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.
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F W
FW
Department of Physiology, University of Toronto, Ont., Canada.
Marin
L
L
Atwood
H L
HL
D000818:Animals; D001369:Axons / Q000502:physiology* / Q000648:ultrastructure; D003386:Brachyura / Q000502:physiology*; D007124:Immunoenzyme Techniques; D008564:Membrane Potentials; D009469:Neuromuscular Junction / Q000502:physiology* / Q000648:ultrastructure; D013569:Synapses / Q000502:physiology* / Q000648:ultrastructure; D009435:Synaptic Transmission