Name: an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins
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Vi takker Irmgard Weiss til teknisk ekspertbistand. Dette arbejde blev støttet af DFG via klyngen af ekspertise til mikroskopi på rækken nanometer og molekylær fysiologi af hjernen (CNMPB, B1-7, at T.D.).

Ingen undersøgelser med levende dyr blev gennemført. Eksperimenter, der involverer aflivede dyr for at opnå celle kulturer blev godkendt af de lokale dyrebeskyttelse myndigheder (Tierschutzkommission der Universitätsmedizin Göttingen) i henhold til godkendelsen nummer T10/30. Forsøgene blev udført med de godkendte protokoller.
1. primære hippocampus cellekultur
<ol><li>Forberede de dissocierede cellekultur af rotte hippocampus embryonale dag 1916,<s...

<ol>
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L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Hippocampal+Neurons+from+Prenatal+Mice.">Isolation and Culture of Hippocampal Neurons from Prenatal Mice.</a> Journal of Visualized Experiments. (65), 4-9 (2012).</li><li>Tsuriel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+sharing+as+a+predominant+determinant+of+synaptic+matrix+molecular+dynamics.">Local sharing as a predominant determinant of synaptic matrix molecular dynamics.</a> PLOS Biology. 4, 1572-1587 (2006).</li><li>Iwabuchi, S., Kakazu, Y., Koh, J., Goodman, K. M., Harata, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Synaptic+Vesicle+Recycling+Using+FM+Dyes+During+Evoked,+Spontaneous,+and+Miniature+Synaptic+Activities.">Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities.</a> Journal of Visualized Experiments. (85), 1-10 (2014).</li><li>Villarreal, S., Lee, S. H., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Synaptic+Vesicle+Endocytosis+in+Cultured+Hippocampal+Neurons.">Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.</a> Journal of Visualized Experiments. (127), 1-8 (2017).</li><li>Kavalali, E. T., Jorgensen, E. 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E., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+is+dispensable+for+sorting+of+synaptotagmin+1+but+is+critical+for+targeting+of+SV2+and+synaptophysin+to+recycling+synaptic+vesicles.">Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV2 and synaptophysin to recycling synaptic vesicles.</a> Journal of Biological Chemistry. 287, 35658-35668 (2012).</li><li>Afuwape, O. A. T., Wasser, C. R., Schikorski, T., Kavalali, E. 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Der er tre assays rutinemæssigt anvendes til at studere synaptic vesikel (SV) genbrug. De første to omfatter anvendelse af en) fluorescerende styryl farvestoffer såsom FM1-43 (som indarbejde membraner, tages i organeller under endocytose og frigives efter exocytose); og b) fluorescently mærkede rekombinante SV proteiner (som ved overekspression, indarbejde de proteinholdige genanvendelse maskiner). Hvis de vedhæftede fluorophores ændrer deres fluorescens afhængigt af pH, kan de bruges til at overvåge ændringer m...

