我们感谢布卢明顿股票中心和维也纳的果蝇的飞行股票 rna 干扰中心。该设备的专利属于中国科学院上海市先进研究院。对 RflyDetection 软件的要求应由富煌 (见作者名单)。这项工作得到了中国国家基础研究项目 (973 项目 2013CB945602) 和中国国家自然科学基金 (31270825 和 31171043) 的资助。我们感谢何议员的讨论和意见。

1. 飞集 <ol> <li> 在标准飞行食物上保持苍蝇在25和 #176; C、70% 湿度和 12 h/12 h 光/暗循环. </li>
	<li> 收集 UAS-Dicer2;DDC-GAL4 飞行 virginsunder 二氧化碳 (CO 2 ) 麻醉. </li>
	<li> 将这些处女带到2天大的成年雄蝇携带以下基因型: UAS-mCD8GFP (控制), auxR 16182 ( 辅助 干扰) , auxGFP ( 辅助 干扰), 和 auxR 16182 ;auxGFP (抢救), 与男性: 女性比率为 1:2. </li>
	<li> 分别为每个组和 #160;p er 实验收集新 eclosed 的雄性和雌性小瓶, 每只普通的苍蝇小瓶中放置10只苍蝇, 标准食物在和 #160; 25 和 #176; C. </li>
	<li> 根据实验, 将收集的苍蝇保持35天, 并将其用于5、15、25和35天的自动环分析. </li>
</ol> 2。自动环形检测 <ol> <li> 将每种基因型的10只收集的苍蝇 (中性) 转移到每个小瓶中, 然后用螺钉固定药瓶。分析控制苍蝇和苍蝇携带不同基因型一起为每组实验 (最多10瓶同时, 每瓶10只苍蝇). </li>
	<li> 打开放置在设备前面的数码相机, 一旦苍蝇都装好就开始录音. </li>
	<li> 在允许1分钟的苍蝇以小瓶结算后, 打开控制步骤驱动程序的步骤控制器; 这将驱动小型电动机控制连接的拉杆, 使其在2秒内连续上升并轻拍设备4次。请参见 图 1 。
	<ol> <li> 敲击后, 请注意, 苍蝇开始爬上墙。确保录制继续进行. </li>
	</ol> </li> <li> 在3到5连续的试验中重复步骤2.3 中描述的60秒间隔中的同步. </li>
	<li> 重复试验5、15、25和35天的老苍蝇。每组至少进行3项独立实验, 每次试验至少30只收集的苍蝇 (3 瓶). </li>
</ol> 3。数据分析 <ol> <li> 将录制的视频导入计算机. </li>
	<li> 在敲击后6秒拍摄视频的快照, 用于每个试用版. </li>
	<li> 将快照映像导入 RflyDetection 软件 (请参见 图 2 ), 使用 #39; 文件和 #39; 菜单. </li>
	<li> 通过使用工具栏上的基线图标, 然后使用光标标记图像上的上、下基线, 精确设置小瓶的上下基线. </li>
	<li> 输入每瓶的苍蝇数 (, 如 , 10 在这里) #39; 飞在矩形和 #39; 在和 #39 中的字段和瓶长 (, 如 , 14 厘米); 管高度和 #39; 设置栏中的字段。
	<ol> <li> 请注意, 每个小瓶都在屏幕上检测到各个飞行位置并标记有点. 
		注意: 图 2 指示所有菜单按钮单击的位置. </li>
	</ol> </li> <li> 注意, 软件自动确定每只苍蝇的爬升距离, 并在右侧面板的表格中显示每瓶10只苍蝇的平均值。(请参见 图 2 ) </li>
	<li> 使用统计软件 ( 如 、棱镜) 处理爬数, 以进行进一步的统计分析. </li>
	<li> 将数据显示为平均值和 #177; SEM. </li>
	<li> 计算 p 值的重要性 (用星号、* p 和 #60; 0.05、 p 和 #60; 0.01、*** p 和 #60; 0.001) 使用 one-way 方差分析 Bonferroni 多项比较试验. </li>
</ol>

