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EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Primary Hippocampal Cell Culture
<ol>
	<li>Prepare the dissociated cell culture of the rat hippocampus on embryonic day 19. 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.</li>
	<li>Culture the neurons for 3 days (day&#160;in vitro&#160;(DIV) 3) in a 24-well plate in the incubator at 37 &#176;C with 5% CO2.</li>
	<li>Assess the coverslips for indicators of cell health using transmitted light microscopy (e.g.,&#160;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.</li>
</ol>
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.
<ol>
	<li>Prepare 500 mL of transfection buffer (274 mM sodium chloride, NaCl, 10 mM potassium chloride, KCl, 1.4 mM disodium hydrogen phosphate, Na2HPO4, 15 mM glucose, 42 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES) in an Erlenmeyer flask.
	<ol>
		<li>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.</li>
		<li>Adjust the pH to 6.95 with 1 M sodium hydroxide, NaOH using a pH meter.</li>
		<li>Adjust the volume with distilled water to 500 mL and check the pH using a pH meter.</li>
		<li>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.</li>
		<li>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 green fluorescent protein (GFP). Determine the number of transfected cells per coverslip for every transfection buffer pH value to assess which buffer works the best.</li>
		<li>Aliquot the buffer with the highest transfection efficiency into 2 mL microcentrifuge tubes Freeze and store the tubes at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Pre-warm the reduced serum medium, cell culture medium, and distilled water to 37 &#176;C in the water bath.</li>
	<li>Prepare the transfection mix in a 1.5 mL microcentrifuge tube. Work under the laminar flow hood to ensure sterile working conditions.
	<ol>
		<li>Mix 7.5 &#181;L of 2 M calcium chloride with 4 &#181;g of each endotoxin-free DNA (Synaptophysin-mOrange and mGFP/GFP-Rogdi). Add water to reach a total volume of 60 &#181;L in a 1.5 mL microcentrifuge tube.</li>
		<li>Add 60 &#181;L 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Under the laminar flow hood, remove the cell culture medium (&#34;conditioned medium&#34;) from the wells using a 1000 &#181;L pipet and store it in a separate container in the incubator.</li>
	<li>Add 500 &#181;L of reduced serum medium to each well. Incubate the cells at 37 &#176;C and 5% CO2&#160;until the 20-minute incubation period (step 2.3.3) is over.</li>
	<li>Add 30 &#181;L of transfection mix to each well by pipetting several drops. Discard the residue at the bottom of the tube.</li>
	<li>After all the wells have been supplied with transfection mix, gently shake the 24-well plate to ensure the distribution of the transfection mix in the medium.</li>
	<li>Incubate the wells for 60 minutes at 37 &#176;C and 5% CO2.</li>
	<li>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 &#181;L of medium and add the same amount of fresh medium. Repeat this three times.&#160;&#160;&#160;&#160; 
	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.</li>
	<li>Remove and discard the cell culture medium and add 450 &#181;L of the conditioned medium well-by-well.</li>
	<li>Let the neurons mature in the incubator at 37 &#176;C and 5% CO2&#160;to DIV 10.</li>
</ol>
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.
<ol>
	<li>Prepare 50 mL of 10x depolarization buffer (640 mM NaCl, 700 mM KCl, 10 mM magnesium chloride, MgCl2, 20 mM calcium chloride, CaCl2, 300 mM glucose, 200 mM HEPES, pH 7.4) in an Erlenmeyer flask.&#160;&#160;&#160;&#160; 
	NOTE: Depolarization buffer can be kept at 4 &#176;C for several weeks. If a non-depolarizing solution is also used, prepare a 10x Tyrode&#39;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 &#181;M of Tetrodotoxin before use to block action potential generation.
	<ol>
		<li>Dissolve 1.87 g of NaCl, 2.61 g of KCl, 0.1 g of MgCl2-6H2O, 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.</li>
	</ol>
	</li>
	<li>Prepare 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) (pH 7.4) for fixation after stimulation.
	<ol>
		<li>For 500 mL of 4% PFA in 1x PBS, dissolve 20 g of paraformaldehyde in 450 mL of distilled H2O.&#160;&#160;&#160; 
		NOTE: Heating the solution may speed up dissolving, but do not heat the solution over 70 &#176;C, as the PFA may disintegrate.&#160; 
		CAUTION: PFA is toxic, potentially carcinogenic, and teratogenic. Wear gloves when working with PFA, work under the fume hood, and avoid ingestion.</li>
		<li>Let the solution cool to RT and add 50 mL of 10x PBS stock solution. Adjust the pH to 7.4 with NaOH/HCl(hydrochloric acid) using a pH meter.</li>
	</ol>
	</li>
	<li>Pre-warm 600 &#181;L of 1x depolarization buffer and 10 mL of cell culture medium to 37 &#176;C in the water bath.</li>
	<li>Add 1 &#181;L of mouse anti-Syt1 antibody (clone 604.2) to the 1x depolarization buffer and vortex for 10 seconds.</li>
	<li>Remove and discard the cell culture medium from the cells. Add 200 &#181;L of the depolarization-antibody mix to each well and incubate for 5 minutes at 37 &#176;C and 5% CO2&#160;in the incubator.</li>
	<li>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 &#181;L of medium and add the same amount of fresh medium. Repeat three times.</li>
	<li>Remove and discard the cell culture medium and add 300 &#181;L of 4% PFA in 1x PBS. Incubate for 20 minutes at 4 &#176;C.</li>
	<li>Wash three times for 5 minutes each with 1x PBS.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The protocol can be paused here.</li>
</ol>
4. Immunocytochemistry
<ol>
	<li>Prepare 50 mL of blocking buffer.&#160;&#160; 
	NOTE: Blocking buffer can be kept at -20 &#176;C for several months.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Dilute the secondary, fluorophore-coupled antibody (directed against the primary Syt1-antibody species) in 200 &#181;L of blocking buffer in each well at a dilution of 1:1000.</li>
	<li>Remove and discard the 1x PBS from each well containing a coverslip.</li>
	<li>Add 200 &#181;L of blocking buffer-antibody mix to each well and incubate for 60 minutes at RT.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	CAUTION: Because the secondary antibodies are light-sensitive, all steps moving forward must be performed in the dark.</li>
	<li>After incubation, wash the cells three times for 5 minutes with 1 mL of 1x PBS.</li>
	<li>Embed the coverslips on microscope slides with the embedding medium.
	<ol>
		<li>Add a 7 &#181;L 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.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		CAUTION: Cells on the surface of the coverslip are easily damaged, so forceps must be handled with care.</li>
		<li>Dip the coverslip into distilled water to remove the PBS and dry it carefully by touching one edge to soft tissue.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>1,08,34,21,000</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>ThermoElectron Corporation</td>
			<td></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>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>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>1,08,60,31,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genius 3</td>
			<td>IKA</td>
			<td>3340001</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>1004</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparing cultured neurons for optical analysis of recycled synaptic vesicles

