We thank Rachelle Kanippayoor for her help in identifying the new wild-type strain as being of the melanogaster species. R.W.F, O.F. and A.F.S were responsible for research design; R.W.F, M.N. and O.F. performed the experiments. R.W.F, M.N., O.F. and A.F.S. analyzed the data; R.W.F., I.S.M. and A.F.S. wrote the manuscript. 
This work was supported by PSC-CUNY research awards, jointly funded by The Professional Staff Congress and The City University of New York to A.F.S.; by internal funding from Western University to A.F.S. and I.S.M.; by a training support from the National Alliance for Hispanic Health&#8217;s Alliance/Merck Ciencia (Science) Hispanic Scholars Program and a University Fellowship from the Yale Graduate School of Arts and Sciences to R.W.F.

1. Ekipman ve In-house düzenlendi Reaktifler (Diğerleri için Malzeme Listesi) <ol><li> Sinek işi gerçekleştirmek için bir Drosophila soğuk anestezi cihazı hazırlayın. <ol><li> Küçük bir plastik kutu (10.2 cm genişliğinde, 12.7 cm uzunluğunda), bir pipet uçları kutusunun tipik üst kapağını (3.8 cm derinliğinde, 10.2 cm genişliğinde, 12.7 cm uzunluğunda) kapsayacak şekilde bir gözenekli polietilen levha kesti. </li><li> Ezilmiş buz ile doldurun kutu, gözenekli polietilen levha ile kaplayın. </li></ol></li><li> T-labirent aparatı hazırlayın; Daha önce ayrıntıları 28-31 açıklanan aparatların adapte olur. </li><li> 32 anlatıldığı gibi bir sinek aspiratör hazırlayın. </li><li> Ve deney şişeleri dışarı stres olmadan sinekler yerinden bir Phototaxis tepki cihazı hazırlayın: <ol><li> 29,33 tarafından tarif edildiği gibi, bir karşı cihazı kullanın. </li></ol></li></ol> NOT: 28 tarafından tarif edildiği gibi T-labirent da başka amaçlar için de kullanılabilir. <ol start="5"><li> Hom olunogeneous aydınlatma koşulları: beyaz tezgah örtüsü ile kaplı bir bankta deney gerçekleştirmek ve bir beyaz tahta önünde. NOT: Bu deneyler için kullanılan sinek suşları Drosophila vahşi türleri melanogaster vardı: Kanton-S, Oregon ve Semerkant laboratuvar stokları 34 uçar; Elwood sinekler Long Island, New York, ABD tarihinde, Huntington Elwood Mahallesi&#39;nde Güz 2011 yılında toplanmıştır. </li></ol> 2. Deney öncesi Sinekler hazırlanması Deney yapmadan önce 1-2 gün: <ol><li> 12 saat ışık / karanlık döngüsü 25 ° C&#39;de standart mısır unu-agar-pekmez-maya ortamda sinekler koruyun. </li><li> Deney yapmadan 1-2 gün önce soğuk anestezi altında cevap sinekler toplayın <ol><li> Bir huni kullanılarak, 50 ml Falcon tüpleri içine yükseltilmiş edildiği şişe sinekler aktarın. </li><li> Bir içine buz seviyesinin altında sinekler şahin tüp yerleştirinyalıtılmış buz kovası. </li><li> En -4 ° C soğuk ° ezilmiş buz ile soğuk anestezi cihazı yerleştirin. </li><li> Sinekler chill komaya girmek için 5 dakika bekleyin. </li><li> Soğuk anestezi cihazının gözenekli polietilen levha üzerine sinekler aktarın. </li><li> Soğuk anestezi cihazında cevap sinekleri toplayın. Oda sıcaklığında, bir stereo mikroskop altında, dişi sinekler 3-7 gün eski erkek ayırmak ve 40 kişilik gruplar halinde bu sinekler korumak. </li></ol></li><li> Deney yapmadan 1-2 gün önce yayıcı sinekleri toplayın. <ol><li> Yavaşça Kanton-S karma kendi numarasını