact, denial, optimistic thought, regret, and realistic thought. Some patients in the breast clinic had concerns about their inherited risk of cancer, despite lacking a family history of breast cancer. Health care professionals should inquire about family histories and provide appropriate support and counseling to patients as needed, regardless of the patient's genetic risk for the disease. © 2011 Springer Science+Business Media B.V.
  • Masahiko Watanabe, Masanobu Kano
    EUROPEAN JOURNAL OF NEUROSCIENCE 34 10 1697 - 1710 2011年11月 [査読有り][通常論文]
     
    Innervation of Purkinje cells (PCs) by multiple climbing fibers (CFs) is refined into mono-innervation during the first three postnatal weeks of rodents lives. In this review article, we will integrate the current knowledge on developmental process and mechanisms of CF synapse elimination. In the creeper stage of CF innervation (postnatal day 0 (P0)similar to), CFs creep among PC somata to form transient synapses on immature dendrites. In the pericellular nest stage (P5 similar to), CFs densely surround and innervate PC somata. CF innervation is then displaced to the apical portion of PC somata in the capuchon stage (P9 similar to), and translocate to dendrites in the dendritic (P12 similar to) stage. Along with the developmental changes in CF wiring, functional and morphological distinctions become larger among CF inputs. PCs are initially innervated by more than five CFs with similar strengths (similar to P3). During P37 only a single CF is selectively strengthened (functional differentiation), and it undergoes dendritic translocation from P9 on (dendritic translocation). Following the functional differentiation, perisomatic CF synapses are eliminated nonselectively; this proceeds in two distinct phases. The early phase (P711) is conducted independently of parallel fiber (PF)PC synapse formation, while the late phase (P1217) critically depends on it. The P/Q-type voltage-dependent Ca2+ channel in PCs triggers selective strengthening of single CF inputs, promotes dendritic translocation of the strengthened CFs, and drives the early phase of CF synapse elimination. In contrast, the late phase is mediated by the mGluR1GaqPLC beta 4PKC signaling cascade in PCs driven at PFPC synapses, whose structural connectivity is stabilized and maintained by the GluRd2Cbln1neurexin system.
  • Shigeki Kato, Masahito Kuramochi, Kenta Kobayashi, Ryoji Fukabori, Kana Okada, Motokazu Uchigashima, Masahiko Watanabe, Yuji Tsutsui, Kazuto Kobayashi
    JOURNAL OF NEUROSCIENCE 31 47 17169 - 17179 2011年11月 [査読有り][通常論文]
     
    The dorsal striatum receives converging excitatory inputs from diverse brain regions, including the cerebral cortex and the intralaminar/midline thalamic nuclei, and mediates learning processes contributing to instrumental motor actions. However, the roles of each striatal input pathway in these learning processes remain uncertain. We developed a novel strategy to target specific neural pathways and applied this strategy for studying behavioral roles of the pathway originating from the parafascicular nucleus (PF) and projecting to the dorsolateral striatum. A highly efficient retrograde gene transfer vector encoding the recombinant immunotoxin (IT) receptor was injected into the dorsolateral striatum in mice to express the receptor in neurons innervating the striatum. IT treatment into the PF of the vector-injected animals caused a selective elimination of neurons of the PF-derived thalamostriatal pathway. The elimination of this pathway impaired the response selection accuracy and delayed the motor response in the acquisition of a visual cue-dependent discrimination task. When the pathway elimination was induced after learning acquisition, it disturbed the response accuracy in the task performance with no apparent change in the response time. The elimination did not influence spontaneous locomotion, methamphetamine-induced hyperactivity, and motor skill learning that demand the function of the dorsal striatum. These results demonstrate that thalamostriatal projection derived from the PF plays essential roles in the acquisition and execution of discrimination learning in response to sensory stimulus. The temporal difference in the pathway requirement for visual discrimination suggests a stage-specific role of thalamostriatal pathway in the modulation of response time of learned motor actions.
  • Maria R. Karlocai, Kinga Toth, Masahiko Watanabe, Catherine Ledent, Gabor Juhasz, Tamas F. Freund, Zsofia Magloczky
    PLOS ONE 6 11 2011年11月 [査読有り][通常論文]
     
    The endocannabinoid system plays a central role in retrograde synaptic communication and may control the spread of activity in an epileptic network. Using the pilocarpine model of temporal lobe epilepsy we examined the expression pattern of the Type 1 cannabinoid receptor (CB1-R) in the hippocampi of CD1 mice at survival times of 2 hours, 1 day, 3 days and 2 months (acute, latent and chronic phases). Based on the behavioral signs of the acute seizures, animals were classified as "weakly'' or "strongly'' epileptic using the modified Racine scale. Mice of the weak group had mild seizures, whereas seizures in the strong group were frequent with intense motor symptoms and the majority of these animals developed sclerosis in the chronic phase. In control samples the most intense staining of CB1-R-positive fibers was found in the molecular layer of the dentate gyrus and in str. pyramidale of the cornu Ammonis. In weak animals no significant changes were seen at any survival time compared to controls. In strong animals, however, in the acute phase, a massive reduction in CB1-R-stained terminals occurred in the hippocampus. In the latent phase CB1-R immunoreactivity gradually recovered. In the chronic phase, CB1-immunostaining in sclerotic samples was stronger throughout the hippocampus. Quantitative electron microscopic analysis showed an increase in the number of CB1-R-positive terminals in the dentate gyrus. Moreover, the number of immunogold particles significantly increased in GABAergic terminals. Our results suggest a proconvulsive downregulation of CB1 receptors in the acute phase most probably due to receptor internalization, followed by compensatory upregulation and sprouting in the chronic phase of epilepsy. In conclusion, the changes in CB1 receptor expression pattern revealed in this study are associated with the severity of hippocampal injury initiated by acute seizures that ultimately leads to sclerosis in the vulnerable regions in the chronic phase.
  • Ryoichi Ichikawa, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Kouichi Hashimoto, Haruyuki Tatsumi, Yoshiro Inoue, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 47 16916 - 16927 2011年11月 [査読有り][通常論文]
     
    In early postnatal development, perisomatic innervation of cerebellar Purkinje cells (PCs) switches from glutamatergic climbing fibers (CFs) to GABAergic basket cell fibers (BFs). Here we examined the switching process in C57BL/6 mice. At postnatal day 7 (P7), most perisomatic synapses were formed by CFs on to somatic spines. The density of CF-spine synapses peaked at P9, when pericellular nest around PCs by CFs was most developed, and CF-spine synapses constituted 88% of the total perisomatic synapses. Thereafter, CF-spine synapses dropped to 63% at P12, 6% at P15, and <1% at P20, whereas BF synapses increased reciprocally. During the switching period, a substantial number of BF synapses existed as BF-spine synapses (37% of the total perisomatic synapses at P15), and free spines surrounded by BFs or Bergmann glia also emerged. By P20, BF-spine synapses and free spines virtually disappeared, and BF-soma synapses became predominant (88%), thus attaining the adult pattern of perisomatic innervation. Parallel with the presynaptic switching, postsynaptic receptor phenotype also switched from glutamatergic to GABAergic. In the active switching period, particularly at P12, fragmental clusters of AMPA-type glutamate receptor were juxtaposed with those of GABA(A) receptor. When examined with serial ultrathin sections, immunogold labeling for glutamate and GABA(A) receptors was often clustered beneath single BF terminals. These results suggest that a considerable fraction of somatic spines is succeeded from CFs to BFs and Bergmann glia in the early postnatal period, and that the switching of postsynaptic receptor phenotypes mainly proceeds under the coverage of BF terminals.
  • Pablo R. Brumovsky, David R. Robinson, Jun-Ho La, Kim B. Seroogy, Kerstin H. Lundgren, Kathryn M. Albers, Michael E. Kiyatkin, Rebecca P. Seal, Robert H. Edwards, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
    JOURNAL OF COMPARATIVE NEUROLOGY 519 16 3346 - 3366 2011年11月 [査読有り][通常論文]
     
    Vesicular glutamate transporters (VGLUTs) have been extensively studied in various neuronal systems, but their expression in visceral sensory and autonomic neurons remains to be analyzed in detail. Here we studied VGLUTs type 1 and 2 (VGLUT(1) and VGLUT(2), respectively) in neurons innervating the mouse colorectum. Lumbosacral and thoracolumbar dorsal root ganglion (DRG), lumbar sympathetic chain (LSC), and major pelvic ganglion (MPG) neurons innervating the colorectum of BALB/C mice were retrogradely traced with Fast Blue, dissected, and processed for immunohistochemistry. Tissue from additional naive mice was included. Previously characterized antibodies against VGLUT(1), VGLUT(2), and calcitonin gene-related peptide (CGRP) were used. Riboprobe in situ hybridization, using probes against VGLUT(1) and VGLUT(2), was also performed. Most colorectal DRG neurons expressed VGLUT(2) and often colocalized with CGRP. A smaller percentage of neurons expressed VGLUT(1). VGLUT(2)-immunoreactive (IR) neurons in the MPG were rare. Abundant VGLUT(2)-IR nerves were detected in all layers of the colorectum; VGLUT(1)-IR nerves were sparse. A subpopulation of myenteric plexus neurons expressed VGLUT2 protein and mRNA, but VGLUT1 mRNA was undetectable. In conclusion, we show 1) that most colorectal DRG neurons express VGLUT(2), and to a lesser extent, VGLUT(1); 2) abundance of VGLUT2-IR fibers innervating colorectum; and 3) a subpopulation of myenteric plexus neurons expressing VGLUT(2). Altogether, our data suggests a role for VGLUT(2) in colorectal glutamatergic neurotransmission, potentially influencing colorectal sensitivity and motility. J. Comp. Neurol. 519:3346-3366, 2011. (C) 2011 Wiley-Liss, Inc.
  • Takatoshi Nakamura, Hiroyuki Mitomi, Wataru Onozato, Takeo Sato, Atsushi Ikeda, Masanori Naito, Naoto Ogura, Hiroki Kamata, Akira Ooki, Masahiko Watanabe
    Hepato-Gastroenterology 58 112 1947 - 1950 2011年11月 [査読有り][通常論文]
     
    Background/Aims: In Japan, the safety and long-term outcomes of laparoscopic surgery for advanced colorectal cancer remains a matter of debate. We studied the safety and outcomes of laparoscopic surgery in patients with pathological stage II and III colon cancer. Methodology: The study group comprised 253 patients with colon cancer who underwent laparoscopic surgery from January 1998 through December 2006. We studied surgical outcomes, invasiveness, safety, recurrence rates, recurrence patterns, and long-term outcomes. Results: Median follow-up was 67 months (range, 7-149). Laparoscopic surgery was converted to open surgery in 5 patients (2%). Postoperative complications occurred in 23 patients (9%) wound infections were most common (11 patients, 4.3%), followed by ileus (5 patients, 1.9%). Recurrence developed in 66 patients (26%). Liver and lung metastases were the most common types of recurrence there was no port-site recurrence. The 10-year recurrence-free survival rate and the overall survival rate were respectively 92.9% and 93.3% in stage II disease, 82.7% and 82.9% in stage IIIA and IIIB disease, and 70.3% and 68.6% in stage IIIC disease. Conclusions: In patients with pathological stage II and III colon cancer, laparoscopic surgery is safe, minimally invasive, and has good surgical outcomes, overall survival rates and recurrence-free survival rates. Our results suggest that laparoscopic surgery is a viable treatment option for pathological stage II and III colon cancer. © H.G.E. Update Medical Publishing S.A., Athens.
  • T. C. P. Sardella, E. Polgar, M. Watanabe, A. J. Todd
    NEUROSCIENCE 192 708 - 720 2011年09月 [査読有り][通常論文]
     
    Nitric oxide produced by neuronal nitric oxide synthase (nNOS) in the spinal cord is required for development of hyperalgesia in inflammatory and neuropathic pain states. nNOS is expressed by some dorsal horn neurons, and an early study that used a histochemical method to identify these cells suggested that they were mainly inhibitory interneurons. We have carried out a quantitative analysis of nNOS-immunoreactivity in laminae I-III of the rat dorsal horn, to determine the proportion of inhibitory and excitatory neurons and axonal boutons that express the protein. nNOS was present in similar to 5% of neurons in laminae I and III, and 18% of those in lamina II. Although most cells with strong nNOS immunostaining were GABA-immunoreactive, two-thirds of the nNOS-positive cells in lamina II and half of those in lamina III were not GABAergic, and some of these expressed protein kinase C gamma (PKC gamma). We estimate that nNOS is present in 17-19% of the inhibitory interneurons in laminae I-II, and 6% of those in lamina III. However, our results suggest that nNOS is also expressed at a relatively low level by a significant proportion (similar to 17%) of excitatory interneurons in lamina II. nNOS was seldom seen in boutons that contained vesicular glutamate transporter 2, which is expressed by excitatory interneurons, but was co-localised with the vesicular GABA transporter (VGAT, a marker for GABAergic and glycinergic axons). nNOS was detected in 13% of VGAT boutons in lamina I and in 7-8% of those in laminae II-III. However, it was only found in 2-4% of the VGAT boutons that were presynaptic to PKC gamma-expressing interneurons in this region. These results indicate that nNOS is more widely expressed than previously thought, being present in both inhibitory and excitatory neurons. They provide further evidence that axons of neurochemically defined populations of inhibitory interneuron are selective in their post-synaptic targets. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Thomas C. P. Sardella, Erika Polgar, Francesca Garzillo, Takahiro Furuta, Takeshi Kaneko, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 7 2011年09月 [査読有り][通常論文]
     
    Background: The opioid peptide dynorphin is expressed by certain neurons in the superficial dorsal horn of the spinal cord, but little is known about the types of cell that contain dynorphin. In this study, we have used an antibody against the dynorphin precursor preprodynorphin (PPD), to reveal the cell bodies and axons of dynorphin-expressing neurons in the rat spinal cord. The main aims were to estimate the proportion of neurons in each of laminae I-III that express dynorphin and to determine whether they are excitatory or inhibitory neurons. Results: PPD-immunoreactive cells were concentrated in lamina I and the outer part of lamina II (IIo), where they constituted 17% and 8%, respectively, of all neurons. Around half of those in lamina I and 80% of those in lamina II were GABA-immunoreactive. We have previously identified four non-overlapping neurochemical populations of inhibitory interneurons in this region, defined by the presence of neuropeptide Y, galanin, parvalbumin and neuronal nitric oxide synthase. PPD co-localised extensively with galanin in both cell bodies and axons, but rarely or not at all with the other three markers. PPD was present in around 4% of GABAergic boutons (identified by the presence of the vesicular GABA transporter) in laminae I-II. Conclusions: These results show that most dynorphin-expressing cells in the superficial dorsal horn are inhibitory interneurons, and that they largely correspond to the population that is defined by the presence of galanin. We estimate that dynorphin is present in similar to 32% of inhibitory interneurons in lamina I and 11% of those in lamina II. Since the proportion of GABAergic boutons that contain PPD in these laminae was considerably lower than this, our findings suggest that these neurons may generate relatively small axonal arborisations.
  • Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
    PLOS ONE 6 9 2011年09月 [査読有り][通常論文]
     
    Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.
  • Hiroshi Katoh, Keishi Yamashita, Guoqin Wang, Takeo Sato, Takatoshi Nakamura, Masahiko Watanabe
    Annals of Surgical Oncology 18 9 2432 - 2441 2011年09月 [査読有り][通常論文]
     
    Background: Previous studies have suggested a detrimental prognostic effect of preoperative obstruction proximal to colorectal cancer (CRC). If such a detrimental effect is preserved in each stage of advanced (stage II or III) CRC, we can identify high-risk patients. Methods: We enrolled 641 patients with pathologically confirmed advanced CRC (stage II, n = 207 stage III, n = 434) who had undergone curative resection of the primary lesion. The association of preoperative obstruction with clinicopathologic parameters was evaluated. Kaplan-Meier analysis and Cox proportional hazard models were used to estimate the effect of preoperative obstruction on disease-free survival in each stage. Results: Preoperative obstruction was seen in 63 patients (9.8%) (stage II, n = 16 stage III, n = 47). Multivariable analysis showed that preoperative obstruction was significantly associated with preoperative elevation of carcinoembryonic antigen level in patients with colon cancer (odds ratio = 3.59 P < 0.001), while it was correlated with poor differentiation in patients with rectal cancer (odds ratio = 3.99 P = 0.016). Preoperative obstruction was a significant prognostic factor in stage III CRC (P < 0.001), but not in stage II disease. Multivariable prognostic analysis showed that preoperative obstruction was a remnant independent prognostic factor in stage III CRC. This finding was confirmed by separate analyses of colon and rectal cancer. Preoperative obstruction was associated with systemic recurrence (P = 0.003) rather than peritoneal or local recurrence. Conclusions: These findings suggest that preoperative obstruction may predict worse long-term prognosis in patients with stage III CRC and may be a potential clinical marker to identify patients with high-risk stage III CRC. © 2011 Society of Surgical Oncology.
  • Hiromitsu Moriya, Makoto Ohbu, Nobuyuki Kobayashi, Satoshi Tanabe, Natsuya Katada, Nobue Futawatari, Shinichi Sakuramoto, Shiro Kikuchi, Isao Okayasu, Masahiko Watanabe
    World Journal of Surgery 35 9 2031 - 2037 2011年09月 [査読有り][通常論文]
     
    Background: Resected specimens of superficial squamous cell carcinoma of the esophagus (SSCCE) underwent D2-40 immunostaining to accurately assess lymphatic tumor emboli (LY) and to analyze correlations between LY and lymph node metastasis (N). This present study was designed to determine the accuracy of LY grade for predicting the risk of N. Materials and methods: We studied 75 patients with SSCCE who underwent surgical resection of their tumors. Resected specimens were sliced into continuous sections at 5 mm intervals. Intramucosal cancers are classified into three groups (m1, m2, m3), and submucosal cancers are also divided into three groups (sm1, sm2, sm3). The numbers of LY present in lymphatic ducts on D2-40 immunostaining, venous tumor emboli (V) on CD34 immunostaining, and lymphatic tumor emboli (ly) and V on hematoxylin-eosin staining (HE) and elastica van Gieson staining (EVG) were counted for each case. The presence of lymphatic tumor emboli was graded according to the total number of LY per case as follows: 0, LY0 1 to 2, LY1 3 to 9, LY2 and 10 or more, LY3. Results: All m1 and m2 cases were LY- and N- Lymphatic tumor emboli were present in 54% of m3 cases, 70% of sm1 cases, 54% of sm2 cases, and 75% of sm3 cases. Determination of N was positive in 18% of m3 cases, 47% of sm1 cases, 36% of sm2 cases, and 62% of sm3 cases. The frequency of LY significantly correlated with the number of N (p < 0.0001). Multiple regression analysis showed that only LY and V significantly correlated with N. When the detection rate of N was compared between LY and ly, LY was superior to ly in terms of specificity, accuracy, positive predictive value, and false positive rate. As for LY grade, N was positive in 39.1% of LY1 cases, 81.8% of LY2 cases, and 100% of LY3 cases. Even in LY-, N was positive in one sm1 case and in two sm2 cases. In the sm1 case, the depth of invasion was 350 μm from the lower margin of the muscularis mucosae. Conclusions: Evaluation of lymphatic invasion on the basis of LY is more accurate for the prediction of N than conventional techniques, and LY grade strongly correlates with N. In patients with SSCCE, mucosal cancers (m1, m2, and m3) and submucosal cancers with a depth of invasion of ≤200 μm from the lower margin of the muscularis mucosae on endoscopic mucosal resection have a low risk of N if the number of LY is 0. Endoscopic mucosal resection alone can provide good treatment outcomes in such patients. © 2011 Société Internationale de Chirurgie.
  • Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
    PLOS ONE 6 9 2011年09月 [査読有り][通常論文]
     
    Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.
  • Keishi Yamashita, Shinichi Sakuramoto, Masayuki Nemoto, Tomotaka Shibata, Hiroaki Mieno, Natsuya Katada, Shiroh Kikuchi, Masahiko Watanabe
    World Journal of Gastroenterology 17 29 3390 - 3397 2011年08月07日 [査読有り][通常論文]
     
    AIM: To investigate the trend in gastric cancer surgery in the context of rapid therapeutic advancement in Japan and East Asia. METHODS: A retrospective analysis was performed on 4163 patients who underwent gastric resection for gastric cancer with histological confirmation between 1971 and 2007 at the surgical unit in Kitasato University Hospital, to determine the trend in gastric cancer requiring surgery. RESULTS: Gastric cancer requiring surgical resection increased in our hospital, but the incidence adjusted for population was constant during the observed period. Interestingly, the ratio of diffuse type/intestinal type gastric cancer was unexpectedly unchanged, and that of advanced/early gastric cancer (EGC) was, however, markedly reduced, while the actual incidence of potentially curative advanced gastric cancer tended to decrease. The incidence of EGC requiring surgery tended to increase as a whole, which is consistent with increased prevalence of endoscopic surveillance. As a result, overall survival and mortality of gastric cancer requiring gastric resection has recently markedly improved. CONCLUSION: In Japan, planned interventions may improve surgical gastric cancer mortality, but an unexpected trend of persistent existence of intestinal type cancer suggests the need for more robust medical intervention. © 2011 Baishideng. All rights reserved.
  • Pablo R. Brumovsky, Kim B. Seroogy, Kerstin H. Lundgren, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
    EXPERIMENTAL NEUROLOGY 230 2 258 - 272 2011年08月 [査読有り][通常論文]
     
    Glutamate is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons. However, to date a glutamatergic phenotype of autonomic neurons has not been described. Therefore, we explored the expression of vesicular glutamate transporter (VGLUT) types 1,2 and 3 in lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) of naive BALB/C mice, as well as after pelvic nerve axotomy (PNA), using immunohistochemistry and in situ hybridization. Colocalization with activating transcription factor-3 (ATF-3), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and calcitonin gene-related peptide was also examined. Sham-PNA, sciatic nerve axotomy (SNA) or naive mice were included. In naive mice, VGLUT(2)-like immunoreactivity (LI) was only detected in fibers and varicosities in LSC and MPG; no ATF-3-immunoreactive (IR) neurons were visible. In contrast, PNA induced upregulation of VGLUT(2) protein and transcript, as well as of ATF-3-LI in subpopulations of LSC neurons. Interestingly, VGLUT(2)-IR LSC neurons coexpressed ATF-3, and often lacked the noradrenergic marker TH. SNA only increased VGLUT(2) protein and transcript in scattered LSC neurons. Neither PNA nor SNA upregulated VGLUT(2) in MPG neurons. We also found perineuronal baskets immunoreactive either for VGLUT(2) or the acetylcholinergic marker VAChT in non-PNA MPGs, usually around TH-IR neurons. VGLUT(1)-LI was restricted to some varicosities in MPGs, was absent in LSCs, and remained largely unaffected by PNA or SNA. This was confirmed by the lack of expression of VGLUT(1) or VGLUT(3) mRNAs in LSCs, even after PNA or SNA. Taken together, axotomy of visceral and non-visceral nerves results in a glutamatergic phenotype of some LSC neurons. In addition, we show previously non-described MPG perineuronal glutamatergic baskets. (C) 2011 Elsevier Inc. All rights reserved.
  • Pablo R. Brumovsky, Kim B. Seroogy, Kerstin H. Lundgren, Masahiko Watanabe, Tomas Hokfelt, G. F. Gebhart
    EXPERIMENTAL NEUROLOGY 230 2 258 - 272 2011年08月 [査読有り][通常論文]
     
    Glutamate is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons. However, to date a glutamatergic phenotype of autonomic neurons has not been described. Therefore, we explored the expression of vesicular glutamate transporter (VGLUT) types 1,2 and 3 in lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) of naive BALB/C mice, as well as after pelvic nerve axotomy (PNA), using immunohistochemistry and in situ hybridization. Colocalization with activating transcription factor-3 (ATF-3), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and calcitonin gene-related peptide was also examined. Sham-PNA, sciatic nerve axotomy (SNA) or naive mice were included. In naive mice, VGLUT(2)-like immunoreactivity (LI) was only detected in fibers and varicosities in LSC and MPG; no ATF-3-immunoreactive (IR) neurons were visible. In contrast, PNA induced upregulation of VGLUT(2) protein and transcript, as well as of ATF-3-LI in subpopulations of LSC neurons. Interestingly, VGLUT(2)-IR LSC neurons coexpressed ATF-3, and often lacked the noradrenergic marker TH. SNA only increased VGLUT(2) protein and transcript in scattered LSC neurons. Neither PNA nor SNA upregulated VGLUT(2) in MPG neurons. We also found perineuronal baskets immunoreactive either for VGLUT(2) or the acetylcholinergic marker VAChT in non-PNA MPGs, usually around TH-IR neurons. VGLUT(1)-LI was restricted to some varicosities in MPGs, was absent in LSCs, and remained largely unaffected by PNA or SNA. This was confirmed by the lack of expression of VGLUT(1) or VGLUT(3) mRNAs in LSCs, even after PNA or SNA. Taken together, axotomy of visceral and non-visceral nerves results in a glutamatergic phenotype of some LSC neurons. In addition, we show previously non-described MPG perineuronal glutamatergic baskets. (C) 2011 Elsevier Inc. All rights reserved.
  • Devinder Arora, Matthew Hearing, Desirae M. Haluk, Kelsey Mirkovic, Ana Fajardo-Serrano, Martin W. Wessendorf, Masahiko Watanabe, Rafael Lujan, Kevin Wickman
    JOURNAL OF NEUROSCIENCE 31 34 12251 - 12257 2011年08月 [査読有り][通常論文]
     
    Enhanced glutamatergic neurotransmission in dopamine (DA) neurons of the ventral tegmental area (VTA), triggered by a single cocaine injection, represents an early adaptation linked to the more enduring effects of abused drugs that characterize addiction. Here, we examined the impact of in vivo cocaine exposure on metabotropic inhibitory signaling involving G-protein-gated inwardly rectifying K+ (Girk) channels in VTA DA neurons. Somatodendritic Girk currents evoked by the GABA(B) receptor (GABA(B)R) agonist baclofen were diminished in a dose-dependent manner in mice given a single cocaine injection. This adaptation persisted for 3-4 d, was specific for DA neurons of the VTA, and occurred in parallel with an increase in spontaneous glutamatergic neurotransmission. No additional suppression of GABA(B)R-Girk signaling was observed following repeated cocaine administration. While total Girk2 and GABA(B)R1 mRNA and protein levels were unaltered by cocaine exposure in VTA DA neurons, the cocaine-induced decrease in GABA(B)R-Girk signaling correlated with a reduction in Girk2-containing channels at the plasma membrane in VTA DA neurons. Systemic pretreatment with sulpiride, but not SCH23390 (7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol), prevented the cocaine-induced suppression of GABA(B)R-Girk signaling, implicating D-2/3 DA receptor activation in this adaptation. The acute cocaine-induced weakening of somatodendritic Girk signaling complements the previously demonstrated cocaine-induced strengthening of glutamatergic neurotransmission, likely contributing to enhanced output of VTA DA neurons during the early stages of addiction.
  • Christoph Straub, David L. Hunt, Miwako Yamasaki, Kwang S. Kim, Masahiko Watanabe, Pablo E. Castillo, Susumu Tomita
    NATURE NEUROSCIENCE 14 7 866 - U83 2011年07月 [査読有り][通常論文]
     
    Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.
  • Takatoshi Nakamura, Hiroyuki Mitomi, Wataru Onozato, Takeo Sato, Atsushi Ikeda, Masanori Naito, Naoto Ogura, Hiroki Kamata, Akira Ooki, Masahiko Watanabe
    Hepato-Gastroenterology 58 109 1200 - 1204 2011年07月 [査読有り][通常論文]
     
