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首页> 外文期刊>Journal of Neuroscience Methods >Focal macromolecule delivery in neuronal tissue using simultaneous pressure ejection and local electroporation.
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Focal macromolecule delivery in neuronal tissue using simultaneous pressure ejection and local electroporation.

机译：使用同时的压力喷射和局部电穿孔在神经元组织中进行局部大分子递送。

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Electroporation creates transient pores in the plasma membrane to introduce macromolecules within a cell or cell population. Generally, electrical pulses are delivered between two electrodes separated from each other, making electroporation less likely to be localised. We have developed a new device combining local pressure ejection with local electroporation through a double-barrelled glass micropipette to transfer impermeable macromolecules in brain slices or in cultured HEK293 cells. The design achieves better targeting of the site of pressure ejection with that of electroporation. With this technique, we have been able to limit the delivery of propidium iodide or dextran amine within areas of 100-200mum diameter. We confirm that local electroporation is transient and show that when combined with pressure ejection, it allows local transfection of EGFP plasmids within HEK293 cells or within cerebellar and hippocampal slice cultures. We further show that local electroporation is less damaging when compared to global electroporation using two separate electrodes. Focal delivery of dextran amine dyes within trapezoid body fibres allowed tracing axonal tracts within brainstem slices, enabling the study of identified calyx of Held presynaptic terminals in living brain tissue. This labelling method can be used to target small nuclei in neuronal tissue and is generally applicable to the study of functional synaptic connectivity, or live axonal tracing in a variety of brain areas.
机译：电穿孔在质膜上产生瞬时孔，以将大分子引入细胞或细胞群内。通常，电脉冲在彼此分离的两个电极之间传递，从而使电穿孔难以定位。我们开发了一种新设备，该设备结合了通过双管式玻璃微量移液器进行局部压力喷射与局部电穿孔的功能，从而将不可渗透的大分子转移到脑切片或培养的HEK293细胞中。该设计实现了与电穿孔更好地对准压力喷射部位。通过这种技术，我们能够将碘化丙锭或右旋糖酐胺的输送限制在100-200μm直径的区域内。我们证实局部电穿孔是瞬时的，并显示当与压力喷射结合时，它允许HEK293细胞内或小脑和海马切片培养物中EGFP质粒的局部转染。我们进一步表明，与使用两个单独的电极进行整体电穿孔相比，局部电穿孔的损害较小。葡聚糖胺染料在梯形身体纤维内的局部递送允许在脑干切片内追踪轴突，从而使得能够研究在活的脑组织中Held突触前末端的花萼。这种标记方法可用于靶向神经元组织中的小核，通常可用于研究功能性突触连通性或各种脑区域的活轴突追踪。

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