Recruitment of synapses in the neurosecretory process during long-term facilitation at the lobster neuromuscular junction.

We investigated long-term facilitation at the lobster neuromuscular synapse employing a combination of FM1-43 staining of synaptic vesicles, electron microscopy analysis, and electrical recordings of synaptic activity. Synaptic terminals were loaded with the fluorescent dye FM1-43 producing clusters of activity-dependent fluorescent spots. Electron microscopy analysis of synaptic ultrastructure suggested that fluorescent spots represent compartments of synaptic terminals filled with vesicles. Excitatory postsynaptic currents were recorded from the stained synaptic terminals using focal macropatch electrodes. Terminals were stained during the nerve stimulation at a low stimulation frequency (2, 5 or 10 Hz) before and after long-term facilitation was elicited by high-frequency stimulation (20 or 30 Hz for 5 min). We found that staining after long-term facilitation results in the appearance of new fluorescent spots, as well as in the increase in fluorescence of the spots that appeared before long-term facilitation. This increase in fluorescence accounted for the increase in quantal release. Activation of individual fluorescent spots was found to be non-uniform. In spite of overall increase in fluorescence, some synaptic compartments decreased their staining after long-term facilitation. Thus, our study demonstrates that long-term facilitation produces non-uniform activation of FM1-43 uptake in synaptic compartments that correlates with the increase in quantal neurosecretion.