Name: use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule
Uploaded: 2015-11-25T15:00:00.000+00:00
Duration: 13 min 30 s
Description: Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

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The authors have nothing to disclose.

Birlikte iyon spesifik elektrotlar ile Ramsay testinin kullanımı, izole böcek Malpighi (böbrek) tübüllerde sıvı salgılanması oranları ve iyon akışlarının ölçülmesini sağlar. Yirmi ya da daha fazla nitro tübüller microperfused tübül bireyin tahlili ile karşılaştırıldığında daha yüksek verim sağlayarak bir zamanda analiz edilebilir. Buna ek olarak, belirli bir iyon elektrotlar ikinci düzeneğe sıvının küçük hacimlerde transferinde sokulabilir hataları sınırlayı...

<html> Böbrek epitel iyon taşıma organizma iono- ve osmotik düzenleme temelini oluşturmaktadır. Drosophila melanogaster Malpighi (böbrek) tübül epitel iyon taşıma moleküler mekanizmaları incelemek için eşsiz bir fırsat sunmaktadır. Bu fizyolojik çalışmaya renal tübüllerin erişilebilirlik ile eşleştirilmiş Drosophila güçlü genetik, birleşimi kaynaklanmaktadır. Tekniği 1 öncülük araştırmacı adını Ramsay deneyi, izole Malpighi tüplerinin sıvı sekresyon oranları ölçen ve Dow ve arkadaşları 2 tarafından 1994 yılında Drosophila kurulmuştur. Bu sıvı salgılanması düzenleyen hücre spesifik sinyal yolları tanımlamak için, Drosophila gibi GAL4 UAS sistemi 3,4 olarak genetik araçları kullanılarak ileri çalışmalar için yol açtı. Bir örnek diğerleri 6,7 arasında bir peptit hormonu 5 yanıt olarak, kalsiyum sinyal içerir. Sinek tübülün ana segmentten bir potasyum klorid zengin sıvının salgılanması ile gerçekleşir in ve_content &quot;&gt; genetik teknikler ve klasik fizyolojik çalışmanın bir kombinasyonu idrar nesil göstermiştir. Bu öncelikle, katyon paralel transepitelyal salgılama yoluyla gerçekleşir K +, Na +, aynı zamanda, ana hücre, ve Cl yoluyla -. stellat hücre 8-12 aracılığıyla salgılama ayrıca transepitelyal K + Na + akıları ölçmek için yeteneği, sıvı sekresyon ölçümünün taşıma mekanizmaları daha ayrıntılı bir karakterizasyonu sağlar yalnız. Örneğin, unstimulated Drosophila tübüllerde Na + / K + ATPaz inhibitörü uvabain sıvı salgılanması 2 üzerinde hiçbir etkisi yoktur, asıl hücrelere onun alımı organik anyon taşıyıcısı inhibitörü torokolat 13 tarafından inhibe bile. Ancak, Linton ve O&#39;Donnell ouabain depolarize gösterdibazolateral membran potansiyeli ve Na + akımına 9 artar. Örnek Sonuçlar gösterildiği gibi, bu bulguları teyit edilmiş, ve eş zamanlı olarak 14 azalma K + akı olduğunu göstermiştir; artan Na + akışı ve K + azalmış akışı salgılanmasında net bir değişikliğe neden sıvı sekresyon karşıt etkileri vardır. . Böylece, &quot;ouabain paradoks,&quot; yani iki çözünürlükleri vardır, Drosophila tübül sıvı salgılanması üzerinde hiçbir etkisi yoktur ouabain ilk gözlem: Birincisi, uyarılmış tübüllerde sıvı salgılanması üzerindeki ouabainin etkisi belirgin değildir nedeniyle organik anyon taşıyıcısı 13 tarafından alımı; ve ikinci, uyarılmamış tübüllerde, uvabain (Temsilci Sonuçlar ve ref bakın. 9) sıvı salgılanması hiçbir net bir değişiklik sonuçlanan transepitelyal Na + ve K + akı üzerindeki etkilerini karşıt vardır. Bu nedenle, Na + birincil rolü / K + ATPuyarılmamış tübüllerde ase Na + bazolateral membran boyunca taşıma işlemleri -coupled için uygun bir konsantrasyon gradyanı oluşturmak için hücre içi Na + konsantrasyonunu düşürmek için. Nitekim, ayrı ayrı Na + ve K + akıları ölçerek, biz tübüller sinek sodyum potasyum-2-klorür kotransportörü (NKCC) eksik ouabain ilavesinden sonra başka bir azalma ile, transepitelyal K + akı azalmış ve transepitelyal hiçbir değişiklik göstermiştir Na + 14 akı. Bu bulgular, NKCC ile hücreye giren Na + Na + / K + -ATPase&#39;ın deveran eden sonucunu desteklemiştir. Başka bir örnekte, Ianowski ve ark., 10 mM mM ile 6 banyo K + konsantrasyonunun azaltılması sıvı sekresyonunda net bir değişiklik ile, Rhodnius prolixus gelen tübüllerde transepitelyal K + akışı ve artan transepitelyal Na + akımına azaldığı görülmüştür <sup&gt; 15. Larva tübüller arasında, Na + ve K + akı akı diferansiyel etkileri de tuzluluk 17 yetiştirme yanıt olarak değişen bir tuzu diyetler 16 ve iki sivrisinek türlerde karşılık olarak Drosophila tübüllerde gözlenmiştir. Ramsay tahlil hazırlığında