Name: a novel method to model chronic traumatic encephalopathy in drosophila
Uploaded: 2017-07-04T13:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

作者没有什么可以披露的。

打击装置装配（ 图1 ） <ol><li>从1 mL结核菌素注射器中取出柱塞。切成1毫升标记的桶。 </li><li>从200μL移液器吸头上取下气溶胶屏障（直径3mm，直径4 mm），并将其用作冲击器。将冲击器放在注射器筒内。轻轻敲击枪管将冲击器移动到前端，平面覆盖喷嘴开口。 </li><li>将枪管末端连接到连接到果蝇麻醉站的二氧化碳（CO 2 ）流量调节器的塑料管上。 </li><li>将桶垂直握住，并将其夹紧到标准的夹持架上，以便冲击器停留在机筒的底部。 </li><li>修改200μL移液器吸头，使飞针座。 <ol><li>从尖端切割4毫米直径为0.8毫米的开口，只允许飞头暴露。 注意:飞行器的所有其他部分的rax和rak将留在移液器吸头内。 </li></ol></li><li>修改1,000μL移液器针头和1 mL注射器针头帽，使连接器。 <ol><li>从尖端的开口切掉44毫米。取6毫米长的1毫升注射器针头帽，并将其紧紧地推入1,000μL移液器吸头的剩余部分。 </li></ol></li></ol> 2.打击装置的操作<ol><li>麻醉一只2日龄的成年雌性飞蝇，使用二氧化碳在飞盘上。 </li><li>使用细微的刷子将其轻轻转移到飞轮支架上。轻轻敲击支架，以便在尖端外部看到飞头。如果将飞翔长鼻暴露在尖端外部，用钝的1 mL注射器针轻轻地将其收回到尖端内。 注意:请确保将持针器内的飞龙鼻子保持在内。否则，飞蝇可能会因吸吮的长鼻子受伤而死亡。 </li><li>拧紧飞翔使用连接器将其装入注射器筒，使飞头朝下。 </li><li>将气体压力设置在100 kPa。根据实验设计调整流量。 </li><li>快速打开和关闭流量调节器拨动开关，以便冲击器撞击飞头一次。 </li><li>抬起飞翼，将其移动到飞盘上。反转飞轮架，轻轻敲击侧面让飞出。将飞行物留在空的小瓶中恢复。 </li></ol>视频辅助运动跟踪<ol><li>用透明硅弹性体填充6厘米直径的陪替氏培养皿，制作跟踪舞台。在硅和盘盖之间留出3毫米的空间，以便苍蝇自由行走，但不能飞行。 </li><li>每次从假手术或治疗组麻醉四只苍蝇，并将它们放置在舞台上。将苍蝇放在22°C 1小时。 </li><li>将电荷耦合设备（CCD）摄像机放置在场地上方并记录5分钟。</li><li>使用Ctrax软件（可从Caltech免费获得）分析记录的移动轨迹16 。以编程语言（例如Matlab）导出跟踪数据 - 兼容格式，并根据每帧移动距离分析数据17 。计算每次飞行的平均步行距离，并将其与所有其他记录的苍蝇/组合，以获得由同一组中的文件群体行进的平均累积距离。 </li></ol>

