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. equipamentos e reagentes criados em casa (Veja a lista de material para os outros) <ol><li> Prepare um aparelho de anestesia Drosophila frio para realizar o trabalho real. <ol><li> Cortar uma folha de polietileno poroso para cobrir uma pequena caixa de plástico (12.7 cm de comprimento, 10,2 cm de largura), normalmente a tampa superior de uma caixa de pontas de pipeta (12.7 cm de comprimento, 10,2 cm de largura, 3,8 cm de profundidade). </li><li> Preencha a caixa com gelo picado, cubra com a folha de polietileno poroso. </li></ol></li><li> Prepare um aparelho T-maze; adaptar o aparelho anteriormente descrito em detalhes 28-31. </li><li> Preparar um aspirador de mosca como descrito 32. </li><li> Prepare um aparelho de resposta phototaxis para deslocar moscas dentro e fora de frascos de teste sem estresse: <ol><li> Usar um aparelho em contracorrente, tal como descrito por 29,33. </li></ol></li></ol> NOTA: O labirinto em T pode também ser utilizado para outros fins, como descrito por 28. <ol start="5"><li> Certifique-homcondições de iluminação ogeneous: realizar o experimento em um banco coberto com uma capa de banco branco, e na frente de um quadro branco. NOTA: Para estas experiências, as linhagens de moscas utilizados foram Drosophila melanogaster tipos selvagens: Canton-S, Oregon e Samarkand voa de nossos estoques de laboratório 34; Elwood moscas foram coletadas no outono de 2011 no bairro de Elwood de Huntington, em Long Island, Nova York, EUA. </li></ol> 2. Preparar as moscas antes do experimento 1-2 dias antes de realizar o experimento: <ol><li> Manter moscas em meio padrão fubá-agar-melaço de levedura a 25 ° C em um ciclo claro / escuro de 12 horas de luz. </li><li> Recolher as moscas que respondem sob anestesia frio 1-2 dias antes de se efectuar a experiência <ol><li> Transferem-se as moscas das garrafas no qual foram colocadas em tubos Falcon de 50 ml, utilizando um funil. </li><li> Coloque o tubo de falcão com as moscas sob nível de gelo, em umbalde de gelo isolado. </li><li> Coloque o aparelho de anestesia fria com gelo picado a -4 ° C para relaxar. </li><li> Espere 5 minutos para as moscas para entrar em um coma frio. </li><li> Transferem-se as moscas para a folha de polietileno poroso do aparelho de anestesia frio. </li><li> Colete as moscas de resposta sobre o aparelho de anestesia frio. No RT, sob microscópio estereoscópico, separe masculina 3-7 dias de idade de moscas fêmeas e manter essas moscas em grupos de 40. </li></ol></li><li> Recolher as moscas emissor 1-2 dias antes de realizar a experiência. <ol><li> Gentilmente recolher Canton-S mixed-sexo voa de suas garrafas, usando um aspirador de boca, controlando o seu número. </li><li> Manter essas moscas no grupo de 60-100, pois eles serão os emissores. </li><li> Preparar todas as amostras de emissores como pontos de dados necessários. </li></ol></li><li> Permitir que pelo menos uma noite de recuperação após a coleta dos respondentes e emissores, para minimizar qualquer efeito de confusão da anestesia frioe coleta de boca aspirador no comportamento. </li><li> Abrir o saco de frascos de teste, para permitir que o ar com cheiro plástico obsoleto em que o saco seja substituído por ar fresco, pelo menos 1 dia antes da experiência. </li></ol> 2 horas antes da realização do ensaio: <ol start="6"><li> Certifique-se de que a temperatura na sala em que a experiência é realizada é de cerca de 23-25 ​​° C, com luz uniforme, e humidade superior a 30%. </li><li> Transfira os respondedores e moscas emissores de frutas em frascos de alimentos frescos para garantir que estas moscas não estão morrendo de fome. </li><li> Deixe que as moscas de fruta ajustar ao ambiente, durante 2 horas, no banco em que o experimento será realizado. </li></ol> 3. a realização do experimento evitação social <ol><li> Executar cada experiência à mesma hora do dia, num intervalo de 3-4 horas, à tarde entre o tempo Zeitgeber ZT5 e ZT9. </li><li> Coloque o T-maze no teclado batendo, e aperte o parafuso de modo clipe tele é estável elevador. </li><li> Transfira as moscas de resposta em um tubo de ensaio fresco. Encaixe este frasco contendo as moscas de resposta sobre a parte superior do