We would like to thank members of the Tessier lab for critical reading of this manuscript and helpful suggestions during the preparation of this protocol.

1. fame <ol><li> Preparare volare fiale fame saturando un batuffolo di cotone con 18,2 MW acqua sul fondo di una fiala mosca standard. In alternativa, allo stesso modo di saturare una piccola striscia di carta da filtro con 18,2 MW acqua e posto in un angolo all&#39;interno della fiala. </li><li> Raccogliere vola in gruppi di circa 100 animali su un rilievo di CO 2 e quindi aggiungere le mosche per una fiala preparato. Nota: i migliori risultati si ottengono con animali che sono meno di 5 giorni. Tuttavia, l&#39;esatta età degli animali può essere controllata come variabile sperimentale per determinare le variazioni di preferenza di gusto nel tempo. </li><li> Utilizzare un batuffolo di cotone o schiuma tappo per fissare le fiale chiuse. Posizionare le fiale dalla loro parte in un incubatore ambiente controllato. Mantenere la temperatura a 25 ° C, e l&#39;umidità superiore al 70%. Lascia fiale intatte per 24 ore. </li></ol> 2. Taste Preferenza Assay <ol><li> Preparare tutti tastants per il saggio lo stesso giorno uns test. Nota: I tastants esatta da utilizzare varia a seconda domanda sperimentale viene chiesto. I seguenti sono esempi di tastants utilizzati in questo protocollo. Vedere la sezione 4 per ottimizzazioni. <ol><li> Preparare il controllo tastant (1 mM saccarosio) mediante la combinazione di 10 ml di 100 mm soluzione di saccarosio, 13 ml di colorante alimentare rosso, e 977 ml di 18,2 MW acqua. </li><li> Preparare tastant sperimentale (5 mm saccarosio) mediante la combinazione di 50 ml di 100 mm soluzione di saccarosio, 10 ml di colorante alimentare blu, e 940 ml di 18,2 MW acqua. </li></ol></li><li> Rendere camere dosaggio usando da 100 mm x 15 mm piatto di plastica standard di petri preparato nel seguente modo: <ol><li> Mettere tre gocce 10 microlitri di controllo tastant più vicino al bordo del piatto a ore 12 e un altro 3 gocce a ore 6. Assicurarsi che la distanza tra gocce è simile. </li><li> Inserire tre gocce 10 microlitri di tastant sperimentale vicina al bordo della piastra a ore 3 e unaltri 3 gocce alle ore 9. Assicurarsi che la distanza tra gocce è simile. </li><li> Ripetere i punti 2.2.1 e 2.2.2 per il numero di repliche, se lo desideri. </li></ol></li><li> Vuoto 1 fiala di ~ 100 mosche di fame su un tappetino di CO 2 giusto il tempo per anestetizzare gli animali (circa 10 sec). Spennellate gli animali in mezzo a una camera di test preparata e coprire con il coperchio piatto. Nota: più lunghi periodi di esposizione CO 2 dovrebbe essere evitato per migliorare i tempi di recupero e l&#39;interferenza con il limite di comportamento alimentare. L&#39;esposizione al ghiaccio (~ 5 min) può essere utilizzato per anestetizzare per evitare di CO 2 effetti comportamentali che possono derivare da un&#39;esposizione limitata. </li><li> Posizionare la camera di test in una scatola di cartone opaco. Assicurarsi di etichettare l&#39;esterno della scatola con la condizione e il genotipo in fase di test. </li><li> Posizionare l&#39;intera impostazione (camera di analisi contenuta in scatola di cartone a partire dal punto 2.4) in un incubatore a 25 ° con almeno il 70% di umidità per 2 ore. </li&gt; <li> Ripetere i passaggi 2.3 tramite 2.5 per tutte le repliche. </li><li> Dopo 2 ore, posizionare le camere di test, ancora contenuti in scatole di cartone, direttamente in un congelatore a -20 ° C fino al momento per la quantificazione. </li></ol> 3. Gusto Preferenza Assay Quantificazione <ol><li> Lasciare una sola camera di test per riscaldare a temperatura ambiente (circa 5 min). </li><li> Sotto un microscopio dissezione, utilizzando un pennello o un paio di pinze, animali di gruppo in base al colore della loro addome: rosso, blu, viola o trasparente (Figura 1). </li><li> Registrare il numero di animali in ciascun gruppo. Considerare gli animali chiare per non hanno partecipato al test e quindi non includerli in tutti i calcoli. </li><li> Calcolare l&#39;indice preferenza, secondo una delle seguenti formule: <ol><li> Se il tastant sperimentale di interesse viene aggiunto il colorante rosso, quindi utilizzare (N + rosso 0,5N viola) / (N + N rosso N purpl blue +e). </li><li> Se il tastant sperimentale viene aggiunto il colorante blu, quindi regolare l&#39;equazione (N blu + 0,5 N viola) / (N + N blu rosso + N viola). </li></ol></li><li> Ripetere i calcoli per tutte le condizioni sperimentali e replicati. </li></ol> 4. Ottimizzazione del Gusto Preferenza Assay <ol><li> Empiricamente determinare la concentrazione di indicatori colorante alimentare per essere utilizzato in modo colorante alimentare non influenza l&#39;esito del test gusto, come segue: <ol><li> Preparare 4 tastants utilizzando la stessa mescola di base (ad esempio, 5 mM saccarosio), come indicato al punto 2.1, ma omettere il colorante alimentare. </li><li> Aggiungere 1,3% colorante alimentare rosso per una delle sostanze sapide. Effettuare le restanti 3 tastants con prodotti alimentari blu coloranti di concentrazioni variabili in ogni tubo (ad esempio 0,6%, 1% e 1,3%). </li><li> protocollo completo passi 2.2 attraverso 3.4 per ogni coppia tastant: 1,3% vs. 0,6% rosso blu; 1,3% rispetto al rosso 1% e il blu1,3% contro 1,3% rosso blu. </li><li> Ripetere il passaggio 4.1.1-4.1.3 con diverse percentuali di colorante alimentare blu fino alle medie indice preferenza un valore pari a 0 (Figura 2). Nota: Come punto di partenza, 1,3% colorante alimentare rosso accoppiato con 1% colorante alimentare blu produce in genere buoni risultati. Se nessuna concentrazione soddisfacente di colorante alimentare blu può essere abbinato al 1,3% di colorante, poi passo 4.1.1 tramite 4.1.3 può essere ripetuta con concentrazioni variabili di colorazione rossa e una costante concentrazione di colorante alimentare blu. </li><li> Analizzare tutte le condizioni per essere testati con le stesse concentrazioni di colorante alimentare ottimizzati. </li></ol></li></ol>

