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

Wij bedanken Dr. Yong-Ling Zhu voor het verstrekken van synaptophysin-pHluorin2x-construct, en Dr. James E. Rothman voor het verstrekken van VAMP2-phluorin. Wij danken Dr. Susan Cheng en Virginia Crocker van NINDS elektronenmicroscopie faciliteit voor hun technische ondersteuning en hulp. Dit werk werd gesteund door de National Institute of Neurological Disorders en beroerte intramurale Research Program in de Verenigde Staten en een subsidie van het KRIBB initiatief onderzoeksprogramma (Korean biomedisch wetenschapper Fellowship Program), Korea Research Institute van Biowetenschappen en biotechnologie, Republiek Korea.

Opmerking: het volgende protocol beschrijft de pHluorin imaging en EM methoden gebruikt bij gekweekte hippocampal neuronen. pHluorin monitoren synaptic vesikel eiwit opname in levende cellen en EM detecteert opname van synaptic blaasje en Ultrastructurele wijzigingen.
 verzorging van de dieren en procedure zijn goedgekeurd door de NIH Animal Care en gebruik Comité NIH richtsnoeren gevolgd.
 1. pHluorin Imaging <ol><li>Hippocampal neuron cultuur ...

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Hier tonen we twee methoden voor de controle van de synaptische vesikel endocytose. In de eerste methode gecontroleerd we pHluorin met een synaptic vesikel eiwit in transfected neuronen gesmolten en vervolgens elektrisch gestimuleerd. Ten tweede hebben we gebruikt EM beeldvorming van HRP opname zoals geïnduceerd door KCl. We gebruikten verschillende prikkels om twee redenen. Ten eerste, toepassing van hoge kalium induceert depolarisatie van alle neuronen in de cultuur. Dit vergemakkelijkt EM onderzoek, gezien het feit d...

Neurotransmitters zijn opgeslagen in synaptische vesikels en uitgebracht door exocytose. De synaptische vesikel membraan eiwit zijn geïnternaliseerd door endocytose en hergebruikt in de volgende ronde van exocytose. Endocytose van synaptische vesikels is belangrijk voor het behoud van synaptic vesikel zwembaden en uitstekende blaasjes verwijdert uit het plasma-membraan. De pH-gevoelige groen fluorescent proteïne pHluorin, die is uitgeblust in zure omstandigheden en dequenched in neutrale pH, is gebruikt om endocytose tijd cursussen in cellen1,2,3 levendete meten. Het pHluorin eiwit is meestal gekoppeld aan de lumenal kant van synaptic vesikel eiwitten, zoals synaptophysin of VAMP2/synaptobrevin. In rust, is de pHluorin in de 5,5 pH lumen van synaptische vesikels uitgeblust. Vesikel fusie aan het plasma-membraan blootstelt de vesiculaire lumen aan de extracellulaire oplossing waar de pH ~ 7.3 is, resulterend in een stijging in pHluorin fluorescentie. Na exocytose vervalt de verhoogde fluorescentie, als gevolg van endocytose van synaptic vesikel eiwitten gevolgd door vesikel opnieuw verzuring binnen deze herstelde blaasjes. Hoewel het verval zowel endocytose en vesiculaire opnieuw verzuring weerspiegelt, hierin meestal endocytose, omdat opnieuw verzuring sneller dan endocytose in de meeste omstandigheden1,4 is. De tijdconstante van opnieuw verzuring is 3-4-s of minder5,6, dat over het algemeen sneller dan de 10 is s of meer vereist voor vesikel endocytose4,5. Als experimenten nodig zijn voor het onderscheiden van endocytose van opnieuw verzuring, kunnen zuur blussen experimenten met behulp van de 4-Morpholineethanesulfonic (MES) zuuroplossing (25 mM) met een pH van 5.5 worden gebruikt om te bepalen of synaptic vesikel eiwitten worden opgehaald uit het plasma-membraan via endocytose1,3,4. Dus, de verhoging van de intensiteit van de fluorescentie van pHluorin weerspiegelt een balans van exo- en endocytose en de daling na zenuwstimulatie specifiek weerspiegelt endocytose.
pHluorin imaging mogen worden gebruikt niet alleen voor het meten van het tijdsverloop van endocytose, maar ook de grootte van synaptic vesikel zwembaden7,8, en de waarschijnlijkheid van evoked introductie en spontane release9. Vele factoren en eiwitten die betrokken zijn bij het reguleren van endocytose, zoals calcium, oplosbare NSF-gehechtheid eiwit receptor (SNARE) eiwitten, hersenen-afgeleide neurotrophic factor(BDNF) en calcineurin zijn geïdentificeerd met behulp van pHluorin imaging1 , 2 , 10 , 11 , 12 , 13 , 14 , 15 , 16. Bovendien, vrijlating van de neurotransmitter in niet alleen primaire neuronen maar in cellen van het neuroblastoom met TIRFM17kon worden opgespoord. Onlangs, pHluorin varianten, dsRed, mOrange en pHTomato werden ontwikkeld voor monitoring van gelijktijdige opnamen van meerdere factoren in een enkele synapse18,19. Bijvoorbeeld, is pHTomato gefuseerd met synaptophysin en gebruikt met een genetisch gecodeerde calcium indicator (GCaMP5K) voor het bewaken van de presynaptische vesikel fusion en Ca2 + toestroom in de postsynaptisch compartiment20. Daarom, pHluorin gekoppeld aan synaptic eiwitten biedt een handige methode voor het analyseren van de relatie tussen endocytose en exocytose.
EM is een andere methode gebruikt bij het bestuderen van endocytose, als gevolg van de hoge ruimtelijke resolutie waarin ultrastructurele wijzigingen tijdens endocytose. Twee algemene gebieden zijn de capaciteit om te visualiseren pathologische veranderingen binnen de neuronale cellen21 en bijhouden van vesikel eiwitten22. In het bijzonder, de waarneming van synaptic vesikel opname, membraan kromming bekleed door clathrin in de periactive zone en endosomal structuren zijn mogelijk met EM3,23,24,25 ,26,27,28. Terwijl EM potentiële artefacten, zoals fixeer-geïnduceerde misvormingen impliceert, die van invloed kunnen zijn op de endocytose en data-analyse is arbeidsintensief, dat de resolutie biedt een aantrekkelijke mogelijkheid om te visualiseren van cellulaire structuur. Potentiële kleefpoeders problemen en de beperking in EM temporele resolutie kunnen worden overwonnen door de hoge druk bevriezing, het verstrekken van een snelle en niet-chemische methode voor het stabiliseren van de delicate structuren aanwezig tijdens 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

