title: Imaging Activity-Dependent Signaling Dynamics at the Neuronal Synapse Using FRET-Based Biosensors.
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.
authors.affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Farsi
authors.affiliation: Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany. andrew.woehler@mdc-berlin.de.|DFG-Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), 37073, Göttingen, Germany. andrew.woehler@mdc-berlin.de.|Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Robert-Rössle-Straße 10, 13125, Berlin-Buch, Germany. andrew.woehler@mdc-berlin.de.
authors.forename: Andrew
authors.initials: A
authors.lastname: Woehler
mesh_terms: D000818:Animals; D015415:Biomarkers; D015374:Biosensing Techniques; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D017930:Genes, Reporter; D007091:Image Processing, Computer-Assisted; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013569:Synapses / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D000242:Cyclic AMP
keywords: FRET; Intracellular signaling; Live-cell imaging; Synaptic plasticity; cAMP