Biz finansman için RIKEN teşekkür ederim. P10-Gal4 Alain Vincent (Université Paul Sabatier, Toulouse, Fransa) bir tür hediye edildi.

1. Reaktiflerin hazırlanması Başlamadan önce Notlar: Diseksiyon ve immünohistokimyasal bir manyetik odasında yapılmakta ve larva özel şekilli böcek pimleri kullanarak aşağı tutturulmuş. Manyetik bir odasının inşaat, ve bu pimlerin hazırlanması ile ilgili ayrıntılı talimatlar, ilgili referanslar 14,15 bulunabilir . Kısaca, bir 1x1cm kare delik bir manyetik levha ve küçük bir oda yapmak için kağıdın arka tarafına yapıştırılmış bir lamel bölünür. Odanın iki epoksi yapıştırıcı ile mühürlenir; sonra bu tutkal odası kullanmadan önce% 70 etanol ile birkaç kez yıkanır. Diseksiyon böcek pimleri istenilen şekil bükme tarafından hazırlanan ve daha sonra bir metal sekmesini 14,15 yapıştırılmış . Alternatif metal bir sekmesinde, bir cut-off sarı ucundan yapılan bir kolu ile ters bir düz başlı çelik raptiye kullandım. Bu manyetik oda düzenleme kullanın, yakın kontrolü ove sağlarr pin konumlandırma ve doku diseksiyonu sırasında germe. DA nöronlar araştırmacılar farklı alt muhabiri gen ekspresyonu sürücü için birkaç farklı Gal4 hatları (Shimono ve arkadaşları 16 tarafından özetlenmiştir) kullanabilirsiniz. Bu satırları çoğu kamu stok merkezlerinden mevcuttur. Bu temsili protokolde, DA nöron iki zıt sınıfların ortak etiketli olduğu bir çizgi immün yürütmek en basit sınıf I ve en karmaşık sınıf IV (P10-Gal4 17,18, UAS-mCD8: Kusabira-Orange (KO)). <ol><li> Ca + + ücretsiz HL3.1 tuzlu su hazırlayın <ol><li> MM: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 Hepes, 115 sakkaroz ve 5 trehaloz pH 7,2 19. Filtre sterilize ve 4 saklamak ° C Not: Ca + + ücretsiz bir çözüm diseksiyonu sırasında kas kasılması önler. </li></ol></li><li> 2x PHEM tampon hazırlayın <ol><li> MM: 130 BORU, 60 Hepes, 20 EGTA, 4 MgSO4, pH 7.0. Filtre sterilize ve 4 saklamak ° C Not: Bu malzemeler, pH 7.0 yaklaşana kadar çözülmez. </li></ol></li><li> Fiksatif taze fiksasyon hemen önce hazırlayın. <ol><li> 50 ml Falcon tüp 25ml çözüm, ilk karışımı 2g paraformaldehid, 100μl 1M NaOH ve 10 ml su hazırlamak için. </li><li> Çözüm netleşene kadar çırpıcı ile 55 ° C su banyosunda fiksatif çalkalayın. (Toplam hacmi 11.5 ml olacaktır.) </li><li> Buz üzerinde soğuk fiksatif. </li><li> 12.5ml 2x PHEM tampon ekleyin. </li><li> 1M HCl ile pH 7.0 &#39;a ayarlayın. </li><li> Çözüm hacmi 25ml kadar su ile doldurun. </li><li> Filtre whatman kağıt kullanılarak çözüm. </li></ol></li></ol> 2. Larvaların diseksiyonu Başlamadan önce Not: duyusal dendritler özellikle Mikrotubul ağları ve arıza diseksiyon başladıktan sonra hızla. Achemen fiksasyonu takiben en az beş dakika içinde hızlı diseksiyon hieving Bu protokolün başarısında önemli faktörlerdir. <ol><li> Larva suda yıkayın ve hızlı bir şekilde saç bir döngü kullanarak bu düşüş içine taşıyabilirsiniz. </li><li> Orient camına larva dorsal yüzü yukarı ve ventral tarafta. Not: Bu yönelim ventral küme nöronlar incelemektir. Dorsal küme nöronlar incelemek için, yönünü ters çevirin. </li><li> Ön ucunu ağzına kanca