Name: a simple technique to assay locomotor activity in drosophila
Uploaded: 2023-02-24T14:00:00
Description: Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

Este trabalho foi apoiado por um fundo de lançamento especial da Universidade Soochow e da Fundação Nacional de Ciência da China (NSFC) (82171414). Agradecemos aos membros do laboratório do Prof. Chunfeng Liu por sua discussão e comentários.

Foram utilizadas 1118 moscas adultas W 1118 e larvas de terceiro ínstar para o presente estudo.
1. Preparação experimental
NOTA: Uma arena de campo aberto para rastreamento de locomoção de Drosophila é feita com sílica gel incolor e inodora.
<ol><li>Misture o reagente A e o reagente B na proporção de 1:10, de acordo com as instruções do fabricante para o kit de sílica (consulte a Tabela de Materiais...

<ol>
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M., 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=Automated+real-time+quantification+of+group+locomotor+activity+in+Drosophila+melanogaster.">Automated real-time quantification of group locomotor activity in Drosophila melanogaster.</a> Scientific Reports. 9 (1), 4427 (2019).</li><li>Werkhoven, Z., Rohrsen, C., Qin, C., Brembs, B., de Bivort, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MARGO+(Massively+Automated+Real-time+GUI+for+Object-tracking),+a+platform+for+high-throughput+ethology.">MARGO (Massively Automated Real-time GUI for Object-tracking), a platform for high-throughput ethology.</a> PLoS One. 14 (11), e0224243 (2019).</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> Experimental Gerontology. 40 (5), 386-395 (2005).</li><li>Cichewicz, K., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ShinyR-DAM:+a+program+analyzing+Drosophila+activity,+sleep+and+circadian+rhythms.">ShinyR-DAM: a program analyzing Drosophila activity, sleep and circadian rhythms.</a> Communications Biology. 1, 25 (2018).</li><li>McParland, A. L., Follansbee, T. L., Ganter, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+larval+activity+in+the+Drosophila+activity+monitor.">Measurement of larval activity in the Drosophila activity monitor.</a> Journal of Visualized Experiments. 98, e52684 (2015).</li><li>Walter, T., Couzin, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRex,+a+fast+multi-animal+tracking+system+with+markerless+identification,+and+2D+estimation+of+posture+and+visual+fields.">TRex, a fast multi-animal tracking system with markerless identification, and 2D estimation of posture and visual fields.</a> eLife. 10, (2021).</li><li>Maia Chagas, A., Prieto-Godino, L. L., Arrenberg, A. B., Baden, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#8364;100+lab:+A+3D-printable+open-source+platform+for+fluorescence+microscopy,+optogenetics,+and+accurate+temperature+control+during+behaviour+of+zebrafish,+Drosophila,+and+Caenorhabditis+elegans.">The &#8364;100 lab: A 3D-printable open-source platform for fluorescence microscopy, optogenetics, and accurate temperature control during behaviour of zebrafish, Drosophila, and Caenorhabditis elegans.</a> PLoS Biology. 15 (7), e2002702 (2017).</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> Journal of Visualized Experiments. (61), (2012).</li><li>Xiao, G. <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+the+behavior+of+Drosophila+melanogaster+for+toxicity+study.">Methods to assay the behavior of Drosophila melanogaster for toxicity study.</a> Methods in Molecular Biology. 2326, 47-54 (2021).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Johnson, M. E., Bobrovskaya, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+the+rotenone+models+of+Parkinson's+disease:+their+ability+to+reproduce+the+features+of+clinical+disease+and+model+gene-environment+interactions.">An update on the rotenone models of Parkinson's disease: their ability to reproduce the features of clinical disease and model gene-environment interactions.</a> Neurotoxicology. 46, 101-116 (2015).</li><li>Coulom, H., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+rotenone+models+sporadic+Parkinson's+disease+in+Drosophila+melanogaster.">Chronic exposure to rotenone models sporadic Parkinson's disease in Drosophila melanogaster.</a> The Journal of Neuroscience. 24 (48), 10993-10998 (2004).</li><li>Kumar, P. P., Bawani, S. S., Anandhi, D. U., Prashanth, K. V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotenone+mediated+developmental+toxicity+in+Drosophila+melanogaster.">Rotenone mediated developmental toxicity in Drosophila melanogaster.</a> Environmental Toxicology and Pharmacology. 93, 103892 (2022).</li><li>Gulyas, M., Bencsik, N., Pusztai, S., Liliom, H., Schlett, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AnimalTracker:+an+ImageJ-based+tracking+API+to+create+a+customized+behaviour+analyser+program.">AnimalTracker: an ImageJ-based tracking API to create a customized behaviour analyser program.</a> Neuroinformatics. 14 (4), 479-481 (2016).</li><li>Qu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EasyFlyTracker:+a+simple+video+tracking+Python+package+for+analyzing+adult+Drosophila+locomotor+and+sleep+activity+to+facilitate+revealing+the+effect+of+psychiatric+drugs.">EasyFlyTracker: a simple video tracking Python package for analyzing adult Drosophila locomotor and sleep activity to facilitate revealing the effect of psychiatric drugs.</a> Frontiers in Behavioral Neuroscience. 15, 809665 (2022).</li><li>Yarwais, Z. H., Najmalddin, H. O., Omar, Z. J., Mohammed, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+data+collection+of+Drosophila+movement+behaviour+assays+using+computer+vision+in+Python.">Automated data collection of Drosophila movement behaviour assays using computer vision in Python.</a> International Journal of Innovative Approaches in Science Research. 4 (1), 15-22 (2020).</li></ol>

