Name: measuring constipation in a drosophila model of parkinson's disease
Uploaded: 2023-09-22T14:40:00.000+00:00
Duration: 3 min 20 s
Description: Watch this Scientific Journal Video about Exploring Non-Motor Symptoms in Parkinson's Disease at JoVE.com

我们感谢布莱根妇女医院和哈佛医学院的 Mel Feany 博士对照和表达 果蝇 系的 α-突触核蛋白的善意礼物。我们感谢以下对 Olsen 博士的资助来源:NINDS K08、美国帕金森病协会 George C. Cotzias 奖学金、国防部帕金森病早期研究者奖。

用于该测定的果蝇:对照:nSyb-QF2、nSyb-Gal4/+;α-突触核蛋白果蝇:nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+;关闭后 1 天和 10 天;雄性和雌性（交配和未交配）苍蝇（见 材料表）。
1.准备染色的苍蝇食品
<ol><li>将蓝色软凝胶糊食用色素（见 材料表）与蒸馏水以 1:1 的比例 （v/v） 混合。 注意:使用仅含有不可吸收染料的商业食用色素，因为一些蓝色染料已被证明具有神经保护作用21.</li>
	<li>以 10-15 秒的间隔用微波炉加热由标准玉米面 - 琼脂（见 材料表）组成的苍蝇食品小瓶，直到食物融化成液体或浆液。不要让食物沸腾。</li>
	<li>将染料混合物添加到每瓶食物中，以获得小瓶之间的均匀着色。混合在染料混合物中，直到食物呈均匀的蓝色（图1）。加入一定量的蓝色染料混合物，使食物的颜色饱和，并且小瓶之间的颜色没有变化。 注意: 此步骤必须在微波炉加热后立即完成，因为食物会很快凝固。如果食物凝固，请再次用微波炉加热小瓶。</li>
	<li>将染色食品小瓶风干直至凝固。干燥时将薄纸巾放在小瓶开口上，以防止任何流浪苍蝇落在小瓶中。</li>
	<li>食物冷却凝固后，将用于测试的苍蝇转移到蓝色食物中。 注意:对于窄瓶（25 毫米），建议在每个小瓶中添加 9-14 只苍蝇。对于宽小瓶（28.5 mm），建议在每个小瓶中加入10-20只苍蝇。苍蝇不按性别或交配经验分开，包括雄性和雌性苍蝇的混合种群，包括交配和未交配的雌性。</li>
	<li>将小瓶与苍蝇一起在培养箱中25°C孵育过夜。</li>
</ol>2. 对苍蝇进行成像
注意:此步骤是可选的（图 2）。
<ol><li>第二天早上（时间0），用二氧化碳麻醉代表苍蝇60秒。放置苍蝇，使腹侧朝上。</li>
	<li>使用相机（见 材料表），每小时捕捉苍蝇的图像，以可视化消化系统中剩余的蓝色食物量。 注意:这些苍蝇不应用于测定的定量部分（步骤3），因为麻醉可能会影响结果。</li>
	<li>每小时对新苍蝇进行麻醉和成像。</li>
</ol>3.进行便秘测定
<ol><li>将剩余的（未麻醉的）苍蝇转移到装有标准 果蝇 食物的小瓶中。对每个小瓶进行编号。</li>
	<li>将苍蝇留在25°C的培养箱中60分钟。</li>
	<li>60分钟后，将苍蝇转移到装有标准 果蝇 食物的新小瓶中。从第一组样品瓶开始数据记录。</li>
	<li>在小瓶的长度上画一条虚线，以标记开始计数的点。</li>
	<li>手动计算每个小瓶壁上小圆点的数量。不要计算在食物或小瓶塞上发现的点。</li>
	<li>首先计算每个小瓶壁上的蓝点数量。</li>
	<li>计算每个小瓶壁上不透明的无色点的数量。 注意:每个点是苍蝇粪便，它是尿液和粪便的组合 22.有时，苍蝇可能会走过粪便并留下痕迹，在这种情况下，只计算原始点，而不是痕迹中的每个标记。</li>
	<li>记录每个小瓶的蓝点与点总数的比率。 注意:点总数是小瓶壁上的蓝点数与同一小瓶壁上的无色点数相加。这将用于计算每小时蓝色粪便的百分比。</li>
	<li>重复步骤 3.2-3.8 七次，每小时收集一次数据，总共 8 小时。</li>
</ol>4. 数据分析
<ol><li>绘制每种情况下每小时蓝色粪便的百分比。</li>
	<li>比较条件之间的数据。 注意:统计显著性可以通过 3 种方式确定:通过比较条件之间的各个时间点、测量线随时间变化的斜率或比较曲线下的面积。</li>
</ol>

