Este trabalho foi financiado pelo National Institutes of Health concede F32 NS078958 (DTB) e R01 AG033620 (BG).

1. Montar vôo Tester <ol><li> Cilindro vôo seguro para Anel Suporte 1 usando grampos cadeia. (Deixe cerca de 3 cm por baixo do cilindro para pesar prato.) (Nota: O cilindro de vôo que usamos é de 90 cm de comprimento, com um diâmetro de 13,5 cm.) </li><li> Insira pesar a cápsula com uma fina camada de óleo mineral por baixo do cilindro de voo. </li><li> Funil Segura para Anel Fique 2, utilizando uma braçadeira de anel e grampo garra. Ajustar a altura do funil de modo a que a parte inferior do funil é alinhada com a parte superior do cilindro de voo. (Nota: o diâmetro da ponta do funil deve ser menor do que o diâmetro exterior dos tubos de ensaio colocados nos tubos de descarga de modo a que os frascos não vai cair.) </li><li> Inserção do tubo de queda para o topo do funil e fixá-lo usando uma braçadeira garra. (Nota: Nós usamos um tubo de queda que é de 25 cm de comprimento Dropping voar contendo frascos dessa altura permite ejeção consistente de todas as moscas com força uniforme O diâmetro interno do tubo de queda deve ser sligh..tly maior do que o diâmetro externo do frasco para permitir que o frasco de cair livremente.) </li><li> Folha de corte (s) de poliacrilamida para o tamanho adequado. (Nota: Para ajudar na colocação e remoção da folha, a largura deve ser ligeiramente menor do que a circunferência interna do cilindro de voo). </li><li> Aplique uma fina camada de Emaranhado-Trap para a folha. Deixe descansar por 1 hora antes de usar. (Nota: Deixe espaço suficiente na parte superior e inferior da folha (aproximadamente 3 cm) não revestido para agarrar a folha para a inserção / remoção.) </li><li> Insira a folha de poliacrilamida no cilindro de vôo. </li><li> Monte a pista câmera usando suportes de pinho. (Nota: garantir que o fundo da pista pode suportar a câmera sem bloquear a lente Consulte a Figura 1B.). </li><li> Adicionar rolhas e parafuso no lugar. (Nota: coloque as rolhas em locais que permitam a câmera para ver toda a folha de plástico no modo panorâmico.) </li></ol> 2. Execute Experiment <ol> &lt;li&gt; Colete frascos de moscas a serem testadas. Para melhores resultados, usar não mais do que 20 moscas / frasco. </li><li> Bata suavemente voa para baixo do frasco, retire e insira em tubo de queda e liberar frasco. (Nota: O frasco cai no tubo de queda até atingir a abertura funil estreito Quando o frasco atinge o funil, as moscas são ejetados para dentro do cilindro de vôo.). </li><li> Levante o tubo de queda para remover o frasco vazio. (Nota: Vários frascos de moscas do mesmo grupo de teste pode ser analisada em uma única folha de poliacrilamida Nós achamos que até 200 moscas (10 frascos de 20 moscas cada) pode ser testada e fotografada prontamente em uma única folha.. </li><li> Retire a folha de plástico e coloque-o sobre uma superfície branca plana. (Nota: cartolina branca pode ser usado se bancadas são de cor escura.) </li><li> Monte a pista câmera sobre a folha de plástico. A câmara deverá ser suficientemente elevado acima da folha ter tanto a parte superior e inferior da folha, no campo de visão. </li><li> Deslize a câmera junto ªe pista, mantendo o botão &quot;capturar&quot; a adquirir uma imagem panorâmica. </li><li> O número de moscas que aterram no óleo podem ser contados manualmente para cada julgamento. </li><li> Repita os passos de 2,2-2,7 para todas as condições de um determinado experimento. As moscas podem ser removidas da folha entre cada ensaio. Alternativamente, pode ser usado várias folhas, com uma nova folha para cada julgamento. </li></ol> 3. Coleta de Dados <ol><li> Abrir arquivos de imagem usando o software ImageJ. </li><li> Corte imagens se necessário incluir apenas a área da superfície de aterragem. (Esta é a área revestida em emaranhado-Trap.) </li><li> Converter imagens de 8-bit em tons de cinza. </li><li> Criar um &quot;Threshold&quot; para filtrar o fundo branco. (Imagem → Ajustar → Threshold). </li><li> Defina os parâmetros para identificar cada mosca utilizando o menu &quot;analisar partículas&quot;. (Analisar → Analisar Partículas) Definir os parâmetros utilizados para identificar uma partícula. Com a nossa criada, nós achamos que utilizar umárea de 5-90 pixels 2 e uma circularidade de 0,4-1,0 irá identificar com precisão todas as amostras. </li><li> Medir a localização de cada mosca utilizando a lista gerada de coordenadas de cada partícula. A coordenada x em pixels pode ser convertida para centímetros para calcular a altura da aterragem. </li><li> Importe a tabela em uma planilha eletrônica (como o Microsoft Excel). </li></ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., 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=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. 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=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

Os autores não têm conflitos de interesse de divulgar.

