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

Lead Research Organisation: University of Leicester
Department Name: Cell Physiology and Pharmacology


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Technical Summary

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.


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Ruenraroengsak P (2007) Cell uptake, cytoplasmic diffusion and nuclear access of a 6.5 nm diameter dendrimer. in International journal of pharmaceutics

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Zhu L (2007) The effects of fear conditioning on cerebellar LTP and LTD. in The European journal of neuroscience

Description We developed a series of fluorescent protein based sensors that can be used to monitor communication between excitable cells in the brain. Through this grant, we we were able to generate a transgenic mouse that has been instrumental in helping us to understand more about brain function in relation to healthy ageing and obtain further grant funding in this area
Exploitation Route Our sensors are available for use by others. We have undertaken a number of collaborations with others who have been interested in our mouse models
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description This grant was started at the School of Pharmacy (BB/C508377/1) and completed in Leicester when i moved in 2007. The work was to produce a new ratiometric pH sensor for monitoring and quantifying transmitter release in neurones. This project stimulated a significant part of my subsequent research into using pairs of fluorescent proteins to quantify either transmitter release, calcium concentrations in specific cellular compartments or receptors on the surface of cells. By developing these sensors, they are being used by us and other groups around the world to address aspects of synaptic signalling. For one of these sensors, we have created a transgenic mouse that allows dynamic measurements of synaptic activity in living brain tissue. This is hugely exciting and we hope that it will be of great use to the bioscience community
First Year Of Impact 2006
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic