Gostaríamos de agradecer a Astha Maltare para gerar os dados rastejantes larvas. Gostaríamos de agradecer ao Dr. Nicholas Lanson Jr. para dar seus comentários sobre o manuscrito. Este trabalho foi financiado pela Robert Packard Centro de ALS em Johns Hopkins (a UBP) e da Associação de Esclerose Lateral Amiotrófica (UBP) e R01MH083689 do National Institutes of Mental Health (CDN).

A. Ensaio Crawling larval 1. Coleta de larvas <ol><li> Configure uma garrafa de 8 onças de moscas machos (10-15 + de 10-15 fêmeas). </li><li> Deixe moscas põem ovos para 24 horas, em seguida, garrafa transparente de moscas. (Transfira os adultos em um novo frasco e repetir se necessário). </li><li> Incubar garrafa durante 3-4 dias, ou até larvas de terceiro estádio são visíveis. </li><li> Adicionar 50 - 100 ml de sacarose a 20% para a garrafa com larvas e deixar assentar durante 20 minutos. As larvas irá flutuar para o topo. </li><li> Coletar larvas utilizando uma pipeta de 25 ml sorológico com a ponta cortada, e coloque em uma cesta de malha. </li><li> Lavar as larvas no cesto de malha duas vezes com desionizada H 2 O. As larvas são agora pronto para os experimentos. </li></ol> 2. Para tratar larvas com droga <ol><li> Usar uma escova para transportar o número desejado de larvas para um copo de 5 ml contendo uma solução de sacarose a 5% + droga. </li><li> Deixe alimentam as larvas por pelo menos 15 minutos.</li><li> Verter droga tratada com larvas para um cesto de malha e enxaguar. Eles estão agora prontos para usar. </li></ol> 3. Ensaio Locomotor (medição da distância total percorrida ou contrações da parede do corpo) <ol><li> Usar uma escova para o transporte de larva indivíduo a uma: <ol><li> 15 centímetros placa de Petri contendo agarose a 2% (previamente vertida e deixou-se endurecer) ao longo de papel de gráfico com uma grade de 0,2 cm 2. <ol><li> Contar o número de linhas de grade cruzados em 1 minuto. </li></ol></li><li> Poço de um prato de dissecção de vidro contendo uma solução diluída de pasta de levedura. <ol><li> Conte peristaltismo (contrações anterior completo para posterior movimento de contração = 1) em um minuto, observando sob um microscópio de dissecção. </li></ol></li></ol></li><li> Repita até que os números desejados de larvas foram contadas. </li></ol> B. rápida iterativo geotaxis negativos (ANEL) Protocolo Este ensaio foi originalmente anulaçõesdescrita por Gargano et al 5. <ol><li> Colete recém-emergidas moscas machos adultos sob luz anestesiados CO 2 e coloque em um alimento frasco contendo padrão (ou alimento droga +). </li><li> Manter as moscas à temperatura ambiente (no topo de bancada. ~ 22 ° C) durante 2-3 dias para permitir a recuperação a partir de CO 2 (e acumulação de níveis de estado estacionário droga se for caso disso). </li><li> Transferir cerca de 25 moscas sem anestesia para frascos de poliestireno preparados. </li><li> Montar frascos com moscas dentro do aparelho ANEL (Figura 1). </li><li> Permitir que as moscas se aclimatar ao ambiente, tranquilo, por 15-20 minutos. </li><li> Durante este tempo câmara lugar ~ digital 1 m em frente do aparelho (em uma plataforma, se necessário, para alinhar o centro da lente a meia-altura do frascos), concentrar e ampliar a câmara para o aparelho, e definir um temporizador para 3,0 segundos. </li><li> Com cuidado, tomar posse do aparelho ANEL com a mão esquerda para não disturb as moscas, e mantenha o timer com sua mão direita. </li><li> Acentuadamente toque no aparelho para baixo sobre a superfície da bancada três vezes, assegurando que a torneira é bastante difícil de abater todas as moscas para a parte inferior dos frascos. </li><li> Simultaneamente com a realização do terceiro toque, iniciar a contagem regressiva de 3 segundos. </li><li> Em três segundos tirar uma foto. </li><li> Redefinir o temporizador para 1 minuto e começar. Durante este tempo, redefinir a câmera eo foco sobre o aparelho, e um outro canal do temporizador para três segundos. </li><li> Após 1 minuto, repita os passos 1,7-1,10 </li><li> Após um total de 5-6 ensaios, fazer upload de imagens para um computador e usar o seu visualizador de imagens favorito abrir e marcar a altura média subiu para cada frasco. </li><li> Realização de análise estatística em seus diferentes grupos, comparando a altura média subiu. </li></ol> C. Corte e Ensaio Mating <ol><li> A primeira coisa de manhã, bem claras produtoras de garrafas de flies a ser usado. </li><li> Durante o curso do dia (cada 3-4 horas), recolher recém-emergidas machos sexualmente ingénuos e fêmeas: <ol><li> Coloque machos individualmente em frascos ou tubos com meio. </li><li> Coloque 5-6 fêmeas em conjunto por frasco / tubo. </li><li> Isolar moscas recolhidos a 25 ° C sob 12 hr luz / escuro durante 5 dias. </li><li> Transferir uma fêmea para a câmara de uma roda de acasalamento. </li><li> Transferir um macho para dentro da câmara de uma roda de acasalamento. </li><li> Observe par sob um microscópio de dissecção para os seguintes comportamentos: <ol><li> Orientação (os orienta masculina para a mulher) </li><li> Tapping (o macho bate o sexo feminino) </li><li> Música Wing (o macho estende e vibra uma asa) </li><li> Lamber (os licks do sexo masculino a genitália feminina) </li><li> Curling (o macho enrola seu abdômen debaixo de si mesmo) </li><li> Tentativa de cópula (Curling atividade ao tentar montar a fêmea) </li></ol></li></ol></li><li> Observar durante 10 minutos ou até que a cópula bem sucedida, observando o tempo em que cada comportamento ocorre (latência), o tempo total envolvida em comportamento de corte até cópula (para calcular o índice de corte), bem como o número de pares que realizar com sucesso um determinado comportamento ( freqüência). 100% dos pares de tipo selvagem, em geral acasalar dentro de 5 minutos. </li><li> Calcular um índice de corte (IC) é calculada dividindo o tempo gasto no corte dividida pelo tempo total de até cópula. Para pares de tipo selvagem isso geralmente varia entre 0,6-0,8. </li></ol> D. Representante Resultados Rastejando ensaio Larva tipo normal selvagem vai passear a 3 centímetros por minuto, e exibem 40-50 ~ de contrações da parede do corpo em um minuto. Recentemente, desenvolvemos um modelo de Drosophila FUS / TLS relacionada com esclerose lateral amiotrófica, que mostra defeito rastejando larval, tempo de vida reduzido e adultos escalada deficiência 6. Visamos a expressão of tipo selvagem e formas mutantes de FUS / TLS para os neurônios motores (OK-371-gal4 motorista) e realizou um ensaio de rastreamento larval. Como mostrado abaixo, as larvas do tipo selvagem rastrear até 12 cm, enquanto que a expressão de FUS tipo selvagem diminuiu a capacidade de larvas de rastreamento a cerca de 6 cm. Animais que expressam o R521C mutação ALS-causando em FUS / TLS demonstram uma deterioração muito grave no seu movimento de rastreamento (Figura 1), arrastando apenas cerca de 1 cm / minuto. Ensaio ANEL negativo geotaxis Jovens moscas do tipo selvagem adultos deve ter uma altura média de escalada de ~ 4-5 cm de um período de tempo de 3 segundos (o tempo pode ser ajustado de 3 segundos para acomodar diferentes estirpes ou os níveis de actividade para definir uma altura média dada para uma dada tensão / tratamento). Moscas que permanecem na parte inferior é atribuído um valor de 0. Não é aconselhável a utilização de mais de 25 moscas por frasco porque, em seguida, torna-se difícil determinar a localização de cada indivíduo mosca para medir a altura. Ndessensibilização o tem sido observada em até os 6 ensaios consecutivos espaçados 1 minuto para além de que têm empregado. É crítico para NÃO reutilizar os frascos de polistireno de teste neste ensaio, após os conjuntos de dados iniciais são recolhidas porque as moscas novos colocados em frascos utilizados não irá subir para a mesma extensão como em frascos frescos. <img alt="A Figura 1" src="/files/ftp_upload/3795/3795fig1.jpg" /> Figura 1. Configuração para o Ensaio RING. A câmera digital é colocado ~ m 1 na frente do aparelho contendo moscas nos frascos de poliestireno, foco e zoom da câmera para o aparelho e definir um temporizador para 3,0 segundos. <img alt="A Figura 2" src="/files/ftp_upload/3795/3795fig2.jpg" /> Dados Figura 2. Representativos do ensaio rastejar larval usando moscas ectopicamente expressando UAS-FUS WT, e UAS-FUS R521C sob o controlo de um controlador de neurónio motor (OK371-gal4). <pclass = &quot;jove_content&quot;&gt;

