Yazarlar, finansman için RIKEN teşekkür ederiz. Ayrıca genetik ve immünhistokimya protokolleri ile ilgili tartışmalar için, Çağrı Başel Yalgin, Caroline Delandre ve Jay Parrish teşekkür ederim.

Reaktiflerin, 1.Preparation <ol><li> Ca + + ücretsiz HL3.1 tuzlu su 28 hazırlayın. <ol><li> MM: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 Hepes, 115 sakkaroz ve 5 trehaloz; pH 7,2. Sterilize Filtre ve 4 saklamak ° C Not: Ca + + ücretsiz bir çözüm diseksiyonu sırasında kas kasılması önler. </li></ol></li><li> Poli-L-lisin (PLL) lamelleri olun. <ol><li> 100mg PLL, -20 ° C&#39;de 4.2ml suda çözülür ve 300μl alikotları Eppendorf tüpleri ve dondurma yapmak </li><li> Kaplama lamelleri, ilk çözülme bir kısım, GKD 2 O 10ml getirmek ve Kodak Photo-Flo 20μl eklemek önce son PLL konsantrasyonu 0.7 mg / ml; </li><li> Batmak lamelleri, 30 dakika için PLL çözüm çıkarın ve kuru, kısa bir süre sonra su ve kuru yıkayın. Iki kez tekrarlayın. Arıtılmış lamelleri son yaklaşık bir ay. </li></ol></li></ol> 2. Genetik haçlar <ol><li> To MARCM klonlar oluşturur. İşte bir örnek çapraz bir pan-DA sürücü 11,27 kullanarak: FRT2A x hsFLP Gal4 109 (2) 80, UAS-mCD8: GFP, küvet-Gal80 FRT2A/SM5-TM6B </li><li> Klonlar Flp-out oluşturmak için. Burada bir sınıf IV-özel sürücü 8 kullanarak bir örnek çapraz: PPK-Gal4 x yw, hsFLP UAS-FRT-CD2, y +-stop-FRT-mCD8: GFP </li></ol> 3. Embriyoların toplanması <ol><li> Drosophila haçlar tutun bir koleksiyon şişesi 25 ° C ve maya yapıştırın 30,31 ince bir tabaka ile bir elma suyu agar plaka 29 yayılmasını embriyolar toplamak. </li></ol> 4. Isı şok tedavi embriyoların <ol><li> Embriyoların atılmış, bunun üzerine elma suyu agar plaka muhalefet boş bir tabak yerleştirin. Parafilm (Şekil 2) ile bu iki tabak etrafında Seal. </li><li> Isı şok sırasında, batığın incie su banyosu plaka ve bir metal ağırlık kullanarak basılı tutun. </li><li> MARCM embriyolar <ol><li> 2s için embriyolar toplayın ve çevrili bir 10cm petri inkübe ek bir 2 saat için 25 ° C&#39;de doku nemlendirilmiş. Not: ısı şok protokolü ayarlama klonları oluşturulur hangi frekans değiştirir. </li><li> 38 1s için su banyosu tek izole nöronların, daldırın ve ısı şok amaçlayan bir klon daha az sayıda ° C için </li><li> Klon daha büyük bir sayı için, 45 dakikaya için ısı şoku 38 ° C, ek bir 30 dakika daha sonra RT 30 dakika, ısı çarpması kurtarmak. </li></ol></li><li> 24 saat embriyolar Flp toplamak ve 38 1s için ısı şok ° C </li></ol> 5. Klonlar için tarama <ol><li> Isı şok sonra, su geçirmez bir düzenlemenin kapağını kaldırın ve elma suyu agar plaka 10cm petri nemlendirilmiş dokuları ile çevrili bir yerde. Kültür dolaşmak kadar embriyo ve sonraki larva 25 ° Cing 3. instar. Not: kültür koşulları, beslenme, özellikle DA dendritik çardak morfolojisi 32,33 değiştirmek için ortaya konmuştur. Büyüyen larva, her zaman maya yapıştırmak için erişimi olduğundan emin olun; gerektiği gibi yapıştırın maya izlemek ve yenilemek. </li><li> Bu noktadan itibaren protokol itibaren, larvaları hafifçe böcek forseps kullanarak işleyebilirsiniz. </li><li> Kısaca ve yavaşça, musluk suyunda larvaların yıkayın ve sonra agar bir tabağa