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

42.6K 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

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 ...

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.

Yüksek üretilen iş analiz Locomotor davranış Drosophila Adası tahlil

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Yanıt koku nöronlar Optogenetic uyarılması olarak Drosophila larva davranışını izlemek

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Drosophila melanogaster Öğrenme Kaynaklı Sinaptik Plastisite Vivo Optik Kalsiyum Görüntüleme

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Farelerde Ağrı Davranışını Ölçmek için Mekanik Çatışmadan Kaçınma Testi

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...