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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 neurotransmitter^1,2. 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 antibodies^4,5,6,7. 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 recycling^6,8.

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 Kohlschütter-Tönz 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 imaging^7,12, and after fixation^6,9 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 protocols^9,12,13,14. 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.

Protokół

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 Universitätsmedizin Göttingen) under the approval number T10/30. The experiments were conducted with the approved protocols.

1. Primary Hippocampal Cell Culture

1. Prepare the dissociated cell culture of the rat hippocampus on embryonic day 19^16,17. Plate the cells on 12 mm coverslips coated with polyethyleneimine (PEI) in 24-well dishes at a density of 50,000 - 60,000 cells/well. Check the density using a cell counting chamber and phase contrast optics.
2. Culture the neurons for 3 days (day in vitro (DIV) 3) in a 24-well plate in the incubator at 37 °C with 5% CO₂.
3. Assess the coverslips for indicators of cell health using transmitted light microscopy (e.g., phase contrast optics at a magnification of 10 - 20X). Check for the following indicators of good health: a clear phase contrast halo, neurites without beaded structures, and no soma clustering or neurite bundling.

2. Transfection

Note: The following protocol refers to a double-transfection for 3 wells. However, the protocol works best when amounts sufficient for 4 wells are prepared.

1. Prepare 500 mL of transfection buffer (274 mM NaCl, 10 mM KCl, 1.4 mM Na₂HPO₄, 15 mM glucose, 42 mM HEPES) in an Erlenmeyer flask.
   1. Dissolve 8.0 g of NaCl, 0.37 g of KCl, 0.095 g of Na₂HPO₄, 1.35 g of glucose, and 5.0 g of HEPES in 400 mL of distilled water in an Erlenmeyer flask.
   2. Adjust the pH to 6.95 with 1 M NaOH using a pH meter.
   3. Adjust the volume with distilled water to 500 mL and check the pH using a pH meter.
   4. Make 20 - 30 mL aliquots of transfection buffer with the following pH values by pipetting 1 M NaOH to the transfection buffer: 6.96, 6.97, 6.98, 6.99, 7.00, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, 7.11.
      Note: The pH of the transfection buffer is crucial for the transfection efficacy.
   5. To test which transfection buffer leads to the highest number of transfected cells, test each pH value from 6.96 to 7.11. Use the transfection method described in 2.2 - 2.11 and a validated plasmid expressing GFP. Determine the number of transfected cells per coverslip for every transfection buffer pH value to assess which buffer works the best.
   6. Aliquot the buffer with the highest transfection efficiency into 2 mL microcentrifuge tubes Freeze and store the tubes at -20 °C.
2. Pre-warm the reduced serum medium, cell culture medium, and distilled water to 37 °C in the water bath.
3. Prepare the transfection mix in a 1.5 mL microcentrifuge tube. Work under the laminar flow hood to ensure sterile working conditions.
   1. Mix 7.5 µL of 2 M calcium chloride with 4 µg of each endotoxin-free DNA (Synaptophysin-mOrange and mGFP/GFP-Rogdi). Add water to reach a total volume of 60 µL in a 1.5 mL microcentrifuge tube.
   2. Add 60 µL of transfection buffer to the mix. To obtain the best results, add the transfection buffer dropwise while shaking the DNA-mix gently on the vortex.
   3. Incubate at room temperature (RT) for 20 minutes. Avoid shaking the incubation tube during the incubation time by placing the tube next to the laminar flow hood.
4. Under the laminar flow hood, remove the cell culture medium (“conditioned medium”) from the wells using a 1000 µL pipet and store it in a separate container in the incubator.
5. Add 500 µL of reduced serum medium to each well. Incubate the cells at 37 °C and 5% CO₂ until the 20 minute incubation period (step 2.3.3) is over.
6. Add 30 µL of transfection mix to each well by pipetting several drops. Discard the residue at the bottom of the tube.
7. 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.
8. Incubate the wells for 60 minutes at 37 °C and 5% CO₂.
9. Remove and discard the transfection mix and wash it three times with cell culture medium. Add 1 mL of cell culture medium to each well and incubate them for 30 seconds at RT. Remove 750 µL of medium and add the same amount of fresh medium. Repeat this three times.
   Note: The washing step is critical. Keep the time that each well has no medium at a minimum (i.e., remove and replace well-by-well) and add the washing medium gently.
10. Remove and discard the cell culture medium and add 450 µL of the conditioned medium well-by-well.
11. Let the neurons mature in the incubator at 37 °C and 5% CO₂ to DIV 10.

3. Stimulation and Syt1 Uptake

Note: The following protocol applies the uptake to 3 wells. For depolarization of any other number of wells, adjust the amounts accordingly.

