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Nonequivalent release sites govern synaptic depression

Significance Activity-dependent depression is a common aspect of synaptic plasticity resulting from an inability to recover full-release competency during bouts of high-frequency stimulation. At the zebrafish neuromuscular synapse, frequencies between 1 and 10 Hz lead to 50% depression, but paradoxically, this synapse can still follow frequencies approaching the refractory period of the action potential without failure. Capitalizing on the advantages offered by this synapse, we report a new mechanism causal to nonlinear dependence of steady-state depression on frequency involving two types of release sites differing in vesicle reloading rates. The slow sites cannot sustain vesicle release at high frequencies, and therefore, it is the number of fast reloading sites that determines the steady-state level of transmission associated with depression. Synaptic depression is prominent among synapses, but the underlying mechanisms remain uncertain. Here, we use paired patch clamp recording to study neuromuscular transmission between the caudal primary motor neuron and target skeletal muscle in zebrafish. This synapse has an unusually low number of release sites, all with high probabilities of release in response to low-frequency stimulation. During high-frequency stimulation, the synapse undergoes short-term depression and reaches steady-state levels of transmission that sustain the swimming behavior. To determine the release parameters underlying this steady state, we applied variance analysis. Our analysis revealed two functionally distinct subclasses of release sites differing by over 60-fold in rates of vesicle reloading. A slow reloading class requires seconds to recover and contributes to depression onset but not the steady-state transmission. By contrast, a fast reloading class recovers within tens of milliseconds and is solely responsible for steady-state transmission. Thus, in contrast to most current models that assign levels of steady-state depression to vesicle availability, our findings instead assign this function to nonuniform release site kinetics. The duality of active-site properties accounts for the highly nonlinear dependence of steady-state depression levels on frequency.