Chapter title |
Imaging Activity-Dependent Signaling Dynamics at the Neuronal Synapse Using FRET-Based Biosensors.
|
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Chapter number | 18 |
Book title |
Synapse Development
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Published in |
Methods in molecular biology, January 2017
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DOI | 10.1007/978-1-4939-6688-2_18 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6686-8, 978-1-4939-6688-2
|
Authors |
Zohreh Farsi, Andrew Woehler |
Editors |
Alexandros Poulopoulos |
Abstract |
In this chapter, we introduce the combined use of FRET-based biosensors and synaptic markers as an effective tool for studying intracellular signaling pathways in small synaptic terminals of neuronal cells. The approach is based on the unmixing of excitation/emission spectral fingerprints of a FRET donor and acceptor pair, as well as a lipophilic styryl dye, FM1-43, loaded into presynaptic terminals. The destaining of FM1-43 during evoked release provides a map to guide the sampling of fluorescence for FRET analysis. In the example presented here, we measure the temporal dynamics of cAMP at the presynaptic terminal using an intramolecular CFP/YFP-based FRET sensor. However, this methodology can be applied to investigate the spatial and temporal regulation of a variety of signaling processes, as well as dynamic changes in protein-protein interaction. |
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