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

• Project/Area Number: 13877365
• Research Category: Grant-in-Aid for Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Physical pharmacy
• Research Institution: The University of Tokyo
• Principal Investigator: 桐野 豊 東京大学, 大学院・薬学系研究科, 教授 (10012668)
• Co-Investigator(Kenkyū-buntansha): 渡辺 恵 東京大学, 大学院・薬学系研究科, 助手 (80302610)
• Project Period (FY): 2001 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 2002: ¥900,000 (Direct Cost: ¥900,000); Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
• Keywords: 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

Research Abstract

ナメクジの嗅覚中枢である脳神経節前脳には多数の神経細胞が存在し,これらが自発的に膜電位の同期振動を生じている.この自発活動は脳神経節の単離後も長時間安定して持続することから,さまざまな測定法のスクリーニングに適した実験系である.これまでの研究により,市販の膜電位感受性色素di-4-ANEPPSによって前脳全体で同期した周期的蛍光変化が記録されることが明らかになった.この蛍光変化は1%程度であり,生理的状態の神経細胞が示す蛍光変化としては極めて大きく,また安定して長時間記録できることから,本実験系は膜電位感受性色素のスクリーニングに適していることがわかる.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を用いて脳神経節を染色し,一次スクリーニングとして染色の状態と自発的蛍光変化の有無を調べた結果,クマリン系色素が強く脳組織を染色することが示されたが,前脳の膜電位振動に伴う蛍光変化を示す色素は存在しなかった.これは用いたクマリン系色素が極性基を持たず,細胞膜内での配向が一定しないためではないかと考え,これにスルホン酸基を付加した色素を合成した.これを用いて脳神経節を染色すると,脳組織は同様に強く染色されたが,やはり膜電位変化を反映した蛍光変化は得られなかった.本研究の結果,ナメクジの単離脳標本が膜電位感受性色素のスクリーニングに有用であること,膜電位感受性色素として機能するためには少なくとも色素の細胞膜への移行と,細胞膜内での配向という2つの要因が重要であること,またリポソームの実験で膜電位応答性のある色素が必ずしも生体で膜電位応答性を示とは限らず,生体を用いたスクリーニングが必要であることが明らかになった.

Research Products

• [Publications] Kanako Takatsuki et al.: "Scopolamine impairs eyeblink conditioning in the cerebellar LTD-deficient mice"NeuroReport. 13. 159-162 (2002)
• [Publications] Kanako Takatsuki et al.: "Hippocampal damage disrupts eyeblink conditioning in mice lacking glutamate receptor subunit δ2"J.Biol.Phys.. 28. 539-547 (2002)
• [Publications] Yasuko Inokuma et al.: "Two Types of Network Oscillations and Their Odor Responses in the Primary Olfactory Center of a Terrestrial Mollusk"J.Neurophysiol.. 87. 3160-3164 (2002)
• [Publications] Kaori Takehara et al.: "Time-limited role of the hippocampus in the memory for trace eyeblink conditioning in mice"Brain Res.. 951. 183-190 (2002)
• [Publications] Sadaharu Kotani et al.: "Classical eyeblink conditioning in the decerebrate guinea pigs"Eur.J.Neurosci.. 15. 1267-1270 (2002)
• [Publications] Hidenori Taru et al.: "Interaction of Alzheimer's β-amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade"J.Biol.Chem.. 277. 20070-20078 (2002)
• [Publications] Hidenori Taru et al.: "Differential roles of JIP scaffold proteins in the modulation of amyloid precursor protein metabolism"J.Biol.Chem.. 277. 27567-27574 (2002)
• [Publications] Sotaro Shibayama et al.: "Identification of a C-terminal region that is required for the nuclear translocation of ERK2 by passive diffusion"J.Biol.Chem.. 277. 37777-37782 (2002)
• [Publications] Ryota Matsuo et al.: "Delayed-onset amnesia caused by protein synthesis inhibition in odor-taste associative memory of the terrestrial slug Limax valentianus"Nurosci.Lett.. 334. 201-205 (2002)
• [Publications] Yasushi Kishimoto et al.: "mGluR1 in cerebellar Purkinje cells is required for normal association of temporally contiguous stimuli in classical conditioning"Eur.J.Neurosci.. 16. 2416-2424 (2002)
• [Publications] Satoshi Shimozono et al.: "Confocal imaging of subcellular Ca^<2+> concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein"Sci.STKE. 2002(125). 14 (2002)
• [Publications] Kanako Takatsuki et al.: "The hippocampus plays an important role in eyeblink conditioning with a short trace interval in glutamate receptor subunit δ2 mutant mice"J.Neurosci. 23. 17-22 (2003)
• [Publications] 桐野 豊: "瞬目反射連合学習を担う脳の領域"パリティ. 18. 52-53 (2003)
• [Publications] 渡辺 恵, 桐野 豊: "ナメクジの匂い学習のメカニズム"Aroma Research. (in press).
• [Publications] Sadaharu Kotani et al.: "Trace eyeblink conditioning in decerebrate guinea pigs"Eur.J.Neurisci.. (in press).
• [Publications] Satoshi Watanabe et al.: "Contribution of excitatory chloride conductance in the determination of the direction of traveling waves in an olfactory center"J. Neurosci. (in press).
• [Publications] 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)
• [Publications] 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)
• [Publications] 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)
• [Publications] 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)
• [Publications] 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)
• [Publications] 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)
• [Publications] 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)
• [Publications] Kanako Takatsuki, et al.: "Effects of the noncompetitive NMDA receptor antagonist MK-801 on classical eyeblink conditioning in mice"Neurophamacol. 41. 618-628 (2001)
• [Publications] 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)
• [Publications] Yasushi Kishimoto, et al.: "Impaired delay but normal trace eyeblink conditioning in PLCβ4 mutant mice"Neuroreport. 12. 2919-2922 (2001)
• [Publications] Yasushi Kishimoto, et al.: "Age-dependent impairment of delay and trace eyeblink conditioning in mice"Neuroreport. 12. 3349-3352 (2001)
• [Publications] 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)
• [Publications] 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)
• [Publications] Satoshi Shimozono, et al.: "Identification and characterization of an output neuron from the oscillatory molluscan olfactory network"Brain Res.. 921. 98-105 (2001)
• [Publications] 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)
• [Publications] Kanako Takatsuki, et al.: "Scopolamine impairs eyeblink conditioning in the cerebellar LTD- deficient mice"NeuroReport. 13. 159-162 (2002)
• [Publications] Kanako Takatsuki, et al.: "Hippocampal damage disrupts eyeblink conditioning in mice lacking glutamate receptor subunit δ2"J. Biol. Phys.. (in press). (2002)
• [Publications] 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)
• [Publications] 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)