Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Summary

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

Abstract

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

Introduction

The functionality of synaptic vesicles is an important determinant of synaptic transmission. These vesicles release neurotransmitters when they fuse with the presynaptic plasma membrane (exocytosis), and they become ready for another cycle of release after being regenerated from the plasma membrane (endocytosis) and reloaded with neurotransmitter. Research into the dynamics of and mechanisms underlying synaptic vesicle recycling has been greatly accelerated by the introduction of styryl FM dyes¹. These amphipathic molecules, which have positively charged hydrophilic head groups and hydrophobic tails (multiple dyes in Figure 1A, stereoview o....

Protocol

1. Primary Culture of Neurons from the Mammalian Brain

All animal procedures performed in this study are approved by the Institutional Animal Care and Use Committee of the University of Iowa.

1. Prepare the dissociated cell culture of the CA3-CA1 regions of hippocampus from mice or rats on postnatal days 0-1^19,20. Plate the hippocampal cells on 12-mm coverslips (thickness number 0) preseeded with the rat glial feeder layer, in 24-well dishes, and at a density of 12,000 cells/well.
2. Culture the hippocampal neurons for at least 8 days for functional nerve terminals to develop²¹. We use neurons on 11-14 ....

Results

As an example, we show representative results for the destaining time course of synaptic vesicles (Figure 4). Cultured hippocampal neurons were stained with FM4-64 using the spontaneous synaptic activity (step 2.3) and washed with dye-free solution (rinsing solution 2). The imaging shows the initial destaining time course using spontaneous activity (step 5.3) (initial part of continuous line, Figure 4A). This is followed by the destaining time course using three rounds of evoked activity.......

Discussion

We have described protocols for staining and destaining synaptic vesicles in response to evoked, spontaneous and miniature synaptic activity, and for imaging during the destaining phase. In addition to the existing protocols, we have included a new protocol of observing the FM destaining based on miniature synaptic activity. Using these protocols, we previously identified abnormalities in cultured neurons from a mouse model of the movement disorder dystonia. In comparison to their counterparts in wild-type mice, those ne.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pulse generator	AMPI	Master-8
Isolated stimulator	Digitimer	DS3
Inverted microscope	Nikon	Eclipse TS100	This is used for assessing the cell morphology at low magnification.
Inverted microscope	Nikon	Eclipse TiE	This is used for high-resolution fluorescence and transmitted light imaging, with minimal focus drift.
Objective lens	Nikon		Water-immersion lens is recommended. Oil-immersion lens is usable unless an imaged structure is deep from the coverslip surface (e.g. >10 μm).
Filter cube	Nikon	77032509	490/20-nm ex, 510-nm dclp, 520-nm-LP em for FM1-43
Filter cube	Nikon	77032809	490/420 nm ex, 510 nm dclp, 650 nm LP em for FM4-64.
EMCCD camera	Andor Technology	iXon EM+ DU-860	This EMCCD camera is used for high-sensitivity detection of fluorescence.
Liquid recirculating chiller	Solid State Cooling Systems	Oasis 160	This is used for continuously perfusing the camera with chilled water for maintaining a temperature of -80°C, and thereby reducing noise.
LED	CoolLED-Custom Interconnect	490 nm	This light source is used for rapid on/off control of fluorescence excitation.
Image acquisition software	Andor Technology	Solis
Imaging chamber	Warner Instruments	RC-21BRFS
Fast perfusion system	Warner Instruments	SF-77B
CNQX	Tocris Bioscience	1045
D,L-AP5	Tocris Bioscience	0106
Tetrodotoxin	Tocris Bioscience	1069	Caution: toxic reagent. Handle with care.
FM1-43	Invitrogen	T35356
Aldehyde-fixable FM1-43 (FM1-43FX)	Invitrogen	F35355
FM4-64	Invitrogen	T13320
Aldehyde-fixable FM4-64 (FM4-64FX)	Invitrogen	F34653
Ionomycin	Sigma-Aldrich	I0634
Hanks’ balanced salt	Sigma-Aldrich	H2387
Minimum Essential Medium	Invitrogen	51200-038	This solution does not contain phenol red that will interfere with fluorescence imaging.
Paraformaldehyde	Electron Microscopy Sciences	15710	Caution: toxic reagent. Handle with care.
Sucrose	Sigma-Aldrich	S7903