Name: measuring synaptic vesicle endocytosis in cultured hippocampal neurons
Uploaded: 2017-09-04T12:30:00.000+00:00
Duration: 7 min 30 s
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>Hipokampal nöron kültür <ol><li>ile birleştirerek 4 mM NaHCO 3 ve 5 mM HEPES hazırla Hippocampus arabellek (HB) ve pH 7.3 ile 5 M NaOH için ayarlayın. Kültür orta neurobasal orta, %2 B27, 0,5 mM L-glutamin ve % 1 penisilin streptomisin karıştırılarak olun. Ayrıca, sert kapak Ciltli arabellek % 20 ile karışımı hazırlamak Fetal sığır Serum (HB/20% FBS). 
		Not: Bu kodlamayla Protokolü Sankaranarayanan, et al. dayanır 29 ve Wu vd. 24</li> <li>fare pups postnatal gün 0 gün 2 kültür orta içine arasında başını kesmek ve beyin 4 ° C HB/20% FBS hulâsa. Beyin sapı ve talamus hippocampi ortaya çıkarmak için kaldırın. Hippocampi çıkarılması beyin sapı ve Talamus, tarafından açığa sonra incelemek ve transfer taze 4 ° C HB/20% FBS. 
		Not: Genellikle, 4 x 10-5 hücre/mL bir yavru (iki hippocampi) için verimidir. Cımbız veya makas kullanılarak yapışan membranlar temizlik sonra taze 4 ° C HB/20% FBS için transfer. Dentat gyrus göz önüne sermek ve subiculum makasla kesme tarafından izole, sonra aktarmak taze 4 ° C HB/20% FBS.</li>
		<li>Her Hipokampus uçtan uca yaklaşık 10 dilimler halinde keserek bölmek ve bir 15 mL polipropilen konik tüp için transfer.</li>
		<li>Doku yerleşmek izin sonra yıkayın HB/20% FBS 10 mL ile ve sonra yıkayın HB ile üç kez 10 mL.</li>
		<li>137 mM NaCl, 5 mM KCl, 7 mM Na2HPO 4 ve 25 mM HEPES sindirim çözüm hazırlamak ve pH 7.2 ile 5 M NaOH için ayarlayın. Süpernatant hippocampi kaldırın. Tripsin 10 mg ve Dnaz 1 mg 2 mL sindirim çözeltisi ekleyin ve filtre aracılığıyla doğrudan örnek Pelet üzerine bir steril 0,22 µm membran.</li>
		<li>Hippocampi 37 ° C'de 5 min için kuluçkaya sonra yıkama HB/20% FBS iki kez 10 mL ile ve sonra bir kez yıkayın HB ile 10 mL.</li>
		<li>MgSO 4 6 mg hücrelerle ayırmak &#8729; 7 H 2 O ve Dnaz 1 mg 2 mL HB ve hippocampi üzerine steril filtre cips steril bir 0,22 µm filtreden. Yavaşça hücreleri dikkatli triturating tarafından hava kabarcıkları tanıtımı önlemek için karşılamaya süre ayırmak. Doku parçacıklar 2 min için yerleşmek izin ve sonra yavaş yavaş süpernatant için başka bir tüp aktarın.</li>
		<li>Ekle 3 mL HB/20% FBS hücre süspansiyon ve santrifüj veya 10 dk 4 ° C'de ve 1000 rpm. Süpernatant atmak ve kültür ortamında resuspend.</li>
		<li>Plaka 60.000 hücreleri 150 µL kültür orta coverslip açıklarında sızıntısına olmadan Poli-D-lizin kaplı 25 mm çapında coverslip üzerinde askıya alındı. Önceden ısıtılmış kültür orta 2 h 2 mL kaplama sonra ekleyin. Coverslips 6-şey kodlamayla plakaları veya steril Petri yemekler sürdürülür.</li>
		<li>Koru hücreleri % 5 37 ° C'de CO 2 14-21 gün önce kayıt için kuluçka makinesine kültür orta nemlendirilmelidir. Kültür büyüme sırasında orta üst yarısı haftada iki kez değiştirin.</li>
	</ol></li> <li>Transfection <ol><li>synaptophysin-pHluorin 2 X 6-7 gün sonra kaplama, transfect (SpH) veya VAMP2-pHluorin Hipokampal nöronların içine. SpH için bir pcDNA3 vektör 30 eklenen bir Sitomegalovirüs (CMV) organizatörü kullanın. VAMP2-pHluorin için bir PCI vektör 31 eklenen bir CMV organizatörü kullanın.</li>
		<li>Lipid taşıyıcı tarafından her iki vektör plazmid 1 µg kullanarak hedef hücrelere transfect. Serum yoksun, kültür orta Protokolü adım 1.1.1, transfection için kullanın. Orta 2 h toksisite azaltmak için transfection sonra değiştirin. 
		Not: hücreleri sağlıksız olmadıkça SpH boutons içinde düşük ifade durumunda 2 µg veya KULUÇKA zaman DNA konsantrasyon lipid taşıyıcı ile artırın. Genellikle 4-10 hücreleri (0,006 %0.008) nöronlar transfected.</li>
	</ol></li> <li>Işık mikroskobu <ol><li>hazırla normal tuzlu çözüm oluşan 119 mM NaCl, 2 mM CaCl 2, 2.5 mM KCl, 25 mM HEPES (pH 7,4), 30 mM glikoz, 2 mM MgCl 2, 0.01 mM 6-cyano-7-nitroquinoxaline-2,3-dione () CNQX) ve 0.05 mM DL-2-amino-5-phosphonovaleric asit (AP5). Bir coverslip kültür orta plaka ve yer alan stimülasyon, sızıntıları önlemek için yağ ve dolgu macunu kullanarak sağlar bir görüntüleme odası üzerinde al. Hemen 750 µL normal tuzlu çözüm ekleyerek odası plaka bir coverslip transfer sırasında kuru cam izin kaçının. 