Projetamos um método, baseado no material de código aberto AnimalTracker API compatível com o programa de processamento de imagens de Fiji, que pode permitir que os pesquisadores avaliem sistematicamente a atividade locomotora rastreando larvas adultas e individuais. O AnimalTracke é uma ferramenta escrita em Java que pode ser facilmente integrada a bancos de dados existentes ou outras ferramentas para facilitar a análise do comportamento de rastreamento de animais projetado por aplicativos<sup cla...

Drosophila melanogaster fornece um excelente organismo modelo para investigar funções celulares e moleculares em modelos de doenças neuronais criadas por modificação gênica1, tratamento medicamentoso2 e senescência3. A alta conservação de vias biológicas, propriedades físicas e genes homólogos associados a doenças entre humanos e Drosophila torna a mosca-da-fruta uma mimética ideal desde o nível molecular até o comportamental4. Em muitos modelos de doenças, a deficiência comportamental é um índice importante, fornecendo um modelo útil para várias neuropatias humanas 5,6. Atualmente, a drosófila é utilizada no estudo de múltiplas doenças humanas, do neurodesenvolvimento e de doenças neurodegenerativas, como a doença de Parkinson e a esclerose lateral amiotrófica7,8. A detecção da habilidade motora dos modelos de doença é crucial para a compreensão do progresso patogênico e pode fornecer uma correlação fenotípica com os mecanismos moleculares subjacentes ao processo da doença.
Recentemente, ferramentas de software disponíveis comercialmente e programas de baixo custo têm sido desenvolvidos para estratégias de detecção locomotora de Drosophila, tais como testes de alto rendimento em moscas agrupadas9,10 e mensuração da locomoção em tempo real11,12. Uma dessas abordagens convencionais é a geotáxi negativa interativa rápida (RING), também chamada de ensaio de escalada, que inclui múltiplos canais que permitem conter uma grande população de moscas com o mesmo sexo e idade, reduzindo a variação durante a coleta de dados 9,13. Outro método de pré-teste para análise do comportamento locomotor é o TriKinetics Drosophila activity monitor (DAM), um dispositivo que utiliza múltiplos feixes para detectar o movimento da atividade das moscas dentro de um tubo de vidro fino14. O aparelho registra a posição continuamente, o que representa a locomoção automatizada por meio do cálculo dos cruzamentos de feixes para estudar a atividade e o ritmo circadiano das moscas em um período maior de tempo15. Embora esses métodos tenham sido amplamente utilizados na análise de defeitos comportamentais em moscas-das-frutas para determinar mudanças na locomoção comportamental, eles sempre requerem equipamentos especiais de teste ou processos complexos de análise, e restringem sua aplicação em alguns modelos com um dispositivo limitado e simples. Estratégias baseadas em grupos de rastreamento animal para testar a Drosophila adulta, como o FlyGrAM11 e o ensaio da ilha de Drosophila 10, implementam o recrutamento social e o rastreamento individual em uma área predefinida. No entanto, a restrição social individual em áreas desafiadas pode ter um efeito negativo sobre as identificações nas imagens, causadas pela colisão ou sobreposição de moscas. Mesmo que alguns métodos baseados em materiais de código aberto, como TRex16, MARGO 12 e FlyPi17, tenham uma emergência, eles podem rastrear rapidamente as moscas com uso flexível em testes comportamentais. Essas abordagens de teste estão associadas a elaboradas instalações de aparelhos experimentais, requisitos especiais de software ou linguagens de computador profissionais. Para as larvas, a mensuração da distância total percorrida ao longo do número de linhas de borda da grade por unidade de tempo18, ou a contagem aproximada das contrações da parede corporal para os indivíduos manualmente19, são os métodos predominantes para avaliar sua capacidade locomotora. Devido à falta de precisão nos equipamentos ou dispositivos e métodos de análise, algumas locomoções comportamentais das larvas podem escapar à detecção, dificultando a avaliação precisa do movimento comportamental, especialmente o movimentofino15.
O presente método desenvolvido utiliza a interface de programação de aplicativos (API) AnimalTracker , compatível com o programa de processamento de imagens Fiji (ImageJ), para avaliar sistematicamente a atividade locomotora de moscas adultas e larvais, analisando seu comportamento de rastreamento a partir de vídeos de alta definição (HD). Fiji é um software de código aberto ImageJ distribuição que pode combinar bibliotecas de software robustas com inúmeras linguagens de script, resultando em prototipagem rápida de algoritmos de processamento de imagem, tornando-o popular entre os biólogos por suas capacidades de análise de imagem20. Na abordagem atual, a integração de Fiji na API AnimalTracker é explorada para desenvolver um ensaio comportamental exclusivo de Drosophila com inserção de algoritmo personalizado, e fornece uma etapa útil para documentação detalhada e tutoriais para apoiar capacidades analíticas robustas do comportamento locomotor (Figura 1). Para contornar a complicação de identificações objetivas nas imagens causadas pela colisão ou sobreposição de moscas, cada arena é restrita a hospedar apenas uma mosca. Ao avaliar a precisão do rastreamento da abordagem, implementou-se o rastreamento e quantificação dos movimentos locomotores de Drosophila que foram administrados com a droga tóxica rotenona, que geralmente é usada para modelos animais da doença de Parkinson, descobrindo o comprometimento da locomoção no tratamento medicamentoso21. Esta metodologia, que emprega software livre e de código aberto, não necessita de instrumentação de alto custo, podendo analisar de forma precisa e reprodutível a locomoção comportamental da Drosophila .

