Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity inreal time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in healthand disease.Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM)provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection canonly excite fluorophores placed in a thin layer (<150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicle tracking and quantitative analysis of fusion events.In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherentcells and for transfecting them with synapto-pHluorin are provided, as well as how to perform TIRFM and imaging. Finally, a strategy aiming toselect, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles have been analyzed under resting andstimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causesa parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior(increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release butalso modifies the mechanism of vesicle fusion and recycling.With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutiveand stimulated secretion.

Tirfm and ph-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: Cell imaging and data analysis / Daniele, Federica; Di Cairano, Eliana S.; Moretti, Stefania; Piccoli, Giovanni; Perego, Carla. - In: JOURNAL OF VISUALIZED EXPERIMENTS. - ISSN 1940-087X. - 2015:95(2015), pp. e52267.1-e52267.13. [10.3791/52267]

Tirfm and ph-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: Cell imaging and data analysis

Piccoli, Giovanni;
2015-01-01

Abstract

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity inreal time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in healthand disease.Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM)provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection canonly excite fluorophores placed in a thin layer (<150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicle tracking and quantitative analysis of fusion events.In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherentcells and for transfecting them with synapto-pHluorin are provided, as well as how to perform TIRFM and imaging. Finally, a strategy aiming toselect, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles have been analyzed under resting andstimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causesa parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior(increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release butalso modifies the mechanism of vesicle fusion and recycling.With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutiveand stimulated secretion.
2015
95
Daniele, Federica; Di Cairano, Eliana S.; Moretti, Stefania; Piccoli, Giovanni; Perego, Carla
Tirfm and ph-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: Cell imaging and data analysis / Daniele, Federica; Di Cairano, Eliana S.; Moretti, Stefania; Piccoli, Giovanni; Perego, Carla. - In: JOURNAL OF VISUALIZED EXPERIMENTS. - ISSN 1940-087X. - 2015:95(2015), pp. e52267.1-e52267.13. [10.3791/52267]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11572/123066
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