Açıklanan projenin içeriği sadece yazarların sorumluluğundadır ve mutlaka, Ulusal Çocuk Sağlığı ve İnsan Gelişimi veya Enstitüsü resmi görüşlerini temsil etmemektedir TAG Hibe Numarası R01HD050725 Ulusal Çocuk Sağlığı ve İnsan Gelişimi Enstitüsü tarafından desteklenen Ulusal Sağlık Enstitüleri. Biz, el yazması ve video girişi için Godenschwege laboratuvar yanı sıra Barbara Schreader üyelerine teşekkür ediyorum.

1. Ventral sinir kablosu diseksiyonu GF doldurarak Diseksiyon ve boya, oda sıcaklığında yapılabilir. Adımlar için tüm diseksiyon soğuk (4 ° C) serum fizyolojik hücre metabolizmasını yavaşlatır ve nöronlar daha uzun süre canlı tutmak için kullanılmalıdır. <ol><li> CO 2 yetişkin sinekler (4 gün veya daha büyük) anestezisi veya buz üzerinde hareketsiz kadar onları soğuk. Buz ile anestezi daha fazla zaman gerektirecektir (30 dakika kadar) ama, daha uzun süre hareketsiz sinekler devam edecektir. </li><li> Sylgard kaplı bir Petri kabındaki bir sinek. Bu diseksiyon yüksek bir büyütme (80-100X) diseksiyonu stereomikroskopta kullanımını gerektirir. </li><li> Vannas makasla, bacaklar, burnumun ve kanatları çıkartın. </li><li> Sylgard çanağı yönlendirmek için sinek dorsal tarafı, kaba forseps ve ince böcek iğne (minutien pin) kullanın. İlk karın Pin ve sonra (bkz. Şekil 2) boyun biraz gergin ve erişilebilir şekilde, başın arkasında, boynun her iki tarafındaki iki iğne yerleştirerek hayvan biraz uzatıyoruz. </li><li> Taze, soğuk Drosophila salin sinek Batmak. En sık kullanılan Drosophila Salines diseksiyon için, ancak biz burada Gu ve O&#39;Dowd açıklanan tuzlu, 2006 4 yeterlidir. Bir hava kabarcığı sinek saracaktır. Bir şırınga ile havayı çıkarın. </li><li> Karın ve sırt ucunu kısa bir yatay kesi olun. Kesi başlayarak, toraks doğru karın dorsal orta hat boyunca sığ, düz kesim yapmak. Sonra bağ doğru orta hat boyunca mümkün olduğunca tam olarak toraks sığ uzunlamasına kesilmiş devam edin. VNC (Şekil 1) zarar olarak sığ kesimler yapmak için kritik önem taşımaktadır. </li><li> Toraks dikkatle açmak için iki güzel böcek pimleri kullanın. Pimleri kullanarak vücut dışında yarı yarıya yaymak ve uçuş kasları (Şekil 2) yoluyla pimleri. Şekil 2 manikür ripping önlemek için tasvir toraks daha fazla açılmamalıdır. VNC düz bir oryantasyon sağlamak için pimleri, her iki tarafında aynı anterior posterior pozisyonda yer almalıdır. </li><li> Boya enjeksiyon ve uygun görüntüleme için VNC Erişim iç organları tarafından tıkalı olabilir. Bu nedenle, mide, bağırsak, kalp ve üreme organları çıkarmak için forseps ve makas kullanın. VNC her iki yakasında uzanan tükürük bezleri dışarı çekin. Dikkatli diseksiyon kayan doku ve yağ çıkarmak için taze tuzlu su ile durulayın. </li><li> Servikal bağ (CVC) altından yağ vücut çıkarın. Yağlı vücut, yüksek büyütme en iyi görünür. Dikkatle yan doku kavrayın ve çekin ince forseps kullanın. </li></ol> Diseksiyonu 5 ila 10 dakika içinde yapılabilir. Taze, soğuk tuzlu su kullanarak disseke, hasar sinir sistemi, en az bir saat ayakta kalacaktır. 2. Hücre içi GF dolgu Bu diseksiyon sonra mümkün olduğunca çabuk dolum boya yapılması tavsiye edilir. <ol><li> Sabit bir aşamada (Şekil 3) ve 10x hava amacı ile dik bir mikroskop altında diseke sinek Sylgard çanak yerleştirin. Merkezi boya dolgu elektrot tutucu doğru konumlandırılmış sinek arka ucu ile örnek. CVC diferansiyel girişim kontrast (DIC) modu ve odak kullanın. GF&#39;lerin büyük aksonlar CVC dorsal yüzey üzerinde açıkça görülebilir olmalıdır. </li><li> % 1 &#39;lik bir konsantrasyon deiyonize H2O Lucifer Sarı CH Dilithium tuz sulandırınız. Yaklaşık bir direnç filament ile borosilikat cam elektrot çekin. M 60-80. Cam elektrot ucu uzun ve keskin olmalıdır. Elektrodun ucu, yaklaşık Lucifer Sarı çözüm içine elektrod ucu ters sonunda elektrot yerleştirerek% 1 Lucifer Sarı solüsyonu ile doldurun. 