果蝇中的便秘测定

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作者没有什么可透露的。

该协议中有几个步骤将有助于成功完成测定。首先，重要的是要确保每个小瓶的每轮之间的时间间隔在整个实验过程中保持一致。用数字标记小瓶将有助于避免冗长的基因型描述，从而节省时间。此外，在整个实验过程中，粪便计数方法22 保持一致至关重要。虽然在食物和小瓶塞上可以看到蓝色粪便，但无色粪便则不然。因此，不要计算食物或小瓶塞上的蓝点。
在行为测定中，由于苍蝇行为的波动或影响测定的未知变量，结果总是存在不一致的可能性。我们建议在所有实验中使用相同的 果蝇 培养基、相同的食用色素和相同品牌的小瓶。有趣的是，在几项试验中，观察到苍蝇在下午早些时候排便的频率较低，这可能是由于苍蝇的昼夜节律23.然而，这种行为在对照果蝇和表达α-突触核蛋白的果蝇中是一致的，因此不应引起关注。
如果苍蝇在测定开始时没有排泄蓝色粪便，则可能使用的染料色素沉着不足。在这种情况下，可以相应地增加染料与蒸馏水的比例。也有可能当苍蝇的消化道中只剩下少量蓝色食物时，可能很难确定粪便是淡蓝色还是无色。发生这种情况时，在小瓶后面放一张白纸将有助于确定粪便点的颜色。即使粪便是很浅的蓝色，最好将其记录为蓝色粪便而不是无色粪便。
该检测的一个局限性是它需要手动计数粪便斑点。为了提高高通量筛选的潜力，该方案将来可能会被修改，以允许自动定量多孔板中单个果蝇产生的蓝色粪便斑点。另一个局限性是，尽管 α-突触核蛋白模型有可能发展成为 PD 的前驱模型，但尚未确定存在便秘而没有神经退行性变的最佳时间点。
总之，这种方法提供了一种简单、直接的方法来模拟果蝇模型中的便秘，便秘是一种未被充分研究的非运动性 PD 症状。

帕金森病 （PD） 是一种进行性神经退行性疾病，临床特征是存在运动迟缓、强直和震颤等运动症状，导致显着的发病率1。在病理学上，PD的定义是黑质中多巴胺能神经元的丢失和α-突触核蛋白的错误折叠，导致路易体和路易神经突的形成。帕金森病的发病机制仍然知之甚少，可能是由遗传和环境因素的复杂相互作用引起的 2,3。目前，尚无改善疾病的疗法，部分原因可能是诊断时病理学已处于晚期。研究表明，黑质中超过 60% 的多巴胺能神经元已经因运动症状的发作而丢失，这强调了探索早期疾病检测的潜在生物标志物的必要性4.便秘就是这样一种临床生物标志物，这在帕金森病患者中很常见 5,6，并且可能在运动症状出现之前数年甚至数十年。
尽管 PD 的临床定义基于运动症状，但便秘是几种非运动症状之一，这些症状对症治疗更具挑战性，并导致患者生活质量的显着损害7.代表大脑和肠神经系统之间双向交流的肠脑轴的改变与帕金森病的发病机制有关。在帕金森病患者胃肠道的组织样本中发现了 α-突触核蛋白聚集体8，动物模型表明肠道神经系统中的 α-突触核蛋白聚集体以朊病毒样方式扩散到中枢神经系统9。此外，帕金森病患者在肠道微生物组中表现出异常10，并可能经历过度的肠道炎症11。帕金森病的便秘研究不足，很少有报道的苍蝇 12,13 或啮齿动物14,15 中帕金森氏症相关便秘的模型。
该测定采用果蝇 PD 模型，其中果蝇在泛神经元驱动因子 n-突触反射蛋白的控制下表达人类 α-突触核蛋白基因。这些果蝇表现出 PD 的所有标志性特征，包括 α-突触核蛋白聚集、运动功能障碍和与年龄相关的神经退行性变，导致多巴胺能神经元的丢失16,17。以前的研究引入了测量果蝇粪便输出量以评估肠道功能障碍，量化果蝇粪便并比较各种遗传系的排泄物量，以揭示消化系统的功能差异18,19,20。在这里，我们演示了使用表达人α-突触核蛋白的果蝇的便秘测定。这个简单而有价值的工具可以研究帕金森病的重要非运动症状。