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal tracker</td>
			<td>Hungarian Brain Research Program</td>
			<td>version: 1.7</td>
			<td>pfficial website: http://animaltracker.elte.hu/main/downloads</td>
		</tr>
		<tr>
			<td>Camera software</td>
			<td>Microsoft</td>
			<td>version: 2021.105.10.0</td>
			<td>built-in windows 10 system</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>DELL</td>
			<td>Vostro-14-5480</td>
			<td>a comupter running win 10 system is available</td>
		</tr>
		<tr>
			<td>Drosophila carbon dioxide anesthesia workstation</td>
			<td>Wu han Yihong technology</td>
			<td>#YHDFPCO2-018</td>
			<td>official website: http://www.yhkjwh.com/</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>Fiji team</td>
			<td>version: 1.53v</td>
			<td>official website: https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Format factory software</td>
			<td>Pcfreetime</td>
			<td>version: X64 5.4.5</td>
			<td>official website: http://www.pcfreetime.com/formatfactory/CN/index.html</td>
		</tr>
		<tr>
			<td>Graph pad prism</td>
			<td>GraphPad Software</td>
			<td>version: 8.0.2</td>
			<td>official website: https://www.graphpad-prism.cn</td>
		</tr>
		<tr>
			<td>Hight definition camera</td>
			<td>TTQ</td>
			<td>Jingwang2 (HD1080P F1.6 6-60mm)</td>
			<td>official website: http://www.ttq100.com/product_show.php?id=35</td>
		</tr>
		<tr>
			<td>Office software</td>
			<td>Microsoft</td>
			<td>version: office 2019</td>
			<td>official website: https://www.microsoftstore.com.cn/software/office</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Bkman</td>
			<td>110301003</td>
			<td>size: 60 mm</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>DOW</td>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Mix well according to the instructions</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Macklin</td>
			<td>#144-55-8</td>
			<td>Mix well with silica gel</td>
		</tr>
	</tbody>
</table>

a simple technique to assay locomotor activity in drosophila

Drosophila melanogaster é um organismo modelo ideal para o estudo de várias doenças devido à sua abundância de técnicas avançadas de manipulação genética e diversas características comportamentais. A identificação de deficiência comportamental em modelos animais é uma medida crucial da gravidade da doença, por exemplo, em doenças neurodegenerativas, onde os pacientes frequentemente experimentam prejuízos na função motora. No entanto, com a disponibilidade de vários sistemas para rastrear e avaliar déficits motores em modelos de moscas, como indivíduos tratados com drogas ou transgênicos, ainda falta um sistema econômico e fácil de usar para avaliação precisa de múltiplos ângulos. Desenvolve-se aqui um método baseado na interface de programação de aplicativos (API) AnimalTracker , compatível com o programa de processamento de imagens de Fiji, para avaliar sistematicamente as atividades de movimento de indivíduos adultos e larvais a partir de vídeos gravados, permitindo assim a análise de seu comportamento de rastreamento. Este método requer apenas uma câmera de alta definição e uma integração de hardware periférico de computador para gravar e analisar o comportamento, tornando-se uma abordagem acessível e eficaz para a triagem de modelos de moscas com deficiências comportamentais transgênicas ou ambientais. Exemplos de testes comportamentais usando moscas tratadas farmacologicamente são dados para mostrar como as técnicas podem detectar mudanças comportamentais em moscas adultas e larvas de maneira altamente repetível.