<ol>
	<li>Herrero, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+fly+behavior+in+response+to+chemosensory+signals.">Fruit fly behavior in response to chemosensory signals.</a> Peptides. 38 (2), 228-237 (2012).</li><li>Vosshall, L. B., Stocker, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+smell+and+taste+in+Drosophila.">Molecular architecture of smell and taste in Drosophila.</a> Annu Rev Neurosci. 30, 505-533 (2007).</li><li>Harris, D. T., Kallman, B. R., Mullaney, B. C., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representations+of+Taste+Modality+in+the+Drosophila+Brain.">Representations of Taste Modality in the Drosophila Brain.</a> Neuron. 86 (6), 1449-1460 (2015).</li><li>Hong, E. J., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+encoding+of+odors+by+channels+with+diverse+sensitivity+to+inhibition.">Simultaneous encoding of odors by channels with diverse sensitivity to inhibition.</a> Neuron. 85 (3), 573-589 (2015).</li><li>Kain, P., Dahanukar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+taste+neurons+that+convey+sweet+taste+and+starvation+in+the+Drosophila+brain.">Secondary taste neurons that convey sweet taste and starvation in the Drosophila brain.</a> Neuron. 85 (4), 819-832 (2015).</li><li>Masek, P., Worden, K., Aso, Y., Rubin, G. M., Keene, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dopamine-modulated+neural+circuit+regulating+aversive+taste+memory+in+Drosophila.">A dopamine-modulated neural circuit regulating aversive taste memory in Drosophila.</a> Curr Biol. 25 (11), 1535-1541 (2015).</li><li>Charlu, S., Wisotsky, Z., Medina, A., Dahanukar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+sensing+by+sweet+and+bitter+taste+neurons+in+Drosophila+melanogaster.">Acid sensing by sweet and bitter taste neurons in Drosophila melanogaster.</a> Nat Commun. 4, 2042 (2013).</li><li>Clyne, P. J., Warr, C. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidate+taste+receptors+in+Drosophila.">Candidate taste receptors in Drosophila.</a> Science. 287 (5459), 1830-1834 (2000).</li><li>Scott, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemosensory+gene+family+encoding+candidate+gustatory+and+olfactory+receptors+in+Drosophila.">A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila.</a> Cell. 104 (5), 661-673 (2001).</li><li>Kim, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+TRPA1+channel+mediates+chemical+avoidance+in+gustatory+receptor+neurons.">Drosophila TRPA1 channel mediates chemical avoidance in gustatory receptor neurons.</a> Proc Natl Acad Sci U S A. 107 (18), 8440-8445 (2010).</li><li>Koh, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+IR20a+clade+of+ionotropic+receptors+are+candidate+taste+and+pheromone+receptors.">The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors.</a> Neuron. 83 (4), 850-865 (2014).</li><li>Zhang, Y. V., Ni, J., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+for+attractive+salt-taste+coding+in+Drosophila.">The molecular basis for attractive salt-taste coding in Drosophila.</a> Science. 340 (6138), 1334-1338 (2013).</li><li>Zhang, Y. V., Raghuwanshi, R. P., Shen, W. L., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+experience-induced+taste+desensitization+modulated+by+the+Drosophila+TRPL+channel.">Food experience-induced taste desensitization modulated by the Drosophila TRPL channel.</a> Nat Neurosci. 16 (10), 1468-1476 (2013).</li><li>Liman, E. R., Zhang, Y. V., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+coding+of+taste.">Peripheral coding of taste.</a> Neuron. 81 (5), 984-1000 (2014).</li><li>Rodrigues, V., Cheah, P. Y., Ray, K., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=malvolio,+the+Drosophila+homologue+of+mouse+NRAMP-1+(Bcg),+is+expressed+in+macrophages+and+in+the+nervous+system+and+is+required+for+normal+taste+behaviour.">malvolio, the Drosophila homologue of mouse NRAMP-1 (Bcg), is expressed in macrophages and in the nervous system and is required for normal taste behaviour.</a> EMBO J. 14 (13), 3007-3020 (1995).</li><li>Tanimura, T., Isono, K., Yamamoto, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+sensitivity+to+trehalose+and+its+alteration+by+gene+dosage+in+Drosophila+melanogaster.">Taste sensitivity to trehalose and its alteration by gene dosage in Drosophila melanogaster.</a> Genetics. 119 (2), 399-406 (1988).</li><li>Weiss, L. A., Dahanukar, A., Kwon, J. Y., Banerjee, D., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+cellular+basis+of+bitter+taste+in+Drosophila.">The molecular and cellular basis of bitter taste in Drosophila.</a> Neuron. 69 (2), 258-272 (2011).</li><li>French, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+mechanism+for+bitter+avoidance+in+Drosophila.">Dual mechanism for bitter avoidance in Drosophila.</a> J Neurosci. 35 (9), 3990-4004 (2015).</li><li>Deshpande, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Drosophila+food+intake:+comparative+analysis+of+current+methodology.">Quantifying Drosophila food intake: comparative analysis of current methodology.</a> Nat Methods. 11 (5), 535-540 (2014).</li></ol>