<table><tbody><tr><td>1400 g sucrose</td><td>MP Biomedicals</td><td>904713</td><td></td></tr><tr><td>1800 g dextrose</td><td>MP Biomedicals</td><td>901521</td><td></td></tr><tr><td>2884 g yeast</td><td>MP Biomedicals</td><td>903312</td><td></td></tr><tr><td>428 g agar</td><td>Fisher Scientific</td><td>10253156</td><td></td></tr><tr><td>4600 mL molasses</td><td>Grandma&#039;s Molasses</td><td>7971942</td><td></td></tr><tr><td>68 L water</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>680 mL tegosept mix (1200 g tegosept in 6 L ethanol)</td><td></td><td></td><td></td></tr><tr><td>6864 g cornmeal</td><td>Pearl Milling</td><td>125045</td><td></td></tr><tr><td>800 mL acid mix (83 mL phosphoric acid in 1 L water + 836 mL propionic acid in 1 L water)</td><td></td><td></td><td></td></tr><tr><td>cellSens Standard software</td><td>Olympus</td><td>N/A</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco-Aper</td><td>111ACS200</td><td></td></tr><tr><td>Flugs for wide plastic vials</td><td>Genesee Scientific</td><td>49-101</td><td></td></tr><tr><td>Flystuff wide Drosophila vials, polystyrene</td><td>Genesee Scientific</td><td>32-117</td><td></td></tr><tr><td>Graphpad Prism</td><td>GraphPad</td><td>N/A</td><td>Version 9.5.1</td></tr><tr><td>Olympus DP23 camera</td><td>Olympus</td><td>N/A</td><td></td></tr><tr><td>Olympus SZX12 Stereo Microscope</td><td>Olympus</td><td>N/A</td><td></td></tr><tr><td>Phosphoric Acid</td><td>Fisher Scientific</td><td>S25470A</td><td></td></tr><tr><td>Propionic Acid</td><td>Fisher Scientific</td><td>A258 - 500&amp;nbsp;</td><td></td></tr><tr><td>Soft gel paste food color, Royal blue</td><td>AmeriColor</td><td>202</td><td></td></tr><tr><td>Tegosept</td><td>Apex</td><td>20-258</td><td></td></tr><tr><td>&lt;em&gt;&lt;strong&gt;Drosophila Stocks&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;nSyb-QF2, nSyb-Gal4&lt;/em&gt;</td><td>All lines provided by Dr. Mel Feany</td><td>N/A</td><td>Lines are available directly from Dr. Feany&amp;nbsp;</td></tr><tr><td>&lt;em&gt;nSyb-QF2, nSyb-Gal4, QUAS-alpha synuclein&lt;/em&gt;</td><td></td><td>N/A</td><td></td></tr><tr><td>&lt;em&gt;w1118&lt;/em&gt;</td><td></td><td>N/A</td><td></td></tr></tbody></table>

