Istiyorum Yazarlar Bloomington stok merkezi, Liqun Luo, Charles Zuker ve Lily ve Yuh Nung Oca sinek stoklarının cömert paylaşımı ve finansman aşağıdaki teşekkür: Lisans Ford Fakültesi Yaz Bursu (JD ve SW) somas-URM (SW), Renee ve Anthony M. Marlon, MD (DL) &#39;63 Yaz Araştırma Bursu (CL ve DL için) BD Kurumlar Yaz Araştırma Bursu James C. &#39;75 ve Jane Colihan Yaz Araştırma Bursu (TO) aracılığıyla Mezunlar / Kutsal Haç Koleji Veli Yaz Araştırma Fonu ve Stransky Vakfı Yaz Araştırma Bursu (TO). Laboratuvarda tüm çalışmaları desteklemek için Kutsal Haç Koleji&#39;nde Biyoloji Bölümü ve Dekanlığa Special thanks.

1. Haçlar MARCM Sinekler oluşturmak için <ol><li> 22 santigrat derece veya standart Drosophila ortam Mısır unu pekmez ortam (% 0.13 Tegosept, 23.5 g / L maya, 60 g / L Mısır unu, 60 mL / L melas;, 4 mL / L asit karışımı 6.5 g / L agar) üzerine sinekler bakımı Bir 12-st ışık / karanlık döngüsünde. Not: Standart sıcaklık daha düşük bir sağlıklı MARCM-stoklarını tutmak için kullanılır. Bu 14 gün yumurta ve yetişkinlik arasındaki gelişimsel süresini uzatır. </li><li> Bir yukarı-aktive dizisi (UAS-GFP), GAL80 bastırıcı protein, Gal4 transkripsiyon faktörü, bir kontrol altında (GFP gerekli genetik elementler içeren bir MARCM hazır stoktan - (8 gün eski, yaklaşık 1) bakire kadın kullanın ısı-şok odaklı rekombinaz ve FRT rekombinasyon sitesi). Not: Bizim MARCM hazır stokları aşağıdaki genotipleri içerir: elavGal4, UAS-CD8-GFP, hs-flipase; FRT40A, tubGal80 / CYO; tubGal4 / TM3Sb. Not: Belirli FRT sitesi mutasyon yere bağlı olacaktırgenomuna. Örneğin, FRT40A gibi NOMPC (MARCM protokolleri ile ilgili daha ayrıntılı bilgi için 10 bakınız) olarak kromozomun 2 sol kolunda, üzerinde mutasyon test etmek için kullanılır. </li><li> Bir ilgi mutasyon ve buna tekabül eden FRT sitesini ihtiva erkeklere Çapraz MARCM hazır bakire kadın (; NOMPC3, FRT40A / CYO, örneğin w- + / +). Etkili MARCM indüksiyonu için kontrol etmek için, aynı FRT bölgesini ihtiva eden bir erkek sinekler kullanılarak bir ikinci çapraz ayarlanır, ancak mutasyonu olmayan. Melezlerde 1 erkek: 5 kadın yaklaşık bir oranını kullanın. </li></ol> Döşeme 2. Zamanlı Yumurta <ol><li> Sinekleri, en az bir gece çiftleşme bırakıldıktan sonra, taze ortam ile yeni şişelere yetişkin hareket eder. Sinekler tekrar yeni şişe içine haçlar aktarmadan önce yaklaşık 4 saat karanlık ortamda yumurtalarını bırakmak için izin verin. Yumurtlama döneminin başlangıç ​​ve bitiş saatlerini not alın. Not: 4-hr interva sonundan önce medyaya yumurta sayısının yüksek olması göründüğünde kısa aralıklarla kabul edilebilirl. <ol><li> Isı, daha sonra şok olması için santigrat 22 derece 12 saat aydınlık / karanlık döngüsünde yeterli yumurta sayısına (yaklaşık 20&#39;den fazla) ve mağaza ile şişeleri tutun. Not: Yetişkin onlar daha fazla test için muhafaza edilmesi tavsiye edilir davranışsal test ve verimlilik çıkarına uçar medya üzerinde yumurta düşük sayıda küçük şişeler (az 20 yaklaşık daha) yeterli sayıda elde etmek olası değildir. Not: Bizim sinekler 8 PM 8 saatleri arasında ışıkları ile yetiştirilir. 