kontrol, bir ağız aspiratörü kullanarak, kendi şişe uçar toplamak. </li><li> Bunlar yayıcılar olacak, 60-100 grubunda bu sinekler koruyun. </li><li> Veri noktaları gerektiği gibi yayıcılar gibi birçok örnekleri hazırlayın. </li></ol></li><li> Soğuk anestezi herhangi karıştırıcı etkisini en aza indirmek için, yanıt ve vericilerin toplandıktan sonra iyileşme en az bir gece izin verdavranış ve ağız aspiratörü koleksiyonu. </li><li> En az 1 gün önce deney, taze hava ile değiştirilmesi çanta bayat plastik kokulu hava sağlamak için, deney şişeleri çanta açın. </li></ol> Deney yapmadan önce 2 saat: <ol start="6"><li> Hatta hafif olan, deney yapıldığı odanın sıcaklığı yaklaşık 23-25 ​​° C olduğundan emin olun, ve% 30 üzerinde nem. </li><li> Bu sinekler hasret değil emin olmak için taze gıda şişeleri içine müdahale ve yayıcılar meyve sinekleri aktarın. </li><li> Meyve sinekleri deney yapılacaktır hangi bankta, 2 saat boyunca çevreye uyum edelim. </li></ol> 3. Sosyal Kaçınma Deneme Sahne <ol><li> Zeitgeber zaman ZT5 ve ZT9 arasında öğleden sonra 3-4 saatlik bir aralıkta, günün aynı zamanda her bir deney yapın. </li><li> Vurma pedi T-labirent yerleştirin ve t böylece vida klibi sıkınO asansör kararlı. </li><li> Taze bir test şişenin içine cevap sinekler aktarın. T-labirent üst kısmına cevap sinekleri içeren bu şişeyi çekin. </li><li> T-labirent aparatı eğik ve meyve sinekleri asansörde düşmek böylece vurma mat üzerine dokunun. </li><li> Sinekler T-labirent üst ve alt kısmı arasında bu yüzden aşağı asansörün üst kısmını taşımak, ancak seçim noktasının üzerinde asansör tutmak için dikkat - kaçmasını sinekleri önlemek için. </li><li> DSO (önceden sinekler tarafından işgal edildi DSO-serbest şişeleri üretmek için bakınız Bölüm 5) ile şişeleri alın. <ol><li> Cevap sinekler bu yeni ortama uyum iken, bir test flakon yayıcı sinekler yerleştirin. </li><li> Meyve sinekleri kaçış yok bu yüzden şişeyi kapatın pamuk bir parça yerleştirin. </li><li> 15 sn için vorteks 5 saniye vorteks gelen flakon kaldırmak: Mekanik aşağıdaki gibi bir mini girdap bu flakon çalkalayın. 55 saniyelik bir toplam 3 kez tekrarlayın. </li><li> RemBir gıda şişe içinde aktarılarak - - taze şişeden dışarı yayıcı sinekleri ove ve hızlı bir şekilde T-labirent tertibatının iki yüzünden birinin içine bu DSO dolu şişe yerleştirin. </li><li> Her yeni dönemde DSO dolu şişenin için yerleştirme tarafını alternatif. </li></ol></li><li> T-labirent diğer tarafında temiz bir test şişe yerleştirin. </li><li> Meyve sinekleri, taze bir test şişeye ve DSO test flakon arasındaki seçti, böylece tamamen asansör getir, ve Sayacı başlatmak. </li><li> Aksi belirtilmediği sürece, sinekler, 1 dakika boyunca, taze şişe DSO şişe arasında seçim olsun. </li><li> 1 dakika sonra, taze flakon, DSO flakon ve asansörde sıkışmış olanlar sinekler ayırmak için asansör yukarı taşımak. </li><li> Her flakon sinek sayısını ve asansörde. </li><li> Her genotip / koşul için bu işlemi tekrarlayın. </li></ol> 4. Sosyal Kaçınma Veri Analizi <ol><li> Her Genot için her flakon meyve sinekleri sayısınıype ve bir elektronik veri aktarmak. </li><li> Bir elektronik tablo programı kullanarak, sineklerin toplam sayısına bölünmesi