Name: an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins
Uploaded: 2018-06-26T15:00:00
Description: Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

We thank Irmgard Weiss for expert technical assistance. This work was supported by the DFG via the Cluster of excellence for microscopy at the nanometer range and molecular physiology of the brain (CNMPB, B1-7, to T.D.).

No studies with live animals were conducted. Experiments involving euthanized animals to obtain cell cultures were approved by the local animal protection authorities (Tierschutzkommission der Universit&#228;tsmedizin G&#246;ttingen) under the approval number T10/30. The experiments were conducted with the approved protocols.
1. Primary Hippocampal Cell Culture
<ol>
	<li>Prepare the dissociated cell culture of the rat hippocampus on embryonic day 1916,<sup ...

<ol>
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There are three assays routinely used to study synaptic vesicle (SV) recycling. The first two include the use of a) fluorescent styryl dyes such as FM1-43 (which incorporate into membranes, are taken up into organelles during endocytosis, and are released after exocytosis); and b) fluorescently tagged recombinant SV proteins (which, upon overexpression, incorporate into the proteinaceous recycling machinery). If the attached fluorophores change their fluorescence depending on the pH, they can be used to monitor changes b...

Studying synaptic vesicle recycling is important in determining how presynaptic properties change, either during synaptic plasticity or in response to perturbation of synaptic function. Studying Synaptotagmin-1 (Syt1) antibody uptake provides one method of measuring the amount of SV recycling. Syt1 is a SV-associated protein that acts as a Ca2+ sensor and is necessary for exocytotic release of the neurotransmitter1,2. It is a transmembrane protein with a C-terminal cytoplasmic domain outside the SV and an N-terminal luminal domain inside the SV3. During exocytosis, the luminal domain of Syt1 becomes exposed to the external medium. To this external medium, we add antibodies directed against the cytoplasmic domain, which becomes internalized during endocytosis. These antibodies can be either pre-conjugated with fluorophores or immunostained with secondary antibodies4,5,6,7. The fluorescence intensity of the resulting immunosignal is proportional to the amount of SV recycling. This approach can be used to determine both constitutive and depolarization-induced SV recycling6,8.
Syt1 uptake assays can be performed after virus-mediated gene transfer to virtually all cells in the dish or after sparse transfection of a small number of cells. Our method combines the assay with sparse double transfection of primary hippocampal neurons using calcium phosphate9. We use a recombinant marker protein known to accumulate at presynapses, fluorescently tagged Synaptophysin, to locate presynaptic terminals and overexpress our protein of interest, Rogdi. This allows us to test whether or not Rogdi targets functional synapses and affects SV recycling. The gene encoding Rogdi was originally identified in a screen for Drosophila mutants characterized by impaired memory10. In humans, mutations in the Rogdi gene cause a rare and devastating disease called Kohlsch&#252;tter-T&#246;nz syndrome. Patients suffer from dental enamel malformations, pharmacoresistant epilepsy, and psychomotor delays; however, the subcellular localization of the gene product remained elusive11. Thus, the Syt1 uptake assay provided key evidence for the localization of GFP-tagged Rogdi at functional synapses9.
This uptake technique has several benefits. First, SV recycling can be observed both in real time by performing live imaging7,12, and after fixation6,9 by measuring the fluorescence intensity of the Syt1 fluorescence label. Additionally, several Syt1 antibody variants have been developed. There are untagged variants that can be labeled with a secondary antibody following a standard immunostaining protocol after fixation, and pre-conjugated variants with a fluorescence label already attached. Finally, antibody-based fluorescence is advantageous due to the large selection of commercially available secondary or conjugated dyes that can be used.
When fixing and immunostaining the neurons, it is also possible to stain for additional proteins and perform colocalization analysis. This can help determine where they are located in relation to recycling SVs. The intensity of the fluorescence label is the direct measure of the amount of SV recycling. In addition, the antibodies selectively label Syt1-containing structures, resulting in high specificity and little background fluorescence4. Different stimulation protocols can also be used, such as depolarization buffers or electric stimulation protocols9,12,13,14. However, basal SV recycling can be measured without stimulating the neuronal cultures15.
Our method specifically addresses Syt1 antibody uptake in double-transfected neurons with secondary antibody immunolabeling after fixation. However, we refer to all routinely used variants of the assay in our discussion to give viewers an opportunity to adapt the protocol to specific needs.

