1996 年度実績報告書

シナプス伝達受容分子と可塑性

研究課題

研究課題/領域番号	07278102
研究機関	東京大学
研究代表者	三品昌美東京大学, 医学部, 教授 (80144351)
研究分担者	崎村建司新潟大学, 脳研究所, 教授 (40162325) 渡辺雅彦北海道大学, 医学部, 助教授 (70210945) 杉山博之九州大学, 理学部, 教授 (20124224) 高橋智幸東京大学, 医学部, 教授 (40092415) 二木宏明東京大学, 大学院・人文,社会系研究科, 教授 (10073074)
キーワード	グルタミン酸受容体 / NMDA受容体 / ノックアウトマウス / シナプス可塑性 / シナプス形成 / 記憶 / 学習 / 運動失調
研究概要	本研究班は、シナプス伝達の長期増強および長期抑圧の機構ならびに記憶・学習との関係を実験的に検証することを目的とし、遺伝子ノックアウトマウスの作成と解析を中心に推進した。NMDA受容体チャネルのε1サブユニットを欠損したマウスにおけるNMDA受容体チャネルの性質をスライスパッチクランプ法で解析することにより、発達の過程でε2型からε1型およびε3型への転換が起こり、成熟加硫細胞ではε1型およびε3型がそれぞれ単一チャネルコンダクタンスを異にするチャネルを形成して共存していることおよびε3型が低Mg^<2+>感受性チャネルを形成することが示唆された。さらに、標的遺伝子組換えによりNMDA受容体チャネルのε2サブユニットを欠損したマウスを作成した。ε2サブユニット欠損マウスは、生後1日で死亡したが、人工哺乳により飼育可能なことから、死因は母乳を摂取できないことに因ると考えられた。欠損マウスは哺乳に必要な吸引反射が消失しており、三叉神経核においてヒゲ知覚神経に対応するバレレット構造の形成が認められなかった。ヒゲ知覚神経を標識し解析した結果、知覚神経は三叉神経核に到達しているが、その神経終末は散漫に分布していることが示唆される。さらに、海馬シナプスにおいてシナプス伝達の長期抑圧が欠損していた。すなわち、ε2サブユニットはシナプス可塑性とシナプス形成の両者に関与し、マウスの発達に必須の分子であることが明らかとなった。小脳プルキニエ細胞に特異的に発現するグルタミン酸受容体チャネルδ2サブユニット欠損マウスのプルキンエ細胞シナプスから記録を行い平行線維刺激の効果を調べたところ、反応の大きさが有意に減弱しており、平行線維-プルキンエ細胞シナプスの形成不全があるという形態学的所見を機能的に裏付けた。また、このマウスでは、登上線維がプルキンエ細胞の遠位樹状突起に、過剰な異所性シナプスを形成することを発見した。これは平行線維シナプス形成不全の結果と考えられ、平行線維と登上線維の間でプルキンエ細胞樹状突起のシナプス後部をめぐる競合があることが示唆された。代謝型グルタミン酸受容体およびプロテインキナーゼCγの欠損マウスでは、平行線維シナプスは正常に形成されたが、登上線維シナプスの成熟に障害があることが判明した。胚幹細胞を用いた遺伝子操作を多角的に効率よく行うためのピューロマイシンとピューロマイシン耐性遺伝子系を開発し、部位および時期特異的標的遺伝子組換えのためにCre-loxPプラスミドによるloxP組換え細胞の濃縮系を開発した。本成果は、多くの計画班員の協同研究で達成されたものであり、重点領域研究の長所を発揮することが出来た。

研究成果
(28件)

すべてその他

すべて文献書誌 (28件)

