Biz larva tarama verilerinin üretilmesi için Astha Maltare teşekkür etmek istiyorum. Biz el yazması üzerine yaptığı yorum vermek için Dr Nicholas Lanson Jr teşekkür etmek istiyorum. Bu çalışma Johns Hopkins ALS Robert Packard Merkezi (UBP) ve Amyotrofik Lateral Skleroz Derneği (UBP) tarafından desteklenen ve Ruh Sağlığı Ulusal Enstitüsü (CDN) den R01MH083689.

A. Larva sürünüyor Testi 1. Larva Koleksiyonu <ol><li> Sinekler (10-15 erkek + 10-15 kadın) bir 8 ons şişe ayarlayın. </li><li> Sinekler sonra 24 saat boyunca yumurta, sineklerin net şişe inşa edelim. (Yeni bir şişeye aktarın ve yetişkinlerin uygun şekilde tekrarlayınız). </li><li> 3-4 gün inkübe şişe, ya da üçüncü evre larvaları görünür olana kadar. </li><li> Larvaları ile şişeye% 20 sakkaroz, 100 ml ve 20 dakika bekletin - 50 ekleyin. Larva başına yüzer. </li><li> Uçlu bir 25 ml serolojik pipet kullanarak toplayın larva kesti ve bir mesh sepet içine yer. </li><li> Iki kez deiyonize H 2 O ile örgü sepet içinde larva yıkayın Larva artık deneyler için hazır. </li></ol> 2. Ilaç ile larva tedavisi için <ol><li> Bir çözüm% 5 sakkaroz + ilaç içeren 5 ml&#39;lik behere larva İstediğiniz numaraya taşımak için bir fırça kullanın. </li><li> En azından 15 dakika süreyle larvaları besleme sağlar.</li><li> Bir örgü sepet içine ilaç ile tedavi edilen larva dökün ve durulayın. Onlar artık kullanıma hazır. </li></ol> 3. Lokomotor Assay (toplam mesafe ölçme seyahat veya vücut duvarı kasılmaları) <ol><li> Bir tek tek larva taşımak için bir fırça kullanın: <ol><li> 0,2 cm 2 ızgara ile milimetrik kağıt üzerinde% 2 agaroz (daha önce dökülür ve sertleşmesine izin) içeren 15 cm Petri. <ol><li> 1 dakika içinde geçti çizgileri sayısını sayın. </li></ol></li><li> Seyreltilmiş maya hamur çözeltisi içeren bir cam diseksiyon çanağı iyi. <ol><li> Bir diseksiyon mikroskobu altında gözlemlerken bir dakika içinde peristaltizm kasılmalar (posterior hareketi = 1 daralma tam anterior) sayın. </li></ol></li></ol></li><li> Larva istenilen sayıda sayılmış kadar tekrarlayın. </li></ol> B. Hızlı negatif geotaxis (HALKA) Protokol iteratif Bu test aslında kaldırdığımızda edildiGargano ark 5 tarafından tarif edildiği. <ol><li> Standart bir flakon içeren yiyecekler (veya gıda + ilaç) içine ışık CO 2 anesthetization ve yer altında yeni ortaya çıkan yetişkin erkek sinekleri toplayın. </li><li> Oda sıcaklığında sinekler Koru (tezgah üstünde. ~ 22 ° C) CO 2 kurtarma (ve kararlı durum ilaç düzeyleri birikimi uygunsa) izin vermek için 2-3 gün. </li><li> Hazırlanan polistiren şişelere anestezi olmadan 25 sinekler hakkında aktarın. </li><li> HALKA aparatı (Şekil 1) içine sinekler şişeleri birleştirin. </li><li> Sinekler 15-20 dakika boyunca rahatsız edilmeden çevre, gelmesini bekleyin. </li><li> Bu süre yerde dijital kamera sırasında ~ cihazları önünde 1 m (bir platform flakon orta yükseklikte lensin merkezine hizalamak için gerekirse üzerine), odaklanma ve aparat üzerine kamera, zum ve bir zamanlayıcı ayarlamak 3.0 saniye. </li><li> Dikkatle dist için sol elinizle HALKA aygıtının tutun şekilde alamayızurb sinekler ve sağ elinizle zamanlayıcı tutun. </li><li> Keskin musluk şişe altına tüm sinekler yıkmak yeterince zor sağlanması, tezgah yüzeyinin üç kez aparatı aşağı dokunun. </li><li> Aynı zamanda üçüncü kademe tamamlanması ile birlikte, 3 saniyelik geri sayım sayacını başlatmak. </li><li> Üç saniyede bir resim çekin. </li><li> 1 dakika zamanlayıcı sıfırlayın ve başlayın. Bu süre boyunca cihaz üzerindeki kamera ve odak sıfırlamak ve üç saniye sayacı başka bir kanala ayarlayın. </li><li> 1 dakika sonra tekrar 1,7-1,10 adımlar </li><li> 5-6 denemenin toplamı sonra, bir bilgisayara resim yüklemek ve açmak için favori resim görüntüleyici kullanın ve ortalama yüksekliği her flakon için tırmandı puan. </li><li> Tırmandı ortalama yüksekliği