koyun. Not: Bu adım, arka planda otomatik floresan larva yüzeyinde yiyecek veya kirden azaltır. </li><li> Güçlü bir floresan diseksiyon mikroskobu altında larvaları inceleyin. Larvaların vücut duvarı GFP pozitif nöronlar / hücreleri ile tanımlayın. </li></ol> Dendritler 6 in vivo görüntüleme <ol><li> Larva küçük bir damla% 80 gliserol ile depresyon slayt bardağın içine yerleştirin. Larva hareketsiz bir slayt lamel yerleştirin. Herhangi bir hava larva ve coversli arasında kalmasını sağlamaks. </li><li> Ilgi nöron görselleştirme izin vermek için larva rulo lamel yavaşça itin. MCD8: konfokal mikroskobi ile GFP etiketli dendritik mili. </li></ol> 7. Larvaların diseksiyonu Başlamadan önce Not: DA nöron dendritler diseksiyon başladıktan sonra hızla zayıflar. Iyi dendrit morfolojisi sağlamak için daha az 5dk her bireyin larva parçalara ayır. <ol><li> 3. instar larvaları dolaşıp, aşağıdaki değişiklikler ile daha önce 34 açıklandığı gibi bir Sylgard plaka üzerinde teşrih : <ol><li> DA nöron akson Termini görüntüleme için, merkezi sinir sistemi sağlam ve segmental sinirler kırık olmadığını garanti eder. Larvaların açtıktan sonra, ilk önce vücut duvarı dikkatle trakeal bağlantıları kesmek ve daha sonra yavaşça tüm bağırsak kaldırmak. </li><li> Eğer dendritler yalnız görüntüleme, düz bir montaj sağlamak için MSS çıkarın. </li></ol></li></ol> 8. Fixatlarva filetosu iyon ve engelleme <ol><li> Hala Sylgard plaka tutturulmuş larva ile yavaşça dönen çalkalayıcı RT 20mins için PBS içinde% 4 PFA sabitleyin. </li><li> Fiksasyon sonra larva dokularının izleri (yağ vücut, trakea, vb.) Çıkarın. </li><li> Sylgard plaka ve çalkalayıcı üzerinde 3x 10mins (% 0.1 Triton X-100 ile PBS) PBST yıkayın. Akson terminallerini inceleyerek varsa fileto, Sylgard plaka üzerinde tutun. (Boyama dendritler Eğer bu adımı 34 larva filetosu 0.5 ml tüp aktarmak mümkün) </li><li> RT 20mins larvaları çalkalamalı PBST% 5 normal eşek serum (NDS) engelleyin. </li></ol> 9 - Larva filetosu boyanması <ol><li> Primer antikor engelleme çözümü ve inkübe çevrili küçük bir Tupperware konteyner ° C dokuların nemlendirilmiş 4 geceleme (NDS / PBST% 5) çıkarın. </li><li> 4 ° C tüpler çıkartın ve oda sıcaklığında bir saat inkübe edin. </li><li> PBST 6x 10mins yıkayın. Ikincil antib ekleody (NDS / PBST% 5) ve kapak fluorofor fotoğraf beyazlatma 34 engellemek için. </li><li> Ya iki saat boyunca oda sıcaklığında inkübe edin, ya da bir gecede 4 ° C oda sıcaklığında bir saat izledi. </li><li> PBST larvaları 6x 10mins yıkayın ve montaj devam. </li></ol> 10. Dendrit çardak muayene için larva filetosu montajı <ol><li> Mount diseksiyon makas veya baş (ağız kanca da dahil olmak üzere) ve 34 (spiracles dahil) posterior kesmek için bir neşter kullanılarak mümkün olduğu kadar düz, her larva fileto. </li><li> % 80 gliserol monte slayt aşağı manikür-yan larva fileto yerleştirin ve &#39;hızlı&#39; mount 34 oje ile lamel iki mühür. </li><li> Kalıcı bir montaj ve daha net bir görüntü için <ol><li> PLL lamel PBS bir damla üzerine disseke larva fileto kas tarafı aşağı gelecek şekilde yerleştirin, hızla lamel uyacaktır. </li><li> Howeve, monte edildikten sonra mümkün olduğunca fazla sıvı olarak Kaldırr tamamen kurumasına izin vermeyin. </li><li> % 35 etanol,% 50,% 70,% 95, ve her biri 10 dakika (2.