1. Prepare 50 mL of 10x depolarization buffer (640 mM NaCl, 700 mM KCl, 10 mM MgCl₂, 20 mM CaCl₂, 300 mM glucose, 200 mM HEPES, pH 7.4) in an Erlenmeyer flask.
   Note: Depolarization buffer can be kept at 4 °C for several weeks. If a non-depolarizing solution is also used, , prepare a 10x Tyrode’s solution consisting of 1290 mM NaCl, 50 mM KCl, 10 mM MgCl₂, 20 mM CaCl₂, 300 mM Glucose, 200 mM HEPES pH 7.4 in order to compare stimulation-induced recycling with spontaneous recycling. After dilution to 1x, add 1 µM of Tetrodotoxin before use to block action potential generation.
   1. Dissolve 1.87 g of NaCl, 2.61 g of KCl, 0.1 g of MgCl₂-6H₂0, 0.15 g of CaCl₂-2H₂O and 3.0 g of glucose-1 H₂O and 2.38 g of HEPES in 50 mL of distilled water in an Erlenmeyer flask. Adjust the pH with NaOH and sterile-filter the solution. Dilute the buffer 1:10 in distilled water to achieve a 1x concentration.
2. Prepare 4% paraformaldehyde (PFA) in 1x PBS (pH 7.4) for fixation after stimulation.
   1. For 500 mL of 4% PFA in 1x PBS, dissolve 20 g of paraformaldehyde in 450 mL of distilled H₂O.
      Note: Heating the solution may speed up dissolving, but do not heat the solution over 70 °C, as the PFA may disintegrate.
      Caution: PFA is toxic, potentially carcinogenic, and teratogenic. Wear gloves when working with PFA, work under the fume hood, and avoid ingestion.
   2. Let the solution cool to RT and add 50 mL of 10x PBS stock solution. Adjust the pH to 7.4 with NaOH/HCl using a pH meter.
3. Pre-warm 600 µL of 1x depolarization buffer and 10 mL of cell culture medium to 37 °C in the water bath.
4. Add 1 µL of mouse anti-Syt1 antibody (clone 604.2) to the 1x depolarization buffer and vortex for 10 seconds.
5. Remove and discard the cell culture medium from the cells. Add 200 µL of the depolarization-antibody mix to each well and incubate for 5 minutes at 37 °C and 5% CO₂ in the incubator.
6. Remove and discard the depolarization-antibody mix and wash it three times with cell culture medium. Add 1 mL of cell culture medium to each well and incubate for 30 seconds at RT. Remove 750 µL of medium and add the same amount of fresh medium. Repeat three times.
7. Remove and discard the cell culture medium and add 300 µL of 4% PFA in 1x PBS. Incubate for 20 minutes at 4 °C.
8. Wash three times for 5 minutes each with 1x PBS.
   Note: The protocol can be paused here.

4. Immunocytochemistry

1. Prepare 50 mL of blocking buffer.
   Note: Blocking buffer can be kept at -20 °C for several months.
   1. Dissolve 2.5 g of sucrose and 1 g of bovine serum albumin (BSA) in 5 mL of 10x PBS stock solution. Add 1.5 mL of 10% detergent stock solution. Stir the solution until all the components have properly dissolved and add distilled H₂O until reaching a final volume of 50 mL. Aliquot the solution and freeze the aliquots for storage.
2. Dilute the secondary, fluorophore-coupled antibody (directed against the primary Syt1-antibody species) in 200 µL of blocking buffer in each well at a dilution of 1:1000.
3. Remove and discard the 1x PBS from each well containing a coverslip.
4. Add 200 µL of blocking buffer-antibody mix to each well and incubate for 60 minutes at RT.
   Caution: Because the secondary antibodies are light-sensitive, all steps moving forward must be performed in the dark.
5. After incubation, wash the cells three times for 5 minutes with 1 mL of 1x PBS.
6. Embed the coverslips on microscope slides with the embedding medium.
   1. Add a 7 µL drop of embedding medium onto the microscope slide. Remove the coverslip from the 24-well plate by lifting it with a syringe and grabbing it with forceps.
      Caution: Cells on the surface of the coverslip are easily damaged, so forceps must be handled with care.
   2. Dip the coverslip into distilled water to remove the PBS and dry it carefully by touching one edge to a soft tissue.
   3. 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.
7. Leave the slides to dry under the hood for 1 - 2 h (cover them to avoid light exposure) and store them in a microscope slide box at 4 °C.
   Note: The protocol can be paused here.

5. Microscopic Analysis

1. After the coverslips dry, place them under the microscope with the objective and camera.
2. Adjust the exposure time for every channel so that few pixels are overexposed to ensure maximum distribution of grey values.
   Note: While the exposure time may vary between the channels, it should be constant for one channel to ensure comparability between the coverslips.
3. Acquire multi-channel images for 10 regions of interest (ROIs) per coverslip. Check that the ROI contains axonal processes from a transfected neuron by checking GFP-fluorescence, which should be punctate.

6. Statistical Analysis

1. Export the images as .tif files. Load the images into OpenView¹⁸ by clicking File | Load image file.
2. Choose the Synaptophysin-mOrange image as channel 1, the Rogdi-GFP/mGFP image as channel 2, and the Alexa647 fluorescence image as channel 3.
3. Threshold the ROIs.
   1. Click Analysis | Place area over puncta. Choose Threshold and Delta intensity values so that upon visual inspection, diffuse fluorescence is excluded, leaving only punctate signals in the image of channel 1 (representing Synaptophysin-mOrange fluorescence). Keep the same threshold for all images.
4. Transfer these ROIs to the corresponding channel 2 (GFP-Rogdi/mGFP fluorescence) and 3 (Syt1-fluorescence) of each coverslip area by clicking Execute now.
   Note: The ROIs should only be considered if the cell is double-transfected. In this method, mOrange- and GFP-fluorescence should be clearly visible.
5. Save the data into the analysis log editor by clicking Log data.
6. Open the analysis log editor under the Windows tab and copy the values for each channel. Paste the values for the separate channels into a spreadsheet.
7. Determine the average fluorescence intensity of the Syt1-channel at the ROIs in both transfection conditions (GFP-Rogdi and mGFP).
8. Apply appropriate statistical tests such as Student’s t-test to determine significant differences.

Wyniki

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 (

Dyskusje

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...

Materiały

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