Use of a ratiometric pH sensor for the live imaging of transmitter release in the CNS

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In this proposal, we will develop a method of visualising transmitter release and vesicle cycling at actively releasing synapses and use it to investigate the mechanisms underlying differences in release properties and transmission characteristics at identified synapses in the central nervous system. A newly created, ratiometric, pH sensitive fluorescent protein construct (VFpH) will be fused to the luminally exposed C-terminus of vesicle associated protein-2 (VAMP-2/synaptobrevin-2). The lumen of non-releasing vesicles is at a low pH. On release, the lumen is briefly exposed to the more alkaline external environment. Since VFpH provides both a pH-insensitive and a pH-sensitive signal, this construct will allow us not only to visualise vesicles actively releasing transmitter but we will also be ale to estimate the proportions of vesicles that are releasing and those that are in the process of recycling. We will first characterise the VAMP-VFpH fusion construct in model cell systems that undergo exocytosis and then express it in cerebellar granule cells maintained in organotypic slice culture. We will then combine this method of imaging transmitter release with electrophysiological measurements of synaptic currents at single synapses to investigate the mechanisms that underpin previously identified differences in transmission properties at synapses formed by separate segments of granule cell axons with Purkinje cells.