No presente estudo, déficits locomotores em moscas adultas e larvas de terceiro ínstar tratadas com rotenona foram examinados e comparados em sua atividade motora com a de uma mosca controle alimentada com o solvente dimetilsulfóxido (DMSO). Demonstrou-se que o tratamento com rotenona em Drosophila causa perda de neurônios dopaminérgicos no cérebro22 e leva a déficits locomotores significativos23. Como mostrado na Figura 11 e na Figura 1...

Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

A Simple Technique to Assay Locomotor Activity in Drosophila

O presente protocolo avalia a atividade locomotora de Drosophila rastreando e analisando o movimento de moscas em uma arena artesanal usando software de código aberto Fiji, compatível com plugins para segmentar pixels de cada quadro com base em gravação de vídeo de alta definição para calcular parâmetros de velocidade, distância, 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 ...

O método é baseado em software livre para avaliar sistematicamente as atividades motoras locais para o comportamento de rastreamento de moscas adultas e larvas a partir de vídeo gravado. Este método requer integração de dispositivos de baixo custo para registrar e analisar o comportamento, tornando-se uma abordagem acessível e eficaz para o rastreamento da locomoção de moscas. Essa tecnologia é empregada para identificar modificação comportamental em um modelo de mosca de doença humana com comprometimento de movimento local, mas não é adequada para o tratamento de doenças, deficiências ou dificuldades.É essencial limpar a janela de vídeo relevante durante o processo de análise, ou seja, a janela de vídeo original e a janela de processamento de vídeo. Para começar, misture o reagente A e o reagente B do kit de sílica na proporção de um para 10, de acordo com as instruções do fabricante, para preparar a arena de campo aberto para o rastreamento da locomoção da drosófila. Em seguida, ajuste a câmera HD em um tripé, ajustando-a para que a lente da câmera fique perpendicular à superfície da arena de sílica.Em seguida, abra o vídeo com Fiji. Arraste a barra de progresso para o quadro inicial e aprove tacitamente. Escolha todo o corpo da mosca usando a ferramenta de seleção à mão livre.Clique na imagem, ajuste e brilho e contraste para ajustar o balanço de branco até que o valor de cinza da área selecionada se aproxime do plano de fundo amplo. Transfira a mosca anestesiada para a arena de campo aberto. Assim que a mosca se recuperar da anestesia, coloque o prato de arena com a mosca sob a câmera e agite-o rapidamente de um lado para o outro para garantir que a mosca esteja em movimento quando a gravação começar.Pressione o botão de gravação no aplicativo da câmera para iniciar a gravação de vídeo. Após a conclusão da gravação, pressione o botão de parada para encerrar a gravação de vídeo. Converta os vídeos gravados em formato AVI com codificação pagã M-J para que possam ser abertos e analisados usando Fiji.Também defina os quadros por segundo do vídeo para 15 fps para moscas adultas e 12 fps para larvas. Para abrir o vídeo com Fiji, selecione as duas opções, use a pilha virtual e converta para escala de cinza, na janela pop-up. Obtenha uma janela de processamento usando a ferramenta de configuração de imagem ativa do plug-in de rastreador de animais e crie uma área de rastreamento que circunda a arena na janela de vídeo original, usando a ferramenta oval.Defina os filtros e os parâmetros dos dois filtros para o primeiro quadro em branco na janela de processamento. Em seguida, selecione o próximo quadro na janela de vídeo original e escolha a superfície filtrada da janela de processamento. Depois que uma janela de processamento filtrada for selecionada, use a ferramenta definir limite para girar o trato com um perfil vermelho, coberto na janela de processamento.Em seguida, use o detector de blob definido para permitir que o computador reconheça a mosca com um perfil vermelho, coberto na janela de processamento. Defina o último quadro para a mosca adulta ou larvas, conforme descrito no manuscrito do texto. Use a ferramenta mostrar blobs para apresentar um retângulo de rastreamento na janela de vídeo original.Inicie o rastreamento e exporte o arquivo de rastreamento após a conclusão do monitoramento. Carregue os arquivos de trilha e zona usando o plugin rastreador de animais e analisador de rastreamento. Selecione o índice desejado usando as configurações de zona e altere as configurações de parâmetro.Calcule o tempo do intervalo de quadros usando a taxa de quadros. Produzir os gráficos de análise quantitativa utilizando software de planilha eletrônica e prisma de gráfico pad, após serem analisados no analisador de rastreamento. Abra o arquivo de faixa.Copie todas as coordenadas para o Microsoft Office Excel e divida as células usando a tecla de espaço. Para calcular o tempo de imobilidade por intervalo de quadros, selecione todos os resultados calculados e insira um gráfico de colunas para exibir visualmente o tempo de imobilidade pela margem de todo o gráfico de colunas. Para ilustrar as mudanças no ângulo de direção, selecione todos os resultados calculados e insira um diagrama de dispersão.Moscas adultas e larvas de terceiro ínstar, tratadas com rotenona, apresentaram déficits locomotores significativos em comparação com moscas controle, alimentadas com solvente DMSO. A análise quantitativa do tratamento com rotenona em moscas adultas mostrou uma diminuição significativa na distância percorrida e na velocidade média. E um aumento significativo no tempo de imobilidade.Gráficos representativos mostraram menos picos para a velocidade de movimento por intervalo de quadros em moscas alimentadas com Rotenone em comparação com aquelas no controle, indicando a severidade do déficit de atividade locomotora. A coluna de imobilidade institucional de pixels movidos por quadro também mostrou movimento significativamente menor dentro de um minuto para moscas alimentadas com rotenona em comparação com moscas controle. Gráficos representativos ilustrando as mudanças de direção do ângulo de movimento em animais alimentados com rotenona e controle também revelaram alterações na direção escolhida pelas moscas.Resultados semelhantes foram observados para as larvas do terceiro ínstar. Os resultados revelaram que o movimento comportamental das larvas rastreadoras alimentadas com rotenona foi significativamente prejudicado em relação ao controle. A etapa mais crucial no processo de análise é formar o primeiro quadro em branco, pois evidencia se o software pode verificar de forma eficaz.Essa tecnologia pode permitir que os pesquisadores se aprofundem no mecanismo subjacente de múltiplos defeitos, como lesões nervosas ou musculares.