The authors declare that they have no competing financial interests.

Abbiamo descritto un protocollo semplice ma efficace per determinare preferenza di gusto in Drosophila. Le versioni di questo test sono abitualmente utilizzati in esperimenti per determinare i contributi di recettori gustativi (GRS) a percepire le diverse qualità (amaro, dolce, acido, salato e umami) di composti di gusto. Il genoma di Drosophila contiene circa 60 geni che codificano 68 individuati recettori gustativi da splicing alternativo 8,9. Tuttavia, altre proteine ​​come recettori...

Gli animali usano chemosensation distinguere condizioni vantaggiose a parte condizioni svantaggiose. Questa percezione può essere fondamentale per cose come determinare la migliore fonte di cibo, evitando sostanze tossiche o determinare il miglior partner di accoppiamento 1. Chemosensation è spesso diviso in due componenti sensoriali: olfatto e sensi gustative. Una principale caratteristica distintiva di questi sensi è che l&#39;olfatto (odore) è usato per campionare l&#39;ambiente chimico gassoso circostante mentre gustation (gusto) richiede il contatto fisico con un substrato non volatile. Entrambe le modalità sensoriali stimolano risposte neurologiche che vengono elaborati e decodificati nel cervello per produrre l&#39;attrattiva o repulsiva comportamento appropriato 2. Questi sensi sono quindi fondamentale per la sopravvivenza degli animali. La mosca della frutta Drosophila melanogaster è un organismo modello che continua a crescere in popolarità per l&#39;uso in capireing come gli insetti percepiscono odore e sapore. Frutta mosche offrono enormi vantaggi rispetto ad altri sistemi modello per la ricchezza di strumenti genetici disponibili per la dissezione di percorsi molecolari, cellulari e comportamentali. Il lavoro nel corso degli ultimi 15 anni è stato particolarmente determinante nel caratterizzare le identità cellulari specifici, recettori neuronali, e meccanismi coinvolti sia in odore e il sapore di segnalazione. Ora, il potere della genetica della Drosophila viene usata per chiarire ulteriormente come questi processi sono codificati al singolo neurone e il circuito unico livello 3-6. Pertanto, le analisi che forniscono facilmente lanciati letture di alterazioni percorsi sensoriali sono di vitale importanza per il continuo avanzamento di questi campi. Mentre molto si sa su come i segnali olfattivi sono codificati ed elaborati nel cervello, e tanto meno è capito su meccanismi simili nel percorso gustativo. Descriviamo qui un protocollo che può essere utilizzato per verificare preferen gustoce in Drosophila. Drosophila, come i mammiferi, in genere preferiscono i composti di degustazione di dolci al contrario di composti sapore amaro. Qualsiasi combinazione di queste fonti di cibo può essere utilizzato in questo progetto sperimentale per determinare come note alterazioni genetiche influenzano la scelta gusto. Inoltre, le strategie di intervento farmacologico può analogamente essere valutati per i loro effetti sulla preferenza di gusto degli animali. La facilità e la flessibilità di questo test lo rende un paradigma utile per comprendere la natura della percezione gustativa in Drosophila.

