Somato-axodendritic release of oxytocin into the brain due to calcium amplification is essential for social memory

子どものこころの発達研究センターOxytocin (OT) is released into the brain from the cell soma, axons, and dendrites of neurosecretory cells in the hypothalamus. Locally released OT can activate OT receptors, form inositol-1,4,5-trisphosphate and elevate intracellular free calcium (Ca2+) concentrations [(Ca2+)i] in self and neighboring neurons in the hypothalamus, resulting in further OT release: i.e., autocrine or paracrine systems of OT-induced OT release. CD38-dependent cyclic ADP-ribose (cADPR) is also involved in this autoregulation by elevating [Ca2+]i via Ca2+ mobilization through ryanodine receptors on intracellular Ca2+ pools that are sensitive to both Ca2+ and cADPR. In addition, it has recently been reported that heat stimulation and hyperthermia enhance [Ca2+]i increases by Ca2+ influx, probably through TRPM2 cation channels, suggesting that cADPR and TRPM2 molecules act as Ca2+ signal amplifiers. Thus, OT release is not simply due to depolarization–secretion coupling. Both of these molecules play critical roles not only during labor and milk ejection in reproductive females, but also during social behavior in daily life in both genders. This was clearly demonstrated in CD38 knockout mice in that social behavior was impaired by reduction of [Ca2+]i elevation and subsequent OT secretion. Evidence for the associations of CD38 with social behavior and psychiatric disorder is discussed, especially in subjects with autism spectrum disorder. © 2015 The Author(s)Embargo Period 12 month

Membrane current responses to intracellular injections of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in NG108-15 hybrid cells

AbstractIontophoretic injections of inositol 1,4,5-trisphosphate inside neuroblastoma × glioma NG108-15 hybrid cells evoked an outward K+ current across the outer cell membrane, probably activated by the release of intracellular Ca2+. No such current was produced by equivalent intracellular injections of inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate. Instead, these compounds evoked an inward current with a reversal potential of about −20 mV, and which may therefore be due to a non-specific cation conductance. This suggests that these derivatives are unable to release sufficient Ca2+ to activate the Ca2+-dependent K+ current in these cells

A personal view from a long-lasting collaborator on the research strategies of Marshall Nirenberg

In this review, I summarized transition in Dr. Marshall Nirenberg\u27s research interests during 1970s, from a view of a long-lasting collaborator. Nirenberg switched his research filed to neurobiology after his success in deciphering genetic code and being honored with the Nobel Prize in Physiology or Medicine in 1968. His targets were to obtain genetically pure population of neurons, i.e. neuroblastoma clones, to make somatic hydrid cells, to culture neuronal and muscle cells, and to produce monoclonal antibodies against whole retinal or neuroblastoma cells. He studied neurotransmitters, receptors, cyclic nucleotides, cell differentiation, secretion, synapse formation, and chemical recognition. Especially he liked his hypothesis for opiate tolerance and dependency as a model of cellular memory. Through these studies, he seemed to devote all his time of about 50 years from 1960s to decoding brain memory processes. © 2011 Elsevier Ltd. All rights reserved

Bradykinin inhibits potassium (M) currents in N1E- 115 neuroblastoma cells Responses resemble those in NG108-15 neuroblastoma x glioma hybrid cells

AbstractApplication of bradykinin to voltage-clamped N1E-115 mouse neuroblastoma cells evoked sequential outward and inward membrane currents, accompanied by an increase and decrease of membrane conductance, respectively. Methacholine produced an inward current with a decreased conductance. The outward current response to bradykinin was imitated by intracellular inositol 1,4,5-trisphosphate (IP3). Bath application of phorbol dibutyrate induced an inward current and potentiated the response to IP3. We conclude that the response of these cells to bradykinin is identical to that of NG108-15 hybrid cells, and therefore may be attributed to the dual effects of inositol trisphosphate and diacylglycerol formed by hydrolysis of phosphatidylinositide

Evidence for a Ca2+-independent hydrolysis of phosphatidylinositol 4,5-bisphosphate in neuron-like cell line NG108-15 cells

