NIH grant R01 MH 099557 tarafından desteklenen

1. imalat kayıt elektrotlar <ol><li><ol><li>kullanımı aşağıdaki cam elektrotlar çekerek protokolü için elektrot çektirme (Tablo malzemeleri görmek): <ol><li>Line 1: ısı 510 çek - hız 30 zaman 250; Line 2: 490 çekme - hız 30 ısı zaman 250. 
			Not: Zaman birimi birim başına 0.5 ms karşılık gelir; diğer birimleri görecelidir. Rampa test gerçekleştirildikten sonra ısı değerini her filaman için ayarlanmalıdır.</li>
		</ol></li> <li>Çekti elektrot iç çapı 7-10 µm ( şekil 1A) aralığında olması için mikroskop (35 x büyütme) kullanın. Kılcal damarlar toz birikimi önlemek için sıkıca kapalı kaplarda saklayın.</li>
	</ol></li> <li>Yangın parlatma <ol><li>yangın Lehçe kılcal damarlar (1-2 s için maksimum ısı değeri % 80-90) mikro-demirhane kullanarak (Tablo malzemeleri görmek). Cilalı elektrot son iç çapı 5 µm ( şekil 1A) 27 olduğundan emin olun.</li>
	</ol></li> <li>Bükme Not: iki virajlı elektrot yüksek büyütme objektif altında kas üstüne konumlandırmak için yapılır.
	<ol><li>Kullanımı şekil 1B adımında gösterilen aparatı. Elektrot manipülatör içinde düzeltmek ve ucu onu değil müessir filament üzerinde konumlandırmak</li>
		<li>Set ısı değeri en fazla 60-70 oranında ve mikro-forge pedal için 1-2 s tuşuna basın (filaman ısı Malzemeleri tablo bkz:). L şeklindeki bir iğne yavaşça (büyütülmüş şekil 1B) elektrot uç çekmek için kullanın. Viraj yaklaşık 90 o ( şekil 1 c) olun.</li>
		<li>Elektrot meşale ateşte forseps kullanarak el ile sık ve ikinci bend bir mesafe üzerinden ilk bend 7-10 mm ve bir açıyla yaklaşık 120 o ( şekil 1 c).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" /> şekil 1. Micropipette imalat son adımlar. (A) elektrot ipuçları çekerek ve yangın parlatma sonra. (B.1) ipucu bükme Kur. (B.2) kutulu alanının sağ tarafta genişlemiş gösterilir. Böylece elektrot ucunu biraz tel üzerinde konumlandırılmış ve değil dokunaklı elektrot ve filament sabittir. (C.1) kayıt elektrot. (C.2) kutulu alanının sağ tarafta genişlemiş gösterilir. İlk bend yaklaşık 90 ° vardır ve ilk bend elektrot ucu arasındaki mesafe yaklaşık 1 mm. ölçek kadar çubuk = 3 mm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
 2. ek hazırlık adımları <ol><li>hazırla heamolymph gibi (HL3) çözüm (mM): 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 trehalose, 115 Sükroz, 5 HEPES ve 1 mM CaCl 2 7.3 için-pH ayarlama; 7.4. çözüm buzdolabında saklayın ve her hafta taze olun.</li>
	<li>Dışında onları bükmeye gerek yok pipets, aynı şekilde kayıt olarak cam stimülasyon pipets olun. 
	Not: Stimülasyon elektrotlar hazırlanması ayrıntılı 28 ve 27 olarak tanımlanır. Yangın parlatma 5-7 µm aralığında olmalıdır sonra son çapı.</li>
	<li>Bir elektrot tutucu bir stimülasyon pipet bağlı bir şırıngaya ekle.</li>
	<li>Üretim diseksiyon plakalar üzerinden küçük petri yemekler (35 x 10 mm) silikon kauçuk epoksi ile kaplı ( 28 içinde açıklandığı gibi Malzemeleri tablo görmek). 
	Not: Silikon kauçuk kullanmadan önce tamamen sertleşmiş olmalıdır.</li>
	<li>Gezgin bir üçüncü-biçim larva almak ve 27 , 28 , 29 , 30 ' açıklandığı gibi incelemek. 
	Not: boutons görselleştirmek için sinek zorlanma CD8-GFP kullanın (bkz. Tablo reçetesi) <ol><li>kullanma forseps, (bkz: Malzemeler tablo) almak 3 rd INSTAR larvaları bir şişe.</li>
		<li>İlk iğneyi posterior yerleştirerek larvalar, pin ve başka bir pin ağız kanca için anterior (Kapat).</li>
		<li>Ekleyin HL3 çözüm.</li>
		<li>(Bkz. Tablo reçetesi) bahar makas ta üst iğnesinden yukarıdan aşağıya doğru bir larva dorsal tarafta kullanarak bir kesim yapmak.</li>
		<li>Üstünde belgili tanımlık sol ve sağ taraflarındaki, larvalar 2 ek pimleri yerleştirerek larva fileto pin.</li>
		<li>Çıkarmak cesaret ve forseps kullanarak tracheas.</li>
		<li>Sadece bahar makas kullanarak ventral sinir kordonu dışında sinirler kesti.</li>
	</ol></li></ol> 3. Elektrik kayıtları EJC'ler <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> Şekil 2. Kayıt kurulum. Örnek NMJ silikon kaplı petri kabına (ok) dik bileşik mikroskop epifluorescence yetenekleri, yüksek büyütme objektif ve iki micromanipulators ile donatılmış hareketli sahne üzerinde konumlandırılmış sabitlenmiş. Mikroskop bir anti-titreşim tablo üzerinde konuşlu olduğunu. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol><li>Kayıt <ol><li>yer petri kabına hazırlık mikroskop sahnede ( Şekil 2). Referans elektrot banyoda takın.</li>