30-60 saniye. Daha sonra boya ve LiCl arasında bir hava kabarcığı bırakarak Hamilton şırınga kullanarak 3M LiCl elektrot dolgu. Kafa aşamasına boya dolgu elektrot bağlayın. </li><li> Tuzlu su içine bir klorür kaplı gümüş tel Toprak elektrodu takın ve 3D hidrolik mikromanipülatör kullanarak VNC sinek paralel üzerindeki boya dolgu elektrot yerleştirmek. Boya dolgu elektrodu, akım, sırasıyla geçen hiper-ya da depolarize elektrottan olumlu veya olumsuz ücret boyalar serbest akımı geçirilerek sağlayan bir amplifikatöre bağlı olmalıdır. </li><li> 40x suya daldırma lens geçin ve GF&#39;lerin üzerine boya dolgu elektrot ucu düşük. GF aksonlar CVC dorsal yüzeyine yakın yatıyordu. GF elektrot eklemek için ileri (doğru) baş hareketi kullanın. </li><li> Epifluorescent ışık ışık kaynağı değiştirin ve Lucifer Sarı filtre geçmek. GF Lucifer Sarı içine enjekte etmek için amplifikatör ile boya dolgu elektrot hiperpolarizan mevcut uygulayın. Miktar boya enjekte th tarafından kontrol edilebilire akım miktarı akım geçirilen süre yanı sıra geçti. Boya enjeksiyon yerinde veya hasar GF dışarı dökülmesini önlemek için, düşük akım (yaklaşık 3-5 nA) ile 1-5 dakika içinde enjekte etmek için tavsiye edilir. </li><li> VNC GF terminali (Şekil 4A) açıkça görülebilir kadar uygulayın. Postsinaptik nöronların trans-sinaptik boyanması için boya da postsinaptik nöron görülebileceği kadar geçerli geçmek için devam edin. Geçerli kapatın ve boya dolgu elektrot kaldırmak. </li></ol> 3. Temsilcisi Sonuçlar: Şekil 4A bir temsilci boya Lucifer Sarı ile bir GF doldurunuz gösterilmiştir. Görüntü mikroskop üzerine monte edilmiş bir nokta kamerayı kullanarak Nikon Elements yazılımı bir görüntü yığını olarak satın alınmıştır. Bu örnek akım yaklaşık 2 dakika boyunca elektrot geçirildi. GF iyice doldurulur ve toraks büyük GF terminali (Şekil 4A, elips) olarak etiketlenir. Lucifer Sarı gap junction geçmesine kadar küçük olmasına rağmen postsinaptik nöronların, bu durumda görünür. Sinyal tespit edilmesi çok zayıf, çünkü boya GF yeterince uzun enjekte değilse trans-postsinaptik nöronların sinaptik doldurur görülmemiş olabilir. GF&#39;lerin (Şekil 4B, ok başları) postsinaptik nöronlar gibi Neurobiotin bile küçük nöronal izleyiciler, daha güvenilir bir şekilde görüntülendi olabilir. 2 dakika boyunca bir depolarizan akımı ile 2 M potasyum asetat çözeltisi% 7 Neurobiotin enjekte güçlü postsinaptik nöron etiketlemek için yeterli. Neurobiotin konfokal mikroskopi için farklı dalga boylarında boyalar birleştiğinde Streptavidin kullanarak görüntülenebilmekte. Neurobiotin floresan boya olmadığından, aynı çözüm Rhodamin Dextran veya Alexa hydrazides ko-enjeksiyon, GF (Şekil 4C, Boerner ve Godenschwege, 2010 5) başarılı bir enjeksiyon izlemek için kullanılan olabilir. <img src="/files/ftp_upload/3080/3080fig1.jpg" alt="Şekil 1" /> Şekil 1 bir sinek şematik çizimi yanal görüntülenebilir. Toraks, iç yapıları görselleştirmek için uzunlamasına bir kesi ile görüntülenir. Diseksiyon sırasında, dorsal longitudinal uçuş kasları (DLMS mavi), uçuş kaslarına ventral uzanan ventral sinir kablosu (VNC, koyu kırmızı), zarar vermeden kesilmiştir. Gut (yeşil) VNC üstünde yatıyor. <img src="/files/ftp_upload/3080/3080fig2.jpg" alt="Şekil 2" /> Şekil 2 disseke bir sineğin Dorsal görünümü. İki pimleri sinek konumuna başın arkasında yerleştirilir. Uçuş kasları (DLM) ile yapıştırılmış pimleri, toraks açık tutun. İç organları ventral sinir kablosu (VNC) ortaya çıkarıldı. <img src="/files/ftp_upload/3080/3080fig3.jpg" alt="Şekil 3" /> Şekil 3 Boya doldurun kurulum. <img src="/files/ftp_upload/3080/3080fig4.jpg" alt="Şekil 4" /> Şekil 4 A) Z-Lucifer Sarı ile dolu bir GF gösteren VNC arka kısmının projeksiyon. Görüntü bir noktaya kamera ile boya dolum hemen sonra elde edildi. Vahşi hayvanlarda toraks terminali (elips) olduğu açıkça görülebilir. B) GF boya Konfokal görüntü yabani tip bir sinek Neurobiotin ile doldurun. GF&#39;lerin (oklar) ve GF&#39;lerin (ok başları) postsinaptik nöronların her ikisi de etiketlenir. Streptavidin Cy3 akuple Neurobiotin görüntülendi. C) vahşi tip GF&#39;lerin Konfokal görüntü Alexa555 ile doldurdu.