yakın pin merkezinde böcek-pin kullanın. En iyi sonuçları elde etmek için sonuna kadar yakın pin yerleştirin. </li><li> Diseksiyonu odasında bir damla serum fizyolojik HL3.1 yerleştirin. </li><li> Microscissors ile larva çok arka ucu kesti. Not: Bu adım, microscissors (adım 2.7) için erişim sağlayacak larva posterior sonunda bir diyafram açılır. </li><li> Forseps larva arka sonunda diyafram dışarı alay bağırsak bölgesi tut. Bütün bağırsak yavaşça çekin. </li><li> Yeriventral orta hat boyunca öne doğru diyafram ve kesme yoluyla microscissors bir bıçak ucu. </li><li> Köşesinde pimleri kullanarak, ilk arka sonra ön artık ücretsiz köşelerinde, pin. Aynı zamanda larva fileto yavaşça açın germek. </li></ol> 3. Fiksasyon, engelleme, boyama, ve larva filetosu montaj Başlamadan önce Notlar: Tüm fiksasyon ve boyama adımları diseksiyonu odasında yürütülür. Bu süreçte, bu doku hasarına yol açabilir larva tutan böcek pinleri devirmek için dikkatli olun. Deney kurumasını önlemek için, boyama nemlendirilmiş dokuları ile çevrili küçük bir Tupperware konteyner bütün adımları uygulayın. <ol><li> Sarı bir uç kullanarak Ca + + ücretsiz HL3.1 tampon aspire. Hemen başka bir pipetteman kullanarak fiksatif ekleyin. </li><li> Ca + + ücretsiz HL3.1 tampon kalan izleri ile yavaşça fiksatif karıştırmak için aşağı yukarı pipet veOdanın. Hemen aspirat ve sonra odasına taze fiksatif ekleyin. </li><li> 20 dakika oda sıcaklığında inkübe edin. </li><li> PBST 6x 10mins (% 0.1 PBS içinde Triton X-100) yıkayın. </li><li> RT 20 dakika PBST% 5 keçi serumu ile engelleyin. </li><li> PBST% 5 keçi serumu seyreltilmiş primer antikorlar ile bloke çözüm değiştirin. Kullanılan birincil antikorlar fare anti-α-tubulin (DM1A) ve sıçan anti-CD8 (5H10) seyreltilmiş 1 / 1000. Not: Araştırmacı, fare anti-Futsch (22C10) 20,21 seyreltilmiş bazı durumlarda 1 / 1000 ile fare anti-α-tubulin (DM1A) yerine (tartışma) isteyebilirsiniz . </li><li> 4 (en az 16h) bir gece inkübe ° C </li><li> PBST 6x 10mins yıkayın. </li><li> PBST% 5 keçi serumu seyreltilmiş ikincil antikor çözüm ekleyin. Kullanılan sekonder antikor Alexa Fluor 488 keçi anti-fare IgG ve Cy3 eşek anti-sıçan IgG. Fluorofor önlemek için kapalı örnek tutun22 foto-kasar. </li><li> Ya iki saat boyunca oda sıcaklığında inkübe edin, ya da bir gecede 4 ° C oda sıcaklığında bir saat izledi. </li><li> PBST 6x 10mins yıkayın. </li><li> % 80 gliserol monte slayt aşağı manikür-yan larva fileto yerleştirin ve bir &#39;hızlı&#39; monte etmek için oje ile lamel iki mühür. Not: gelişmiş doku temizleme ve kalıcı istikrarlı bir örnek için, DPX monte edilir, daha önce 23 açıklandığı gibi. </li></ol> 4. Temsilcisi Sonuçlar: Floresan boyama konfokal bir mikroskop altında incelendi. Sayılarla dendrit bir çardak içinde 1-2 farklı dallarında farklı sitoskeletal organizasyon var. Şekil 1, 1. instar larva aşamasında sınıf IV DA nöron mili bir bölge gösterir . KO ve ikincil bir anti-CD8 antikor ve floresan (Cy3) kullanarak tespit: bütün mili mCD8 ile işaretlenir. Tubulin anti-α-tubulin antikor ve Florlu kullanarak tespitescent ikincil (Alexa Fluor 