transepitelyal iyon akışının ölçümünde büyük zorluk salgılanan sıvı içinde iyon konsantrasyonlarının belirlenmesidir. Bu meydan okuma alev photometery 18, radyoaktif iyonların 19 kullanımı ve elektron prob dalga boyu dağıtıcı spektroskopisi 20 olmak üzere değişen çözümleri ile karşılandı. Bu teknikler iyon konsantrasyonlarının ölçümü için bir enstrümana salgıladığı sıvı damla transferini gerektirir. Uyarılmamış Drosophila tübül salgılanan sıvının miktarı az olduğundan, genelde ~ 0.5 nl / min, bu teknik bir sorun teşkil etmektedir ve salgılanan sıvının bir kısmını ise de hata getirmektedirtransferi üzerine kaybetti. Buna karşılık, iyon belirli elektrotların kullanılması in situ (iyon konsantrasyonu hesaplanabilir olan) iyonu aktivitesinin ölçülmesini sağlar. Geçerli protokol K + iyonoforu 21 olarak Valinomycin kullanılarak Rhodnius tübül boyunca transepitelyal K + akının ölçülmesi için iş Maddrell ve arkadaşlarının kullandığı uyarlanmıştır ve aynı zamanda, bir 4-tert--butylcalix [4] aren-tetraasetik asit kullanımını tarif edilmiştir tetraetil ester bazlı Na Messerli et ile karakterize -spesifik iyon spesifik elektrot +. ark. 22. İyon spesifik elektrotlar erişkin 9,23 ve larva 16 Drosophila melanogaster Ramsay tahlilinde Malpighian tübüller tarafından salgılanan sıvının iyon konsantrasyonunu ölçmek için kullanılmıştır, Yeni Zelanda Alpine Weta (Hemideina maori) 24 ve sivrisinek 17. Burada, biz ayrıntılı olarak Ramsay kullanımını diye tanımladıklarıSıvı salgılanması Drosophila melanogaster gelen Malpighian tübüllerde oranları, hem de salınan sıvı içinde, K +, Na + &#39;nın konsantrasyonlarını belirlemek için iyon belirli elektrotların kullanılmasını ve böylece transepitelyal iyonu akılarının hesaplama ölçmek için demek. Tahlilinde bir genel görünüşü Şekil 1&#39;de verilmektedir. <img alt="figür 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Malpighi Pipet Şekil 1. şematik ve İyon Konsantrasyonları Tedbir İyon özel elektrotlar kullanımı ile Ramsay Deneyi. Bu rakam Ramsay testi için kurulum göstermektedir. (A) Her bir sinek, dört tübüller, ön tübüllerin bir çift ve arka tübüller bir çift hemolimf çevrili karın boşluğunda yüzer sahiptir. Her bir çiftin iki tübülleri sonra midgut ve hindgu kavşakta idrar üreter boşaltır, at katılmakt. Tübüller kör uçlu. İdrar (kırmızı ile gösterilen) sıvı salgılayan ana kesimi tarafından üretilen ve bağırsak içine üreter doğru ve dışarı akar. Diseksiyon sonra tübül çifti üreter bölücü ile barsaktan ayrılır. (B) tübül çifti sonra deney kabına içinde banyo salin damlacık içine aktarılır. Burada adlandırılan iki tübül biri &quot;çapa tübül,&quot; bir metal pim etrafında sarılır ve inert olan bir. Diğer tübül salgılayan tubule olduğunu. İlk bölüm (sıvı salgılar değildir) ve salgılayan tübülün ana segmenti yıkanma tuzlu su damlacık içinde kalır. In vivo oluşacak şekilde daha sonra İyonlar ve banyo tuzlu ve ana segment tübül lümenine su taşımak ve, üreter doğru hareket. Alt segment (mavi) banyo tuzlu ve bu nedenle atıl dışında. Üreter kesilir yana, salgılanan sıvı üreter kesik ucundan bir damlacık olarak ortaya çıkmaktadır. TO salgılanması devam ederken sıvı damlacık zamanla genişletir salgılanır ve çapı oküler mikrometre kullanılarak ölçülür. Mineral yağ tabakası salgılanan sıvının buharlaşmasını önler. Referans ve iyon spesifik elektrotlar salgılanan sıvının iyon konsantrasyonunu ölçmek. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td><td>May be purchased from multiple distributors</td></tr><tr><td>Petri dish, polystyrene, 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Specific brand is not important</td></tr><tr><td>Petri dish, polystyrene, 35 mm x 10 mm</td><td>Corning Life Sciences</td><td>Fisher 08-757-100A</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Blades #1</td><td>Fine Science Tools</td><td>10011-00</td><td>Specific brand is not important; use appropriate sharps precautions</td></tr><tr><td>Needle, 30 G x 1/2</td><td>Becton Dickinson</td><td>305106</td><td>Use appropriate sharps precautions</td></tr><tr><td>Minutien pins, black anodized, 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Stereomicroscope with ocular micrometer</td><td>Nikon</td><td>SMZ800</td><td>Specific brand is not important; this is given as an example</td></tr><tr><td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td><td>Hobby