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

Sinir sistemi, anında vücuttan ayıklanan olmadan disseke edilebilir. Bu, iki avantajı vardır, ilk diseksiyon sinir sistemi için çok az hasara neden olur, ve ikincisi, sinirler, kaslar ve duyu organları bağlı kalmak. Olarak tanımlanan diseksiyonu, Sahne GF&#39;lerin yalındır boya-etiketleme için örnek hazırlar. Elverişli, GF&#39;lerin postsinaptik motor nöronlar kaslara bağlı kalmak. Aksonlar, bu nedenle, nöronlar daha uzun süre canlı tutmak, hasarsız. Buna ek olarak, sinir sistemine zarar gap jun...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Sylgard 184 Silikon Elastomer Seti </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> Diseksiyon Mikroskop </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> Vannas Makas Süper İnce </td><td> Akademik Aletleri </td><td> VS1023 </td><td></td></tr><tr><td> Dumont Forseps Dumontstar 55 </td><td> Güzel Bilim Araçları </td><td> 11295-51 </td><td></td></tr><tr><td> Austerlitz Böcek pimleri </td><td> Güzel Bilim Araçları </td><td> 26002-10 </td><td> Ø 0.1mm </td></tr><tr><td> Borosilikat Elektrotlar </td><td> Dünya Hassas Aletler </td><td> 1B100F-4 </td><td></td></tr><tr><td> Dikey Pipet Çektirme 700C </td><td> David Kopf Aletleri </td><td> Model 700C </td><td></td></tr><tr><td> Lucifer Sarı CH Dilithium tuz </td><td> Sigma </td><td> L0259 </td><td> H 2 O% 1 </td></tr><tr><td> Sabit Sahne Dikey Mikroskop ve Kamera </td><td> Nikon </td><td> FN-1 ve DS-U2 Kamera </td><td></td></tr><tr><td> Plan Fluor 10X Hava Amaç </td><td> Nikon </td><td> CFI Planı Fluor 16mm 10X NA 0.3 WD </td><td></td></tr><tr><td> Fluor 40X su Amaç daldırma </td><td> Nikon </td><td> CFI Fluor 40X W NA 0.80 WD 2.0mm </td><td></td></tr><tr><td> 3D hidrolik Mikromanipülatör </td><td> Narishige Uluslararası </td><td> Model MW0-3 </td><td></td></tr><tr><td> Amplifikatör </td><td> Araçların Başlarken, Inc. </td><td> Model 5A </td><td></td></tr></tbody></table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

