Focal macromolecule delivery in neuronal tissue using simultaneous pressure ejection and local electroporation.
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 100-200 micrometer. 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.
Barker
Matthew
M
Department of Cell Physiology and Pharmacology, Leicester University, Medical Sciences Building, P.O. Box 138, University Road, Leicester LE1 9HN, UK.
Billups
Brian
B
Hamann
Martine
M
D000818:Animals; D001306:Auditory Pathways / Q000166:cytology; D001933:Brain Stem / Q000166:cytology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D004396:Coloring Agents; D003911:Dextrans; D004582:Electronics, Medical / Q000295:instrumentation / Q000379:methods; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D008839:Microelectrodes; D008845:Microinjections / Q000295:instrumentation / Q000379:methods*; D009445:Neuroanatomy / Q000295:instrumentation / Q000379:methods*; D009924:Organ Culture Techniques; D011312:Pressure; D017729:Presynaptic Terminals / Q000648:ultrastructure; D011419:Propidium; D051381:Rats; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*
D004396:Coloring Agents; D003911:Dextrans; D005456:Fluorescent Dyes; D011419:Propidium