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. Attrezzature e reagenti creati in-house (vedi elenco dei materiali per gli altri) <ol><li> Preparare una Drosophila apparecchiatura anestesia fredda per svolgere un lavoro mosca. <ol><li> Tagliare un foglio di polietilene porosa per coprire una piccola scatola di plastica (lungo 12,7 centimetri, larghezza 10,2 centimetri), in genere la copertura superiore di una scatola punte per pipette (lungo 12,7 centimetri, larghezza 10,2 centimetri, 3,8 centimetri di profondità). </li><li> Riempire la scatola con ghiaccio tritato, coprire con il foglio di polietilene porosa. </li></ol></li><li> Preparare un apparato T-labirinto; adattare l&#39;apparecchiatura precedentemente descritta in dettaglio 28-31. </li><li> Preparare un aspiratore mosca come descritto 32. </li><li> Preparare un apparato di risposta fototassi per spostare le mosche dentro e fuori di flaconi di prova, senza stress: <ol><li> Utilizzare un apparato in controcorrente, come descritto da 29,33. </li></ol></li></ol> NOTA: La T-maze può essere utilizzato anche per altri scopi, come descritto da 28. <ol start="5"><li> Garantire homcondizioni di luce ogeneous: eseguire l&#39;esperimento su un banco coperto con un coperchio panchina bianca, e di fronte a un bordo bianco. NOTA: per questi esperimenti, i ceppi utilizzati sono stati mosca Drosophila melanogaster wild-tipo: Canton-S, Oregon e Samarcanda vola dalle nostre scorte di laboratorio 34; Mosche Elwood sono stati raccolti in autunno 2011 nel quartiere Elwood di Huntington, a Long Island, New York, Stati Uniti d&#39;America. </li></ol> 2. Preparare le mosche prima dell&#39;esperimento 1-2 giorni prima di eseguire l&#39;esperimento: <ol><li> Mantenere mosche sul medio farina di mais-agar-melassa-lievito di serie a 25 ° C su un 12 ore di luce / buio ciclo. </li><li> Raccogliere linea risponditore sotto anestesia freddi 1-2 giorni prima di eseguire l&#39;esperimento <ol><li> Trasferire le mosche dalle bottiglie in cui sono stati raccolti in 50 ml provette Falcon, con un imbuto. </li><li> Posizionare il tubo falco con le mosche sotto il livello del ghiaccio, in unsecchiello per il ghiaccio isolato. </li><li> Collocare l&#39;apparecchio di anestesia fredda con ghiaccio tritato a -4 ° C per rilassarsi. </li><li> Attendere 5 minuti per le mosche di entrare in un coma freddo. </li><li> Trasferire le mosche sul foglio di polietilene porosa dell&#39;apparato anestesia freddo. </li><li> Raccogliere le mosche risponditore sull&#39;apparato anestesia freddo. A RT, sotto uno stereomicroscopio, separare i 3-7 giorni vecchio maschio da mosche femmine e mantenere queste mosche in gruppi di 40. </li></ol></li><li> Raccogliere linea emettitore 1-2 giorni prima di eseguire l&#39;esperimento. <ol><li> Raccoglie delicatamente Canton-S mixed-sex aria dal loro bottiglie, con un aspiratore bocca, controllando per il loro numero. </li><li> Mantenere queste mosche in gruppo di 60-100, in quanto saranno gli emettitori. </li><li> Preparare tutti i campioni di emettitori come punti dati necessari. </li></ol></li><li> Lasciare almeno una notte di recupero dopo la raccolta dei soccorritori e per gli emettitori, per ridurre al minimo qualsiasi effetto confondente dell&#39;anestesia freddoe la raccolta della bocca di aspirazione sul comportamento. </li><li> Aprire la busta di fiale di prova, per permettere all&#39;aria di plastica maleodoranti stantio nel sacchetto per essere sostituito da aria fresca, almeno 1 giorno prima dell&#39;esperimento. </li></ol> 2 ore prima di eseguire l&#39;esperimento: <ol start="6"><li> Assicurarsi che la temperatura nel locale in cui viene eseguita l&#39;esperimento è di circa 23-25 ​​° C, con anche la luce, e umidità superiore al 30%. </li><li> Trasferire i soccorritori e mosche emettitori di frutta in fiale di cibo fresco per garantire che queste mosche non sono affamate. </li><li> Lasciate che i moscerini della frutta regolare per l&#39;ambiente per 2 ore, sulla panchina su cui verrà eseguita l&#39;esperimento. </li></ol> 3. Eseguire l&#39;esperimento sociale Prevenzione <ol><li> Eseguire ogni esperimento allo stesso tempo del giorno, in un intervallo di 3-4 ore nel pomeriggio tra tempo Zeitgeber ZT5 e ZT9. </li><li> Posizionare il T-labirinto sul pad martellante, e stringere la clip vite così tegli ascensore è stabile. </li><li> Trasferire linea risponditore in un test flacone fresco. Snap questo flaconcino contenente linea risponditore sulla parte