karşılaştırırken, farklı grup üzerinde istatistiksel analiz. </li></ol> C. Kur yapma ve çiftleşme Testi <ol><li> Sabah ilk iş, fli net iyi üreten şişeleres kullanılır. </li><li> Gün içerisinde (her 3-4 saat) içinde, yeni ortaya çıkan cinsel naif erkek ve kadın toplamak: <ol><li> Orta şişelerde veya tüpleri tek tek erkeklerde yerleştirin. </li><li> Şişe / tüp başına birlikte 5-6 dişi koyun. </li><li> 5 gün boyunca karanlık 12 saat ışık altında / 25 ° C &#39;de toplanan sinekler kesin. </li><li> Bir çiftleşme tekerleğin odasına bir kadın aktarın. </li><li> Bir çiftleşme tekerleğin odasına bir erkek aktarın. </li><li> Aşağıdaki davranışlardan bir diseksiyon mikroskobu altında çifti gözlemleyin: <ol><li> Oryantasyon (kadın doğru erkek yönlendirir) </li><li> (Erkek kadın yararlanıyor) dokunulduğunda </li><li> Kanat şarkı (erkek genişletir ve bir kanat titreşir) </li><li> (Erkek yalıyor kadın genital organlarının) Licking </li><li> Curling (erkek kendisi altında karnı kıvrılıyor) </li><li> (Kadın monte çalışırken aktivite Curling) birleşme girişimi </li></ol></li></ol></li><li> 1 dikkat0 dakika veya daha başarılı bir birleşme kadar, her davranışın meydana geldiği zaman not (gecikme), kur birleşme kadar davranış (kur endeksi hesaplamak için) yanı sıra, başarılı bir şekilde belirli bir davranışı gerçekleştirmek çiftlerinin numarası yapan toplam süre ( frekans). Vahşi tip çiftlerinin% 100 genellikle 5 dakika içinde çiftleşirler olacaktır. </li><li> Hesaplamak zaman bölerek bir kur endeksine (CI) çiftleşmeye kadar toplam süre bölünmesiyle kur geçirdi. Vahşi tip çiftleri için bu genellikle 0.6-0.8 arasında değişmektedir. </li></ol> D. Örnek Sonuçlar Assay Crawling Normal vahşi tip larva ~ 3 cm / dakika ve sergi ~ bir dakika içinde 40-50 vücut duvarı kasılmaları başıboş dolaşırlar. Biz son zamanlarda larva sürünen, kusur, düşük kullanım ömrü ve değer düşüklüğü 6 tırmanma yetişkin gösterir FUS / TLS ile ilgili Amyotrofik lateral skleroz bir Drosophila modeli geliştirdik. Biz ifadesi o hedefMotor nöronların (OK-371-gal4 sürücü) ve f yabani tip ve FUS / TLS mutant formları bir larva tarama testi yapıldı. Aşağıda görüldüğü gibi vahşi tip FUS ifadesi yaklaşık 6 cm larva tarama yeteneği azalmış ise, vahşi tip larva, en fazla 12 cm sürün. FUS / TLS ALS neden olan mutasyon R521C ifade Hayvanlar sadece yaklaşık 1 cm / dakika, emekleme, onların tarama hareketi çok ciddi bozulma (Şekil 1) göstermektedir. Negatif geotaxis HALKA assay Genç vahşi tip ergin sinek 3 saniyelik bir zaman diliminde ~ 4-5 cm (zaman belirli bir için belirli bir ortalama yüksekliği tanımlamak için farklı suşları veya aktivite seviyelerini karşılamak için 3 saniye arasında ayarlanabilir ortalama tırmanma yükseklikte olmalıdır / tedavi) süzün. Altta kalan sinekler 0 değeri tahsis edilir. Onu daha sonra yüksekliğini ölçmek için her bir sinek yerini belirlemek zor olur, çünkü vialde 25&#39;ten fazla sinek kullanılması tavsiye edilmez. No duyarsızlaştırma istihdam ediyor olması dışında 1 dakika aralıklı 6 ardışık denemeler için yukarı görülmüştür. Bu kullanılan vialler içine yerleştirilen yeni sinekler taze şişeler içerisine aynı ölçüde tırmanmaya çünkü toplanmış veri kümesi başlangıç ​​sonra bu tahlilde test polistiren şişeleri yeniden edilmeyen kritik önem taşır. <img alt="Şekil 1" src="/files/ftp_upload/3795/3795fig1.jpg" /> Şekil 1. HALKA Assay Kur. Odaklanma ve aparat üzerine kamera yakınlaştırmak;; dijital kamera polistiren flakonlarda sinekler içeren cihazları önünde ~ 1 m yerleştirilir ve 3.0 saniye için zamanlayıcıyı ayarlayabilirsiniz. <img alt="Şekil 2" src="/files/ftp_upload/3795/3795fig2.jpg" /> Kullanarak larva tarama testte Şekil 2. Temsilcisi veri ektopik UAS-FUS WT ifade ve motor nöron sürücüsü (OK371-gal4) kontrolü altında UAS-FUS R521C sinekleri. <pclass = &quot;jove_content&quot;&gt;