    Background/Aims: We compared the results of laparoscopic resection of colon cancer between patients 75 years or older and those 64 years or younger, to confirm whether this procedure is warranted in elderly patients. Methodology: The study group was comprised of patients with stage I to III colon cancer treated by laparoscopic surgery from 1995 through 2006. Oncologic outcomes were compared between 74 patients 75 years or older (elderly group) and 74 patients 64 years or younger (younger group) who were matched for gender, tumor location and pathological tumor-node-metastasis (TNM) stage. Results: In patients with stage I or II disease, the disease-free survival rate and overall survival rate were similar in the elderly group (100% and 100%, respectively) and the younger group (95.6% and 95.8%, respectively). In patients with stage III disease, the disease-free survival rate and overall survival rate were also similar in the elderly group (76.7% and 88.5%, respectively) and the younger group (88.5% and 88.5%, respectively). Conclusions: Postoperative complications and long-term oncologic outcomes were similar in elderly patients and younger patients with colon cancer who underwent laparoscopic colectomy in our hospital. These results demonstrate that laparoscopic resection of colon cancer is warranted in patients 75 years or older. © H.G.E. Update Medical Publishing S.A.
  • Yamato Hida, Masahiro Fukaya, Akari Hagiwara, Maki Deguchi-Tawarada, Toshinori Yoshioka, Isao Kitajima, Eiji Inoue, Masahiko Watanabe, Toshihisa Ohtsuka
    JOURNAL OF BIOCHEMISTRY 149 6 693 - 700 2011年06月 [査読有り][通常論文]
     
    The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.
  • Duane Allen, Chris T. Bond, Rafael Lujan, Carmen Ballesteros-Merino, Mike T. Lin, Kang Wang, Nathan Klett, Masahiko Watanabe, Ryuichi Shigemoto, Robert W. Stackman, James Maylie, John P. Adelman
    NATURE NEUROSCIENCE 14 6 744 - U344 2011年06月 [査読有り][通常論文]
     
    SK2-containing channels are expressed in the postsynaptic density (PSD) of dendritic spines on mouse hippocampal area CA1 pyramidal neurons and influence synaptic responses, plasticity and learning. The Sk2 gene (also known as Kcnn2) encodes two isoforms that differ only in the length of their N-terminal domains. SK2-long (SK2-L) and SK2-short (SK2-S) are coexpressed in CA1 pyramidal neurons and likely form heteromeric channels. In mice lacking SK2-L (SK2-S only mice), SK2-S-containing channels were expressed in the extrasynaptic membrane, but were excluded from the PSD. The SK channel contribution to excitatory postsynaptic potentials was absent in SK2-S only mice and was restored by SK2-L re-expression. Blocking SK channels increased the amount of long-term potentiation induced in area CA1 in slices from wild-type mice but had no effect in slices from SK2-S only mice. Furthermore, SK2-S only mice outperformed wild-type mice in the novel object recognition task. These results indicate that SK2-L directs synaptic SK2-containing channel expression and is important for normal synaptic signaling, plasticity and learning.
  • Kouichi Hashimoto, Mika Tsujita, Taisuke Miyazaki, Kazuo Kitamura, Maya Yamazaki, Hee-Sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 24 9987 - 9992 2011年06月 [査読有り][通常論文]
     
    Neural circuits are initially redundant but rearranged through activity-dependent synapse elimination during postnatal development. This process is crucial for shaping mature neural circuits and for proper brain function. At birth, Purkinje cells (PCs) in the cerebellum are innervated by multiple climbing fibers (CFs) with similar synaptic strengths. During postnatal development, a single CF is selectively strengthened in each PC through synaptic competition, the strengthened single CF undergoes translocation to a PC dendrite, and massive elimination of redundant CF synapses follows. To investigate the cellular mechanisms of this activity-dependent synaptic refinement, we generated mice with PC-selective deletion of the Ca(v)2.1 P/Q-type Ca2+ channel, the major voltage-dependent Ca2+ channel in PCs. In the PC-selective Ca(v)2.1 knockout mice, Ca2+ transients induced by spontaneous CF inputs are markedly reduced in PCs in vivo. Not a single but multiple CFs were equally strengthened in each PC from postnatal day 5 (P5) to P8, multiple CFs underwent translocation to PC dendrites, and subsequent synapse elimination until around P12 was severely impaired. Thus, P/Q-type Ca2+ channels in postsynaptic PCs mediate synaptic competition among multiple CFs and trigger synapse elimination in developing cerebellum.
  • Yamato Hida, Masahiro Fukaya, Akari Hagiwara, Maki Deguchi-Tawarada, Toshinori Yoshioka, Isao Kitajima, Eiji Inoue, Masahiko Watanabe, Toshihisa Ohtsuka
    JOURNAL OF BIOCHEMISTRY 149 6 693 - 700 2011年06月 [査読有り][通常論文]
     
    The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.
  • Kouichi Hashimoto, Mika Tsujita, Taisuke Miyazaki, Kazuo Kitamura, Maya Yamazaki, Hee-Sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 24 9987 - 9992 2011年06月 [査読有り][通常論文]
     
    Neural circuits are initially redundant but rearranged through activity-dependent synapse elimination during postnatal development. This process is crucial for shaping mature neural circuits and for proper brain function. At birth, Purkinje cells (PCs) in the cerebellum are innervated by multiple climbing fibers (CFs) with similar synaptic strengths. During postnatal development, a single CF is selectively strengthened in each PC through synaptic competition, the strengthened single CF undergoes translocation to a PC dendrite, and massive elimination of redundant CF synapses follows. To investigate the cellular mechanisms of this activity-dependent synaptic refinement, we generated mice with PC-selective deletion of the Ca(v)2.1 P/Q-type Ca2+ channel, the major voltage-dependent Ca2+ channel in PCs. In the PC-selective Ca(v)2.1 knockout mice, Ca2+ transients induced by spontaneous CF inputs are markedly reduced in PCs in vivo. Not a single but multiple CFs were equally strengthened in each PC from postnatal day 5 (P5) to P8, multiple CFs underwent translocation to PC dendrites, and subsequent synapse elimination until around P12 was severely impaired. Thus, P/Q-type Ca2+ channels in postsynaptic PCs mediate synaptic competition among multiple CFs and trigger synapse elimination in developing cerebellum.
  • Takeshi Izumi, Shuken Boku, Weng Shinmin, Takeshi Inoue, Kotaro Konno, Taku Yamaguchi, Takayuki Yoshida, Machiko Matsumoto, Masahiko Watanabe, Tsukasa Koyama, Mitsuhiro Yoshioka
    JOURNAL OF NEUROSCIENCE RESEARCH 89 5 773 - 790 2011年05月 [査読有り][通常論文]
     
    The amygdala is one of the crucial brain structures for conditioned fear, in which conditioned stimuli are received by the basolateral nucleus of the amygdala (BLA), inducing a fear reaction via the central nucleus of the amygdala (CeA). Whereas BLA sends glutamatergic projections into CeA, the intercalated nucleus of the amygdala (ITC) sends GABAergic projections into CeA, which is doubly regulated by BLA and ITC. In the present study, we investigated the characteristics of the neural cells activated by retrieval of conditioned fear in BLA and ITC using immunohistochemistry, in situ hybridization, and Western blot analysis of transcription factors and neural cell markers. Because most conditioned fear-induced c-Fos-positive cells in BLA were glutaminase positive and 67-kDa isomer of glutamic acid decarboxylase (GAD67) negative, these cells are speculated to be glutamatergic. Seventy-eight percent of the phosphorylated CREB (pCREB)positive cells were glutaminase double positive and 13% of the pCREB-positive cells were GAD67 double positive, indicating that many of the conditioned fear-induced pCREB-positive cells in BLA were glutamatergic, but at least some of the pCREB-positive cells were GABAergic. These results suggested that CREB phosphorylation was increased both in glutamatergic and in GABAergic neurons, but c-Fos expression was increased mainly in glutamatergic neurons in BLA. CREB phosphorylation but not c-Fos expression in ITC was specifically increased by retrieval of conditioned fear. It is therefore speculated that ITC GABAergic neurons were activated by retrieval of conditioned fear and that transcription factors other than c-Fos were relevant to the activation. (C) 2011 Wiley-Liss, Inc.
  • Davor Stanic, Jan Mulder, Masahiko Watanabe, Tomas Hokfelt
    JOURNAL OF COMPARATIVE NEUROLOGY 519 7 1219 - 1257 2011年05月 [査読有り][通常論文]
     
    Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling-related molecules, which previously have not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R-like immunoreactivity (-LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY- and Y1R-LI was found in the HiFo. Following colchicine treatment, Y2R-LI accumulated in cell bodies that coexpressed gamma-aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R-positive nerve terminals appeared to surround GABA-immunoreactive (ir) cells in the HiFo and other neuronal populations, e. g., NPY-ir cells in HiFo and tyrosine hydroxylase-ir cells in the hypothalamus. In the HiFo, Y2R-ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R-LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release. J. Comp. Neurol. 519: 1219-1257, 2011. (C) 2011 Wiley-Liss, Inc.
  • Shunya Suzuki, Keiichi Tamai, Masahiko Watanabe, Masanao Kyuuma, Masao Ono, Kazuo Sugamura, Nobuyuki Tanaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 408 4 582 - 588 2011年05月 [査読有り][通常論文]
     
    Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH(-1-) mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH(-1-) mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates. (C) 2011 Elsevier Inc. All rights reserved.
  • Motokazu Uchigashima, Maya Yamazaki, Miwako Yamasaki, Asami Tanimura, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 21 7700 - 7714 2011年05月 [査読有り][通常論文]
     
    2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)-granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase alpha (DGL alpha) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC-GC synapses, as they were abolished in DGL alpha-knock-out mice. Immunohistochemistry revealed that DGL alpha was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB(1) receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC-GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC-GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC-GC synapses by escaping from enzymatic degradation. This molecular-anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.
  • Motokazu Uchigashima, Maya Yamazaki, Miwako Yamasaki, Asami Tanimura, Kenji Sakimura, Masanobu Kano, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 21 7700 - 7714 2011年05月 [査読有り][通常論文]
     
    2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the 2-AG signaling system at mossy cell (MC)-granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that 2-AG produced by diacylglycerol lipase alpha (DGL alpha) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC-GC synapses, as they were abolished in DGL alpha-knock-out mice. Immunohistochemistry revealed that DGL alpha was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB(1) receptors accumulated in the terminal portion of MC axons. On the other hand, the major 2-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC-GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for 2-AG-mediated retrograde suppression of MC-GC synaptic transmission and also suggest that 2-AG released from activated GC spines is readily accessible to nearby MC-GC synapses by escaping from enzymatic degradation. This molecular-anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.
  • Sheena Y. X. Tiong, Erika Polgar, Josie C. van Kralingen, Masahiko Watanabe, Andrew J. Todd
    MOLECULAR PAIN 7 2011年05月 [査読有り][通常論文]
     
    Background: Inhibitory interneurons constitute 30-40% of neurons in laminae I-III and have an important anti-nociceptive role. However, because of the difficulty in classifying them we know little about their organisation. Previous studies have identified 3 non-overlapping groups of inhibitory interneuron, which contain neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin, and have shown that these differ in postsynaptic targets. Some inhibitory interneurons contain galanin and the first aim of this study was to determine whether these form a different population from those containing NPY, nNOS or parvalbumin. We also estimated the proportion of neurons and GABAergic axons that contain galanin in laminae I-III. Results: Galanin cells were concentrated in laminae I-IIo, with few in laminae IIi-III. Galanin showed minimal co-localisation with NPY, nNOS or parvalbumin in laminae I-II, but most galanin-containing cells in lamina III were nNOS-positive. Galanin cells constituted similar to 7%, 3% and 2% of all neurons in laminae I, II and III, and we estimate that this corresponds to 26%, 10% and 5% of the GABAergic neurons in these laminae. However, galanin was only found in similar to 6% of GABAergic boutons in laminae I-IIo, and similar to 1% of those in laminae IIi-III. Conclusions: These results show that galanin, NPY, nNOS and parvalbumin can be used to define four distinct neurochemical populations of inhibitory interneurons. Together with results of a recent study, they suggest that the galanin and NPY populations account for around half of the inhibitory interneurons in lamina I and a quarter of those in lamina II.
  • Shunya Suzuki, Keiichi Tamai, Masahiko Watanabe, Masanao Kyuuma, Masao Ono, Kazuo Sugamura, Nobuyuki Tanaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 408 4 582 - 588 2011年05月 [査読有り][通常論文]
     
    Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH(-1-) mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH(-1-) mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates. (C) 2011 Elsevier Inc. All rights reserved.
  • Davor Stanic, Jan Mulder, Masahiko Watanabe, Tomas Hokfelt
    JOURNAL OF COMPARATIVE NEUROLOGY 519 7 1219 - 1257 2011年05月 [査読有り][通常論文]
     
    Neuropeptide Y (NPY) is widely expressed in the brain and its biological effects are mediated through a variety of receptors. We examined, using immunohistochemistry, expression of the Y2 receptor (R) protein in the adult mouse brain and its association with NPY and the Y1R, as well as a range of additional neurotransmitters and signaling-related molecules, which previously have not been defined. Our main focus was on the hippocampal formation (HiFo), amygdaloid complex, and hypothalamus, considering the known functions of NPY and the wide expression of NPY, Y1R, and Y2R in these regions. Y2R-like immunoreactivity (-LI) was distributed in nerve fibers/terminal endings throughout the brain axis, without apparent colocalization with NPY or the Y1R. Occasional coexistence between NPY- and Y1R-LI was found in the HiFo. Following colchicine treatment, Y2R-LI accumulated in cell bodies that coexpressed gamma-aminobutyric acid (GABA) in a population of cells in the amygdaloid complex and lateral septal nucleus, but not in the HiFo. Instead, Y2R-positive nerve terminals appeared to surround GABA-immunoreactive (ir) cells in the HiFo and other neuronal populations, e. g., NPY-ir cells in HiFo and tyrosine hydroxylase-ir cells in the hypothalamus. In the HiFo, Y2R-ir mossy fibers coexpressed GABA, glutamic acid decarboxylase 67 and calbindin, and Y2R-LI was found in the same fibers that contained the presynaptic metabotropic glutamate receptor 2, but not together with any of the three vesicular glutamate transporters. Our findings provide further support that Y2R is mostly presynaptic, and that Y2Rs thus have a modulatory role in mediating presynaptic neurotransmitter release. J. Comp. Neurol. 519: 1219-1257, 2011. (C) 2011 Wiley-Liss, Inc.
  • Takeshi Izumi, Shuken Boku, Weng Shinmin, Takeshi Inoue, Kotaro Konno, Taku Yamaguchi, Takayuki Yoshida, Machiko Matsumoto, Masahiko Watanabe, Tsukasa Koyama, Mitsuhiro Yoshioka
    JOURNAL OF NEUROSCIENCE RESEARCH 89 5 773 - 790 2011年05月 [査読有り][通常論文]
     
    The amygdala is one of the crucial brain structures for conditioned fear, in which conditioned stimuli are received by the basolateral nucleus of the amygdala (BLA), inducing a fear reaction via the central nucleus of the amygdala (CeA). Whereas BLA sends glutamatergic projections into CeA, the intercalated nucleus of the amygdala (ITC) sends GABAergic projections into CeA, which is doubly regulated by BLA and ITC. In the present study, we investigated the characteristics of the neural cells activated by retrieval of conditioned fear in BLA and ITC using immunohistochemistry, in situ hybridization, and Western blot analysis of transcription factors and neural cell markers. Because most conditioned fear-induced c-Fos-positive cells in BLA were glutaminase positive and 67-kDa isomer of glutamic acid decarboxylase (GAD67) negative, these cells are speculated to be glutamatergic. Seventy-eight percent of the phosphorylated CREB (pCREB)positive cells were glutaminase double positive and 13% of the pCREB-positive cells were GAD67 double positive, indicating that many of the conditioned fear-induced pCREB-positive cells in BLA were glutamatergic, but at least some of the pCREB-positive cells were GABAergic. These results suggested that CREB phosphorylation was increased both in glutamatergic and in GABAergic neurons, but c-Fos expression was increased mainly in glutamatergic neurons in BLA. CREB phosphorylation but not c-Fos expression in ITC was specifically increased by retrieval of conditioned fear. It is therefore speculated that ITC GABAergic neurons were activated by retrieval of conditioned fear and that transcription factors other than c-Fos were relevant to the activation. (C) 2011 Wiley-Liss, Inc.
  • Akira Ooki, Keishi Yamashita, Shiro Kikuchi, Shinichi Sakuramoto, Natsuya Katada, Mina Waraya, Hiroshi Kawamata, Hiroshi Nishimiya, Kazunori Nakamura, Masahiko Watanabe
    BMC Cancer 11 2011年04月06日 [査読有り][通常論文]
     
    Background: Phosphatase of regenerating liver-3 (PRL-3) has deserved attention as a crucial molecule in the multiple steps of metastasis. In the present study, we examined the mechanisms regulating PRL-3 expression, and assessed the clinical potential of PRL-3-targeted therapy in gastric cancer.Methods: PRL-3 genomic amplification was analyzed using quantitative-polymerase chain reaction and/or fluorescence in situ hybridization in 77 primary gastric tumors. The anticancer activity of PRL-3 inhibitor (1-4-bromo-2-benzylidene rhodanine) treatment was evaluated against cancer cells with different genetic and expression status.Results: PRL-3 genomic amplification was closely concordant with high level of its protein expression in cell lines, and was found in 20% (8/40) among human primary tumors with its expression, which were all stage III/IV disease (40%, 8/20), but in none (0/37) among those without expression. Additionally, PRL-3 genomic amplification was associated with metastatic lymph node status, leading to advanced stage and thereby poor outcomes in patients with lymph node metastasis (P = 0.021). PRL-3 small interfering RNA robustly repressed metastatic properties, including cell proliferation, invasion, and anchorage-independent colony formation. Although neither PRL-3 genomic amplification nor expression level was responsible for the sensitivity to PRL-3 inhibitor treatment, the inhibitor showed dose-dependent anticancer efficacy, and remarkably induced apoptosis on all the tested cell lines with PRL-3 expression.Conclusions: We have for the first time, demonstrated that PRL-3 genomic amplification is one of the predominant mechanisms inducing its expression, especially in more advanced stage, and that PRL-3-targeted therapy may have a great potential against gastric cancer with its expression. © 2011 Ooki et al licensee BioMed Central Ltd.
  • Erika Polgar, Thomas C. P. Sardella, Masahiko Watanabe, Andrew J. Todd
    JOURNAL OF COMPARATIVE NEUROLOGY 519 6 1007 - 1023 2011年04月 [査読有り][通常論文]
     
    Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cc (PKC gamma)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron. J. Comp. Neurol. 519: 1007-1023, 2011. (C) 2010 Wiley-Liss, Inc.
  • Jan Mulder, Misha Zilberter, Susana J. Pasquare, Alan Alpar, Gunnar Schulte, Samira G. Ferreira, Attila Koefalvi, Ana M. Martin-Moreno, Erik Keimpema, Heikki Tanila, Masahiko Watanabe, Ken Mackie, Tibor Hortobagyi, Maria L. de Ceballos, Tibor Harkany
    BRAIN 134 1041 - 1060 2011年04月 [査読有り][通常論文]
     
    Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimer's disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer's disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer's disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-beta-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase alpha and beta isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer's (Braak stage VI), with ectopic sn-1-diacylglycerol lipase beta expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer's disease (Braak stage III). However, Alzheimer's pathology differentially impacts serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase alpha/beta-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer's tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer's disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-beta. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer's disease.
  • Eszter Szabadits, Csaba Cserep, Andras Szonyi, Yugo Fukazawa, Ryuichi Shigemoto, Masahiko Watanabe, Shigeyoshi Itohara, Tamas F. Freund, Gabor Nyiri
    JOURNAL OF NEUROSCIENCE 31 16 5893 - 5904 2011年04月 [査読有り][通常論文]
     
    GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)-cGMP cascade triggered by NMDA receptor(NMDAR) activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that-similar to glutamatergic synapses-the Ca2+ transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 mu M NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze-fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition via NO-cGMP signaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus.
  • Eszter Szabadits, Csaba Cserep, Andras Szonyi, Yugo Fukazawa, Ryuichi Shigemoto, Masahiko Watanabe, Shigeyoshi Itohara, Tamas F. Freund, Gabor Nyiri
    JOURNAL OF NEUROSCIENCE 31 16 5893 - 5904 2011年04月 [査読有り][通常論文]
     
    GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)-cGMP cascade triggered by NMDA receptor(NMDAR) activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that-similar to glutamatergic synapses-the Ca2+ transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 mu M NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze-fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition via NO-cGMP signaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus.
  • Erika Polgar, Thomas C. P. Sardella, Masahiko Watanabe, Andrew J. Todd
    JOURNAL OF COMPARATIVE NEUROLOGY 519 6 1007 - 1023 2011年04月 [査読有り][通常論文]
     
    Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cc (PKC gamma)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron. J. Comp. Neurol. 519: 1007-1023, 2011. (C) 2010 Wiley-Liss, Inc.
  • Jan Mulder, Misha Zilberter, Susana J. Pasquare, Alan Alpar, Gunnar Schulte, Samira G. Ferreira, Attila Koefalvi, Ana M. Martin-Moreno, Erik Keimpema, Heikki Tanila, Masahiko Watanabe, Ken Mackie, Tibor Hortobagyi, Maria L. de Ceballos, Tibor Harkany
    BRAIN 134 1041 - 1060 2011年04月 [査読有り][通常論文]
     
    Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimer's disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer's disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer's disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-beta-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase alpha and beta isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer's (Braak stage VI), with ectopic sn-1-diacylglycerol lipase beta expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer's disease (Braak stage III). However, Alzheimer's pathology differentially impacts serine hydrolase alpha/beta-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase alpha/beta-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer's tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer's disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-beta. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer's disease.
  • Atsuko Tsutsui, Takatoshi Nakamura, Hiroyuki Mitomi, Wataru Onozato, Takeo Sato, Heita Ozawa, Masanori Naito, Atsushi Ikeda, Atsushi Ihara, Masahiko Watanabe
    Surgery Today 41 4 572 - 575 2011年04月 [査読有り][通常論文]
     
    Sacrococcygeal teratoma is a relatively rare congenital retroperitoneal tumor in adults. The standard treatment is a complete tumor resection. This report describes the successful laparoscopic resection of a sacrococcygeal teratoma. The patient was a 27-year-old woman with a well-demarcated cystic mass, 6 cm in diameter, in the retroperitoneum overlying the anterior surface of the sacrum. The mass was resected laparoscopically. A histopathological examination showed a mature teratoma. The magnifying function of the laparoscope allowed an en bloc resection in the narrow pelvic cavity, without damaging the tumor. The aesthetic outcome was excellent. The patient remains relapse-free at 1 year 6 months after surgery. © Springer 2011.
  • Miyazaki T, Watanabe M
    Anatomical science international 86 1 10 - 18 1 2011年03月 [査読有り][通常論文]
     
    In the adult cerebellum, a single climbing fiber (CF) innervates proximal dendrites of Purkinje cells (PCs). This monoinnervation is established by the developmental elimination of surplus CFs through homosynaptic competition among multiply innervating CFs and heterosynaptic competition between CFs and parallel fibers, i.e., granule cell axons innervating distal PC dendrites. Although the developmental process of CF monoinnervation and defects in it in mutant and experimental animal models have been extensively studied by electrophysiological techniques, for quite some time this subject was poorly understood from a morphological perspective due to a lack of neuroanatomical methods that could distinguish CFs with different neuronal origins. Soon after the identification of type 2 vesicular glutamate transporter (VGluT2) that selectively detects CF terminals in the molecular layer, we developed a novel method of combined anterograde tracer labeling and VGluT2 immunohistochemistry. This method enables us to identify the mode (mono vs. multiple) of CF innervation and the site of multiple innervation. Since then, we have applied this method to various kinds of gene-manipulated mice manifesting ataxia and other cerebellar phenotypes. In this review, we summarize experimental procedures for the combined tracer/VGluT2 labeling method, and then introduce what we have learned by applying this method in studies on the role of GluR delta 2 and Ca(v)2.1 in CF monoinnervation. This method has provided informative anatomical correlates to electrophysiological data and vice versa, and will extend our knowledge of the molecular and cellular mechanisms for the development, plasticity, degeneration, and repair of the CF-PC projection system.
  • Tayo Katano, Takanobu Nakazawa, Terumasa Nakatsuka, Masahiko Watanabe, Tadashi Yamamoto, Seiji Ito
    NEUROPHARMACOLOGY 60 4 609 - 616 2011年03月 [査読有り][通常論文]
     
    Previously we demonstrated that phosphorylation of NR2B subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor at Tyr1472 is increased in a neuropathic-pain model and that this phosphorylation is required for the maintenance of neuropathic pain by L5-spinal nerve transection. We obtained these results by using a selective NR2B antagonist and mice deficient in Fyn, which is an Src-family tyrosine protein kinase. However, how Tyr1472 phosphorylation of NR2B is involved in the maintenance of neuropathic pain was unclear. Here, we demonstrated that neuropathic pain was markedly attenuated in the spared nerve injury model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of NR2B (Y1472F-KI). While phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) at its Thr286 and that of the GluR1 subunit of the AMPA receptor at its Ser831 was enhanced in the spinal dorsal horn after spared nerve injury in wild-type mice, such phosphorylation was markedly impaired in Y1472F-KI mice. Inhibition of CaMKII by intrathecal injection of KN93, an inhibitor of CaMKII, reduced mechanical allodynia and phosphorylation of CaMKII at its Thr286 and that of GluR1 at its Ser831 in the spinal cord 7 days after spared nerve injury. These results demonstrate that the phosphorylation of CaMKII and GluR1 occurs downstream of the Tyr1472 phosphorylation of NR2B subunits in the spinal cord and give the first suggestion that activation of CaMKII and GluR1-AMPA receptors may be involved in mechanical allodynia caused by peripheral nerve injury. (C) 2011 Elsevier Ltd. All rights reserved.
  • Masahiro Fukaya, Akifumi Kamata, Yoshinobu Hara, Hideaki Tamaki, Osamu Katsumata, Naoki Ito, Shin'ichi Takeda, Yutaka Hata, Tatsuo Suzuki, Masahiko Watanabe, Robert J. Harvey, Hiroyuki Sakagami
    JOURNAL OF NEUROCHEMISTRY 116 6 1122 - 1137 2011年03月 [査読有り][通常論文]
     
    P>SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both beta-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.
  • Rylan S. Larsen, Rebekah J. Corlew, Maile A. Henson, Adam C. Roberts, Masayoshi Mishina, Masahiko Watanabe, Stuart A. Lipton, Nobuki Nakanishi, Isabel Perez-Otano, Richard J. Weinberg, Benjamin D. Philpot
    NATURE NEUROSCIENCE 14 3 338 - 344 2011年03月 [査読有り][通常論文]
     
    Recent evidence suggests that presynaptic-acting NMDA receptors (preNMDARs) are important for neocortical synaptic transmission and plasticity. We found that unique properties of the NR3A subunit enable preNMDARs to enhance spontaneous and evoked glutamate release and that NR3A is required for spike timing-dependent long-term depression in the juvenile mouse visual cortex. In the mature cortex, NR2B-containing preNMDARs enhanced neurotransmission in the absence of magnesium, indicating that presynaptic NMDARs may function under depolarizing conditions throughout life. Our findings indicate that NR3A relieves preNMDARs from the dual-activation requirement of ligand-binding and depolarization; the developmental removal of NR3A limits preNMDAR functionality by restoring this associative property.
  • Jae-Yeol Joo, Sung-Jin Lee, Takeshi Uemura, Tomoyuki Yoshida, Misato Yasumura, Masahiko Watanabe, Masayoshi Mishina
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 406 4 627 - 632 2011年03月 [査読有り][通常論文]
     