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Comportamento Drosophila é estreitamente regulada por fatores genéticos e ambientais. Nós, e outros, já utilizado nos ensaios aqui descritos para coletar dados para analisar genes relacionados a comportamentos e voar para humanos doenças neurodegenerativas modelados em Drosophila 5-19. Para o ensaio de rastreamento, seleção criteriosa de 3 º ínstar é um passo crítico. Se o tratamento com um medicamento, levará 10-15 minutos (ou mais dependendo do tipo e da natureza da ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Sacarose </td><td> Fisher Scientific </td><td> S5-500 </td></tr><tr><td> Agarose </td><td> Invitrogen </td><td> 16500-500 </td></tr><tr><td> 6 oz garrafa de Drosophila </td><td> Genesee Científico </td><td> 32-130 </td></tr><tr><td> Paint Brush (# 1) </td><td> Ted Pella, Inc. </td><td> 11859 </td></tr><tr><td> Fly componentes dos alimentos </td><td></td><td></td></tr><tr><td> Farinha de milho </td><td> Fisher Scientific </td><td> NC9109741 </td></tr><tr><td> Agar </td><td> Genesee Científico </td><td> 66-104 </td></tr><tr><td> Melaço </td><td> Fisher Scientific </td><td> NC9349176 </td></tr><tr><td> Ácido propiônico </td><td> Acros </td><td> 14930-0010 </td></tr><tr><td> Tegosept </td><td> Ápice </td><td> 20-258 </td></tr><tr><td> Etanol </td><td> PescadorCientífico </td><td> BP2818-4 </td></tr><tr><td> Levedura </td><td> Genesee Científico </td><td> 62-107 </td></tr></tbody></table>

