A method of introducing large molecules into neurons.

希望将一些标记分子或生物活性物质引入神经元而不对细胞膜造成任何损伤。该技术应该易于使用，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。我们使用了一个充满荧光染料的小吸头吸管，并将吸头压在细胞膜上。随着对移液管轻轻施加负压，将一片细胞膜吞入移液管尖端，然后在移液管内施加三角波形的0.1-1伏峰到峰的电压脉冲。这些电压脉冲将荧光染料引入神经元。成功地引入了3 k至10 k的分子，这些神经元存活了几天。然而，我们不能将更大的分子引入神经元，如质粒DNA。这可能是由于神经膜的脆弱性，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相对较高（最高50%过夜存活），因此我们仍然可以应用该技术来标记体外制剂中的神经元通路。通过标记神经束，应该更容易找到和记录特定的突触后神经元。虽然这项技术有尺寸限制，但它也可以将一些活性物质引入神经元，以促进突触功能的分析。

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.