Vital imaging and ultrastructural analysis of individual axon terminals labeled by iontophoretic application of lipophilic dye

We describe a method for in vivo confocal fluorescence imaging of synaptic terminals and subsequent electron microscopic reconstructions of the same t erminals. By iontophoretically applying lipophilic dye to nerve terminals a t a single neuromuscular junction with a sharp microelectrode in living neo natal mice, we were able to quickly label other synaptic terminals of the s ame motor unit. This vital labeling technique allows the same synapses to b e imaged in living animals for several days. By using two dyes applied to s eparate junctions we could visualize competing axons converging at the same site. We also show that similar approaches can be used to study synaptic i nputs to neurons. Following photoconversion, the dye labeled axons and syna pses were easily identified and distinguished from unlabeled synapses of ot her axons ultrastructurally. This new labeling technique thus provides a us eful means to study reorganization of synaptic structure at high temporal a nd spatial resolution. (C) 1999 Elsevier Science B.V. All rights reserved.