Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Abstract

The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K + ) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. Reagents FM dye(s)Dye concentrations are usually 2-10 µM for FM1-43 or FM4-64, and 25-40 µM for FM2-10. The dyes readily dissolve in water; stocks of 1-5 mM concentration should be prepared and can be stored at 4˚C. Neuronal tissue or cell sample of interest Equipment Imaging setupAn upright epifluorescence microscope equipped with a 40×-60× water-immersion objective is optimal. When working with cell cultures, the advantages of high-resolution oil-immersion objectives (e.g., 100×/1.4 numerical aperture [NA]) can be exploited by using an inverted setup. For FM1-43, maximal fluorescence is at 465-nm excitation/560-nm emission (Henkel et al. 1996); a conventional fluorescein filter set (435-nm excitation) can also be used. Because the major concern in FM imaging is phototoxicity (which appears usually before any clear signs of photobleaching and alters the ability of the preparations to recycle vesicles), keep the illumination to a minimum by including neutral-density filters. For example, when using a 40×/0.75-NA water-immersion objective with a 100-W mercury lamp, 5%-15% transmission is sufficient. On confocal setups, an argon 488-nm laser line is typically used for excitation, but special care has to be taken to avoid phototoxicity. Images are typically acquired by charge-coupled device (CCD) cameras or photomultiplier tubes, which allows for fast acquisition rates and software-based quantitative image analysis.Neuronal stimulator (see Step 3)

Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

“…Similar results were observed using FM4-64 to label membrane ( Supplementary Fig. 7a ) 28 and Atto 488 to identify fusion modes (n = 75 spots, 6 cells, Supplementary Fig. 7b–d ).…”

Synaptic vesicles transiently dock to refill release sites

“…For the FM 1-43FX (Invitrogen) uptake assay, we used a modified version of a previously published protocol 72 . Neurons on sapphire disks were first incubated with 30 µM Pitstop 2 (Sigma) in physiological saline (1 mM Ca 2+ ) for 2 min.…”

Modeling Human Neural FunctionalityIn Vitro: Three-Dimensional Culture for Dopaminergic Differentiation

“…7A). Synaptic activity was assessed using the fluorescent probe FM1-43, 32,33 by the ability of differentiated neurospheres to respond to depolarizing stimuli. Neurospheres at different stages of differentiation were loaded with FM1-43, and their destaining kinetics upon depolarization indicated that the same depolarizing stimuli led to a modest decrease in the fluorescence intensity of 7Diff and 21Diff neurospheres, when compared with neurospheres at the end of the differentiation process (18Mat), in which fluorescence dropped to less than half of its initial value (Fig.…”