This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1.固定在项圈的头和石蜡包埋注:所有的在定影过程中的步骤应在通风橱中进行。甲基苯甲酸甲酯，而不是构成一个健康风险，具有高度不同的气味，这可以是压倒性如果在通风橱中不进行处理。 <ol><li>麻醉苍蝇之前，加入15毫升的氯仿和5毫升冰醋酸30毫升99％乙醇（不要混合的冰醋酸和氯仿）占50毫升卡诺的解决方案。在具有平坦底部，玻璃容器倒如一个晶化盘，以确保该环可以平放和由该溶液完全覆盖。 </li><li>麻醉与CO 2或乙醚苍蝇。 </li><li>螺纹蝇（最多20个与大多数环）可以通过颈部进入使用镊子的衣领。记对准所有磁头的相同的方向，如在图1A，并轻柔，保证不发生向头部或眼睛没有损害。 </li><li>包括随机位置，使苍蝇的顺序可以在部分很容易确定正弦oculis苍蝇（箭头， 图1A）。此外，如果实验苍蝇具有光或白色眼睛的颜色，螺纹一些红眼蝇，如野生型，在它们之间，以确保有足够的颜料是本染色的幻灯片。如果使用多个领衣领号码记录蝇量级的协议片一起。 </li><li>一旦领已经完成，将其放置在3.5准备的卡诺的解决方案 - 4小时。 </li><li> dump出来卡诺液到适当的处理罐，并开始清洗乙醇。请一定要慢慢倒入，以免扰乱容器中环的位置。 </li><li>洗套环在99％乙醇30分钟两次。 </li><li>洗在100％乙醇套环1小时。一定要改变时间的洗涤，以防overdehydration。 </li><li>放入甲基苯甲酸衣领O / N在室温。密封容器用石蜡膜以防止甲基苯甲酸甲酯的蒸发。 </li><li>倒入methylbenozate到通风柜适当一次性容器。加1的预先准备的混合物:1熔点低（56 - 57°C）石蜡和苯甲酸甲酯。从这点上来说，轴环需要在65℃下被保持在培养箱中以确保石蜡不硬化。 </li><li>倾出methylbenozate和石蜡混合物到适当的一次性容器，并倒入熔融的纯石蜡，保持在65℃，到套环。 </li><li>改变石蜡后30分钟，并重复此至少5次。至少6 - 8次洗涤应该执行。 </li><li>一旦洗涤完成后，放置套环与槽大约轴环的尺寸的橡胶冰块托盘。倒入熔化的石蜡过他们，直到完全覆盖，并允许它变硬O / N（尽量避免气泡）。 </li><li>取出石蜡块，包含3宁从冰块托盘的衣领。从使用刀片领子分开石蜡块，轻轻掰的衣领。该负责人将在蜡块，而机构会留在衣领。该块可以在室温下保持。 </li><li>清洁套环，在65℃下浸泡他们在一个deparafinization剂以除去石蜡，清洁光擦洗，并在重复使用之前在乙醇洗净。 </li></ol> 2.切割和安装<ol><li>暖加热板到50℃。将上盘对象持有人（或金属固定块）和刀片，让他们热身。 </li><li>取决于用于切片所需的方向，连接或者与朝向侧磁头（对于水平段）的石蜡块或朝上（对于正面部），以加热安装块（简要地熔融在接触侧的块）。从加热板上取下块并让其冷却至少10英里n至确保该石蜡硬化够适当的密封到安装块。保持平行，尽可能防止不均匀的部分中的块的表面对准头的行。 </li><li>取一个刀片并修剪多余的石蜡从蝇头远，使得只有一小行与嵌入式头保持（剃须刀刀片可以温热更容易修整）。确保不要修剪过多，使石蜡不切片（更多微调可以切片过程中进行）时断裂。 </li><li>安装块放入切片机的对象保持器，并确保磁头行的取向是尽可能平行于叶片的边缘。 </li><li>通过覆盖他们的聚-L-赖氨酸（PLL）的溶液的薄层准备显微镜载玻片并让它们干燥5分钟。覆盖它们与水之前不久使用。 </li><li>削减7微米的部分和部分的色带转移到幻灯片浮在水面上。&lt;BR /&gt;注:要获取水平段整个大脑，我们将收集从开始切入眼睛，直到头已经完全切断（从长鼻切入脑）当色带。可能需要对整个头部多个幻灯片。 </li><li>放置在37℃的热板的滑动，并允许色带展开约1分钟。 </li><li>去除多余的水（浇其关闭或使用组织）和干燥的幻灯片O / N。 </li><li>通过将玻片在填充有deparafinization剂（完全覆盖部分）一高，垂直滑动染色缸中删除从滑动的石蜡。执行的30分钟洗涤3次 - 每次60分钟。 </li><li>取下最后洗的幻灯片。将2滴嵌入媒体到幻灯片上，并用大盖玻片覆盖。 </li></ol> 3.拍摄和分析章节<ol><li>允许制备的玻片干燥1 - 2天。然后，检查它们放在荧光microsc在蓝光下单操作。 </li><li>使用较低的放大率确定苍蝇的方向，如果着眼于一个特定区域，以找到感兴趣的区域。 注:对于SWS苍蝇（ 见图2），我们发现，含有大合缝的部分，并拍摄图像（通常在40倍放大倍率）。当分析整个脑（ 如图3），我们通过从头部所有部分滚动，要么具有最严重的表型，或显示空泡所有节拍摄部分。 </li><li>为双盲分析，采取与数字图像，而不知道基因型并记录行中的套环数和头部的位置以后，以确定它们。 </li><li>一旦图像已被拍摄，使用成像软件进行分析。 </li><li>每计数或部分每头空泡的数量。为了测量液泡的大小，在一个软件程序中打开的图像，并选择与选择的空泡太湖确定在所选择的液泡的像素的量。 </li><li>转换为微米2，取在用于采集照片放大一个阶段微米校准幻灯片的照片。确定象素的数量在100微米2以计算换算因子。 </li><li>通过在上述步骤中计算出的换算因子除以象素数目转换的总像素数成微米2。 </li></ol>

