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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicles+interchange+their+membrane+proteins+with+a+large+surface+reservoir+during+recycling.">Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling.</a> Neuron. 51 (2), 179-186 (2006).</li><li>Gimber, N., Tadeus, G., Maritzen, T., Schmoranzer, J., Haucke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusional+spread+and+confinement+of+newly+exocytosed+synaptic+vesicle+proteins.">Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins.</a> Nat Commun. 6, 8392 (2015).</li><li>Nicholson-Fish, J. C., Smillie, K. J., Cousin, M. 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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&#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 ...

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

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 vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

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 ...

Nörobilim

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 in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

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 Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of 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

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

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

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 ...

Inducing Dendritic Growth in Cultured Sympathetic 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 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

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

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 ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and 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 of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

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

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

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

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 extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

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

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 ...