1) Hazırlık Drosophila melanogaster nöronal kas kavşağı (NMJ) <ol><li> Standart Drosophila tuzlu su hazırlayın (130 mM NaCl, 36 mM sukroz, 5 mM KCl, 2 mM CaCl 2, 2 mM MgCl 2, 5 mM Hepes, pH 7,3 4. </li><li> Serum fizyolojik (1.1) hazırlık parçalara ayır. Drosophila larvaları bir Sylgard tabak içinde dorsal yan pined; dorsal tarafına uzunlamasına kesitli ve iç organlar çıkarılır. Hazırlık sonra gergin ve pined. Birkaç karın kasları daha sonra kullanılabilir. </li></ol> 2) Uyarılma ve FM boyama <ol><li> Ağartma FM boyalar korumak amacıyla, düşük ışık koşullarında aşağıdaki adımları yapmanız tavsiye edilir. </li><li> Sinirleri kimyasal uyarımı için, yüksek oranda potasyum tampon kullanılır. Drosophila salin (1.1), 90 mM KCl FM1-43 boya 10 mcM içeren hazırlayın. Işıktan koruyarak çözüm tutun. </li><li> Oda sıcaklığında 1 dakika için önceden hazırlanmış bir tamponu ile hazırlık Banyosu. </li><li> Ekstrasellüler FM1-43 boya kaldırmak için standart Drosophila serum fizyolojik (1.1) ile yıkayın. Fiksasyon hızla devam edin. </li></ol> 3) Sabitleme <ol><li> Fosfat tamponlu salin (PBS) oda sıcaklığında 45 dakika süreyle% 2.5 lik gluteraldehit ile hazırlık Hamamı. Morfolojik özellikleri iyi korunması için, glutaraldehid paraformaldehid tercih edilir. </li><li> PBS ile bir kez yıkayın ve sonra 15 dakika süreyle 100 mM NH 4 Cl batık hazırlık bırakın . Bu adım, kalan glutaraldehid fiksatif serbest aldehit grupları gidermek için yapılır. </li><li> NH 4 Cl çözüm normal PBS ile yıkayın. </li></ol> 4) Fotoçevrim <ol><li> 4 NMJ hazırlık az 30 dakika boyunca inkübe ° C PBS içinde diaminobenzidin (DAB), 1.5 mg ml-1 içeren. </li><li> Floresan mikroskop altında örnek yerleştirin. Nispeten düşük büyütme suya daldırma objektif (20x 0.5 NA) kullanarak floresan sinyali bulun ve odaklanır. </li><li> FM boya tamamen beyazlatılmış (Şekil 1C) kadar maksimum lamba yoğunluğu ile örnek aydınlatın. Fotoçevrim oluştu olup olmadığını kontrol etmek için, iletim ışığı altında FM boya adım beyazlatma öncesi ve sonrası örnek kontrol edilmesi tavsiye edilmektedir. Fotoçevrim yer alıyorsa, koyu kahverengi bir çökelti (Şekil 1C) bekleniyor. Aydınlatma süresi de hazırlık içine DAB penetrasyon üzerine aydınlatma gücü ve bağlı olarak değişkendir. En hazırlıkları için, 20x objektif kullanırken ~ 30-45 dakika aydınlatma süreleri optimum olarak bulundu. Kısa aydınlatma süreleri, yüksek büyütme hedefleri (60x) için kullanılır. </li><li> Aydınlatma için geri saçılan ışık ışınları toplar ve bu nedenle toplam yoğunluğunu artırır arka ayna içeren bir lamba konut, bir cıva lambası kullanmayı tercih. </li></ol> 5) EM için örnek İşleme <ol><li> Osmication <ol class="lalpha"><li> Bir hacim su ağacın hacmi osmium tetroksit (örnek başına yaklaşık 300 ul) ile bir çözüm hazırlayın. Osmiyum tetroksit kullanarak, eldiven ve aye koruma araçları kullanarak başlık altında yapılması gerekir. Çalışma </li><li> Osmiyum tetroksit çözüm olarak hazırlanması (Kaputun altında) oda sıcaklığında 1 saat süreyle inkübe edin. </li><li> PBS (4-5 kez 5 dakika) ile iyice yıkayın ve temiz bir cam şişeyi her örnekler aktarmak. </li></ol></li><li> Kurutma <ol class="lalpha"><li> 30, 50, 70 90, 95 ve% 100 etanol içeren ayrı çözümler hazırlayın. </li><li> 5 dakika boyunca her bir örnek için 1 ml% 30 etanol ekleyin. </li><li> 5 dakika boyunca her bir örnek için 1 ml% 50 etanol ekleyin. </li><li> 5 dakika boyunca her bir örnek için 1 ml% 70 etanol ekleyin. </li><li> 5 dakika boyunca her bir örnek için 1 ml% 90 etanol ekleyin. </li><li> (3x tekrar) 5 dakika boyunca her bir örnek için 1 ml% 95 etanol ekleyin. </li><li> (3x tekrar) 5 dakika boyunca her bir örnek için 1 ml% 100 etanol ekleyin. </li></ol></li><li> Gömme ve daha fazla işlem <ol class="lalpha"><li> EPON reçine bir hacim etanol ile 1 hacim karıştırın. (2-4 saat) sürekli dönme altında hazırlanması ekleyin. </li><li> % 100 EPON açık şişeler hazırlık yerleştirin, kalan etanol buharlaşmasına izin (4-6 saat). </li><li> 60 ° C&#39;de 36 saat için hazırlık kalıplara yerleştirin. </li><li> Elektron mikroskobu işleme. Standart ultramicrotomy prosedürleri kullanarak, 50-80 nm ince bölümlerde hazırlık süreci. </li></ol></li><li> Elektron mikroskobu görüntüleme <ol class="lalpha"><li> Hazırlık geleneksel EM prosedürlerini kullanarak görüntülü. </li></ol></li></ol> 6) Temsilcisi Sonuçlar Beklenen sonuçlar Şekil 1C ve 2 de özetlenmiştir. Bro oluşumunu aydınlatma işlemi sonuçlarıwn DAB çökelti, floresan ve transmisyon görüntüleme hem de görünür olacaktır. FM boya boyama ile ilişkili floresan aydınlatma sırasında ilk geçtiği ortadan kalkması. Aldehit fiksatif tarafından uyarılan eşiğe, aksine, deney boyunca görünür olacaktır. Ağartma ~ aydınlatma başladıktan sonra 10-20 dakika tipik olarak yer alır. Genellikle hiçbir Fotoçevrim ürün bu noktada gözlenebilir. Aydınlatma devam edilmelidir ve hazırlıkları ~ 10 dakika sonra aynı anda yer almayan neden DAB yağış ve FM boya kasar belirsiz DAB birikimi nedeniyle kahverengi bir gölge (Şekil 1C iv) dönecek, ki nispeten yüksek mümkün okside DAB miktarı toplama ve yağış önce birikmesine ihtiyaç duyar ve bu nedenle bu reaksiyon ağartma işlemi daha yavaş. Bu aşamada, DAB yağış, büyük olasılıkla eksik Bununla birlikte, hazırlanması, elektron mikroskobu işlem için hazır değildir. Biz hazırlanması sırasında (presinaptik sinir terminal) tam bir dönüşüm gösteren bir koyu kahverengi veya siyah renk, varsayar, 5-10 dakika daha uzun süre beklemek tercih ederler. Genel yüzey hazırlama (örneğin, nöromüsküler bileşke kas lifleri) hafif bir dönüşüm bu aşamada görülebilir. Büyük olasılıkla otofloresans (ve / veya fiksatif floresan) ile DAB uyarılan bir dönüşüm için ilgili ve bu işlem için zararlı değildir. Sonra elektron mikroskobu için hazırlık işlenir ve koyu (etiketli) veziküllerin varlığı için kontrol edilmelidir. 5,6 önce gösterdiğimiz gibi, bu veziküller olmayan etiketli olanlardan daha çok yoğun ve bu nedenle kolayca ayırt edilir (Şekil 2B). Veziküller farklı türleri ayırt şansını artırmak için, bölümden hiçbir kontrast tutan yazılan boyama (hiçbir uranil asetat ve kurşun sitrat boyama) yapılabilir; osmiyum boyama en hücresel elemanları gözlemlemek için yeterli ve gibi değildir DAB çökelek gibi karanlık. <img src="/files/ftp_upload/1790/1790fig1.jpg" alt="Şekil 1" /> Şekil 1: FM boyalar ve Fotoçevrim için kullanılacak. (A) uyarımı altında FM boya kullanarak veziküller yükleme ve distaining için klasik bir deney adımları temsil etmek. (I) FM boya içeren tampon örnek inkübe edin. (Ii) FM boya varlığı vezikül füzyon ikna etmek için (elektriksel ya da kimyasal) geliştirin . (Iii) stimülasyon sonra altdizi endositoz ekstrasellüler tampon hala mevcut FM boya ile bazı veziküller yük olacaktır. (Iv) tamponu ile yıkanarak ekstraselüler FM boya değiştirin. (V) yeni bir stimülasyon ekzositoz üzerine onların FM boya serbest yüklü veziküller neden olacaktır. (B) Şematik başkanı, köprü ve FM1-43 boya kuyruk gösteriyor. (C), Drosophila NMJ bir Fotoçevrim deney örneği . (I) dış boya molekülleri ve sabitleme yıkama FM1-43, NMJ yükledikten sonra, bir sinir terminal floresan geleneksel bir Epifloresans mikroskobu (63x) ile tespit edilebilir. (Ii-iii) sürekli aydınlatma altında boya tamamen beyazlatılmış. (Iv) aydınlatma devam ederken, siyah renk diaminobenzidin yağış nedeniyle görünür. Ölçek çubuğu 10 mm temsil eder. <img src="/files/ftp_upload/1790/1790fig2.jpg" alt="Şekil 2" /> Şekil 2: photoconverted EM photoconverted örnekleri örnekleri (A) görüntü sinaptik vezikül içeren bir sinir terminal gösterir, ama bu, yeterli aydınlatma veya fakir doku diaminobenzidin penetrasyonu olmaması neden olabilir . (B) FM Fotoçevrim geçti birkaç koyu (dolu) veziküller Synaptic bouton. (C), herhangi bir vezikül veya organelleri ayırt genel bir karanlık terminal aydınlatma sonuçları Fazla . Ölçek çubuklar 200 nm temsil eder.