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<li>Kretzschmar, D., Hasan, G., Sharma, S., Heisenberg, M., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+swiss+cheese+mutant+causes+glial+hyperwrapping+and+brain+degeneration+in+Drosophila%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila.</a> J Neurosci. 17, 7425-7432 (1997).</li>
<li>Dutta, S., Rieche, F., Eckl, N., Duch, C., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glial+expression+of+Swiss+cheese+%28SWS%29%2C+the+Drosophila+orthologue+of+neuropathy+target+esterase+%28NTE%29%2C+is+required+for+neuronal+ensheathment+and+function%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">Glial expression of Swiss cheese (SWS), the Drosophila orthologue of neuropathy target esterase (NTE), is required for neuronal ensheathment and function.</a> Dis Model Mech. 9, 283-294 (2016).</li>
<li>Davis, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucocerebrosidase+Deficiency+in+Drosophila+Results+in+alpha-Synuclein-Independent+Protein+Aggregation+and+Neurodegeneration%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration.</a> PLoS Genet. 12, e1005944(2016).</li>
<li>Dias-Santagata, D., Fulga, T. A., Duttaroy, A., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidative+stress+mediates+tau-induced+neurodegeneration+in+Drosophila%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Oxidative stress mediates tau-induced neurodegeneration in Drosophila.</a> J Clin Invest. 117, 236-245 (2007).</li>
<li>Kretzschmar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glial+and+neuronal+expression+of+polyglutamine+proteins+induce+behavioral+changes+and+aggregate+formation+in+Drosophila%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila.</a> Glia. 49, 59-72 (2005).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Drosophila+model+for+TDP-43+proteinopathy%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">A Drosophila model for TDP-43 proteinopathy.</a> Proc Natl Acad Sci U S A. 107, 3169-3174 (2010).</li>
</ol>

作者什么都没有透露。

所描述的方法提供了在果蝇的脑量化的神经变性的方法。而其它的方法，如计数特定的细胞类型，可用于鉴定神经变性，这种方法的优点是，它可被更一般地应用。细胞计数要求这些细胞可以使用一个特定的抗体或细胞特异性标记的表达，这并不总是可用可靠地识别。此外，已经表明，显着不同的结果可以与方法24中获得，这可能是由于用于标记和检测的条件。检测衰退的细胞的另...

