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.
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.