measuring constipation in a drosophila model of parkinson's disease

帕金森病 （PD） 的非运动症状很常见，难以治疗，并且会严重损害生活质量。一种普遍的非运动症状是便秘，它可能比诊断为帕金森病早数年甚至数十年。便秘在帕金森病的动物模型中尚未得到充分探索，并且缺乏特异性治疗。该测定利用了 PD 的 果蝇 模型，其中人 α-突触核蛋白在泛神经元驱动下表达。表达α-突触核蛋白的果蝇具有帕金森病的标志性特征:多巴胺能神经元的丧失、运动障碍和α-突触核蛋白包涵体。
该协议概述了一种研究这些苍蝇便秘的方法。将苍蝇放在带有蓝色添加剂的苍蝇食物上过夜，然后在第二天转移到标准食物中。随后将它们移至装有标准苍蝇食品的新小瓶中，每小时持续 8 小时。在每次转移之前，计算蓝色粪便斑点与小瓶壁上总粪便斑点的百分比。缺乏α-突触核蛋白的对照果蝇在表达α-突触核蛋白的果蝇之前数小时排出所有蓝色染料。此外，蓝色食物从肠道的通过可以通过简单的摄影来监测。该测定的简单性使其能够用于正向遗传或化学筛选，以鉴定 果蝇便秘的修饰剂。

因为果蝇的腹部是透明的，所以在活的麻醉果蝇中，可以在肠道内看到蓝色食物。可以通过在不同时间点拍摄苍蝇的图像来评估肠道运输的定性差异。在对照果蝇中，蓝色食物会迅速被排出，而在α-突触核蛋白果蝇中，蓝色食物在肠道中停留长达8小时（图2）。
α-突触核蛋白果蝇表现出与年龄相关的神经变性，在闭合后 10 天形成健壮的表型10。在这个时间点，通过比较条件之间的各个时间点，通过测量线随时间变化的斜率或比较曲线下的面积，可以看到与对照果蝇相比肠道运输时间的差异（图3）。在较早的时间点，即闭合后第1天，在对照果蝇和α-突触核蛋白果蝇之间的肠道运输中没有观察到差异，此时运动功能障碍和神经退行性变也尚未存在（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65966/65966fig1v2.jpg"> 图 1:蓝色食品瓶和粪便斑点。 （A） 所有小瓶都含有蓝色食物。此时，苍蝇刚刚被引入蓝色食物，看不到蓝色粪便。（B） 将 w1118 苍蝇转移到新鲜食物中 1 小时后的小瓶，然后取出。左边的插图在（C）中放大，右边的插图在（D）中放大。（C） 由于存在苍蝇食物而被排除在分析之外的区域。（D） 粪便斑点的放大视图，表示蓝色斑点（黑色箭头）和非蓝色斑点（黄色箭头）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65966/65966fig1v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65966/65966fig02.jpg"> 图2:染色食品肠道运输随时间变化的可视化。 对照果蝇和在神经元中表达人类α-突触核蛋白的果蝇在染色的食物上过夜，然后在第二天转移到标准培养基中。苍蝇每小时被连续转移到新的标准苍蝇食物中。图中显示了时间零和转印后 2、4、6 和 8 小时的代表性图像。对照组和α-突触核蛋白果蝇在零点时在肠道中显示出类似的蓝色食物斑块。在随后的时间点，对照果蝇的蓝色食物比α-突触核蛋白果蝇少，并且在对照果蝇的8小时时间点，所有蓝色食物都消失了。各蝇系的基因型如下:对照（nSyb-QF2、nSyb-Gal4/+）;α-突触核蛋白苍蝇（nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+）。雌性苍蝇用于成像，因为它们的体型较大，更容易可视化腹部。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65966/65966fig2v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65966/65966fig3v2.jpg"> 图 3:苍蝇闭合后第 10 天的便秘测定。 对照苍蝇和在神经元中表达人类α-突触核蛋白的果蝇在染成蓝色的食物上过夜，然后在第二天转移到标准苍蝇食物中。苍蝇每小时连续转移到新的标准培养基上。（A） 每小时蓝色粪便的百分比。误差线表示标准偏差。N = 每个基因型 6 瓶;每个小瓶含有 9-14 只苍蝇。所有统计分析均在Graphpad Prism中进行（参见 材料表）。使用双因素方差分析计算每个时间点的统计显着性。使用线性回归分析计算线的斜率和斜率之间的差值。（B） 计算每种基因型的曲线下面积。误差线表示标准偏差。各蝇系的基因型如下:对照（nSyb-QF2、nSyb-Gal4/+）;α-突触核蛋白苍蝇（nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+）。雄性和雌性苍蝇的数量大致相等。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65966/65966fig3v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65966/65966fig4v2.jpg"> 图 4:苍蝇闭合后第 1 天的便秘测定。 对照果蝇和在神经元中表达人类α-突触核蛋白的果蝇在染色的食物上过夜，然后在第二天转移到标准培养基中。苍蝇每小时被连续转移到新的标准苍蝇食物中。（A） 每小时蓝色粪便的百分比。误差线表示标准偏差。N = 每种基因型至少 5 瓶;每个小瓶含有 9-14 只苍蝇。所有统计分析均在Graphpad Prism中进行。使用双因素方差分析计算每个时间点的统计显着性。使用线性回归分析计算线的斜率和斜率之间的差值。（B） 计算每种基因型的曲线下面积。误差线表示标准偏差。各蝇系的基因型如下:对照（nSyb-QF2、nSyb-Gal4/+）;α-突触核蛋白苍蝇（nSyb-QF2、nSyb-Gal4、QUAS-α-突触核蛋白/+）。雄性和雌性苍蝇的数量大致相等。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65966/65966fig4v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Exploring Non-Motor Symptoms in Parkinson's Disease at JoVE.com

