Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

Summary

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Abstract

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye ¹; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K⁺). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1A_i-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1A_iv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy ^2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

Protocol

1) Preparation of Drosophila melanogaster neuronal muscular junction (NMJ)

1. Prepare standard Drosophila saline (130 mM NaCl, 36 mM sucrose, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 5 mM Hepes, pH 7.3 ⁴.
2. Dissect the preparation in saline (1.1). The Drosophila larvae is pined dorsal side-up in a Sylgard dish; the dorsal side is sectioned longitudinal, and the internal organs are removed. The preparation is then stretched and pined. Several ventral muscles can be then used.

2) Stimulation and FM staining

1. It is advisable to do all the following steps in low light con....

Discussion

A few critical steps should be taken into account:

• The DAB incubation should be performed only after thorough washing and quenching of the preparations. Otherwise the non-reacted glutaraldehyde will interact with DAB and cause its precipitation (typically in the form of flat crystals, which are not electron dense). Preparations where this precipitation takes place abundantly are rarely usable for electron microscopy.
• Illumination times should be optimized, by testing several identical preparation.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FM 1-43	Invitrogen	F10317
Epon resin	Plano GmbH	R1030
di-aminobenzidine hydrochloride	Sigma-Aldrich	D5905
50% Glutaraldehyde	AppliChem	A3166	EM grade
Sylgard	Dow Corning	104186298
Axioskop 2 FS plus	Carl Zeiss, Inc.
Objective 20x 0.5 NA	Olympus Corporation		Dry objective
100W Hg Lamp	Carl Zeiss, Inc.
Lamp housing with back mirror	Carl Zeiss, Inc.	1007-980
MRm camera	Carl Zeiss, Inc.	0445-554	Image acquisition
Ex. Filter (HQ 470/40)	AHF	F49-671
Dichroic (495 DCLP)	AHF	F33-100
Em. Filter (HQ 500 LP)	AHF	F42-018
EM	Carl Zeiss, Inc.
Proscan CCD HSS	Proscan Electronic Sys.		1024 x 1024