488). Tubulin ana dalları için olumlu, bazı ince yan dalları tubulin negatif. Film 1 sınıf I DA nöron benzer bir boyama ile seri bölümleri bir dizi. Şekil 2, 1. instar larva aşamasında Futsch ve CD8 karşı antikorlar ile boyanarak sınıf IV DA nöronun mili bir bölge gösterir . Ana dalları Futsch pozitif, bazı ince yan dalları Futsch negatif. Şekil 3. Trakea larva vücut duvarı karmaşık bir mikrotübül organizasyonu göstermektedir. Filmler 2 ve 3, vücut duvarı kasları ve trakea ile boyama seri bölümleri göstermektedir. <img alt="Şekil 1" src="/files/ftp_upload/3662/3662fig1.jpg" /> Şekil 1. Şekil 1, 1. instar larva aşamasında sınıf IV DA nöron mili bir bölge gösterir . Bütün mili mCD8 ile işaretlenir: KO ve anti-CD8 antikor ve floresan ikincil (Cy3) kullanarak algılandı. Tubulin anti-&amp;-Tubuli kullanarak tespitn antikor ve floresan sekonder (Alexa 488). Altta yatan Panelleri AC sıralı konfokal z bölümler (0.5μm) C&#39;-C &quot;, panel C. Örnek (sarı ok ucu) veya mikrotübül vurgulanır (mor ok ucu) olmadan şubeleri tek antikor boyama Kırmızı ok uçları vurgulamak mikrotübül epitel hücreleri. <img alt="Şekil 2" src="/files/ftp_upload/3662/3662fig2.jpg" /> Şekil 2. Şekil 2, 1. instar larva aşamasında Futsch ve CD8 karşı antikorlar ile boyanarak sınıf IV da nöronun mili benzer bir bölgeyi gösterir . Bütün mili mCD8 ile işaretlenir: KO ve anti-CD8 antikor ve floresan ikincil (Cy3) kullanarak algılandı. Futsch Futsch anti-antikor ve floresan ikincil (Alexa 488) ile tespit edilir. Ana dalları Futsch pozitif, bazı ince yan dalları Futsch negatif. (Sarı ok ucu) ya da (mor ok ucu) Futsch olmadan şubeleri örneği vurgulanır. Şekil 3. Trakea, yukarıda açıklanan anti-tubulin protokolü kullanılarak boyandı. Bu larva, üçüncü instar ve daha önce 23,24 açıklandığı gibi disseke. Movie 3, bir kare ile işaretlenmiş alan genişlemiş seri bölümleri gösterir. Film 1 Seri bölümleri ben nöron bir sınıfın dendritik mili boyunca tubulin boyama izleme. KO (Kırmızı) ve ikincil anti-CD8 antikor ve floresan (Cy3) kullanarak tespit: bütün mili mCD8 ile işaretlenir. Tubulin (Yeşil) bir anti-α-tubulin antikor ve floresan ikincil (Alexa Fluor 488) kullanılarak tespit edilir. Ölçek: video görüntüsünün bir yan bölümünde 46.88μm karşılık gelir. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">Movie görmek için buraya tıklayın.</a> Bod tubulin boyama izleme Film 2 Seri bölümleriüçüncü bir instar larva y duvarı kasları. Tubulin anti-α-tubulin antikor ve floresan ikincil (Alexa Fluor 488) kullanılarak tespit edilmiştir. Ölçek: video görüntüsünün bir yan bölümünde 46.88μm karşılık gelir. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">Movie görmek için buraya tıklayın.</a> Film 3 Seri üçüncü bir instar larva Şekil.3 bir kare ile işaretlenmiş bir vücut duvar trakea, tubulin boyama izleme bölümleri. Tubulin anti-α-tubulin antikor ve floresan ikincil (Alexa Fluor 488) kullanılarak tespit edilmiştir. Ölçek: video görüntüsünün bir yan bölümünde 46.88μm karşılık gelir. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">Movie görmek için buraya tıklayın.</a>

