2001 年度実績報告書

新規高感度膜電位感受性色素の探索と細胞内染色法の開発

研究課題

研究課題/領域番号	13877365
研究種目	萌芽的研究
研究機関	東京大学
研究代表者	桐野豊東京大学, 大学院・薬学系研究科, 教授 (10012668)
研究分担者	渡辺恵東京大学, 大学院・薬学系研究科, 助手 (80302610)
キーワード	膜電位感受性色素 / 光学測定 / 脳神経節 / ナメクジ / 軟体動物 / 嗅覚中枢 / di-4-ANEPPS / クマリン
研究概要	ナメクジ脳神経節前脳では多数の神経細胞が同期して自発的に約1Hzで膜電位振動を生じており,単離後も長時間にわたって活性を維持することから光学測定に適した材料である.従ってナメクジ脳神経節を用いることにより,神経組織における膜電位感受性色素の評価を行うことが可能であると考えられる.はじめにポジティブコントロールとして,既に膜電位の光学測定に用いられている色素であるdi-4-ANEPPSを用いた.ナメクジ脳神経節をdi-4-ANEPPS(0,005%)で40-60分間染色後,高速CCDカメラを用いて測定すると,前脳の全体で同期した周期的蛍光変化が生じた.これは局所場電位や,パッチクランプ法で記録される前脳神経細胞の膜電位の振動と同期していたことから,神経細胞の膜電位を反映したものであることが示された.このときの蛍光変化率は0.5%程度であった.そこで次に,リポソーム-バリノマイシン系による予備実験で膜電位依存性を持つことが示されたクマリン系およびロドシアニン系の色素(計9種類)を用いて同様に脳神経節を染色し,染色の状態と自発的蛍光変化の有無を調べた.その結果,クマリン系色素では,神経節全体が一様に強く染色されたが,自発的蛍光変化は観測されなかった.一方ロドシアニン系色素では,神経節組織の染色が全般的に弱く,自発的蛍光変化も観測されなかった.本研究の結果から,染色性に優れたクマリン系色素について極性基の配置を再検討した誘導体を作成して用いることより,膜電位測定ができる可能性があると考えられた.

研究成果
(19件)

すべてその他

すべて文献書誌 (19件)

[文献書誌] Tadashi Nakaya, et al.: "Expression and Function of a Novel Gene in Odor-taste Associative Learning in the Terrestrial Slug"Genes Cells. 6. 43-56 (2001)
[文献書誌] Yasushi Kishimoto, et al.: "Long-trace Interval Eyeblink Conditioning Is Impaired in Mutant Mice Lacking NMDA Receptor Subunit ε1"Eur. J. Neurosci.. 13. 1221-1227 (2001)
[文献書誌] Yasushi Kishimoto, et al.: "Classical Eyeblink Conditioning in Glutamate Receptor Subunitδ2 Mutant Mice Is Impaired in Delay Paradigm But Not in Trace Paradigm"Eur. J. Neurosci.. 13. 1249-1253 (2001)
[文献書誌] Satoshi Watanabe, et al.: "Modulation of Oscillatory Neural Activities by Cholinergic Activation of Interneurons in the Olfactory Center of a Terrestrial Slug"Brain Res.. 896. 30-35 (2001)
[文献書誌] Kazuyasu Shindo, et al.: "Solution Structure of Micelle-bound H5 Peptide(427-452): A Primary Structure Corresponding to the Pore-Forming Region of the Voltage Dependent Potassium Channel"Biochim. Biophys. Acta.. 1545. 153-159 (2001)
[文献書誌] Yasushi Satoh, et al.: "β-Amyloid peptides inhibit acetylcholine release from cholinergic presynaptic nerve endings isolated from an electric ray"Neurosci. Lett.. 302. 97-100 (2001)
[文献書誌] Tsuyoshi Inoue, et al.: "Serotonin and NO Complementarily Regulate Generation of Oscillatory Activity in the Olfactory CNS of a Terrestrial Mollusk"J. Neurophysiol.. 85. 2634-2638 (2001)
[文献書誌] Kanako Takatsuki, et al.: "Effects of the noncompetitive NMDA receptor antagonist MK-801 on classical eyeblink conditioning in mice"Neurophamacol. 41. 618-628 (2001)
[文献書誌] Kanae Ando, et al.: "Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of β-amyloid"J. Biol. Chem.. 276. 40353-40361 (2001)
[文献書誌] Yasushi Kishimoto, et al.: "Impaired delay but normal trace eyeblink conditioning in PLCβ4 mutant mice"Neuroreport. 12. 2919-2922 (2001)
[文献書誌] Yasushi Kishimoto, et al.: "Age-dependent impairment of delay and trace eyeblink conditioning in mice"Neuroreport. 12. 3349-3352 (2001)
[文献書誌] Yasushi Kishimoto, et al.: "Impairment of eyeblink conditioning in GluRδ2 mutant mice depends on the temporal overlap between conditioned and unconditioned stimuli"Fur. J. Neurosci.. 14. 1515-1521 (2001)
[文献書誌] Moritoshi Hirono, et al.: "Phospholipase Cβ4 and Protein Kinase Cα and/or Protein Kinase CβI Are Involved in the Induction of Long-Term Depression in Cerebellar Purkinje Cells"J. Biol. Chem.. 276. 45236-45242 (2001)
[文献書誌] Satoshi Shimozono, et al.: "Identification and characterization of an output neuron from the oscillatory molluscan olfactory network"Brain Res.. 921. 98-105 (2001)
[文献書誌] Yoshinori Muto, et al.: "Blepharismin, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes"FEBS Lett. 508. 423-426 (2001)
[文献書誌] Kanako Takatsuki, et al.: "Scopolamine impairs eyeblink conditioning in the cerebellar LTD- deficient mice"NeuroReport. 13. 159-162 (2002)
[文献書誌] Kanako Takatsuki, et al.: "Hippocampal damage disrupts eyeblink conditioning in mice lacking glutamate receptor subunit δ2"J. Biol. Phys.. (in press). (2002)
[文献書誌] Yasuko Inokuma, et al.: "Two Types of Network Oscillations and Their Odor Responses in the Primary Olfactory Center of a Terrestrial Mollusk"J. Neurophysiol.. (in press). (2002)
[文献書誌] 桐野豊: ""働く脳と変化する脳"「新生物物理の最前線」生物物理学会編,講談社ブルーバックス,pp.197-237"講談社. 203 (2001)

