Name: quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders
Uploaded: 2015-06-13T15:00:00.000+00:00
Duration: 6 min 30 s
Description: Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

研究果蝇在遵守阿尔伯塔省的研究准则大学。 1.收集飞行<ol><li>收集20苍蝇使用CO 2（克）麻醉并置于含有食品25毫米×95毫米收集瓶中。 </li><li>含蝇水平商店小瓶，以避免在那些在底部积聚小瓶任何液体捕集苍蝇。 </li><li>在22℃在培养箱中孵育苍蝇为至少21小时，在45％的湿度下约15小时。以12小时光照设置孵化器:黑暗周期。 </li></ol> 2.登山分析<ol><li>第二天早上，转移20苍蝇从单一到小瓶250毫升的玻璃量筒。标记滚筒的位置，以保持它恒定日常生活。使用每个基因型一层玻璃圆筒，以防止基因型之间的交叉污染。在每个实验和RO的清洗结束基因型之间优化射孔他们。 <ol><li>进行中的环境光的实验（或红光是否有视力潜在缺陷）在温度和湿度为22℃和40％分别。为了避免昼夜节律惊慌失措，总是在每天的同一时间进行实验。 </li></ol></li><li>密封圆筒的顶部用阻挡膜（蜡膜），以防止任何苍蝇的逃逸（ 图2）。 </li><li>设置在三脚架上的摄像机。在190毫升线的250毫升对焦摄像头量筒（17.5厘米）。 </li><li>计数死蝇数在圆筒的底部，并在食品小瓶中。记录该号码作为死亡率。 </li><li>很轻多次敲击对闭孔泡沫垫气缸以足够的力量以置换苍蝇的内底表面。轻按5 - 10倍，而用另一只手按在相机上记录。 </li><li>按相机上的"录制"按钮。 </li><li>启动VIDEO相机记录和挖掘缸六次在不同的非节奏模式。 </li><li>进行每次试验用于从苍蝇最后抽头的时间2分钟，并记录蝇渡17.5厘米（190毫升）中选择每个时间点（量化每10秒）的高度的数量。注意:毫升标记在缸体会有所不同，从一个气缸模型到另一个视直径。为了避免错误，测量所用每个气缸的高度。 </li><li>一旦审判已经结束，处理苍蝇在95％乙醇。 </li><li>重复步骤2.1至2.9，直到所有的重复每次都过测试，新鲜苍蝇。 注:虽然5次重复可能足以与具有运动有很强的影响的突变，20蝇（200苍蝇）的10次生物学重复，建议检测更小的差异。 </li><li>在实验完成后，洗气瓶在实验室洗碗机和干燥O / N被重新使用。 </li></ol> 3.分析<ol><li> ANALY每个视频泽飞试验。每10秒，记录果蝇通过目标线的总数。 <ol><li>如果一只苍蝇爬上回落或下降，飞为-1和记录算下飞行越过目标线为相同数量的攀升是回落或下降飞。例如，如果在15 日飞落在目标线下方，下一飞越线（16 日飞）被认为是15 日飞，而不是16 个 。 </li></ol></li><li>从苍蝇（20）的总人数减去死亡获得苍蝇停留在试验的数量。在每个时间点，获得蝇目标线以上的分数。 </li><li>绘制每个百分比在每个时间点（参见图3）。 </li><li>在120秒的数据点进行分析的性能和执行学生t检验时2基团存在或ANOVA和一个检测后多重比较（和Bonferroni修改为计划和Tukey计划外协mparisons）。柯尔莫哥洛夫-斯米尔诺夫测试17也进行确定正态性和方差相等，但也给突变组的分布比较控制。 </li><li>呈现的数据过时效，画出蝇的百分比在120秒苍蝇不同年龄的攀爬（2天，1周，2周），以查看是否有一个渐进赤字（ 图4）。 </li></ol>

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The authors have nothing to disclose.

果蝇已经被证明是在帕金森氏病14和其他神经变性病症1,2-极好模型。除了 ​​在果蝇中可用的遗传工具，其基因组中是高度保守的对参与神经障碍19基因。全基因组基因筛查方法（包括全外显子组测序）的出现很可能继续提供与人类运动障碍相关的候选基因较大名单。的治疗这些疾病的发展将需要的动物模型，以增加我们涉及神经变性的早期阶段的病理学的...