		Not: CNQX ve AP5 tekrarlayan etkinlik için potansiyele sahiptir postsinaptik faaliyetleri engellemek için kullanıldı. Bir oda bir ters Floresans mikroskobu yerleştirmeden önce temizlik doku odası değil kaçak onaylamak için kullanın. Yavaş sızıntı neden olan odak değişiklikleri kayıt sırasında Kırılma indisi değişimler sonucunda normal salin ve daldırma yağ karışımı ile.</li>
		<li>Uyarım ve <ol><li>görüntü üzerinde bir ters widefield mikroskobu ile bir 60 X (1.4 sayısal diyafram) yağı daldırma kayıt lens ile bir metal halide lamba. 480 bir uyarma zirve için ayarla bir filtre ile pHluorin görselleştirmek nm, 490 nm uzun pası ayna, 500-550 nm emisyon filtre ve bir el ile fiske vurmak çekim. Esir alma imge her 100 ile 2 x 2 binning, ms, bir elektron çarparak şarj birleştiğinde aygıt (EMCCD) kamera kullanarak. 
			Not: Photobleaching görüntü yakalama aralığı da dahil olmak üzere, önlemek için uygun koşullar elde etmek ve filtre uygulamak için çeşitli özellikler düşünülmesi gereken ve binning, hangi ekipman üzerinde bağımlı. Confocal görüntüleme durumunda lazer güç ve pozlama zaman photobleaching önlemek için düşünülmelidir. En az 3 dk uyaran olmadan kaydettikten sonra kurulum belirlendi ve kayıt için en az 10 gerçekleştirildi s photobleaching için kontrol etmek için uyaran olmadan.</li>
			<li>Her deneyde boutons analiz kolaylığı için yüksek yoğunluğu ile bir alanı seçin. Yeşil floresan sinyallerini bouton ve boutons arasında sürekli ifade yuvarlak veya oval ifade desen üzerinde durularak tarafından transfected hücrelerini tanımlamak.</li>
			<li>Yol endositoz 1 kullanarak alan stimülasyon tarafından ms darbesi, 20 mA eylem bir darbe uyarıcı sağlanan ve platin elektrot uyarıcı yalıtım birimi içinde aracılığıyla teslim potansiyel (AP). Görüntü Floresans etkinlik boyunca stimülasyon ve hücre kurtarma sırasında. 
			Not: Ölü hücreleri, söz konusu olduğunda ifade yaşam boutons güçlü olduğunu ve boutons arasındaki düzensiz bir desen gösterdi.</li>
		</ol></li></ol></li> <li>Görüntü analizi <ol><li>Bir tek bouton floresan yoğunluğu çözümlemek için bir bölge, faiz (yatırım Getirisi kadar) bir 1.5 x 1,5 µm kare olarak ayarlayın; bir bouton yaklaşık 1,5 µm. kullanım izleri photobleaching için kontrol ve arka plan olarak çıkarmak stimülasyon önce içinde boyutudur.</li>
		<li>Normale floresan değiştirme (ΔF) ile denklem: <img alt="Equation" height="11" src="/files/ftp_upload/55862/55862eq1.jpg" width="172" /> nereye F maksimum F 0 basvurunuz maksimal artış stimülasyon sonra ve taban çizgisi Floresan, anılan sıraya göre. Endositoz çürüme oranı ilk 4-10 s pHluorin floresan en yüksek noktadan sonra izleyen. Zaman sabiti (τ) temel bir mono Üstel fonksiyon ile en yüksek artış dan pHluorin floresan çürüme yaklaştırarak endositoz ile elde edilir.</li>
	</ol></li></ol> 2. Elektron mikroskobu <ol><li>hazırla bir Poli-D-lizin her şey oda sıcaklığında 1 h için 1,5 mL % 0,01 Poly-D-lizin çözeltisi steril filtre uygulamak, sonra üç kere steril su ile yıkama tarafından 6-şey plaka kaplı. İncelemek, kültür ve Hipokampal nöronlar olduğu gibi 1.1.1, 1.1.2 ve 1.1.3, adımları sırasıyla korumak.</li>
	<li>Hazırla yüksek K + stimülasyon Çözümle 31.5 mm NaCl, 2 mM CaCl 2, 90 mM KCl, 25 mM HEPES (pH 7,4), 30 mM glikoz, HRP 2 mM MgCl 2, 0.01 mM CNQX, 0.05 mM AP5 ve 5 mg/mL HRP, sonra ayarlamak için pH 7.4 ile 5 M NaOH.
	<ol><li>Stimulate Hipokampal nöron kültürü ile 1,5 mL yüksek K + stimülasyon çözüm (+ K olarak tarafından her şey için 1,5 mL ilavesi başvurulan) 90 için oda sıcaklığında s. 5 mg/mL HRP 90 için aynı konsantrasyonu (R adlandırılır) istirahat durumda uygulamak s, ama normal salin solüsyonu geçirin. Kurtarma örnek için K + örnek ile yüksek K + stimülasyon çözümü uygulamak sonra hızla yıkama ve normal salin ile değiştirin ve 10 dk. için kuluçkaya</li>