[文献書誌] Kutsuwada,T.: "Impairment of suckling response,trigeminal neuronal pattem formation and hippocampal LTD in NMDA receptor ε2 subunit mutant mice." Neuron. 16. 333-344 (1996)
[文献書誌] Nagasawa,M.: "Gene structure and chromosomal localization of the mouse NMDA receptor channel subunits." Mol.Brain Res.36. 1-11 (1996)
[文献書誌] Ito,I.: "Age-dependent reduction of hippocampal LTP in mice lacking N-methyl-D-asparatte receptor ε1 subunit." Neurosci.Lett.203. 69-71 (1996)
[文献書誌] Takayama,C.: "Developmental changes in expression and distribution of the glutamate receptor channel δ2 subunit according to the Purkinje cell maturation." Dev.Brain Res.92. 147-155 (1996)
[文献書誌] Takahashi,T.: "Functional correlation of NMDA receptor ε subunits expression with the properties of single-channel and synaptic currents in the developing cerebellum." J.Neurosci.16. 4376-4382 (1996)
[文献書誌] Asada,H.: "Mice lacking the 65 kDa isoform of glutamic acid decarboxylase(GAD65)maintain normal levels of GAD67 and GABA in their brain but are susceptible to seizures." Biochemical and Biophysical Research Communications. 229. 891-895 (1996)
[文献書誌] Miyakawa,T.: "Susceptibility of drug-induced seizures of Fyn tyrosine kinase deficient mice." NeuroReport. 7. 2723-2726 (1996)
[文献書誌] Miyakawa,T.: "Radial maze performance,open-field and elevated plus-maze behaviors in Fyn-kinase deficient mice:Further evidence for increased fearfulness." Mol.Brain Res.37. 145-150 (1996)
[文献書誌] Nakagawa,S.: "Altered gene expression of the N-methyl-D-aspartate receptor channel subunits in Purkinje cells of the staggerer mutant mouse." Eur.J.Neurosci.8. 2644-2651 (1996)
[文献書誌] Yamada,K.: "EAAT4 is a post-synaptic glutamate transporter at Purkinje cell synapses." Neuroreport. 7. 2013-2017 (1996)
[文献書誌] Nakagawa,S.: "Regional variation in expressions of calbindin and inositol 1,4,5-trisphosph ate receptor typel mRNAs in the cerebellum of the staggerer mutant mouse." Eur.J.Neurosci.8. 1401-1407 (1996)
[文献書誌] Shibata,T.: "Dynamic changes in expression of glutamate transporter mRNAs in developing brain." Neuroreport. 7. 705-709 (1996)
[文献書誌] Hori,Y.: "Long-lasting facilitation induced by serotonin in superficial dorsal horn neurones of rat spinal cord." J.Physiol.492. 867-876 (1996)
[文献書誌] Silver,R.A.: "Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites." J.Physiol.494. 231-250 (1996)
[文献書誌] Yokoi,M.: "Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2." Science. 273. 645-647 (1996)
[文献書誌] Kobayashi,K.: "Presynaptic long-term depression at the hippocampal mossy fiber-CA3 synapse." Science. 273. 648-650 (1996)
[文献書誌] Takahashi,T.: "Presynaptic calcium current modulation by a glutamate receptor." Science. 274. 594-597 (1996)
[文献書誌] Tempia,F.: "The fractional calcium current through neuronal AMPA receptor channels with a ′low′calcium permeability." J.Neurosci.15. 456-466 (1996)
[文献書誌] Kano,M.: "Calcium-induced rebound potentiation of GABA-mediated currents is mediated by calcium/calmodulin dependent kinase II." Proc.Natl.Acad.Sci.USA.93. 13351-13356 (1996)
[文献書誌] Uchino,S.: "Inducible expression of N-methy-D-aspartate(NMDA)receptor channels from cloned cDNAs in CHO cells." Mol.Brain Res.(in press.).
[文献書誌] Uchino,S.: "Analysis of the glycine binding domain of the NMDA receptor channel ζ1 subunit." NeuroReport. (in press.).
[文献書誌] Ito,I.: "Synapse-selective impairment of NMDA receptor functions in mice lacking NMDA receptor ε1 or ε2 subunit." J.Physiol.(in press.).
[文献書誌] Kai,N.: "Molecular cloning of Fyn-associated molecules in the mouse central nervous system." J.Neurosci.Res.(in press.).
[文献書誌] Minami,T.: "Absence of prostglandin E_2-induced hyperalgesia in NMDA receptor ε subunit knockout mice." Brit.J.Phamacal.(in press.).
[文献書誌] Hayashi,Y.: "Calcium and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the postsynaptic density." Mol.Brain Res.(in press.).
[文献書誌] Yamada K.: "Changes in expression and distribution of the glutamate transporter EAAT4 in developing mouse Purkinje cells." Neurosci.Res.(in press.).
[文献書誌] Kano,M.: "Persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking mGluR1." Neuron. (in press.).
[文献書誌] Kano,M.: "Long-lasting potentiation of GABA ergic inhibitory synaptic transmission in cerebellar Purkinje cells:its properties and possible mechanisms." Behav.Brain Sci.(in press.).

