A method of introducing large molecules into neurons.

在不破坏细胞膜的情况下，将某些标记分子或生物活性物质引入神经元是可取的。这项技术应该易于使用，目标应该是任何单个神经元。我们已经操纵了电穿孔技术，以便将其应用于单个神经元。我们已经使用了一个充满荧光染料的小针尖移液管，并将尖端压在细胞膜上。在移液器上轻轻施加负压，在移液器顶端吞下一片细胞膜，然后在移液器内施加0.1-1V的三角波形的峰间电压脉冲。这些电压脉冲给神经元注入了荧光染料。3K到10K的分子被成功地导入，这些神经元在接下来的几天里存活了下来。然而，我们不能向神经元中引入更大的分子，如质粒DNA。这可能是由于神经膜的脆性，因为我们可以通过这种技术将相同的质粒DNA导入cos-7。由于微电穿孔后神经元的存活率较高(最高可达50%的过夜存活率)，我们仍可应用这一技术在体外制备中标记神经元的通路。通过标记神经束，应该更容易从特定的突触后神经元中找到和记录。虽然这项技术有大小的限制，但在神经元中引入一些活性物质也应该是有用的，以便于分析突触功能。

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.