AbstractThe addition of bradykinin to 32Pi-labeled neuroblastoma × glioma hybrid NG108-15 cells caused a substantial loss of radioactivity from phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2). The bradykinin-induced hydrolysis of PI-4,5-P2 was almost equally observed even when extracellular Ca2+ was depleted with EGTA (100μM). On the other hand, high K+ depolarization of the cells, which allows Ca2+ influx through voltage-dependent Ca2+ channels, failed to induce any significant decrease in the radioactivity of PI-4,5-P2. These data indicate that the bradykinin-stimulated PI-4,5-P2 hydrolysis in NG108-15 cells is independent of extracellular Ca2+ and also that PI-4,5-P2 hydrolysis is not stimulated by an elevation of intracellular Ca2+ concentration

Remission of social behavior impairment by oral administration of a precursor of NAD in CD157, but not in CD38, knockout mice

Nicotinamide adenine dinucleotide (NAD) is a substrate of adenosine diphosphate (ADP)-ribosyl cyclase and is catalyzed to cyclic ADP-ribose (cADPR) by CD38 and/or CD157. cADPR, a Ca2+ mobilizing second messenger, is critical in releasing oxytocin from the hypothalamus into the brain. Although NAD precursors effectively play a role in neurodegenerative disorders, muscular dystrophy, and senescence, the beneficial effects of elevating NAD by NAD precursor supplementation on brain function, especially social interaction, and whether CD38 is required in this response, has not been intensely studied. Here, we report that oral gavage administration of nicotinamide riboside, a perspective NAD precursor with high bioavailability, for 12 days did not show any suppressive or increasing effects on sociability (mouse’s interest in social targets compared to non-social targets) in both CD157KO and CD38KO male mice models in a three-chamber test. CD157KO and CD38KO mice displayed no social preference (that is, more interest towards a novel mouse than a familiar one) behavior. This defect was rescued after oral gavage administration of nicotinamide riboside for 12 days in CD157KO mice, but not in CD38KO mice. Social memory was not observed in CD157KO and CD38KO mice; subsequently, nicotinamide riboside administration had no effect on social memory. Together with the results that nicotinamide riboside had essentially no or little effect on body weight during treatment in CD157KO mice, nicotinamide riboside is less harmful and has beneficial effect on defects in recovery from social behavioral, for which CD38 is required in mice

コリン性シナプス形成の調節機構

金沢大学がん研究所研究課題/領域番号:57213010, 研究期間(年度):1982出典：研究課題「コリン性シナプス形成の調節機構」課題番号57213010（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-57213010/）を加工して作

脳型ADPリボシールシクラーゼの精製とcDNAクローニング

神経腫瘍細胞NG108-15細胞のADPリボシルシクラーゼは、ムスカリン受容体とカップルしていたが、脳のADPリボシルシクラーゼは、カップルする受容体が、数多くの試みの中からは検出できなかった。一方、心臓のADPリボシルシクラーゼは、イソプロテレノール、ノルアドレナリンやアンデオテンシンII刺激により、活性化された。私達の目的は受容体とカップルする膜型ADPリボシルシクラーゼの分子を決めることにあるので、さしあたり、ラット心室心筋を出発材料にすることにした。心筋を細分して低調液で、30分間浸け、テフロンガラスホモゲナイザーで均一化した後、1000g、10分の遠心により核を取り除いた。上澄みを105000g、15分の超遠心にかけ、膜分画を得た。それを2%のトライトンXで可溶化し、105000g、30分にて遠心し沈渣を除いた後、上澄みをDEAEカラムにとうした。ADPリボシルシクラーゼ活性は、DEAEカラムを1M NaClで溶出すると、タンパクのピークと一致して検出できた。この分画をさらにゲル濾過すると、アルブミンの溶出よりも少し分子量の低い分画にADPリボシルシクラーゼ活性が回収できた。ブルーセファロースカラスにより単一タンパクまでにさらに精製できた。一方、DNAシークエンスのデータベースの中より、ホモロジー検索によりCD38/ADPリボシールシクラーゼの類似DNAを検出することを試みた。よく保存された20マーをプライマーとし、心臓RNAをプレートとし、RT-PCRを試み期待される長さ、及びそうではないものを得た。これらのDNA断片の塩基配列を現在決めている。新規のADPリボシルシクラーゼを検索しているところである。We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [3H]cADP-ribose formation from [3H]NAD+ in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 mM increased ADP-ribosyl cyclase activity by 1.7-fold in venticular muscle ; This increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 mM GTP-g-S, while 10 mM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from b-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells, and suggest that increased cADP-ribose synthesis is involved in upregulation of cardiac function by sympathetic stimulation.研究課題/領域番号:09480221, 研究期間(年度):1997-1999出典：「脳型ADPリボシールシクラーゼの精製とcDNAクローニング」研究成果報告書　課題番号09480221 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Aキナーゼアンカータンパク(AKAP)依存的シナプス後電位: 終止の分子メカニズム