8 PM ve 8:00-gece yarısı - Biz puan daha yüksek yumurta yoğunlukları yol akşam saat, bu nedenle genellikle 4 ila zamanlı haç yapmak bulduk. Inkübatör düzenli aydınlık-karanlık döngüsü rahatsız etmemek için sinekler ertesi gün kuluçka iade edilir daha sonra karanlık bir kabine oda sıcaklığında kendi yumurtalarını ve. </li></ol></li></ol> 3. Mitotik Birleşimi tetikleyebilecek Isı Şok gerçekleştirin <ol><li> Yaklaşık 85 - yumurtlama sonrasında 100 saat, yer v1 saat boyunca 37 ° C&#39;de bir su banyosu içine IALS. Su seviyesi şişeleri medyanın yüksekliğinin üstünde ulaşır, ancak tam boğulmaktan larvaları önlemek için flakon daldırın olmadığından emin olun. </li><li> Su banyosu şişeleri çıkarın ve oda sıcaklığında 1 saatlik toparlanma dönemine izin verir. </li><li> Geri 37 santigrat derece su banyosu içine 1 saat yerleştirin şişeleri. </li><li> Eclosion kadar 22 derece santigratta 12 saat ışık / karanlık döngüsünde inkübatör su banyosunda deposundan şişeleri çıkarın. </li></ol> Davranışsal Test 4. Hazırlık <ol><li> Eclosion ardından, buz üzerinde sinekler uyutmak. MARCM için tüm genetik öğeleri içeren ve GFP-işaretli klonlar muhtemelen üst stoklarında kullanılan gözlemlenebilir fenotip belirteçler dayalı görülecektir işaret genotipleri ile sinekleri seçin. Iridektomi makasla sinekler başını kesmek. Not: Örneğin, genetik belirteçler olmadan sinekler için CYO (kıvırcık kanatlar) ve Sb (anız kıl) olduklarını gösteren seçilenmozaik bölgeleri oluşturmak için gerekli tüm genetik unsurlar içerecektir fy sinekler GFP ile işaretlenmiş. Not: Decapitation uyarıldığında uzak arasındaki yetişkin önlemek için gereklidir. </li><li> Kapalı, nemli ortamda başsız sinekler yerleştirin ve yaklaşık 10 izin - kurtarma 20 saat. Sadece kullanım hakkı kendilerini daha fazla test için tedirgin o zaman uçar. Deney Dekapitasyon 30 saat içinde uçar. </li></ol> 5. Hareket Testleri <ol><li> Bir floresan diseksiyon mikroskop altında decapitated sinekler gözlemleyerek, kıl dış duyu organları da GFP ile işaretlenmiş homozigot klonları tanımlamak. Kıl adını ve sol veya sağ tarafına kaydedin. Herhangi bir potansiyel önyargı ortadan kaldırmak için, kıl stimülasyonu gerçekleştirmek laboratuar elemanı kıl, vahşi tip veya mutant genotipine kör olmalıdır. </li><li> Sert saç veya ince forseps ile sinek vücudun doğru kıl GFP saptırma bir tımar refleksini işaretli ortaya ve bacak yeniden gözlemlemekyanıtlarının yokluğu. Stimülasyon ve bacaklarını hareket etmiyor sinekler sıfır puan kıl cevaben kendi bacak kaldırma sinekler birinin bir puan verin. Not: Daha önceki çalışmalarda 6,7 belirli bir kıl uyararak ortaya bacak tepkisini karakterize var. Bu, her bir fırça kılı yanıt bacak ve söz konusu kıl uyarılmasına yanıt sinekler oranını içerir. Biz sadece Vandervorst sınıflamasına 6 dayalı cevap bekleniyor bacak hareketleri attı. </li><li> Birbirinden 2 dk aralıklı beş deneyler. </li></ol>