		<li>Kayıt elektrot HL3 çözüm ile doldurun. 10 x amaç altında (bkz. Tablo reçetesi) elektrot banyo içine sokmak ve 6 ve 7 2, 3 veya 4 micromanipulator kullanarak karın segmentlerinin kaslar yerleştirin (bkz. Tablo reçetesi) () şekil 3 A.1 ve A.2).</li>
		<li>Geçiş yapma amacı için 60 x (Tablo malzemeleri görmek). Epi-floresan veya DIC optik kullanarak ilgi alan üzerinde odaklanın. Sinaptik bouton ( şekil 3 B.1-3) üstüne elektrot ucu yerleştirin.</li>
		<li>Elektrot kas üzerine hafifçe basın. Aşırı basınç NMJ zarar veya spontan sinirsel aktivite artışı teşvik. Elektrot ucu tıkanmış - Eğer o, bunu yerine dikkat edin</li>
		<li>Amplifikatör, A/D yönetim kurulu ve bilgisayar geçiş.</li>
		<li>Amplifikatör gerilim kelepçe konumunda seçin.</li>
		<li>Başlangıç Alım yazılım ve &#39; boşluk içermeyen &#39; modu.</li>
		<li>MEJCs bilgisayar ekranında görünümünü gözlemlemek.</li>
		<li>MEJCs genliği 0,2 - aralıkta olduğunu emin olun 0,7 na 
		Not: Daha küçük EJC'ler kayıt elektrot kusurları vardır veya bu doğru yerleştirilmiyor gösteriyor.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> şekil 3. Görsel olarak sinaptik boutons. (A) A aydınlık alan görüntü, bir hemi-kesim 10 x büyütme (A.1) ve kas 6 ve 7 gösterilen genişlemiş kutulu alanının altında (A.2, ok işaretleri kayıt elektrot). Ölçek çubuğu = 50 µm. (B) sinaptik boutons genetik olarak kodlanmış nöronal marker (CD8-GFP) ile bir Drosophila satırda görüntülenir epi-floresan görüntüleme (B.1) veya DIC optik (B.2) kullanarak. Görüntüleri ile hedef ve filtre küp GFP görüntüleme için x 60 alınır (Tablo malzemeleri görmek). Synaptic boutons oklarla işaretlenmiş ve floresan ve DIC görüntülerin bir bindirme B.3 içinde gösterilir. Ölçek çubuğu 10 µm. = <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol start="2"> <li>stimülasyon <ol><li>görsel bir denetim altında bir micromanipulator kullanarak yer stimülasyon elektrot karın kesimleri 2-4 innerve axon yakınındaki.</li>
		<li>Şırınga piston çekerek negatif basınç uygula bağlı elektrot sahibine böylece akson elektrot ( Şekil 2) içinde çekti.</li>
		<li>Uyarıcı açın. Vana yalıtım Üniteyi açar (bir sıfır akım ayarlamak ve sonra yavaşça, EJC'ler görününceye kadar (veya eşik değerine ulaşılana kadar) artırmak için Malzemeler tablo bkz:).</li>
		<li>Bir suprathreshold rejim stimülasyon geçerli stimülasyon ile artmış yaklaşık iki kez, EJC'ler gözlenmesi için eşik göre gerçekleştir. 
		Not: deneyim, böyle stimülasyon yoğunluğu aksiyon potansiyeli başarısızlıklar ve aksiyon potansiyeli ateş önlemek için en iyi yöntemdir. Örneğin, EJC'ler 0.2 stimülasyon mevcut görünür anne, 0,4 mevcut kullanmak deneme boyunca anne.</li>
	</ol></li> <li>Direnç mühür <ol><li>amplifikatör için elektrot direnç anahtarı çevirerek kayıt macropatch elektrot mühür dayanıklılığını ölçmek &#34; test mühür &#34; pozisyon. GΩ mühür direnç değeri içinde görüntülenir gözlemlemek &#34; geçerli &#34; penceresi.</li>
		<li>0.5 - 2 M aralığında dirençtir mühür emin olun &#8486;. Bu sınırların dışında değerler gösterir elektrot düzgün cilalı veya uygun şekilde konumlandırılmış.</li>
		<li>Mühür direnç kaydı boyunca izlemek, deney boyunca sabit kalır emin olmak istedim.</li>
	</ol></li></ol> 4. Analiz <ol><li>analiz kayıtları istihdam özelleştirilmiş yazılım analiz için (bkz. Tablo reçetesi). 
	Not: Bizim laboratuvar içi yazılım Quantan 31 EJC'ler ve focally kayıt mEJCs ( şekil 4) tespiti için özelleştirilmiş, kullanır. Bu yazılım bir Gauss dijital filtre içerir ve çok quantal olaylar ( şekil 4A) üst üste içinde quantal doruklarına algılama sağlar. Diğer yaklaşımlar 17 ' açıklanan.</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> şekil 4. Quantal analizi. , MEJCs (a) ve EJC'ler (B) Quantan yazılım tarafından. Olay alan yeşil işaretlenir ve tepeler tarafından kırmızı ok uçları işaretlenir. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction.">FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.</a> Methods Mol Biol. 440, 349-369 (2008).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Imlach, W., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li><li>Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li><li>Huntwork, S., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li><li>Zhong, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