a-simple-technique-to-assay-locomotor-activity-in-drosophila

Research

JoVE Journal

Behavior

A Simple Technique to Assay Locomotor Activity in Drosophila

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Rato de curto e longo prazo da atividade locomotora analisados ​​pelo software Rastreamento Vídeo

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

Comportamento

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

Medidas comportamentais e do aparelho locomotor Usando um sistema aberto Activity Monitoring Campo de Doenças músculo-esquelético

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

A utilização do campo aberto Maze medir Locomotor e Ansiedade-like Comportamento no Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

A esteira de fadiga de teste: Um ensaio simples, de alta taxa de transferência de Fadiga-like Comportamento para o Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. However, studying human locomotor learning can be challenging. During infancy and childhood, the neuromuscular system is quite immature, and it is unlikely that locomotor learning during early stages of development is governed by the same mechanisms as in adulthood. By the time humans reach maturity, they are so proficient at walking that it is difficult to come up with a sufficiently novel task to study de novo locomotor learning. The split-belt treadmill, which has two belts that can drive each leg at a different speed, enables the study of both short- (i.e., immediate) and long-term (i.e., over minutes-days; a form of motor learning) gait modifications in response to a novel change in the walking environment. Individuals can easily be screened for previous exposure to the split-belt treadmill, thus ensuring that all experimental participants have no (or equivalent) prior experience. This paper describes a typical split-belt treadmill adaptation protocol that incorporates testing methods to quantify locomotor learning and generalization of this learning to other walking contexts. A discussion of important considerations for designing split-belt treadmill experiments follows, including factors like treadmill belt speeds, rest breaks, and distractors. Additionally, potential but understudied confounding variables (e.g., arm movements, prior experience) are considered in the discussion.

We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments).

Usando uma escada rolante Split-cinto para avaliar a generalização de adaptação de locomoção humana

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

Um método para testar o efeito de sugestões ambientais no comportamento de acoplamento em<em&gt; Drosophila melanogaster</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

Um método computacional para quantificar a atividade Circadian voar

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Aplicação de técnica de FlpOut MultiColor para estudar a alta resolução única célula morfologias e interações de células da Glia em Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

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. ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Um ensaio simples e de baixo custo para medir a deambulação em modelos de Mouse de Distrofia Muscular

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...