Name: measuring synaptic vesicle endocytosis in cultured hippocampal neurons
Uploaded: 2017-09-04T12:30:00
Description: Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

Vi takker Dr. Yong-Ling Zhu for å gi synaptophysin-pHluorin2x konstruksjon og Dr. James E. Rothman for å gi VAMP2-phluorin. Vi takker Dr. Susan Cheng og Virginia Crocker NINDS elektronmikroskop anlegg for kundestøtte og hjelp. Dette arbeidet ble støttet av National Institute av nevrologiske lidelser og hjerneslag Intramural Research Program i USA og et stipend fra KRIBB initiativ forskningsprogrammet (koreansk biomedisinsk forsker Fellowship Program), Korea forskningsinstitutt Bioscience og bioteknologi, Sør-Korea.

Merk: følgende protokollen beskriver pHluorin tenkelig metoder og EM-metoder som brukes i kulturperler hippocampus neurons. pHluorin skjermer synaptic vesicle protein opptak i levende celler og EM oppdager opptak av synaptic vesicle og ultrastructural endringer.
 dyr omsorg og prosedyre følges NIH retningslinjer ble godkjent av NIH dyr omsorg og bruk komiteen.
 1. pHluorin Imaging <ol><li>hippocampus Nevron kultur <ol><li>forberede Hippocampus ...

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Her viser vi to metoder for å kartlegge synaptic vesicle endocytose. I den første metoden overvåket vi pHluorin smeltet sammen med et synaptic vesicle protein i transfekterte nevroner og senere påtrykkes. Dernest brukte vi EM avbilding av HRP opptak som forårsaket av KCl. Vi brukte ulike stimuli for to grunner. Først høyt kalium programmet induserer depolarization for alle neurons i kulturen. Dette forenkler EM undersøkelse, gitt at vår EM-metode ikke kan skille mellom ikke-stimulert og stimulert neurons. Andre,...

Nevrotransmittere er lagret i synaptic blemmer og utgitt av exocytosis. Den synaptiske vesicle membran protein er internalisert endocytose, og på nytt i neste runde av exocytosis. Endocytose av synaptic blemmer er viktig for å opprettholde synaptic vesicle bassenger og fjerner utstående blemmer fra plasma membranen. PH-sensitive grønne fluorescerende protein pHluorin, som er slukket under Sure forhold og dequenched i nøytral pH, har blitt brukt til å måle endocytose tiden kurs i lever celler1,2,3. PHluorin protein er vanligvis knyttet til lumenal siden av synaptic vesicle proteiner, for eksempel synaptophysin eller VAMP2/synaptobrevin. Ved hvile, er pHluorin slukket i 5,5 pH lumen av synaptic blemmer. Vesicle fusion å plasma membranen eksponerer den vesicula lumen til ekstracellulære løsning der pH er ~ 7.3, resulterer i en økning i pHluorin fluorescens. Etter exocytosis, økt fluorescens henfaller, på grunn av endocytose synaptic vesicle proteiner etterfulgt av vesicle re forsuring innenfor de gjenopprettede blemmer. Selv om forfallet gjenspeiler både endocytose og vesicula re forsuring, gjenspeiler hovedsakelig endocytose, fordi re forsuring er raskere enn endocytose i de fleste forhold1,4. Tiden konstant av re forsuring er 3-4 s eller mindre5,6, som er generelt raskere enn 10 s eller mer kreves for vesicle endocytose4,5. Hvis eksperimenter er nødvendig å skille endocytose fra re forsuring, kan acid slukke eksperimenter med 4-Morpholineethanesulfonic acid (MES) løsningen (25 mM) med en pH på 5.5 brukes til å bestemme om synaptic vesicle proteiner er Hentet fra plasma membranen via endocytose1,3,4. Dermed pHluorin fluorescens intensitet økningen gjenspeiler en balanse av exo- og endocytose, og nedgangen etter nervestimulering spesifikt gjenspeiler endocytose.
pHluorin imaging kan brukes ikke bare til å måle tid selvfølgelig endocytose, men også størrelsen på synaptic vesicle bassenger7,8, og sannsynligheten for vakte utgivelsen og spontan slipp9. Mange faktorer og proteiner involvert i å regulere endocytose, som kalsium, løselig NSF-vedlegg protein reseptor (SNARE) proteiner, hjerne-avledet nevrotropisk factor(BDNF) og calcineurin har blitt identifisert ved hjelp av pHluorin bildebehandling1 , 2 , 10 , 11 , 12 , 13 , 14 , 15 , 16. videre utgivelsen av nevrotransmitter kan oppdages i ikke bare primære neurons men i neuroblastom celler med TIRFM17. Nylig ble pHluorin varianter, dsRed, mOrange og pHTomato utviklet for å overvåke samtidig opptak av flere faktorer i en enkelt synapse18,19. For eksempel er pHTomato smeltet sammen med synaptophysin og brukes med en genetisk kodet kalsium indikator (GCaMP5K) til å overvåke presynaptic vesicle fusion og Ca2 + tilstrømningen i postsynaptic rommet20. Derfor gir pHluorin knyttet til synaptic proteiner en nyttig metode for å analysere forholdet mellom endocytose og exocytosis.
EM er en annen metode brukes vanligvis til å studere endocytose, på grunn av høy romlig oppløsning som viser ultrastructural endringer under endocytose. To generelle områder er muligheten til å visualisere patologiske forandringer i neuronal celler21 og spore vesicle proteiner22. Spesielt observasjon av synaptic vesicle opptak, membran kurvatur belagt med clathrin i den periactive sonen, og endosomal strukturer er mulig med EM3,23,24,25 ,26,27,28. Mens EM involverer potensielle artefakter som bindemiddel-indusert misdannelser, som kan påvirke endocytose og dataanalyse er arbeidsintensiv, oppløsningen gir en attraktiv mulighet å visualisere cellestruktur. Potensielle etappe, den stabiliserende problemer og begrensning i EM midlertidig løsning kan overvinnes ved høyt trykk frysing, gir en rask og ikke-kjemisk metode til å stabilisere skjøre strukturer under endocytose27.