<table border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:543px;">Blue Food Coloring (Water, Propylene Glycol, FD&#38;C Blue 1 and Red 40, Propylparaben)</td>
			<td style="width:128px;">McCormick</td>
			<td style="width:111px;">N/A</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cryo/Freezer Boxes w/o Dividers</td>
			<td>Fisher</td>
			<td>03-395-455</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glacial Acetic Acid</td>
			<td>Fisher</td>
			<td>BP2401-500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Leica S6 E Stereozoom 0.63x-4.0x microscope</td>
			<td>W. Nuhsbaum, Inc.</td>
			<td>10446294</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Petri Dish (100 x 15 mm)</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td>Reuseable if thoroughly washed and dried</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Quick-Snap Microtubes</td>
			<td>Alkali Scientific Inc.</td>
			<td>C3017</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Red Food Coloring (Water, Propylene Glycol, FD&#38;C Reds 40 and 3, Propylparaben)</td>
			<td>McCormick</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sucrose</td>
			<td>IBI Scientific</td>
			<td>IB37160</td>
			<td></td>
		</tr>
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</table>

taste preference assay for adult drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Alcuni risultati tipici da saggi di preferenza di gusto sono riportati di seguito. Nella maggior parte degli esperimenti qualche variazione di intensità di colorazione addominale sarà visto (Figura 1). Qualsiasi colorazione nell&#39;addome se intensa o debole è considerato un ingestione positivo. E &#39;quindi consigliabile per i ricercatori a segnare animali, mentre ciechi alla condizione sperimentale in modo da limitare i potenziali errori. <p class="jove_conten...

Watch this Scientific Journal Video about Taste Preference Assay for Adult Drosophila at JoVE.com

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Gusto Preferenza Assay per adulti<em&gt; Drosophila</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

taste-preference-assay-for-adult-drosophila

Research

JoVE Journal

Neuroscience

Taste Preference Assay for Adult Drosophila

9.5K Views.  Indiana University School of Medicine-South Bend. Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Video: Taste Preference Assay for Adult Drosophila

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Metodi per test Drosophila Comportamento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Ricerca

Neuroscienze

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila adulti olfattiva Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Luce Preferenza Assay per studiare Innata e circadiano Photobehavior Regolamentato<em&gt; Drosophila</em&gt; Larve

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

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.

A Single-fly saggio per comportamento alimentare in<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 ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Potenziale di membrana Dye Imaging di ventromediale dell&#39;ipotalamo neuroni da topi adulti per studiare glucosio Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Elettrofisiologici Registrazione da<em&gt; Drosophila</em&gt; Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

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.

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Nuovo metodo di analisi per Cold nocicezione in<em&gt; Drosophila</em&gt; Le larve

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

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.

Gusto Preferenza Assay per adulti

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Capitoli in questo video

Metodi per test Drosophila Comportamento

Drosophila adulti olfattiva Shock Learning

Luce Preferenza Assay per studiare Innata e circadiano Photobehavior Regolamentato

A Single-fly saggio per comportamento alimentare in

Potenziale di membrana Dye Imaging di ventromediale dell'ipotalamo neuroni da topi adulti per studiare glucosio Sensing

Elettrofisiologici Registrazione da

Assay Semplice per la quantificazione della Social Prevenzione in

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti

Nuovo metodo di analisi per Cold nocicezione in

High-throughput analisi del comportamento motorio nell'analisi di Isola della drosofila