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High resolution single molecule imaging to determine the spatio-temporal rules of glutamate receptor trafficking during synaptic plasticity


The regulation of neurotransmitter receptors localization at synaptic sites plays a crucial role for efficient neuronal transmission and plasticity. The molecular mechanisms that regulate receptor trafficking in and out synapses just begin to be unravelled . They include exo and endocytosis to insert and remove receptors from the plasma membrane, as well as receptor surface trafficking through lateral diffusion between the synaptic and extrasynaptic sites. These processes are highly regulated by neuronal act ivity, adaptor proteins and phosphorylation. The recent development of high resolution single molecule imaging in live neurons by the host laboratory has allowed to attain unprecedented precision in the determination of individual receptor movements in and out synapses. This has led to the demonstration that receptor diffusion in the plane of the membrane is a key process in regulating receptor numbers at synapses, and that the different types of glutamate receptors display differential activity-dependent r egulation of mobility.The aim of the present proposal is to further develop imaging approaches to provide a comprehensive insight into glutamate receptor trafficking paying particular attention to a real-time description of AMPA and NMDA receptor insertion and removal from the membrane. We will use innovative molecular tools (pH sensitive GFPs) and develop a combination of new imaging approaches (multi-color evanescent wave microscopy combined with single molecule tracking to investigate the relative contri butions of receptor exocytosis, endocytosis and lateral diffusion to the regulation of receptor number at synapses. By overcoming technical limitations others have encountered till now, our aim is to provide for the first time high resolution characterizat ion of the localization, kinetics and regulation of receptor exo and endocytosis. Ultimately, completion of this project will help understanding the mechanisms that link GluR dynamics to synaptic plasticity.

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