% 100 EtOH çözüm sık değiştirilmesi olmalıdır nihayet 2x% 100 etanol: etanol bir dizi lamel (ekli larva fileto) atın ); nihayet, 2-3x Ksilen 10mins yıkayın. </li><li> DPX mountant (Distyrene Plastifiyan Ksilen) temiz bir slayt üzerinde bir damla koyun ve üstüne dikkatlice lamel fileto tarafı aşağı yatıyordu. Karanlıkta tutun ve görüntüleme önce ayarlamak için DPX için bir gün bekleyin. </li></ol></li></ol> 11. Akson Termini muayene için larva filetosu montajı <ol><li> Segmental sinirler diseksiyonu, fiksasyon sırasında sağlam PNS ve MSS arasında çalışan ve periferik duyusal nöron hücre gövdesi VNC aksonlar izleme izin boyama tutun. </li><li> Bir depresyon slayt% 80 gliserol her larva fileto manikür tarafı yere monte edin. DA hücre gövdesi aksonlar İzKonfokal mikroskop altında VNC. Not: VNC cognate segmentine her vücut duvarı segmentinde projesi PNS nöronlar. </li></ol> 12. Temsilcisi Sonuçlar: Temsilcisi sonuçları rakamlar 3-5 gösterilmiştir. Şekil 3 konfokal mikroskop altında in vivo olarak yakalanan bir sınıf IV da nöron, tüm mili gösterir. Şekil 4 doğru morfolojisi korumak için sabit olmuştur immünohistokimyasal etiketli bir sınıf III nöron dendrit çardak bir parçası kadar yakın gösterir. Ilişkili içerlek (anti-GFP antikor ile hem boyanmış), başarısız bir diseksiyon ve fiksasyon sonra oluşabilir bozulma gösterir. Şekil 5 MSS tek bir sınıf IV akson terminus (anti-GFP, yeşil), tüm sınıf IV Termini anti-CD2 (kırmızı) ile birlikte lekeli. <img alt="Şekil 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> Şekil 1 Protokolü genel bakış. a) toplayın ve sonra ısı-şok embriyolar. b)<html> Canlı larva sonra bir GFP pozitif DA nöron klon ile larva, ve görüntü GFP etiketli dendrit çardak seçin. c) larva ayır ve sonra immünohistokimyasal fileto leke. d) Mount lekeli fileto ve ardından görüntü, dendritik mili ve aksonal projeksiyonlar DA nöron klonlar. <img alt="Şekil 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> Şekil 2 elma suyu agar plaka ısı şok Hazırlık. Plaka (beyaz ok, ab) (kırmızı ok b) üstüne atın, ikinci bir petri ekleyin ve Parafilm (mavi ok, b) ile mühür. <img alt="Şekil 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> Bir mCD8 dendrit çardak in vivo görüntü Şekil 3: 3. instar larva bir sınıf IV (v&#39;ada) nöron temsil GFP etiketli MARCM klon (2.1 olarak genotip). Ölçek çubuğu 50μm. d/3111/3111fig4.jpg &quot;/&gt; Şekil 4 mCD8: başarılı diseksiyon ve fiksasyonun (yeşil) sonra iyi morfoloji gösteren anti-GFP antikor ile boyandı sınıf III nöron (ddaA), GFP etiketli MARCM klonu. Başarısız diseksiyonu ve sabitleme (kırmızı) sonra ortaya çıkan içerlek gösterir dendrit bozulması (beyaz ok). Ölçek çubuğu 25μm. <img alt="Şekil 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> Şekil 5 3. instar larva VNC. Tek bir sınıf VI (vdaB) akson terminus (2.2 olarak genotip) bir Flp-out klonu anti-GFP antikor (yeşil) kullanarak tespit edilmiştir. Anti-CD2 tüm sınıf IV Termini (kırmızı) etiket. Ölçek çubuğu 25μm.

