TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Summary

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Abstract

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and 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 can only 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 vesicles 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 adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique to perform TIRFM and imaging. Finally, a strategy aiming to select, 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 are analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a 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 but also 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 constitutive and stimulated secretion.

Introduction

Chemical synaptic transmission between neurons is a major mechanism of communication in the nervous system. It relies on the release of neurotransmitters through a dynamic cycle of vesicle fusion and retrieval at the presynaptic site. Many of the proteins involved in vesicle dynamics have been identified; however, their specific contribution to the phenomenon remains to be clarified¹.

Our understanding is partly limited by the fact that the most widely used assays for exo/endocytosis are not always the most appropriate. Several studies related to vesicle fusion and dynamics rely on electrophysiological techniques. This technique ....

Protocol

1. Cell Culture and Transfection

1. SH-SY5Y cell culture
   NOTE: The experiments have been performed using the human neuroblastoma SH- SY5Y (ATCC# CRL-2266)¹⁵. SH-SY5Y cells grow as a mixture of floating clusters and adherent cells. Follow the instructions reported in the protocol (cell density, splitting ratio, etc.) to have cells that grow firmly attached to glass cover, which is crucial for TIRFM.
   1. Before starting, under the laminar flow biosafety cabinet, make the opportune volume of sterile phosphate buffer saline solution (PBS) and culture medium.
      1. Make 50 ml of PBS with concentrations of....

Results

The TIRF imaging and data analysis procedures described are designed to study vesicles dynamics in cellular systems. This technique can be used to determine the effects of signaling molecules and drugs on fusion events and neurotransmitter vesicle dynamics¹⁷. Using GFP-tagged plasma membrane proteins, the TIRFM analysis has been employed to characterize the constitutive trafficking of GFP-tagged glutamate transporters in glial and epithelial cells^18,19.

To validate the im.......

Discussion

This paper presents a protocol to image and analyze vesicles dynamics in secreting cells, using fluorescent cDNA-encoded vectors and TIRFM. Key elements of successful imaging by TIRFM are the selection of the cellular model and cell transfection with genetically-encoded optical indicators of vesicle release and recycling.

TIRFM is ideally suited for cells growing adherent to a glass cover and sufficiently flat to allow stable visualization of membranes and fusion events. Vesicles should ideall.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Axio Observer Z1	Zeiss	491912-9850-000	inverted microscope
Multiline Argon Laser Lasos 77	Lasos	00000-1312-752	multi-line (458/488/514 nm), 100 mW argon-ion laser
Laser TIRF slider	Zeiss	423681-9901-000
100X Objective	Zeiss	421190-9900-000	Oil, NA 1.45 Alpha-Plan
CCD Camera RetigaSRV Fast 1394	QImaging
LAMBDA 10-3 optical filter changer with SmartShutter	Sutter Instrument Company
Software
Image ProPlus 6.3 Software	Media Cybernetics		spot selection, ROI selection, fluorescence intensity determination
Excel	Microsoft		photobleaching correction, whole-cell and single-vesicle analyses
GraphPad Prism 4.00	GraphPad Software, Inc.		statistical analysis