methods to assay drosophila behavior

Drosophila melanogaster, a mosca da fruta, tem sido utilizada para estudar os mecanismos moleculares de uma ampla gama de doenças humanas, tais como o cancro, doença cardiovascular e várias doenças neurológicas 1. Nós otimizamos simples e robusto ensaios comportamentais para determinar a locomoção das larvas, a capacidade de escalar adulto (ensaio ANEL), e os comportamentos de corte de Drosophila. Estes ensaios comportamentais são amplamente aplicáveis ​​para o estudo do papel dos fatores genéticos e ambientais sobre o comportamento da mosca. Capacidade de rastreamento das larvas pode ser confiavelmente utilizado para determinar alterações em início de carreira nas habilidades de rastreamento de larvas de Drosophila e também para examinar efeito de drogas ou de genes de doenças humanas (em moscas transgénicas) em sua locomoção. O ensaio de rastreamento larval torna-se mais aplicável se a expressão ou a supressão de um gene provoca letalidade nas fases de pupa ou adulto, como estas moscas não chegam à idade adulta, onde de outra forma poderia ser avaliado. Esta base umssay também pode ser utilizado em conjunto com luz brilhante ou stress para examinar respostas comportamentais adicionais em larvas de Drosophila. Comportamento de corte tem sido amplamente utilizada para investigar base genética do comportamento sexual, e também pode ser usado para examinar a actividade e coordenação, bem como a aprendizagem ea memória. Drosophila comportamento de corte envolve a troca de vários estímulos sensoriais incluindo visual, auditivo, e os sinais de quimio entre homens e mulheres que levam a uma série complexa de condutas motoras bem caracterizadas, culminando na cópula bem-sucedida. Ensaios adultos tradicionais de escalada (geotaxis negativos) são de trabalho, tedioso intensivo e demorado, com variação significativa entre os diferentes estudos 2-4. O rápido iterativo geotaxis negativos (ANEL) ensaio 5 tem muitas vantagens sobre os protocolos mais amplamente empregadas, oferecendo uma abordagem de transferência reproduzível, sensível e de alta para quantificar locomotor adulto e negativos geotaxis behaviors. No ensaio de RING, vários genótipos ou tratamentos de drogas podem ser testados simultaneamente utilizando grande número de animais, com a abordagem de alto rendimento tornando-o mais favorável para as experiências de rastreio.

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Methods to Assay Drosophila Behavior

Drosophila melanogaster é um sistema modelo de genética e comportamentalmente tratável que tem sido usada para compreender a base molecular e celular de muitos processos biológicos importantes para mais de um século 1. Drosophila tem sido bem explorado para obter insights sobre a base genética do comportamento da mosca.

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 ...

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Methods to Assay Drosophila Behavior

42.6K visualizações.  Louisiana State University Health Sciences Center. Drosophila melanogaster é um sistema modelo de genética e comportamentalmente tratável que tem sido usada para compreender a base molecular e celular de muitos processos biológicos importantes para mais de um século 1. Drosophila tem sido bem explorado para obter insights sobre a base genética do comportamento da mosca.

Vídeo: Methods to Assay Drosophila Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Único Recordings Sensillum na Insetos Drosophila melanogaster E Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

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 ...

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 Ensaio para comportamento de forrageamento em<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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Usando Canais Linear de agarose para o Estudo<em&gt; Drosophila</em&gt; Comportamento larval Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Uma maneira simples para medir alterações na Recompensa comportamento de busca Usando<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Quantificação do comportamento de higienização da Drosophila

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

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.

Análise do elevado-throughput de comportamento Locomotor no ensaio de ilha de Drosophila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Rastreamento do comportamento larval de Drosophila em resposta à estimulação optogenética de neurônios olfativos

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

In vivo Optical cálcio Imaging de aprendizagem induzida por plasticidade sináptica em Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Ensaio mecânico de prevenção de conflitos para medir o comportamento da dor em camundongos

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...