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

Dr. Yong-Ling Zhu synaptophysin pHluorin2x yapı ve Dr James E. Rothman VAMP2-phluorin sağlamak için verdiğiniz için teşekkür ederiz. Biz Dr Susan Cheng ve Virginia Crocker NINDS elektron mikroskobu tesisin kendi teknik destek ve yardım için teşekkür ederiz. Bu eser sinir hastalıkları Ulusal Enstitüsü ve kontur Intramural araştırma programının ABD ve bir hibe programından KRIBB araştırma girişimi (Korece biyomedikal bilim adamı Fellowship programı) Kore Araştırma Enstitüsü tarafından desteklenen Bioscience ve biyoteknoloji, Kore.

Not: pHluorin görüntüleme yöntemleri ve kültürlü Hipokampal nöronlar. pHluorin monitörler sinaptik vezikül protein anlamazdın canlı hücreler içinde kullanılan bir EM yöntem aşağıdaki iletişim kuralı tanımlar ve EM sinaptik vezikül alımını algılar ve ultrastructural değişiklikleri.
 hayvan bakımı ve yordam NIH yönergeleri takip ve NIH hayvan bakım ve kullanım Komitesi tarafından kabul edildi.
 1. pHluorin görüntüleme <ol><li><...

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Burada biz sinaptik vezikül endositoz izlemek için iki yöntem göstermek. İlk yöntemde, bir sinaptik vezikül protein transfected nöronlar içinde erimiş ve sonradan ortaya çıkan elektriksel olarak uyarılmış pHluorin izlenir. İkinci olarak, biz EM görüntüleme HRP Alım KCl tarafından indüklenen olarak kullanılır. Biz iki nedenden dolayı farklı uyaranlara kullanılır. İlk olarak, yüksek potasyum uygulaması depolarizasyon kültür bütün nöronların neden olmaktadır. EM metodolojimiz sigara uya...

Nörotransmitter sinaptik veziküller içinde depolanan ve ekzositozu tarafından yayımlanan. Sinaptik vezikül membran ve protein o zaman endositoz tarafından içselleştirilmiş ve ekzositozu sonraki turda yeniden. Sinaptik veziküller endositoz sinaptik vezikül havuzları korumak için önemlidir ve çıkıntılı veziküller plazma zarı kaldırır. Asidik şartlar altında su ve nötr pH dequenched, pH-duyarlı yeşil flüoresan protein pHluorin derslerde hücreleri1,2,3canlı endositoz zamanı ölçmek için kullanılmaktadır. PHluorin protein genellikle sinaptik vezikül proteinler, synaptophysin veya VAMP2/synaptobrevin gibi lumenal tarafına eklenir. İstirahat, sinaptik veziküller 5,5 pH Lümen içinde pHluorin söndürülür. Plazma zarı vezikül fusion sunar veziküler Lümen ekstraselüler çözüm pH ~ 7.3, nerede bir artış pHluorin floresan kaynaklanan. Ekzositozu sonra artan Floresans bozunmaları, endositoz vezikül yeniden asitleştirme kurtarılan Bu veziküller içinde ardından sinaptik vezikül proteinlerin nedeniyle. Endositoz ve veziküler yeniden asitleştirme çürüme yansıtan rağmen yeniden asitleştirme endositoz çoğu koşulları1,4daha hızlı olduğu için çoğunlukla endositoz, yansıtır. 3-4 s veya genellikle daha hızlı 10'dan daha az5,olan6, yeniden asitleştirme zaman sabiti olan s veya daha vezikül endositoz4,5için gerekli. Deneyler endositoz yeniden asitleştirme ayırt etmek gerekiyorsa, pH 5.5 ile 4-Morpholineethanesulfonic asit (MES) çözüm (25 mM) kullanarak asit su verme deneyleri sinaptik vezikül proteinler alınır olup olmadığını belirlemek için kullanılabilir endositoz1,3,4üzerinden plazma zarı. Böylece, pHluorin floresan yoğunluk artmak exo - ve endositoz bir denge yansıtır ve sinir stimülasyonu sonra düşüş özellikle endositoz yansıtır.
pHluorin görüntüleme sadece endositoz zaman tabii, aynı zamanda sinaptik vezikül havuzları7,8boyutunu ölçmek için kullanılabilir ve olasılık uyarılmış yayın ve spontan bırakın9. Birçok faktörler ve proteinler endositoz, kalsiyum, çözünür NSF-ek protein reseptörü (tuzak) proteinler, beyin kaynaklı Nörotrofik factor(BDNF) ve kalsinörin gibi düzenlenmesinde ilgili pHluorin görüntüleme1 kullanarak belirlendi , 2 , 10 , 11 , 12 , 13 , 14 , 15 , 16. Ayrıca, nörotransmitter sürümü yalnızca birincil nöronlar ama Nöroblastom hücreleri TIRFM17ile tespit edilemedi. Son zamanlarda, pHluorin türevleri, dsRed, mOrange ve pHTomato bir tek synapse18,19Multipl faktörler eşzamanlı kayıtları izlemek için geliştirilmiştir. Örneğin, pHTomato synaptophysin ile erimiş edilmiş ve genetik olarak kodlanmış kalsiyum göstergesiyle (GCaMP5K) presynaptic vezikül fusion ve Ca2 + akını postsinaptik yuvası20izlemek için kullanılır. Bu nedenle, sinaptik proteinler için bağlı pHluorin ekzositozu endositoz arasındaki ilişkiyi analiz etmek için yararlı bir yöntem sağlar.
EM endositoz ultrastructural değişiklikleri sırasında endositoz gösterir yüksek Uzaysal çözünürlük nedeniyle çalışma için yaygın olarak kullanılan başka bir yöntemdir. İki genel patolojik değişiklikler nöronal hücre21 içinde görselleştirmek ve vezikül proteinler22takip yeteneği alanlardır. Özellikle, clathrin periactive bölgesinde tarafından gözlem sinaptik vezikül Alım, membran eğriliği kaplı ve endosomal yapıları EM3,23,24ile,25 mümkündür ,26,27,28. Bu endositoz etkileyebilir EM malformasyonlar, sabitleştirici indüklenen gibi potansiyel yapılar içerir ve veri analizidir emek yoğun, çözünürlük hücresel yapısı görselleştirmek için cazip bir fırsat sağlar. Olası sabitleştirici sorunları ve EM zamansal çözünürlük sınırlaması dondurma, hassas yapıları endositoz27sırasında mevcut sabitleme hızlı ve kimyasal olmayan bir yöntem sağlayan yüksek basınç tarafından üstesinden gelebilir.