	</ol></li> <li>Fiksasyon ve boyama <ol><li>0.1 M Na cacodylate arabellek 21.4 g/L Na cacodylate pH 7.4 kullanarak hazırlayın. Hücreleri 0.1 M Na cacodylate arabellek için 1S oda sıcaklığında en az % 4 oxazolidin ile düzeltmek. Üç kez ile 0.1 M Na cacodylate arabellek 7 dakika yıkayın.</li>
		0,5 mg/mL oluşan <li>hazırlamak Diaminobenzidine (DAB) çözüm, H 2 O 2 GKD 2 O ve 0,22 µm filtre ile filtre %0,3 ile KURULAMAK. Üç kez ile 0.1 M Na cacodylate arabellek için 7 dk. 37 ° c yıkama 30 dk için 1,5 mL DAB çözüm uygulamak. 
		Dikkat: DAB şüpheli ve zehirli kanserojen olduğu. Lütfen eldiven ve laboratuvar mont kullanın. 
		Not: DAB ile etiketleme, oksidasyon nedeniyle tarafından H 2 O 2, HRP tarafından katalize oluşur. Örnekteki artan bir sinyal bileşenleri sonucu küçük artışlar. HRP konsantrasyonu artan katalizör etkisi kadar hızlandırır. H 2 O 2 yeterince yüksek konsantrasyonlarda HRP, yan etki zayıflatıcı için 32 etiketleme etkisini inhibe izin verir. Bu çalışmada, bu etiketleme sistemi konsantrasyonları mevcut araştırma 33 , 34 dayalı seçildi.</li>
		<li>Nöronlar ile 1,5 mL 0.1 M Na cacodylate arabellek için 1S fiksasyon sonrası gibi 4 ° C'de % 1 OsO 4 kuluçkaya. Üç kez ile 1,5 mL 0.1 M Na cacodylate arabelleği 7 dakika yıkayın. 
		Dikkat: toksisite ve reaktivite OsO 4 nedeniyle örnek buz üzerinde kimyasal bir mahallede tutmak için kuluçka buzdolabı kullanılması çoğu durumda tercih.</li>
		<li>Hazırlama 0.1 M sodyum asetat arabellek 13.61 g/L sodyum asetat ve 11,43 mL/L buzul Asetik asit pH 5.0 ile. Üç kez 1.5 mL 0.1 M asetat arabelleği pH 7 min için 5.0 ile yıkama ve 0.1 M asetat tampon pH 5,0 4 1 h için 1,5 mL % 1'uranyl asetat ile kuluçkaya ° C. yıkama üç kez ile 1,5 mL 0.1 M asetat arabellek 7 dk için.</li>
	</ol></li> <li>Epoksi gömme <ol><li>nöron kültürü tek 1,5 mL yıkar her 7 min için % 50, % 70 ve % 90 etanol ile kurutmak ve 1,5 mL % 100 etanol 7 dk her bir duman başlıklı 3 yıkar.</li>
		<li>Mix 485 mL/L bisphenol-A-(epichlorhydrin) epoksi reçine, 160 mL/L dodecenyl süksinik anhidrit (DDSA), 340 mL/L metil-5-Norbornene-2,3-Dikarboksilik anhidrit (NMA) ve 15 mL/L 2,4,6-tris (dimethylaminomethyl) fenol (DMP-30) epoksi oluşturmak için reçine. Karışımı iyice, sonra saklamak hava kabarcıkları kaldırmak için vakum altında. 
		Not: onlar kesit sırasında reçine çürükler neden olabilir çünkü bu reçine, özellikle de çıplak gözle, daha küçük hava kabarcıkları kaldırmak için önemlidir.</li>
		<li>% 50 epoksi ile etanol değiştirerek örnek reçine etanol üzerinde bir shaker, oda sıcaklığında 30 dakika içinde % 70 epoksi reçine etanol bir shaker üzerinde oda sıcaklığında 30 dakika sonra infiltrat.</li>
		<li>Epoksi çözüm % 100 epoksi reçine ile reçine ve incubations için 1 h 50 ° C. Ekle taze % 100 epoksi ile için taze % 100 epoksi reçine 10 min 50 ° c gerçekleştir iki değişimleri, kuluçkaya anahtarı reçine ve 50 ° C gece ve daha sonra 60 ve #17 sertleşmesine izin 6; C sertleşmesine 36 h üzerinden için.</li>
	</ol></li> <li>Her örnek bir kuyumcu ile muti-şey plaka kaldırmak &#39; s el testeresi. Faiz, bölgelerinde yoğun bir ters ışık mikroskop kullanarak ve sonra microtome tarafından kesit için 70-80 nm blok kesme hücreleri, konsantrasyonları seçin. Microtome chuck kesme bölgede dağ ve microtome yük. Chuck microtome konumlandırın ve blok yüzeyine paralel kenar ile bir elmas bıçak mount. 70-80 nm kalınlığı doğrudan bireysel Izgaralar üzerine bölümlerini toplamak.</li>
	<li>Ağırlık olarak % 1 çözüm için suya uranyl asetat dağıtılması ve ayrı ayrı kurşun sitrat ağırlık olarak % 3 çözüm için suya geçiyoruz. %1 sulu uranyl asetat 15dk için ve sonra %3 sulu kurşun sitrat örnekleri karşıtlığı artırmak 5 min için batma tarafından bölümleri counterstain.</li>
	<li>EM görüntüleme <ol><li>transmisyon elektron mikroskop bölümlerle ve birincil büyütmede 10.000-20, 000 X 3 CCD dijital fotoğraf makinesi ile kayıt görüntüleri inceleyin.</li>
	</ol></li> <li>İstatistik <ol><li>gerçekleştirmek bir t-test kontrol ve deneysel örnekleri arasında ortalama ve standart hata ölçü (s.e.m.) karşılaştırarak önemli farkları belirlemek için.</li>
	</ol>
	</li>
</ol>