<ol>
	<li>Betz, W. J., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+analysis+of+synaptic+vesicle+recycling+at+the+frog+neuromuscular+junction.">Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.</a> Science. 255, 200-203 (1992).</li><li>Henkel, A. W., L&#252;bke, J., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM1-43+dye+ultrastructural+localization+in+and+release+from+frog+motor+nerve+terminals.">FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.</a> Proc Natl Acad Sci USA. 93, 1918-1923 (1996).</li><li>Sandell, J. H., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+of+some+fluorescent+markers+to+a+diaminobenzidine+product.">Photoconversion of some fluorescent markers to a diaminobenzidine product.</a> J Histochem Cytochem. 36, 555-559 (1988).</li><li>Kuromi, H., Kidokoro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optically+determined+size+of+exo/endo+cycling+vesicle+pool+correlates+with+the+quantal+content+at+the+neuromuscular+junction+of+Drosophila+larvae.">The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.</a> J Neurosci. 19, 1557-1565 (1999).</li><li>Denker, A., Kr&#246;hnert, K., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+synaptic+vesicle+pool+localization+in+the+Drosophila+neuromuscular+junction.">Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction.</a> J Physiol (Lond). 587, 2919-2926 (2009).</li><li>Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+organization+of+the+readily+releasable+pool+of+synaptic+vesicles.">The structural organization of the readily releasable pool of synaptic vesicles.</a> Science. 303, 2037-2039 (2004).</li><li>Darcy, K. J., Staras, K., Collinson, L. M., Goda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+sharing+of+recycling+synaptic+vesicles+between+presynaptic+boutons.">Constitutive sharing of recycling synaptic vesicles between presynaptic boutons.</a> Nat Neurosci. 9, 315-321 (2006).</li><li>Harata, N., Ryan, T. A., Smith, S. J., Buchanan, J., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+recycling+synaptic+vesicles+in+hippocampal+neurons+by+FM1-43+photoconversion.">Visualizing recycling synaptic vesicles in hippocampal neurons by FM1-43 photoconversion.</a> Proc Natl Acad Sci USA. 98, 12748-12753 (2001).</li><li>Lange, R. P. J. d. e., de Roos, A. D. G., Borst, J. G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+vesicle+recycling+in+the+rat+calyx+of+Held.">Two modes of vesicle recycling in the rat calyx of Held.</a> J Neurosci. 23, 10164-10173 (2003).</li><li>Richards, D. A., Guatimosim, C., Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pools+at+the+frog+neuromuscular+junction.">Synaptic vesicle pools at the frog neuromuscular junction.</a> Neuron. 39, 529-541 (2003).</li></ol>