Yazarlar ifşa gerek yok.

Drosophila sinaptik iletimi çalışmaya bir avantajlı model organizma temsil eder. Sinaptik potansiyelleri, kayıtları iki elektrot gerilimi kelepçe33,34ve Fokal macropatch ile sinaptik akıntılarının hücre içi kayıtları da dahil olmak üzere larva NMJ, birkaç kayıt yapılandırma kullanılmıştır Burada açıklanan sinaptik akıntılarının kayıtları. İkinci tekniği görüntülenmeyecektir boutons kesin miktar sinaptik iletimi sağlar.
Açıklanan protokol başarısı eleştirel açıkça ilgi alanı görselleştirmek ve elektrotlar kayıt hazırlık özelleştirmek için yeteneği üzerinde bağlıdır. Böylece, kalite DIC optik, bir yüksek büyütme su daldırma amacı bir parlatma ve elektrotlar kayıt bükme yangın için özelleştirilmiş donatım ve uzun çalışma mesafesi ile eleştirel önemlidir.
Bu yaklaşımın avantajı bu kayıt elektrot altında konumlandırılmış bir kaç sinapslarda etkinliğini izlemeye olanak sağlamasıdır. Bu unutulmamalıdır, ancak, elektrot RIM konumlandırılmış boutons da kayıtlı etkinlik için katkıda bulunabilir. Bu nedenle, elektrot yanı sıra mühür direnç konumunu sırasında deney değişmez, çok önemlidir.
Bir tek bouton etkinliğini izlemek için yeteneği potansiyel olarak son görüntüleme teknolojileri ile kombine edilebilir. Örneğin, bireysel etkin bölgeler35 aktivitesinin optik algılama EJC'ler ve mEJC Fokal kayıtları ile kombine edilebilir ve optik algılama Uzaysal çözünürlük elektrik kayıtlar zamansal çözünürlük ile bir çift.