<ol>
	<li>McKee, A. C., 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=The+first+NINDS/NIBIB+consensus+meeting+to+define+neuropathological+criteria+for+the+diagnosis+of+chronic+traumatic+encephalopathy.">The first NINDS/NIBIB consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy.</a> Acta Neuropathol. 131, 75-86 (2016).</li><li>Martland, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Punch+drunk.">Punch drunk.</a> JAMA. 91 (15), 1103-1107 (1928).</li><li>Millspaugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dementia+pugilistica.">Dementia pugilistica.</a> US Naval Med Bull. 35, 297-303 (1937).</li><li>Omalu, B. I., 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=Chronic+traumatic+encephalopathy+in+a+national+football+league+player:+part+II.">Chronic traumatic encephalopathy in a national football league player: part II.</a> Neurosurgery. 59 (5), 1086-1092 (2006).</li><li>Goldstein, L. E., 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=Chronic+traumatic+encephalopathy+in+blast-exposed+military+veterans+and+a+blast+neurotrauma+mouse+model.">Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model.</a> Sci Transl Med. 4 (134), (2012).</li><li>Mez, J., Stern, R. A., McKee, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+traumatic+encephalopathy:+where+are+we+and+where+are+we+going?.">Chronic traumatic encephalopathy: where are we and where are we going?.</a> Curr Neurol Neurosci Rep. 13 (12), 407 (2013).</li><li>McKee, A. C., 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=The+spectrum+of+disease+in+chronic+traumatic+encephalopathy.">The spectrum of disease in chronic traumatic encephalopathy.</a> Brain. 136 (Pt 1), 43-64 (2013).</li><li>Petraglia, A. 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=The+spectrum+of+neurobehavioral+sequelae+after+repetitive+mild+traumatic+brain+injury:+a+novel+mouse+model+of+chronic+traumatic+encephalopathy.">The spectrum of neurobehavioral sequelae after repetitive mild traumatic brain injury: a novel mouse model of chronic traumatic encephalopathy.</a> J Neurotrauma. 31 (13), 1211-1224 (2014).</li><li>Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+in+the+study+of+human+neurodegeneration.">Drosophila melanogaster in the study of human neurodegeneration.</a> CNS Neurol Disord Drug Targets. 9 (4), 504-523 (2010).</li><li>Littleton, J. T., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+and+synaptic+organization:+analysis+of+the+Drosophila+genome.">Ion channels and synaptic organization: analysis of the Drosophila genome.</a> Neuron. 26 (1), 35-43 (2000).</li><li>Appel, L. F., 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=The+Drosophila+Stubble-stubbloid+gene+encodes+an+apparent+transmembrane+serine+protease+required+for+epithelial+morphogenesis.">The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis.</a> Proc Natl Acad Sci USA. 90 (11), 4937-4941 (1993).</li><li>Piyankarage, S. C., Featherstone, D. E., Shippy, 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=Nanoliter+hemolymph+sampling+and+analysis+of+individual+adult+Drosophila+melanogaster.">Nanoliter hemolymph sampling and analysis of individual adult Drosophila melanogaster.</a> Anal Chem. 84 (10), 4460-4466 (2012).</li><li>Katzenberger, R. 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=A+Drosophila+model+of+closed+head+traumatic+brain+injury.">A Drosophila model of closed head traumatic brain injury.</a> Proc Natl Acad Sci USA. 110 (44), E4152-E4159 (2013).</li><li>Barekat, A., 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=Using+Drosophila+as+an+integrated+model+to+study+mild+repetitive+traumatic+brain+injury.">Using Drosophila as an integrated model to study mild repetitive traumatic brain injury.</a> Sci Rep. 6, 25252 (2016).</li><li>Katzenberger, R. 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=A+Method+to+Inflict+Closed+Head+Traumatic+Brain+Injury+in+Drosophila.">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila.</a> J Vis Exp. (e52905), (2015).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat Methods. 6 (6), 451-457 (2009).</li><li>Straw, A. D., Dickinson, M. 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=Motmot,+an+open-source+toolkit+for+realtime+video+acquisition+and+analysis.">Motmot, an open-source toolkit for realtime video acquisition and analysis.</a> Source Code Biol Med. 4 (5), 1-10 (2009).</li><li>Talavage, T. 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=Functionally-detected+cognitive+impairment+in+high+school+football+players+without+clinically-diagnosed+concussion.">Functionally-detected cognitive impairment in high school football players without clinically-diagnosed concussion.</a> J Neurotrauma. 31 (4), 327-338 (2014).</li><li>Theadom, A., 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=Frequency+and+impact+of+recurrent+traumatic+brain+injury+in+a+population-based+sample.">Frequency and impact of recurrent traumatic brain injury in a population-based sample.</a> J Neurotrauma. 32 (10), 674-681 (2015).</li><li>Drobysheva, D., 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+optimized+method+for+histological+detection+of+dopaminergic+neurons+in+Drosophila+melanogaster.">An optimized method for histological detection of dopaminergic neurons in Drosophila melanogaster.</a> J Histochem Cytochem. 56 (12), 1049-1063 (2008).</li></ol>

这项工作得到了约翰霍普金斯大学医学院教师创业基金会的支持

忠实地建模CTE特征的动物模型，包括神经生理学改变，神经病理学特征和神经行为缺陷，对于揭示疾病机制和开发诊断和治疗目标至关重要。可以理解，人类疾病的动物模型在模拟所有临床相关终点方面是完美的。然而，我们认为，强大的CTE模型应该满足以下三个要求:（1）影响必须直接适用于具有完整的头皮和颅骨保护的头部; （2）在冲击暴露期间头部不应固定，以便允许快速加速 - 减速和旋?...