Birkaç kritik adımlar dikkate alınmalıdır: <ul><li> DAB inkübasyon sadece hazırlıkları iyice yıkama ve soğutma sonra yapılmalıdır. Aksi takdirde olmayan tepki glutaraldehid DAB ile etkileşim ve yağış yoğun elektron değildir şeklinde düz kristaller, (genellikle) neden olacaktır. Bu yağış bol bol yer alır elektron mikroskobu için hazırlıklar nadiren kullanılabilir. </li><li> Aydınlatma kez farklı aydınlatma kez birkaç özdeş hazırlıkları test optimize edilmelidir. Bizim tecrübel...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>FM 1-43</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F10317</td>
<td>&nbsp;</td>
<tr>
<td>Epon resin</td>
<td>&nbsp;</td>
<td>Plano</td>
<td>R1030</td>
<td>&nbsp;</td>
<tr>
<td>di-aminobenzidine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde</td>
<td>&nbsp;</td>
<td>AppliChem</td>
<td>A3166</td>
<td>EM grade</td>
</tr>
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>104186298</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop 2 FS plus</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Objective 20x 0.5 NA</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Dry objective</td>
</tr>
<tr>
<td>100W Hg Lamp</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lamp housing with back mirror</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>1007-980</td>
<td>&nbsp;</td>
<tr>
<td>MRm camera</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>0445-554</td>
<td>Image acquisition</td>
</tr>
<tr>
<td>Ex. Filter (HQ 470/40)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F49-671</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic (495 DCLP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F33-100</td>
<td>&nbsp;</td>
<tr>
<td>Em. Filter (HQ 500 LP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F42-018</td>
<td>&nbsp;</td>
<tr>
<td>EM</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Proscan CCD HSS</td>
<td>&nbsp;</td>
<td>Proscan Electronic Sys.</td>
<td>&nbsp;</td>
<td>1024 x 1024</td>
</tr>		</tbody>
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studying synaptic vesicle pools using photoconversion of styryl dyes