    Trans-synaptic interaction of postsynaptic glutamate receptor 62 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates synapse formation in the cerebellum [T. Uemura, S.J. Lee, M. Yasumura, T. Takeuchi, T. Yoshida, M. Ra, R. Taguchi, K. Sakimura, M. Mishina, Cell 141 (2010) 1068-1079]. This finding raises a question whether other Cbln family members interact with NRXNs to regulate synapse formation in the forebrain. Here, we showed that Cbln1 and Cbln2 induced presynaptic differentiation of cultured cortical neurons, while Cbln4 exhibited little activity. When compared with neuroligin 1, Cbln1 and Cbln2 induced preferentially inhibitory presynaptic differentiation rather than excitatory one in cortical cultures. The synaptogenic activities of Cbln1 and Cbln2 were suppressed by the addition of the extracellular domain of NRXN1 beta to the cortical neuron cultures. Consistently, Cbln1 and Cbln2 showed robust binding activities to NRXN1 alpha and three beta-NRXNs, while only weak interactions were observed between Cbln4 and NRXNs. The interactions of Cbln1, Cbln2 and Cbln4 were selective for NRXN variants containing splice segment (S) 4. Affinities for NRXNs estimated by surface plasmon resonance analysis were variable among Cbln subtypes. Cbln1 showed higher affinities to NRXNs than Cbln2, while the binding ability of Cbln4 was much lower than those of Cbln1 and Cbln2. The affinities of Cbln1 and Cbln2 were comparable between NRXN1 alpha and NRXN1 beta, but those for NRXN2 beta and NRXN3 beta were lower. These results suggest that Cbln subtypes exert synaptogenic activities in cortical neurons by differentially interacting with NRXN variants containing S4. (C) 2011 Elsevier Inc. All rights reserved.
  • Yamasaki M, Miyazaki T, Azechi H, Abe M, Natsume R, Hagiwara T, Aiba A, Mishina M, Sakimura K, Watanabe M
    The Journal of neuroscience : the official journal of the Society for Neuroscience 31 9 3362 - 3374 9 2011年03月 [査読有り][通常論文]
     
    The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor delta 2 (GluR delta 2 or G1uD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRS2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluR delta 2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluR delta 2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluR52. These results suggest that GluR delta 2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.
  • Miwako Yamasaki, Taisuke Miyazaki, Hirotsugu Azechi, Manabu Abe, Rie Natsume, Teruki Hagiwara, Atsu Aiba, Masayoshi Mishina, Kenji Sakimura, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 31 9 3362 - 3374 2011年03月 [査読有り][通常論文]
     
    The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor delta 2 (GluR delta 2 or G1uD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRS2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluR delta 2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluR delta 2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluR52. These results suggest that GluR delta 2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.
  • H. Yamamoto, Y. Takamatsu, K. Imai, E. Kamegaya, Y. Hagino, M. Watanabe, T. Yamamoto, I. Sora, H. Koga, K. Ikeda
    CURRENT NEUROPHARMACOLOGY 9 1 73 - 78 2011年03月 [査読有り][通常論文]
     
    Previously, we found fluoxetine reduces methamphetamine preference in mice. However, effects of fluoxetine on developed methamphetamine preference and on methamphetamine induced gene expression changes have been largely unknown. The present study investigates effects of post-treatment with fluoxetine on methamphetamine dependence and on gene expressions after long-term withdrawal in mice. First, we examined whether chronic post-treatment with fluoxetine attenuated methamphetamine-conditioned place preference. Next, we examined the changes in gene expression levels after long-term withdrawal (with saline or fluoxetine treatment) following chronic methamphetamine treatment. Using mRNA from the pooled frontal cortices of 10 mice per group, gene expression analyses were performed using a custom-developed cDNA array and a real-time quantitative reverse transcription-PCR. Chronic post-treatments with fluoxetine abolished the conditioned place preference developed by methamphetamine administrations. Even after long-term withdrawal from repeated methamphetamine administration, -opioid receptor (MOP) gene expression was significantly reduced in the frontal cortex. The reduced MOP gene expression in the frontal cortex was restored by chronic administration with fluoxetine. These changes were confirmed by Western blot analyses. These findings suggest that the chronic post-treatments with fluoxetine might be effective for restoring the reduction of MOP levels in the frontal cortex following long-term abstinence from methamphetamine.
  • Taisuke Miyazaki, Masahiko Watanabe
    ANATOMICAL SCIENCE INTERNATIONAL 86 1 10 - 18 2011年03月 [査読有り][通常論文]
     
    In the adult cerebellum, a single climbing fiber (CF) innervates proximal dendrites of Purkinje cells (PCs). This monoinnervation is established by the developmental elimination of surplus CFs through homosynaptic competition among multiply innervating CFs and heterosynaptic competition between CFs and parallel fibers, i.e., granule cell axons innervating distal PC dendrites. Although the developmental process of CF monoinnervation and defects in it in mutant and experimental animal models have been extensively studied by electrophysiological techniques, for quite some time this subject was poorly understood from a morphological perspective due to a lack of neuroanatomical methods that could distinguish CFs with different neuronal origins. Soon after the identification of type 2 vesicular glutamate transporter (VGluT2) that selectively detects CF terminals in the molecular layer, we developed a novel method of combined anterograde tracer labeling and VGluT2 immunohistochemistry. This method enables us to identify the mode (mono vs. multiple) of CF innervation and the site of multiple innervation. Since then, we have applied this method to various kinds of gene-manipulated mice manifesting ataxia and other cerebellar phenotypes. In this review, we summarize experimental procedures for the combined tracer/VGluT2 labeling method, and then introduce what we have learned by applying this method in studies on the role of GluR delta 2 and Ca(v)2.1 in CF monoinnervation. This method has provided informative anatomical correlates to electrophysiological data and vice versa, and will extend our knowledge of the molecular and cellular mechanisms for the development, plasticity, degeneration, and repair of the CF-PC projection system.
  • Rylan S. Larsen, Rebekah J. Corlew, Maile A. Henson, Adam C. Roberts, Masayoshi Mishina, Masahiko Watanabe, Stuart A. Lipton, Nobuki Nakanishi, Isabel Perez-Otano, Richard J. Weinberg, Benjamin D. Philpot
    NATURE NEUROSCIENCE 14 3 338 - 344 2011年03月 [査読有り][通常論文]
     
    Recent evidence suggests that presynaptic-acting NMDA receptors (preNMDARs) are important for neocortical synaptic transmission and plasticity. We found that unique properties of the NR3A subunit enable preNMDARs to enhance spontaneous and evoked glutamate release and that NR3A is required for spike timing-dependent long-term depression in the juvenile mouse visual cortex. In the mature cortex, NR2B-containing preNMDARs enhanced neurotransmission in the absence of magnesium, indicating that presynaptic NMDARs may function under depolarizing conditions throughout life. Our findings indicate that NR3A relieves preNMDARs from the dual-activation requirement of ligand-binding and depolarization; the developmental removal of NR3A limits preNMDAR functionality by restoring this associative property.
  • Tayo Katano, Takanobu Nakazawa, Terumasa Nakatsuka, Masahiko Watanabe, Tadashi Yamamoto, Seiji Ito
    NEUROPHARMACOLOGY 60 4 609 - 616 2011年03月 [査読有り][通常論文]
     
    Previously we demonstrated that phosphorylation of NR2B subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor at Tyr1472 is increased in a neuropathic-pain model and that this phosphorylation is required for the maintenance of neuropathic pain by L5-spinal nerve transection. We obtained these results by using a selective NR2B antagonist and mice deficient in Fyn, which is an Src-family tyrosine protein kinase. However, how Tyr1472 phosphorylation of NR2B is involved in the maintenance of neuropathic pain was unclear. Here, we demonstrated that neuropathic pain was markedly attenuated in the spared nerve injury model of mice with a knock-in mutation of the Tyr1472 site to phenylalanine of NR2B (Y1472F-KI). While phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) at its Thr286 and that of the GluR1 subunit of the AMPA receptor at its Ser831 was enhanced in the spinal dorsal horn after spared nerve injury in wild-type mice, such phosphorylation was markedly impaired in Y1472F-KI mice. Inhibition of CaMKII by intrathecal injection of KN93, an inhibitor of CaMKII, reduced mechanical allodynia and phosphorylation of CaMKII at its Thr286 and that of GluR1 at its Ser831 in the spinal cord 7 days after spared nerve injury. These results demonstrate that the phosphorylation of CaMKII and GluR1 occurs downstream of the Tyr1472 phosphorylation of NR2B subunits in the spinal cord and give the first suggestion that activation of CaMKII and GluR1-AMPA receptors may be involved in mechanical allodynia caused by peripheral nerve injury. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shinya Kasai, Hideko Yamamoto, Etsuko Kamegaya, George R. Uhl, Ichiro Sora, Masahiko Watanabe, Kazutaka Ikeda
    CURRENT NEUROPHARMACOLOGY 9 1 219 - 222 2011年03月 [査読有り][通常論文]
     
    Increasing evidence suggests that mu opioid receptor (MOP) expression is altered during the development of and withdrawal from substance dependence. Although anti-MOP antibodies have been hypothesized to be useful for estimating MOP expression levels, inconsistent MOP molecular weights (MWs) have been reported in studies using anti-MOP antibodies. In the present study, we generated a new anti-MOP antibody (N38) against the 1-38 amino acid sequence of the mouse MOP N-terminus and conducted Western blot analysis with wildtype and MOP knockout brain lysates to determine the MWs of intrinsic MOP. The N38 antibody detected migrating bands with relative MWs of 60-67 kDa in the plasma membrane fraction isolated from wildtype brain, but not from the MOP knockout brain. These migrating bands exhibited semi-linear density in the range of 3-30 mu g membrane proteins/lane. The N38 antibody may be useful for quantitatively detecting MOP.
  • Jae-Yeol Joo, Sung-Jin Lee, Takeshi Uemura, Tomoyuki Yoshida, Misato Yasumura, Masahiko Watanabe, Masayoshi Mishina
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 406 4 627 - 632 2011年03月 [査読有り][通常論文]
     
    Trans-synaptic interaction of postsynaptic glutamate receptor 62 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates synapse formation in the cerebellum [T. Uemura, S.J. Lee, M. Yasumura, T. Takeuchi, T. Yoshida, M. Ra, R. Taguchi, K. Sakimura, M. Mishina, Cell 141 (2010) 1068-1079]. This finding raises a question whether other Cbln family members interact with NRXNs to regulate synapse formation in the forebrain. Here, we showed that Cbln1 and Cbln2 induced presynaptic differentiation of cultured cortical neurons, while Cbln4 exhibited little activity. When compared with neuroligin 1, Cbln1 and Cbln2 induced preferentially inhibitory presynaptic differentiation rather than excitatory one in cortical cultures. The synaptogenic activities of Cbln1 and Cbln2 were suppressed by the addition of the extracellular domain of NRXN1 beta to the cortical neuron cultures. Consistently, Cbln1 and Cbln2 showed robust binding activities to NRXN1 alpha and three beta-NRXNs, while only weak interactions were observed between Cbln4 and NRXNs. The interactions of Cbln1, Cbln2 and Cbln4 were selective for NRXN variants containing splice segment (S) 4. Affinities for NRXNs estimated by surface plasmon resonance analysis were variable among Cbln subtypes. Cbln1 showed higher affinities to NRXNs than Cbln2, while the binding ability of Cbln4 was much lower than those of Cbln1 and Cbln2. The affinities of Cbln1 and Cbln2 were comparable between NRXN1 alpha and NRXN1 beta, but those for NRXN2 beta and NRXN3 beta were lower. These results suggest that Cbln subtypes exert synaptogenic activities in cortical neurons by differentially interacting with NRXN variants containing S4. (C) 2011 Elsevier Inc. All rights reserved.
  • Masahiro Fukaya, Akifumi Kamata, Yoshinobu Hara, Hideaki Tamaki, Osamu Katsumata, Naoki Ito, Shin'ichi Takeda, Yutaka Hata, Tatsuo Suzuki, Masahiko Watanabe, Robert J. Harvey, Hiroyuki Sakagami
    JOURNAL OF NEUROCHEMISTRY 116 6 1122 - 1137 2011年03月 [査読有り][通常論文]
     
    P>SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both beta-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.
  • Yasuhiko Yamamoto, Ai Harashima, Hidehito Saito, Koichi Tsuneyama, Seiichi Munesue, So Motoyoshi, Dong Han, Takuo Watanabe, Masahide Asano, Shin Takasawa, Hiroshi Okamoto, Satoshi Shimura, Tadahiro Karasawa, Hideto Yonekura, Hiroshi Yamamoto
    JOURNAL OF IMMUNOLOGY 186 5 3248 - 3257 2011年03月 [査読有り][通常論文]
     
    Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-alpha secretion from peritoneal macrophages and an NF-kappa B promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE(-/-) mice. After LPS injection, RAGE(+/+) mice showed a higher mortality, higher serum levels of IL-6, TNF-alpha, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE(-/-) mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE(-/-) as well as RAGE(+/+) mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock. The Journal of Immunology, 2011, 186: 3248-3257.
  • Kenta Kobayashi, Tomoyuki Masuda, Masanori Takahashi, Jun-ichi Miyazaki, Masahiro Nakagawa, Motokazu Uchigashima, Masahiko Watanabe, Hiroyuki Yaginuma, Noriko Osumi, Kozo Kaibuchi, Kazuto Kobayashi
    EUROPEAN JOURNAL OF NEUROSCIENCE 33 4 612 - 621 2011年02月 [査読有り][通常論文]
     
    Cranial motor neurons, which are divided into somatic motor (SM), branchiomotor (BM) and visceral motor (VM) neurons, form distinct axonal trajectories to innervate their synapse targets. Rho GTPase regulates various neuronal functions through one of the major effector proteins, Rho-kinase. Here, we addressed the in vivo role of the Rho/Rho-kinase signaling pathway in axon patterning of cranial motor neurons. We performed conditional expression of a dominant-negative mutant for RhoA or Rho-kinase in transgenic mice by using the Cre-loxP system to suppress the activity of these molecules in developing cranial motor neurons. Blockade of the Rho/Rho-kinase signaling pathway caused defects in the patterning of SM axons but not in that of BM/VM axons, in which defects were accompanied by reduced muscle innervation and reduced synapse formation by SM neurons. In addition, blockade of the signaling pathway shifted the trajectory of growing SM axons in explant cultures, whereas it did not appear to affect the rate of spontaneous axonal outgrowth. These results indicate that the Rho/Rho-kinase signaling pathway plays an essential role in the axon patterning of cranial SM neurons during development.
  • Takayuki Yoshida, Motokazu Uchigashima, Miwako Yamasaki, Istvan Katona, Maya Yamazaki, Kenji Sakimura, Masanobu Kano, Mitsuhiro Yoshioka, Masahiko Watanabe
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 7 3059 - 3064 2011年02月 [査読有り][通常論文]
     
    2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn-1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB(1) receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, it has been demonstrated that CB(1) is particularly enriched in axon terminals of cholecystokinin (CCK)-positive GABAergic interneurons, induces short-and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here, we clarified a unique molecular convergence of DGL alpha, CB(1), and MGL at specific inhibitory synapses in the basal nucleus (BA), but not lateral nucleus, of the basolateral amygdala. The synapses, termed invaginating synapses, consisted of conventional symmetrical contact and unique perisynaptic invagination of nerve terminals into perikarya. At invaginating synapses, DGL alpha was preferentially recruited to concave somatic membrane of postsynaptic pyramidal neurons, whereas invaginating presynaptic terminals highly expressed CB(1), MGL, and CCK. No such molecular convergence was seen for flat perisomatic synapses made by parvalbumin-positive interneurons. On the other hand, DGL alpha and CB(1) were expressed weakly at axospinous excitatory synapses. Consistent with these morphological data, thresholds for DGL alpha-mediated depolarization-induced retrograde suppression were much lower for inhibitory synapses than for excitatory synapses in BA pyramidal neurons. Moreover, depolarization-induced suppression was readily saturated for inhibition, but never for excitation. These findings suggest that perisomatic inhibition by invaginating synapses is a key target of 2-AG-mediated control of the excitability of BA pyramidal neurons.
  • A. Ludanyi, S. -J. Hu, M. Yamazaki, A. Tanimura, D. Piomelli, M. Watanabe, M. Kano, K. Sakimura, Z. Magloczky, K. Mackie, T. F. Freund, I. Katona
    NEUROSCIENCE 174 50 - 63 2011年02月 [査読有り][通常論文]
     
    Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-alpha (DGL-alpha), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-alpha-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-alpha-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-alpha in dendritic spine heads. Similar DGL-alpha-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-alpha knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Toshiaki Mishima, Yoshiya Ito, Kanako Hosono, Yukio Tamura, Yasushi Uchida, Mitsuhiro Hirata, Tatsunori Suzsuki, Hideki Amano, Shintaro Kato, Yukiko Kurihara, Hiroki Kurihara, Izumi Hayashi, Masahiko Watanabe, Masataka Majima
    American Journal of Physiology - Heart and Circulatory Physiology 300 2 H431 - H439 2011年02月 [査読有り][通常論文]
     
    It is known that the neural system plays a fundamental role in neovascularization. A neuropeptide, calcitonin gene-related peptide (CGRP), is widely distributed in the central and peripheral neuronal systems. However, it remains to be elucidated the role of CGRP in angiogenesis during ischemia. The present study examined whether endogenous CGRP released from neuronal systems facilitates revascularization in response to ischemia using CGRP knockout mice (CGRP-/-). CGRP-/- or their wild-type littermates (CGRP+/+) were subjected to unilateral hindlimb ischemia. CGRP -/- exhibited impaired blood flow recovery from ischemia and decreased capillary density expressed in terms of the number of CD-31-positive cells in the ischemic tissues compared with CGRP+/+. In vivo microscopic studies showed that the functional capillary density in CGRP -/- was reduced. Hindlimb ischemia increased the expression of pro-CGRP mRNA and of CGRP protein in the lumbar dorsal root ganglia. Lack of CGRP decreased mRNA expression of growth factors, including CD31, vascular endothelial growth factor-A, basic fibroblast growth factor, and transforming growth factor-β, in the ischemic limb tissue. The application of CGRP enhanced the mRNA expression of CD31 and VEGF-A in human umbilical vein endothelial cells (HUVECs) and fibroblasts. Subcutaneous infusion of CGRP8-37, a CGRP antagonist, using miniosmotic pumps delayed angiogenesis and reduced the expression of proangiogenic growth factors during hindlimb ischemia. These results indicate that endogenous CGRP facilitates angiogenesis in response to ischemia. Targeting CGRP may provide a promising approach for controlling angiogenesis related to pathophysiological conditions. Copyright © 2011 the American Physiological Society.
  • Taisuke Miyazaki, Miwako Yamasaki, Motokazu Uchigashima, Ayano Matsushima, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 33 1 82 - 94 2011年01月 [査読有り][通常論文]
     
    Secretogranin II (SgII), or chromogranin C, is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and large dense-core vesicle (LDCVs), and also functions as a precursor of neuropeptide secretoneurin. Although SgII is widely distributed in the brain and is predominantly localized at terminals of mossy fibers in the hippocampus and cerebellum and climbing fibers in the cerebellum, its cellular expression and ultrastructural localization remain largely unknown. In the present study, we addressed this issue in the adult mouse brain by multiple-labeling fluorescence in situ hybridization and immunofluorescence and by preembedding and postembedding immunoelectron microscopies. SgII was expressed in various neurons, distributed as either tiny puncta or coarse aggregates in the neuropil, and intensely accumulated in perikarya of particular neurons, such as parvalbumin-positive interneurons and mossy cells in the hippocampus and Purkinje cells in the cerebellum. Coarse aggregates were typical of terminals of mossy fibers and climbing fibers. In these terminals, numerous immunogold particles were clustered on individual LDCVs, and one or two particles also fell within small synaptic vesicle-accumulating portions. SgII was further detected as tiny puncta in neural elements lacking LDCVs, such as parallel fibers of cerebellar granule cells, somatodendritic elements of various neurons and Bergmann glia. Thus, SgII is present in LDCV and non-LDCV compartments of various neural cells. The wide subcellular localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non-LDCV compartments.
  • Taisuke Miyazaki, Miwako Yamasaki, Motokazu Uchigashima, Ayano Matsushima, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 33 1 82 - 94 2011年01月 [査読有り][通常論文]
     
    Secretogranin II (SgII), or chromogranin C, is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and large dense-core vesicle (LDCVs), and also functions as a precursor of neuropeptide secretoneurin. Although SgII is widely distributed in the brain and is predominantly localized at terminals of mossy fibers in the hippocampus and cerebellum and climbing fibers in the cerebellum, its cellular expression and ultrastructural localization remain largely unknown. In the present study, we addressed this issue in the adult mouse brain by multiple-labeling fluorescence in situ hybridization and immunofluorescence and by preembedding and postembedding immunoelectron microscopies. SgII was expressed in various neurons, distributed as either tiny puncta or coarse aggregates in the neuropil, and intensely accumulated in perikarya of particular neurons, such as parvalbumin-positive interneurons and mossy cells in the hippocampus and Purkinje cells in the cerebellum. Coarse aggregates were typical of terminals of mossy fibers and climbing fibers. In these terminals, numerous immunogold particles were clustered on individual LDCVs, and one or two particles also fell within small synaptic vesicle-accumulating portions. SgII was further detected as tiny puncta in neural elements lacking LDCVs, such as parallel fibers of cerebellar granule cells, somatodendritic elements of various neurons and Bergmann glia. Thus, SgII is present in LDCV and non-LDCV compartments of various neural cells. The wide subcellular localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non-LDCV compartments.
  • Noriko Ando, Yumi Iwamitsu, Masaru Kuranami, Shigemi Okazaki, Yuki Nakatani, Kenji Yamamoto, Masahiko Watanabe, Hitoshi Miyaoka
    Psychosomatics 52 1 9  2011年 [査読有り][通常論文]
     
    Objective: The objective of this study was to determine how age and psychological characteristics assessed prior to diagnosis could predict psychological distress in outpatients immediately after disclosure of their diagnosis. Methods: This is a longitudinal and prospective study, and participants were breast cancer patients and patients with benign breast problems (BBP). Patients were asked to complete questionnaires to determine levels of the following: trait anxiety (State-Trait Anxiety Inventory), negative emotional suppression (Courtauld Emotional Control Scale), life stress events (Life Experiences Survey), and psychological distress (Profile of Mood Status) prior to diagnosis. They were asked to complete a questionnaire measuring psychological distress after being told their diagnosis. We analyzed a total of 38 women diagnosed with breast cancer and 95 women diagnosed with a BBP. Results: A two-way analysis of variance (prior to, after diagnosis × cancer, benign) showed that psychological distress after diagnosis among breast cancer patients was significantly higher than in patients with a BBP. The multiple regression model accounted for a significant amount of variance in the breast cancer group (model adjusted R2 = 0.545, p < 0.001), and only trait anxiety was statistically significant (β = 0.778, p < 0.001). In the BBP group, the multiple regression analysis yielded a significant result (model adjusted R2 = 0.462, p < 0.001), with trait anxiety and negative life changes as statistically significant factors (β = 0.449 and 0.324 respectively p < 0.01). Conclusions: In both groups, trait anxiety assessed prior to diagnosis was the significant predictor of psychological distress after diagnosis, and might have prospects as a screening method for psychologically vulnerable women. © 2011 The Academy of Psychosomatic Medicine.
  • T. Sanada, K. Hashimoto, A. Hayashida, M. Watanabe
    ECS Transactions 41 5 59 - 66 2011年 [査読有り][通常論文]
     
    In the LED manufacturing process, a thin Au film is deposited on a wafer, and then most of it is removed with small pieces of Au film left for electrodes. For this removal, we apply physical cleaning method, which can remove the metal film without chemicals. In this study, we investigate a removal process of a thin aluminum film deposited on a glass surface by steam-water mixed spray. At the initial stage of this process, tiny spots, whose sizes are the order of droplet diameter, are observed. And then very characteristic removal profiles, which are typically lines with sub-millimeter width, are observed. We discuss the removal mechanism due to high-speed droplet impact. ©The Electrochemical Society.
  • Hiroshi Katoh, Keishi Yamashita, Guoqin Wang, Takeo Sato, Takatoshi Nakamura, Masahiko Watanabe
    Journal of Gastrointestinal Surgery 15 1 120 - 129 2011年 [査読有り][通常論文]
     
    Purpose: In stage II colorectal cancer (CRC), high-risk patient selection is required, but no candidate markers have been elucidated. Our concern was whether anastomotic leakage (Lk) is a potential available clinicopathological factor for selecting high-risk stage II. Methods: Two hundred seven patients with stage II CRC who underwent curative resection were analyzed. Clinical variables were tested for their relationship to survival. Results: The 5-year disease-free survival rate (DFS) was 87.0%. The univariable prognostic analyses indicated that Lk (P = 0.003) was the only significant factor. The multivariable prognostic analysis revealed that Lk remained to be potently independent [hazard ratio (HR), 4.21, P = 0.021), and the DFS was 58.3% in cases with Lk, while 88.7% in the counterpart. The multivariable logistic regression analysis revealed perioperative blood transfusion (P = 0.001) was independently associated with Lk. Intriguingly, Lk was closely associated with hematogenic recurrence (P = 0.003) rather than peritoneal or local recurrence. Although sustained increase of the serum C-reactive protein at 2 weeks after operation predicted poor prognosis, the mutitivariable analysis including the C-reactive protein level revealed that Lk still indicated the prognostic potential (HR, 3.70, P = 0.075). Conclusions: The findings concluded that Lk may be a high risk for systemic recurrence in stage II CRC. © 2010 The Society for Surgery of the Alimentary Tract.
  • Wataru Onozato, Keishi Yamashita, Kazuya Yamashita, Tatsuru Kuba, Hiroshi Katoh, Takatoshi Nakamura, Takeo Sato, Atsushi Ihara, Isao Okayasu, Masahiko Watanabe
    Journal of Surgical Oncology 103 1 25 - 33 2011年01月01日 [査読有り][通常論文]
     
    Purpose Genetic alterations that are closely associated with patient prognosis can be landmarks of definitive therapeutic targets as well as useful biomarkers in human cancer clinics. Methods Three hundred seventy-eight colorectal cancer (CRC) patients were examined for K-ras mutations by single-strand conformation polymorphism (SSCP), with a subsequent 144 young colon cancer (YCC) patients added to validate its prognostic significance. Results K-ras mutations were identified in 161 (43%) of the 378 CRC patients and were significantly associated with tumor location (colon vs. rectum 80/218 = 37% vs. 81/160 = 51% P = 0.0068) and age (≥60 vs. < 60 103/220 = 47% vs. 58/158 = 37% P = 0.049). The incidence of K-ras mutations was 30% in YCC patients as compared to 55% in elderly rectal cancer patients (P = 0.0004). K-ras mutations significantly correlated with a worse prognosis (P = 0.0014) only in 73 curatively resected YCC with stages I-III, but not in other CRCs, which was further validated in the independent set of the corresponding 144 YCC patients (P = 0.024). Both univariate and multivariate analyses identified K-ras mutations as an independent prognostic factor (HR = 5.5, P = 0.029 HR = 3.6, P = 0.011) in both learning and validation sets of the curatively resected YCC with stages I-III, respectively, and the prognostic relevance was marked in stage III YCC patients (P = 0.002), but not in stages I, II, and IV. Conclusion In curative YCC, K-ras mutations could have excellent prognostic value. Hence, the K-ras mutation status could be a good indicator to predict the clinical outcome in curatively resected stage III YCC patients, and K-ras pathway inhibition may be a relevant therapeutic target in CRC, excluding YCC patients with no K-ras mutation. Copyright © 2010 Wiley-Liss, Inc.
  • Yohei Okubo, Masato Mashimo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
    NEUROSCIENCE RESEARCH 71 E327 - E327 2011年 [査読有り][通常論文]
  • Takayuki Yoshida, Motokazu Uchigashima, Miwako Yamasaki, Istvan Katona, Maya Yamazaki, Kenji Sakimura, Masanobu Kano, Mitsuhiro Yoshioka, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E93 - E94 2011年 [査読有り][通常論文]
  • Takehiro Kudo, Motokazu Uchigashima, Taisuke Miyazaki, Miwako Yamasaki, Masabumi Minami, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E322 - E322 2011年 [査読有り][通常論文]
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Hashimoto, Keiko Shimamoto, Kazuhisa Kohda, Michisuke Yuzaki, Kohichi Tanaka, Masanobu Kano, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E63 - E63 2011年 [査読有り][通常論文]
  • Mari Hondo, Naoki Furutani, Miwako Yamasaki, Masahiko Watanabe, Takeshi Sakurai
    NEUROSCIENCE RESEARCH 71 E170 - E170 2011年 [査読有り][通常論文]
  • Kohtarou Konno, Miwako Yamasaki, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E213 - E213 2011年 [査読有り][通常論文]
  • Ryoichi Ichikawa, Miwako Yamasaki, Taisuke Miyazaki, Haruyuki Tatsumi, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E215 - E215 2011年 [査読有り][通常論文]
  • Miwako Yamasaki, Taisuke Miyazaki, Hirotsugu Azechi, Manabu Abe, Rie Natsume, Teruki Hagiwara, Atsu Aiba, Masayoshi Mishina, Kenji Sakimura, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 71 E93 - E93 2011年 [査読有り][通常論文]
  • Izumi Watanabe, Hirotsugu Azechi, Kaori Akashi, Maya Yamazaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
    NEUROSCIENCE RESEARCH 71 E315 - E316 2011年 [査読有り][通常論文]
  • Imre Farkas, Imre Kallo, Levente Deli, Barbara Vida, Erik Hrabovszky, Csaba Fekete, Suzanne M. Moenter, Masahiko Watanabe, Zsolt Liposits
    ENDOCRINOLOGY 151 12 5818 - 5829 2010年12月 [査読有り][通常論文]
     