ardından DSO-serbest şişe içinde sinekler sayısından DSO şişe içinde sinekler sayısını çıkartarak, ve her genotip için Performans Endeksi (PI) hesaplayın. </li><li> Sonuçların analizi için istatistiksel analiz yazılımı kullanılarak pis &#39;anlamına gelir karşılaştırın. <ol><li> İdeal olarak, her bir deney için, 2-3 iç tekrarlar ve 3 bağımsız tekrarlar hazırlamak, farklı şişe, farklı günlerde yapılan ya da 30 geçer. </li><li> Veriler normal dağılıma takip gibi, Gauss dağılımı istatistiksel testler kullanın. </li><li> Bir etkisi sınarken (yani, yoğunluk - Şekil 2), bir kontrol için her sonucu karşılaştırmak için (kolon sütun) çoklu karşılaştırma için post-testi, veya basit bir t-testi ile, bir tek-yönlü ANOVA kullanın. </li><li> Grup deneyleri yapıldığında, koşulları ve cinsiyetleri (boş şişeleri hem test vurguladı bird un-vurguladı sinekler; ve cinsiyet etkisi - Şekil 1), iki yönlü ANOVA kullanılmıştır. </li></ol></li></ol> 5. Daha önce gerilmemiş Sinekler tarafından işgal küçük şişeler oluşturun - yerine 3,5-3,6 arasında gerçekleştirin <ol><li> Bir kontrol olarak, stresli değil sinekler tarafından işgal şişeleri hazırlamak için phototaxis kullanın. </li><li> Ters konumda taze boş şişeleri ile, bir test flakon sinekler aktarın ve karşı akımlı aparatı oturtun. </li><li> Doğrudan 15 W floresan soğuk-beyaz ışığın önünde uzak şişeye yatay cihazı Lay ve siyah bir bezle örtün. </li><li> Sinekler hafifçe taze flakon hareket edelim. Bu işlem birkaç dakika sürebilir. Sineklerin en uzak flakon ışığa doğru yürüyorlar sonra, 1 dakika için Sayacı başlatmak. </li><li> Yavaşça, ışığın konumunu değiştirmek, siyah bez kaldırmak siyah bezle kurtarmak ve sinekler testi flakon bırakmak için izin. </li><li> O zaman, ben3,2-3,5 gibi seçim deneyi nitiate. 3.6 ve 3.7 yap. </li><li> Diğer taraftan deney şişesini boşalan sinekler ayırmak için, cihazın kaydırıcıyı hareket ettirin. </li><li> Hemen seçim deney için, 3.7 gibi T-labirent boşalan deney şişesini kullanın. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+methods:+Know+when+your+numbers+are+significant.">Research methods: Know when your numbers are significant.</a> Nature. 492, 180-181 (2012).</li><li>Stowers, L., Logan, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+olfactory+signaling.">Sexual dimorphism in olfactory signaling.</a> Curr. Opin. Neurobiol. 20, 770-775 (2010).</li><li>Simon, A. F., Liang, D. T., Krantz, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+decline+in+behavioral+performance+of+Drosophila+melanogaster+with+age.">Differential decline in behavioral performance of Drosophila melanogaster with age.</a> Mechanisms of Ageing and Development. , 127-647 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Bu protokol, sosyal kaçınma tahlil için ayrıntılı bir yordam açıklanır. Kanton-S sadece daha önce mekanik olmuştur vurguladı var uçar hangi bir şişe önlemek ve yanıt verenlerin seks ve sayı sosyal kaçınma performansı olduğunu etkilemez. Ancak, yanıt genetik arka önemli bir etkisi vardır. Bu denemeyi başarılı gerçekleştirmek için pek çok kritik adımlar şunlardır: 1) her zaman deneyden önce Sinekler 2 saat aktarmak ve çevrelerini rahatsız değil emin olun;...