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S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+in+Drosophila.">A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila.</a> Nature Protocols. 1, 2583-2589 (2006).</li><li>Eberl, D. F., Hardy, R. W., Kernan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+similar+transduction+mechanisms+for+touch+and+hearing+in+Drosophila.">Genetically similar transduction mechanisms for touch and hearing in Drosophila.</a> J Neurosci. 20, 5981-5988 (2000).</li><li>Walker, R. G., Willingham, A. T., Zuker, 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=A+Drosophila+mechanosensory+transduction+channel.">A Drosophila mechanosensory transduction channel.</a> Science. 287, 2229-2234 (2000).</li><li>Yan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+NOMPC+is+a+mechanotransduction+channel+subunit+for+gentle-touch+sensation.">Drosophila NOMPC is a mechanotransduction channel subunit for gentle-touch sensation.</a> Nature. 493, 221-225 (2013).</li><li>Arnadottir, J., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+Mechanosensitive+Channels.">Eukaryotic Mechanosensitive Channels.</a> Annual Review of Biophysics. 39, 111-137 (2010).</li><li>Christensen, A. P., Corey, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRP+channels+in+mechanosensation:+direct+or+indirect+activation?.">TRP channels in mechanosensation: direct or indirect activation?.</a> Nature Reviews Neuroscience. 8, 510-521 (2007).</li><li>Cheng, L. E., Song, W., Looger, L. L., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+TRP+Channel+NompC+in+Drosophila+Larval+and+Adult+Locomotion.">The Role of the TRP Channel NompC in Drosophila Larval and Adult Locomotion.</a> Neuron. 67, 373-380 (2010).</li><li>Lee, J., Moon, S., Cha, Y., Chung, Y. 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+TRPN(=NOMPC)+channel+localizes+to+the+distal+end+of+mechanosensory+cilia.">Drosophila TRPN(=NOMPC) channel localizes to the distal end of mechanosensory cilia.</a> PLoS One. 5, e11012 (2010).</li><li>Liang, X., Madrid, J., Saleh, H. S., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOMPC,+a+Member+of+the+TRP+Channel+Family,+Localizes+to+the+Tubular+Body+and+Distal+Cilium+of+Drosophila+Campaniform+and+Chordotonal+Receptor+Cells.">NOMPC, a Member of the TRP Channel Family, Localizes to the Tubular Body and Distal Cilium of Drosophila Campaniform and Chordotonal Receptor Cells.</a> Cytoskeleton. 68, 1-7 (2011).</li><li>de Vries, S. E., Clandinin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loom-sensitive+neurons+link+computation+to+action+in+the+Drosophila+visual+system.">Loom-sensitive neurons link computation to action in the Drosophila visual system.</a> Curr Biol. 22, 353-362 (2012).</li><li>Jarman, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+mechanosensation+using+the+fly.">Studies of mechanosensation using the fly.</a> Hum Mol Genet. 11, 1215-1218 (2002).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarını var.

Mutantlar toplama yetişkinler olarak tarama engel olur ölümcül p-element mutasyonları içerdiğinden bu protokol, bu protokol ilk olarak Lee ve Luo tarafından açıklanan karmaşık genetik tekniği kullanır. Drosophila mechanosensation etkileyen mutasyonların taranması için yetişkin bir davranış deneyi kullanır (1999) ve yetişkin ölümcül aşmak için Wu ve Luo tarafından bir protokol, (2006) gibi ayrıntılı. MARCM homozigot hücrelerinin klonal bölgelerini oluşturmak için homolog kromoz...

