"The mechanism of neurotransmitter release is a fundamental aspect of nervous system function, but it is still highly controversial. There is overwhelming evidence that vesicles can release their contents by fully collapsing into the cell surface, after which they are retrieved slowly (10-20 s) by formation of a clathrin cage around the membrane. But it has also been suggested that there is a second mode of exocytosis in which vesicular content is released through a transitory fusion pore and then quickly retrieved (“kiss-and-run”). Several lines of evidence indicate that a fast mechanism of endocytosis (~1 s) does indeed operate at some synapses, but the idea that the vesicle does not merge with the surface membane remains controversial. We will investigate synaptic vesicle endocytosis in two types of retinal synapse (conventional and ribbon-type), using genetically modified zebrafish expressing fluorescent fusion proteins that report synaptic vesicle fusion and retrieval. Neurons will be isolated and total internal reflection fluorescence microscopy (TIRFM) used to monitor the synaptic vesicle cycle in real-time at the level of individual vesicles. A key aim of this project will be to establish the role of fast and slow endocytosis during both spontaneous vesicle fusion and ongoing synaptic activity and identify proteins recruited to the membrane during these processes (including clathrin, dynamin and endophilin)."