<table>
	<tbody>
		<tr>
			<td>Lipofectamine LTX with Plus</td>
			<td>Thermo Fisher</td>
			<td>15338-100</td>
			<td>Transfection of plasmid DNA including synaptophysin or VAMP2-pHluorin</td>
		</tr>
		<tr>
			<td>neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td>Growth medium for neuron, Warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermo Fisher</td>
			<td>17504-044</td>
			<td>Gradient for neuronal differentiation</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td>Gradient for neuronal culture</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine coated coverslip</td>
			<td>Neuvitro</td>
			<td>GG-25-pdl</td>
			<td>Substrate for neuronal growth and imaging of pHluorin</td>
		</tr>
		<tr>
			<td>Trypsin XI from bovine pancrease</td>
			<td>Sigma</td>
			<td>T1005</td>
			<td>Neuronal culture-digest hippocampal tissues</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td>Neuronal culture-inhibits viscous cell suspension</td>
		</tr>
		<tr>
			<td>pulse stimulator</td>
			<td>A-M systems</td>
			<td>model 2100</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>Slotted bath with field stimulation</td>
			<td>Warner Instruments</td>
			<td>RC-21BRFS</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>stimulus isolation unit</td>
			<td>Warner Instruments</td>
			<td>SIU102</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>lubricant</td>
			<td>Dow corning</td>
			<td>111</td>
			<td>pHluorin imaging-seal with coverslip and imaging chamber, avoid leak from chamber</td>
		</tr>
		<tr>
			<td>AP5</td>
			<td>Tocris</td>
			<td>3693</td>
			<td>Gradient for normal saline, selective NMDA receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>190</td>
			<td>Gradient for normal saline, competitive AMPA/kainate receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Nikon</td>
			<td>C-HGFI</td>
			<td>Metal halide light source for pHluorin</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3</td>
			<td>pHluorin imaging, detect pHluorin fluorescence intensity</td>
		</tr>
		<tr>
			<td>Inverted microscopy</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td>Imaging for synaptophysin or VAMP2 pHluorin transfected cells</td>
		</tr>
		<tr>
			<td>NIS-Elements AR</td>
			<td>Nikon</td>
			<td>NIS-Elements Advanced Research</td>
			<td>Software for imaging acquisition and analysis</td>
		</tr>
		<tr>
			<td>Igor Pro</td>
			<td>WaveMetrics</td>
			<td>Igor pro</td>
			<td>Software for imaging analysis and data presentation</td>
		</tr>
		<tr>
			<td>imaging chamber</td>
			<td>Warner Instruments</td>
			<td>RC21B</td>
			<td>pHluorin imaging, apply field stimulation on living cells</td>
		</tr>
		<tr>
			<td>poly-l-lysine</td>
			<td>Sigma</td>
			<td>P4832</td>
			<td>Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase(HRP)</td>
			<td>Sigma</td>
			<td>P6782</td>
			<td>Electron microscopy, labeling of endocytosed synaptic vesicles by catalyzing DAB in presence hydrogen peroxide, final concentration is 5 mg/mL in normal saline, make fresh before use</td>
		</tr>
		<tr>
			<td>Na cacodylate</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td>Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N</td>
		</tr>
		<tr>
			<td>3,3&#8242;-Diaminobenzidine(DAB)</td>
			<td>Sigma</td>
			<td>D8001</td>
			<td>Electron microscopy, labeling of endocytosed synaptic vesicles, substrate for HRP, final concentration is 0.5 mg/mL in DDW and filtered, make fresh before use</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16365</td>
			<td>Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use</td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>JEOL</td>
			<td>200CX</td>
			<td>Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes</td>
		</tr>
		<tr>
			<td>CCD digital camera</td>
			<td>AMT</td>
			<td>XR-100</td>
			<td>Electron microscopy, capturing images</td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Leica microsystems</td>
			<td>16707235</td>
			<td>Electron microscopy, grid staining</td>
		</tr>
	</tbody>
</table>