superiore della T-labirinto. </li><li> Inclinare l&#39;apparecchio T-labirinto e toccare sul tappeto martellante in modo che i moscerini della frutta caduta in ascensore. </li><li> Spostare la parte superiore del l&#39;ascensore in modo in linea sono tra la parte superiore e inferiore del T-labirinto, ma attenzione per mantenere l&#39;ascensore sopra del punto scelta - per evitare le mosche fuoriuscita. </li><li> Ottenere fiale con DSO (vedi sezione 5 per generare fiale DSO-libero che in precedenza occupati da mosche). <ol><li> Mentre le mosche risponditore stanno adeguando a questo nuovo ambiente, mettere in linea emettitore in un flaconcino di prova. </li><li> Posizionare un pezzo di cotone per chiudere la fiala in modo che i moscerini non sfuggono. </li><li> Meccanicamente Agitare questo flaconcino su una mini-vortice come segue: vortex per 15 secondi, togliere il flacone dal vortice per 5 sec. Ripetere 3 volte per un totale di 55 sec. </li><li> Remil sobrio linea emettitore dal flaconcino fresca - trasferendo in una fiala alimentare - e rapidamente posizionare questo DSO fiala riempita in uno dei due lati dell&#39;apparato T-labirinto. </li><li> Alternare il lato di posizionamento per il flacone DSO compilato in ogni nuova esecuzione. </li></ol></li><li> Inserire un test flaconcino fresca sul lato della T-labirinto. </li><li> Portare l&#39;ascensore fino in fondo, in modo che i moscerini della frutta possono scegliere tra il flacone di prova fresco e la fiala test con DSO, e avviare il timer. </li><li> Lasciate che i mosche scegliere tra la fiala fresco e la fiala DSO, per 1 minuto, se non diversamente specificato. </li><li> Dopo 1 min, spostare l&#39;ascensore fino a separare le mosche nel flacone fresco, DSO flacone, e quelli bloccati in ascensore. </li><li> Contare il numero di mosche in ogni fiala, e in ascensore. </li><li> Ripetere questa operazione per ciascun genotipo / condizione. </li></ol> 4. Analizzare i dati sociale Prevenzione <ol><li> Contare il numero di moscerini della frutta in ogni fiala per ogni genotipo, e trasferire i dati in un foglio di calcolo. </li><li> Utilizzando un foglio di calcolo, calcolare il Performance Index (PI) per ciascun genotipo sottraendo il numero di mosche in flaconcino DSO dal numero di mosche in flacone DSO-libera, e dividendo per il numero totale di mosche. </li><li> Confrontare Pis &quot;: mediante software di analisi statistica per l&#39;analisi dei risultati. <ol><li> Idealmente, preparare 2-3 ripetizioni interne per ogni esperimento, e 3 ripetizioni indipendenti, effettuate in giorni diversi, con diverse bottiglie o croci 30. </li><li> Utilizzare test statistici distribuzione gaussiana, come i dati seguono una distribuzione normale. </li><li> Durante il test di un effetto (cioè, densità - figura 2), utilizzare un ANOVA, con post-test di confronto multiplo (colonna per colonna), o un semplice t-test per confrontare ogni risultato a un controllo. </li><li> Quando gli esperimenti di gruppo vengono eseguiti, testare entrambe le condizioni e generi (flaconi vuoti, ha sottolineato und vola non-ha sottolineato; ed effetto genere - figura 1), utilizzando una ANOVA a due vie. </li></ol></li></ol> 5. Generare fiale precedentemente occupato da mosche non accentate - Eseguire Invece di 3,5-3,6 <ol><li> Utilizzare fototassi preparare fiale occupati da mosche che non sono stati sottolineati, come controllo. </li><li> Trasferire le mosche in un flaconcino di prova, e farlo scattare in dell&#39;apparato controcorrente, con fiale freschi vuoti nella posizione di fronte. </li><li> Posare l&#39;apparecchio orizzontalmente con la fiala distale direttamente di fronte a 15 W fluorescente bianca fredda, e coprire con un panno nero. </li><li> Lasciate linea si muovono delicatamente nel flaconcino fresco. Questo potrebbe richiedere alcuni minuti. Una volta che la maggior parte delle mosche camminano verso la luce nel flacone distale, avviare il timer per 1 min. </li><li> Rimuovere delicatamente il panno nero, cambiare la posizione della luce, il recupero con il panno nero, e permettono in linea di lasciare il flacone di prova. </li><li> A quel tempo, initiate l&#39;esperimento scelta in 3.2 a 3.5. Salta 3.6 e 3.7. </li><li> Spostare il cursore dell&#39;apparecchiatura, per separare le mosche che liberate il flaconcino di prova dall&#39;altro lato. </li><li> Utilizzare il flacone di prova sgomberato nel T-labirinto subito per l&#39;esperimento scelta, come in 3.7. </li></ol>