shop</td><td></td><td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td></tr><tr><td>Glass cutting tools (glass cutter, glass cutting pliers)</td><td>Hobby shop</td><td></td><td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td></tr><tr><td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0042</td><td></td></tr><tr><td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0044</td><td></td></tr><tr><td>Nitric acid, 70%</td><td>Sigma</td><td>438073</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td></tr><tr><td>Cimarec 7 in x 7 in hotplate</td><td>Fisher</td><td>11675911Q</td><td>Specific brand is not important; caution when heated</td></tr><tr><td>Selectophore dichlorodimethylsilane</td><td>Sigma</td><td>40136-1ML</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Two-step vertical pipet puller</td><td>Narishige</td><td>PC-10</td><td>Other pipet pullers can be used; this is given as an example</td></tr><tr><td>Glass petri dish, 150 mm diameter x 15 mm height</td><td>Fisher</td><td>08-748E</td><td>Specific brand is not important; only one dish needed</td></tr><tr><td>World Precision Instruments E210 1 mm micropipette storage jar</td><td>Fisher</td><td>50-821-852</td><td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td></tr><tr><td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td><td>Fisher</td><td>S161-500</td><td>Indicating silica useful for determining whether silica gel retains desiccating ability</td></tr><tr><td>World Precision Instruments MicroFil, 34G</td><td>Fisher</td><td>50-821-914</td><td>May be available from other distributors.</td></tr><tr><td>1 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309659</td><td>May be available from other distributors.</td></tr><tr><td>3 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309657</td><td>May be available from other distributors.</td></tr><tr><td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td><td>Cole-Parmer</td><td>UX-30600-02</td><td>Specific brand is not important</td></tr><tr><td>Female Luer Locking Connector</td><td>4 Medical Solutions</td><td>ADC 9873-10</td><td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td></tr><tr><td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td><td>Fisher</td><td>14-179-110</td><td>Specific brand is not important</td></tr><tr><td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td><td>Fisher</td><td>14171208</td><td>Specific brand is not important</td></tr><tr><td>Modeling clay</td><td></td><td></td><td>Specific brand is not important</td></tr><tr><td>Selectophore potassium ionophore I, cocktail B</td><td>Sigma</td><td>99373</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Selectophore sodium ionophore X</td><td>Sigma</td><td>71747</td><td>Sodium ionophore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td></tr><tr><td>Selectophore 2-nitrophenyl octyl ether</td><td>Sigma</td><td>73732</td><td></td></tr><tr><td>Selectophore sodium tetraphenylborate</td><td>Sigma</td><td>72018</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Schneider&#039;s Drosophila medium</td><td>Life Technologies</td><td>21720024</td><td></td></tr><tr><td>High impedance electrometer</td><td>World Precision Instruments</td><td>FD223a</td><td></td></tr><tr><td>Microelectrode holder 1 mm with 45&amp;deg; body, vented, with handle</td><td>Warner Instruments</td><td>64-1051</td><td></td></tr><tr><td>Microelectrode holder 1 mm with straight body, vented</td><td>Warner Instruments</td><td>64-1007</td><td></td></tr><tr><td>Silver wire</td><td>Warner Instruments</td><td>64-1318</td><td></td></tr><tr><td>Micromanipulators, pair</td><td>Leitz</td><td></td><td>Various brands/models will work; this is an example</td></tr><tr><td>Faraday cage</td><td>Technical Manufacturing Corporation</td><td>81-334-03</td><td>This is an example; any Faraday cage will work</td></tr><tr><td>Single gooseneck fiberoptic light</td><td>Nikon</td><td></td><td>Specific brand is not important</td></tr><tr><td>mineral oil</td><td>Fisher</td><td>BP-2629</td><td>Specific brand is not important</td></tr><tr><td>forceps, Dumont #5 with Biologie tip</td><td>Fine Science Tool</td><td>11295-10</td><td>May be available from other distributors.</td></tr></tbody></table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