Usando os métodos descritos aqui, temos sido capazes de avaliar rapidamente o desempenho de vôo de um grande número de mutantes de Drosophila, proporcionando maior eficiência do que era possível anteriormente. Para nossos experimentos, nós rotineiramente separar machos e fêmeas e criá-los em baixa densidade (menos de 20 moscas / frasco) para limitar a agressão que possa danificar asas. Outra consideração importante é controlar adequadamente as diferenças de desempenho de vôo, devido a diferenças ...

Drosophila tem sido utilizado para estudar a base genética de comportamento 1, e os investigadores desenvolveram um número de maneiras para analisar vários tipos de comportamento 2-6. As moscas têm sido particularmente úteis no fornecimento de modelos úteis de distúrbios neuromusculares 7. Um ensaio comum usado para estudar o comportamento locomotor é o desempenho de vôo. O testador vôo original é útil para identificar vôo mutantes defectivos e para a avaliação quantitativa da capacidade voo 1, mas tem várias deficiências que limitam a sua aplicação para as telas de alto rendimento: o uso de cilindros revestidos de petróleo é confuso e complicado, algumas funções, como o comprimento do cilindro e introdução de moscas para dentro do tubo com força variável reduzir a precisão quantitativa e é difícil de recuperar moscas vivo do testador. Para superar essas limitações, nós modificamos o testador vôo para incluir uma série de melhorias. Nós adicionamos uma &quot;gota tuser &quot;a introdução de moscas para eliminar a variabilidade entre experiências e os utilizadores. Usamos folhas de acrílico removível revestido com um adesivo que permite uma mais fácil limpeza e recuperação de moscas individuais. Temos aumentado o comprimento do tubo de voo para melhorar a exactidão quantitativa e fiabilidade. Finalmente , usamos uma câmera digital e software de imagem para calcular as alturas de pouso de moscas. Acreditamos que essas melhorias serão úteis para qualquer laboratório interessado na realização de telas genéticos de larga escala por defeitos no desempenho de vôo.

<table><tbody><tr><td>Putty knife</td><td>Home Depot</td><td>630147</td><td>&lt;a href=&quot;http://www.homedepot.com&quot;&gt;www.homedepot.com&lt;/a&gt;</td></tr><tr><td>Pine back band moulding (2x)</td><td>Home Depot</td><td>156469</td><td>&lt;a href=&quot;http://www.homedepot.com&quot;&gt;www.homedepot.com&lt;/a&gt;</td></tr><tr><td>Furring Strip Board</td><td>Home Depot</td><td>164704</td><td>&lt;a href=&quot;http://www.homedepot.com&quot;&gt;www.homedepot.com&lt;/a&gt;</td></tr><tr><td>Tangle-Trap Insect Trap Coating</td><td>BioControl Network</td><td>268941</td><td>&lt;a href=&quot;http://www.biconet.com&quot;&gt;www.biconet.com&lt;/a&gt;</td></tr><tr><td>Laptop Computer&amp;nbsp;</td><td>Apple</td><td></td><td>&lt;a href=&quot;http://www.apple.com/mac/&quot;&gt;www.apple.com/mac/&lt;/a&gt;</td></tr><tr><td>Mineral oil</td><td>Fisher Scientific</td><td>BP26291</td><td>&lt;a href=&quot;http://www.fishersci.com&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>White poster board</td><td>Staples</td><td>247403</td><td>&lt;a href=&quot;http://www.staples.com&quot;&gt;www.staples.com&lt;/a&gt;</td></tr><tr><td>Polystyrene weighing dish</td><td>Fisher Scientific</td><td>S67091A</td><td>&lt;a href=&quot;http://www.fishersci.com&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>ImageJ Software</td><td>National Institutes of Health</td><td></td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij/&quot;&gt;http://rsb.info.nih.gov/ij/&lt;/a&gt;</td></tr><tr><td>Digital camera</td><td>Sony</td><td>DSC-TX7</td><td>&lt;a href=&quot;http://www.store.sony.com&quot;&gt;www.store.sony.com&lt;/a&gt;</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td></td><td>&lt;a href=&quot;http://www.finescience.com&quot;&gt;www.finescience.com&lt;/a&gt;</td></tr><tr><td>Polycarbonate cylinder (drop tube)</td><td>McMaster-Carr</td><td>8585K62</td><td>&lt;a href=&quot;http://www.mcmaster.com&quot;&gt;www.mcmaster.com&lt;/a&gt;</td></tr><tr><td>Flight cylinder (acrylic)</td><td>McMaster-Carr</td><td>8486K943</td><td>&lt;a href=&quot;http://www.mcmaster.com&quot;&gt;www.mcmaster.com&lt;/a&gt;</td></tr><tr><td>Polycarbonate sheets</td><td>McMaster-Carr</td><td>85585K25</td><td>&lt;a href=&quot;http://www.mcmaster.com&quot;&gt;www.mcmaster.com&lt;/a&gt;</td></tr><tr><td>ring stand (2x)</td><td>Fisher Scientific</td><td>S47808</td><td>&lt;a href=&quot;http://www.fishersci.com&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>Ring support</td><td>Fisher Scientific</td><td>S47791</td><td>&lt;a href=&quot;http://www.fishersci.com&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>Three-prong extension clamps (x2)</td><td>Fisher Scientific</td><td>05-769-7Q</td><td>&lt;a href=&quot;http://www.fishersci.com&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>Funnel</td><td>Fisher Scientific</td><td>10-500-3</td><td>&lt;a href=&quot;http://www.fishersci.com&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>chain clamps (2x)</td><td>VWR</td><td>21573-275</td><td>&lt;a href=&quot;http://www.vwr.com&quot;&gt;www.vwr.com&lt;/a&gt;</td></tr><tr><td>Glass vials</td><td>VWR</td><td>66020-198</td><td>&lt;a href=&quot;http://www.vwr.com/&quot;&gt;www.vwr.com&lt;/a&gt;</td></tr></tbody></table>