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18, 377-402 (2004).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Kaczynski, T. J., Gunawardena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Embryonic+Nervous+System+in+Whole-mount+Drosophila+Embryos.">Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos.</a> J. Vis. Exp. (46), e2150-e2150 (2010).</li><li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures.">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> J Vis Exp. , (2009).</li><li>Medina, P. M., Swick, L. L., Andersen, R., Blalock, Z., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+forward+genetic+screen+for+identifying+mutations+affecting+larval+neuronal+dendrite+development+in+Drosophila+melanogaster.">A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster.</a> Genetics. 172, 2325-2335 (2006).</li><li>Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+and+AMPK+maintain+epithelial+cell+polarity+under+energetic+stress.">LKB1 and AMPK maintain epithelial cell polarity under energetic stress.</a> J Cell Biol. 177, 387-392 (2007).</li><li>Brent, J., Werner, K., 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+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. 25, e1108-e1108 (2009).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

Çıkar çatışması ilan etti.

Drosophila larva DA nöron modeli mekanizmalarının araştırılması için mükemmel bir genetik sistem sağladığı kontrolü nöron morfolojisi ve devre oluşumu. MARCM genel etiketleme ve mutant DA nöron klonları üretmek için kullanılır. MARCM için bir pan-nöral (örneğin Gal4 C155) ya da nöron spesifik bir sürücü kullanır . Bir pan-nöral sürücüsü kullanarak doğrudan kamu stok merkezlerinde yaygın olarak çeşitli stokları kullanmak mümkündür. MSS işar...

<table border="1"><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> SZX16 floresan diseksiyon mikroskobu (GFPHQ filtre ile) </td><td> Olympus </td><td> SZX16 </td><td></td></tr><tr><td> Canlı Böcek Forseps </td><td> FST </td><td> 26030-10 </td><td></td></tr><tr><td> 26mm x 76mm depresyon slayt cam </td><td> Toshinriko Co. </td><td> T8-R004 </td><td></td></tr><tr><td> Sylgard 184 (veya Silpot 184) </td><td> Dow Corning </td><td> 3097358-1004 </td><td></td></tr><tr><td> Poli-L-lisin </td><td> Sigma </td><td> P-1524 </td><td> Bu ürün en etkili olduğu kanıtlanmıştır </td></tr><tr><td> DPX montaj orta </td><td> Sigma </td><td> 44581 </td><td></td></tr><tr><td> Tavşan, anti-GFP </td><td> Invitrogen </td><td> A-11122 </td><td> Seyreltme 1:500 </td></tr><tr><td> Sıçan, anti-CD8 </td><td> Caltag </td><td> 5H10 </td><td> Seyreltme 1:200 </td></tr><tr><td> Fare, anti-CD2 </td><td> AbD serotec </td><td> MCA443R </td><td> Seyreltme 1:700 </td></tr><tr><td> Fare, anti-Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td> Seyreltme 01:10 </td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