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W., Tyrer, M., Shepherd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+and+tau+reporters+disrupt+central+projections+of+sensory+neurons+in+Drosophila.">Tau and tau reporters disrupt central projections of sensory neurons in Drosophila.</a> J. Comp. Neurol. 428, 630-640 (2000).</li></ol>

Biz ifşa etmek başka bir şey var.

Ne kadar karmaşık hücre şekiller elde edilir anlamak için, doğru tahlil mikrotübül organizasyonu edebilmek için önemlidir. Burada dendritik arborization duyusal nöron dendritler tahlil mikrotübül organizasyonu sağlam bir immünohistolojik etiketleme yöntemi açıklar. Boyama duyusal nöronlar ek olarak, bu yöntem, trakea, kaslar ve diğer vücut duvarı dokuları sağlam immünohistolojik boyama ulaşır. Biz DA nöronlarının gelişmekte olan duyusal dendritler mikrotübül...

<table border="1"><tbody><tr><th> Reaktifi Adı </th><th> Şirket </th><th> Katalog numara </th><th> Yorumlar (Isteğe bağlı) </th></tr><tr><td> Forseps </td><td> Dumont </td><td> 11251-20 </td><td></td></tr><tr><td> Microscissors </td><td> FST </td><td> 15000-08 </td><td></td></tr><tr><td> Fare anti-α-tubulin (Klonlama: DM1A) </td><td> Sigma </td><td> T9026 </td><td> Seyreltme 1 / 1000 </td></tr><tr><td> Mouse anti-Futsch (Klonlama: 22C10) süpernatant </td><td> Gelişim Çalışmalar Hibridoma Bankası </td><td> 22C10 </td><td> Seyreltme 1 / 1000 </td></tr><tr><td> Sıçan anti-CD8 (Klonlama: 5H10) </td><td> Caltag </td><td> MCD0800 </td><td> Seyreltme 1 / 1000 </td></tr><tr><td> Alexa Fluor 488 anti-fare IgG </td><td> Invitrogen </td><td> A-11001 </td><td> Seyreltme 1 / 500 </td></tr><tr><td> Cy3 anti-Rat IgG </td><td> Jackson Immunoresearch </td><td> 712-166-150 </td><td> Seyreltme 1 / 200 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

Karmaşık hücre şekilleri farklılıklar nasıl elde edilir anlamak için, doğru mikrotübül organizasyonu takip etmek önemlidir. Drosophila larva vücut duvarı, hücre ve doku morfolojilerinden çalışma modelleri çeşitli hücre tipleri içerir . Örneğin trakea tüp morfolojilerinden 1 ve Drosophila larva duyusal nöronlar, genel ve dendritik farklılaşma 2-5 ve 6 dejenerasyon nöron sınıf özel mekanizmaların aydınlatılması için birincil sistem haline gelmiştir dendritik arborization (DA) incelemek için kullanılır . . Dendrit dallarının şekli nöron sınıflar arasında önemli farklılıklar ve hatta tek bir nöron 7,8 farklı dalları arasında. DA nöronların genetik çalışmalar diferansiyel sitoskeletal organizasyon dendritik şube şekli 4,9-11 morfolojik farklılıklar altında olduğunu göstermektedir. Biz sağlam bir immünolojik etiketleme yöntemiDA duyusal nöron dendrit çardak in vivo mikrotübül organizasyonu (Şekil 1, 2, Movie 1) ssay. Bu protokol ilk instar larva diseksiyonu ve immün aktif duyusal nöron dendrit akıbet ve dallanma organizasyon 12,13 meydana gelen bir aşama göstermektedir. Duyusal nöronlar boyama ek olarak, bu yöntem güçlü kas mikrotübül organizasyonu etiketleme (Film 2, 3), soluk borusu (Şekil 3, Film 3), ve diğer vücut duvarı dokulara ulaşır. Kontrol doku ve hücre şekli, bu mekanizmaların araştırılması vücut duvarı in situ mikrotübül organizasyonu analiz etmek isteyen araştırmacılar için değerli .

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

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

Nöron dendritler gibi karmaşık hücre şekilleri, geliştirme sırasında nasıl elde edilir anlamak için, bu, doğru tahlil mikrotübül organizasyonu edebilmek için önemlidir. Burada dendritik arborization duyusal nöron dendritler mikrotübül organizasyonu, trakea, kas incelemek için sağlam bir immünohistolojik bir etiketleme yöntemi açıklamak ve diğer<em&gt; Drosophila</em&gt; Larva vücut duvar dokuları.

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 ...

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JoVE Journal

Neuroscience

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

12.3K Görüntüleme.  RIKEN Brain Science Institute. Nöron dendritler gibi karmaşık hücre şekilleri, geliştirme sırasında nasıl elde edilir anlamak için, bu, doğru tahlil mikrotübül organizasyonu edebilmek için önemlidir. Burada dendritik arborization duyusal nöron dendritler mikrotübül organizasyonu, trakea, kas incelemek için sağlam bir immünohistolojik bir etiketleme yöntemi açıklamak ve diğer Drosophila Larva vücut duvar dokuları.

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Nörobilim

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

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

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

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Içinde proprioseptörlere Görselleştirmesi<em&gt; Drosophila</em&gt; Larva ve Pupa

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Merkez Sinir Sistemi Tek Hücreler Etiketleme<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of 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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Of Bilgisayar İzleme tarafından Analiz Edilmiş Olfaktor Davranışlar<em&gt; Drosophila</em&gt; Dört kadranda olfactometer içinde

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Beyin Dilimlerinde ve Birincil Hücre Kültüründe Piramidal Nöronların Balistik Etiketlemesi

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Otomatik Görüntü Analizi Kullanılarak Kemirgen Modellerinin Substantia Nigra'sında Dopaminerjik Nöron Yoğunluğunun Yarı Nicel Belirlenmesi

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Drosophila Nöromüsküler Kavşaklarda ve Kas Hücrelerinde Mikrotübül Ağlarının Görüntülenmesi

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Yazar Spotlight: Drosophila nöromüsküler kavşaklarında mikrotübül ağını anlamak 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.