Humans rely on the ability to convert mechanical stimuli from their environment, such as touch, pressure, vibration, or sound waves, into sensory information that can be processed by the nervous system, in a process termed mechanotransduction. Many of the overall mechanistic features of mechanotransduction between humans and invertebrates are the same1, making Drosophila a useful model to study the molecular mechanisms of mechanotransduction. Drosophila melanogaster contain two sets of specialized sensory organs (Type I and II) that are capable of converting mechanical stimuli into action potentials. Type I mechanoreceptors have a neuron with a single dendrite or sensory process, surrounded by three support cells2. The Type I mechanoreceptors include bristle mechanoreceptors, hearing sensitive chordotonal organs (the Johnston's organ), and campaniform sensilla that convey information about wing beats3. The bristles that cover the dorsal side of the fly are the most abundant and easily accessible of the Type I organs. Similar to the extracellular environment of hair cells associated with hearing and balance in vertebrates, the support cells surrounding fly mechanosensitive neurons secrete a high potassium endolymph that creates an unusual concentration gradient for potassium1. Mechanosensitive neurons, of both the mammalian and fly systems, utilize this high extracellular potassium to depolarize the cell. In response to mechanical stimulation of the macrochaete bristles towards the body wall, the sensory neurons respond with a burst of action potentials driven by this potassium depolarization of the cell membrane4. The Drosophila sensory neurons that innervate bristles resemble the mechanosensitive cells of other organisms, including vertebrate hair cells, in both structure and function1,5. The accessibility of Drosophila external sense organs to experimental manipulation and the abundance of genetic techniques available to researchers make Drosophila an excellent model system to investigate the molecular underpinnings of mechanosensation. 
In the fly, stimulation of a single sensory neuron that innervates a bristle leads to an observable behavioral response. Stimulation of different bristles evokes specific, reproducible behavioral responses, depending on the bristle that is stimulated. Upon tactile stimulation, decapitated wild-type flies exhibit a complex grooming reflex wherein they clean the area near the stimulated bristle with a patterned set of leg movements5-7. When homozygous for known single-gene mutations that interfere with learning7 or coordination and locomotor activity5, flies respond abnormally to mechanical stimulation. This grooming reflex is therefore a useful tool to study the effects of single-gene mutations on a specific, replicable behavior.
The robust behavioral response to stimulation of a single macrochaete bristle holds the potential to assist in identifying new genes involved in mechanotransduction. This protocol being used to test a collection of mutant flies for the absence of a behavioral response to indicate that the mutation interferes with mechanosensation. In the mutant collection selected for screening, the mutations cause lethality before adulthood, and therefore would be impossible to test using traditional adult behavior screens. Originally, this collection of lethal p-elements was combined with FRT recombination sites to test cell growth defects in clones. The clones were made specifically in the eye because adult flies can survive in the lab setting without functional vision8. However, removal of all mechanosensation can cause adults to be severely uncoordinated or die before eclosion5. This protocol uses a mosaic approach to circumvent the lethality of the mutations and allow for adult stage testing. A genetic technique called Mosaic Analysis with a Repressible Cell Marker (MARCM)9 is used to generate homozygous mutant cells in a limited number of adult fly sensory organs, while the rest of the organism remains heterozygous. These MARCM flies readily survive until adulthood, yet the bristles are homozygous for the lethal genes of interest.
MARCM allows for regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP), while the rest of the organism remains heterozygous at that particular locus and unmarked9. MARCM combines individual p-element mutations with five common genetic elements: GFP under the control of an upstream-activating sequence (UAS-GFP), Gal80 repressor protein under control of a general promoter (tub-Gal80), Gal4 transcription factor under control of a general promoter (tub-Gal4), the FRT recombinase enzyme expressed through a heat-shock controlled promoter, and a FRT recombination site10. By driving mitotic recombination through a heat-shock activated recombinase, a limited number of cells are made homozygous for the mutation and marked with GFP. GFP expression is repressed in heterozygous cells by the presence of Gal80 on the wild-type copy of the chromosome. 
A heat-shock protocol for MARCM was optimized to induce recombination in the fly bristle external sense organs, while much of the organism remains heterozygous, and thus unmarked with GFP. Mosaic flies generated using this protocol contained homozygous mutations most frequently in the post alar or dorsal central bristles on the surface of the notum. 
We have tested the utility of this combination of MARCM and the grooming behavior screen with a known mechanosensitive mutant, NOMPC. The ion channel, no mechanoreceptor potential C (NOMPC), is an essential component of the mechanotransduction pathway in Drosophila5,11-13. NOMPC belongs to the transient receptor potential (TRP) superfamily of cation channels5 and satisfies all of the criteria to qualify as a mechanosensitive channel in Drosophila14,15: 1) NOMPC is expressed in the ciliate tips of type 1 sensory neurons of Drosophila 13,16-18, 2) NOMPC null larvae do not have an electrical response to tactile stimulation13, 3) Ectopic expression of NOMPC in touch insensitive cells can induce sensitivity to mechanical stimulation13, 4) heterologous expression of NOMPC in Schneider 2 cells yields a mechanosensitive channel13, and 5) NOMPC adult mutants display defects in their response to mechanical stimulation5. Given this evidence, we predicted that NOMPC mutant clones would show an altered or inhibited grooming response in response to mechanical stimulation of bristles.
A MARCM-stock containing the NOMPC mutation was developed for use in a proof of principle experiment of the grooming assay. Mosaic flies were stimulated at macrochaete bristles containing homozygous NOMPC mutant cells. We expected an inhibition of the grooming response following stimulation of the macrochaete bristle. We found that only 2 of 14 mutant bristle flies tested gave a single response to repeated stimulation; most did not respond to stimulation of the homozygous mutant bristle. Having confirmed that this MARCM-based behavioral assay produces an abnormal grooming reflex in a known mechanosensitive mosaic mutant, this technique can be used in a screen for additional mechanosensitive mutations.