随着预期寿命的延长，神经退行性疾病如阿尔茨海默或帕金森已成为普通人群越来越健康威胁。根据美国国立卫生研究院，1.15亿人的全球预计将被老年痴呆症在2050年的影响虽然显著进展，在确定至少参与了一些这些疾病的基因和风险因素，其中许多已经取得，底层分子机制仍未知的或不充分的了解。 像线虫和果蝇简单无脊椎动物模式生物提供了多种实验的优势来研究神经退行性疾病，包括生命周期短，大量的后代，以及行之有效的，有时独特的遗传和分子生物学方法1的可用性机制-12。此外，这些生物是适合于偏互动屏幕，可识别通过神经退行性表型的加重或改善效果有助于这些疾病的因素。 分析这些基因的相互作用和评估老化的影响需要量化的协议，以检测神经功能障碍和衡量其严重程度。测量在果蝇行为方面，如嗅觉学习，负趋地性，或快趋光性，这提供了一个数值性能值13-21时，这种评估可以比较容易地完成。另外，也可以通过计数神经元，以确定对神经元存活的影响。然而，专注于特定人群，这显然是可识别的，像在PD的影响，即使这样，结果一直存在争议22-24多巴胺能神经元时，这是唯一可能的。 这里所描述的协议使用的衣领方法执行石蜡连续切片中，一种方法最初由海森堡和博尔，谁用它在果蝇 25分离解剖大脑突变体的发展。使用该套环方法的随后被改编，包括在冷冻切片，vibratome部分和塑料部分26-28。这里，采用这种方法，得到了整个飞头，然后可将其用于测量在苍蝇开发具有神经变性表型16,21,29-32液泡的连续切片。这些测量可以在特定脑区来完成，或者可以覆盖整个大脑;后一种方法可以让老化过程中观察到一个识别即使很弱的退行性表型。最后，使用套环时，最多20苍蝇可被处理为一个准备，这不仅耗时少，而且还允许用于控制和在相同的制备实验苍蝇的分析，最小化由于在微小变化的工件的制备方法。

<table><tbody><tr><td>Name of the Reagent/Equipment</td><td>Company</td><td>Catalog Number</td><td></td></tr><tr><td>Collar</td><td>Genesee Scientific</td><td>TS 48-100</td><td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A description on how to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&amp;nbsp;</td></tr><tr><td>Rubber ice cube tray for embedding</td><td>Household store</td><td></td><td>The size can be made-to-fit by glueing in additional walls&amp;nbsp;</td></tr><tr><td>Crystallizing dish</td><td>Fisher Scientific company</td><td>08-762-3</td><td></td></tr><tr><td>Ether</td><td>Fisher Scientific Company</td><td>E138-1</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories Inc.</td><td>2701</td><td></td></tr><tr><td>Choloroform</td><td>Fisher Scientific Company</td><td>C298-500</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher Scientific Company</td><td>A38-212</td><td></td></tr><tr><td>Methylbenzoate</td><td>Fisher Scientific Company</td><td>M205-500</td><td>Distinct Odor - Use in fume hood!</td></tr><tr><td>Low Melting Point Paraffin Wax</td><td>Fisher Scientific Company</td><td>T565</td><td>Make sure to keep extra melted in a 65&amp;deg;C waterbath</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>Reichert Jung 2040 Autocut</td><td></td></tr><tr><td>Microscope Slide</td><td>Fisher Scientific Company</td><td>12-550D</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific Company</td><td>12-545-M</td><td></td></tr><tr><td>SafeClear</td><td>Fisher Scientific Company</td><td>314-629</td><td>Three different containers for washes</td></tr><tr><td>Vertical Staining Jar with Cover</td><td>Ted Pella Inc.&amp;nbsp;</td><td>432-1</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific Company</td><td>SP15-500</td><td></td></tr><tr><td>Poly-L-Lysine Solution (PLL)</td><td>Sigma Life Science</td><td>P8290-500</td><td></td></tr></tbody></table>

mass histology to quantify neurodegeneration in drosophila

进行性神经变性疾病如阿尔茨海默氏病（AD）或帕金森氏病（PD）是全世界人类健康日益严重的威胁。虽然哺乳动物模型提供了重要的见解致病的内在机制，在哺乳动物系统与他们的成本高一起的复杂性限制了其使用。因此，简单而完善的果蝇模型的系统提供了用于调查了在这些疾病影响的分子通路的替代方案。除了行为缺陷，神经变性疾病是由组织学表型，如神经元死亡和轴突病变表征。量化神经元变性，并确定它是如何由遗传和环境因素的影响，我们使用一个基于在成年果蝇大脑测量液泡组织学方法。为了尽量减少系统误差的影响，并直接比较从控制和记录部分在一个制备erimental苍蝇，我们使用石蜡切片的"衣领"方法。神经变性，然后通过测量已经在飞脑开发液泡的大小和/或数量的评估。这可以通过关注感兴趣的特定区域，或通过获得跨越完整头连续切片分析整个大脑来完成。因此，该方法允许人们测量不仅严重退化，而且在相对温和的表型是只在少数部分检测的，正常的老化期间发生。

