Direct Observation Of Vesicle Transport On The Synaptic Ribbon Provides Evidence That Vesicles Are Mobilized And Prepared Rapidly For Release

Abstract

Synaptic ribbons are thought to provide vesicles for continuous synaptic transmission in some retinal non-spiking neurons, yet recent studies indicate that genetic removal of the ribbon has little effect on vesicle release kinetics. To investigate vesicle replenishment at synaptic ribbons, we imaged synaptic vesicles and ribbons in retinal bipolar cells with TIRF microscopy during stimulation with trains of 30-ms depolarizations. Analysis of vesicles released by the stimuli revealed that the vast majority of releasable vesicles reside within 300 nm of the ribbon center. A single 30-ms step to 0 mV was sufficient to deplete the most membrane-proximal vesicle pool, while triggering rapid stepwise movements of distal vesicles along the ribbon and toward the plasma membrane. Replenishment only becomes rate-limiting for recovery from paired-pulse depression for interstimulus intervals shorter than 250 ms. For longer interstimulus intervals, vesicle movement down the ribbon is fast enough to replenish released vesicles, but newly arrived vesicles are not release-ready. Notably, vesicle re-supply is 40-to 50-fold faster than previously measured in non-ribbon conventional synapses, whereas vesicle maturation rate is comparable. Moreover, in contrast to conventional synapses, vesicles docked at the base of the ribbon release with high fidelity. Lastly, our data show that with multiple stimuli, the delay in vesicle departure increases. Our results support a role for ribbons in the rapid supply and efficient preparation of vesicles for release, provide direct measurements of vesicle movement down the synaptic ribbon and suggest that multiple factors contribute to paired-pulse depression.