Sinir sistemi gelişimi doğru nöron sınıfa özel dendritik gelişim ve aksonal kablo takip nöron konumu ve kimliği, doğru özellikleri gerektirir. Son zamanlarda Drosophila larva periferik sinir sistemi (PNS) dendritik arborization (DA) duyusal nöron nöron farklılaşma iki genel ve özel sınıf mekanizmaları aydınlatmak için güçlü bir genetik model haline gelmiştir . Dört ana DA nöron sınıfları (I-IV) 1 vardır. Dendrit çardak karmaşıklığı artan sırasına göre adlandırılır ve kendi farklılaşma 2-10 genetik kontrol özel sınıf farklılıkları vardır. 1) DA nöron dendrit çardak yayılır meyve sineği güçlü genetik araçlar, 2) yararlanmak: Çünkü DA duyusal sistem dendritik morfoloji 11-13 kontrol arkasındaki moleküler mekanizmalarının araştırılması için pratik bir model bir optik cle altında sadece 2 boyutlu olarakar larva manikür) kolay in vivo yüksek çözünürlüklü, 3 görselleştirmek için sınıfa özel çeşitliliğin dendritik morfoloji) basit vs yüksek dallı dendritik ağaçlar oluşumunu kontrol anahtar öğeleri bulmak için karşılaştırmalı bir analizini kolaylaştırır ve 4 basmakalıp dendritik çardak şekilleri farklı DA nöronlarının morfometrik istatistiksel analizler kolaylaştırır. DA nöron aktivitesi, larva lokomosyon merkezi desen jeneratör 14-16 çıkış değiştirir . Farklı DA nöron sınıfları farklı duyusal yöntemleri var ve bunların aktivasyon 14,16-20 farklı davranışsal tepkiler ortaya çıkarır. Ayrıca farklı sınıflar, ventral sinir kablosunu Drosophila larva merkezi sinir sistemi (VNC) 21 basmakalıp aksonal projeksiyonlar gönderir . Bu tahminler, dendritik alanında 7,22 vücut duvarı DA nöron duyusal yöntemidir ve pozisyon hem topografik gösterimleri ile sona erdirme, 23. DA aksonal projeksiyonlar Bu nedenle muayene topografik harita 7,22,23 yanı sıra, basit bir devre modüle larval lokomosyon 14-17 kablolama altında yatan mekanizmaları aydınlatmak için kullanılabilir . Biz burada MARCM (Mozaik Repressible Hücre Marker Analizi) 1,10,25 ve Flp-22,26,27 teknikleri (Şekil 1 özetlenmiştir) ile 24 işaretleme DA nöronlar oluşturmak ve genetik mozaikler analiz için pratik bir rehber sunuyoruz.

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Dendritik arborization duyusal nöronlar<em&gt; Drosophila</em&gt; Larva periferik sinir sisteminin genel ve nöron nöron farklılaşma sınıfa özel mekanizmaları aydınlatmak için faydalı model vardır. Biz dendritik arborization nöron genetik mozaikleri oluşturmak ve analiz etmek için pratik bir rehber sunuyoruz.

Morfolojik analiz Drosophila</emGenetik Mozaikler> Larvaların Periferik Duyusal Nöron Dendritler ve Aksonlar

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Research

JoVE Journal

Neuroscience

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

15.2K Görüntüleme.  RIKEN Brain Science Institute. Dendritik arborization duyusal nöronlar Drosophila Larva periferik sinir sisteminin genel ve nöron nöron farklılaşma sınıfa özel mekanizmaları aydınlatmak için faydalı model vardır. Biz dendritik arborization nöron genetik mozaikleri oluşturmak ve analiz etmek için pratik bir rehber sunuyoruz.

Video: Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

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

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Immünohistolojik Duyusal Nöron Dendritler Mikrotübüller Etiketleme, trakea ve kaslar Drosophila Larva Vücut Duvar

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Aksonlar, Dendrit ve Beyin Dilimleri Bireysel Nöron Dendritik Spine kaynaklanan voltaj duyarlı Boya Kaydı

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Içinde larvaların Devre Fonksiyonel Analiz Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Biyokimyasal Analiz için Duyu Nöronlar gelen üretim ve Aksonlar izolasyonu gözenekli Filtre Kullanma

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Kaldırılması<em&gt; Drosophila</emDuyu Nöronlar ve Epidermal Hücre İmmünofloresan Analizi Larvaların Fileto dan&gt; Kas Dokusu

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Doğrusal Agaroz Kanallar kullanarak Okuyacak<em&gt; Drosophila</em&gt; Larva Tarama Davranış

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Drosophila melanogaster Larval ve yetişkin beyin metabolik Analizi

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Morfolojik analiz Drosophila Larvaların Periferik Duyusal Nöron Dendritler ve Aksonlar