Drosophila sinaptik iletimi denetleme moleküler mekanizmaları incelemek için mükemmel model bir sistemdir. Drosophila nöromüsküler sistemde glutamatergic ve bu nedenle Drosophila nöromüsküler kavşak (NMJ) glutamatergic yayın korunmuş özelliklerini incelemek için kullanılabilir. Jan Jan'ın çalışma ve1beri üçüncü INSTAR larva geniş eksitatör kavşak potansiyeller (EJPs) veya akımları (EJC'ler) izleyerek uyarılmış ve spontan sinaptik iletimi çalışma için kullanılmıştır. EJPs yaygın olarak intracellularly bir keskin cam mikro elektrot ile kaydedilir ve onlar sinapslarda belirli kas lif yapım tüm boutons de dahil olmak üzere tüm NMJ etkinliğini yansıtır.
Buna ek olarak, sınırlı sayıda sürüm site etkinliğini focally nöronal terminalleri veya sinaptik varicosities yakınındaki bir micropipette ipucu konumlandırma tarafından kaydedilir. Bu teknik aslında Katz ve Miledi2tarafından istihdam edildi ve başarıyla kurbağa3,4,5, fare6 dahil olmak üzere birkaç NMJ hazırlıklar istihdam Fokal hücre dışı kayıtları , 7 , 8, kabuklu9,10,11,12,13,14,15,16ve Drosophila17,18,19,20,21,22,23. Bu yaklaşım daha fazla elektrotlar24,25recoding macropatch en iyi duruma getirilmiş Dudel tarafından geliştirilmiştir. Dudel'ın uygulamasında bu teknik yakından gevşek-yama-kelepçe yöntemi26eşleştirdi.
Drosophila larva NMJ sinaptik boutons açıkça tanımladığı ve genetik olarak kodlanmış nöronal floresan Etiketler ( Tablo malzemelerigörmek) transgenik çizgilerle kolayca kullanılabilir. Bu avantajları bize EJC'ler ve seçili sinaptik bouton20,21,22mEJCs kaydetmek etkin. Burada, bu teknik ayrıntılı olarak açıklanmaktadır.

<table width="1251">
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		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sutter P-97</td>
			<td style="width:288px;">Sutter instrument</td>
			<td style="width:180px;">P-97</td>
			<td style="width:495px;">Microelectrode puller</td>
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			<td height="21" style="height:21px;">Narishige MF-830</td>
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			<td>Microforge</td>
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			<td height="21" style="width:288px;height:21px;">WPI MF200</td>
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			<td height="21" style="width:288px;height:21px;">Glass capilaries</td>
			<td style="width:288px;">WPI</td>
			<td>B150-86-10</td>
			<td style="width:495px;">Glass capilaries</td>
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			<td height="21" style="height:21px;">Microtorch 1WG61</td>
			<td style="width:288px;">Grainer</td>
			<td style="width:180px;">1WG61</td>
			<td>Microtorch</td>
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			<td height="21" style="width:288px;height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:288px;">Dow Corning</td>
			<td style="width:180px;">SYLGARD 184</td>
			<td>Silicone for dissection plates preparation</td>
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		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Dissection pins</td>
			<td style="width:288px;">Amazon</td>
			<td>B00J5PMPJA</td>
			<td>Pins for larvae positioning</td>
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			<td height="21" style="width:288px;height:21px;">Tweezers</td>
			<td style="width:288px;">WPIINC</td>
			<td>500342</td>
			<td>Tweezers for placing pins, removing the guts and tracheas.&#160;</td>
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			<td height="21" style="width:288px;height:21px;">Scissors</td>
			<td style="width:288px;">WPIINC</td>
			<td>501778</td>
			<td>Scissors for cutting the cuticula of the larvae and nerves.</td>
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		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus BX61WI</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">BX61WI</td>
			<td>Upright microscope</td>
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		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus Lumplan FL N 60x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">UPLFLN 60X</td>
			<td>Microscope objective 60X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus UPlan FL N 10x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">Uplanfl N 10X</td>
			<td>Microscope objective 10X</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Narishige Micromanipulator</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MHW-3</td>
			<td>Three-axis Water Hydraulic Micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">npi Electronic GmbH ELC-03XS</td>
			<td style="width:288px;">npi Electronic GmbH</td>
			<td style="width:180px;">ELC-03XS</td>
			<td>Electrophysiological amplifier</td>
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		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Master 8</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Master 8</td>
			<td>Electrical stimulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Iso-Flex</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Iso-Flex</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">TMC antivibration table</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">63-9090</td>
			<td>Antivibration table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMC Faraday cage</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">81-333-90</td>
			<td>Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Digidata 1322A</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">Digidata 1322A</td>
			<td>Digidata</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Computer</td>
			<td style="width:288px;">Dell</td>
			<td style="width:180px;">Dell Dimension 5150</td>
			<td>Computer with Win XP OS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Electrode holder</td>
			<td style="width:288px;">WPI</td>
			<td>MEH3SW&#160;</td>
			<td style="width:495px;">Electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Optical filter</td>
			<td style="width:288px;">Omega optical</td>
			<td style="width:180px;">XF 115-2</td>
			<td style="width:495px;">Filter cube for Green Fluorescent Protein (GFP) detection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">pCLAMP 8</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">8.0.0.81</td>
			<td>Software for signal recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Quantan</td>
			<td style="width:288px;">In-house software</td>
			<td style="width:180px;">-</td>
			<td>Software for signal processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Canton-S (Wildtype)</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td>64349</td>
			<td>Control fly line</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">cpx SH1</td>
			<td style="width:288px;">Generous Gift of J.T. Littleton</td>
			<td style="width:180px;">-</td>
			<td>Complexin knock-out fly line with increased spontaneous exocytosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">CD8-GFP</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td style="width:180px;">5137</td>
			<td>Fly line with neuronal fluorescent (GFP) Tag</td>
		</tr>
	</tbody>
</table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