Source: Riemann, D. et al., <a href="https://app.jove.com/v/58043">An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure for preparing cultured neurons for optical analysis of recycled synaptic vesicles. The process involves fluorescence tagging of synaptic vesicle proteins and immunostaining to label and visualize the recycled vesicles within the neurons.

Preparing Cultured Neurons for Optical Analysis of Recycled Synaptic Vesicles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Primary Hippocampal Cell ...

Begin with a coverslip containing cultured neurons expressing synaptic vesicle membrane proteins, including fluorescent-tagged synaptophysin and synaptotagmin-1.
Remove the medium, add a mix of depolarization buffer and primary antibodies, then incubate.
The buffer ions enter the cells, stimulating the fusion of synaptic vesicles with the membrane, which exposes synaptotagmin-1.
The primary antibodies selectively bind to the exposed synaptotagmin-1.
Discard the buffer and wash with the medium to remove unbound antibodies.
Introduce fresh medium and incubate to facilitate membrane internalization, forming recycled vesicles with labeled synaptophysin and antibody-bound synaptotagmin-1.
Remove the medium, fix the cells, and wash.
Introduce a detergent-based blocking buffer with fluorophore-coupled secondary antibodies.
The detergent permeabilizes the cells, allowing the secondary antibodies to interact with antibody-bound synaptotagmin-1.
Lift the coverslip, wash it with water, and dry it.
Place the coverslip face-down on a slide with an embedding medium for optical analysis of the dual-labeled recycled vesicles.

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31 vues. Source : Riemann, D. et al., An Optical Assay for Synaptic Vesicule Recycling in Cultured Neuros Overexpression Presynaptic Proteins. J. Vis. Exp. (2018) Cette vidéo montre la procédure de préparation des neurones en culture pour l’analyse optique des vésicules synaptiques recyclées. Le processus implique le marquage par fluorescence des protéines des vésicules synaptiques et l’immunomarquage pour marquer et visualiser les vésicules recyclées dans les neurones. 

Vidéo: Préparation de neurones cultivés pour l’analyse optique de vésicules synaptiques recyclées - Concept

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Mesurer l’endocytose de la vésicule synaptique dans des neurones Hippocampal cultivés

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

FM colorant cyclisme au niveau de la Synapse : en comparant la dépolarisation élevés de Potassium, électrique et Stimulation de la Channelrhodopsin

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Le test basé sur la microscopie pour étudier et analyser les endosomes de recyclage à l’aide du trafic SNARE

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

Preparing Cultured Neurons for Optical Analysis of Recycled Synaptic Vesicles

Transcription

Preparing Cultured Neurons for Optical Analysis of Recycled Synaptic Vesicles