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Use of FM1‐43 and Other Derivatives to Investigate Neuronal Function

Michael A. Cousin¹

¹University of Edinburgh, Edinburgh, Scotland

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 2.6

DOI:

10.1002/0471142301.ns0206s43

Online Posting Date:

April, 2008

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Mike Cousin

Abstract

The fluorescent dye FM1-43 and its derivatives can be used to monitor the physiology of synaptic vesicle turnover in central nerve terminals. They do so by their ability to reversibly partition into membranes, a process that results in a huge increase in fluorescence in comparison to their quantum yield in solution. This unit provides protocols for quantifying total synaptic vesicle turnover, the kinetics and extent of synaptic vesicle exocytosis, and the kinetics and mode of synaptic vesicle endocytosis. Descriptions of other ways these protocols have been used to derive information about the life cycle of the synaptic vesicle are also provided. Curr. Protoc. Neurosci. 43:2.6.1-2.6.12. © 2008 by John Wiley & Sons, Inc.

Keywords: FM1-43; synaptic vesicle; exocytosis; endocytosis; fluorescence; FM2-10; FM4-64

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Introduction
Basic Protocol: Visualization and Quantification of Synaptic Vesicle Exocytosis
Alternate Protocol: Visualization and Quantification of Synaptic Vesicle Endocytosis
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol: Visualization and Quantification of Synaptic Vesicle Exocytosis

Materials

Cultured neurons grown on glass coverslips (poly-lysine-coated or otherwise; e.g., Chapter 3)
Saline⁺ solution (see recipe), room temperature
10 µM FM1-43 (Molecular Probes) in saline⁺ solution (see recipe for saline⁺ solution)
10 µM FM1-43 in saline⁺ solution (see recipe for saline⁺ solution) supplemented with 50 to 100 mM KCl (reduce NaCl accordingly to maintain osmolarity)

Perfusion chamber with parallel platinum electrodes (RC-21 BRFS, Warner Instrument) and perfusion apparatus (VC-66CS, Warner Instrument)
Inverted epi-fluorescence microscope with attached imaging system: light source (monochromator or filter wheel), cooled CCD camera, computer, and imaging software (see unit 2.1)
Stimulator (D330-Multistim System, Digitimer Ltd.)

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Figures

Figure 2.6.1

Structure of FM1-43 and common derivatives. Styryl dyes have a hydrophilic head of two quaternary nitrogens that prevents them from crossing membranes. They also all have a hydrophobic hydrocarbon tail that allows insertion into membranes. The length of the hydrocarbon tail (in number of carbons, n) determines the speed of departitioning from the membrane. In the figure, the solid line represents FM2-10 (n = 2); the dashed line represents FM1-43 (n = 4); and the dotted line represents FM1-84 (n = 5).

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Figure 2.6.2

(A) Loading and unloading of neurons with FM1-43, as performed in the Basic Protocol. (i) For loading, FM1-43 is added to neurons and partitions into the lipid bilayer via its hydrocarbon tail, causing an increase in its fluorescence. It cannot cross the lipid bilayer due to its hydrophilic head groups. Nerve terminals are stimulated (S1), either by induction of action potentials or by exposure to KCl, evoking SV exocytosis. (ii) During the subsequent SV endocytosis, the FM1-43 that partitioned into the plasma membrane is internalized. SVs are now loaded with FM1-43. Noninternalized FM1-43 is then washed off the lipid bilayer; this does not affect internalized FM1-43. (iii) For unloading, the loaded neurons are stimulated again (S2) and the resulting exocytosis unloads FM1-43 from the SV. FM1-43 is unloaded by departitioning from the lumen of the SV. Unloading is observed as a loss of fluorescence as FM1-43 comes off membrane and into solution. (B) Bright-field image of neurite field of primary culture of cerebellar granule cells (63× magnification). Scale bar represents 50 µm. (C) Fluorescence image of same field now loaded with FM1-43. Note the punctate appearance of dye loading. (D) High-resolution image of same neurite field. Nerve terminals are highlighted by asterisks. (E) High-resolution image following FM1-43 unloading evoked by a second stimulation. Note that the bright punctate areas have lost their fluorescence (white asterisks).

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Figure 2.6.3

Schematic of the Basic Protocol for SV exocytosis. For dye loading, FM1-43 is applied to the cells on the initiation of stimulation (see Basic Protocol, step 3) and is kept present for 1 min after the termination of stimulation (step 4). Noninternalized FM1-43 is washed away over a 15 min period (steps 5 and 6). FM1-43 is unloaded from SVs by a second stimulation (step 9). If the effect of a pharmacological antagonist is to be investigated, it should be applied before and during the second stimulation (unloading), i.e., during steps 5 to 9 of the Basic Protocol.