Drosophila nöromüsküler kavşak (NMJ) glutamatergic sinaptik iletimi çalışmaya bir mükemmel model sistemidir. Biz odak macropatch kayıtları görüntülenmeyecektir boutons Drosophila larva NMJ, sinaptik akımlardan tekniğini tanımlamak. Bu teknik MİKROPİPETLER yanı sıra bileşik yüksek büyütme, uzun mesafe su daldırma amacı, farklı girişim kontrast (DIC) optik ve bir floresan mikroskop kayıt özelleştirilmiş imalat gerektirir Eki. Kayıt elektrot DIC optik, epi-floresan veya her ikisi ile görüntülenir seçilen bir sinaptik bouton üst kısmında konumlandırılmış. Bu teknik avantajı bu sınırlı sayıda site açıklaması sinaptik etkinliğini izlemeye olanak sağlamasıdır. Kayıt elektrot birkaç mikron çapında ve elektrot RIM dışında konumlandırılmış yayın siteleri kayıtlı akımları önemli ölçüde etkilemez. Kaydedilen sinaptik akımlar hızlı Kinetik var ve kolayca çözülebilir. Bu avantajlar özellikle mutasyona uğramış sinek hatları ile gelişmiş kendiliğinden ya da zaman uyumsuz sinirsel aktivite çalışmaları için önemlidir.

