Single Vesicle Recording in Hippocampal ‘Xenapses’ Reveals Diffusional Dispersion of SV Proteins After Fusion

In order to maintain neuronal transmission after exocytosis of synaptic vesicles (SVs), the vesicular proteins have to be cleared away from the active zone. Until now it remained controversial whether SV components remain clustered during translocation from sites of exocytosis or disperse by free diffusion. To adress this question, we developed a novel purely presynaptic neuronal preparation which enables single vesicle recording by TIRFM.Using click-chemistry we functionalized micropatterned coverglasses with protein domains of synaptic cell adhesion molecules, serving as artificial postsynapses. On these host substrates purely presynaptic boutons form ‘en face’ directly onto the coverslip, termed ‘xenapses’. Serial section TEM as well as focused-ion-beam SEM showed that xenapses contain a few hundred SVs, many of them docked in several clusters at the bottom membrane. 4Pi and TIRF-STORM confirmed the existence of several active zones. Thus, xenapses offer the unique opportunity to record exocytosis of single vesicles by TIRFM. Using fusion constructs of the pH-sensitive pHluorin, single fusion events were visible as diffraction-limited spots on stimulation with single action potentials. We could localize fusion events synchronous to action potentials with ∼20 nm precision and follow the fate of released SV proteins. We observed diffusional dispersion of vesicular proteins post fusion with diffusion constants in the range of 0.1 - 1 µm²/s. Thus, our results point to free diffusion as mechanism for fast clearance.