慢性创伤性脑病（CTE）最近被认为是一种独特的神经变性疾病，与其他tau病，如阿尔茨海默病1分离 。与阿尔茨海默病和其他常见的tau蛋白病（其最重要的危险因素是年龄增长和痴呆家族史）不同，CTE如其名称所示，意味着与脑外伤历史密切相关，很可能与体育运动员接触，例如拳击手和足球运动员，以及2,3,4,5的退伍军人。它被认为是通过反复的震荡和冲击打击脑袋。患者可能会出现症状和体征，如认知缺陷，情绪和行为变化以及运动功能障碍，其与阿尔茨海默病，额颞叶痴呆，路易体痴呆和帕金森病6 。相比之下，脑组织的死后检查显示在皮层sulci深度周围的小血管周围的超磷酸化tau积累的不同模式，在其他退化病症中未观察到的病理特征7 。然而，到目前为止，关于导致疾病表现的发病机制知之甚少。这在很大程度上是由于缺乏一个忠实的动物模型 - 只是最近才有了5,8的鼠类模型。这些模型生物体具有成本密集型护理和相对较长使用寿命的缺点，不太适用于神经退行性疾病研究。 与哺乳动物相比，无脊椎动物如果蝇是一种很好的替代方案，具有成本效益的维护，广泛的解剖遗传决定因素的工具，寿命相对较短9 。值得注意的是，飞行和人类大脑分享进化上保守的分子和细胞通路，以及解剖学相似性10,11,12 。已经报道了两个巧妙的果蝇模型来研究创伤性脑损伤。由Katzenberger及其同事设计的第一个"高冲击创伤"（HIT）装置将塑料瓶中的自由移动的苍蝇与金属弹簧13,15的自由端相连。当塑料小瓶倾斜直立并释放时，它会撞到聚氨酯垫，并在苍蝇弹到小瓶壁并反弹时给野生动物施加创伤。相比之下，Barekat和同事们设计了一种不同的送货方式使用Omni Bead Ruptor-24匀浆机平台14 。苍蝇与CO 2失活，并置于2mL螺旋管中，将其固定在均化器上并进行预编程的摇动条件。使用组织匀浆器系统的一个好处是，实验者可以调节受伤的强度，受伤持续时间和伤害发作次数。然而，这两个制度都有同样的缺点:头部的主要伤害在冲击位置和强度方面是随机的。此外，这两种方法导致相当大的死亡率，这是由身体和内脏其他部位的不可避免的附带损害造成的。在这里，我们描述了一种在果蝇中诱导mTBI的新方法。我们的装置由气体推进的弹道冲击器组成。与现有的果蝇模型14,15相比，我们的方法具有独特的优势可引起影响，仅针对自由移动的飞头，从而允许准确控制各种因素，如影响严重性，影响之间的时间间隔以及持续影响的总数。

<table><tbody><tr><td>Aerosol Barrier</td><td>USA Scientific</td><td>1120-8810</td><td>Used as an impactor</td></tr><tr><td>200 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1111-0706</td><td>Used as a fly head holder</td></tr><tr><td>1000 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1122-1830</td><td>Used as a connector</td></tr><tr><td>1 mL Tuberculin Syringe</td><td>Becton Dickinson</td><td>309625</td><td></td></tr><tr><td>60 mm Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713A</td><td>Used as a tracking arenas</td></tr><tr><td>Flow Regulator</td><td>Genesee Scientific</td><td>59-122WC</td><td></td></tr><tr><td>Standard Clamp Holder/stand</td><td>EISCO Scientific</td><td>CH0688</td><td></td></tr><tr><td>Fine Brush</td><td>Genesee Scientific</td><td>59-204</td><td></td></tr><tr><td>Flypad</td><td>Genesee Scientific</td><td>59-114</td><td></td></tr><tr><td>Sylgard Silicone Elastomer</td><td>Dow Corning</td><td>4019862</td><td></td></tr><tr><td>CCD Camera</td><td>Microsoft&amp;nbsp;</td><td>HD-5000</td><td></td></tr><tr><td>Ctrax Walking Fly Tracker</td><td>Caltech</td><td>Ctrax 0.2.11</td><td></td></tr><tr><td>MATLAB Image Processing Toolbox</td><td>MATLAB</td><td>R2015b</td><td></td></tr></tbody></table>