破坏性神经变性疾病如帕金森氏病，肌萎缩性侧索硬化，和遗传性痉挛性截瘫日益认识到。不幸的是，大多数这些神经变性疾病的仍然没有治疗方法。的广泛临床应用的全基因组，无偏基因测试，如全基因组测序已导致了越来越多的基因受到牵连在人类运动障碍。尽管这样的进步，从早期到晚期的病理进展，仍然难以在这些疾病。 果蝇提供一个具有遗传工具来研究基因在需求控制的空间和时间的方式。此外， 果蝇已经证明在筛选药物对神经系统疾病，如帕金森氏1，阿尔茨海默2，智力障碍3,4-和癫痫5,6-等等是有用的。我们的目标是建立一个符合成本效益可靠的检测，将允许高通量分析，将仍然非常敏感，可以检测电机性能的微小变化。 有用于量化对果蝇攀登行为的基因突变和/或环境条件的影响几个实验。大多数的测定的利用苍蝇爬，称为负趋地性，或攀登测定的自然趋势。本泽7在1967年提出，用于趋光性的研究逆流装置也可以用来研究gravitaxis。自那时以来，Ganetsky 8和许多其他9 -12已经建立在初始测定。其原理是放置在小瓶中的苍蝇的已知数量和强烈挖掘小瓶在硬表面上，造成苍蝇下降到小瓶的底部。因为它是一个先天行为，苍蝇将试图攀爬到小瓶的顶部，相对的重力。该测定是定量的测量仪表水库有多少苍蝇在一个规定的时间段过去攀上小瓶标记。速度，而不是对苍蝇攀登总数的测量已成为一个可靠的参数和的情况下的苍蝇准则的数目并不显著13中所示的缺陷。 攀登测定在许多神经变性疾病，包括帕金森氏病14的研究证明是有益的。然而，我们注意到，机车的缺陷未必在时间被检测到，其中神经变性是已经看到在病理研究14。因此，使用传统的测定法的可能限制，研究疾病发病的早期阶段的能力。机车缺陷在以后的病理阶段的出现可能反映了其疾病进展是太先进完整的救援。 这就提出了与传统的攀登测定的灵敏度的潜在问题。的俗的电位不能tional爬坡法检测温和机车缺陷可以归因于向其中的苍蝇都需要攀登高度。传统的试验15,16措施蝇的数目，成功地翻越2至5厘米的高度在10至20秒。

<table><tbody><tr><td>Drosophila stocks</td><td></td><td></td><td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td></tr><tr><td>Video camera</td><td></td><td></td><td>Any digital camcorder will do. Make sure they can focus on close object.</td></tr><tr><td>Graduated cylinder</td><td>Kimble</td><td>20028W</td><td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td></tr><tr><td>Computer</td><td></td><td></td><td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point.</td></tr></tbody></table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

从神经变性疾病造成机车的缺陷可以是疾病的晚期发作症状，随后的几年亚临床变性的，因此目前的治疗策略是不能治愈。通过使用全基因组测序的，越来越多的基因已被确定，以在人类运动的作用。尽管确定这些基因中，它是不知道这些基因如何是至关重要的正常机车运作。因此，可靠的测定法，其利用模式生物以阐明，以便确定治疗性利益的新靶标，这些基因的作用，需要比以往更。我们设计了负趋地性测定在先允许对较温和的缺陷的检测，并具有随着时间的推移，以评估这些缺陷的能力的致敏版本。在玻璃进行测定量筒中，被密封与蜡阻挡膜。通过提高门槛的距离要攀升至17.5Ç米和增加实验的持续时间为2分钟，我们观察到在检测温和流动性功能障碍更大的灵敏度。该测定是成本有效的，不需要大量的训练，以获得高度可重复的结果。这使得它一个很好的技术来筛选候选药物在果蝇突变体运动缺陷。

攀岩是一个强大的和可重复的行为。事实上，有一天老野生型果蝇达到目标的距离爬坡性能迅速（25 - 30秒）。突变果蝇呈现范围从轻微（或延迟）的性能来完成无力攀登到目标。我们说明这个这里有两个不同的突变等位基因。第一个是由一个完全缺失的spastin基因（温泉5.75）18的基因spastin的一个严重的等位基因。在这条线（5.75温泉与TM6b）一天的小苍蝇甚至2分钟后达不到WT爬坡?...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

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

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 ...

quantitative-analysis-climbing-defects-drosophila-model

Research

JoVE Journal

Neuroscience

18.5K 次观看.  University of Alberta. 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. 

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

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

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 ...

遗传学

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.

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. ...

行为学

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 ...

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 ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

生物学

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

与神经退化有关的成人 果 蝇的可视化突触变性

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

攀登缺陷的神经退行性疾病的果蝇模型定量分析

摘要

探索更多视频

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

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

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

评估神经退行性表型

一种测定果蝇运动活性的简单技术

大众组织学量化在神经退行性变

嗜德维人神经元树突状树突状复杂度的定量分析

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

成绩单和代谢产物的分离纯化

与神经退化有关的成人果蝇的可视化突触变性

攀登缺陷的神经退行性疾病的果蝇模型定量分析

摘要

探索更多视频

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

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

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

评估神经退行性表型

一种测定果蝇运动活性的简单技术

大众组织学量化在神经退行性变

嗜德维人神经元树突状树突状复杂度的定量分析

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

成绩单和代谢产物的分离纯化

与神经退化有关的成人 果 蝇的可视化突触变性

与神经退化有关的成人果蝇的可视化突触变性