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Figure 2.6.4

Analysis of FM1-43 data obtained from the Basic Protocol. (A) Typical raw data from FM1-43 unloading experiments. Graph is plotted as fluorescence against time and individual lines correspond to the fluorescence of individual nerve terminals. (B) The same data normalized to an arbitrary value. In this way, FM1-43 unloading can be quantified as an absolute decrease in fluorescence (F). (C) The same data normalized for total dye loss. The fluorescence at the start of unloading is normalized to 1 and that at the end of unloading is normalized to 0. In this format, FM1-43 unloading is quantified as the rate () of unloading.

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Figure 2.6.5

Schematic of the Alternate Protocol for studying fast SV endocytosis. For dye loading, FM1-43 is applied to the cells on the initiation of stimulation and is removed upon termination of stimulation. Noninternalized FM1-43 is washed away over a 15-min, period and the amount of FM1-43 loading is quantified by its unloading, evoked by a second stimulation. If the effect of a pharmacological antagonist is to be investigated it should be applied before and during the first stimulation (loading).

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Figure 2.6.6

Schematic of the Alternate Protocol for slow SV endocytosis. For dye loading, FM1-43 is applied to the cells upon termination of stimulation and is kept present for 1 min. Noninternalized FM1-43 is washed away over a 15-min period and the amount of FM1-43 loading is quantified by its unloading, evoked by a second stimulation. If the effect of a pharmacological antagonist is to be investigated it should be applied before the first stimulation and kept present until the removal of the FM1-43 (i.e., until the end of the loading step).

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Figure 2.6.7

(A) Schematic of the Alternate Protocol for monitoring SV endocytosis kinetics. For dye loading, FM1-43 is applied to the cells upon termination of stimulation and is kept present for 1 min. This protocol is then repeated, but the addition of FM1-43 is delayed by 10- to 20-sec intervals. Noninternalized FM1-43 is washed away over a 15-min period and the amount of FM1-43 loading is quantified by its unloading, evoked by a second stimulation. If the effect of a pharmacological antagonist is to be investigated it should be applied before the first stimulation and maintained until the removal of FM1-43 (i.e., until the end of the loading step). (B) Endocytosis kinetics are calculated as (unloaded fluorescence delay time)/(unloaded fluorescence at time 0). Typical results are shown.

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Literature Cited

Literature Cited
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Mike Cousin

Posted on September 17, 2009 - 10:07am

Hi Paul,

It is possible to do multiple loads and unloads of dye however we have found that you have to give the cells time to recover (we use cultured cerebellar granule neurones). We have been able to get 3 rounds of reproducable loading and unloading, providing that we give the cells a 20 min wait between load/unload protocols.

The FM dyes have been published to be phototoxic to cells during continual illumination, however the sampling conditions you describe shouldn't cause this. It does sound that the cells are damaged from what you describe however (neurones take up dye excessively when they are dying). Can you get loading and unloading from a different region in the same coverslip? If you can it does suggest that the illumination is damaging the cells.

We also see enhanced background staining with multiple loads/unloads (sometimes a 2 fold increase in intensity), however the puncta are still visable and they release the same amount of dye across different rounds.

I would suggest letting the cells recover for at least 20 mins before trying another round of load/unload. Everything else with your setup sounds OK.

hope this helps,

Mike

Anonymous (not verified)

Posted on September 17, 2009 - 7:19am

Thank you very much for this helpful article.

I just started using this technique and have problems when i try to do repeated loading/unloading cycles on the same set of synapses. The first stimulation run (load+unload) gives nice punctae and destaining curves that match very well the results reported in literature. But when i try to repeat the experiments on the very same set of synapses (without changing to another region on the same cover slip) things get messed up. The whole preparation becomes super bright, no punctae are discernible neither destaining (apart of photobleaching). Also i have the impression that the synapses look pretty damaged.

Is there a general problem with this technique when one tries to do multiple experiments? Can it be that the dye is somehow toxic after it entered the vesicles?

Any comment on this is highly appreciated!

Best,

Paul

Here is a short summary of the experimental conditions:
FM1-43 (Biotium) at 2-5 microM; for electric stimulation i use 600AP at 20 Hz (or alternatively 30 s with 50 mM KCl); Images are acquired with 1 Hz samplerate, i take 20 images before/after the stimulation to get baselines; the light source is a mercury lamp with neutral density filters (6% transmittance). I use hippocampal cultures made according to standard protocols, e.g. postnatal P0/P1 pups, grown on PDL/laminin coated cover slips and supplied with Neurobasal-B27-glutamax medium.