a novel method to model chronic traumatic encephalopathy in drosophila

慢性创伤性脑病（CTE）是一种既定的神经退行性疾病，与暴露于重度轻度创伤性脑损伤（mTBI）密切相关。尽管最近有一个界定神经病理学标准的共识，但其复杂的病理变化的机制仍然很难捉摸。在这里，我们描述了一种在黑腹果蝇 （ 果蝇 ）中开发CTE模型的新方法，旨在鉴定导致大脑中特征性过度磷酸化tau积累和神经元死亡的关键基因和途径。对可能受到轻微封闭伤害的强度影响直接传递给飞头，使头部快速加速和减速。我们的方法消除了其他果蝇 mTBI模型固有的潜在问题（ 例如，动物死亡可能是由于损伤引起的身体其他部位或内脏）。较少的劳动力和成本密集的动物护理，短寿命和广泛的遗传工具使果蝇成为研究CTE发病机理的理想选择，并且可以进行大规模，全基因组的前期遗传和药理筛选。我们预计，模型的持续表征将为疾病预防和治疗方法产生重要的机械观点。

为了建立使用成年果蝇的CTE模型，我们确定了我们的装置在单次闭合性头部受伤时的有效性。为了消除与基因型，性别或年龄有关的变化，我们在实验中使用了2日龄的Canton-S WT雌性苍蝇。通过在100 kPa的恒定气压下调节CO 2的流量，可以很容易地控制冲击器的强度。以最高流速（15L / min）暴露于单次冲击的苍蝇显示出最小的外部缺陷（ 图2</stron...

Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

在这里，我们描述了一种在黑腹果蝇中引起封闭头部创伤性脑损伤的新方法。我们的方法的优点是可以直接将可重复的冲击强度提供给头部。无脊椎动物系统的进一步探索将有助于阐明慢性创伤性脑病的发病机制。

慢性创伤性脑病模型的一种新方法<em&gt;果蝇</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

a-novel-method-to-model-chronic-traumatic-encephalopathy-in-drosophila

Research

JoVE Journal

Neuroscience

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

8.2K 次观看.  Johns Hopkins University School of Medicine. 在这里，我们描述了一种在黑腹果蝇中引起封闭头部创伤性脑损伤的新方法。我们的方法的优点是可以直接将可重复的冲击强度提供给头部。无脊椎动物系统的进一步探索将有助于阐明慢性创伤性脑病的发病机制。 

视频: A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

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

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

发育生物学

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 ...

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 ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

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 ...

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

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

Source: Sun, M. and Chen, L. L. <a href="https://www.jove.com/video/55602/" target="_blank">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila</a>. J. Vis. Exp. (2017).
This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking software.

追踪自由行走的果蝇:一种评估果蝇运动的方法

Source: Sun, M. and Chen, L. L. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. J. Vis. Exp. (2017).
This video describes how to ...

JoVE Encyclopedia of Experiments

黑腹果蝇（果蝇）

成虫

A Technique to Inflict Closed-Head Traumatic Brain Injury in Drosophila Flies

Source: Sun, M., et al. <a href="https://app.jove.com/v/55602">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure to inflict a closed-head traumatic injury in anesthetized Drosophila flies using a strike device with a gas-propelled impactor. The injury affects neuronal function and impairs walking ability.

一种对果蝇造成闭合头部创伤性脑损伤的技术

1. Assembly of the Strike Device (Figure 1)

	Remove the plunger from a 1 mL tuberculin syringe. Cut the barrel at the 1 mL mark.
	Remove an aerosol ...

慢性创伤性脑病模型的一种新方法

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

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

评估神经退行性表型

攀登缺陷的神经退行性疾病的果蝇模型定量分析

方法对敌人造成闭合性颅脑创伤性脑损伤

大众组织学量化在神经退行性变

成绩单和代谢产物的分离纯化

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

追踪自由行走的果蝇:一种评估果蝇运动的方法

一种对果蝇造成闭合头部创伤性脑损伤的技术