measuring synaptic vesicle endocytosis in cultured hippocampal neurons

Under endocytose hentes smeltet synaptic blemmer på nerve terminaler, slik at vesicle resirkulering og dermed vedlikehold av synaptic overføring under repeterende nerve avfyring. Nedsatt endocytose patologiske forhold fører til nedgang i synaptiske styrke og hjernen fungerer. Her beskriver vi metoder brukes til å måle synaptic vesicle endocytose på pattedyr hippocampus synapse i neuronal kultur. Vi overvåket synaptic vesicle protein endocytose kombinerer en synaptic vesicula membran protein, inkludert synaptophysin og VAMP2/synaptobrevin, på vesicula lumenal side, med pHluorin, en pH-sensitive grønne fluorescerende protein som øker sin fluorescens intensiteten som pH øker. Under exocytosis, vesicula lumen pH øker, mens under endocytose vesicula lumen pH er nytt sur. Dermed indikerer en økning av pHluorin fluorescens fusion, mens en nedgang angir endocytose av merket synaptic vesicle protein. I tillegg til metoden pHluorin imaging for å registrere endocytose, overvåket vi vesicula membran endocytose av elektronmikroskop (EM) målinger av pepperrot peroxidase (HRP) opptak av blemmer. Til slutt, vi overvåket dannelsen av nerve terminalen membran groper i ulike tider etter høy kalium-indusert depolarization. Time course of HRP opptak og membran grav formasjon angir time course of endocytose.

Med metoden lipid carrier ble SpH uttrykt i hippocampus neurons, tillater for identifisering av boutons (figur 1a). Elektrisk stimulering av cellene indusert exocytosis, og en tilsvarende økning i fluorescens intensitet. Økningen i fluorescens (ΔF) ble stoppet av slutter stimulans (figur 1b). Den økte fluorescensen ble etterfulgt av en langsom nedgang, på grunn av endocytose. I VAMP2-pHluorin diffunderer VAMP2 langs axon fra...

Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Synaptiske vesicle endocytose oppdages ved * lys av pHluorin smeltet sammen med synaptic vesicle protein og elektronmikroskop for vesicle opptak.

Måle Synaptic Vesicle endocytose i kulturperler hippocampus nerveceller

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

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 ...

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 av Synaptic vesicle Pool Etterfylling i Cultured lillehjernen Granule Neurons bruker FM Fargestoffer

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

Inducing dendrittiske Veksten i dyrkede sympatiske 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

Lateral Diffusjon og eksocytose av membran proteiner i dyrkede Neurons vurdert ved hjelp av Fluorescens Recovery og fluorescens-tap photobleaching

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

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

Påvisning av Protein palmitoylation i Cultured hippocampus neurons ved Immunoutfellingsanalyse og Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Analysere DNA-skader i hippocampus Neurons kometsystemet

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Post-embedding immunogold merking av Synaptic Proteiner i hippocampus Slice kulturer

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

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

Undersøkelse av Synaptic Vesikkel Recycling Bruke FM Fargestoffer Under fremkalt, spontan, og miniatyr Synaptic Aktiviteter

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...