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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.

Questo protocollo descrive una procedura dettagliata per il saggio evitamento sociale. Canton-S eviterà solo una fiala in che vola in precedenza stato meccanicamente sottolineato, e che il sesso e il numero di responder non influenza che le prestazioni evitamento sociale. Tuttavia, background genetico dei soccorritori ha una grande influenza. Di seguito sono riportati alcuni passaggi critici per l&#39;esecuzione di questo esperimento con successo: 1) il trasferimento sempre in linea 2 ore p...

L&#39;obiettivo di questo metodo è di quantificare facilmente un nuovo aspetto del semplice comportamento sociale in Drosophila melanogaster, indipendente dalla corteggiamento e aggressività. Interazioni sociali sono fondamentali per il corretto sviluppo e la salute delle persone all&#39;interno di una società, così come la funzionalità di un gruppo sociale nel suo complesso. La complessità di queste interazioni richiede campioni di grandi dimensioni e di un sistema che consente una semplificazione del comportamento come basi genetiche e neurali del comportamento sociale sono ancora poco conosciuti. Drosophila melanogaster è un modello genetico potente che può essere utilizzato per identificare il genetica e basi neurali delle interazioni sociali. Infatti, D. melanogaster ha un repertorio di comportamenti sociali complessi e alcune misure dirette di socializzazione sono già stato fatto 1-7. Tuttavia, la maggior parte di questi sforzi sono stati concentrati sui comportamenti sociali relativamente complessi, come aggresinterazioni sive 3,6, vari aspetti del corteggiamento 3,8-12, e come i social esperienza influisce altri comportamenti quali l&#39;apprendimento, o il ritmo circadiano 13-17. Inoltre, molti di questi test si basano su analisi di modelli di interazione complesse di gruppi di mosche, con il monitoraggio di video e software per analizzare l&#39;abbondanza risultante dei dati. Tali analisi hanno un valore inestimabile, e portare a nuove importanti informazioni quali la dinamica delle interazioni fly-fly in gruppi 7. Un limite, però, è l&#39;inaccessibilità di questi test per la comunità in generale, e la limitata conoscenza dei meccanismi alla base il riconoscimento degli altri. In altre parole, la base dell&#39;emissione di un segnale da un individuo e il suo riconoscimento da un altro è ancora scarsamente compreso 18. Al contrario, vola anche mostrare un comportamento semplice, evitamento sociale, in cui gli individui si muovono da un segnale emesso da mosche sollecitate: la D. Melanogaster stress odorizzante o DSO 19. In un saggio ad alta prestazione, questo comportamento può essere quantificato come la prevenzione di un segnale di stress emesso da altre mosche, o evitamento sociale 19. Mosche sono posti in un apparecchio T-labirinto e data la scelta per evitare una fiala contenente DSO. Utilizzando questo test, CO 2 ha dimostrato di essere un componente del DSO, e parte del circuito neuronale necessario per rispondere a CO 2 è stato dissezionato 19. Il saggio evitamento sociale qui presentato è simile concettualmente ai semplici test comportamentali sviluppati nel laboratorio di Seymour Benzer che hanno permesso generazioni di ricercatori per analizzare comportamenti complessi 20. Analisi di evitamento sociale può essere effettuata conveniente utilizzando il test T-maze, consentendo