<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

Endositoz sırasında erimiş sinaptik veziküller vezikül geri dönüşüm ve böylece sinaptik iletimi sırasında tekrarlayan sinir ateş bakım sağlayan sinir terminallerinde alınır. Patolojik durumlarda Engelli endositoz sinaptik gücü ve beyin fonksiyonları düşüşler yol açar. Burada, sinaptik vezikül endositoz memeli Hipokampal synapse nöronal kültür, ölçmek için kullanılan yöntemleri açıklar. Sinaptik vezikül protein endositoz veziküler lumenal yan pHluorin, artar bir pH duyarlı yeşil flüoresan protein ile synaptophysin ve VAMP2/synaptobrevin, dahil olmak üzere bir sinaptik veziküler membran protein eritme tarafından izlenen onun pH olarak floresan yoğunluğu artar. Ekzositozu sırasında veziküler Lümen pH artırır, veziküler endositoz Lümen sırasında pH yeniden acidified ise. Endositoz etiketli sinaptik vezikül protein bir azalma gösterir, ancak böylece, fusion, pHluorin floresan yoğunluğu bir artış gösterir. Endositoz kaydetmek için pHluorin görüntüleme yöntemi kullanmaya ek olarak, biz membran veziküler endositoz Horseradish peroksidaz (HRP) Alım elektron mikroskobu (EM) ölçümleri tarafından veziküller tarafından izlenir. Son olarak, yüksek potasyum kaynaklı depolarizasyon sonra çeşitli zamanlarda sinir terminal membran çukurlar oluşumu izlenir. HRP alımı ve membran çukur oluşumu zaman tabii endositoz zaman elbette gösterir.

Lipid taşıyıcı yöntemi kullanarak, SpH Hipokampal nöronlarda boutons (Şekil 1a) tanımlanması için izin ifade edildi. Hücrelerin elektriksel stimülasyon ekzositozu ve karşılık gelen bir artış Floresans şiddeti indüklenen. Floresan (ΔF) artış uyarıcı (Şekil 1b) bitiş tarafından durduruldu. Artan Floresans endositoz nedeniyle yavaş bir düşüş izledi. VAMP2-pHluorin durumunda VAMP2 uyarıcı<sup class="...

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

Sinaptik vezikül endositoz sinaptik vezikül protein ile erimiş pHluorin ışık mikroskobu ve elektron mikroskobu vezikül Alım tarafından algılanır.

Sinaptik vezikül endositoz kültürlü Hipokampal nöronlar içinde ölçme

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

Styryl Boyaların Fotoçevrim kullanarak Synaptic veziküllü Havuzları incelenmesi

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

Kültür Hipokampal Nöronlar Dendritik Spine Floresan Takip edilen Proteinler Photobleaching sonra Floresan Kurtarma (sıkı bağlamak)

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

Kültür MSS Nöronlar Dendritik Omurga Morfoloji Analizi

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

FM Boyalar Kültür Serebellar Granül Nöronlar Synaptic veziküllü Havuzu Yenileme Kantitatif Analiz

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

Kültür sempatik nöronlar bulunan dendritik Büyüme indükleyen

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

Kültür Nöronlar Membran Proteinlerin Yanal Difüzyon ve ekzositoz Floresans Kurtarma ve Floresans kaybı Photobleaching kullanarak Değerlendirilen

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

Immünopresipitasyon ve Açil-Biotin Borsası (ABE) tarafından Yetiştirilmiş Hipokampal Nöronlar Protein Palmitoylation tespiti

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

Comet Assay kullanma Hipokampal Nöronlar DNA Hasarı assaying

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

Hipokampal Dilim Kültürleri Synaptic Proteinler Post-gömme immunogold Etiketleme

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

Uyarılmış, Spontan ve Minyatür Synaptic Faaliyetleri Sırasında FM boyalar kullanarak sinaptik vezikül Geri Dönüşüm Sınavı

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