Böbrek epitel iyon taşıma modülasyonu organizmalar dış koşullar değişen yüzüne iyonik ve osmotik homeostazı korumak için olanak sağlar. Drosophila melanogaster Malpighi (böbrek) tübül dolayı bu organizmanın ve fizyolojik çalışmaya renal tübüllerin erişilebilirlik güçlü genetiğe, epitel iyon taşıma moleküler mekanizmaları incelemek için eşsiz bir fırsat sunmaktadır. Burada, salgılanan sıvının sodyum ve potasyum konsantrasyonları ölçmek için iyon belirli elektrotların kullanımı ile izole edilmiş uçucu renal tüplerin sıvı sekresyon oranları ölçmek için Ramsay tahlilinin kullanımı açıklanmaktadır. Bu deney iyonu konsantrasyonunu ölçmek için ayrı bir cihaz ile salgılanan sıvıyı transfer etmeye gerek kalmadan, 20 tübüller bir kerede transepitelyal sıvısı ve iyon akılarının çalışma ~ sağlar. Genetik olarak farklı tübüller taşıma süreçlerinde belirli genlerin rolünü değerlendirmek için analiz edilebilir. Buna ek olarak, bathing tuzlu su ilave kimyasal özellikleri ya da ilaçlar ya da hormonların etkilerini incelemek için modifiye edilebilir. Özetle, bu teknik, bu taşıma mekanizmaları epitel iyon Drosophila tübülde ulaştırma yanı sıra, düzenlemenin temel mekanizmaları moleküler karakterizasyonu sağlar.