使用在由眼睛颜料33包括整个飞头染色的连续切片所描述的方法的结果。这方面的一个部分示于图1B中 ，其中，来自个体头的部分被示出从上到下。从不同的苍蝇的部分被视为左至右在本实施例。为了便于取向和苍蝇的鉴定，无眼蝇（ 正弦oculis）被插入为在3位（箭头， 图1B）的标志物。 <p class="jove_content" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

果蝇被广泛用作模型系统来研究神经变性。这个协议描述由退化，如由大脑液泡形成确定的，可量化的方法。它也最大限度地减少的效果因的实验程序由处理和切片对照和实验苍蝇，因为一个样本。

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 ...

mass-histology-to-quantify-neurodegeneration-in-drosophila

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

10.1K 次观看.  Oregon Health & Sciences University.  果蝇被广泛用作模型系统来研究神经变性。这个协议描述由退化，如由大脑液泡形成确定的，可量化的方法。它也最大限度地减少的效果因的实验程序由处理和切片对照和实验苍蝇，因为一个样本。 

视频: Mass Histology to Quantify Neurodegeneration 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

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

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 ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

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 ...

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 ...

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 ...

Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression patterns. Traditional methods often involve challenges such as performing complex dissection, maintaining tissue integrity, and visualizing specific expression patterns during high-resolution imaging. We present an optimized protocol that combines cryosectioning with fluorescence staining and immunostaining. This method improves tissue preservation and signal clarity and reduces the need for laborious dissection for Drosophila brain imaging. The method entails rapid dissection, optimal fixation, cryoprotection, and cryosectioning, followed by fluorescent staining and immunostaining. The protocol significantly reduces tissue damage, enhances antibody penetration, and yields sharp, well-defined images. We demonstrate the effectiveness of this approach by visualizing specific neural populations and synaptic proteins with high fidelity. This versatile method allows for the analysis of various protein markers in the adult brain across multiple z-planes and can be adapted for other tissues and model organisms. The protocol provides a reliable and efficient tool for researchers conducting high-quality immunohistochemistry in Drosophila neurobiology studies. This method's detailed visualization facilitates comprehensive analysis of neuroanatomy, pathology, and protein localization, making it particularly valuable for neuroscience research.

This study presents a simplified protocol for tissue processing involving decapitation, fixation, cryosectioning, fluorescence staining, immunostaining, and imaging, which can be extended to confocal and multiphoton imaging. The method maintains efficacy comparable to complex dissections, bypassing the need for advanced motor skills. Quantitative image analysis provides extensive investigative potential.

果 蝇 头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression ...

Assessing the Structural Integrity of the Dorsal Longitudinal Muscle Neuromuscular Junctions in Drosophila

Source: Sidisky, J. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61363">Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration.</a> J. Vis. Exp. (2020).
This video demonstrates a protocol for assessing the structural integrity of neuromuscular junctions in the dorsal longitudinal muscles of Drosophila. The procedure involves staining neurons and muscles with two distinct antibodies and comparing fluorescence patterns between mutant and wild-type flies. A reduction in neuronal staining in mutant flies indicates neurodegeneration, while the muscle structure remains intact.

评估果蝇背纵肌神经肌肉接头的结构完整性

1. Flash freezing and thorax bisection

	Before beginning the bisections, fill a Dewar flask with liquid nitrogen wearing proper cryo-protective gloves ...

大众组织学量化在神经退行性变

摘要

探索更多视频

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

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

一个简单的神经元机械损伤方法学研究

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

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

与神经退化有关的成人果蝇的可视化突触变性

果蝇头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

评估果蝇背纵肌神经肌肉接头的结构完整性

大众组织学量化在神经退行性变

摘要

探索更多视频

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

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

一个简单的神经元机械损伤方法学研究

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

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

与神经退化有关的成人 果 蝇的可视化突触变性

果 蝇 头部的直接冷冻切片，用于增强脑荧光染色和免疫染色

评估果蝇背纵肌神经肌肉接头的结构完整性

与神经退化有关的成人果蝇的可视化突触变性

果蝇头部的直接冷冻切片，用于增强脑荧光染色和免疫染色