labirinto-T. </li><li> Inclinar o aparelho T-maze e coloque-a sobre o tapete batendo tão moscas da fruta cair no elevador. </li><li> Mover a parte superior do elevador para baixo para que as moscas são entre a secção superior e inferior do labirinto em T, mas prestar atenção para manter o elevador acima do ponto de escolha - para impedir que as moscas de escapar. </li><li> Obter frascos com DSO (ver seção 5 para gerar DSO-free frascos que foram anteriormente ocupadas por moscas). <ol><li> Enquanto as moscas de resposta estão se ajustando a esse novo ambiente, coloque as moscas de emissor em um tubo de ensaio. </li><li> Coloque um pedaço de algodão para fechar o frasco de modo que as moscas de fruta não escapam. </li><li> Mecanicamente isto agitar o frasco numa mini-vórtice do seguinte modo: em vórtice durante 15 seg, remover o frasco de vórtice durante 5 segundos. Repita 3 vezes para um total de 55 seg. </li><li> Remove as moscas emissor para fora do frasco fresco - por transferência num frasco de alimentos - e rapidamente colocar esse frasco cheio DSO em um dos dois lados do aparelho de labirinto-T. </li><li> Alterne o lado de colocação para o frasco DSO preenchidos em cada nova execução. </li></ol></li><li> Coloque um tubo de ensaio fresco no outro lado do labirinto-T. </li><li> Traga o elevador completamente para baixo, de modo que as moscas de fruta pode escolher entre o tubo de ensaio fresco eo tubo de ensaio com DSO, e iniciar o timer. </li><li> Deixe as moscas escolher entre o frasco fresco e o frasco DSO, por 1 min, salvo indicação em contrário. </li><li> Após 1 min, mover o elevador para cima para separar as moscas no frasco fresco, DSO frasco, e os que estão presos no elevador. </li><li> Contar o número de moscas em cada frasco, e no elevador. </li><li> Repita esse procedimento para cada genótipo / condição. </li></ol> 4. Analisando os dados evitação social <ol><li> Contar o número de moscas da fruta em cada frasco para cada Genotipo, e transferir os dados para uma planilha. </li><li> Usando um programa de planilha, calcular o Índice de Desempenho (PI) para cada genótipo, subtraindo o número de moscas no frasco DSO do número de moscas no frasco DSO-livre, e dividindo pelo número total de moscas. </li><li> Compare dos IPs que consiste em utilizar o software de análise estatística para análise dos resultados. <ol><li> Idealmente, preparar 2-3 repetições internas de cada experiência, e três repetições independentes, realizadas em dias diferentes, com diferentes garrafas ou 30 atravessa. </li><li> Use testes estatísticos de distribuição de Gauss, como os dados seguem uma distribuição normal. </li><li> Ao testar um efeito (isto é, densidade - Figura 2), utilizar um ANOVA de uma via, com o pós-teste de comparação múltipla (coluna por coluna), ou um t-teste simples para comparar cada resultado de um controlo. </li><li> Quando experimentos do grupo são realizadas, testar ambas as condições e sexos (frascos vazios, destacou umd moscas un-esforçado; e efeito de gênero - Figura 1), usando uma ANOVA de duas vias. </li></ol></li></ol> 5. Gerar frascos anteriormente ocupado por Flies átonas - Realizar Em vez de 3,5-3,6 <ol><li> Use fototaxia para preparar frascos ocupados por moscas que não têm sido destacadas, como um controlo. </li><li> Transfira as moscas em um tubo de ensaio, e encaixe-o aparelho em contracorrente, com frescos frascos vazios no local oposto. </li><li> Coloque o aparelho na horizontal, com o frasco distal diretamente em frente a 15 W fluorescente luz cool-branco, e cubra com um pano preto. </li><li> Deixe as moscas mover suavemente no frasco fresco. Isso pode levar vários minutos. Uma vez que a maioria das moscas estão caminhando em direção à luz no frasco distal, iniciar o temporizador para 1 min. </li><li> Remova cuidadosamente o pano preto, mudar a localização da luz, recuperar com o pano preto, e permitir que as moscas de deixar o tubo de ensaio. </li><li> Naquela época, eunitiate o experimento escolha como em 3.2 a 3.5. Ir 3.6 e 3.7. </li><li> Mover o cursor do aparelho, para separar as moscas que desocupado o tubo de ensaio a partir do outro lado. </li><li> Use o frasco teste desocupado na T-labirinto de imediato para o experimento escolha, como em 3.7. </li></ol>

