Optical Monitoring of Individual Release Sites Tests a New Mechanism for Synaptic Depression

Abstract

We recently used paired motoneuron muscle recordings in zebrafish to explore frequency-dependent synaptic depression. Most models for synaptic depression focus on vesicle heterogeneity. However, our variance analysis of steady state endplate current (EPC) amplitudes has turned up a new mechanism that assigns depression to the dropout of a release site class with ultra-slow vesicle reloading rates. This assignment was made on the basis of 1) high release probability of all release sites at low frequency; 2) low variance of EPC amplitude at steady state transmission, suggesting that only a subset of release sites can sustain fast release at high frequencies; 3) greater than 60 fold difference in vesicle reloading rate of for the two classes of release sites. As a direct test of these processes, we generated a fish line where the calcium indicator GCaMP6f was fused to postsynaptic rapsyn to optically monitor the activity of postsynaptic ACh receptors. Both the density and calcium permeability of the receptors provide detection of single release events. Because the ∼15 synaptic boutons are physically separated along the length of the muscle, each can be individually monitored for optical response to a motoneuron action potential. Data thus far indicates that stimulus frequencies that produce no depression (e.g., 0.2 Hz) show faithful GCaMP6f fluorescence responses to each stimulus. Stimulation at frequencies associated with synaptic depression has been more difficult to analyze due to the slow kinetics of GCaMP6f. However, preliminary recordings obtained at 10 Hz revealed all synapses responding to the first stimulation and with select synapses consistently dropping out during the stimulus train. These results are consistent with two classes of active sites bearing distinct release kinetics underlying frequency-dependent depression. Firm conclusions require further testing at high frequencies.