    Cannabinoids suppress fertility via reducing hypothalamic GnRH output. gamma-Aminobutyric acid (GABA)(A) receptor (GABA(A)-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABA(A)-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABA(A)-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABA(A)-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl2 in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs. (Endocrinology 151:5818-5829,2010)
  • Jung Hoon Yang, Akira Wada, Kazuyuki Yoshida, Yurika Miyoshi, Tomoko Sayano, Kayoko Esaki, Masami O. Kinoshita, Shozo Tomonaga, Norihiro Azuma, Masahiko Watanabe, Kenji Hamase, Kiyoshi Zaitsu, Takeo Machida, Albee Messing, Shigeyoshi Itohara, Yoshio Hirabayashi, Shigeki Furuya
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 53 41380 - 41390 2010年12月 [査読有り][通常論文]
     
    In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L-and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.
  • Jung Hoon Yang, Akira Wada, Kazuyuki Yoshida, Yurika Miyoshi, Tomoko Sayano, Kayoko Esaki, Masami O. Kinoshita, Shozo Tomonaga, Norihiro Azuma, Masahiko Watanabe, Kenji Hamase, Kiyoshi Zaitsu, Takeo Machida, Albee Messing, Shigeyoshi Itohara, Yoshio Hirabayashi, Shigeki Furuya
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 53 41380 - 41390 2010年12月 [査読有り][通常論文]
     
    In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L-and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.
  • Imre Farkas, Imre Kallo, Levente Deli, Barbara Vida, Erik Hrabovszky, Csaba Fekete, Suzanne M. Moenter, Masahiko Watanabe, Zsolt Liposits
    ENDOCRINOLOGY 151 12 5818 - 5829 2010年12月 [査読有り][通常論文]
     
    Cannabinoids suppress fertility via reducing hypothalamic GnRH output. gamma-Aminobutyric acid (GABA)(A) receptor (GABA(A)-R)-mediated transmission is a major input to GnRH cells that can be excitatory. We hypothesized that cannabinoids act via inhibiting GABAergic input. We performed loose-patch electrophysiological studies of acute slices from adult male GnRH-green fluorescent protein transgenic mice. Bath application of type 1 cannabinoid receptor (CB1) agonist WIN55,212 decreased GnRH neuron firing rate. This action was detectable in presence of the glutamate receptor antagonist kynurenic acid but disappeared when bicuculline was also present, indicating GABA(A)-R involvement. In immunocytochemical experiments, CB1-immunoreactive axons formed contacts with GnRH neurons and a subset established symmetric synapses characteristic of GABAergic neurotransmission. Functional studies were continued with whole-cell patch-clamp electrophysiology in presence of tetrodotoxin. WIN55,212 decreased the frequency of GABA(A)-R-mediated miniature postsynaptic currents (mPSCs) (reflecting spontaneous vesicle fusion), which was prevented with the CB1 antagonist AM251, indicating collectively that activation of presynaptic CB1 inhibits GABA release. AM251 alone increased mPSC frequency, providing evidence that endocannabinoids tonically inhibit GABA(A)-R drive onto GnRH neurons. Increased mPSC frequency was absent when diacylglycerol lipase was blocked intracellularly with tetrahydrolipstatin, showing that tonic inhibition is caused by 2-arachidonoylglycerol production of GnRH neurons. CdCl2 in extracellular solution can maintain both action potentials and spontaneous vesicle fusion. Under these conditions, when endocannabinoid-mediated blockade of spontaneous vesicle fusion was blocked with AM251, GnRH neuron firing increased, revealing an endogenous endocannabinoid brake on GnRH neuron firing. Retrograde endocannabinoid signaling may represent an important mechanism under physiological and pathological conditions whereby GnRH neurons regulate their excitatory GABAergic inputs. (Endocrinology 151:5818-5829,2010)
  • Parajuli LK, Fukazawa Y, Watanabe M, Shigemoto R
    The Journal of comparative neurology 518 21 4362 - 4374 21 2010年11月 [査読有り][通常論文]
     
    T-type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T-type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the alpha 1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the alpha 1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the alpha 1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane-associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type-specific expression levels and for uniform expression density of the alpha 1G subunit over the plasma membrane of dLGN cells. J. Comp. Neurol. 518: 4362-4374, 2010. (C) 2010 Wiley-Liss, Inc.
  • Masato Mashimo, Yohei Okubo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 10 1668 - 1677 2010年11月 [査読有り][通常論文]
     
    The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we describe a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5-trisphosphate (IP(3)) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber-Purkinje cell synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP(3) signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum.
  • Miyazaki T, Yamasaki M, Takeuchi T, Sakimura K, Mishina M, Watanabe M
    The Journal of neuroscience : the official journal of the Society for Neuroscience 30 45 15196 - 15209 45 2010年11月 [査読有り][通常論文]
     
    Glutamate receptor GluR delta 2 is exclusively expressed in Purkinje cells (PCs) from early development and plays key roles in parallel fiber (PF) synapse formation, elimination of surplus climbing fibers (CFs), long-term depression, motor coordination, and motor learning. To address its role in adulthood, we previously developed a mouse model of drug-induced GluR delta 2 ablation in adult PCs (Takeuchi et al., 2005). In that study, we demonstrated an essential role to maintain the connectivity of PF-PC synapses, based on the observation that both mismatching of presynaptic and postsynaptic specializations and disconnection of PF-PC synapses are progressively increased after GluR delta 2 ablation. Here, we pursued its role for CF wiring in adult cerebellum. In parallel with the disconnection of PF-PC synapses, ascending CF branches exhibited distal extension to innervate distal dendrites of the target and neighboring PCs. Furthermore, transverse CF branches, a short motile collateral rarely forming synapses in wild-type animals, displayed aberrant mediolateral extension to innervate distal dendrites of neighboring and remote PCs. Consequently, many PCs were wired by single main CF and other surplus CFs innervating a small part of distal dendrites. Electrophysiological recording further revealed that surplus CF-EPSCs characterized with slow rise time and small amplitude emerged after GluR delta 2 ablation, and increased progressively both in number and amplitude. Therefore, GluR delta 2 is essential for maintaining CF monoinnervation in adult cerebellum by suppressing aberrant invasion of CF branches to the territory of PF innervation. Thus, GluR delta 2 fuels heterosynaptic competition and gives PFs the competitive advantages over CFs throughout the animal's life.
  • Laxmi Kumar Parajuli, Yugo Fukazawa, Masahiko Watanabe, Ryuichi Shigemoto
    JOURNAL OF COMPARATIVE NEUROLOGY 518 21 4362 - 4374 2010年11月 [査読有り][通常論文]
     
    T-type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T-type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the alpha 1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the alpha 1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the alpha 1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane-associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type-specific expression levels and for uniform expression density of the alpha 1G subunit over the plasma membrane of dLGN cells. J. Comp. Neurol. 518: 4362-4374, 2010. (C) 2010 Wiley-Liss, Inc.
  • Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 30 45 15196 - 15209 2010年11月 [査読有り][通常論文]
     
    Glutamate receptor GluR delta 2 is exclusively expressed in Purkinje cells (PCs) from early development and plays key roles in parallel fiber (PF) synapse formation, elimination of surplus climbing fibers (CFs), long-term depression, motor coordination, and motor learning. To address its role in adulthood, we previously developed a mouse model of drug-induced GluR delta 2 ablation in adult PCs (Takeuchi et al., 2005). In that study, we demonstrated an essential role to maintain the connectivity of PF-PC synapses, based on the observation that both mismatching of presynaptic and postsynaptic specializations and disconnection of PF-PC synapses are progressively increased after GluR delta 2 ablation. Here, we pursued its role for CF wiring in adult cerebellum. In parallel with the disconnection of PF-PC synapses, ascending CF branches exhibited distal extension to innervate distal dendrites of the target and neighboring PCs. Furthermore, transverse CF branches, a short motile collateral rarely forming synapses in wild-type animals, displayed aberrant mediolateral extension to innervate distal dendrites of neighboring and remote PCs. Consequently, many PCs were wired by single main CF and other surplus CFs innervating a small part of distal dendrites. Electrophysiological recording further revealed that surplus CF-EPSCs characterized with slow rise time and small amplitude emerged after GluR delta 2 ablation, and increased progressively both in number and amplitude. Therefore, GluR delta 2 is essential for maintaining CF monoinnervation in adult cerebellum by suppressing aberrant invasion of CF branches to the territory of PF innervation. Thus, GluR delta 2 fuels heterosynaptic competition and gives PFs the competitive advantages over CFs throughout the animal's life.
  • Masato Mashimo, Yohei Okubo, Toshiko Yamazawa, Miwako Yamasaki, Masahiko Watanabe, Toshihiko Murayama, Masamitsu Iino
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 10 1668 - 1677 2010年11月 [査読有り][通常論文]
     
    The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we describe a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5-trisphosphate (IP(3)) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber-Purkinje cell synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP(3) signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum.
  • Mayumi Sugimoto, Shinji Sasaki, Toshio Watanabe, Shota Nishimura, Atsushi Ideta, Maya Yamazaki, Keiko Matsuda, Michisuke Yuzaki, Kenji Sakimura, Yoshito Aoyagi, Yoshikazu Sugimoto
    PLOS ONE 5 11 e13817  2010年11月 [査読有り][通常論文]
     
    Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.
  • Francisco Ciruela, Victor Fernandez-Duenas, Kristoffer Sahlholm, Laura Fernandez-Alacid, Joel C. Nicolau, Masahiko Watanabe, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 8 1265 - 1277 2010年10月 [査読有り][通常論文]
     
    The stimulation of inhibitory neurotransmitter receptors, such as gamma-aminobutyric acid type B (GABA(B)) receptors, activates G protein-gated inwardly-rectifying K+ (GIRK) channels, which influence membrane excitability. There is now evidence suggesting that G protein-coupled receptors and G protein-gated inwardly-rectifying K+ [GIRK/family 3 of inwardly-rectifying K+ (Kir3)] channels do not diffuse freely within the plasma membrane, but instead there are direct protein-protein interactions between them. Here, we used bioluminescence resonance energy transfer, co-immunoprecipitation, confocal and electron microscopy techniques to investigate the oligomerization of GABA(B) receptors with GIRK channels containing the GIRK3 subunit, whose contribution to functional channels is still unresolved. Co-expression of GABA(B) receptors and GIRK channels in human embryonic kidney-293 cells in combination with co-immunoprecipitation experiments established that the metabotropic receptor forms stable complexes with GIRK channels. Using bioluminescence resonance energy transfer, we have shown that, in living cells under physiological conditions, GABA(B) receptors interact directly with GIRK1/GIRK3 heterotetramers. In addition, we have provided evidence that the receptor-effector complexes are also found in vivo and identified that the cerebellar granule cells are one neuron population where the interaction probably takes place. Altogether, our data show that signalling complexes containing GABA(B) receptors and GIRK channels are formed shortly after biosynthesis, probably in the endoplasmic reticulum and/or endoplasmic reticulum/Golgi apparatus complex, suggesting that this might be a general feature of receptor-effector ion channel signal transduction and supporting a channel-forming role for the GIRK3 subunit.
  • Chihiro Takasaki, Miwako Yamasaki, Motokazu Uchigashima, Kohtarou Konno, Yuchio Yanagawa, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 8 1326 - 1336 2010年10月 [査読有り][通常論文]
     
    Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2',3'-Cyclic nucleotide 3'-phosphodiesterase-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2+ glia. However, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and l-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.
  • Erik Keimpema, Klaudia Barabas, Yury M. Morozov, Giuseppe Tortoriello, Masaaki Torii, Gary Cameron, Yuchio Yanagawa, Masahiko Watanabe, Ken Mackie, Tibor Harkany
    JOURNAL OF NEUROSCIENCE 30 42 13992 - 14007 2010年10月 [査読有り][通常論文]
     
    Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB1 cannabinoid receptors (CB(1)Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGL alpha/beta) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB(1)Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGL alpha and CB(1)Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGL alpha undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB1R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.
  • Chihiro Takasaki, Miwako Yamasaki, Motokazu Uchigashima, Kohtarou Konno, Yuchio Yanagawa, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 8 1326 - 1336 2010年10月 [査読有り][通常論文]
     
    Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2',3'-Cyclic nucleotide 3'-phosphodiesterase-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2+ glia. However, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and l-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.
  • Erik Keimpema, Klaudia Barabas, Yury M. Morozov, Giuseppe Tortoriello, Masaaki Torii, Gary Cameron, Yuchio Yanagawa, Masahiko Watanabe, Ken Mackie, Tibor Harkany
    JOURNAL OF NEUROSCIENCE 30 42 13992 - 14007 2010年10月 [査読有り][通常論文]
     
    Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB1 cannabinoid receptors (CB(1)Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGL alpha/beta) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB(1)Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGL alpha and CB(1)Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGL alpha undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB1R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.
  • Francisco Ciruela, Victor Fernandez-Duenas, Kristoffer Sahlholm, Laura Fernandez-Alacid, Joel C. Nicolau, Masahiko Watanabe, Rafael Lujan
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 8 1265 - 1277 2010年10月 [査読有り][通常論文]
     
    The stimulation of inhibitory neurotransmitter receptors, such as gamma-aminobutyric acid type B (GABA(B)) receptors, activates G protein-gated inwardly-rectifying K+ (GIRK) channels, which influence membrane excitability. There is now evidence suggesting that G protein-coupled receptors and G protein-gated inwardly-rectifying K+ [GIRK/family 3 of inwardly-rectifying K+ (Kir3)] channels do not diffuse freely within the plasma membrane, but instead there are direct protein-protein interactions between them. Here, we used bioluminescence resonance energy transfer, co-immunoprecipitation, confocal and electron microscopy techniques to investigate the oligomerization of GABA(B) receptors with GIRK channels containing the GIRK3 subunit, whose contribution to functional channels is still unresolved. Co-expression of GABA(B) receptors and GIRK channels in human embryonic kidney-293 cells in combination with co-immunoprecipitation experiments established that the metabotropic receptor forms stable complexes with GIRK channels. Using bioluminescence resonance energy transfer, we have shown that, in living cells under physiological conditions, GABA(B) receptors interact directly with GIRK1/GIRK3 heterotetramers. In addition, we have provided evidence that the receptor-effector complexes are also found in vivo and identified that the cerebellar granule cells are one neuron population where the interaction probably takes place. Altogether, our data show that signalling complexes containing GABA(B) receptors and GIRK channels are formed shortly after biosynthesis, probably in the endoplasmic reticulum and/or endoplasmic reticulum/Golgi apparatus complex, suggesting that this might be a general feature of receptor-effector ion channel signal transduction and supporting a channel-forming role for the GIRK3 subunit.
  • Shinji Matsumura, Shunji Kunori, Tamaki Mabuchi, Tayo Katano, Takanobu Nakazawa, Tetsuya Abe, Masahiko Watanabe, Tadashi Yamamoto, Emiko Okuda-Ashitaka, Seiji Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 5 798 - 810 2010年09月 [査読有り][通常論文]
     
    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N-methyl-d-aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C gamma at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain.
  • Mike T. Lin, Rafael Lujan, Masahiko Watanabe, Matthew Frerking, James Maylie, John P. Adelman
    JOURNAL OF NEUROSCIENCE 30 35 11726 - 11734 2010年09月 [査読有り][通常論文]
     
    Small conductance Ca(2+)-activated K(+) type 2 (SK2) channels are expressed in the postsynaptic density of CA1 neurons where they are activated by synaptically evoked Ca(2+) influx to limit the size of EPSPs and spine Ca(2+) transients. At Schaffer collateral synapses, the induction of long-term potentiation (LTP) increases the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated contribution to synaptic transmission and decreases the synaptic SK2 channel contribution through protein kinase A-dependent channel endocytosis. Using a combination of electrophysiology and immunoelectron microscopy in mice, the relationship between the dynamics of spine SK2 channels and AMPARs was investigated. Unlike AMPARs, synaptic SK2 channels under basal conditions do not rapidly recycle. Furthermore, SK2 channels occupy a distinct population of endosomes separate from AMPARs. However, blocking vesicular exocytosis or the delivery of synaptic GluA1-containing AMPARs during the induction of LTP blocks SK2 channel endocytosis. By similar to 2 h after the induction of LTP, synaptic SK2 channel expression and function are restored. Thus, LTP-dependent endocytosis of SK2 is coupled to LTP-dependent AMPA exocytosis, and the similar to 2 h window after the induction of LTP during which synaptic SK2 activity is absent may be important for consolidating the later phases of LTP.
  • Shinji Matsumura, Shunji Kunori, Tamaki Mabuchi, Tayo Katano, Takanobu Nakazawa, Tetsuya Abe, Masahiko Watanabe, Tadashi Yamamoto, Emiko Okuda-Ashitaka, Seiji Ito
    EUROPEAN JOURNAL OF NEUROSCIENCE 32 5 798 - 810 2010年09月 [査読有り][通常論文]
     
    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N-methyl-d-aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C gamma at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain.
  • Mike T. Lin, Rafael Lujan, Masahiko Watanabe, Matthew Frerking, James Maylie, John P. Adelman
    JOURNAL OF NEUROSCIENCE 30 35 11726 - 11734 2010年09月 [査読有り][通常論文]
     
    Small conductance Ca(2+)-activated K(+) type 2 (SK2) channels are expressed in the postsynaptic density of CA1 neurons where they are activated by synaptically evoked Ca(2+) influx to limit the size of EPSPs and spine Ca(2+) transients. At Schaffer collateral synapses, the induction of long-term potentiation (LTP) increases the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated contribution to synaptic transmission and decreases the synaptic SK2 channel contribution through protein kinase A-dependent channel endocytosis. Using a combination of electrophysiology and immunoelectron microscopy in mice, the relationship between the dynamics of spine SK2 channels and AMPARs was investigated. Unlike AMPARs, synaptic SK2 channels under basal conditions do not rapidly recycle. Furthermore, SK2 channels occupy a distinct population of endosomes separate from AMPARs. However, blocking vesicular exocytosis or the delivery of synaptic GluA1-containing AMPARs during the induction of LTP blocks SK2 channel endocytosis. By similar to 2 h after the induction of LTP, synaptic SK2 channel expression and function are restored. Thus, LTP-dependent endocytosis of SK2 is coupled to LTP-dependent AMPA exocytosis, and the similar to 2 h window after the induction of LTP during which synaptic SK2 activity is absent may be important for consolidating the later phases of LTP.
  • Heita Ozawa, Masaaki Iwatsuki, Koshi Mimori, Tetsuya Sato, Fredrik Johansson, Hiroyuki Toh, Masahiko Watanabe, Masaki Mori
    Annals of Surgical Oncology 17 9 2341 - 2348 2010年09月 [査読有り][通常論文]
     
    Background: Lymph node metastasis is widely accepted as one of the most important determinants of prognosis in colorectal cancer (CRC) patients. Therefore, there is an urgent need to identify molecular markers that can be used to predict lymph node metastasis. Materials and Methods: Candidate genes were found using LMD and cDNA microarrays in a large-scale study of CRC, followed by Penalized Canonical Correlation Analysis (PCCA). We focused on the Fanconi anemia, complementation group D2 (FANCD2) gene and evaluated FANCD2 mRNA expression in 133 CRC cases to determine the clinicopathological significance of FANCD2 expression. Results: The mean level of FANCD2 mRNA expression in tumor tissue specimens was significantly higher than in nontumor tissue. FANCD2 expression was found to be a significant factor affecting lymph node metastasis: the high FANCD2 expression group had a significantly poorer prognosis than the low expression group. Conclusions: This study suggests that PCCA can be used to identify genes related to clinicopathological features. Furthermore, high FANCD2 expression was a significant independent factor for lymph node metastasis. © 2010 Society of Surgical Oncology.
  • Clementina M. van Rijn, Silvana Gaetani, Ines Santolini, Aleksandra Badura, Aleksandra Gabova, Jin Fu, Masashiko Watanabe, Vincenzo Cuomo, Gilles van Luijtelaar, Ferdinando Nicoletti, Richard T. Ngomba
    EPILEPSIA 51 8 1511 - 1521 2010年08月 [査読有り][通常論文]
     
    P>Purpose: Genetically epileptic WAG/Rij rats develop spontaneous absence-like seizures after 3 months of age. We used WAG/Rij rats to examine whether absence seizures are associated with changes in the expression of type-1 cannabinoid (CB1) receptors. Methods: Receptor expression was examined by in situ hybridization and western blot analysis in various brain regions of "presymptomatic" 2-month old and "symptomatic" 8-month-old WAG/Rij rats relative to age-matched nonepileptic control rats. Furthermore, we examined whether pharmacologic activation of CB1 receptor affects absence seizures. We recorded spontaneous spike-wave discharges (SWDs) in 8-month old WAG/Rij rats systemically injected with the potent CB1 receptor agonist, R(+)WIN55,212-2 (3-12 mg/kg, s.c.), given alone or combined with the CB1 receptor antagonist/inverse agonist, AM251 (12 mg/kg, s.c.). Results: Data showed a reduction of CB1 receptor mRNA and protein levels in the reticular thalamic nucleus, and a reduction in CB1 receptor protein levels in ventral basal thalamic nuclei of 8-month-old WAG/Rij rats, as compared with age-matched ACI control rats. In vivo, R(+)WIN55,212-2 caused a dose-dependent reduction in the frequency of SWDs in the first 3 h after the injection. This was followed by a late increase in the mean SWD duration, which suggests a biphasic modulation of SWDs by CB1 receptor agonists. Both effects were reversed or attenuated when R(+)WIN55,212-2 was combined with AM251. Discussion: These data indicate that the development of absence seizures is associated with plastic modifications of CB1 receptors within the thalamic-cortical-thalamic network, and raise the interesting possibility that CB1 receptors are targeted by novel antiabsence drugs.
  • Hassan Marzban, Seung-Hyuk Chung, Maryam Kherad Pezhouh, Hans Feirabend, Masahiko Watanabe, Jan Voogd, Richard Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 518 12 2221 - 2239 2010年06月 [査読有り][通常論文]
     
    The The chick is a well-understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens zebrin II/aldolase C and phospholipase C beta 4 (PLC beta 4)-has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains ("transverse zones"), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II-immunopositive/PLC beta 4-immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II-Via) were distinguished. A central zone (CZ; similar to lobules Vla-VIIIa,b) and a posterior zone (PZ; similar to lobules VIIIa,b-IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II-immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II-immunonegative P3- stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3-. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds. J. Comp. Neurol. 518:2221-2239, 2010. (C) 2010 Wiley-Liss, Inc.
  • Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Mika Tsujita, Makoto Itakura, Manabu Abe, Rie Natsume, Masami Takahashi, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 12 2204 - 2220 2010年06月 [査読有り][通常論文]
     
    The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, gamma-2 and gamma-7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, gamma-2 and gamma-7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in gamma-2-knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in gamma-7-KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in gamma-2/gamma-7 double-KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in gamma-2-KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber-Purkinje cell synapses, and AMPA receptor-mediated currents at climbing fiber-Purkinje cell synapses were all reduced to approximately 10% of the wild-type levels in DKO mice. On the other hand, the reduction in GluA4 in gamma-7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas that of GluA1 and GluA4 in gamma-7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, gamma-2 and gamma-7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.
  • Takuma Kitanishi, Jun Sakai, Shinichi Kojima, Yoshito Saitoh, Kaoru Inokuchi, Masahiro Fukaya, Masahiko Watanabe, Norio Matsuki, Maki K Yamada
    Genes to cells : devoted to molecular & cellular mechanisms 15 7 737 - 47 2010年06月 [査読有り][通常論文]
     
    Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.
  • Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Mika Tsujita, Makoto Itakura, Manabu Abe, Rie Natsume, Masami Takahashi, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 12 2204 - 2220 2010年06月 [査読有り][通常論文]
     
    The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, gamma-2 and gamma-7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, gamma-2 and gamma-7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in gamma-2-knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in gamma-7-KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in gamma-2/gamma-7 double-KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in gamma-2-KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber-Purkinje cell synapses, and AMPA receptor-mediated currents at climbing fiber-Purkinje cell synapses were all reduced to approximately 10% of the wild-type levels in DKO mice. On the other hand, the reduction in GluA4 in gamma-7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas that of GluA1 and GluA4 in gamma-7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, gamma-2 and gamma-7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.
  • Hassan Marzban, Seung-Hyuk Chung, Maryam Kherad Pezhouh, Hans Feirabend, Masahiko Watanabe, Jan Voogd, Richard Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 518 12 2221 - 2239 2010年06月 [査読有り][通常論文]
     
    The The chick is a well-understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens zebrin II/aldolase C and phospholipase C beta 4 (PLC beta 4)-has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains ("transverse zones"), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II-immunopositive/PLC beta 4-immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II-Via) were distinguished. A central zone (CZ; similar to lobules Vla-VIIIa,b) and a posterior zone (PZ; similar to lobules VIIIa,b-IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II-immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II-immunonegative P3- stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3-. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds. J. Comp. Neurol. 518:2221-2239, 2010. (C) 2010 Wiley-Liss, Inc.
  • Takuma Kitanishi, Jun Sakai, Shinichi Kojima, Yoshito Saitoh, Kaoru Inokuchi, Masahiro Fukaya, Masahiko Watanabe, Norio Matsuki, Maki K Yamada
    Genes to cells : devoted to molecular & cellular mechanisms 15 7 737 - 47 2010年06月 [査読有り][通常論文]
     
    Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.
  • Takatoshi Iijima, Eriko Miura, Masahiko Watanabe, Michisuke Yuzaki
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 9 1606 - 1615 2010年05月 [査読有り][通常論文]
     
    Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q-like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans-neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1-C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase-polymerase chain reaction and high-resolution in-situ hybridization. In the adult brain, C1ql1-C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia-like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher-order complexes via their N-terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system.
  • Takatoshi Iijima, Eriko Miura, Masahiko Watanabe, Michisuke Yuzaki
    EUROPEAN JOURNAL OF NEUROSCIENCE 31 9 1606 - 1615 2010年05月 [査読有り][通常論文]
     
    Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q-like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans-neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1-C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase-polymerase chain reaction and high-resolution in-situ hybridization. In the adult brain, C1ql1-C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia-like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher-order complexes via their N-terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system.
  • Shinichi Sakuramoto, Shiro Kikuchi, Nobue Futawatari, Hiromitsu Moriya, Natsuya Katada, Keishi Yamashita, Masahiko Watanabe
    SURGERY 147 5 742 - 747 2010年05月 [査読有り][通常論文]
     