7 Şekil 8 genetik ve farmakolojik farklı K + ve Na + akıları, yalnız sıvı sekresyon oranları ölçülerek yakalanan değildir bilgileri ayırt edebilir K + Na + konsantrasyonlarını ölçmek için iyon spesifik elektrotlar ile Ramsay testinin bu kullanımını göstermek. Şekil Na + akımı (Şekil 7B) değişmez ise NKCC bir homozigot mütasyon taşıyan sinekler tübüllerdeki sıvı sekresyonunda azalma...

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Bu protokol, Drosophila melanogaster izole Malpighian (renal) tübüllerin sıvı sekresyon oranları ölçmek için Ramsay tahlilinin kullanımı açıklanmaktadır. Buna ek olarak, belirli bir iyon elektrotların kullanımı transepitelyal iyon akışının hesaplanmasını sağlayan, salgılanan sıvının içinde sodyum ve potasyum konsantrasyonlarını ölçmek için, tarif edilmektedir.

Ramsay Testi Kullanımı sıvı salgılanması ve İyon Flux fiyatlar Tedbir<em&gt; Drosophila melanogaster</em&gt; Malpighi Tübül

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

use-ramsay-assay-to-measure-fluid-secretion-ion-flux-rates-drosophila

Research

JoVE Journal

Biology

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

11.8K Görüntüleme.  University of Texas Southwestern Medical Center.  Bu protokol, Drosophila melanogaster izole Malpighian (renal) tübüllerin sıvı sekresyon oranları ölçmek için Ramsay tahlilinin kullanımı açıklanmaktadır. Buna ek olarak, belirli bir iyon elektrotların kullanımı transepitelyal iyon akışının hesaplanmasını sağlayan, salgılanan sıvının içinde sodyum ve potasyum konsantrasyonlarını ölçmek için, tarif edilmektedir. 

Video: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

Belirlenen Nöronlar ve Beslenme Davranışı Kalsiyum sinyaller aynı anda kayıt Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

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 ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Drosophila melanogaster Kan-beyin Bariyer Bütünlüğü için Tahse

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Gelişim Biyolojisi

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Fizyolojik Yanıtları Ölçüm<em&gt; Drosophila</em&gt; Canlı Kalsiyum Görüntüleme kullanma Lipit Feromonlar Sensory Nöronlar

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

İmmünoloji ve Enfeksiyon

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

Kılcal Besleyici Testi Gıda Alımını içinde Tedbirler<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Drosophila Larvaları Kullanılarak Termogenetik Nöronal Ekranda Makro besin alımının nicelleştirilmesi

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Larvaların Faaliyet Ölçümü<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Biyoloji

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Drosophila melanogaster Malpighi tübül epiteli optik Quantification intrasellüler pH floresan genetik olarak kodlanmış pH göstergesi

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Yetişkin Sivrisinekte Nöropeptitlerin ve Boşaltım Sisteminin Diğer Düzenleyicilerinin Aktivitesinin Incelenmesi

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ

Source: Rossano, A. J. and Romero, M. F. <a href="https://www.jove.com/video/55698/" target="_blank">Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator</a>. J. Vis. Exp. (2017).
This article describes the dissection of the anterior Malpighian tubules (MT) from a female Drosophila fly and an example protocol from Rossano et al. (2017) in which the MTs are prepared for live imaging in a perfusion chamber.

Ramsay Testi Kullanımı sıvı salgılanması ve İyon Flux fiyatlar Tedbir

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Belirlenen Nöronlar ve Beslenme Davranışı Kalsiyum sinyaller aynı anda kayıt Drosophila melanogaster

Test için Yöntemler Drosophila Davranış

Drosophila melanogaster Kan-beyin Bariyer Bütünlüğü için Tahse

Fizyolojik Yanıtları Ölçüm

Kılcal Besleyici Testi Gıda Alımını içinde Tedbirler

Drosophila Larvaları Kullanılarak Termogenetik Nöronal Ekranda Makro besin alımının nicelleştirilmesi

Larvaların Faaliyet Ölçümü

Drosophila melanogaster Malpighi tübül epiteli optik Quantification intrasellüler pH floresan genetik olarak kodlanmış pH göstergesi

Yetişkin Sivrisinekte Nöropeptitlerin ve Boşaltım Sisteminin Diğer Düzenleyicilerinin Aktivitesinin Incelenmesi

Yetişkin Bir Sinekten Malpighian Tübül Diseksiyonu: Osmoregülasyon ve Boşaltım Organının İzole Edilmesi