Studerer synaptic vesikel genanvendelse er vigtigt ved fastsættelsen, hvordan præsynaptiske egenskaber ændre, enten under synaptisk plasticitet eller svar til undertrykkelse af netbårne synaptiske funktion. At studere Synaptotagmin-1 (Syt1) giver antistof optagelse én metode til måling af mængden af SV genanvendelse. Syt1 er en SV-forbundet protein, der fungerer som en Ca2 + -sensor og er nødvendige for at exocytotic udgivelsen af neurotransmitter1,2. Det er en transmembrane protein med en C-terminale cytoplasmatisk domæne uden SV og en N-terminale luminale domæne inde SV3. Under exocytose, bliver den luminale domæne af Syt1 udsat for den eksterne medium. Til denne eksterne medium tilføje vi antistoffer rettet mod den cytoplasmatisk domæne, som bliver internaliseret under endocytose. Disse antistoffer kan være enten pre konjugeret med fluorophores eller immunostained med sekundær antistoffer4,5,6,7. Fluorescens-intensiteten af den resulterende immunosignal er proportional med mængden af SV genanvendelse. Denne fremgangsmåde kan bruges til at bestemme både konstituerende og depolarisering-induceret SV genanvendelse6,8.
Syt1 optagelse assays kan udføres efter virus-medieret genoverførsel til stort set alle celler i skålen eller sparsomme Transfektion af et lille antal celler. Vores metode kombinerer analysen med sparsom dobbelt Transfektion af primære hippocampus neuroner ved hjælp af calciumphosphat9. Vi bruger en rekombinant markør protein kendt at akkumulere på presynapses, fluorescently mærket Synaptophysin, til at finde præsynaptiske terminaler og overexpress vores protein af interesse, Rogdi. Dette giver os mulighed for at teste eller ej Rogdi mål funktionelle synapser og påvirker SV genanvendelse. Det gen, der koder Rogdi blev oprindeligt identificeret i en skærm for Drosophila mutanter karakteriseret ved nedsat hukommelse10. Hos mennesker forårsage mutationer i Rogdi genet en sjælden og ødelæggende sygdom kaldet Kohlschütter-Tönz syndrom. Patienter lider af dental emalje misdannelser, pharmacoresistant epilepsi og psykomotoriske forsinkelser; dog forblev subcellulært lokaliseringen af produktets gen undvigende11. Således givet Syt1 optagelse assay afgørende bevis for lokalisering af normal god landbrugspraksis-mærket Rogdi på funktionelle synapser9.
Denne optagelse teknik har flere fordele. Først kan SV genanvendelse observeres både i realtid ved at udføre live imaging7,12, og efter fiksering6,9 ved at måle fluorescens-intensiteten af Syt1 fluorescens etiket. Derudover er flere Syt1 antistof varianter blevet udviklet. Der er ukodede varianter, der kan være mærket med en sekundær antistof efter en standard immunfarvning protokol efter fiksering og pre konjugeret varianter med et fluorescens etiket. Endelig er-antistof-baserede fluorescens fordelagtig på grund af det store udvalg af kommercielt tilgængelige sekundære eller konjugeret farvestoffer, der kan bruges.
Når fastsættelse og immunfarvning neuroner, det er også muligt at pletten for ekstra proteiner og udføre colocalization analyse. Dette kan hjælpe med at bestemme, hvor de er placeret i forhold til genanvendelse SVs. Intensiteten af fluorescens etiket er et direkte mål for mængden af SV genanvendelse. Derudover etiket antistoffer selektivt Syt1-holdige strukturer, hvilket resulterer i høj specificitet og lidt baggrund fluorescens4. Forskellige stimulation protokoller kan også bruges som depolarisering buffere eller elektrisk stimulation protokoller9,12,13,14. Basal SV genanvendelse kan dog måles uden stimulere neuronal kulturer15.
Vores metode omhandler specifikt Syt1 antistof optagelse i dobbelt-transfekteret neuroner med sekundær antistof immunolabeling efter fiksering. Men, vi henviser til alle rutinemæssigt anvendes varianter af analysen i vores diskussion at give seerne mulighed for at tilpasse protokollen til specifikke behov.

<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

På aktive præsynaptiske nerve terminaler gennemgå synaptiske vesikler cyklusser af exo- og endocytose. Under genanvendelse, bliver de luminale domæner af SV transmembrane proteiner eksponeret på cellens overflade. En af disse proteiner er Synaptotagmin-1 (Syt1). Et antistof rettet mod den luminale domæne af Syt1, når de er tilføjet til næringssubstratet, er taget i løbet af exo-endocytotic cyklus. Denne optagelse er proportional med mængden af SV genbrug og kan kvantificeres ved immunfluorescens. Her, kombinerer vi Syt1 antistof optagelse med dobbelt Transfektion af kulturperler hippocampus neuroner. Dette giver os (1) lokalisere præsynaptiske websteder baseret på udtryk for rekombinante præsynaptiske markør Synaptophysin, (2) bestemme deres funktionalitet ved hjælp af Syt1 udbredelse og (3) karakteriserer målretning og virkningerne af en protein af interesse, normal god landbrugspraksis-Rogdi.

En forventet resultat af denne tilgang er at finde ca 50 dobbelt-transfekteret neuroner pr. coverslip med en tæthed på 50.000 neuroner pr. brønd. Axon af hvert neuron forventes at vise flere hotspots fluorescently markeret Synaptophysin akkumulering, der angiver klynger ofSVs. På funktionelle præsynaptiske websteder colocalizes den rekombinante Synaptophysin signal med punktformet Syt1 fluorescens. Brug dobbelt Transfektion, er enten normal god landbrugspraksis-Rogdi som protein af i...

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

Vi beskriver en optisk assay for synaptic vesikel (SV) genbrug i kulturperler neuroner. Kombinere denne protokol med dobbelt Transfektion udtrykke en præsynaptiske markør og protein af interesse tillader os at lokalisere præsynaptiske websteder, deres synaptic vesikel genanvendelse kapacitet, og bestemme rollen af protein af interesse.

En optisk Assay for Synaptic vesikel genanvendelse i kulturperler neuroner overekspression præsynaptiske proteiner

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

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

Kvantitativ analyse af Synaptic vesikel Pool Replenishment i dyrkede cerebellare Granule Neuroner bruger FM Farvestoffer

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

Fremkalde dendritisvækst i dyrkede sympatiske neuroner

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

Lateral Diffusion og exocytose af membranproteiner i dyrkede neuroner vurderes ved hjælp af Fluorescens Recovery og Fluorescens-tab fotoblegning

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In vivo to-foton billedbehandling af oplevelsesbaseret afhængige molekylære ændringer i kortikal neuroner

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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

Undersøgelse af synaptiske vesikel Genbrug Brug af FM Farvestoffer Under Evoked, spontan, og Miniature Synaptiske aktiviteter

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

Differential Mærkning af Celle-overflade og internaliseres Proteiner efter Antibody Fodring Live dyrkede neuroner

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