1996 年度 実績報告書

シナプス伝達受容分子と可塑性

研究代表者

三品 昌美 東京大学, 医学部, 教授 (80144351)

研究成果

[文献書誌] Kutsuwada,T.: "Impairment of suckling response,trigeminal neuronal pattem formation and hippocampal LTD in NMDA receptor ε2 subunit mutant mice." Neuron. 16. 333-344 (1996)

[文献書誌] Nagasawa,M.: "Gene structure and chromosomal localization of the mouse NMDA receptor channel subunits." Mol.Brain Res.36. 1-11 (1996)

[文献書誌] Ito,I.: "Age-dependent reduction of hippocampal LTP in mice lacking N-methyl-D-asparatte receptor ε1 subunit." Neurosci.Lett.203. 69-71 (1996)

[文献書誌] Takayama,C.: "Developmental changes in expression and distribution of the glutamate receptor channel δ2 subunit according to the Purkinje cell maturation." Dev.Brain Res.92. 147-155 (1996)

[文献書誌] Takahashi,T.: "Functional correlation of NMDA receptor ε subunits expression with the properties of single-channel and synaptic currents in the developing cerebellum." J.Neurosci.16. 4376-4382 (1996)

[文献書誌] Asada,H.: "Mice lacking the 65 kDa isoform of glutamic acid decarboxylase(GAD65)maintain normal levels of GAD67 and GABA in their brain but are susceptible to seizures." Biochemical and Biophysical Research Communications. 229. 891-895 (1996)

[文献書誌] Miyakawa,T.: "Susceptibility of drug-induced seizures of Fyn tyrosine kinase deficient mice." NeuroReport. 7. 2723-2726 (1996)

[文献書誌] Miyakawa,T.: "Radial maze performance,open-field and elevated plus-maze behaviors in Fyn-kinase deficient mice:Further evidence for increased fearfulness." Mol.Brain Res.37. 145-150 (1996)

[文献書誌] Nakagawa,S.: "Altered gene expression of the N-methyl-D-aspartate receptor channel subunits in Purkinje cells of the staggerer mutant mouse." Eur.J.Neurosci.8. 2644-2651 (1996)

[文献書誌] Yamada,K.: "EAAT4 is a post-synaptic glutamate transporter at Purkinje cell synapses." Neuroreport. 7. 2013-2017 (1996)

[文献書誌] Nakagawa,S.: "Regional variation in expressions of calbindin and inositol 1,4,5-trisphosph ate receptor typel mRNAs in the cerebellum of the staggerer mutant mouse." Eur.J.Neurosci.8. 1401-1407 (1996)

[文献書誌] Shibata,T.: "Dynamic changes in expression of glutamate transporter mRNAs in developing brain." Neuroreport. 7. 705-709 (1996)

[文献書誌] Hori,Y.: "Long-lasting facilitation induced by serotonin in superficial dorsal horn neurones of rat spinal cord." J.Physiol.492. 867-876 (1996)

[文献書誌] Silver,R.A.: "Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites." J.Physiol.494. 231-250 (1996)

[文献書誌] Yokoi,M.: "Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2." Science. 273. 645-647 (1996)

[文献書誌] Kobayashi,K.: "Presynaptic long-term depression at the hippocampal mossy fiber-CA3 synapse." Science. 273. 648-650 (1996)

[文献書誌] Takahashi,T.: "Presynaptic calcium current modulation by a glutamate receptor." Science. 274. 594-597 (1996)

[文献書誌] Tempia,F.: "The fractional calcium current through neuronal AMPA receptor channels with a ′low′calcium permeability." J.Neurosci.15. 456-466 (1996)

[文献書誌] Kano,M.: "Calcium-induced rebound potentiation of GABA-mediated currents is mediated by calcium/calmodulin dependent kinase II." Proc.Natl.Acad.Sci.USA.93. 13351-13356 (1996)

[文献書誌] Uchino,S.: "Inducible expression of N-methy-D-aspartate(NMDA)receptor channels from cloned cDNAs in CHO cells." Mol.Brain Res.(in press.).

[文献書誌] Uchino,S.: "Analysis of the glycine binding domain of the NMDA receptor channel ζ1 subunit." NeuroReport. (in press.).

[文献書誌] Ito,I.: "Synapse-selective impairment of NMDA receptor functions in mice lacking NMDA receptor ε1 or ε2 subunit." J.Physiol.(in press.).

[文献書誌] Kai,N.: "Molecular cloning of Fyn-associated molecules in the mouse central nervous system." J.Neurosci.Res.(in press.).

[文献書誌] Minami,T.: "Absence of prostglandin E_2-induced hyperalgesia in NMDA receptor ε subunit knockout mice." Brit.J.Phamacal.(in press.).

[文献書誌] Hayashi,Y.: "Calcium and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the postsynaptic density." Mol.Brain Res.(in press.).

[文献書誌] Yamada K.: "Changes in expression and distribution of the glutamate transporter EAAT4 in developing mouse Purkinje cells." Neurosci.Res.(in press.).

[文献書誌] Kano,M.: "Persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking mGluR1." Neuron. (in press.).

[文献書誌] Kano,M.: "Long-lasting potentiation of GABA ergic inhibitory synaptic transmission in cerebellar Purkinje cells:its properties and possible mechanisms." Behav.Brain Sci.(in press.).

1996 年度実績報告書

三品昌美東京大学, 医学部, 教授 (80144351)