作者聚焦:探索帕金森病的非运动症状 

该协议提出了一种用于在基于α-突触核蛋白的帕金森病 果蝇 模型中模拟便秘的测定方法。

在帕金森病的 果蝇 模型中测量便秘

Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom ...

帕金森病的定义是运动迟缓、震颤和僵硬等运动症状。但许多帕金森病患者患有非运动症状，这些症状更难治疗，并导致更大的生活质量损害。这些尚未在该疾病的动物模型中得到很好的研究，这限制了我们为它们寻找新疗法的能力。该协议简单且价格低廉，但可产生可重复的结果。它易于学习，不需要专门的设备。通过这种简单的检测，我们可以评估患有神经退行性变的果蝇的便秘。未来，我们希望开发一种模型，其中苍蝇在神经退行性变发作之前会出现便秘。这将使我们能够更好地了解帕金森病的发病机制。

measuring-constipation-in-a-drosophila-model-of-parkinson-s-disease

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Measuring Constipation in a Drosophila Model of Parkinson's Disease

Unit 1

Drugs Affecting Gastrointestinal Motility

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1.4K 次观看.  University of Pittsburgh. 帕金森病的定义是运动迟缓、震颤和僵硬等运动症状。但许多帕金森病患者患有非运动症状，这些症状更难治疗，并导致更大的生活质量损害。这些尚未在该疾病的动物模型中得到很好的研究，这限制了我们为它们寻找新疗法的能力。该协议简单且价格低廉，但可产生可重复的结果。它易于学习，不需要专门的设备。通过这种简单的检测，我们可以评估患有神经退行性?...

视频: 作者聚焦:探索帕金森病的非运动症状 

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

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

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 ...

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

方法来描述自发和惊吓引起的步态中的鱼藤酮诱导的帕金森病模型<em&gt;果蝇</em

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

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 ...

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 ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

测量果蝇的作物运动性和食物通度

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

在帕金森病的果蝇模型中测量便秘

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含量的运动能力，学习和记忆的赤字果蝇模型

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

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

方法来描述自发和惊吓引起的步态中的鱼藤酮诱导的帕金森病模型

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

大众组织学量化在神经退行性变

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

测量果蝇的作物运动性和食物通度

在帕金森病的 果蝇 模型中测量便秘

副本

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

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

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

方法来描述自发和惊吓引起的步态中的鱼藤酮诱导的帕金森病模型

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

大众组织学量化在神经退行性变

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

测量果蝇的作物运动性和食物通度

在帕金森病的果蝇模型中测量便秘

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取