Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice.
We describe a method for filling presynaptic terminals and cell dendrites in adult brain slices with the fluorescent calcium indicator fura-2 by localized perfusion of the acetoxymethyl (AM) ester derivative. The method provides labeling selectivity, similar to that produced by intracellular microinjection of fura-2, with the simplicity of bath application of membrane-permeant AM esters. Application of the method to mossy fiber tracts in hippocampal region CA3 and parallel fiber tracts in cerebellum resulted in distant presynaptic terminals well labelled with fura-2 without concomitant postsynaptic labelling, allowing optical measurements of calcium concentration in individual presynaptic terminals. Application of the method to CA1 pyramidal cells produced intracellular loading of apical dendrites with fura-2. Dendritic calcium changes produced by afferent fiber stimulation were similar to those determined from cells filled with fura-2 by intracellular microinjection. The method appears to be general, and should provide a means to fill projecting axons and dendritic processes in many areas of the brain with fluorescent indicators, allowing optical measurements of ion concentration dynamics to be performed in brain slice that were previously impractical.
Regehr
W G
WG
Biophysics Research Department, AT & T Bell Laboratories, Murray Hill, NJ 07974.
Tank
D W
DW
D000818:Animals; D001921:Brain / Q000166:cytology*; D002118:Calcium / Q000378:metabolism; D005453:Fluorescence; D016257:Fura-2; D006168:Guinea Pigs; D006624:Hippocampus / Q000166:cytology; D066298:In Vitro Techniques; D009411:Nerve Endings / Q000187:drug effects*; D010477:Perfusion
D002118:Calcium; D016257:Fura-2