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Yazarlar ifşa gerek yok.

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&amp;deg;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&amp;prime;-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 ...

measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons

Research

JoVE Journal

Neuroscience

9.8K Görüntüleme.  National Institute of Neurological Disorders and Stroke. Sinaptik vezikül endositoz sinaptik vezikül protein ile erimiş pHluorin ışık mikroskobu ve elektron mikroskobu vezikül Alım tarafından algılanır.

Video: Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Nöronlar FM Etiketleme kullanarak ölçüm ekzositoz

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Biyoloji

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 interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Kemirgen Beyin Dilimleri Nöronlar Vibrodissociation Sinaptik İletim ve Görüntü Presinaptik Terminalleri Eğitim

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Nörobilim

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

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 ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

İlköğretim Kültür Hipokampal Nöronlar Yoğun çekirdekli veziküller Canlı Görüntüleme

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

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

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

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

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

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 ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Floresan Probları Kullanan Kültürlü Nöronlarda Endozom ve Lizozom Hareketlerinin Nicelendirilmesi

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Sinaptik vezikül Presynaptic protein Overexpressing kültürlü nöronlarda geri dönüşüm için optik bir tahlil

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

Transmission Electron Microscopic Imaging of Synaptic Vesicle Endocytosis in Mouse Hippocampal Neurons 

<a href='https://app.jove.com/v/55862/measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons'>Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)</a>This video demonstrates the analysis of synaptic vesicle endocytosis in cultured hippocampal neurons through horseradish peroxidase labeling and electron microscopy.

Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)This video demonstrates the ...