Nöronların aksonal ve dendritik morfoloji analiz etmek, doğru etiketleme nöronal yapıları elde etmek için esastır. Az doku hasarı iyi etiketli numunelerin hazırlanması dolayısıyla mutant anomalilerin kimlik sağlayan, bize hücre morfolojisi analiz etmek ve bireysel örnekleri birbirleriyle karşılaştırmak için sağlar. Sinir sistemi göstermiştir diseksiyon yöntemi daha çok erişkin sinek içinde kalır. Dorsal bir kesiden karın ve göğüs açılır ve iç organların çoğu kaldırılır. GF hücre gövdesi ve dendritler içeren beyin sağlam bir kafa 2 kalırken ventral sinir kablosu (VNC) ve dev lifleri (GDS) 1 büyük aksonları içeren servikal bağ (CVC) sadece sırt tarafında açıkta . Bu hazırlanmasında VNC en sinirlerin kaslara bağlı kalmalıdır. Diseksiyonu takiben, floresan boya ile dev bir lif (GF) hücre içi dolum gösterilmiştir. CVC GF aksonlar dorsal yüzeyinde bulunan ve bu nedenle diferansiyel girişim kontrast (DIC) optik bir mikroskop altında kolayca görüntülenebilmekte. Bu VNC akson ve kendi terminalleri dahil olmak üzere tüm GF GF akson boya ile bu sitede enjeksiyon etiket sağlar. Bu yöntem, nöronların bir epifluorescent mikroskop ile doldurduktan sonra hemen yansıması izin GF&#39;lerin güvenilir ve güçlü boyama ile sonuçlanır. Alternatif olarak, floresan sinyali, yüksek çözünürlüklü konfokal mikroskopi için uygun standart immünohistokimya prosedürleri 3 kullanarak gelişmiş olabilir .

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Bir<em&gt; In vivo</em&gt; Yetişkin diseksiyonu<em&gt; Drosophila</em&gt; Ventral sinir kablosu (VNC) gösterilmiştir. Bu özel diseksiyon yöntemi, yüksek çözünürlüklü görüntüleme için floresan boya ile dev bir fiber nöronların sonraki etiketleme sağlayan VNC az zarar neden olur.

Yetişkin Tüm Dağı Hazırlık Drosophila Dev Fiber Boya Enjeksiyon Ventral Sinir Kablosu

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

whole-mount-preparation-adult-drosophila-ventral-nerve-cord-for-giant

Research

JoVE Journal

Neuroscience

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

18.9K Görüntüleme.  Florida Atlantic University. Bir In vivo Yetişkin diseksiyonu Drosophila Ventral sinir kablosu (VNC) gösterilmiştir. Bu özel diseksiyon yöntemi, yüksek çözünürlüklü görüntüleme için floresan boya ile dev bir fiber nöronların sonraki etiketleme sağlayan VNC az zarar neden olur.

Video: Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Tüm montaj Embriyonik Sinir Sistemi Görselleştirme Drosophila Embriyolar

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Nörobilim

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Dev Fiber Pathway Elektrofizyolojik Kayıtlar D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Kullanarak Bileşiklerin Biyoaktivite için eşli Nanoinjection ve Ekran için Elektrofizyoloji Testi Drosophila melanogaster Dev Elyaf Sistemi

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

Fare Embriyonik Omurilik Tüm Dağı Hazırlık aksonal dallanma Eğitim DRG Nöronlar DII-Etiketleme

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Tüm Montajlı Nöromüsküler Kavşak Analizi için Enine Abdominis Kasının Diseksiyonu

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

In Koku Reseptör Nöronlar Tüm Dağı ile immün<em&gt; Drosophila</em&gt; Anten

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Bir<em&gt; İn Vitro</emKas afferentlerin tahrik Properties incelenmesi için&gt; Yetişkin Fare kas-sinir Hazırlık

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Tüm Dağı Diseksiyon ve Yetişkin Fare Kohlea immünofloresan

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Ventral Kök Stimulation için eğik Omurilik Dilimleri hazırlanması

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...