, Plazma zarı (ekzositoz) ile sinaptik veziküllerin füzyon nörotransmitter salınımı ve nöron iletişim içinde gerekli bir adım. Veziküller daha sonra yeni bir exo-endositoz döngüsü (vezikül geri dönüşüm) geçmesi kadar, plazma zarı (endositoz) alınır ve sinir terminali içinde vezikül genel havuzu ile birlikte gruplandırılmıştır. Bu süreçler, elektron mikroskobu, elektrofizyoloji kayıtları, amperometri ve kapasite ölçümleri gibi çeşitli teknikler kullanılarak çalışılmıştır. Önemlisi, son yirmi yılda, floresan ile işaretlenmiş belirteçlerin bir dizi ortaya optik teknikleri, geri dönüşüm dinamikleri veziküller izlemenize olanak sağlayacak. En sık kullanılan belirteçler Bir styryl veya FM boya 1; yapısal, tüm FM boyalar Hidrofilik bir baş ve aromatik bir halka ve bir veya daha fazla çift bağ (Şekil 1B) ile bağlı bir lipofilik kuyruk içerir. Veziküller bir havuz etiket klasik FM boya deneyi sinirin uyarılması sırasında boya ile hazırlanması (Şekil 1Ai) banyo (elektrik veya yüksek K +) oluşur. Bu son endocytosed veziküller (Şekil 1A i-iii) vezikül boya, geri dönüşüm ve sonraki yükleme neden olur. Boya, boya banyosuna stimülasyon ikinci turda veziküller yükledikten sonra ekzositoz yoluyla FM sürümü (Şekil 1A iv-v), floresan yoğunluğu azaltmak (destaining) izleme ile takip edilebilir süreci tetikleyebilir. FM boyalar vezikül geri dönüşüm alanında büyük katkıları olmasına rağmen, geleneksel floresan mikroskobu kullanarak, bireysel veziküller tam lokalizasyonu ya da morfolojisi belirlemek mümkün değil. Bu nedenle, biz burada Fotoçevrim yoluyla nasıl FM boyalar, elektron mikroskobu kullanılarak endositik belirteç olarak da kullanılabilir. Açıklar. Fotoçevrim tekniği, yoğun aydınlatma altında reaktif oksijen türleri oluşturmak için floresan boyaların özelliği patlatır. Floresan etiketli hazırlıkları diaminobenzidin (DAB) içeren bir çözüm batık ve ışıklı. Boya molekülleri tarafından oluşturulan reaktif türler, karanlık bir görünüme sahiptir ve elektron mikroskobu 2,3 kolayca ayırt edilebilir, istikrarlı, çözünmez çökelti oluşturur DAB, okside . DAB sadece floresan moleküller (reaktif oksijen türleri kısa ömürlü olduğu gibi) hemen yakınında okside olduğu gibi, bu teknik sadece floresan etiketli yapıları elektron-yoğun bir çökelti içeren olmasını sağlar. Bu teknik böylece aktif organelleri geri dönüşüm tam yeri ve morfolojisi çalışma sağlar.