<table>
	<tbody>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3306-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSNAP HQ2</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>Invitrogen</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Merck</td>
			<td>8.03456.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti mouse Alexa 647</td>
			<td>Jackson-Immunoresearch</td>
			<td>715605151</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41966</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>multiwell 24 well</td>
			<td>Fisher Scientific</td>
			<td>087721H</td>
			<td></td>
		</tr>
		<tr>
			<td>tube (50 mL)</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS superior</td>
			<td>BiochromAG</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>1,083,421,000</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14170</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Hera Cell 150 (Inkubator)</td>
			<td colspan="2">ThermoElectron Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Honeywell</td>
			<td>M0250-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>Fisher Scientific</td>
			<td>10144633CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol4-88</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioFroxx</td>
			<td>1394KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>BioFroxx</td>
			<td>5155KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OpenView Experiment Analysis Application</td>
			<td>Free software, see comments</td>
			<td></td>
			<td>written by Noam E. Ziv, Technion &#8211; Israel Institute of Technology, Haifa, Israel</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Penstrep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma</td>
			<td>P6148-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>safety hood</td>
			<td>ThermoElectron</td>
			<td></td>
			<td>Serial No. 40649111</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>neoFroxx</td>
			<td>1104kg001</td>
			<td></td>
		</tr>
		<tr>
			<td>Synaptotagmin1</td>
			<td>Synaptic Systems</td>
			<td>105311</td>
			<td>mouse monoclonal; clone 604.2</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>1,086,031,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genius 3&#160;</td>
			<td>IKA</td>
			<td>3340001</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Observer. Z1&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

An expected result of this approach is locating approximately 50 double-transfected neurons per coverslip at a density of 50,000 neurons per well. The axon of each neuron is expected to show multiple hotspots of fluorescently-tagged Synaptophysin accumulation, indicating clusters ofSVs. At functional presynaptic sites, the recombinant Synaptophysin signal colocalizes with punctate Syt1 fluorescence. Using double transfection, either GFP-Rogdi as the protein of interest (<strong class="xfi...

Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

The main advantage of this technique is that it is very simple, requires only standard equipment, and benefits from the primary advantage of antibodies, which is their specificity. The protocol was originally introduced by Michela Matteoli and colleagues, and has proven highly effective. I've used it to test whether a certain protein cordocti is localized to active synapses.Begin this procedure by culturing primary hippocampal cells on coverslips in a 24-well plate for three days, as described in the text protocol. Prewarm the reduced-serum medium, cell-culture medium, and distilled water to 37 degrees celsius in the water bath. Working under the laminar flow hood to ensure sterile working conditions, prepare the transfection mix in a 1.5-milliliter microcentrifuge tube.First, mix 7.5 microliters of two-molar calcium chloride with four micrograms of each endotoxin-free DNA. Add water to reach a total volume of 60 microliters in a 1.5-milliliter microcentrifuge tube. Now, add 60 microliters of transfection buffer to the mix.To obtain the best results, add the transfection buffer dropwise while shaking the DNA mixture gently on the vortex. Incubate the resulting transfection mix at room temperature for 20 minutes. Avoid shaking the tube during the incubation time.Under the laminar flow hood, remove the cell-culture medium from the wells using a 1, 000-microliter pipette, store it in a separate container, and immediately add 500 microliters of reduced-serum medium to each well. Store the conditioned medium in the incubator. Incubate the cells at 37 degrees celsius and 5%CO2 until the 20-minute incubation period for the transfection mix is over.Then, add 30 microliters of transfection mix to each well by pipetting several drops. Discard the residue at the bottom of the tube. After all the wells have been supplied with transfection mix gently shake the 24-well plate to ensure distribution of the transfection mix in the medium.Now incubate the wells for 60 minutes at 37 degrees celsius and 5%CO2. Following incubation, remove and discard the transfection mix, and wash the cells three times with cell-culture medium. The washing step is critical.Minimize the time that each well has no medium by removing and replacing well-by-well. Also, make sure to add the medium gently. To wash the cells, add one milliliter of cell-culture medium to each well, and incubate them for 30 seconds at room temperature.Then, remove 750 microliters of medium, and add the same amount of fresh medium. After repeating this process three times, remove and discard the cell-culture medium, and add 450 microliters of the conditioned medium well-by-well. Now let the neurons mature in the incubator at 37 degrees celsius and 5%CO2 to DIV 10.Prewarm 600 microliters of 1X depolarization buffer and 10 milliliters of cell-culture medium to 37 degrees celsius in the water bath. Add one microliter of mouse Anti-Syt1 antibody to the 1X depolarization buffer, and vortex for 10 seconds. After removing and discarding the cell-culture medium from the cells, add 200 microliters of the depolarization antibody mix to each well.Incubate the plate for five minutes at 37 degrees celsius and 5%CO2 in the incubator. Remove and discard the depolarization antibody mix, and wash the cells three times with cell-culture medium. Add one milliliter of cell-culture medium to each well, and incubate for 30 seconds at room temperature.Then, remove 750 microliters of medium, and add the same amount of fresh medium. Repeat this process three times. Next, remove and discard the cell-culture medium, and add 300 microliters of 4%PFA in 1X PBS.Finally, incubate for 20 minutes at four degrees celsius before washing three times for five minutes each with 1X PBS. Dilute the secondary fluorophore-coupled antibody in 200 microliters of blocking buffer for each well at a dilution of one to 1, 000. Remove and discard the 1X PBS from each well containing coverslips with the cultured neurons.Add 200 microliters of blocking buffer antibody mix to each well, and incubate for 60 minutes at room temperature. After incubation, wash the cells three times for five minutes with one milliliter of 1X PBS. Next, add a seven-microliter drop of embedding medium onto the microscope slide.Remove the coverslip from the 24-well plate by lifting it with a canula and grabbing it with forceps. Dip the coverslip into distilled water to remove the PBS, and dry it carefully by touching one edge to a soft tissue. Flip the coverslip onto the embedding-medium droplet so that the surface carrying the cells faces the microscope slide, thereby embedding the cells into the embedding medium.Leave the slides to dry under the hood for one to two hours, covering them to avoid light exposure. Store the dried slides in a microscope slide box at four degrees celsius. After immunolabeling the Syt1 antibody with a fluorescently-labeled secondary antibody, the extent of Syt1 uptake is finally quantified using the immunosignal.Punctate immunofluorescence indicates sites of Syt1 uptake in the field of view. These sites thus contain synaptic vesicles that recycled during the experiment. Synaptophysin-mOrange fluorescence indicates accumulations of synaptic vesicles in the axon of a transfected neuron.GFP-Rogdi fluorescence indicates accumulations of Rogdi in the axon of the transfected neuron. This part of the experiment reveals extensive colocalization of GFP-Rogdi and Synaptophysin-mOrange, suggesting that GFP-Rogdi accumulates at presynaptic sites within the axon. The overlay reveals which of the coaccumulations of GFP-Rogdi and Synaptophysin-mOrange colocalize with sites of Syt1 antibody uptake.The sites marked by arrows represent accumulations of recombinant Rogdi and recombinant synaptophysin at functional synapses. The protocol depicts a technically straightforward way to determine the extent of synaptic residue recycling. This method can help answer key questions in molecular and cellular science, such as where presynaptic nerve terminals allocated in an axon of a certain neuron and whether a recombinant or endogenous protein of interest accumulates at these terminals.After its development, this technique has been used in many ways to monitor synaptic vesicle recycling in presynaptic terminals. For example, it can be used to determine synaptic vesicle recycling in neurons after overexpression on adaptive proteins as well as in neurons obtained from animals or from stem-cell-derived neurons.

Research

JoVE Journal

Neuroscience

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

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 ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...