    Background. During esophagojejunostomy using a circular stapler after laparoscopy-assisted gastrectomy, placement of the anvil head via the transabdominal approach proved difficult. The authors report on a method modified for laparoscopy-assisted, esophagojejunostomy performed by placing the pretilted anvil head via the transoral approach. Methods. Between November 2007 and December 2008, esophagojejunostomy was performed using the transoral, pretilted anvil head in 27 patients after laparoscopy-assisted gastrectomy. The anesthesiologist introduced the anvil while observing its passage through the pharynx. During the anastomosis, we kept the jejunum fixed in position with a silicone band Lig-A-Loops, thereby preventing the intestine from slipping off the shaft, of the stapler. Results. Esophagojejunal anastomosis Using the transoral anvil head was achieved successfully in 26 patients; for 1 patient, passage of the anvil head was difficult owing to esophageal stenosis. No other complications, such as hypopharyngeal perforation and/or esophageal mucosal injury, occurred during passage. No postoperative complications occurred, except for 1 patient who developed anastomotic stenosis, in whom mild relief was achieved using a bougie. Conclusion. Esophagojejunostomy using the transoral pretilted anvil head is a simple and safe technique. (Surgery 2010;147:742-7)
  • Carmelo Quarta, Luigi Bellocchio, Giacomo Mancini, Roberta Mazza, Cristina Cervino, Luzie J. Braulke, Csaba Fekete, Rocco Latorre, Cristina Nanni, Marco Bucci, Laura E. Clemens, Gerhard Heldmaier, Masahiko Watanabe, Thierry Leste-Lassere, Marlene Maitre, Laura Tedesco, Flaminia Fanelli, Stefan Reuss, Susanne Klaus, Raj Kamal Srivastava, Krisztina Monory, Alessandra Valerio, Annamaria Grandis, Roberto De Giorgio, Renato Pasquali, Enzo Nisoli, Daniela Cota, Beat Lutz, Giovanni Marsicano, Uberto Pagotto
    CELL METABOLISM 11 4 273 - 285 2010年04月 [査読有り][通常論文]
     
    The endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB1) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB1 modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB1 receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB1 expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB1 signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance.
  • Keiko Matsuda, Eriko Miura, Taisuke Miyazaki, Wataru Kakegawa, Kyoichi Emi, Sakae Narumi, Yugo Fukazawa, Aya Ito-Ishida, Tetsuro Kondo, Ryuichi Shigemoto, Masahiko Watanabe, Michisuke Yuzaki
    SCIENCE 328 5976 363 - 368 2010年04月 [査読有り][通常論文]
     
    Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor delta 2 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cbln1 binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.
  • Carmelo Quarta, Luigi Bellocchio, Giacomo Mancini, Roberta Mazza, Cristina Cervino, Luzie J. Braulke, Csaba Fekete, Rocco Latorre, Cristina Nanni, Marco Bucci, Laura E. Clemens, Gerhard Heldmaier, Masahiko Watanabe, Thierry Leste-Lassere, Marlene Maitre, Laura Tedesco, Flaminia Fanelli, Stefan Reuss, Susanne Klaus, Raj Kamal Srivastava, Krisztina Monory, Alessandra Valerio, Annamaria Grandis, Roberto De Giorgio, Renato Pasquali, Enzo Nisoli, Daniela Cota, Beat Lutz, Giovanni Marsicano, Uberto Pagotto
    CELL METABOLISM 11 4 273 - 285 2010年04月 [査読有り][通常論文]
     
    The endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB1) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB1 modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB1 receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB1 expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB1 signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance.
  • Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, Masamitsu Iino
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 14 6526 - 6531 2010年04月 [査読有り][通常論文]
     
    Glutamate is the major neurotransmitter in the brain, mediating point-to-point transmission across the synaptic cleft in excitatory synapses. Using a glutamate imaging method with fluorescent indicators, we show that synaptic activity generates extrasynaptic glutamate dynamics in the vicinity of active synapses. These glutamate dynamics had magnitudes and durations sufficient to activate extrasynaptic glutamate receptors in brain slices. We also observed crosstalk between synapses-i.e., summation of glutamate released from neighboring synapses. Furthermore, we successfully observed that sensory input from the extremities induced extrasynaptic glutamate dynamics within the appropriate sensory area of the cerebral cortex in vivo. Thus, the present study clarifies the spatiotemporal features of extrasynaptic glutamate dynamics, and opens up an avenue to directly visualizing synaptic activity in live animals.
  • Keiko Matsuda, Eriko Miura, Taisuke Miyazaki, Wataru Kakegawa, Kyoichi Emi, Sakae Narumi, Yugo Fukazawa, Aya Ito-Ishida, Tetsuro Kondo, Ryuichi Shigemoto, Masahiko Watanabe, Michisuke Yuzaki
    SCIENCE 328 5976 363 - 368 2010年04月 [査読有り][通常論文]
     
    Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor delta 2 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cbln1 binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.
  • Kenichiro Wakabayashi, Masato Fujioka, Sho Kanzaki, Hirotaka James Okano, Shinsuke Shibata, Daisuke Yamashita, Masatsugu Masuda, Masahiko Mihara, Yoshiyuki Ohsugi, Kaoru Ogawa, Hideyuki Okano
    NEUROSCIENCE RESEARCH 66 4 345 - 352 2010年04月 [査読無し][通常論文]
     
    Hearing impairment can be the cause of serious socio-economic disadvantages. Recent studies have shown inflammatory responses in the inner ear co-occur with various damaging conditions including noise-induced hearing loss. We reported pro-inflammatory cytokine interleukin-6 (IL-6) was induced in the cochlea 6 h after noise exposure, but the pathophysiological implications of this are still obscure. To address this issue, we investigated the effects of IL-6 inhibition using the anti-IL-6 receptor antibody (MR16-1). Noise-exposed mice were treated with MR16-1 and evaluated. Improved hearing at 4 kHz as measured by auditory brainstem response (ABR) was noted in noise-exposed mice treated with MR16-1. Histological analysis revealed the decrease in spiral ganglion neurons was ameliorated in the MR16-1-treated group, while no significant change was observed in the organ of Corti. Immunohistochemistry for Iba1 and CD45 demonstrated a remarkable reduction of activated cochlear macrophages in spiral ganglions compared to the control group when treated with MR16-1. Thus, MR16-1 had protective effects both functionally and pathologically for the noise-damaged cochlea primarily due to suppression of neuronal loss and presumably through alleviation of inflammatory responses. Anti-inflammatory cytokine therapy including IL-6 blockade would be a feasible novel therapeutic strategy for acute sensory neural hearing loss. (C) 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Yuichi Nakahara, Shigeo Imanishi, Kanako Mitsumasu, Yasushi Kanamori, Ken-ichi Iwata, Masahiko Watanabe, Takahiro Kikawada, Takashi Okuda
    CRYOBIOLOGY 60 2 138 - 146 2010年04月 [査読有り][通常論文]
     
    Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210 Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply We also attempted dry-preservation of organs isolated from P vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation (C) 2009 Elsevier Inc All rights reserved
  • Miwako Yamasaki, Minoru Matsui, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 30 12 4408 - 4418 2010年03月 [査読有り][通常論文]
     
    Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called " volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M-1, the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M-1 was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M-1 was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M-1 and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M-1 is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M-1-mediated cortical cholinergic signaling.
  • Ikuo Matsuda, Masahiro Fukaya, Harumi Nakao, Kazuki Nakao, Hideyuki Matsumoto, Kensaku Mori, Masahiko Watanabe, Atsu Aiba
    NEUROSCIENCE RESEARCH 66 3 321 - 329 2010年03月 [査読有り][通常論文]
     
    Semaphorin 3F (Sema3F) is a secreted type of the semaphorin family of axon guidance molecules Sema3F and its receptor Neuropilin-2 (Npn-2) mRNAs were distributed in a mutually exclusive manner throughout mouse brain development In order to examine physiological roles of Sema3F, we generated Sema3F knockout mice (KO) by gene targeting in embryonic stem (ES) cells We found that the loss of Sema3F expression did not significantly affect the mRNA expression of Npn-2 or the other putative Npn-2 ligands, namely. Sema3B, Sema3C, or Sema3G. The barrel structure of the somatosensory cortex and the cerebellar neuroanatomy were not significantly altered in Sema3F KO Finally, optical imaging of intrinsic signals of the dorsal olfactory bulb showed no significant differences in odor map between wildtype mice and Sema3F KO These data suggest that Sema3F plays a relatively restricted. if any, role in its receptor expression and postnatal development of these brain Structures (C) 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society All rights reserved
  • Miwako Yamasaki, Minoru Matsui, Masahiko Watanabe
    JOURNAL OF NEUROSCIENCE 30 12 4408 - 4418 2010年03月 [査読有り][通常論文]
     
    Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called " volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M-1, the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M-1 was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M-1 was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M-1 and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M-1 is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M-1-mediated cortical cholinergic signaling.
  • M. Hondo, K. Nagai, K. Ohno, Y. Kisanuki, J. T. Willie, T. Watanabe, M. Yanagisawa, T. Sakurai
    ACTA PHYSIOLOGICA 198 3 287 - 294 2010年03月 [査読無し][通常論文]
     
    Aim: The effect of orexin on wakefulness has been suggested to be largely mediated by activation of histaminergic neurones in the tuberomammillary nucleus (TMN) via orexin receptor-2 (OX(2)R). However, orexin receptors in other regions of the brain might also play important roles in maintenance of wakefulness. To dissect the role of the histaminergic system as a downstream mediator of the orexin system in the regulation of sleep/wake states without compensation by the orexin receptor-1 (OX(1)R) mediated pathways, we analysed the phenotype of Histamine-1 receptor (H(1)R) and OX(1)R double-deficient (H(1)R(-/-);OX(1)R(-/-)) mice. These mice lack OX(1)R-mediated pathways in addition to deficiency of H(1)R, which is thought to be the most important system in downstream of OX(2)R. Methods: We used H(1)R deficient (H(1)R(-/-)) mice, H(1)R(-/-);OX(1)R(-/-) mice, OX(1)R and OX(2)R double-deficient (OX(1)R(-/-);OX(2)R(-/-)) mice, and wild type controls. Rapid eye movement (REM) sleep, non-REM (NREM) sleep and awake states were determined by polygraphic electroencephalographic/electromyographic recording. Results: No abnormality in sleep/wake states was observed in H(1)R(-/-) mice, consistent with previous studies. H(1)R(-/-);OX(1)R(-/-) mice also showed a sleep/wake phenotype comparable to that of wild type mice, while OX(1)R(-/-); OX(2)R(-/-) mice showed severe fragmentation of sleep/wake states. Conclusions: Our observations showed that regulation of the sleep/wake states is completely achieved by OX(2)R-expressing neurones without involving H(1)R-mediated pathways. The maintenance of basal physiological sleep/wake states is fully achieved without both H(1) and OX(1) receptors. Downstream pathways of OX(2)R other than the histaminergic system might play an important role in the maintenance of sleep/wake states.
  • Asami Tanimura, Maya Yamazaki, Yuki Hashimotodani, Motokazu Uchigashima, Shinya Kawata, Manabu Abe, Yoshihiro Kita, Kouichi Hashimoto, Takao Shimizu, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    NEURON 65 3 320 - 327 2010年02月 [査読有り][通常論文]
     
    Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGL alpha) and beta (DGL beta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGL alpha knockout mice, whereas the retrograde suppression was intact in DGL beta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGL alpha knockout brains, whereas the 2-AG content was normal in DGL beta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGL alpha, but not by DGL beta, mediates retrograde suppression at central synapses.
  • Norifumi Shioda, Yui Yamamoto, Masahiko Watanabe, Bert Binas, Yuji Owada, Kohji Fukunaga
    JOURNAL OF NEUROSCIENCE 30 8 3146 - 3155 2010年02月 [査読有り][通常論文]
     
    Fatty acid binding proteins (FABPs) are essential for energy production and long-chain polyunsaturated fatty acid-related signaling in the brain and other tissues. Of various FABPs, heart-type fatty acid binding protein (H-FABP, FABP3) is highly expressed in neurons of mature brain and plays a role in arachidonic acid incorporation into brain and heart cells. However, the precise function of H-FABP in brain remains unclear. We previously demonstrated that H-FABP is associated with the dopamine D-2 receptor long isoform (D2LR) in vitro. Here, we confirm that H-FABP binds to dopamine D-2 receptor (D2R) in brain extracts and colocalizes immunohistochemically with D2R in the dorsal striatum. We show that H-FABP is highly expressed in acetylcholinergic interneurons and terminals of glutamatergic neurons in the dorsal striatum of mouse brain but absent in dopamine neuron terminals and spines in the same region. H-FABP knock-out (KO) mice showed lower responsiveness to methamphetamine-induced sensitization and enhanced haloperidol-induced catalepsy compared with wild-type mice, indicative of D2R dysfunction. Consistent with the latter, aberrant increased acetylcholine (ACh) release and depolarization-induced glutamate (Glu) release were observed in the dorsal striatum of H-FABP KO mice. Furthermore, phosphorylation of CaMKII (Ca2+/calmodulin-dependent protein kinase II) and ERK (extracellular signal-regulated kinase) was significantly increased in the dorsal striatum. We confirmed elevated ERK phosphorylation following quinpirole-mediated D2R stimulation in H-FABP-overexpressing SHSY-5Y human neuroblastoma cells. Together, H-FABP is highly expressed in ACh interneurons and glutamatergic terminals, thereby regulating dopamine D2R function in the striatum.
  • Zsuzsa S. Kocsis, Csilla S. Molnar, Masahiko Watanabe, Guy Daneels, Dieder Moechars, Zsolt Liposits, Erik Hrabovszky
    NEUROCHEMISTRY INTERNATIONAL 56 3 479 - 486 2010年02月 [査読有り][通常論文]
     
    Recent immunohistochemical studies of the rat adenohypophysis identified type-2 vesicular glutamate transporter (VGLUT-2), a marker for glutamatergic neuronal phenotype, in high percentages of adenohypophysial gonadotrophs and thyrotrophs. The presence and molecular identity of amino acid neurotransmitters in the remaining hormone producing cell types are unknown. In the present study we addressed the putative synthesis of another glutamatergic marker, VGLUT1 by adenohypophysial cells. Immunohistochemical studies revealed VGLUT1 immunoreactivity in a small subset of polygonal medium-sized cells in the anterior lobe. Western blot analysis revealed a single major 60 kDa protein band in the adenohypophysis. Furthermore, the expression of VGLUT1 mRNA was confirmed by reverse transcription-polymerase chain reaction followed by sequence analysis of the amplicon. In contrast with rats which only showed VCLUT1 signal in the anterior lobe of the pituitary, mice contained high levels of VGLUT1 immunoreactivity in the intermediate, in addition to the anterior lobe. No signal was present in VGLUT1-knockout mice, providing evidence for specificity. In rats, results of colocalization studies with dual-immunofluorescent labeling provided evidence for VGLUT1 immunoreactivity in 45.9% of corticotrophs and 7.7% of luteinizing hormone beta-immunopositive gonadotrophs. Cells of the other peptide hormone phenotypes were devoid of VGLUT1 signal. A few cells in the adenohypophysis expressed both VGLUT1 and VGLUT2 immunoreactivities. The presence of the glutamate markers VGLUT1 and VGLUT2 in distinct populations of peptide hormone-secreting hypophysial cells highly indicates the involvement of endogenous glutamate release in autocrine/paracrine regulatory mechanisms. The biological function of adenohypophysial glutamate will require clarification. (C) 2009 Elsevier Ltd. All rights reserved.
  • Yasukazu Hozumi, Masahiko Watanabe, Kaoru Goto
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 58 2 119 - 129 2010年02月 [査読有り][通常論文]
     
    The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase beta (DGK beta) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGK beta in dopaminergic transmission. However, it remains elusive whether DGK beta is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGK beta and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLC beta 4 and PKC alpha. are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119-129, 2010)
  • Keiko Fujikawa, Takeshi Iwata, Kaoru Inoue, Masakazu Akahori, Hanako Kadotani, Masahiro Fukaya, Masahiko Watanabe, Qing Chang, Edward M. Barnett, Wojciech Swat
    PLOS ONE 5 2 2010年02月 [査読有り][通常論文]
     
    Background: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. Methodology/Principal Findings: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. Conclusions/Significance: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.
  • Keiko Fujikawa, Takeshi Iwata, Kaoru Inoue, Masakazu Akahori, Hanako Kadotani, Masahiro Fukaya, Masahiko Watanabe, Qing Chang, Edward M. Barnett, Wojciech Swat
    PLOS ONE 5 2 e9050  2010年02月 [査読有り][通常論文]
     
    Background: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. Methodology/Principal Findings: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. Conclusions/Significance: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.
  • Yiqing Zhao, Xiaodong Zhang, Kishore Guda, Earl Lawrence, Qun Sun, Toshio Watanabe, Yoichiro Iwakura, Masahide Asano, Lanlan Wei, Zhirong Yang, Weiping Zheng, Dawn Dawson, Joseph Willis, Sanford D. Markowitz, Masanobu Satake, Zhenghe Wang
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 6 2592 - 2597 2010年02月 [査読有り][通常論文]
     
    Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis.
  • Hidefumi Kubo, Kanako Hosono, Tatsunori Suzuki, Yasufumi Ogawa, Hiroshi Kato, Hiroki Kamata, Yoshiya Ito, Hideki Amano, Tetsuki Kato, Hiroyuki Sakagami, Izumi Hayashi, Yukihiko Sugimoto, Shuh Narumiya, Masahiko Watanabe, Masataka Majima
    BIOMEDICINE & PHARMACOTHERAPY 64 2 101 - 106 2010年02月 [査読有り][通常論文]
     
    Prostaglandin E(2) (PGE(2)) and prostaglandin E (EP) receptor signaling pathways have been implicated in the promotion Of tumor growth and angiogenesis. However, little is known about their roles in lymphangiogenesis during tumor development. The present study evaluates whether endogenous PGE2 exhibits a critical role in tumor-associated lymphangiogenesis. Treatment of male C57BL/6 mice with a cyclooxygenase-2 inhibitor, celecoxib, for seven days resulted in a 52.4% reduction in tumor size induced by subcutaneous injection of murine Lewis lung cells. Celecoxib treatment down-regulated the expression of vascular endothelial growth factor receptor (VEGFR)-3 in stromal tissues by 73.9%, and attenuated expression of podoplanin, a marker for lymphatic endothelial cells. To examine the role of host PGE receptor signaling, we tested four kinds of EP receptor knockout mice. At Day 7 after tumor cell implantation, EP3 receptor knockout mice, but not EP receptor knockout mice lacking EP1, EP2, or EP4. exhibited a 53.3% reduction in tumor weight, which was associated with a 74.5% reduction in VEGFR-3 mRNA expression in tumor stromal tissues. At Day 14, VEGFR-3 expression in EP3-/- mice remained significantly lower than that of their wild-type (WT) counterparts. The expression of VEGF-C in the tumor stromal tissues in EP3-/- mice were also reduced by 22.1% (Day 7) and 44.1% (Day 14), respectively. In addition, the level Of immunoreactive podoplanin in the tumor tissues from EP3-/- mice was less than that of WT. These results suggest that host EP3 receptor signaling regulates tumor-associated lymphangiogenesis by up-regulating expression of VEGF-C and its receptor, VEGFR-3, in tumor stromal tissues. Host EP3 blockade together with COX-2 inhibition may be a novel therapeutic strategy to suppress tumor-associated lymphangiogenesis. (C) 2009 Elsevier Masson SAS. All rights reserved.
  • Keiko Hayashi, Norihiko Sengoku, Yoshimasa Kosaka, Takumo Enomoto, Sabine Kajita, Yasushi Kondo, Masaru Kuranami, Masahiko Watanabe
    Japanese Journal of Cancer and Chemotherapy 37 12 2756 - 2759 2010年 [査読有り][通常論文]
     
    A 51 -year-old postmenopausal woman was diagnosed as having adenocarcinoma (gastric cancer type 4) from gastric biopsy by upper endoscopy. Her chief complaint was abdominal dilatation. Meanwhile, a breast CT suggested tumor in her left breast and was diagnosed as an invasive lobular carcinoma based on a core needle biopsy. After gastric biopsy, tissues are stained by ER and PgR in immunohistochemistry. The diagnosis was modified from gastric cancer to T2N1M1, stage IV left breast cancer, accompanied by a treatment. Chemotherapy with EC 6 course consisted of a weekly PTX 4 course (epi-rubicin, cyclophosphamide-weekly paclitaxel) was performed. After the chemotherapy, breast mass, ascites and tumor marker were dramatically improved. Then hormonal therapy was administered. She passed away 2 and 1/2 years after her first visit to the hospital. Metastatic gastric tumors simulating type 4 advanced gastric cancer (MGTS type 4) and invasive lobular carcinoma are known to have an unfavorable prognosis. There is no doubt, however, that the multidisciplinary treatments have brought a satisfaction to her and family. We should keep in mind a possibility of gastric metastasis of breast cancer, when consulting a female patient with gastric cancer type 4.
  • Kanako Mitsumasu, Yasushi Kanamori, Mika Fujita, Ken-Ichi Iwata, Daisuke Tanaka, Shingo Kikuta, Masahiko Watanabe, Richard Cornette, Takashi Okuda, Takahiro Kikawada
    FEBS Journal 277 20 4215 - 4228 2010年 [査読有り][通常論文]
     
    Larvae of an anhydrobiotic insect, Polypedilum vanderplanki, accumulate very large amounts of trehalose as a compatible solute on desiccation, but the molecular mechanisms underlying this accumulation are unclear. We therefore isolated the genes coding for trehalose metabolism enzymes, i.e. trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) for the synthesis step, and trehalase (TREH) for the degradation step. Although computational prediction indicated that the alternative splicing variants (PvTps) obtained encoded probable functional motifs consisting of a typical consensus domain of TPS and a conserved sequence of TPP, PvTps did not exert activity as TPP, but only as TPS. Instead, a distinct gene (PvTpp) obtained expressed TPP activity. Previous reports have suggested that insect TPS is, exceptionally, a bifunctional enzyme governing both TPS and TPP. In this article, we propose that TPS and TPP activities in insects can be attributed to discrete genes. The translated product of the TREH ortholog (PvTreh) certainly degraded trehalose to glucose. Trehalose was synthesized abundantly, consistent with increased activities of TPS and TPP and suppressed TREH activity. These results show that trehalose accumulation observed during anhydrobiosis induction in desiccating larvae can be attributed to the activation of the trehalose synthetic pathway and to the depression of trehalose hydrolysis. © 2010 FEBS.
  • Taisuke Miyazaki, Miwako Yamasaki, Tomonori Takeuchi, Kenji Sakimura, Masayoshi Mishina, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 68 E86 - E86 2010年 [査読有り][通常論文]
  • Maya Yamazaki, Masahiro Fukaya, Kouichi Hashimoto, Miwako Yamasaki, Makoto Itakura, Masami Takahashi, Masanobu Kano, Masahiko Watanabe, Kenji Sakimura
    NEUROSCIENCE RESEARCH 68 E223 - E224 2010年 [査読有り][通常論文]
  • Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, Masamitsu Iino
    JOURNAL OF PHARMACOLOGICAL SCIENCES 112 110P - 110P 2010年 [査読有り][通常論文]
  • Miwako Yamasaki, Masahiro Fukaya, Manabu Abe, Kenji Sakimura, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 68 E56 - E56 2010年 [査読有り][通常論文]
  • 岡野 栄之
    NEUROSCIENCE RESEARCH 68 E61 - E61 2010年 [査読無し][通常論文]
  • Nobue Futawatari, Shiro Kikuchi, Hiromitsu Moriya, Natsuya Katada, Shinichi Sakuramoto, Masahiko Watanabe
    SURGERY TODAY 40 1 64 - 67 2010年01月 [査読有り][通常論文]
     
    We herein report our experience in performing a laparoscopy-assisted distal gastrectomy involving standard lymph node dissection for a patient with early gastric cancer and situs inversus. A superficial elevated lesion was found on the posterior wall of the antrum. The preoperative diagnosis was cStage IA (cT1, cN0, cH0, cP0, cM0). A laparoscopy-assisted distal gastrectomy with standard lymph node dissection (D1 + beta) for early gastric cancer was successfully performed by shifting the monitor to the right and left, and by applying sufficient care and time. The course of blood vessels was not abnormal except for the right/left inversion. Billroth I reconstruction was performed through a delta-shaped anastomosis. The postoperative course was favorable and the patient was discharged on postoperative day 12. Based on a histopathological examination, a 5.0 x 5.0-cm, mucosal, poorly differentiated adenocarcinoma (pT1, pN0, sH0, sP0, sM0, Stage IA) was diagnosed.
  • Takeshi Izumi, Yu Omura, Yukino Futami, Taku Yamaguchi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
    JOURNAL OF PHARMACOLOGICAL SCIENCES 112 98P - 98P 2010年 [査読有り][通常論文]
  • Weiqiang Kong, Tomohiro Shiraishi, Yuki Mizushima, Noriyuki Katahira, Akira Fukuda, Masahiko Watanabe
    12TH INTERNATIONAL CONFERENCE ON ADVANCED COMMUNICATION TECHNOLOGY: ICT FOR GREEN GROWTH AND SUSTAINABLE DEVELOPMENT, VOLS 1 AND 2 1003 - 1008 2010年 [査読有り][通常論文]
     
    State Transition Matrix (STM) is a flexible table-like modeling language that has been frequently used for specifying behavior of distributed systems. In this paper, we first present a formalization of the static and dynamic aspects of a STM design (i.e., design written in STM). Consequentially, based on this formalization, we investigate how a STM design can be formally analyzed using SAL, precisely SAL infinite bounded model checker, through a language translation. Specifically, the formal analysis is conducted focusing on four kinds of safety properties related to: (1) Invalid Cells, (2) Static Constraints, (3) Dynamic Constraints, and (4) Deadlock, respectively, since the fulfillment of these properties is commonly desired by industrial practitioners for a STM design. A simple Internet Connection Control system is used as our demonstration example.
  • Kouichi Hashimoto, Mika Tsujita, Kazuo Kitamura, Taisuke Miyazaki, Maya Yamazaki, Hee-Sup Shin, Masahiko Watanabe, Kenji Sakimura, Masanobu Kano
    NEUROSCIENCE RESEARCH 68 E37 - E38 2010年 [査読有り][通常論文]
  • Jonghan Shin, Gangadharan Gireesh, Seong-Wook Kim, Duk-Soo Kim, Sukyung Lee, Yeon-Soo Kim, Masahiko Watanabe, Hee-Sup Shin
    JOURNAL OF NEUROSCIENCE 29 49 15375 - 15385 2009年12月 [査読有り][通常論文]
     
    Anxiety is among the most prevalent and costly diseases of the CNS, but its underlying mechanisms are not fully understood. Although attenuated theta rhythms have been observed in human subjects with increased anxiety, no study has been done on the possible physiological link between these two manifestations. We found that the mutant mouse for phospholipase C beta 4 (PLC-beta 4(-/-)) showed attenuated theta rhythm and increased anxiety, presenting the first animal model for the human condition. PLC-beta 4 is abundantly expressed in the medial septum, a region implicated in anxiety behavior. RNA interference-mediated PLC-beta 4 knockdown in the medial septum produced a phenotype similar to that of PLC-beta 4(-/-) mice. Furthermore, increasing cholinergic signaling by administering an acetylcholinesterase inhibitor cured the anomalies in both cholinergic theta rhythm and anxiety behavior observed in PLC-beta 4(-/-) mice. These findings suggest that (1) PLC-beta 4 in the medial septum is involved in controlling cholinergic theta oscillation and (2) cholinergic theta rhythm plays a critical role in suppressing anxiety. We propose that defining the cholinergic theta rhythm profile may provide guidance in subtyping anxiety disorders in humans for more effective diagnosis and treatments.
  • Kenji Ishido, Mizutomo Azuma, Wasaburo Koizumi, Atsuko Takeuchi, Shinichi Sakuramoto, Masahiko Watanabe, Isao Okayasu
    Pharmacogenetics and Genomics 19 12 955 - 964 2009年12月 [査読有り][通常論文]
     