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The authors declare that they have no competing financial interests.

Este protocolo descreve um procedimento detalhado para o ensaio de evitação social. Canton-S só vai evitar um frasco em que voa tenham sido previamente stress mecânico, e que o sexo e número de respondentes que não afeta o desempenho evitação social. No entanto, a base genética dos respondentes tem uma grande influência. Seguem-se vários passos críticos para a realização deste experimento com sucesso: 1) sempre transferir as moscas 2 horas antes do experimento e certifique-se d...

O objetivo deste método é quantificar facilmente um novo aspecto do comportamento social simples em Drosophila melanogaster, independente do namoro e agressão. As interações sociais são cruciais para o bom desenvolvimento e saúde dos indivíduos dentro de uma sociedade, bem como a funcionalidade de um grupo social como um todo. A elevada complexidade destas interacções exige grandes tamanhos de amostra e um sistema que permite a simplificação do comportamento como as bases genéticas e neurais do comportamento social são ainda pouco compreendidos. Drosophila melanogaster é um poderoso modelo genético que pode ser usada para identificar os factores genéticos e bases neurais de interações sociais. Com efeito, D. melanogaster tem um repertório de comportamentos sociais complexos e algumas medições diretas de socialização já foram feitas 1-7. No entanto, a maioria desses esforços têm sido focados em comportamentos sociais relativamente complexas, como aggresinterações sive 3,6, vários aspectos do namoro 3,8-12, e como experiência social afeta outros comportamentos como a aprendizagem, ou ritmo circadiano 13-17. Além disso, muitos desses ensaios dependem de analisar os padrões de interacção complexas de grupos de moscas, usando o acompanhamento de vídeo e software de computador para analisar a abundância resultante de dados. Essas análises são de valor inestimável, e levar a novos conhecimentos importantes, como a dinâmica das interações fly-fly em grupos de sete. Uma limitação, no entanto, é a inacessibilidade destes ensaios para a comunidade em geral, e o limitado conhecimento dos mecanismos subjacentes reconhecimento dos outros. Em outras palavras, com base na emissão de um sinal por um indivíduo e o seu reconhecimento por outro ainda está mal compreendida 18. Em contraste, as moscas também exibem um comportamento simples, evitação social, onde os indivíduos se afastar de um sinal emitido por moscas estressados: o D. Melanogaster Estresse Odorant ou DSO 19. Em um ensaio de elevado débito, este comportamento pode ser quantificada como a prevenção de um sinal de tensão emitida por outros, moscas ou evitação social 19. As moscas são colocadas num aparelho de labirinto em T e dada a opção de evitar um frasco contendo DSO. Utilizando este ensaio, CO 2 mostrou ser um componente do DSO, e parte do circuito neural necessário para responder ao CO 2 foi dissecada 19. O ensaio de evitação social aqui apresentada é semelhante conceitualmente para os ensaios comportamentais simples desenvolvido no laboratório de Seymour Benzer que permitiram gerações de pesquisadores para dissecar comportamentos complexos 20. Análise de evitação social pode ser levada a cabo de forma eficaz em termos de custos, utilizando o ensaio de labirinto em T, para permitir um estudo mais generalizada do comportamento social. Por exemplo, com este ensaio que recentemente demonstrou que diferentes riscos genéticos para autismo têm efeitos contrastantes em b sociaisensaios ehavior. Mutantes para um gene candidato para o autismo - neurobeachin 21,22 - deficiências presentes tanto no espaço social (descritos em outros 23) e evitação social 24. Sinalização dopaminérgica anormal também é proposto para desempenhar um papel na etiologia de autismo em humanos 25,26. Em contraste com os resultados obtidos com neurobeachin, descobrimos que o desempenho evitação social não foi afectada por um aumento ou diminuição dos níveis de Drosophila vesicular da monoamina transportador (VMAT) em células dopaminérgicas, embora espaço social foi directamente correlacionada com estes níveis de VMAT 27. Os resultados contrastantes obtidos com neurobeachin e VMAT ressaltam a possibilidade de identificar várias formas de comportamento anti-social e, assim, os diferentes circuitos neurais subjacentes modulando a resposta para os outros.

<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.

O ensaio de evitação social é um ensaio robusto quantificar a capacidade de Drosophila melanogaster para reconhecer um sinal de tensão (DSO) emitidos por outras moscas, e avaliando assim um aspecto de interacções sociais. O ensaio é realizado utilizando um aparelho comumente usado em vários ensaios de comportamento conhecido como T-labirinto, que apresentam as moscas com uma escolha entre duas opções diferentes - a esquerda ou direita 19,28-31. Neste caso, a eficiência com que as moscas ev...

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.

Ensaio simples para Quantificação de Prevenção Social<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 visualizações.  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.

Vídeo: 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 emparelhado e Eletrofisiologia Ensaio para a tela de bioatividade dos compostos utilizando os Drosophila melanogaster Sistema Fibra Gigante

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 ...

Neurociência

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.

Optogenética estimulação do comportamento de fuga em<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;

Determinação da atividade locomotora espontânea em<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.

Condições que afetam Espaço Social em<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.

De alta resolução Quantificação do Comportamento Odor-guiada em<em&gt; Drosophila melanogaster</em&gt; Usando a<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.

Clones em um ensaio comportamental para Mechanosensation de MarcM baseada em<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 Ensaio para examinar Preferências de postura de Pessoa<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.

Forrageamento Protocolo Caminho de comprimento para<em&gt; Drosophila melanogaster</em&gt; Larvas

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.

O capilar alimentador ensaio mede ingestão alimentar em<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.

Desenvolvimento de ensaio para quantificação de conteúdo elevado de Sod1 proteína mutante formação agregada em células vivas

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