un più ampio studio del comportamento sociale. Ad esempio, utilizzando questo test abbiamo recentemente dimostrato che i diversi rischi genetici per l&#39;autismo hanno effetti contrastanti in b socialesaggi ehavior. Mutanti di un gene candidato per l&#39;autismo - neurobeachin 21,22 - carenze presenti sia nello spazio sociale (descritte altrove 23) e l&#39;evitare sociale 24. Segnalazione dopaminergico anomala si propone anche di svolgere un ruolo nell&#39;eziologia dell&#39;autismo negli esseri umani 25,26. In contrasto con i risultati ottenuti con neurobeachin, abbiamo scoperto che le prestazioni evitamento sociale è stata influenzata da un aumento o diminuzione dei livelli di Drosophila vescicolare Monoamino Transporter (VMAT) in cellule dopaminergiche, anche se lo spazio sociale è direttamente correlata a questi livelli di VMAT 27. I risultati ottenuti con contrasto neurobeachin e VMAT sottolineano la possibilità di individuare varie forme di comportamento asociale, e quindi le differenti circuiterie neurali sottostanti modulano la risposta ad altri.

<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.

Il saggio evitamento sociale è una prova robusta quantificare la capacità di Drosophila melanogaster riconoscere un segnale di stress (DSO) emessa da altri mosche, e valutare quindi un aspetto delle interazioni sociali. Il test viene eseguito utilizzando un apparecchio comunemente usato in vari saggi comportamento noto come T-labirinto, che presentano in linea con una scelta tra due diverse opzioni - a sinistra oa destra 19,28-31. In questo caso, l&#39;efficienza con cui linea evitano il DSO è qua...

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.

Assay Semplice per la quantificazione della Social Prevenzione in<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 Views.  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.

Nanoinjection associato e test Elettrofisiologia allo schermo per la bioattività dei composti con il Drosophila melanogaster Sistema Fiber Giant

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 ...

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Neuroscienze

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.

Stimolazione Optogenetic del comportamento in Fuga<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;

Determinazione del locomotore attività spontanea in<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.

Condizioni che influiscono spazio sociale in<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.

Ad alta risoluzione quantificazione del comportamento Odor-guidata<em&gt; Drosophila melanogaster</em&gt; Utilizzo della<em&gt; Flywalk</em&gt; Paradigm

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.

Un test comportamentale per Mechanosensation di cloni in marcm-based<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.

High Throughput test per esaminare Preferenze deposizione delle uova dei singoli<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.

Foraggiamento protocollo percorso di lunghezza per<em&gt; Drosophila melanogaster</em&gt; Le larve

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.

Il capillare alimentatore Assay Misure assunzione di cibo in<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.

Sviluppo di analisi per la quantificazione alto contenuto di formazione aggregata di Sod1 mutante della proteina in cellule viventi

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