Tracking newly released synaptic vesicle proteins at ribbon active zones

Summary Clearance of synaptic vesicle proteins from active zones may be rate limiting for sustained neurotransmission. Issues of clearance are critical at ribbon synapses, which continually release neurotransmitter for prolonged periods of time. We used synaptophysin-pHluorin (SypHy) to visualize protein clearance from active zones in retinal bipolar cell ribbon synapses. Depolarizing voltage steps gave rise to small step-like changes in fluorescence likely indicating release of single SypHy molecules from fused synaptic vesicle near active zones. Temporal and spatial fluorescence profiles of individual responses were highly variable, but ensemble averages were well-fit by clearance via free-diffusion using Monte Carlo simulations. The rate of fluorescence decay of ensemble averages varied with the time and location of the fusion event, with clearance being most rapid at the onset of a stimulus when release rate is highest.