Bu yöntemin amacı kolayca kur ve saldırganlık bağımsız Drosophila melanogaster basit sosyal davranış, yeni bir yönünü ölçmek için. Sosyal etkileşimler doğru gelişme ve sağlık bireylerin toplum içinde, hem de bir bütün olarak bir toplumsal grubun işlevselliğine önemlidir. Bu etkileşimlerin yüksek karmaşıklığı büyük örnek boyutları ve sosyal davranış genetik ve nöral temelleri hala tam olarak anlaşılamamıştır gibi davranış basitleştirilmesi sağlayan bir sistem gerektirir. Drosophila melanogaster genetik tanımlamak için kullanılabilecek güçlü bir genetik model ve Sosyal etkileşim nöral temelleri. Gerçekten de, D. melanogaster karmaşık sosyal davranışların bir repertuara sahiptir ve sosyalleşme bazı direkt ölçümler zaten 1-7 yapılmıştır. Ancak bu çabaların çoğu gibi saldırganlığı gibi oldukça karmaşık sosyal davranışlar üzerinde odaklanmıştırsive etkileşimler 3,6, kur 3,8-12 çeşitli yönleri ve nasıl sosyal bir deneyim gibi öğrenme, ya da sirkadiyen ritim 13-17 gibi diğer davranışları etkiler. Buna ek olarak, bu deneylerin çoğuna sineklerin gruplarının karmaşık etkileşimi örüntüye verilerin elde edilen bolluk analiz video izleme ve bilgisayar yazılımı kullanılarak dayanır. Bu tür analizler çok değerli ve bu gruplarda 7 sinek sinek etkileşimlerinin dinamik olarak önemli yeni anlayışlar yol. Bir sınırlama, ancak, geniş topluma bu analizlerin erişilememesi, ve diğerleri tanınmasını altında yatan mekanizmaların sınırlı bilgidir. Başka bir deyişle, bir birey ve bir başka tarafından tanınması bir sinyalin emisyon temeli hala tam olarak 18 anlaşılmaktadır. D.: Buna karşılık, aynı zamanda basit bir davranış, bireylerin stresli sinekler tarafından yayılan bir sinyal uzaklaşmaya sosyal kaçınma, sergiler sineklerin melanStres Odorant veya DSO 19 ogaster. Bir yüksek verimli bir deney, bu davranış, diğer sinekler veya kaçınma 19 tarafından yayılan bir gerilim sinyalinin önlenmesi olarak belirlenebilir. Sinekler T-labirent aparatı yerleştirilmiş ve DSO içeren bir şişe önlemek için seçim verilir. Bu deneme kullanılarak, CO2 2 19 kesitlere ayrılmıştır CO yanıt için gerekli nöral DSO bileşeni, ve bir parçası olduğu gösterilmiştir. Burada sunulan sosyal kaçınma tahlil karmaşık davranışları 20 incelemek için araştırmacıların nesiller etkin Seymour BENZER laboratuvarında geliştirilen basit davranış deneyleri kavramsal benzer. Kaçınma analizi maliyet etkin sosyal davranışlarının daha yaygın çalışma için izin T-labirent tahlili kullanılarak gerçekleştirilebilir. Örneğin, bu deneyde kullanılan son zamanlarda ortaya otizm, farklı genetik riski sosyal b zıt etkilere sahip olduğuehavior deneyler. 21,22 neurobeachin - - otizm için bir aday gen için mutantlar mevcut eksiklikler hem sosyal alanda (başka yerde 23 tarif) ve sosyal kaçınma 24. Anormal dopaminerjik sinyal de insanlarda 25,26 otizm etiyolojisinde rol oynadığı ileri sürülmektedir. Neurobeachin ile elde edilen sonuçların aksine, artmış ya da dopaminerjik hücrelerin Drosophila veziküler Monoamin Taşıyıcı (VMAT) düzeylerini azaltırken sosyal alanı ile doğrudan VMAT 27 bu seviyelere ilişkili, ancak kaçınma performansı, etkilenmemiş olduğunu ortaya koymuştur. neurobeachin ve VMAT ile elde edilen zıt sonuçlar böylece çeşitli asosyal davranış biçimlerini ve başkalarına yanıt modüle farklı altta yatan nöral devreler belirlenmesi olasılığını altını çiziyor.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereo Zoom Microscope&#160;</td>
			<td>Nikon&#160;&#160;</td>
			<td>SMZ-645</td>
			<td>Any other standard scope for fly handling would work</td>
		</tr>
		<tr>
			<td>Small paint brushes&#160;</td>
			<td></td>
			<td></td>
			<td>for pushing flies</td>
		</tr>
		<tr>
			<td>Porous Polyethylene, 12&#34; x 12&#34; Sheet</td>
			<td>Flystuff - Genesee</td>
			<td>46-100</td>
			<td>http://www.flystuff.com/ProductInfo.php?productID=46-100 
			Porous Plastic sheet for the cold anesthesia box</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Alarm Timer/Stopwatch</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sharpie pens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini vortex</td>
			<td>Fisher</td>
			<td>14-955-151&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail 
			For mechanical agitation of the flies - any vortex would work.</td>
		</tr>
		<tr>
			<td>Corning Life Sciences DL No.:352017, Falcon test tube; round bottom; disposable; no closure, 14mL;&#160; 17 x 100mm</td>
			<td>Fisher</td>
			<td>14-959-8 &#160;&#160;</td>
			<td>http://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalogId=29104&#38;productId=2771811&#38;distype=0&#38;highlightProductsItemsFlag=Y&#38;fromSearch=1&#38;searchType=PROD&#38;hasPromo=0 
			These snap in place in the in-house made T-maze and counter-current apparatus (see text)</td>
		</tr>
		<tr>
			<td>cotton balls</td>
			<td></td>
			<td></td>
			<td>to close the vials after the experiment.</td>
		</tr>
		<tr>
			<td>trifold board and white bench cover</td>
			<td></td>
			<td></td>
			<td>to provide a white background, and a homogeneous light.</td>
		</tr>
		<tr>
			<td>white bench cover</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pounding pad</td>
			<td></td>
			<td></td>
			<td>any mouse pad works.</td>
		</tr>
		<tr>
			<td>large black cloth</td>
			<td></td>
			<td></td>
			<td>to cover the counter-current apparatus in phototaxis response.</td>
		</tr>
		<tr>
			<td>cool-white light&#160;</td>
			<td>Home Depot</td>
			<td>1000516563</td>
			<td>http://www.homedepot.ca/product/illume-26-fluorescent-plug-in-linear/911423 
			any similar linear light with fluorescent light bulb cool-white at 13-15W would work</td>
		</tr>
	</tbody>
</table>

