Focal labeling of axonal terminals with active synapses recorded by an extracellular macro-patch electrode

A technique to label active synaptic terminals, whose electrophysiology had been monitored by a macro-patch electrode, was developed for a crustacean neuromuscular preparation. The active synaptic terminals were labeled by release-dependent uptake of horseradish peroxidase (HRP) into synaptic vesicles. The focal labeling technique involved the following steps: (1) locating a site where evoked synaptic currents could be recorded at a subset of neuromuscular synapses by a macro-patch electrode; (2) reducing synaptic transmission by bathing the preparation in a solution containing low [Ca2+] and high [Mg2+]; (3) introducing HRP as an extracellular marker into the solution bathing the preparation; (4) restoring synaptic release focally by ejection of a solution containing Ca2+ from the macro-patch electrode. The muscle fibre with labeled synapses was fixed for electron microscopy and processed for HRP histochemistry. The distribution of HRP-labeled vesicles was documented by electron microscopy and semi-serial sectioning. A significant increase in labeled vesicles was found within a maximum radius of 10-15 micron from the lumen of the macro-path electrode. This maximum radius of labeling set an upper limit to the number of active synapses recorded by the macro-patch electrode.