Odak macropatch kayıtları seçili sinaptik boutons (şekil 5) üzerinden izleme sinirsel aktivite etkinleştirin. Elektrot bir sinaptik bouton üst kısmında (şekil 5A, 1 sitesi) getirildiğinde kaydedilen mEJCs (şekil 5C, 1 sitesi) gürültü seviyesini önemli ölçüde aşan bir genlikleri var ve aşamaları (alt milisaniyelik mesafeden) yükselen keskin. Ne zaman kayıt elektrot uzak sinaptik bouton tarafından birkaç mikron (şekil 5A, site 2), kayıtlı mEJCs düşüş genlikleri neredeyse gürültü seviyesi (şekil 5B, site 2) taşınır. Kaydedilen EJC'ler zar zor gelen gürültü ayırt edilebilir ve aşamaları (şekil 5C, site 1 ve site 2) yükselen uzun.
Sınırlı sayıda yayın site hızlı Kinetik sinaptik akımlarının yanı sıra kaydedilen EJC'ler ve mEJCs katkıda focally kayıt, mutantlar yayın olayları doğru tespiti ile yükseltilmiş sinirsel aktivite sağlar. Bu açıkça mEJCs complexin null mutant (şekil 6A) gelen kayıtları tarafından gösterilmiştir. Spontan aktivite bu mutant32yılında büyük ölçüde yükselmiş ve bu nedenle mEJPs intracellularly kaydedilen örtüşme ve odak kayıtları22 etkinleştirmek iken doğru birbirinden (şekil 6B), açıkça ayırt edici olamaz spontan yayın olaylar (şekil 4A) olarak algılanması.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56493/56493fig5.jpg"> 
Şekil 5. EJC'ler ve mEJCs seçili bouton gelen kayıtları. (A)elektrot bir seçili bouton (sayfa 1) yerleştirerek ve bouton (site 2) uzak hareket. Elektrot (A.2), kas fiber üzerinden kayıt (A.1), epi-floresan ile Etiketlenmiş NMJ görselleştirildiği CD8-GFP resimleri göster ve yer paylaşımları (A.3, A.4), elektrot ile sitesi üzerinden 1 (A.3) veya site 2 () konumlandırılmış A.4). ölçek çubuğu = 10 bouton (sayfa 1) kaydedilen µm. (B) mEJCs kayıt gürültü açıkça ayırt edici olup hızlı yükselen aşamaları vardır. Buna ek olarak, mEJCs sitesinden 2 kaydedilen güvenilir bir şekilde kayıt gürültü ayırt edemez, onların genlikleri several-fold azaltılır ve onlar-si olmak daha yavaş bir zaman ders. (C) genlikleri in bouton (sayfa 1) kaydedilen EJC'ler çok katlı 2 siteden EJC'ler genlikleri kaydedildi tarafından ve onlar da daha hızlı Kinetik var. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig5large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56493/56493fig6.jpg"> 
Şekil 6. Hücre içi kayıtları karşı odak macropatch. Hücre içi kayıtları (B) bu mutant sergi örtüşme ve güvenilir bir şekilde algılanamaz olayları sürümünde çalışırken odak kayıtları complexin null mutant(a)mEJCs doğru algılanmasını etkinleştirin. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig6large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila larva nöromüsküler kavşak sinaptik akımlardan Fokal Macropatch kayıtları

Sinaptik akımları, Drosophila Üçüncü INSTAR larva nöromüsküler kavşak görüntülenmeyecektir sinaptik boutons üzerinden focally kaydedilebilir. Bu teknik, tek bir sinaptik bouton etkinliğini izleme sağlar.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

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Universidad San Sebastian

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Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

6.0K Görüntüleme.  Wayne State University. Sinaptik akımları, Drosophila Üçüncü INSTAR larva nöromüsküler kavşak görüntülenmeyecektir sinaptik boutons üzerinden focally kaydedilebilir. Bu teknik, tek bir sinaptik bouton etkinliğini izleme sağlar.

Video: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila Larvaların segmental Aksonlar içinde Sinaptik veziküller in vivo Görselleştirme

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Nörobilim

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Aktif Bölgeleri Diseksiyon ve Görüntüleme Drosophila Nöromüsküler Junction

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sıralı Nöromusküler Kavşağı&#39;nda Tek Schwann Hücreleri belirginleştiren Photo-beyazlatma

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

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 ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Algılama<em&gt; In Situ</emEn&gt; Protein-protein kompleksleri<em&gt; Drosophila</emProximity ligasyon Testi Kullanımı&gt; Larva Nöromüsküler Junction

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

Nöromusküler Kavşak: Ölçme Synapse Boyut, Konfokal Floresan Mikroskobu kullanılarak sinaptik Protein Yoğunluk Parçalanma ve Değişiklikler

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

En fenotip Karakterizasyonu: Niceleme Matters Neden<em&gt; Drosophila</em&gt; Larva Nöromüsküler Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

Kurbağa Sinir Uçlarına Sinir Kütüğü Vasıtasıyla Kalsiyum Boyası Yükleme: Kurbağa Nöromüsküler Kavşağında Kalsiyum Geçici Kayıt

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Nöromüsküler kavşak işlevleri ölçme

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Yüksek throughput ve Çok parametrik Nicelik için iki Algoritmalar<em&gt; Drosophila</em&gt; Nöromusküler Junction Morfoloji

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...