    OBJECTIVES: Many studies have reported that low intratumoral mRNA expression of thymidylate synthase (TS) is an important biomarker of response to chemotherapy in patients with unresectable advanced gastric cancer. However, the role of gene expression profile of patients who received postoperative adjuvant chemotherapy remains unclear. In this study, we evaluated how TS and other associated genes related to outcome. METHODS: Seventy-nine patients with stage II or III advanced gastric cancer who underwent gastrectomy were analyzed. Thirty-nine patients received adjuvant chemotherapy with S-1 after surgery (S-1 group) and 40 patients underwent surgery only (surgery group). Formalin-fixed, paraffin-embedded tumor tissues were dissected by the laser-captured microdissection technique and analyzed for target gene expressions using a quantitative real-time polymerase chain reaction. RESULTS: There were no significant differences between the S-1 group and the surgery group in gene expressions except TS (P=0.034). In the S-1 group, recurrence-free survival (RFS) and overall survival (OS) were significantly longer in patients with low TS expression compared with patients with high TS expression (P=0.021 and 0.016), whereas there were no correlations in the surgery group. Furthermore, RFS and OS were both correlated with extent of lymph node metastasis (N) (P=0.038 and 0.020) and TS expression (P=0.021 and 0.032). On multivariate analysis it was found that TS expression and N were significant independent prognostic factors of RFS and OS (TS: P=0.027 and 0.050, N: P=0.048 and 0.032). CONCLUSION: Our results suggested that intratumoral TS expression is an independent prognostic factor in patients with gastric cancer who received postoperative adjuvant chemotherapy with S-1. © 2009 Lippincott Williams & Wilkins.
  • Ji-Young Kim, Hassan Marzban, Seung-Hyuk Chung, Masahiko Watanabe, Leonard M. Eisenman, Richard Hawkes
    JOURNAL OF COMPARATIVE NEUROLOGY 517 2 193 - 209 2009年11月 [査読有り][通常論文]
     
    Transverse boundaries divide the mammalian cerebellar cortex into transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. This topography is highly conserved across the Mammalia. Bats have a remarkable cerebellum with presumed adaptations to flight and to echolocation, but nothing is known of its compartmentation. We have therefore used two Purkinje cell compartmentation antigens, zebrin II/aldolase C and phospholipase C beta 4, to reveal the topography of the cerebellum in microchiropteran bats. Three species of bat were studied, Lasiurus cinereus, Lasionycteris noctivagans, and Eptesicus fuscus. A reproducible pattern of zones and stripes was revealed that is similar across the three species. The architecture of the bat cerebellum conforms to the ground plan of other mammals. However, two exceptions to the highly conserved mammalian architectural plan were revealed. First, many Purkinje cells in lobule I express zebrin II. A zebrin II-immunopositive lobule I has not been seen previously in mammals but is characteristic of the avian cerebellum. Second, lobules VI-VII comprise the large central zone. Within the central zone two subdomains are evident, a small anterior subdomain (lobule VI) in which Purkinje cells are predominantly zebrin II-immunopositive/PLC beta 4-immunonegative, as in other mammals, and a posterior subdomain (lobule VII), in which alternating zebrin II/phospholipase C beta 4 stripes are prominent. J. Comp. Neurol. 517:193-209, 2009. (C) 2009 Wiley-Liss, Inc.
  • Koichi Yoshida, Masashi Watanabe, Shigetsugu Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 389 3 506 - 511 2009年11月 [査読有り][通常論文]
     
    The ubiquitin-proteasome system has been implicated in neuronal degeneration and regeneration. We demonstrated that overexpression of ZNRF1, which has been identified as a crucial molecule in nerve regeneration, causes morphological changes such as neurite-like elongation. Molecular dissections showed that both the RING finger domain and zinc finger domain are required for morphological changes. Furthermore, we identified beta-tubulin type 2 (Tubb2) as a ZNRF1-binding protein by yeast two-hybrid screening. In vivo binding assay showed that ZNRF1 interacts with Tubb2 and immunofluorescent staining Suggests that ZNRF1 is colocalized with Tubb2. These results suggest that ZNRF1 mediates regulation of neuritogenesis via interaction with tubulin. (C) 2009 Elsevier Inc. All rights reserved.
  • Noriko Ando, Yumi Iwamitsu, Masaru Kuranami, Shigemi Okazaki, Mei Wada, Kenji Yamamoto, Keiko Todoroki, Masahiko Watanabe, Hitoshi Miyaoka
    SUPPORTIVE CARE IN CANCER 17 11 1361 - 1370 2009年11月 [査読有り][通常論文]
     
    The objective of this study was to determine how psychological characteristics, subjective symptoms, a family history of breast cancer, and age impact psychological distress in outpatients at the first hospital visit prior to breast cancer diagnosis. Participants were prediagnosed women with complaints of breast symptoms who either came to our hospital directly, or with a referral from another clinic. Patients were asked to complete questionnaires to determine the following: trait anxiety (State-Trait Anxiety Inventory), negative emotional suppression (Courtauld Emotional Control Scale), life stress events (Life Experiences Survey), and psychological distress (Profile of Mood States). We examined subjective symptoms (lumps, pain, abnormal nipple discharge, or deformed nipple) and family history of breast cancer based on answers to the interview sheet filled out by patients on their first visit. We analyzed a total of 154 patients who completed the questionnaires out of 237 eligible patients. A significant model was obtained by multiple regression analysis (adjusted R (2) = 0.574, p < 0.01) in which the standard partial regression coefficients for trait anxiety, suppression of anxiety, negative life change events, positive life change events, and subjective symptoms were statistically significant (beta = 0.623, 0.133, 0.155, 0.108, and 0.124, respectively; p < 0.05). Psychological distress prior to diagnosis was higher in patients who had high trait anxiety, suppression of anxiety, many life stress events, and subjective symptoms. In particular, trait anxiety had a large impact on psychological distress, underscoring the need for and importance of adequate psychological care.
  • Masashi Watanabe, Tadasuke Tsukiyama, Shigetsugu Hatakeyama
    Biochemical and Biophysical Research Communications 388 2 422 - 427 2009年10月16日 [査読有り][通常論文]
     
    Tripartite motif-containing protein (TRIM) family proteins are involved in a broad range of biological processes and, consistently, their alterations result in diverse pathological conditions such as genetic diseases, viral infection and cancer development. In this study, we found that one of the TRIM family proteins, TRIM31, is highly expressed in the gastrointestinal tract and interacts with p52Shc, one of the signal transducers. We also found by a binding assay that almost the whole region other than the RING domain is required for the binding to p52Shc but found by pulse-chase analysis that overexpression of TRIM31 does not affect the stability of p52Shc. Moreover, we found that overexpression of TRIM31 suppresses anchorage-independent cell growth induced by the active form of c-Src. These results suggest that TRIM31 attenuates c-Src signaling via p52Shc under anchorage-independent growth conditions and is potentially associated with growth activity of cells in the gastrointestinal tract. © 2009 Elsevier Inc. All rights reserved.
  • Toshiharu Yasaka, David I. Hughes, Erika Polgar, Gergely G. Nagy, Masahiko Watanabe, John S. Riddell, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 29 42 13401 - 13409 2009年10月 [査読有り][通常論文]
     
    Pure NMDA receptor (NMDAr)-mediated EPSCs, thought to correspond to "silent" glutamatergic synapses that lack AMPA receptors (AMPArs), have been observed in superficial spinal dorsal horn of neonatal but not adult rats. Recent anatomical studies suggest that AMPArs are present at virtually all glutamatergic synapses in this region in adults. We used antigen retrieval to examine colocalization of AMPArs and PSD-95 (a marker for glutamatergic synapses) in laminae I-II of neonatal and adult rats. We found a high degree of colocalization in all cases, which suggests that AMPArs are present in the great majority of glutamatergic synapses even in neonatal animals. We therefore reexamined evidence for silent synapses by performing blind whole-cell recordings from superficial dorsal horn neurons in slices from neonatal or adult rats, with focal stimulation to activate glutamatergic synapses. On some occasions in both neonatal (10 of 109, 9%) and adult (9 of 77, 12%) slices, NMDAr-mediated EPSCs were observed when the holding potential was raised to +50 mV at a stimulus strength that had failed to evoke AMPAr-mediated EPSCs. However, in all cases tested, AMPAr-mediated EPSCs were then observed when the cell was returned to-70 mV; this and other properties of the EPSCs suggest that they do not represent genuine silent synapses. When compared with previous findings, our results indicate that the appearance of silent synapses depends on experimental protocol. This suggests that pure NMDAr-mediated EPSCs seen in previous studies do not correspond to AMPAr-lacking synapses but result from another mechanism, for example, loss of labile AMPArs from recently formed synapses.
  • Etsuko Tarusawa, Ko Matsui, Timotheus Budisantoso, Elek Molnar, Masahiko Watanabe, Minoru Matsui, Yugo Fukazawa, Ryuichi Shigemoto
    JOURNAL OF NEUROSCIENCE 29 41 12896 - 12908 2009年10月 [査読有り][通常論文]
     
    To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.
  • Osamu Katsumata, Naoki Ohara, Hideaki Tamaki, Tomoko Niimura, Hideaki Naganuma, Masahiko Watanabe, Hiroyuki Sakagami
    EUROPEAN JOURNAL OF NEUROSCIENCE 30 8 1509 - 1516 2009年10月 [査読有り][通常論文]
     
    IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), which are implicated in membrane trafficking and actin cytoskeleton dynamics. In this study, we examined the immunohistochemical localization of IQ-ArfGEF/BRAG1 in the adult mouse retina using light and electron microscopy. IQ-ArfGEF/BRAG1 was distributed in a punctate manner and colocalized well with RIBEYE in both the outer and inner plexiform layers. Immunoelectron microscopic analysis showed that IQ-ArfGEF/BRAG1 was localized at the synaptic ribbons of photoreceptors. When heterologously expressed in HeLa cells, IQ-ArfGEF/BRAG1 was recruited to RIBEYE-containing clusters and formed an immunoprecipitable complex with RIBEYE. Furthermore, immunoprecipitation analysis showed that anti-IQ-ArfGEF/BRAG1 antibody efficiently pulled down RIBEYE from retinal lysates. These findings indicate that IQ-ArfGEF/BRAG1 is a novel component of retinal synaptic ribbons and forms a protein complex with RIBEYE.
  • Laia Canela, Victor Fernandez-Duenas, Catarina Albergaria, Masahiko Watanabe, Carme Lluis, Josefa Mallol, Enric I. Canela, Rafael Franco, Rafael Lujan, Francisco Ciruela
    JOURNAL OF NEUROCHEMISTRY 111 2 555 - 567 2009年10月 [査読有り][通常論文]
     
    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.
  • Hozumi Y, Watanabe M, Goto K
    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2009年10月01日 [査読有り][通常論文]
  • Etsuko Tarusawa, Ko Matsui, Timotheus Budisantoso, Elek Molnar, Masahiko Watanabe, Minoru Matsui, Yugo Fukazawa, Ryuichi Shigemoto
    JOURNAL OF NEUROSCIENCE 29 41 12896 - 12908 2009年10月 [査読有り][通常論文]
     
    To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.
  • Naoyuki Yamasaki, Masahiko Watanabe, Kimihiro Masuyama, Yasuo Miyake, Hitoshi Kubo, Akihiko Fujii, Masanori Ozaki
    JAPANESE JOURNAL OF APPLIED PHYSICS 48 10 2009年10月 [査読有り][通常論文]
     
    The optical and electrical properties of polymer composite films based on poly(2-methoxy-5-dodecyloxy-p-phenylenevinylene) (MDDOPPV) and poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT) have been studied. We investigated the time-resolved photoluminescence of the composite films and clarified the rapid intermolecular energy transfer from F8BT to MDDOPPV. As a result of the utilization of the polymer composite thin film as the emission layer of a light-emitting diode, a 23-fold increase in luminance was observed in comparison to that with a non composite MDDOPPV emission layer. (C) 2009 The Japan Society of Applied Physics
  • Akira Iizuka, Kiyohiko Takayama, Takashi Torashima, Miwako Yamasaki, Chiho Koyama, Kazuhiro Mitsumura, Masahiko Watanabe, Hirozaku Hirai
    NEUROBIOLOGY OF DISEASE 35 3 457 - 465 2009年09月 [査読有り][通常論文]
     
    Hotfoot5J mice are spontaneously occurring ataxic mice that lack delta 2 glutamate receptor (GluR82) protein in cerebellar Purkinje cells. Here we aimed to rescue the ataxic phenotype of hotfoot5J mice by lentiviral vector-mediated expression of recombinant GIuR82 in Purkinje cells. Lentiviral vectors expressing GluR delta 2 were injected into the cerebellar cortex of hotfoot5J mice 6 or 7 days after birth, and the effects were studied on postnatal day 30. The motor behavior of hotfoot5J mice treated with vectors expressing GluR82 was markedly rescued, whereas the ataxia of hotfoot5J mice treated with vectors expressing GFP was comparable to that of non-injected hotfoot5J littermates. Furthermore, the impaired release probability of glutamate from parallel fiber terminals and the failure of developmental elimination of surplus climbing fibers from Purkinje cells in hotfoot5J mice were completely rescued by GIuR82 expression. These results indicate the therapeutic potential of viral vector-based gene therapy for hereditary cerebellar ataxia and other neuronal disorders. (C) 2009 Elsevier Inc. All rights reserved.
  • K. Hashimoto, T. Yoshida, K. Sakimura, M. Mishina, M. Watanabe, M. Kano
    NEUROSCIENCE 162 3 601 - 611 2009年09月 [査読有り][通常論文]
     
    The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum provides an ideal model for the study of developmental rearrangements of neural circuits. At birth, each PC is innervated by multiple CFs. These surplus CFs are eliminated during postnatal development, and mono innervation is attained by postnatal day 20 (P20) in mice. Earlier studies on spontaneous mutant mice and animals with "hypogranular" cerebella indicate that regression of surplus CFs requires normal generation of granule cells and their axons, parallel fibers (PFs), and normal formation of PF-PC synapses. Our understanding of how PF-PC synapse formation affects development of CF-PC synapse has been greatly advanced by analyses of mutant mice deficient in glutamate receptor 82 subunit (GluR delta 2), an orphan receptor expressed selectively in PCs. Deletion of GluR delta 2 results in impairment of PF-PC synapse formation, which leads to defects in development of CF-PC synapses. In this article, we review how impaired PF-PC synapse formation affects wiring of CFs to PCs based mostly on our data on GluR delta 2 knockout mice. We propose a new scheme that CF-PC synapses are shaped by the three consecutive events, namely functional differentiation of multiple CFs into one strong and a few weak inputs from P3 to P7, "early phase" of CF synapse elimination from P7 to around P11, and "late phase" of CF synapse elimination from around P12. Normal PF-PC synapse formation is required for the "late phase" of CF synapse elimination. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Seung-Hyuk Chung, Hassan Marzban, Masahiko Watanabe, Richard Hawkes
    CEREBELLUM 8 3 267 - 276 2009年09月 [査読有り][通常論文]
     
    Unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons of the granular layer. Previous studies have shown that there are two distinct subsets of UBCs present in the mice cerebellar cortex: calcium-binding protein calretinin (CR) positive and metabotropic glutamate receptor (mGluR)1 alpha positive. In this study, we identify phospholipase C (PLC) beta 4 as an antigenic marker of a novel subset of UBCs. Double immunolabeling reveals that none of the CR+ subset expresses PLC beta 4. In contrast, most members of the mGluR1 alpha subset also express PLC beta 4. In addition, 65% of the PLC beta 4+ subset does not express mGluR1 alpha. Thus, there are three distinct UBC subsets in the mouse cerebellum: CR+/PLC beta 4-/mGluR1 alpha-, PLC beta 4+/mGluR1 alpha-/CR-, and mGluR1 alpha+/PLC beta 4+/CR-. Each has a different topographical distribution, both between lobules and mediolaterally within the vermis. The development of PLC beta 4 expression in UBCs is exclusively postnatal-first seen only at P12 and mature at about 3 weeks. A distinct subset of PLC beta 4+ UBCs is also present in primary cerebellar cultures.
  • Kaori Akashi, Toshikazu Kakizaki, Haruyuki Kamiya, Masahiro Fukaya, Miwako Yamasaki, Manabu Abe, Rie Natsume, Masahiko Watanabe, Kenji Sakimura
    JOURNAL OF NEUROSCIENCE 29 35 10869 - 10882 2009年09月 [査読有り][通常論文]
     
    GluN2B (GluR epsilon 2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluR zeta 1/NR1) was moderately reduced, whereas GluN2A (GluR epsilon 1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.
  • Shigemi Okazaki, Yumi Iwamitsu, Kuranami Masaru, Keiko Todoroki, Shimako Suzuki, Kenji Yamamoto, Masashi Hagino, Masahiko Watanabe, Hitoshi Miyaoka
    Palliative and Supportive Care 7 3 307 - 314 2009年09月 [査読有り][通常論文]
     
    Objective: The purpose of this study was to qualitatively examine the content of the psychological responses in interviews with breast cancer outpatients receiving initial medical consultation.Method: The participants were 180 people who visited the breast cancer outpatient clinic at Kitasato University Hospital between November 2004 and August 2005. The remaining 176 participants (39 breast cancer patients and 137 benign tumor patients average ageSD: 50.712.4 years) were analyzed. Two clinical psychologists carried out the interview, asking the participants to speak freely about their anxieties, worries, thoughts, and feelings up until the medical examination. This study used a content analysis of interviews to chronologically examine psychological response of cancer patients seeking medical consultation at three points in time.Results: Patients at the time of their first outpatient breast cancer consultation experience negative feelings before the examination, directly influenced by the suspicion of cancer. These include anxiety and worries, fear, evasion, depression, and impatience. These tendencies do not change at the time of consultation. However, in addition to negative feelings, some people also possess positive feelings, either simultaneously or at a different point in time. Further, many patients tend to talk at length about psychological responses before seeking treatment, understanding the process they went through to come to seek treatment as an important event.Significance of results: It is important for medical workers to bear in mind the psychological conflicts that patients may undergo before seeking treatment and ensure that sufficient communication takes place. © 2009 Cambridge University Press.
  • Shinichi Sakuramoto, Keishi Yamashita, Shiro Kikuchi, Nobue Futawatari, Natsuya Katada, Hiromitsu Moriya, Kazuya Hirai, Masahiko Watanabe
    Journal of the American College of Surgeons 209 3 344 - 351 2009年09月 [査読有り][通常論文]
     
    Background: Laparoscopy-assisted proximal gastrectomy (LAPG) has become prevalent for early gastric cancer in the upper stomach, but standard esophagogastrostomy is sometimes complicated with reflux esophagitis. Clinical outcomes are described here in patients with reconstruction by esophagogastrostomy with Toupet-like partial fundoplication (TPF) in LAPG. Study Design: From November 2005 through December 2008, LAPG was performed in 36 patients with early gastric cancer, 26 (72.2%) of whom could have reconstruction with the TPF because the remnant stomach was sufficiently large. Results: In LAPG with TPF, mean operation time was 293 minutes, mean blood loss was 119 g, and the mean number of dissected lymph nodes was 25.1. Regarding postoperative complications, anastomotic leakage occurred in two patients. More than 1 year after operation, 3 (15.0%) of the 20 patients had heartburn and 6 (30.0%) had reflux esophagitis (Los Angeles classification grade A, n = 2 grade B, n = 4) proton pump inhibitors were effective in these patients. Conclusions: Esophagogastrostomy with TPF could be a simple, safe, and useful technique for reconstruction after LAPG in patients with early gastric cancer, and its clinical usefulness is worthwhile for the prospective validation. © 2009 American College of Surgeons.
  • Daisuke Ichihara, Eijiro Okada, Kazuhiro Chiba, Yoshiaki Toyama, Hirokazu Fujiwara, Suketaka Momoshima, Yuji Nishiwaki, Takeshi Hashimoto, Jun Ogawa, Masahiko Watanabe, Takeshi Takahata, Morio Matsumoto
    JOURNAL OF ORTHOPAEDIC SCIENCE 14 5 602 - 610 2009年09月 [査読有り][通常論文]
     
    Background. We conducted a prospective long-term follow-up study to assess associations between magnetic resonance imaging (MRI) findings and changes in clinical symptoms, as well as factors relating to the prognosis of symptoms. Methods. A total of 133 patients with acute whiplash injury between 1993 and 1996 participated in this follow-up study. They underwent neurological examinations by spine surgeons and second MRI scans of the cervical spine were obtained. They also filled out a questionnaire regarding cervical symptoms and the accident details. The items evaluated by MRI were (1) a decrease in the signal intensity of the intervertebral disc; (2) anterior compression of the dura and the spinal cord; (3) posterior disc protrusion; (4) disc space narrowing; and (5) foraminal stenosis. Relations between the presence/absence of degenerative changes on MRI, accident details, and patients' symptoms were assessed by calculating the adjusted odds ratio (OR). Results. Progression of some degenerative changes was recognized on MRI in 98.5% of the 133 whiplash injury patients, and clinical symptoms diminished in more than a half of the 133 patients. There were no statistically significant associations between MRI findings and changes in clinical symptoms. The prognosis for neck pain tended to be poor after accidents with double collisions (rear-end collision followed by front-end collision) [adjusted OR 5.83, 95% confidence interval (CI) 1.15-29.71] and accidents with serious car damage (2.87, 1.03-7.99). The prognosis for stiff shoulders tended to be poor in women (2.83, 1.23-6.51); and the prognosis for numbness in the upper extremities tended to be poor after accidents with serious car damage (3.39, 1.14-10.06). Conclusions. This study demonstrated that progression of degenerative changes of the cervical spine on MRI was not associated with clinical symptoms during the 10-year period after whiplash injury.
  • Takashi Torashima, Akira Iizuka, Hajime Horiuchi, Kazuhiro Mitsumura, Miwako Yamasaki, Chiho Koyama, Kiyohiko Takayama, Masae Iino, Masahiko Watanabe, Hirozaku Hirai
    EUROPEAN JOURNAL OF NEUROSCIENCE 30 3 355 - 365 2009年08月 [査読有り][通常論文]
     
    The delta 2 glutamate receptor (GluR delta 2) is expressed predominantly in cerebellar Purkinje cells. GluR delta 2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluR delta 2 function, we produced various GluR delta 2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluR delta 2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluR delta 2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluR delta 2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluR delta 2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluR delta 2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluR delta 2, which functions substantially without conventional ligand binding and ion channel structures.
  • Alejandro J. Pernia-Andrade, Ako Kato, Robert Witschi, Rita Nyilas, Istvan Katona, Tamas F. Freund, Masahiko Watanabe, Joerg Filitz, Wolfgang Koppert, Juergen Schuettler, Guangchen Ji, Volker Neugebauer, Giovanni Marsicano, Beat Lutz, Horacio Vanegas, Hanns Ulrich Zeilhofer
    SCIENCE 325 5941 760 - 764 2009年08月 [査読有り][通常論文]
     
    Diminished synaptic inhibition in the spinal dorsal horn is a major contributor to chronic pain. Pathways that reduce synaptic inhibition in inflammatory and neuropathic pain states have been identified, but central hyperalgesia and diminished dorsal horn synaptic inhibition also occur in the absence of inflammation or neuropathy, solely triggered by intense nociceptive (C-fiber) input to the spinal dorsal horn. We found that endocannabinoids, produced upon strong nociceptive stimulation, activated type 1 cannabinoid (CB1) receptors on inhibitory dorsal horn neurons to reduce the synaptic release of gamma-aminobutyric acid and glycine and thus rendered nociceptive neurons excitable by nonpainful stimuli. Our results suggest that spinal endocannabinoids and CB1 receptors on inhibitory dorsal horn interneurons act as mediators of heterosynaptic pain sensitization and play an unexpected role in dorsal horn pain-controlling circuits.
  • Hirokazu Matsuzaki, Takeshi Izumi, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
    EUROPEAN JOURNAL OF PHARMACOLOGY 615 1-3 76 - 82 2009年08月 [査読有り][通常論文]
     
    Traumatic events in early life are associated with an increased risk of psychiatric diseases in adulthood. 5-hydroxytryptamine (5-HT)(1A) receptors are known to play a pivotal role in the 5-HTergic mechanisms associated with the etiology of stress-related disorders. The goal of the present study was to investigate whether early postnatal stress influences 5-HT1A receptor function in the medial prefrontal cortex in adult rats. Rats were subjected to aversive foot shock (FS) during the third week of the postnatal period (3wFS group). During the postadolescent period (10-14 weeks postnatal), immunohistochemical experiments were carried out to investigate c-Fos expression following the administration of R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a 5-HT1A receptor agonist. In the 3wFS group, the 8-OH-DPAT-induced c-Fos expression in the medial prefrontal cortex was significantly attenuated compared to that in the non-FS control group. A dual immunofluorescence study revealed that a small proportion of c-Fos positive cells co-express parvalbumin, and a relatively large proportion of c-Fos positive cells co-express glutaminase, suggesting that most c-Fos positive cells are glutamatergic neurons. We found that local perfusion of 8-OH-DPAT via a dialysis probe decreased extracellular 5-HT levels in the medial prefrontal cortex of the non-FS group, but not in the 3wFS group. However, the levels of 8-OH-DPAT-induced 5-HT syndrome were not significantly different between the non-FS and 3wFS groups. Therefore, aversive stress in the third week of the postnatal period attenuates 5-HT1A receptor function in the medial prefrontal cortex in adulthood and produces feedback inhibition of the raphe nuclei via postsynaptic 5-HT1A receptors. (C) 2009 Elsevier B.V. All rights reserved.
  • Yasukazu Hozumi, Masahiko Watanabe, Koichi Otani, Kaoru Goto
    BMC NEUROSCIENCE 10 2009年08月 [査読有り][通常論文]
     
    Background: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to phosphatidic acid and comprises multiple isozymes of distinct properties. Of DGKs, mRNA signal for DGK beta is strongly detected in the striatum, and one of the transcripts derived from the human DGK beta locus is annotated in GenBank as being differentially expressed in bipolar disorder patients. Recently, we have reported that DGK beta is expressed in medium spiny neurons of the striatum and is highly concentrated at the perisynapse of dendritic spines. However, it remains elusive how DGK beta is implicated in pathophysiological role in neurons at the cellular level. Results: In the present study, we investigated the expression and subcellular localization of DGK beta in the hippocampus, together with its functional implication using transfected hippocampal neurons. DGK beta is expressed not only in projection neurons but also in interneurons and is concentrated at perisynaptic sites of asymmetrical synapses. Overexpression of wild-type DGK beta promotes dendrite outgrowth at 7 d in vitro (DIV) and spine maturation at 14 DIV in transfected hippocampal neurons, although its kinase-dead mutant has no effect. Conclusion: In the hippocampus, DGK beta is expressed in both projection neurons and interneurons and is accumulated at the perisynapse of dendritic spines in asymmetrical synapses. Transfection experiments suggest that DGK beta may be involved in the molecular machineries of dendrite outgrowth and spinogenesis through its kinase activity.
  • Kenjiro Hisano, Masahiko Watanabe, Yuji Morimoto
    JOURNAL OF ANESTHESIA 23 3 363 - 369 2009年08月 [査読有り][通常論文]
     
    Edaravone, a free radical scavenger, has shown neuroprotection in both animals and humans. To evaluate the mechanism of this protection, we examined the effect of edaravone on neurons themselves against glutamate neurotoxicity. Neurons were collected from 18-day fetal rat brains and a culture of almost pure neurons was obtained after 14-day culture. The neurons were exposed to 50 mu M glutamate for 10 min, followed by normal culture for 24 h. Edaravone was added to the medium during the glutamate insult (prophylactic effect) or after the insult (treatment effect). First, the cell survival rate was measured by staining with trypan blue. Second, the cells were stained with 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di-(acetoxymethyl ester) (C-DCDHF-DA) and the relative amount of reactive oxygen species (ROS) was measured by flow cytometry. Third, the cells were stained with Hoechst 33342 and propidium iodide and the numbers of apoptotic and necrotic cells were counted. A dose-dependent prophylactic effect was observed and the cell survival rate in 500 mu M edaravone was significantly higher than that without it. However, there was no treatment effect beyond 2 h after the insult. The amount of ROS under 500 mu M edaravone at 4 h after the glutamate insult was significantly lower than the control amount. Necrosis, but not apoptosis, was significantly inhibited by edaravone. Edaravone mainly showed a prophylactic effect on neurons against glutamate neurotoxicity, possibly through the inhibition of necrosis via the suppression of ROS production. However, for a protective effect, a higher, supraclinical concentration was required, compared to the concentrations producing a protective effect in glial and endothelial cells in previous studies.
  • Michael E. Hildebrand, Philippe Isope, Taisuke Miyazaki, Toshitaka Nakaya, Esperanza Garcia, Anne Feltz, Toni Schneider, Juergen Hescheler, Masanobu Kano, Kenji Sakimura, Masahiko Watanabe, Stephane Dieudonne, Terrance P. Snutch
    JOURNAL OF NEUROSCIENCE 29 31 9668 - 9682 2009年08月 [査読有り][通常論文]
     