straightforward assay for quantification of social avoidance in drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

sosyal kaçınma tahlil diğer sinekler tarafından yayılan bir stres sinyali tanımak için Drosophila melanogaster yeteneğini (DSO) ölçülmesi ve böylece sosyal etkileşimlerin bir yönünü değerlendiren sağlam bir testtir. Deney genellikle iki farklı seçenek arasında bir seçim ile sinekler mevcut T-labirent olarak bilinen çeşitli davranış deneylerinde kullanılan bir aygıt kullanılarak gerçekleştirilir - sola veya sağa 19,28-31. Bu durumda, sinekler DSO önlemek hangi verimlil...

Watch this Scientific Journal Video about Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster at JoVE.com

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Sosyal Kaçınma Niceleme için Basit Testi<em&gt; Drosophila melanogaster</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

straightforward-assay-for-quantification-social-avoidance-drosophila

Research

JoVE Journal

Neuroscience

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

11.1K Görüntüleme.  Yale University. Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Video: Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Kullanarak Bileşiklerin Biyoaktivite için eşli Nanoinjection ve Ekran için Elektrofizyoloji Testi Drosophila melanogaster Dev Elyaf Sistemi

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

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 ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Olarak spontan lokomotor aktivitenin belirlenmesi<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

Sosyal Space Etkileyen Koşullar<em&gt; Drosophila melanogaster</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

Koku kılavuzluğunda Davranış Yüksek çözünürlüklü Niceleme<em&gt; Drosophila melanogaster</emKullanma&gt;<em&gt; Flywalk</em&gt; Paradigma

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

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

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

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

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

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

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

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Yüksek Verimli Testi Bireyin Yumurta Yumurtlayan Tercihler incelemek için<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

için yiyecek arama yolu uzunluğu Protokolü<em&gt; Drosophila melanogaster</em&gt; Larva

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Yüksek içerik miktar Sod1 canlı hücreler Mutant Protein toplama oluşumu için tahlil geliştirme

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...