金沢大学医薬保健研究域医学系中枢、末梢神経の受容体刺激による膜興奮性変調に関与するシナプス電位のうちでも、特にslow EPSPの発生機序の解明は短期記憶のメカニズムを考える上で重要である。しかし、長らく未解決のままであった。この課題に対し、申請者らは、上頚神経節神経細胞の電位依存性カリウムチャネルであるM(KCNQ2/3)チャネルのムスカリン性アセチルコリン受容体刺激による電流抑制のシグナル伝達経路でのAKAP150の役割を解析してきた。今回、アセチルコリンにより活性化されるポストシナプス側でのムスカリン受容体反応での分子メカニズムを理解し、この知識を痴呆症の治療に役立てるため、Mチャンネルの受容体による抑制機序の解明と、そこに関わるAKAP依存的シグナル伝達の解明をし、記憶、学習の分子基盤として新しい視点を提供することとした。KCNQ2/3チャンネルのアミノ酸配列上PKCによるリン酸化サイトは12ケ所(セリンの7ヶ所とスレオニンの5ヶ所)あり、カルチニューリンが(PKC依存的な)セリンのリン酸を脱リン酸化するか否かをアラニン置換(アラニンスキャンニング)で検討し、PKCとカルチニューリンとKCNQチャンネルの相互作用部位を確定した。その部位での脱リン酸化を実験した。また、IQ配列の変異によるチャンネル抑制の変化を観察した。チャンネルのIQ部位のトリプトファンによる蛍光変化により、両者の親和性の強さ、Ca依存性を測定した。最後に、KCNQ抗体でプルダウンされる分子の中にムスカリン受容体とAKAPがが存在する事を見い出した。研究課題/領域番号:17024020, 研究期間(年度):2005出典：「Aキナーゼアンカータンパク(AKAP)依存的シナプス後電位: 終止の分子メカニズム」研究成果報告書　課題番号17024020（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-17024020/)を加工して作

ニュ-ロブラスト-マ・グリオ-マ雑種細胞におけるシナプス形成・伝達機構

金沢大学医学部1.前年度の調査で、ジプチリ-ルcAMPやプロスタグランディンが効率よくシナプス形成を行う事を知り得たので、それらを用いてNG108-15筋スナプスを形成した。2.イノシト-ル1,4,5三リン酸の細胞内注入。ホルボ-ルジプチレ-トとブラジキニンの細胞外からの投与によるシナプス伝達効率の変化を観察し、いずれも上昇させた。3.細胞外と細胞内のCaの役割を、細胞外からのCaの流入を抑える条件下でシナプス伝達を調査した。4.脳型(m1タイプ)のムスカリン性アセチ-ルコリン受容体遺伝子を、トランスフェクトしたNG108-BM8が、PI代謝を高める事を確認したので、それらの細胞相互間で、シナプス(ムスカリン性)結合が生じないかを調査したが結果はネガティブであった。研究課題/領域番号:01638508, 研究期間(年度):1989出典：研究課題「ニュ-ロブラスト-マ・グリオ-マ雑種細胞におけるシナプス形成・伝達機構」課題番号01638508（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-01638508/）を加工して作