1999 Fiscal Year Final Research Report Summary
A method of introducing large molecules into neurons.
| Project/Area Number |
09558103
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
神経・脳内生理学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
OHMORI Harunori Kyoto Univ. Dept. Physiology, Professor, 医学研究科, 教授 (30126015)
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| Co-Investigator(Kenkyū-buntansha) |
KOYANO Konomi Kyoto Univ. Dept. Physiology, Lecturer, 医学研究科, 講師 (50183041)
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| Project Period (FY) |
1997 – 1999
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| Keywords | neural network / electroporation / synapse / molowlnr qite / macromolecule / voltage pulse |
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| Research Abstract |
It is desirable to introduce some marker molecules or biological active substances into neurons without any damage to the cell membrane. The technique should be easy to use and the target should be any individual neurons. We have manipulated the electroporation technique in order to apply it to a single neuron. We have utilized a small tip pipette filled with fluorescent dyes and pressed the tip against the cell membrane. With gentle application of negative pressure to the pipette a patch of cell membrane was swallowed into the pipette tip and then some voltage pulses of 0.1-1 Volt peak to peak of triangular waveform were applied within the pipette. These voltage pulses introduced neurons with fluorescence dyes. Molecules of 3k to 10k were successfully introduced and these neurons survived for next few days. However, we could not introduce much larger molecules into neurons such as plasmid DNAs. This is probably due to the fragility of neural membrane because we could introduce the same plasmid DNA into cos-7 by this technique. Since survival rate of neurons after microelectroporation was relatively high (maximum 50% overnight survival), we could still apply this technique in order to mark the neuronal pathway in the in vitro preparations. By marking neural tracts it should be easier to find and to record from the specific postsynaptic neurons. Although there are size limitation to this technique, it should also be useful to introduce some active substances into neurons to facilitate analyses of synaptic functions.
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