<ol>
	<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. (49), (2011).</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. (61), (2012).</li><li>Madabattula, S. T., 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=Quantitative+Analysis+of+Climbing+Defects+in+a+Drosophila+Model+of+Neurodegenerative+Disorders.">Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders.</a> J Vis Exp. (100), e52741 (2015).</li><li>Podratz, J. L., 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=An+automated+climbing+apparatus+to+measure+chemotherapy-induced+neurotoxicity+in+Drosophila+melanogaster.">An automated climbing apparatus to measure chemotherapy-induced neurotoxicity in Drosophila melanogaster.</a> Fly (Austin). 7 (3), 187-192 (2013).</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 (5), 386-395 (2005).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+rapid+iterative+negative+geotaxis+assay+and+its+use+in+a+genetic+screen+for+modifiers+of+Abeta(42)-induced+locomotor+decline+in+Drosophila.">Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Abeta(42)-induced locomotor decline in Drosophila.</a> Neurosci Bull. 31 (5), 541-549 (2015).</li><li>Shen, Y., 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=SH2B1+is+Involved+in+the+Accumulation+of+Amyloid-beta42+in+Alzheimer's+Disease.">SH2B1 is Involved in the Accumulation of Amyloid-beta42 in Alzheimer's Disease.</a> J Alzheimers Dis. 55 (2), 835-847 (2017).</li><li>Song, L., 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=Auxilin+Underlies+Progressive+Locomotor+Deficits+and+Dopaminergic+Neuron+Loss+in+a+Drosophila+Model+of+Parkinson's+Disease.">Auxilin Underlies Progressive Locomotor Deficits and Dopaminergic Neuron Loss in a Drosophila Model of Parkinson's Disease.</a> Cell Rep. 18 (5), 1132-1143 (2017).</li><li>Zhang, X., 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=Downregulation+of+RBO-PI4KIII&#945;+Facilitates+A&#946;42+Secretion+and+Ameliorates+Neural+Deficits+in+A&#946;42-Expressing+Drosophila.">Downregulation of RBO-PI4KIII&#945; Facilitates A&#946;42 Secretion and Ameliorates Neural Deficits in A&#946;42-Expressing Drosophila.</a> J Neurosci. 37 (19), 4928-4941 (2017).</li><li>Riemensperger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+dopamine+pathway+underlies+progressive+locomotor+deficits+in+a+Drosophila+model+of+Parkinson+disease.">A single dopamine pathway underlies progressive locomotor deficits in a Drosophila model of Parkinson disease.</a> Cell Rep. 5 (4), 952-960 (2013).</li></ol>

作者没有什么可透露的。

在这里描述的自动环分析可以同时对数以百计的苍蝇的飞行负 geotaxis 行为进行高通量研究。以前的分析成人攀登的策略涉及单个小瓶中单飞的观察, 而苍蝇的位置则由眼睛手动检测。这个相当繁琐的过程有时可能会导致对数据的误读或误解, 以及 labor-intensive 的工作。我们的自动环分析从一个简单的点击开始, 该仪器自动同步和评估高达100苍蝇的攀登能力。电同步提供了更精确的控制时间和其他参?...

为分析果蝇的成人攀爬行为制定了各种协议和方法。比较费力的是, 传统的分析主要涉及将一只苍蝇放入单个小瓶中, 并使用手动的力量来为同步1,2,3,4。这是单调乏味和费时, 不适合大规模高通量的研究, 并有潜在的变化, 手动力量, 用来打击苍蝇以及其他限制。为了改进这种检测, 我们开发了一种快速的迭代负 Geotaxis (环) 试验, 它允许高通量分析在同一时间的大量苍蝇5。然而, 该检测仍需要人工施加力来同步飞行动作。我们的版本的环化验, 修订后, 先前的化验, 包括一个金属基地承载多个装有苍蝇的小瓶自动控制由电机驱动飞行同步6。录音后, 同步后立即爬升, 然后使用自行设计的软件进行分析。我们的自动环试验已经消除了繁琐和 labor-intensive 的过程, 收集数据从一个单一的苍蝇, 一次一个, 并使数据采集过程更有效。此外, 在一些研究中使用了自动环法来阐明阿尔茨海默氏症和帕金森病的发病机制, 并以高效率的方法验证了7,8,9.
在本文中, 我们演示了使用DDC-Gal4驱动的 rna 干扰飞行的自动环检测。DDC-Gal4是在多巴胺能 (DA) 和素神经元中特别表达的 Gal4 线, 因此它代表了分析与神经10相关的运动缺陷的靶基因效应的一个很好的工具。此外, 我们还合并了UAS-Dicer2, 这是一条提高 rna 干扰效率的飞行线, 用于生成UAS-Dicer2;DDC-Gal4工具行。我们选择使用的 rna 干扰的苍蝇是auxilin (辅助) 的 rna 干扰 v16182 (auxR16182), 一个我们先前确定的基因, 以展示对飞行运动活动的影响8。auxGFP苍蝇也准备用于分析在辅助上的效果. 我们将展示如何使用自动环法测量苍蝇负 geotaxis, 提出结果, 并讨论从结果获得的任何影响。