an improved method for accurate and rapid measurement of flight performance in drosophila

Drosophila provou ser um sistema modelo útil para a análise de comportamento, incluindo o vôo. O testador vôo caindo envolvido inicial voa em uma revestido a óleo cilindro graduado; altura de desembarque prevista uma medida de desempenho de vôo, avaliando como moscas longe vai cair antes de produzir o suficiente impulso para fazer contato com a parede do cilindro. Aqui nós descrevemos uma versão atualizada do testador de vôo com quatro grandes melhorias. Primeiro, nós adicionamos um &quot;tubo de queda&quot; para garantir que todas as moscas entrar no cilindro de vôo a uma velocidade semelhante entre ensaios, eliminando a variabilidade entre os usuários. Em segundo lugar, substituiu o revestimento de óleo com folhas de plástico removíveis revestidas em Emaranhado-Trap, um adesivo projetado para capturar insetos vivos. Em terceiro lugar, nós usamos um cilindro de mais tempo para permitir a discriminação mais precisa da capacidade de vôo. Em quarto lugar, usamos uma câmera digital e software de imagem para automatizar a marcação de desempenho de vôo. Estas melhorias permitem o rapid, avaliação quantitativa do comportamento de vôo, útil para grandes conjuntos de dados e telas genéticos de larga escala.

<html> Figura 1A mostra um esquema da montagem testador vôo atualizado. Figura 1B ilustra o projeto de trilha permitem a câmera para tirar uma imagem panorâmica sem bloquear o campo de visão. Os resultados representativos são mostrados na Figura 2, em que o desempenho de voo de moscas mutantes Slowpoke, que têm um voo conhecidos defeito 8-10, são comparados com o tipo selvagem Canton-S voa. Controle voa consistentemente pousar perto do...

Watch this Scientific Journal Video about An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila at JoVE.com

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Aqui nós descrevemos um método para a medição rápida e precisa do desempenho de vôo em Drosophila, permitindo seleção de alto rendimento.

Um método melhorado para rápido e preciso de medição de desempenho em vôo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

an-improved-method-for-accurate-rapid-measurement-flight-performance

Research

JoVE Journal

Biology

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

11.8K visualizações.  University of Wisconsin-Madison.  Aqui nós descrevemos um método para a medição rápida e precisa do desempenho de vôo em Drosophila, permitindo seleção de alto rendimento. 

Vídeo: An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

A TreadWheel: Protocolo de treinamento de intervalo para suavemente induzido por exercício em Drosophila melanogaster.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Genética

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

Um ensaio automatizado rápida iterativo Geotaxis negativo para analisar o comportamento de adultos de escalada em um modelo de drosófila de neurodegeneração

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Comportamento

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

De alta resolução de vídeo de Rastreamento Locomotion em Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Biologia

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.

Métodos de Ensaio para 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 ...

Neurociência

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Um método automatizado para determinar o desempenho de Drosophila em resposta às mudanças de temperatura no espaço e no tempo

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

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 ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Um método de baixo custo para análise de Apreensão-como atividade e movimento em<em&gt; Drosophila</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Uma técnica simples para avaliar a atividade locomotora em Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

A medição da taxa metabólica em<em&gt; Drosophila</em&gt; Usando respirometria

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Medição da Actividade larval no<em&gt; Drosophila</em&gt; Monitor de Atividade

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Um método melhorado para rápido e preciso de medição de desempenho em vôo

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