<table><tbody><tr><td>Brewers Yeast (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90331225&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Corn (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90141125&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Agar (1 lb)</td><td>MoorAgar Inc.</td><td>41004</td><td>Fly Food</td></tr><tr><td>Tegosept (5 kg)</td><td>Genesee</td><td>20-259</td><td>Fly Food</td></tr><tr><td>Molasses (1 Gallon)</td><td>Sugarmill Brand - Thomsen Food Service</td><td>0 2625</td><td>Fly Food</td></tr><tr><td>Propionic Acid</td><td>Fisher</td><td>A258-500</td><td>Fly Food</td></tr><tr><td>Phosphoric acid</td><td>Fisher</td><td>A260-500</td><td>Fly Food</td></tr><tr><td>Drosophila Vials, Narrow (PS)</td><td>Genesee</td><td>32-109</td><td>Fly Cultures</td></tr><tr><td>6oz Square Bottom Bottle (PP)</td><td>Genesee</td><td>32-130</td><td>Fly Cultures</td></tr><tr><td>Flugs - Plastic Fly Bottles</td><td>Genesee</td><td>49-100</td><td>Fly Cultures</td></tr><tr><td>Rayon Balls, Large</td><td>Genesee</td><td>51-100</td><td>Fly Cultures</td></tr><tr><td>Droso-Filler, Narrow</td><td>Genesee</td><td>59-168</td><td>Fly Food Preparation</td></tr><tr><td>Droso-Filler, Bottles</td><td>Genesee</td><td>59-170</td><td>Fly Food Preparation</td></tr><tr><td>8A-C / gear driven lab stirrer with c-clamp mount&amp;nbsp; 1/15 HP, 700 rpm variable speed, 115 V, 50/60 Hz</td><td>Cleveland Mixer</td><td>8A-C</td><td>Fly Food Preparation</td></tr><tr><td>Water jacketed Kettle</td><td></td><td></td><td>Fly Food Preparation</td></tr><tr><td>Diurnal Growth Chamber</td><td>Forma Scientific</td><td></td><td>Temperature and light/dark cycle controlled</td></tr><tr><td>Water bath</td><td>VWR</td><td></td><td>For heat shock</td></tr><tr><td>MicroScissors&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;15000-08</td><td>For removing heads</td></tr><tr><td>Fluroscence Dissecting Microscope</td><td>Zeiss</td><td>SteREO Discovery V8&amp;nbsp;</td><td>With GFP cube (KSC295-814D) band pass filter</td></tr><tr><td>Fluroscence Light Source</td><td>Zeiss</td><td>X-Cite 120</td><td>Fiber optic light pipe makes this easy to configure&amp;nbsp;</td></tr><tr><td>Camera for Scope</td><td>Zeiss</td><td>AxioCam ICc1</td><td></td></tr><tr><td>Image acquistion software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Ice bucket</td><td></td><td></td><td>for cold anthesia</td></tr><tr><td>Homemade cold anthesia tray</td><td></td><td></td><td>for cold anthesia decapitation</td></tr><tr><td>Plastic boxes</td><td></td><td></td><td>for recovery of decaptitated flies in humid environment</td></tr></tbody></table>

