Single synapse glutamate imaging reveals multiple levels of release mode regulation in mammalian synapses

Mammalian central synapses exhibit vast heterogeneity in signaling strength. To understand the extent of this diversity, how it is achieved, and its functional implications, characterization of a large number of individual synapses is required. Using glutamate imaging, we characterized the evoked release probability and spontaneous release frequency of over 24,000 individual synapses. We found striking variability and no correlation between action potential-evoked and spontaneous synaptic release strength, suggesting distinct regulatory mechanisms. Subpixel localization of individual evoked and spontaneous release events reveals tight spatial regulation of evoked release and enhanced spontaneous release outside of evoked release region. Using on-stage post hoc immune-labeling of vesicle-associated proteins, Ca²⁺-sensing proteins, and soluble presynaptic proteins we were able to show that distinct molecular ensembles are associated with evoked and spontaneous modes of synaptic release.