Tijdens endocytose, worden gesmolten synaptische vesikels opgehaald bij zenuw terminals, waardoor voor vesikel recycling en dus het onderhoud van synaptische transmissie tijdens herhaalde zenuw afvuren. Verminderde endocytose in pathologische omstandigheden leidt tot afname van de synaptische sterkte en hersenen functies. Hier beschrijven we methoden voor het meten van synaptic vesikel endocytose in de synaps zoogdieren hippocampal in neuronale cultuur. We synaptic vesikel eiwit endocytose gecontroleerd door het smelten van een synaptic vesiculaire membraan eiwit, inclusief synaptophysin en VAMP2/synaptobrevin, de vesiculaire lumenal kant, met pHluorin, een pH-gevoelige groen fluorescent proteïne dat verhoogt haar de intensiteit van de fluorescentie aangezien de pH stijgt. Tijdens exocytose, vesiculaire lumen pH verhoogt, terwijl tijdens endocytose vesiculaire lumen pH opnieuw aangezuurde is. Dus geeft een stijging van pHluorin fluorescentie intensiteit fusion, overwegende dat een daling endocytose van het gelabelde synaptic vesikel-eiwit blijkt. Naast het gebruik van de beeldvorming methode pHluorin opnemen endocytose, gecontroleerd we vesiculaire membraan endocytose door elektronenmicroscopie (EM) metingen van Horseradish peroxidase (HRP) opname door blaasjes. Tot slot, wij gecontroleerd de vorming van zenuw terminal membraan kuilen op verschillende tijdstippen na hoog kalium-geïnduceerde depolarisatie. Het tijdsverloop van de vorming van HRP opname en membraan pit geeft aan het tijdsverloop van endocytose.

Met behulp van de methode van de vervoerder lipide, is SpH uitgedrukt in de hippocampal neuronen, waardoor voor de identificatie van Los (Figuur 1a). Elektrische stimulatie van de cellen geïnduceerde exocytose en een overeenkomstige stijging van de intensiteit van de fluorescentie. De verhoging van fluorescentie (ΔF) werd gestopt door beëindiging van de stimulus (Figuur 1b). De verhoogde fluorescentie werd gevolgd door een lan...

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

Synaptic vesikel endocytose is gedetecteerd door de lichte microscopie van pHluorin gefuseerd met synaptic vesikel eiwit en elektronenmicroscopie van vesikel opname.

Meten van Synaptic vesikel endocytose in gekweekte Hippocampal neuronen

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

Studeren Synaptic Blaasjes zwembaden met behulp van Photoconversion van styrylkleurstoffen

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

Fluorescence Recovery After Photobleaching (FRAP) van de fluorescentie gemerkte eiwitten in Dendritische stekels van Cultured hippocampus Neuronen

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

Analyse van de dendritische Spine Morfologie in gekweekte CNS Neuronen

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

Kwantitatieve analyse van synaptische Blaasjes Pool Replenishment in gekweekte Cerebellaire Korrel Neuronen met behulp van FM-kleurstoffen

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

Het induceren van dendritische groei in gekweekte sympathische neuronen

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

Laterale diffusie en Exocytose van membraaneiwitten in gekweekte zenuwcellen gemeten met behulp van Fluorescence Recovery en fluorescentie-verlies fotobleken

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

Detectie van eiwit palmitoylering in gekweekte hippocampale neuronen door immunoprecipitatie en 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

Analyseren DNA schade in hippocampale neuronen Met behulp van de Comet Assay

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-inbedding immunogold Etikettering van Synaptic eiwitten in de hippocampus slice culturen

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

Onderzoek van de synaptische vesikel Recycling FM Kleurstoffen Tijdens Evoked, Spontaan, en Miniature Synaptic Activiteiten

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