2001 年度 実績報告書

新規高感度膜電位感受性色素の探索と細胞内染色法の開発

研究代表者

桐野 豊 東京大学, 大学院・薬学系研究科, 教授 (10012668)

研究成果

[文献書誌] Tadashi Nakaya, et al.: "Expression and Function of a Novel Gene in Odor-taste Associative Learning in the Terrestrial Slug"Genes Cells. 6. 43-56 (2001)

[文献書誌] Yasushi Kishimoto, et al.: "Long-trace Interval Eyeblink Conditioning Is Impaired in Mutant Mice Lacking NMDA Receptor Subunit ε1"Eur. J. Neurosci.. 13. 1221-1227 (2001)

[文献書誌] Yasushi Kishimoto, et al.: "Classical Eyeblink Conditioning in Glutamate Receptor Subunitδ2 Mutant Mice Is Impaired in Delay Paradigm But Not in Trace Paradigm"Eur. J. Neurosci.. 13. 1249-1253 (2001)

[文献書誌] Satoshi Watanabe, et al.: "Modulation of Oscillatory Neural Activities by Cholinergic Activation of Interneurons in the Olfactory Center of a Terrestrial Slug"Brain Res.. 896. 30-35 (2001)

[文献書誌] Kazuyasu Shindo, et al.: "Solution Structure of Micelle-bound H5 Peptide(427-452): A Primary Structure Corresponding to the Pore-Forming Region of the Voltage Dependent Potassium Channel"Biochim. Biophys. Acta.. 1545. 153-159 (2001)

[文献書誌] Yasushi Satoh, et al.: "β-Amyloid peptides inhibit acetylcholine release from cholinergic presynaptic nerve endings isolated from an electric ray"Neurosci. Lett.. 302. 97-100 (2001)

[文献書誌] Tsuyoshi Inoue, et al.: "Serotonin and NO Complementarily Regulate Generation of Oscillatory Activity in the Olfactory CNS of a Terrestrial Mollusk"J. Neurophysiol.. 85. 2634-2638 (2001)

[文献書誌] Kanako Takatsuki, et al.: "Effects of the noncompetitive NMDA receptor antagonist MK-801 on classical eyeblink conditioning in mice"Neurophamacol. 41. 618-628 (2001)

[文献書誌] Kanae Ando, et al.: "Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of β-amyloid"J. Biol. Chem.. 276. 40353-40361 (2001)

[文献書誌] Yasushi Kishimoto, et al.: "Impaired delay but normal trace eyeblink conditioning in PLCβ4 mutant mice"Neuroreport. 12. 2919-2922 (2001)

[文献書誌] Yasushi Kishimoto, et al.: "Age-dependent impairment of delay and trace eyeblink conditioning in mice"Neuroreport. 12. 3349-3352 (2001)

[文献書誌] Yasushi Kishimoto, et al.: "Impairment of eyeblink conditioning in GluRδ2 mutant mice depends on the temporal overlap between conditioned and unconditioned stimuli"Fur. J. Neurosci.. 14. 1515-1521 (2001)

[文献書誌] Moritoshi Hirono, et al.: "Phospholipase Cβ4 and Protein Kinase Cα and/or Protein Kinase CβI Are Involved in the Induction of Long-Term Depression in Cerebellar Purkinje Cells"J. Biol. Chem.. 276. 45236-45242 (2001)

[文献書誌] Satoshi Shimozono, et al.: "Identification and characterization of an output neuron from the oscillatory molluscan olfactory network"Brain Res.. 921. 98-105 (2001)

[文献書誌] Yoshinori Muto, et al.: "Blepharismin, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes"FEBS Lett. 508. 423-426 (2001)

[文献書誌] Kanako Takatsuki, et al.: "Scopolamine impairs eyeblink conditioning in the cerebellar LTD- deficient mice"NeuroReport. 13. 159-162 (2002)

[文献書誌] Kanako Takatsuki, et al.: "Hippocampal damage disrupts eyeblink conditioning in mice lacking glutamate receptor subunit δ2"J. Biol. Phys.. (in press). (2002)

[文献書誌] Yasuko Inokuma, et al.: "Two Types of Network Oscillations and Their Odor Responses in the Primary Olfactory Center of a Terrestrial Mollusk"J. Neurophysiol.. (in press). (2002)

[文献書誌] 桐野 豊: ""働く脳と変化する脳"「新生物物理の最前線」生物物理学会編,講談社ブルーバックス,pp.197-237"講談社. 203 (2001)

2001 年度実績報告書

桐野豊東京大学, 大学院・薬学系研究科, 教授 (10012668)

[文献書誌] 桐野豊: ""働く脳と変化する脳"「新生物物理の最前線」生物物理学会編,講談社ブルーバックス,pp.197-237"講談社. 203 (2001)