a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster

Çünkü memeli iç kulak saç hücrelerinin yapısal ve işlevsel homoloji, Drosophila dış duyu organlarını innerve nöronların mechanosensation çalışma için mükemmel bir model sistemi sağlar. Bu protokol, mechanosensation müdahale mutasyonları tespit etmek için kullanılabilir meyve sineği basit bir dokunmatik davranışını tarif etmektedir. Sineklerin toraks ya birinci ya da üçüncü ayağı bir tımar refleksini ortaya çıkarır bir macrochaete dokunsal uyarım kıl. Mechanotransduction (örneğin NOMPC gibi) müdahale mutasyonlar ya da refleks yayın diğer yönleri ile, bakım tepkisini inhibe edebilir. Yetişkin davranışların geleneksel ekran gelişimi sırasında gerekli rolleri vardır mutantlar cevapsız olurdu. Bunun yerine, bu protokol İfade tarafından oluşturulur ve işaretlenmesi için homozigoz mutant hücrelerin sadece sınırlı bölgeler için izin vermek için bir bastırılabilir hücre işaretleyici (MARCM) mozaik analizi ile dokunmatik ekran birleştirenYeşil floresan proteini (GFP) salgılamanın. Anormal davranış yanıtları için MARCM klonlar test ederek, daha geleneksel yöntemlerle cevapsız olurdu mechanosensation katılan yeni genlerin aramak için öldürücü p-element mutasyonlarının bir koleksiyon taranması mümkündür.

Bu protokolün başarısı büyük ölçüde test edilebilir sineklerin toplam verim bağlıdır, ısı şoku etkinliği mitotik rekombinasyon ve 2 dakika aralıklarla uyarılan sinekler sağlam bir davranışsal yanıt elde yeteneği teşvik etmek. Embriyoların yaklaşık% 12.5, gerekli MARCM elemanları ve GFP tanımı için potansiyele sahip Eclose yetişkin yaklaşık% 16,67 ihtiva göz önüne alındığında, bu zamana bırakır sırasında yumurta yüksek sayıda elde etmek için...

Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Arasında Mechanosensation için bir davranış tahlilinde klonlarda MARCM tabanlı<em&gt; Drosophila melanogaster</em

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

a-behavioral-assay-for-mechanosensation-marcm-based-clones-drosophila

Research

JoVE Journal

Neuroscience

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

10.2K Görüntüleme.  College of the Holy Cross. In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Video: A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

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 ...

Nörobilim

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Test için Yöntemler Drosophila Davranış

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Termal nosisepsiyon değerlendirilmesi Yerel ve Küresel Yöntemleri<em&gt; Drosophila</em&gt; Larva

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

Toplayıcılık Davranış için Tek-sinek Deneyi<em&gt; Drosophila</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Drosophila Larvalarında Mekanik Nosiception Ölçümü için Geliştirilmiş Bir Tahlil ve Araçlar

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Davranış

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Test<em&gt; Drosophila</emBir Y-labirenti testi ile&gt; koku alma

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

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 ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Soğuk nosisepsiyon için Roman Tahlili<em&gt; Drosophila</em&gt; Larva

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Drosophila melanogaster'de Nöral Devrelerin Optogenetik Manipülasyonu için Davranışsal Testler

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Fly Climbing Assay: A Method to Test General Locomotion in Drosophila melanogaster

Source: Gevedon, O., et al. <a href="https://www.jove.com/video/59720/in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective">In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes a simple method to quantify fly locomotion and shows the climbing assay performed.&#160; The fly climbing assay is a general test of motor function which relies on a negative geotaxis response.

Sinek Tırmanma Deneyi: Drosophila melanogaster'de Genel Hareketi Test Etmek İçin Bir Yöntem

Source: Gevedon, O., et al. In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster. J. Vis. Exp. (2019).
This ...

Arasında Mechanosensation için bir davranış tahlilinde klonlarda MARCM tabanlı

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Morfolojik analiz Drosophila Larvaların Periferik Duyusal Nöron Dendritler ve Aksonlar

Test için Yöntemler Drosophila Davranış

Termal nosisepsiyon değerlendirilmesi Yerel ve Küresel Yöntemleri

Toplayıcılık Davranış için Tek-sinek Deneyi

Drosophila Larvalarında Mekanik Nosiception Ölçümü için Geliştirilmiş Bir Tahlil ve Araçlar

Test

Of Bilgisayar İzleme tarafından Analiz Edilmiş Olfaktor Davranışlar

Soğuk nosisepsiyon için Roman Tahlili

Drosophila melanogaster'de Nöral Devrelerin Optogenetik Manipülasyonu için Davranışsal Testler

Sinek Tırmanma Deneyi: Drosophila melanogaster'de Genel Hareketi Test Etmek İçin Bir Yöntem