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Biz ifşa hiçbir şey yok.

Drosophila davranış sıkı genetik ve çevresel faktörler tarafından düzenlenir. Biz ve diğerleri, daha önce davranış ve Drosophila 5-19 modellenmiş insan nörodejeneratif hastalıklar uçmak için ilgili genler incelemek için veri toplamak için burada açıklanan testleri kullandılar. Tarama testi için, 3. Larvalar dikkatli seçimi kritik bir adımdır. Bir ilaç ile tedavi etmek, bu da iyi bir erime kabiliyetine sahip olmadığını maksimal etkisi elde etmek için...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Sakaroz </td><td> Fisher Scientific </td><td> S5-500 </td></tr><tr><td> Agaroz </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> 6 oz Drosophila şişe </td><td> Genesee Bilimsel </td><td> 32-130 </td></tr><tr><td> Boya Fırçası (# 1) </td><td> Ted Pella, Inc </td><td> 11859 </td></tr><tr><td> Gıda bileşenleri Fly </td><td></td><td></td></tr><tr><td> Mısır unu </td><td> Fisher Scientific </td><td> NC9109741 </td></tr><tr><td> Agar </td><td> Genesee Bilimsel </td><td> 66-104 </td></tr><tr><td> Melas </td><td> Fisher Scientific </td><td> NC9349176 </td></tr><tr><td> Propiyonik asit </td><td> Acros </td><td> 14930-0010 </td></tr><tr><td> Tegosept </td><td> Tepe </td><td> 20-258 </td></tr><tr><td> Etanol </td><td> BalıkçıBilimsel </td><td> BP2818-4 </td></tr><tr><td> Maya </td><td> Genesee Bilimsel </td><td> 62-107 </td></tr></tbody></table>