    T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Ca(v)3.1 T-type currents via a G-protein-and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Ca(v)3.1 gene knock-out animals show that Ca(v)3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Ca(v)3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.
  • Laura Fernandez-Alacid, Carolina Aguado, Francisco Ciruela, Ricardo Martin, Jose Colon, Maria Jose Cabanero, Martin Gassmann, Masahiko Watanabe, Ryuichi Shigemoto, Kevin Wickman, Bernhard Bettler, Jose Sanchez-Prieto, Rafael Lujan
    JOURNAL OF NEUROCHEMISTRY 110 4 1363 - 1376 2009年08月 [査読有り][通常論文]
     
    Activation of G protein-gated inwardly-rectifying K+ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABA(B)) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABA(B) receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABA(B) receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABA(B) receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA(B) receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA(B) receptors. The association of GIRK channels and GABA(B) receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.
  • Hirokazu Matsuzaki, Takeshi Izumi, Machiko Matsumoto, Hiroko Togashi, Taku Yamaguchi, Takayuki Yoshida, Masahiko Watanabe, Mitsuhiro Yoshioka
    EUROPEAN JOURNAL OF PHARMACOLOGY 615 1-3 76 - 82 2009年08月 [査読有り][通常論文]
     
    Traumatic events in early life are associated with an increased risk of psychiatric diseases in adulthood. 5-hydroxytryptamine (5-HT)(1A) receptors are known to play a pivotal role in the 5-HTergic mechanisms associated with the etiology of stress-related disorders. The goal of the present study was to investigate whether early postnatal stress influences 5-HT1A receptor function in the medial prefrontal cortex in adult rats. Rats were subjected to aversive foot shock (FS) during the third week of the postnatal period (3wFS group). During the postadolescent period (10-14 weeks postnatal), immunohistochemical experiments were carried out to investigate c-Fos expression following the administration of R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a 5-HT1A receptor agonist. In the 3wFS group, the 8-OH-DPAT-induced c-Fos expression in the medial prefrontal cortex was significantly attenuated compared to that in the non-FS control group. A dual immunofluorescence study revealed that a small proportion of c-Fos positive cells co-express parvalbumin, and a relatively large proportion of c-Fos positive cells co-express glutaminase, suggesting that most c-Fos positive cells are glutamatergic neurons. We found that local perfusion of 8-OH-DPAT via a dialysis probe decreased extracellular 5-HT levels in the medial prefrontal cortex of the non-FS group, but not in the 3wFS group. However, the levels of 8-OH-DPAT-induced 5-HT syndrome were not significantly different between the non-FS and 3wFS groups. Therefore, aversive stress in the third week of the postnatal period attenuates 5-HT1A receptor function in the medial prefrontal cortex in adulthood and produces feedback inhibition of the raphe nuclei via postsynaptic 5-HT1A receptors. (C) 2009 Elsevier B.V. All rights reserved.
  • Kenjiro Hisano, Masahiko Watanabe, Yuji Morimoto
    JOURNAL OF ANESTHESIA 23 3 363 - 369 2009年08月 [査読有り][通常論文]
     
    Edaravone, a free radical scavenger, has shown neuroprotection in both animals and humans. To evaluate the mechanism of this protection, we examined the effect of edaravone on neurons themselves against glutamate neurotoxicity. Neurons were collected from 18-day fetal rat brains and a culture of almost pure neurons was obtained after 14-day culture. The neurons were exposed to 50 mu M glutamate for 10 min, followed by normal culture for 24 h. Edaravone was added to the medium during the glutamate insult (prophylactic effect) or after the insult (treatment effect). First, the cell survival rate was measured by staining with trypan blue. Second, the cells were stained with 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di-(acetoxymethyl ester) (C-DCDHF-DA) and the relative amount of reactive oxygen species (ROS) was measured by flow cytometry. Third, the cells were stained with Hoechst 33342 and propidium iodide and the numbers of apoptotic and necrotic cells were counted. A dose-dependent prophylactic effect was observed and the cell survival rate in 500 mu M edaravone was significantly higher than that without it. However, there was no treatment effect beyond 2 h after the insult. The amount of ROS under 500 mu M edaravone at 4 h after the glutamate insult was significantly lower than the control amount. Necrosis, but not apoptosis, was significantly inhibited by edaravone. Edaravone mainly showed a prophylactic effect on neurons against glutamate neurotoxicity, possibly through the inhibition of necrosis via the suppression of ROS production. However, for a protective effect, a higher, supraclinical concentration was required, compared to the concentrations producing a protective effect in glial and endothelial cells in previous studies.
  • Kouichi Hashimoto, Ryoichi Ichikawa, Kazuo Kitamura, Masahiko Watanabe, Masanobu Kano
    NEURON 63 1 106 - 118 2009年07月 [査読有り][通常論文]
     
    Functional neural circuits are formed by eliminating early-formed redundant synapses and strengthening necessary connections during development. In newborn mouse cerebellum, each Purkinje cell (PC) is innervated by multiple climbing fibers (CFs) with similar strengths. Subsequently, a single CF is selectively strengthened by postnatal day 7 (P7). We find that this competition among multiple CFs occurs on the soma before CFs form synapses along dendrites. Notably, inmost PCs, the single CF that has been functionally strengthened (the "winner" CF) undergoes translocation to dendrites while keeping its synapses on the soma. Synapses of the weaker CFs (the "loser" CFs) remain around the soma and form "pericellular nests" with synapses of the winner CFs. Then most perisomatic synapses are eliminated nonselectively by P15. Thus, our results suggest that the selective translocation of the winner CF to dendrites in each PC determines the single CF that survives subsequent synapse elimination and persistently innervates the PC.
  • Shingo Furuse, Tomoyuki Kawamata, Jun Yamamot, Yukitoshi Niiyama, Keiichi Omote, Masahiko Watanabe, Akiyoshi Namiki
    ANESTHESIOLOGY 111 1 173 - 186 2009年07月 [査読有り][通常論文]
     
    Background: Bone cancer pain has a strong impact on the quality of life of patients, but it is difficult to treat. Therefore, development of a novel strategy for the treatment of bone cancer pain is needed for improvement of patient quality of life. This study examined whether selective spinal cannabinoid receptor 1 (CB1) activation alleviates bone cancer pain and also examined the spinal expression of CBI. Methods: A bone cancer pain model was made by implantation of sarcoma cells into the intramedullary space of the mouse femur. In behavioral experiments, the authors examined the effects of activation of spinal CB1 and inhibition of metabolism of endocannabinoid on bone cancer-related pain behaviors. Immunohistochemical experiments examined the distribution and localization of CB1 in the superficial dorsal horn of the spinal cord using specific antibodies. Results: Spinal CBI activation by exogenous administration of a CB1 agonist arachidonyl-2-chloroethylamide reduced bone cancer-related pain behaviors, including behaviors related to spontaneous pain and movement-evoked pain. in immunohistochemical experiments, although mu-opioid receptor 1 expression was reduced in the superficial dorsal horn ipsilateral to the site of implantation of sarcoma cells, CB1 expression was preserved. In addition, CBI was mainly expressed in the axon terminals, but not in the dendritic process in the superficial dorsal horn. Conclusion: Spinal CBI activation reduced bone cancer-related pain behavior. Presynaptic inhibition may contribute to the analgesic effects of spinal CBI activation. These findings may lead to novel strategies for the treatment of bone cancer pain.
  • Shingo Furuse, Tomoyuki Kawamata, Jun Yamamot, Yukitoshi Niiyama, Keiichi Omote, Masahiko Watanabe, Akiyoshi Namiki
    ANESTHESIOLOGY 111 1 173 - 186 2009年07月 [査読有り][通常論文]
     
    Background: Bone cancer pain has a strong impact on the quality of life of patients, but it is difficult to treat. Therefore, development of a novel strategy for the treatment of bone cancer pain is needed for improvement of patient quality of life. This study examined whether selective spinal cannabinoid receptor 1 (CB1) activation alleviates bone cancer pain and also examined the spinal expression of CBI. Methods: A bone cancer pain model was made by implantation of sarcoma cells into the intramedullary space of the mouse femur. In behavioral experiments, the authors examined the effects of activation of spinal CB1 and inhibition of metabolism of endocannabinoid on bone cancer-related pain behaviors. Immunohistochemical experiments examined the distribution and localization of CB1 in the superficial dorsal horn of the spinal cord using specific antibodies. Results: Spinal CBI activation by exogenous administration of a CB1 agonist arachidonyl-2-chloroethylamide reduced bone cancer-related pain behaviors, including behaviors related to spontaneous pain and movement-evoked pain. in immunohistochemical experiments, although mu-opioid receptor 1 expression was reduced in the superficial dorsal horn ipsilateral to the site of implantation of sarcoma cells, CB1 expression was preserved. In addition, CBI was mainly expressed in the axon terminals, but not in the dendritic process in the superficial dorsal horn. Conclusion: Spinal CBI activation reduced bone cancer-related pain behavior. Presynaptic inhibition may contribute to the analgesic effects of spinal CBI activation. These findings may lead to novel strategies for the treatment of bone cancer pain.
  • Yoko Yamagata, Shizuka Kobayashi, Tatsuya Umeda, Akihiro Inoue, Hiroyuki Sakagami, Masahiro Fukaya, Masahiko Watanabe, Nobuhiko Hatanaka, Masako Totsuka, Takeshi Yagi, Kunihiko Obata, Keiji Imoto, Yuchio Yanagawa, Toshiya Manabe, Shigeo Okabe
    JOURNAL OF NEUROSCIENCE 29 23 7607 - 7618 2009年06月 [査読有り][通常論文]
     
    Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII alpha) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKII alpha, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKII alpha, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKII alpha knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKII alpha (K42R) mice, kinase activity was reduced to the same level as in CaMKII alpha-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral-CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKII alpha was preserved. In addition, CaMKII alpha (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKII alpha is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.
  • Nuria Cabello, Jorge Gandia, Daniela C. G. Bertarelli, Masahiko Watanabe, Carme Lluis, Rafael Franco, Sergi Ferre, Rafael Lujan, Francisco Ciruela
    JOURNAL OF NEUROCHEMISTRY 109 5 1497 - 1507 2009年06月 [査読有り][通常論文]
     
    G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu(5) receptors (mGlu(5)R) and dopamine D-2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu(5)R, D2R and adenosine A(2A) receptor (A(2A)R). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu(5)R, D2R and A(2A)R in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu(5)R/D2R/A(2A)R oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.
  • Akira Ooki, Keishi Yamashita, Shiro Kikuchi, Shinichi Sakuramoto, Natsuya Katada, Masahiko Watanabe
    ONCOLOGY REPORTS 21 6 1467 - 1475 2009年06月 [査読有り][通常論文]
     
    Phosphatase of regenerating liver-3 (PRL-3) has received attention as a molecule associated with metastasis ill Various tumor types, including gastric cancer. However, its clinical utility its a biomarker remains unclear in primary gastric cancer. The present study included 173 patients with primary gastric cancer Who underwent gastrectomy with regional lymphadenectomy Lit the Kitasato University Hospital. All patients were informative for the well-characterized clinicopathological variables. including patient outcome. We assessed the potential as a clinically applicable biomarker using immunohistochemistry. PRL-3 overexpression was detected in 78 (45%) of 173 primary tumor tissues and was an independent predictor of lymph node metastasis on multivariate logistic regression analysis (odds ratio=9.32 P<0.0001). PRL-3 overexpression in primary tumor had significant prognostic implication (P=0.0009) and was also an independent prognostic factor (hazard ratio=4.39 P=0.006) in the histologically node-negative patients after Curative resection, but 1101 in the histologically node-positive patients, Moreover, ill advanced gastric cancer with stage I disease, PRL-3 overexpression inversely affected patient Outcome (P=0.02) and showed,I characteristic of stage 11 disease from a prognostic point of view. We demonstrated for the first time that PRL-3 expression in primary tumor could predict the outcome of patients with histologically node-negative gastric cancer. We propose that PRL-3 expression can have a clinical potential as a prognostic biomarker that may facilitate the development Of adjuvant chemotherapy for advanced gastric cancer with stage I disease.
  • Yoko Yamagata, Shizuka Kobayashi, Tatsuya Umeda, Akihiro Inoue, Hiroyuki Sakagami, Masahiro Fukaya, Masahiko Watanabe, Nobuhiko Hatanaka, Masako Totsuka, Takeshi Yagi, Kunihiko Obata, Keiji Imoto, Yuchio Yanagawa, Toshiya Manabe, Shigeo Okabe
    JOURNAL OF NEUROSCIENCE 29 23 7607 - 7618 2009年06月 [査読有り][通常論文]
     
    Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII alpha) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKII alpha, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKII alpha, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKII alpha knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKII alpha (K42R) mice, kinase activity was reduced to the same level as in CaMKII alpha-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral-CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKII alpha was preserved. In addition, CaMKII alpha (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKII alpha is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.
  • Rita Nyilas, Laura C. Gregg, Ken Mackie, Masahiko Watanabe, Andreas Zimmer, Andrea G. Hohmann, Istvan Katona
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 10 1964 - 1978 2009年05月 [査読有り][通常論文]
     
    Cannabinoid administration suppresses pain by acting at spinal, supraspinal and peripheral levels. Intrinsic analgesic pathways also exploit endocannabinoids; however, the underlying neurobiological substrates of endocannabinoid-mediated analgesia have remained largely unknown. Compelling evidence shows that, upon exposure to a painful environmental stressor, an endocannabinoid molecule called 2-arachidonoylglycerol (2-AG) is mobilized in the lumbar spinal cord in temporal correlation with stress-induced antinociception. We therefore characterized the precise molecular architecture of 2-AG signaling and its involvement in nociception in the rodent spinal cord. Nonradioactive in situ hybridization revealed that dorsal horn neurons widely expressed the mRNA of diacylglycerol lipase-alpha (DGL-alpha), the synthesizing enzyme of 2-AG. Peroxidase-based immunocytochemistry demonstrated high levels of DGL-alpha protein and CB(1) cannabinoid receptor, a receptor for 2-AG, in the superficial dorsal horn, at the first site of modulation of the ascending pain pathway. High-resolution electron microscopy uncovered postsynaptic localization of DGL-alpha at nociceptive synapses formed by primary afferents, and revealed presynaptic positioning of CB(1) on excitatory axon terminals. Furthermore, DGL-alpha in postsynaptic elements receiving nociceptive input was colocalized with metabotropic glutamate receptor 5 (mGluR(5)), whose activation induces 2-AG biosynthesis. Finally, intrathecal activation of mGluR(5) at the lumbar level evoked endocannabinoid-mediated stress-induced analgesia through the DGL-2-AG-CB(1) pathway. Taken together, these findings suggest a key role for 2-AG-mediated retrograde suppression of nociceptive transmission at the spinal level. The striking positioning of the molecular players of 2-AG synthesis and action at nociceptive excitatory synapses suggests that pharmacological manipulation of spinal 2-AG levels may be an efficacious way to regulate pain sensation.
  • Wataru Kakegawa, Taisuke Miyazaki, Kazuhisa Kohda, Keiko Matsuda, Kyoichi Emi, Junko Motohashi, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 29 18 5738 - 5748 2009年05月 [査読有り][通常論文]
     
    The delta 2 glutamate receptor (GluR delta 2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2(wt)) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2(wt), but not of a mutant GluD2 lacking the NTD (GluD2(Delta NTD)), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2(NTD)-GluK2) did. Similarly, GluD2(wt) and GluD2(NTD)-GluK2, but not GluD2(Delta NTD), induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF -Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively.
  • Wataru Kakegawa, Taisuke Miyazaki, Kazuhisa Kohda, Keiko Matsuda, Kyoichi Emi, Junko Motohashi, Masahiko Watanabe, Michisuke Yuzaki
    JOURNAL OF NEUROSCIENCE 29 18 5738 - 5748 2009年05月 [査読有り][通常論文]
     
    The delta 2 glutamate receptor (GluR delta 2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2(wt)) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2(wt), but not of a mutant GluD2 lacking the NTD (GluD2(Delta NTD)), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2(NTD)-GluK2) did. Similarly, GluD2(wt) and GluD2(NTD)-GluK2, but not GluD2(Delta NTD), induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF -Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively.
  • Keishi Yamashita, Akira Ooki, Masaki Mori, Myoung Sook Kim, David Sidransky, Masahiko Watanabe
    CANCER RESEARCH 69 2009年05月 [査読有り][通常論文]
  • Jingping Zhang, Tai-Xiang Xu, Penelope J. Hallett, Masahiko Watanabe, Seth G. N. Grant, Ole Isacson, Wei-Dong Yao
    JOURNAL OF NEUROSCIENCE 29 9 2948 - 2960 2009年03月 [査読有り][通常論文]
     
    Classical dopaminergic signaling paradigms and emerging studies on direct physical interactions between the D-1 dopamine (DA) receptor and the NMDA glutamate receptor predict a reciprocally facilitating, positive feedback loop. This loop, if not controlled, may cause concomitant overactivation of both D1 and NMDA receptors, triggering neurotoxicity. Endogenous protective mechanisms must exist. Here, we report that PSD-95, a prototypical structural and signaling scaffold in the postsynaptic density, inhibits D-1-NMDA receptor subunit 1 (NR1) NMDA receptor association and uncouples NMDA receptor-dependent enhancement of D-1 signaling. This uncoupling is achieved, at least in part, via a disinhibition mechanism by which PSD-95 abolishes NMDA receptor-dependent inhibition of D-1 internalization. Knockdown of PSD-95 immobilizes D-1 receptors on the cell surface and escalates NMDA receptor-dependent D-1 cAMP signaling in neurons. Thus, in addition to its role in receptor stabilization and synaptic plasticity, PSD-95 acts as a brake on the D-1-NMDA receptor complex and dampens the interaction between them.
  • Masami O. Kinoshita, Yoko Shinoda, Kazuhisa Sakai, Tsutomu Hashikawa, Masahiko Watanabe, Takeo Machida, Yoshio Hirabayashi, Shigeki Furuya
    NEUROSCIENCE LETTERS 453 1 21 - 26 2009年03月 [査読有り][通常論文]
     
    In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-phosphoglycerate dehydrogenase (Phgdh), an enzyme involved in de novo synthesis Of L-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that L-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. in the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of L-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain. (C) 2009 Elsevier Ireland Ltd. All rights reserved.
  • Yuriko Kawakami, Kazuyuki Yoshida, Jung Hoon Yang, Takeshi Suzuki, Norihiro Azuma, Kazuhisa Sakai, Tsutomu Hashikawa, Masahiko Watanabe, Kaori Yasuda, Satoru Kuhara, Yoshio Hirabayashi, Shigeki Furuya
    Neuroscience Research 63 3 184 - 193 2009年03月 [査読有り][通常論文]
     
    Mutations in the d-3-phosphoglycerate dehydrogenase (PHGDH; EC 1.1.1.95) gene, which encodes an enzyme involved in de novo l-serine biosynthesis, are shown to cause human serine deficiency disorder. This disorder has been characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. Our previous work demonstrated that targeted disruption of mouse Phgdh leads to a marked decrease in serine and glycine, severe growth retardation of the central nervous system, and lethality after embryonic day 13.5. To clarify how a serine deficiency causes neurodevelopmental defects, we characterized changes in metabolites, gene expression and morphological alterations in the spinal cord of Phgdh knockout mice. BeadChip microarray analysis revealed significant dysregulation of genes involved in the cell cycle. Ingenuity Pathway Analysis also revealed a significant perturbation of regulatory networks that operate in the cell cycle progression. Moreover, morphological examinations of the knockout spinal cord demonstrated a marked deficit in dorsal horn neurons. Radial glia cells, native neural stem/progenitor cells, accumulated in the dorsal ventricular zone, but they did not proceed to a G0-like quiescent state. The present integrative study provides in vivo evidence that normal cell cycle progression and subsequent neurogenesis of radial glia cells are severely impaired by serine deficiency. © 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Seiichiro Yamamoto, Masaki Fukunaga, Nobuyoshi Miyajima, Junji Okuda, Fumio Konishi, Masahiko Watanabe
    Journal of the American College of Surgeons 208 3 383 - 389 2009年03月 [査読有り][通常論文]
     
    Background: In laparoscopic operations for rectal carcinoma, only a few multicenter studies of a large number of patients have examined the impact of conversion on outcomes and determined risk factors for conversion. This study was designed to evaluate short-term outcomes and risk factors for conversion to open operation in laparoscopic operations for rectal carcinoma. Study Design: A total of 1,073 patients with carcinoma of the rectum and anus who underwent laparoscopic operations were reviewed retrospectively. Patients were collected from 28 institutions. Patients who required conversion during laparoscopic operation were compared with those with completed laparoscopic resection. Results: Conversion rate was 7.3% (n = 78), and patients requiring conversion were considerably heavier (mean body mass index 24.6 versus 22.7) and had a substantially higher rate of low anterior resection (94.9% versus 83.5%). Conversion was also associated with longer operation time (median 295 minutes versus 270 minutes), greater blood loss (median 265 mL versus 80 mL), longer median postoperative hospital stay (20 days versus 14 days), and higher rates of intraoperative (32.1% versus 3.5%) and postoperative (43.6% versus 21.1%) complications. In multivariate analysis, body mass index and rate of low anterior resection were predictive of conversion. Conclusions: Conversion to open operation is associated with greater morbidity than completed laparoscopic resection. Body mass index and the particular laparoscopic procedure are risk factors for conversion, indicating that appropriate patient selection is essential in laparoscopic operations for rectal carcinoma. © 2009 American College of Surgeons.
  • Andrew J. Todd, Erika Polgar, Christine Watt, Mark E. S. Bailey, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 4 718 - 726 2009年02月 [査読有り][通常論文]
     
    alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPArs), which mediate fast excitatory glutamatergic transmission, are tetramers made from four subunits (GluR1-4 or GluRA-D). Although synaptic AMPArs are not normally detected by immunocytochemistry in perfusion-fixed tissue, they can be revealed by using antigen retrieval with pepsin. All AMPAr-positive synapses in spinal cord are thought to contain GluR2, while the other subunits have specific laminar distributions. GluR4 can be alternatively spliced such that it has a long or short cytoplasmic tail. We have reported that < 10% of AMPAr-containing synapses in lamina II have the long form of GluR4, and that these are often arranged in dorsoventrally orientated clusters. In this study, we test the hypothesis that GluR4-containing receptors are associated with dorsal dendrites of projection neurons in laminae III and IV that express the neurokinin 1 receptor (NK1r). Immunostaining for NK1r was carried out before antigen retrieval, and sections were then reacted to reveal GluR2 and either GluR4 (long form), GluR3 or GluR1. All NK1r-positive lamina III/IV neurons had numerous GluR2-immunoreactive puncta in their dendritic plasma membranes, and virtually all (97%) of the puncta tested were labelled (usually strongly) with the GluR4 antibody. Sizes of puncta varied, but many were elongated and they were significantly larger than nearby puncta that were not associated with the NK1r cells. None of the GluR2 puncta on these cells was positive for GluR1, while 85% were GluR3-immunoreactive. These results show that synaptic AMPArs on the dendrites of the lamina III/IV NK1r projection neurons contain GluR2, GluR3 and GluR4, but not GluR1 subunits.
  • Eriko Miura, Keiko Matsuda, James I. Morgan, Michisuke Yuzaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 4 693 - 706 2009年02月 [査読有り][通常論文]
     
    Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluR delta 2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluR delta 2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluR delta 2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum.
  • Keiko Matsuda, Tetsuro Kondo, Takatoshi Iijima, Shinji Matsuda, Masahiko Watanabe, Michisuke Yuzaki
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 4 707 - 717 2009年02月 [査読有り][通常論文]
     
    Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis.
  • Susana Mato, Elena Alberdi, Catherine Ledent, Masahiko Watanabe, Carlos Matute
    GLIA 57 3 295 - 306 2009年02月 [査読有り][通常論文]
     
    Regulation of Ca2+ homeostasis plays a critical role in oligodendrocyte function and survival. Cannabinoid CB1 and CB2 receptors have been shown to regulate Ca2+ levels and/or K+ currents in a variety of cell types. In this study we investigated the effect of cannabinoid compounds on the Ca2+ influx elicited in cultured oligodendrocytes by transient membrane depolarization with an elevated extracellular K+ concentration (50 mM). The CB1 receptor agonist arachidonoyl-chloro-ethanolamide (ACEA) elicited a concentration -dependent inhibition of depolarization-evoked Ca2+ transients in oligodendroglial somata with a maximal effect (94 +/- 3)% and an EC50 of 1.3 +/- 0.03 mu M. This activity was mimicked by the CB1/CB2 agonist CP55,940, as well as by the endocannabinoids N-arachidonoyl-ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), whereas the CB2 receptor selective agonist JWH133 was ineffective. The CB1 receptor antagonist AM251 (1 mu M) also reduced the Ca2+ response evoked by high extracellular K+ and did not prevent the inhibition elicited by ACEA (3 mu M). Nevertheless, the ability of ACEA and AEA to reduce depolarization-evoked Ca2+ transients was significantly reduced in oligodendrocytes from CB1 receptor knockout mice, as well as by pretreatment with pertussis toxin. Bath application of the inwardly rectifying K+ channels (Kir channels) blockers BaCl2 (300 mu M) and CsCl2 (1 mM) reduced the size of voltage-induced Ca2+ influx and partially prevented the inhibitory effect of ACEA. Our results indicate that cannabinoids inhibit depolarization-evoked Ca2+ transients in oligodendrocytes via CB1 receptor -dependent mechanisms that involve the activation of PTX-sensitive G(i/o) proteins and the blockade of Kir channels. (C) 2008 Wiley-Liss, Inc.
  • Eriko Miura, Keiko Matsuda, James I. Morgan, Michisuke Yuzaki, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 29 4 693 - 706 2009年02月 [査読有り][通常論文]
     
    Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluR delta 2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluR delta 2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluR delta 2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum.
  • Masashi Sanda, Akifumi Kamata, Osamu Katsumata, Kohji Fukunag, Masahiko Watanabe, Hisatake Kondo, Hiroyuki Sakagami
    BRAIN RESEARCH 1251 7 - 15 2009年01月 [査読有り][通常論文]
     
    IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor for Arf1 and Arf6, is localized at the postsynaptic density (PSD) and interacts with PSD-95. In this study, we identified a novel interaction of IQ-ArfGEF/BRAG1 with insulin receptor tyrosine kinase substrate of 53 kDa (IRSp53), also known as brain-specific angiogenesis inhibitor 1-associated protein 2. The interaction was mediated by the binding of the C-terminal proline-rich sequence of IQArfGEF/BRAG1 to the SH3 domain of IRSp53. IQ-ArfGEF/BRAG1 and IRSp53 were colocalized at the PSD of excitatory synapses of certain neuronal populations. Our present findings suggest that IQ-ArfGEF/BRAG1 may play roles downstream of NMDA receptors through the interaction with multivalent PSD proteins such as IRSp53 and PSD-95. (c) 2008 Elsevier B.V. All rights reserved.
  • Cesar Quiroz, Rafael Lujan, Motokazu Uchigashima, Ana Patricia Simoes, Talia N. Lerner, Janusz Borycz, Anil Kachroo, Paula M. Canas, Marco Orru, Michael A. Schwarzschild, Diane L. Rosin, Anatol C. Kreitzer, Rodrigo A. Cunha, Masahiko Watanabe, Sergi Ferre
    THESCIENTIFICWORLDJOURNAL 9 1321 - 1344 2009年 [査読有り][通常論文]
     
    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D-1 and D-2 receptors, respectively. Adenosine A(2A) receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D-2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A(2A) receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A(2A) receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A(2A) receptors could provide a new target for the treatment of neuropsychiatric disorders.
  • Masanobu Kano, Takako Ohno-Shosaku, Yuki Hashimotodani, Motokazu Uchigashima, Masahiko Watanabe
    PHYSIOLOGICAL REVIEWS 89 1 309 - 380 2009年01月 [査読有り][通常論文]
     
    Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe M. Endocannabinoid-Mediated Control of Synaptic Transmission. Physiol Rev 89: 309-380, 2009; doi: 10.1152/physrev.00019.2008. - The discovery of cannabinoid receptors and subsequent identification of their endogenous ligands (endocannabinoids) in early 1990s have greatly accelerated research on cannabinoid actions in the brain. Then, the discovery in 2001 that endocannabinoids mediate retrograde synaptic signaling has opened up a new era for cannabinoid research and also established a new concept how diffusible messengers modulate synaptic efficacy and neural activity. The last 7 years have witnessed remarkable advances in our understanding of the endocannabinoid system. It is now well accepted that endocannabinoids are released from postsynaptic neurons, activate presynaptic cannabinoid CB 1 receptors, and cause transient and long-lasting reduction of neurotransmitter release. In this review, we aim to integrate our current understanding of functions of the endocannabinoid system, especially focusing on the control of synaptic transmission in the brain. We summarize recent electrophysiological studies carried out on synapses of various brain regions and discuss how synaptic transmission is regulated by endocannabinoid signaling. Then we refer to recent anatomical studies on subcellular distribution of the molecules involved in endocannabinoid signaling and discuss how these signaling molecules are arranged around synapses. In addition, we make a brief overview of studies on cannabinoid receptors and their intracellular signaling, biochemical studies on endocannabinoid metabolism, and behavioral studies on the roles of the endocannabinoid system in various aspects of neural functions.
  • Andrew N. Iwaniuk, Hassan Marzban, Janelle M. P. Pakan, Masahiko Watanabe, Richard Hawkes, Douglas R. W. Wylie
    JOURNAL OF CHEMICAL NEUROANATOMY 37 1 55 - 63 2009年01月 [査読有り][通常論文]
     
    The parasagittal organization of the mammalian cerebellar cortex into zones has been well characterized by immunohistochemical, hodological and physiological studies in recent years. The pattern of these parasagittal bands across the cerebellum is highly conserved across mammals, but whhether a similar conservation of immunohistochemically defined parasagittal bands occurs within birds has remained uncertain. Here, we examine the compartmentation of the cerebellar cortex of a group of birds with unique cerebellar morphology-hummingbirds (Trochilidae). Immunohistochemical techniques were used to characterize the expression of zebrin II (aldolase C) and phospholipase C beta 4 (PLC beta 4) in the cerebellar cortex of two hummingbird species. A series of zebrin II immunopositive/immunonegative parasagittal stripes was apparent across most folia representing three major transverse zones: an anterior zone with a central stripe flanked by three lateral stripes on either side; a central zone of high/low immunopositive stripes; and a posterior zone with a central stripe flanked by four to six lateral stripes on either side. In addition, both folia I and X were uniformly immunopositive. The pattern of PLC beta 4 immunoreactivity was largely complementary-PLC beta 4 positive stripes were zebrin II negative and vice versa. The similarity of zebrin II expression between the hummingbirds and the pigeon indicates that the neurochemical compartmentation of the cerebellar cortex in birds is highly conserved, but species differences in the number and width of stripes do occur. (C) 2008 Elsevier B.V. All rights reserved.
  • Daniel Garcia-Ovejero, Angel Arevalo-Martin, Stefania Petrosino, Fabian Docagne, Carlos Hagen, Tiziana Bisogno, Masahiko Watanabe, Carmen Guaza, Vincenzo Di Marzo, Eduardo Molina-Holgado
    NEUROBIOLOGY OF DISEASE 33 1 57 - 71 2009年01月 [査読有り][通常論文]
     
    Endocannabinoids are lipid mediators with protective effects in many diseases of the nervous system. We have studied the modulation of the endocannabinoid system after a spinal cord contusion in rats. In early stages, lesion induced increases of anandamide and palmitoylethanolamide (PEA) levels, an upregulation of the synthesizing enzyme NAPE-phospholipase D and a downregulation of the degradative enzyme FAAH. In delayed stages, lesion induced increases in 2-arachidonoylglycerol and a strong upregulation of the synthesizing enzyme DAGL-alpha. that is expressed by neurons, astrocytes and immune infiltrates. The degradative enzyme MAGL was also moderately increased but only 7 days after the lesion. We have studied the cellular targets for the newly formed endocannabinoids using RT-PCR and immunohistochemistry against CB(1) and CB(2) receptors. We observed that CB, was constitutively expressed by neurons and oligodendrocytes and induced in reactive astrocytes. CB(2) receptor was strongly upregulated after lesion, and mostly expressed by immune infiltrates and astrocytes. The endocannabinoid system may represent an interesting target for new therapeutical approaches to spinal cord injury. (c) 2008 Elsevier Inc. All rights reserved.
  • Fumiaki Fukushima, Kazuhito Nakao, Toru Shinoe, Masahiro Fukaya, Shin-ichi Muramatsu, Kenji Sakimura, Hirotaka Kataoka, Hisashi Mori, Masahiko Watanabe, Toshiya Manabe, Masayoshi Mishina
    PLOS ONE 4 1 2009年01月 [査読有り][通常論文]
     
    Synchronized discharges in the hippocampal CA3 recurrent network are supposed to underlie network oscillations, memory formation and seizure generation. In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole in vivo by restricting synchronous firing of CA3 neurons.
  • Levente Deli, Gabor Wittmann, Imre Kallo, Ronald M. Lechan, Masahiko Watanabe, Zsolt Liposits, Csaba Fekete
    ENDOCRINOLOGY 150 1 98 - 103 2009年01月 [査読有り][通常論文]
     
    Hypophysiotropic TRH-synthesizing neurons of the hypothalamic paraventricular nucleus (PVN) have a critical role in the regulation of the energy homeostasis through control of the hypothalamic-pituitary-thyroid axis. Recently, endocannabinoids have been shown to exert inhibitory effects on TRH neurons via the type 1 cannabinoid receptor (CB1). To understand the anatomical basis for this regulatory mechanism, we determined whether CB1 is contained in axons innervating hypophysiotropic TRH neurons using a recently developed antiserum against the C-terminal portion of mouse CB1. CB1-immunoreactive axons densely innervated the parvicellular subdivisions of the PVN where the hypophysiotropic TRH neurons are located. By double-labeling immunocytochemistry, CB1-immunoreactive varicosities were observed in juxtaposition to the vast majority of TRH neurons in the PVN. At the ultrastructural level, CB1-immunoreactivity was observed in the preterminal portion of axons establishing both symmetric and asymmetric synaptic specializations with the perikarya and dendrites of TRH neurons in the PVN. These data demonstrate that CB1 is abundantly present in axons that are in synaptic association with hypophysiotropic TRH neurons, indicating an important role for endocannabinoids in the regulation of the hypothalamic-pituitarythyroid axis. The presence of both symmetric and asymmetric type CB1 synapses on TRH neurons in the PVN suggests that endocannabinoids may influence both excitatory and inhibitory inputs of these neurons. (Endocrinology 150: 98-103, 2009)
  • Keiko Hayashi, Norihiko Sengoku, Yoshimasa Kosaka, Yasushi Kondou, Naoko Sugiyama, Takumo Enomoto, Masaru Kuranami, Masahiko Watanabe
    Japanese Journal of Cancer and Chemotherapy 36 12 2480 - 2483 2009年 [査読有り][通常論文]
     
    An 83-year-old postmenopausal woman was referred to our hospital in order to get the treatment for ER positive advanced breast cancer (T4aN2M0, stage IIIb). The patient was diagnosed as the dissected aortic aneurysm and the hypertension. It was decided that an operation and chemotherapy would be too difficult with high existing risks. As a result, she was treated with anastrozole of PST, a 3rd-generation aromatase inhibitor, which led to the marked regression of the left breast cancer within a month. The cancer wasn't visualized after eighteen months. With the ongoing breast-hormone Therapy, no remote metastasis has been found after thirty months to this day. The patient hasn't experienced any adverse effects with the above-mentioned therapy. We concluded that the hormone (anastrozole) therapy as PST is a useful treatment for elderly postmenopausal woman with ER positive advanced breast cancer.
  • Nobue Futawatari, Shinichi Sakuramoto, Hiromitsu Moriya, Natsuya Katada, Shiro Kikuchi, Masahiko Watanabe, Atsuko Hara
    Japanese Journal of Gastroenterological Surgery 42 8 1377 - 1383 2009年 [査読有り][通常論文]
     
    We report a case of gastric granular cell tumor that was completely resected en bloc by laparoscopy-assisted partial gastrectomy. A 56-year-old man seen for a stomach tumor found 1 year earlier in a medical checkup was found in upper gastrointestinal endoscopy to have a 25-mm submucosal tumor growing inside the upper lesser gastric curvature. The tumor grew to 30mm within 10 months, necessitating ultrasonography. The diagnosis was a 36mm submucosal tumor originating in the fourth layer. Fine-needle aspiration biopsy done under endoscopic ultrasonography yielded a diagnosis of gastrointestinal stromal tumor (GIST). Abdominal ultrasonography and computed tomography did not show metastasis and the patient was scheduled to undergo surgery. The postoperative course following laparoscopy-assisted partial gastrectomy was favorable, and the man was discharged 8 days after surgery. Histopathological analysis showed the neoplasm to be a 4.5×3.5×3.5cm submucosal tumor. Histological analysis revealed tumor cells with eosinophilic cytoplasm that proliferated by forming large and small lesions, and immunological staining detected S-100 and synaptophysin, but no KIT, CD34, smooth muscle actin, or epithelial markers. The definitive diagnosis was a granular cell tumor. © 2009 The Japanese Society of Gastroenterological Surgery.
  • Yoko Yamagata, Shizuka Kobayashi, Tatsuya Umeda, Akihiro Inoue, Hiroyuki Sakagami, Masahiro Fukaya, Masahiko Watanabe, Nobuhiko Hatanaka, Masako Totsuka, Takeshi Yagi, Kunihiko Obata, Keiji Imoto, Yuchio Yanagawa, Toshiya Manabe, Shigeo Okabe
    NEUROSCIENCE RESEARCH 65 S144 - S144 2009年 [査読有り][通常論文]
  • Eriko Miura, Takatoshi Iijima, Tetsuro Kondo, Masahiko Watanabe, Michisuke Yuzaki
    NEUROSCIENCE RESEARCH 65 S83 - S83 2009年 [査読有り][通常論文]
  • Hiroyuki Kameda, Masashi Watanabe, Miyuki Bohgaki, Tadasuke Tsukiyama, Shigetsugu Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 378 4 744 - 749 2009年01月 [査読有り][通常論文]
     
    Cytoplasmic zinc finger protein A20 functionally dampens inflammatory signals and apoptosis via inhibition of NF-kappa B activation. We have reported that Ymer interacts with A20 and lysine (K)-63-linked polyubiquitin chain and that Ymer inhibits NF-kappa B signaling in collaboration with A20. It has also been reported that Ymer is phosphorylated by EGF stimulation. We found that Ymer was considerably phosphorylated on tyrosine residues also via Src family kinases such as Lck. A luciferase reporter assay showed that mutation of tyrosines on Ymer (YmerY217/279/304F) results in loss of the inhibitory activity for NF-kappa B signaling. Furthermore, a soft agar colony formation assay showed that the combination of SFCY527F and YmerY217/279/304F has no ability for anchorage-independent growth, suggesting that tyrosine phosphorylation of Ymer is important for inhibition of the NF-kappa B-mediated apoptotic pathway. These findings demonstrate that Ymer is likely to be a negative regulator for the NF-kappa B signaling pathway. (C) 2008 Elsevier Inc. All rights reserved
  • Taisuke Miyazaki, Miwako Yamasaki, Kouichi Tanaka, Masahiko Watanabe
    JOURNAL OF PHYSIOLOGICAL SCIENCES 59 198 - 198 2009年 [査読有り][通常論文]
  • Yohei Okubo, Hiroshi Sekiya, Shigeyuki Namiki, Hirokazu Sakamoto, Sho Iinuma, Miwako Yamasaki, Masahiko Watanabe, Kenzo Hirose, Masamitsu Iino
    NEUROSCIENCE RESEARCH 65 S142 - S143 2009年 [査読有り][通常論文]
  • Kaori Akashi, Toshikazu Kakizaki, Haruyuki Kamiya, Masahiro Fukaya, Miwako Yamasaki, Manabu Abe, Masahiko Watanabe, Kenji Sakimura
    NEUROSCIENCE RESEARCH 65 S139 - S140 2009年 [査読有り][通常論文]
  • Masahiko Watanabe, Masahiro Fukaya, Miwako Yamasaki
    NEUROSCIENCE RESEARCH 65 S140 - S141 2009年 [査読有り][通常論文]
  • Miwako Yamasaki, Masahiko Watanabe
    NEUROSCIENCE RESEARCH 65 S47 - S47 2009年 [査読有り][通常論文]
  • Miwako Yamasaki, Masahiko Watanabe
    JOURNAL OF PHYSIOLOGICAL SCIENCES 59 142 - 142 2009年 [査読有り][通常論文]
  • Hiroaki Shiraishi, Tetuo Mikami, Junko Aida, Ken-Ichi Nakamura, Naotaka Izumiyama-Shimomura, Tomio Arai, Masahiko Watanabe, Isao Okayasu, Kaiyo Takubo
    SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY 44 5 538 - 544 2009年 [査読有り][通常論文]
     
    Objective. Telomere shortening is thought to be associated with genetic instability. The purpose of this study was to measure telomere length in a series of Barrett's adenocarcinomas (BAs), focusing on the telomere/centromere fluorescent intensity ratio (TCR) with tissue quantitative fluorescent in situ hybridization (Q-FISH). Material and methods. A total of 11 cases of BA were evaluated for upper esophagus (UE), lower esophagus (LE), Barrett's mucosa (BM), BA, and gastric cardiac mucosa (GC). Q-FISH was performed using two kinds of peptide nucleic acid probe, specific for telomeres and centromeres. The sections were analyzed with a CCD camera and original software (Tissue Telo) for measuring TCR. In addition, Laser Capture Microdissection and GeneScan were implemented for evaluation of genetic instability. Results. The TCR values in BM and, to a lesser extent, BA were significantly lower than those in the other tissues, particularly in heterozygosity (LOH)-positive cases. However, no significant difference was evident between microsatellite instability (MSI)-positive and -negative groups. Conclusions. In our study of BA series, telomere length appeared to change with the degree of histological atypia, with decreases linked to LOH.
  • Keiichi Tamai, Masafumi Toyoshima, Nobuyuki Tanaka, Noriko Yamamoto, Yuji Owada, Hiroshi Kiyonari, Kazuko Murata, Yoshiyuki Ueno, Masao Ono, Tooru Shimosegawa, Nobuo Yaegashi, Masahiko Watanabe, Kazuo Sugamura
    AMERICAN JOURNAL OF PATHOLOGY 173 6 1806 - 1817 2008年12月 [査読有り][通常論文]
     
    The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the brs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked brs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs(flox/flox);SynI-cre mice. Notably, the hrs(flox/flox);SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs(flox/flox);SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates. (Am J Pathol 2008, 173:1806-1817; DOI: 10.2353/ajpath.2008.080684)
  • Yoshiaki Shinohara, Hajime Hirase, Masahiko Watanabe, Makoto Itakura, Masami Takahashi, Ryuichi Shigemoto
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 49 19498 - 19503 2008年12月 [査読有り][通常論文]
     
    Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits.
  • Tie-Jun Sten Shi, Su-Xing Leslie Liu, Henrik Hammarberg, Masahiko Watanabe, Zhi-Qing David Xu, Tomas Hokfelt
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 50 20004 - 20008 2008年12月 [査読有り][通常論文]
     
    Treatment of neuropathic pain is a major clinical problem. This study shows expression of phospholipase beta 3 (PLC beta 3) in mouse and human DRG neurons, mainly in small ones and mostly with a nonpeptidergic phenotype. After spared nerve injury, the pain threshold was strongly reduced, and systemic treatment of such animals with the unselective PLC inhibitor U73122 caused a rapid and long-lasting (48-h) increase in pain threshold. Thus, inhibition of PLC may provide a way to treat neuropathic pain.
  • Erika Polgar, Khulood M. Al-Khater, Safa Shehab, Masahiko Watanabe, Andrew J. Todd
    JOURNAL OF NEUROSCIENCE 28 49 13150 - 13160 2008年12月 [査読有り][通常論文]
     
    Although most projection neurons in lamina I express the neurokinin 1 receptor (NK1r), we have identified a population of large multipolar projection cells that lack the NK1r, are characterized by the high density of gephyrin puncta that coat their cell bodies and dendrites, and express the transcription factor Fos in response to noxious chemical stimulation. Here we show that these cells have a very high density of glutamatergic input from axons with strong immunoreactivity for vesicular glutamate transporter 2 that are likely to originate from excitatory interneurons. However, they receive few contacts from peptidergic primary afferents or transganglionically labeled A delta nociceptors. Unlike most glutamatergic synapses in superficial laminas, those on the gephyrin-coated cells contain the GluR4 subunit of the AMPA receptor. A noxious heat stimulus caused Fos expression in 38% of the gephyrin-coated cells but in 85% of multipolar NK1r-immunoreactive cells. These findings are consistent with the suggestion that there is a correlation between function and morphology for lamina I neurons but indicate that there are at least two populations of multipolar neurons that differ in receptor expression, excitatory inputs, and responses to noxious stimulation. Although there are only similar to 10 gephyrin-coated cells on each side per segment in the lumbar enlargement, they constitute similar to 18% of the lamina I component of the spinothalamic tract at this level, which suggests that they play an important role in transmission of nociceptive information to the cerebral cortex. Our results also provide the first evidence that postsynaptic GluR4-containing AMPA receptors are involved in spinal nociceptive transmission.
  • Carolina Ladera, Maria del Carmen Godino, Maria Jose Cabanero, Magdalena Torres, Masahiko Watanabe, Rafael Lujan, Jose Sanchez-Prieto
    JOURNAL OF NEUROCHEMISTRY 107 6 1506 - 1517 2008年12月 [査読有り][通常論文]
     
    Neuronal G protein-gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post-synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA(B) receptors. In this study, we show for the first time that GABA(B) receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA(B) receptors reduces glutamate release and the Ca(2+) influx mediated by N-type Ca(2+) channels in a mode insensitive to the GIRK channel blocker tertiapin-Q and consistent with direct inhibition of this voltage-gated Ca(2+) channel. However, by means of weak stimulation protocols, we reveal that GABA(B) receptors also reduce glutamate release mediated by P/Q-type Ca(2+) channels, and that these responses are reversed by the GIRK channel blocker tertiapin-Q. Consistent with the functional interaction between GABA(B) receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre-synaptic boutons of asymmetric synapses co-express GABA(B) receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post-synaptic level, also occurs at glutamatergic nerve terminals.
  • Yasukazu Hozumi, Masahiro Fukaya, Naoko Adachi, Naoaki Saito, Koichi Otani, Hisatake Kondo, Masahiko Watanabe, Kaoru Goto
    EUROPEAN JOURNAL OF NEUROSCIENCE 28 12 2409 - 2422 2008年12月 [査読有り][通常論文]
     
    Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers, i.e. diacylglycerol (DAG) and inositol 1,4,5-trisphosphate. The former activates protein kinase C and the latter mobilizes Ca(2+) from intracellular store. DAG kinase (DGK) then phosphorylates DAG to produce another second messenger (phosphatidic acid). Of 10 mammalian DGK isozymes, DGK beta is expressed in dopaminergic projection fields with the highest level in the striatum and its particular splice variant is differentially expressed in patients with bipolar disorder. To gain molecular anatomical evidence for its signaling role, we investigated the cellular expression and subcellular localization of DGK beta in the striatum of rat brain. DGK beta was expressed in medium spiny neurons constituting the striatonigral and striatopallidal pathways, whereas striatal interneurons were below the detection threshold. DGK beta was distributed in somatodendritic elements of medium spiny neurons and localized in association with the smooth endoplasmic reticulum and plasma membrane or in the narrow cytoplasmic space between them. In particular, DGK beta exhibited dense accumulation at perisynaptic sites on dendritic spines forming asymmetrical synapses. The characteristic anatomical localization was consistent with exclusive enrichment of DGK beta in the microsomal and postsynaptic density fractions. Intriguingly, DGK beta was very similar in immunohistochemical and immunochemical distribution to Gq-coupled receptors, such as metabotropic glutamate receptors 1 and 5, and also to other downstream molecules involving DAG metabolism, such as phospholipase C beta and DAG lipase. These findings suggest that abundant DGK beta is provided to perisynaptic sites of medium spiny neurons so that it can effectively produce phosphatidic acid upon activation of Gq-coupled receptors and modulate the cellular state of striatal output neurons.
  • Masanori Tachikawa, Jun Fujinawa, Masato Takahashi, Yasuyuki Kasai, Masahiro Fukaya, Kazuhisa Sakai, Maya Yamazaki, Masatoshi Tomi, Masahiko Watanabe, Kenji Sakimura, Tetsuya Terasaki, Ken-ichi Hosoya
    JOURNAL OF NEUROCHEMISTRY 107 3 768 - 778 2008年11月 [査読有り][通常論文]
     
    Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 mu M/412 mu M which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of D-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.
  • Ruben Nogueiras, Christelle Veyrat-Durebex, Paula M. Suchanek, Marcella Klein, Johannes Tschoep, Charles Caldwell, Stephen C. Woods, Gabor Wittmann, Masahiko Watanabe, Zsolt Liposits, Csaba Fekete, Ofer Reizes, Francoise Rohner-Jeanrenaud, Matthias H. Tschoep
    DIABETES 57 11 2977 - 2991 2008年11月 [査読有り][通常論文]
     
    OBJECTIVE-Blockade of the CB1 receptor is one of the promising strategies for the treatment of obesity. Although antagonists suppress food intake and reduce body weight, the role of central versus peripheral CB1 activation on weight loss and related metabolic parameters remains to be elucidated. We therefore specifically assessed and compared the respective potential relevance of central nervous system (CNS) versus peripheral CB1 receptors in the regulation of energy homeostasis and lipid and glucose metabolism in diet-induced obese (DIO) rats. RESEARCH DESIGN AND METHODS-Both lean and DIO rats were used for our experiments. The expression of key enzymes involved in lipid metabolism was measured by real-time PCR, and euglycemic-hyperinsulinemic clamps were used for insulin sensitivity and glucose metabolism studies. RESULTS-Specific CNS-CB1 blockade decreased body weight and food intake but, independent of those effects, had no beneficial influence on peripheral lipid and glucose metabolism. Peripheral treatment with CB1 antagonist (Rimonabant) also reduced food intake and body weight but, in addition, independently triggered lipid mobilization pathways in white adipose tissue and cellular glucose uptake. Insulin sensitivity and skeletal muscle glucose uptake were enhanced, while hepatic glucose production was decreased during peripheral infusion of the CB1 antagonist. However, these effects depended on the antagonist-elicited reduction of food intake. CONCLUSIONS-Several relevant metabolic processes appear to independently benefit from peripheral blockade of CB I, while CNS-CB1 blockade alone predominantly affects food intake and body weight. Diabetes 57:2977-2991, 2008
  • Masahiro Fukaya, Motokazu Uchigashima, Sachi Nomura, Yuta Hasegawa, Hisaya Kikuchi, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 28 9 1744 - 1759 2008年11月 [査読有り][通常論文]
     
    Upon activation of receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC)beta hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Of four PLC beta isoforms, PLC beta 1 is transcribed predominantly in the telencephalon and its gene inactivation in mice impairs metabotropic glutamate receptor- and muscarinic acetylcholine receptor-dependent hippocampal oscillations, endocannabinoid production in the hippocampus and barrel formation in the somatosensory cortex. Here we examined cellular and subcellular distributions of PLC beta 1 in adult mouse brains. In the telencephalon, high levels of PLC beta 1 were observed in principal neurons, including pyramidal cells in the cortex and hippocampus, granule cells and mossy cells in the dentate gyrus, and medium spiny neurons in the caudate-putamen, whereas most interneurons had low levels of or were negative for PLC beta 1 and, instead, expressed PLC beta 4. By immunofluorescence, tiny clusters of PLC beta 1 were distributed in somatodendritic compartments of principal neurons and positioned close to those of metabotropic glutamate receptor 5, muscarinic acetylcholine receptor M1 and diacylglycerol lipase-alpha, respectively. Immunoelectron microscopy revealed that PLC beta 1 was often associated with the smooth endoplasmic reticulum, cell membrane or postsynaptic density. In particular, it was highly accumulated at the perisynapse of dendritic spines forming asymmetrical synapses. In the cerebellum, PLC beta 1 was generally low but was enriched in axons and dendrites of basket cells. These results suggest that PLC beta 1 is the key effector in telencephalic principal neurons and cerebellar interneurons. Furthermore, the well-orchestrated molecular arrangement appears to be the anatomical basis for the specificity, efficiency and convergence of the neuronal phosphoinositide signaling system.
  • Masahiro Fukaya, Motokazu Uchigashima, Sachi Nomura, Yuta Hasegawa, Hisaya Kikuchi, Masahiko Watanabe
    EUROPEAN JOURNAL OF NEUROSCIENCE 28 9 1744 - 1759 2008年11月 [査読有り][通常論文]
     
    Upon activation of receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC)beta hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Of four PLC beta isoforms, PLC beta 1 is transcribed predominantly in the telencephalon and its gene inactivation in mice impairs metabotropic glutamate receptor- and muscarinic acetylcholine receptor-dependent hippocampal oscillations, endocannabinoid production in the hippocampus and barrel formation in the somatosensory cortex. Here we examined cellular and subcellular distributions of PLC beta 1 in adult mouse brains. In the telencephalon, high levels of PLC beta 1 were observed in principal neurons, including pyramidal cells in the cortex and hippocampus, granule cells and mossy cells in the dentate gyrus, and medium spiny neurons in the caudate-putamen, whereas most interneurons had low levels of or were negative for PLC beta 1 and, instead, expressed PLC beta 4. By immunofluorescence, tiny clusters of PLC beta 1 were distributed in somatodendritic compartments of principal neurons and positioned close to those of metabotropic glutamate receptor 5, muscarinic acetylcholine receptor M1 and diacylglycerol lipase-alpha, respectively. Immunoelectron microscopy revealed that PLC beta 1 was often associated with the smooth endoplasmic reticulum, cell membrane or postsynaptic density. In particular, it was highly accumulated at the perisynapse of dendritic spines forming asymmetrical synapses. In the cerebellum, PLC beta 1 was generally low but was enriched in axons and dendrites of basket cells. These results suggest that PLC beta 1 is the key effector in telencephalic principal neurons and cerebellar interneurons. Furthermore, the well-orchestrated molecular arrangement appears to be the anatomical basis for the specificity, efficiency and convergence of the neuronal phosphoinositide signaling system.
  • Peizhong Mao, Yuan-Xiang Tao, Masahiro Fukaya, Feng Tao, Dechun Li, Masahiko Watanabe, Roger A. Johns
    IUBMB LIFE 60 10 684 - 692 2008年10月 [査読有り][通常論文]
     
    Membrane-associated guanylate kinases (MAGUKs) act as scaffolds to coordinate signaling events through their multiple domains at the plasma membrane. The MAGUK SH3 domain is noncanonical and its function remains unclear. To identify potential binding partners of MAGUK SH3, the synapse-associated protein 102 (SAP102) SH3 domain was used as bait in a yeast two-hybrid screen of a mouse embryonic cDNA library. A mouse homologue of the Drosophila discs large tumor suppressor (Dig, also known as SAP97) bound preferentially to SAP102 SH3. The 4347bp cDNA sequence encoded an 893 amino acid protein with 94% identity to mouse SAP97. A deleted region (33-aa) strongly suggests this is a novel splice variant, which we call Embryonic-dlg/SAP97 (E-dlg). The interaction of SAP102 and F-dig was confirmed in mammalian cells. E-dig can also hind to potassium channel Kvl.4 in a pull-down assay. E-dlg wits highly expressed in embryonic and some adult mouse tissues, such as brain, kidney, and ovary. Furthermore. in situ hybridization showed that E-dlg was mostly express