The authors thank the members of the Harata lab for helpful discussions throughout the execution of this work. This work was funded by grants from the American Heart Association, the Dystonia Medical Research Foundation, the Edward Mallinckrodt, Jr. Foundation, the National Science Foundation, and the Whitehall Foundation to N.C.H.

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.
<ol>
	<li>Prepare the dissociated cell culture of the CA3-CA1 regions of hippocampus from mice or rats on postnatal days 0-119,20. 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 cell...

The authors declare no competing financial interests.

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...

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 of FM1-43 in Figure 1B), can reversibly enter and exit lipid membranes without permeating them. Groups of FM dyes share similar features that influence the range of light that they emit. For example, FM2-10, FM1-43, and FM1-84 have one double bond between two cyclic compounds and show green emission. The difference between them is the length of the hydrophobic tail, which determines its hydrophobicity and therefore the rate of exit from the membrane (departitioning). In the cases of FM5-95 and FM4-64, three double bonds link the cyclic compounds, and they show red emission. These dyes differ with respect to their hydrophilic parts. In all FM dyes, the fluorescence intensity increases when they are inserted into biological membranes, due to an increase in quantum yield in the hydrophobic environment relative to the hydrophilic environment. Thus the changes in FM intensity represent the changes in membrane turnover. The different colors (emission spectra) and hydrophobicities make the FM dyes a versatile research tool in synaptic vesicle recycling.

Based on these features, the FM dyes are mostly used according to the following scheme when analyzing synaptic vesicle recycling (Figure 2). Neurons are bathed in an extracellular solution containing the FM dye, enabling it to be taken up into synaptic vesicles (SVs) as they form via endocytosis (staining). The dye is then washed out by applying a dye-free extracellular solution; this reveals the functional nerve terminals, i.e. only those actively undergoing endocytosis will contain a cluster of synaptic vesicles that are loaded with the dye (Figure 2 bottom). Subsequent exocytosis leads to loss of the FM dye to the extracellular space and a concomitant loss of fluorescence (destaining; due to both the departitioning to a hydrophilic environment and diffusion away from the site of exocytosis). Therefore the changes in FM fluorescence intensity are indicators of synaptic vesicle exo- and endocytosis.

FM dyes have been used to stain and destain the synaptic vesicles in various organisms and preparations2,3. Examples include mammalian neuronal cultures4-9, mammalian brain slices10,11, neuromuscular junctions12,13, retinal bipolar neurons14,15, and hair cells of cochlea16.

Typically in such experiments, both staining and destaining are triggered by extensively stimulating the neurons (evoked activity). Recently, however, synaptic vesicle recycling in response to weak stimulation has also been analyzed, as has recycling in the absence of an external stimulus (spontaneous and miniature synaptic activity)9,17-19. Spontaneous and miniature synaptic activities are defined as those that occur in the absence of external stimuli, with the former involving the spontaneous firing of action potentials (Figure 3). These weak synaptic activities are associated with smaller changes in FM signals than those triggered by extensive stimulation. The measurement requires that the changes in FM fluorescence intensity accurately reflect synaptic vesicle exocytosis or endocytosis but not artifactual changes in intensity. One cause of the artifact is the presence of nonspecific staining of the plasma membrane by FM dyes. Gradual washout of this component will lead to a gradual change in the measured fluorescence intensity, which will be erroneously ascribed to synaptic activities. This factor can be reduced by appropriate methods (see Protocol). The most notable cause of the artifact is the photobleaching of FM dye retained within synaptic vesicles. The photobleaching-related changes in FM intensity must be small in comparison to the biological (synaptic) changes that are measured. The recent development of sensitive cameras, e.g. the electron-multiplying charge-coupled device (EMCCD) camera, makes it possible to minimize photobleaching by shortening exposure time and weakening the intensity of the light used to excite the fluorophore. Another cause of the artifact is a drift in the focusing level of light microscope. The focus drift during an imaging session can be caused by mechanical or thermal effects, and will erroneously lead to a change in the measured fluorescence intensity.

Here we describe protocols and equipment that make it possible to use FM dyes to analyze synaptic vesicle recycling even in the context of weak or no stimulation, in particular, the miniature synaptic activity. We show examples of the staining and destaining of vesicles during evoked and spontaneous synaptic events, using cultured rodent hippocampal neurons, and imaging the destaining phase. We also demonstrate how to evaluate the degree of FM dye photobleaching, in the absence of any FM dye loss due to synaptic activities.

<table border="0" cellpadding="0" cellspacing="0" width="1767">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Pulse generator</td>
			<td>AMPI</td>
			<td>Master-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated stimulator</td>
			<td>Digitimer</td>
			<td>DS3</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>This is used for assessing the cell morphology at low magnification.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TiE</td>
			<td>This is used for high-resolution fluorescence and transmitted light imaging, with minimal focus drift.</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>Water-immersion lens is recommended. Oil-immersion lens is usable unless an imaged structure is deep from the coverslip surface (e.g. &#62;10 &#956;m).</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032509</td>
			<td>490/20-nm ex, 510-nm dclp, 520-nm-LP em for FM1-43</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032809</td>
			<td>490/20-nm ex, 510-nm dclp, 650-nm-LP em for FM4-64.&#160;</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor Technology</td>
			<td>iXon EM+ DU-860</td>
			<td>This EMCCD camera is used for high-sensitivity detection of fluorescence.</td>
		</tr>
		<tr>
			<td>Liquid recirculating chiller</td>
			<td>Solid State Cooling Systems</td>
			<td>Oasis 160</td>
			<td>This is used for continuously perfusing the camera with chilled water for maintaining a temperature of -80&#176;C, and thereby reducing noise.</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>CoolLED-Custom Interconnect</td>
			<td>490 nm&#160;</td>
			<td>This light source is used for rapid on/off control of fluorescence excitation.</td>
		</tr>
		<tr>
			<td>Image acquisition software</td>
			<td>Andor Technology</td>
			<td>Solis</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>Warner Instruments</td>
			<td>RC-21BRFS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast perfusion system</td>
			<td>Warner Instruments</td>
			<td>SF-77B</td>
			<td></td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris Bioscience</td>
			<td>1045</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-AP5</td>
			<td>Tocris Bioscience</td>
			<td>0106</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris Bioscience</td>
			<td>1069</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T35356</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM1-43 (FM1-43FX)</td>
			<td>Invitrogen</td>
			<td>F35355</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Invitrogen</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM4-64 (FM4-64FX)</td>
			<td>Invitrogen</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217; balanced salt</td>
			<td>Sigma-Aldrich</td>
			<td>H2387</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>51200-038</td>
			<td>This solution does not contain phenol red that will interfere with fluorescence imaging.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of synaptic vesicle recycling using fm dyes during evoked, spontaneous, and miniature synaptic activities

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.

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...

Watch this Scientific Journal Video about Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities at JoVE.com

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

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.

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

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20.9K Views.  University of Iowa Carver College of Medicine.  Descriviamo l'uso di coloranti stirilico FM per l'immagine riciclo delle vescicole sinaptiche nei terminali nervosi funzionali. Questo protocollo può essere applicato non solo evocata, ma anche spontanea e attività sinaptiche in miniatura. Il protocollo espande la varietà di eventi sinaptici che possono essere adeguatamente valutati. 