Visible evidence for differences in synaptic effectiveness with activity-dependent vesicular uptake and release of FM1-43

Activity-dependent uptake and release of the fluorescent probe FM 1-43 were used to compare synaptic performance (rates of transmitter release and syn aptic vesicle turnover) at different frequencies in phasic and tonic motor neurons innervating the crayfish leg extensor muscle and in the tonic motor neuron of the opener muscle. The phasic extensor motor neuron, which has a high quantal content of transmitter release, accumulated and released FM1- 43 more rapidly than the tonic motor neuron, especially at low frequencies of stimulation. Individual bright spots appeared on the varicosities of the junctional terminals during stimulation in FM1-43; these spots corresponde d to zones of immunostaining for the synaptic Vesicle associated protein sy naptotagmin, but they were larger and less numerous than synapses identifie d by electron microscopy and appear to represent one to several synapses wi th their associated clusters of synaptic vesicles. The number of bright spo ts observed on varicosities of the tonic terminal after stimulation at grea ter than or equal to 20 Hz is generally similar to values for responding un its (n) calculated from binomial distributions derived from quantal analysi s. At frequencies of less than or equal to 10 Wt, bright spots did not usua lly appear on tonic extensor varicosities, and the quantal release patterns were best fitted with Poisson distributions. Another tonic motor neuron, t he exciter of the opener muscle, showed individual bright spots at lower fr equencies of stimulation, consistent with its higher quantal output at thes e frequencies and corresponding with the binomial fits for quantal release distributions. In this axon, the number of distinctive bright spots increas ed with frequency in the 2- to 20-Hz range, indicating increased participat ion of synapses during frequency facilitation. In the tonic extensor neuron terminals, the brightness and the size of the individual spots increased w ith frequency, and new foci of dye uptake appeared at the edges of preexist ing spots. Relative intensity change varied considerably among individual s pots during dye loading at different frequencies. Similarly, individual spo ts on a single tonic terminal destained at different rates when stimulated after previous loading with FM1-43. These results suggest differential perf ormance of individual synapses or small groups of synapses, some being more effective in transmitter release than others, as inferred from previous ul trastructural and quantal analysis studies. The large overall differences b etween phasic and tonic synapses suggest differential regulation of transmi tter release at individual synapses in the two neurons.