methods to assay drosophila behavior

Drosophila melanogaster, meyve sineği, kanser, kardiyovasküler hastalıklar ve bazı nörolojik hastalıklar 1. sıra insan hastalıklarının geniş bir moleküler mekanizmaları incelemek için kullanılmıştır. Biz larva hareket, yetişkin tırmanma yeteneği (HALKA testi), ve Drosophila kur davranışlarının belirlenmesi için basit ve sağlam davranışsal testler optimize edilmiştir. Bu davranış testleri sinek davranışı üzerinde genetik ve çevresel faktörlerin rolünü incelemek için yaygın olarak uygulanabilir. Larva tarama yeteneği güvenilir ve aynı zamanda lokomosyon ilaçlar ya da genetik hastalık etkisi (transgenik sineklerde) incelenmesi için Drosophila larva tarama yetenekleri erken evre değişiklikleri belirlemek için kullanılabilir. Aksi takdirde değerlendirilebilir nerede bu sinekler yetişkinliğe hayatta yok gibi bir genin ifadesi veya kaldırılması, pupa veya ergin dönemlerinde ölümcül neden olursa larva tarama testi daha uygun olur. Bu temel birssay Drosophila larva ek davranışsal tepkileri incelemek için parlak ışık veya stres ile birlikte de kullanılabilir. Kur davranışı yaygın cinsel davranış genetik temeli araştırmak için kullanılmıştır ve aynı zamanda da öğrenme ve bellek gibi, etkinlik ve koordinasyon incelemek için kullanılabilir. Drosophila Kur davranışı görsel, işitsel ve duyusal sinyaller dahil olmak üzere çeşitli uyarımlar değişimi içerir erkek ve başarılı cinsel birleşme sonuçlanan iyi karakterize motor hareketlerin karmaşık bir dizi neden kadınlar arasında. Geleneksel yetişkin tırmanma testler (negatif geotaxis) farklı çalışmalarda 2-4 arasında belirgin bir farklılaşma ile sıkıcı, yoğun emek ve zaman alıcı vardır. Hızlı (HALKA) testi 5 yetişkin lokomotor ve negatif geotaxis b ölçmek için, tekrarlanabilir duyarlı ve yüksek verimlilik yaklaşımı sağlayan, yaygın kullanılan protokolleri üzerinden pek çok avantajı vardır negatif geotaxis iteratifehaviors. HALKA deneyde, birkaç genotiplerinin veya ilaç tedavileri yüksek verimli tarama deneyleri yaklaşımı için daha uygun hale ile birlikte, aynı zamanda hayvanları, çok sayıda kullanılarak test edilebilir.

Watch this Scientific Journal Video about Methods to Assay Drosophila Behavior at JoVE.com

Methods to Assay Drosophila Behavior

Drosophila melanogaster bir yüzyıl 1 aşkın süredir birçok önemli biyolojik işlemlerin moleküler ve hücresel temelini anlamak için kullanılan bir genetik ve davranışsal uysal modeli sistemidir. Drosophila iyi sinek davranışının genetik temeli içgörü kazanmak için istismar edilmiştir.

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 ...

Research

JoVE Journal

Neuroscience

Methods to Assay Drosophila Behavior

42.7K Görüntüleme.  Louisiana State University Health Sciences Center. Drosophila melanogaster bir yüzyıl 1 aşkın süredir birçok önemli biyolojik işlemlerin moleküler ve hücresel temelini anlamak için kullanılan bir genetik ve davranışsal uysal modeli sistemidir. Drosophila iyi sinek davranışının genetik temeli içgörü kazanmak için istismar edilmiştir.

Video: Methods to Assay Drosophila Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Böcekler Tek Sensillum Kayıtlar Drosophila melanogaster Ve Anofel gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Nörobilim

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Nöronal Morphogenesis Çalışması Yöntemleri:<em&gt; Ex vivo</emEmbriyonik Mürin Serebral Korteks olarak&gt; RNAi Elektroporasyon

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Yılında Escape Davranış Optogenetic Uyarım<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Davranış Tahliller için Boyama Böcekler: anestezi ve Çiftleşme Davranışı<em&gt; Drosophila</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

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

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

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

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

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

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Kullanımı Ödül arayan davranış değişiklikleri Tedbir Basit Yolu<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila Bakım Davranışının Nicelleştirilmesi

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.