<table><tbody><tr><td>Forma Environmental Chamber</td><td>Thermo</td><td>3949</td><td></td></tr><tr><td>Carbon dioxide cylinders</td><td>FuLian GAS Technology</td><td>GB/T6052</td><td></td></tr><tr><td>HDR-Camcorder</td><td>SONY</td><td>HDR-CX220E</td><td></td></tr><tr><td>Binocular stereomicroscope</td><td>Xin Zhen</td><td>SMZ-168BL</td><td></td></tr><tr><td>Electronic scales</td><td>MinQiao</td><td>SL1002N</td><td></td></tr><tr><td>Refrigerator</td><td>Haier</td><td>SC-350</td><td></td></tr><tr><td>Agar-agar powder</td><td>Sinopharm</td><td>10000561</td><td></td></tr><tr><td>Glucose</td><td>Sinopharm</td><td>10010518</td><td></td></tr><tr><td>Corn meal</td><td>Sinopharm</td><td>5464654</td><td></td></tr><tr><td>Brown sugar</td><td>LiuCaiYuan</td><td>45467936</td><td></td></tr><tr><td>Instant dry yeast</td><td>AB MAURI</td><td>20886</td><td></td></tr><tr><td>AuxR&lt;sup&gt;16182&lt;/sup&gt;</td><td>VDRC</td><td>7187</td><td></td></tr><tr><td>UAS-Dicer2</td><td>Bloomington</td><td>24650</td><td></td></tr><tr><td>UAS-mCD8GFP</td><td>Bloomington</td><td>32185</td><td></td></tr><tr><td>DDC-Gal4</td><td></td><td></td><td>A gift from Fude Huang</td></tr><tr><td>AuxGFP</td><td></td><td></td><td>A gift from Henry Chang</td></tr></tbody></table>

an automated rapid iterative negative geotaxis assay for analyzing adult climbing behavior in a drosophila model of neurodegeneration

神经退行性疾病往往与运动能力的逐渐丧失, 缩短寿命, 和年龄相关的神经。为了了解这些细胞事件的机制, 以及它们之间的因果关系,果蝇, 用其复杂的遗传工具和多样的行为特征, 作为疾病模型来评估神经退行性表型。在这里, 我们描述了一个高通量的方法来分析果蝇成人负 geotaxis 行为, 作为可能的运动缺陷的迹象, 与神经。一个自动化的机器是设计和开发, 以驱动飞行同步使用最初的电脉冲, 后来允许记录的负 geotaxis 行为的过程中秒到分钟。然后用自行设计的 RflyDetection 软件对数字记录的视频图像进行统计处理。不同于基于单飞的人工控制的负 geotaxis 检测, 这种精确、快速和高通量的协议允许同时获取超过数百个苍蝇的数据, 提供了一种有效的方法来推进我们与神经相关的运动缺陷的基本机制的理解。

本文说明了在评估苍蝇负 geotaxis 行为的使用自动环法。与以前的环分析不同的是, 我们的检测包括一个自动装置, 它提供了一种同步飞行动作的电动力量, 并同时对数以百计的苍蝇进行解析 (图 1)。分析了Dicer2;DDC 和 #62; auxR16182 苍蝇在6秒的时间内显示出在攀爬距离上的年龄依赖性下降, 这表明在 DA 神经元中, 辅助表达对飞行活动是至关?...

Watch this Scientific Journal Video about An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration at JoVE.com

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

此 step-by 步骤协议使用自动缸系统来分析果蝇的负 geotaxis 行为, 它承载数以百计的苍蝇并使其动作与电动机同步。在同步时, 采用自行设计的 RflyDetection 软件对飞行负 geotaxis 行为进行检测、数字记录和分析。

一种自动快速迭代阴性 Geotaxis 分析方法在神经的果蝇模型中的成人攀爬行为

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

an-automated-rapid-iterative-negative-geotaxis-assay-for-analyzing

Research

JoVE Journal

Behavior

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

9.7K 次观看.  Tongji University School of Medicine. 此 step-by 步骤协议使用自动缸系统来分析果蝇的负 geotaxis 行为, 它承载数以百计的苍蝇并使其动作与电动机同步。在同步时, 采用自行设计的 RflyDetection 软件对飞行负 geotaxis 行为进行检测、数字记录和分析。

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

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

含量的运动能力，学习和记忆的赤字果蝇</em神经退行性疾病>模型

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

科研

神经科学

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.

TreadWheel:果蝇轻度诱导运动的间歇训练协议

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

遗传学

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

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.

对检测方法果蝇行为

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

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

评估神经退行性表型<em&gt;果蝇</em&gt;爬坡试验和全脑多巴胺能神经元免疫组化染色

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

行为学

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;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

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.

一种测定果蝇运动活性的简单技术

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

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

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.

一种自动快速迭代阴性 Geotaxis 分析方法在神经的果蝇模型中的成人攀爬行为

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此视频中的章节

含量的运动能力，学习和记忆的赤字果蝇模型

TreadWheel:果蝇轻度诱导运动的间歇训练协议

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

对检测方法果蝇行为

评估神经退行性表型

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

在自发活动的测定

一种测定果蝇运动活性的简单技术

攀登缺陷的神经退行性疾病的果蝇模型定量分析

果蝇岛试验中运动行为的高通量分析