Watch this Scientific Journal Video about Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes at JoVE.com

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

FM boyalar sinaptik dinamiklerin anlaşılmasına çok değerli yardım edilmiştir. FMS normalde farklı stimülasyon sırasında floresan mikroskop altında takip edilmektedir. Ancak, elektron mikroskobu ile kombine FM boyaların Fotoçevrim sinaptik BOUTONS, diğer ultrastrüktür bileşenleri arasında, farklı sinaptik vezikül havuzları görselleştirme sağlar.

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 ...

studying-synaptic-vesicle-pools-using-photoconversion-of-styryl-dyes

Research

JoVE Journal

Neuroscience

11.6K Görüntüleme.  European Neuroscience Institute Göttingen. FM boyalar sinaptik dinamiklerin anlaşılmasına çok değerli yardım edilmiştir. FMS normalde farklı stimülasyon sırasında floresan mikroskop altında takip edilmektedir. Ancak, elektron mikroskobu ile kombine FM boyaların Fotoçevrim sinaptik BOUTONS, diğer ultrastrüktür bileşenleri arasında, farklı sinaptik vezikül havuzları görselleştirme sağlar.

Video: Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

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 ...

Nörobilim

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 ...

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 ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Astrositik Transporter Akımlar bir Dekonvolüsyon Analizi ile Glutamat Gümrükleme gün Dersin türetilmesi

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

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 ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Vivokültür-tarihlerde hızlı, basit ve ucuz AWESAM iletişim kuralını kullanarak fare Astrocytes

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole kaynaklı çıra fare modeli

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Optogenetics gen nakavt etkileri olduğu gibi nöronal devreler içinde Presynaptic işlevi karakterize etmek için yapay mikroRNA'lar birleştirme

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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 ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Beyin fonksiyonları çocuklarda Magnetoencephalography kullanarak eğitim

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...