Measuring Exocytosis in Neurons Using FM Labeling

Summary

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Abstract

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release. FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated. Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence. Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.

Protocol

Cells: Rat (or mouse) primary hippocampal neuronal cultures (14-28 days in vitro).

Stimulation: Electrical, delivered via two platinum electrodes; 70-90 mV

Microscopy: 60x oil lens on an inverted CCD fluorescent microscope.

Software: Slidebook (Intelligent Imaging Innovations, Santa Monica, CA)

See supplemental methods page for further details

1. Warm HEPES-buffered saline (HBS) to room temperature.  Add the glutamate receptor antagonists APV (50 µM final concentration) and CNQX (10 µM final).  Each FM experiment usua....

Discussion

As 300 action potentials (APs) is sufficient to induce at least one round of vesicle recycling, a loading stimulus of 300 APs or more is often given to label the recycling pool of vesicles. Though more intense loading stimuli, such as 900 APs, may allow a nominal number of additional vesicles to be labeled, this also increases the amount of time the dye can embed in extracellular membranes, leading to greater non-specific staining. I have found the wash step to be critical to ensure proper destaining. It is important .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
APV	Sigma-Aldrich	A5282
FM 4-64	Molecular Probes, Life Technologies	T-3166
CNQX disodium salt	Sigma-Aldrich	C-239