Name: dissection and imaging of active zones in the drosophila neuromuscular junction
Uploaded: 2011-04-27T16:00:00
Description: Watch this Scientific Journal Video about Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction at JoVE.com

我们感谢他们对这个手稿的有益的意见Nael阿拉米博士和博士南澈金。

1。对免疫的筹备工作： <ol><li>要创建一个解剖面，倒入一个小的组织培养板Sylgard 184硅橡胶基地。确保不来填充板完全使清扫面积低于轮辋。 </li><li>修剪清扫引脚易于操纵增加了约3.5毫米长度，并确保至少有6％的幼虫引脚。 </li><li>您将需要钝钳，让你掌握清扫引脚。 </li></ol> 2。解剖的幼虫： <ol><li>选择流浪三龄幼虫蠕动着小瓶双方还没有开始化蛹。 </li><li>广场上的立体显...

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	<li>Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+maturation+and+structural+plasticity+at+Drosophila+neuromuscular+junctions.">Synapse maturation and structural plasticity at Drosophila neuromuscular junctions.</a> Curr Opin Neurobiol. 6, 858-867 (1996).</li><li>Fouquet, W., Owald, D., Wichmann, C., Mertel, S., Depner, H., Dyba, M., Hallermann, S., Kittel, R. J., Eimer, S., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+active+zone+assembly+by+Drosophila+Bruchpilot.">Maturation of active zone assembly by Drosophila Bruchpilot.</a> J Cell Biol. 186, 129-145 (2009).</li><li>Hoang, B., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Analysis+of+Drosophila+Larval+Neuromuscular+Synapses.">Single-Cell Analysis of Drosophila Larval Neuromuscular Synapses.</a> Developmental Biology. 229, 55-70 (2001).</li><li>Keshishian, H., Kim, Y. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+development+and+function:+retrograde+BMP+signaling+in+the+Drosophila+nervous+system.">Orchestrating development and function: retrograde BMP signaling in the Drosophila nervous system.</a> Trends in Neurosciences. 27, 143-147 (2004).</li><li>Kittel, R. J., Wichmann, C., Rasse, T. M., Fouquet, W., Schmidt, M., Schmid, A., Wagh, D. A., Pawlu, C., Kellner, R. R., Willig, K. I., Hell, S. W., Buchner, E., Heckmann, M., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruchpilot+Promotes+Active+Zone+Assembly,+Ca2++Channel+Clustering,+and+Vesicle+Release.">Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release.</a> Science. 312, 1051-1054 (2006).</li><li>Koh, Y. H., Gramates, L. S., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+neuromuscular+junction:+Molecular+components+and+mechanisms+underlying+synaptic+plasticity.">Drosophila larval neuromuscular junction: Molecular components and mechanisms underlying synaptic plasticity.</a> Microscopy Research and Technique. 49, 14-25 (2000).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann N Y Acad Sci. 1184, e1-e20 (2010).</li><li>Ratnaparkhi, A., Lawless, G. M., Schweizer, F. E., Golshani, P., Jackson, G. R. <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+ALS:+human+ALS-associated+mutation+in+VAP33A+suggests+a+dominant+negative+mechanism.">A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism.</a> PLoS One. 3, e2334-e2334 (2008).</li><li>Wagh, D. A., Rasse, T. M., Asan, E., Hofbauer, A., Schwenkert, I., D&#252;rrbeck, H., Buchner, S., Dabauvalle, M. -. C., Schmidt, M., Qin, G., Wichmann, C., Kittel, R., Sigrist, S. J., Buchner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruchpilot,+a+Protein+with+Homology+to+ELKS/CAST,+Is+Required+for+Structural+Integrity+and+Function+of+Synaptic+Active+Zones+in+Drosophila.">Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila.</a> Neuron. 49, 833-844 (2006).</li><li>Zweier, C., Jong, E. K. d. e., Zweier, M., Orrico, A., Ousager, L. B., Collins, A. L., Bijlsma, E. K., Oortveld, M. A., Ekici, A. B., Reis, A., Schenck, A., Rauch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNTNAP2+and+NRXN1+are+mutated+in+autosomal-recessive+Pitt-Hopkins-like+mental+retardation+and+determine+the+level+of+a+common+synaptic+protein+in+Drosophila.">CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila.</a> Am J Hum Genet. 85, 655-666 (2009).</li></ol>

神经元突触终端领域是至关重要的，是适当的沟通后，突触前细胞之间的桥梁。调查疾病模型的神经元的健康，是一种强有力的方式分析，免疫荧光蛋白质的突触终端。这里介绍的免疫荧光法，使研究者检查许多幼虫的同时，限制群体之间的环境差异。 果蝇三龄幼虫的中央神经系统有许多优点，包括谷氨酸的突触，突触末端，交通方便，重现性和遗传操作的权力映射和重复。特别在果?...

<table border="1"><tbody><tr><td>试剂名称</td><td>公司</td><td>目录编号</td><td>评论</td></tr><tr><td> Sylgard 184有机硅弹性体基地</td><td>道康宁</td><td> 68037-59-2 </td><td>混合后让气泡上升缓慢肩或允许坐30分钟或更慢了。 </td></tr><tr><td>不锈钢Minutien引脚</td><td>精细科学的工具</td><td> 26002-10 </td><td>修剪到约。 3 - 4mm的长度与普通剪刀</td></tr><tr><td>椎板钳（抓销钝） </td><td>精细科学工具</td><td> 11223-20 </td><td>使用钝钳抓销</td></tr><tr><td>解剖钳</td><td>世界精密仪器</td><td> 501985 </td><td></td></tr><tr><td>超炫Vannas剪刀，8厘米长</td><td>世界精密仪器</td><td> 501778 </td><td></td></tr><tr><td>鼠抗BRP抗体</td><td> DSHB </td><td> NC82 </td><td>使用1:50稀释</td></tr><tr><td> Cy3标记Affinipure山羊抗辣根过氧化物酶</td><td>杰克逊Immunoresearch </td><td> 123-165-021 </td><td>用1:200稀释</td></tr><tr><td>的Alexa Fluor 488山羊抗鼠IgG </td><td> Invitrogen公司</td><td> A11001 </td><td>使用约。 1:200稀释</td></tr></tbody></table>

dissection and imaging of active zones in the drosophila neuromuscular junction

果蝇幼虫神经肌肉接头（NMJ）突触的结构和功能研究的一个很好的模型。果蝇是缓解强大的遗传操作和幼虫的神经系统已被证明不仅在学习的正常功能特别有用，但也扰动伴随一些神经系统疾病（Lloyd和泰勒，2010）。在果蝇中发现的许多关键的突触分子还发现，在哺乳动物中，最喜欢在哺乳动物中枢神经系统兴奋性突触，果蝇NMJ是谷氨酸和演示活动依赖的重塑（Koh等 ， 2000）。此外， 果蝇的神经细胞可以单独确定，因为他们的神经支配模式都是千篇一律和重复，使我们能够研究确定的突触终端，如运动神经元和体壁肌纤维之间，他们支配（Keshishian和Kim ，2004）。进化上保守的突触组件以及与遗传和体质操纵缓解的存在， 使得果蝇调查的内在机制，突触功能的（Budnik 1996）的理想模型。 在突触终端的活动区域特别感兴趣，因为这些神经递质释放的网站。 NC82是一种单克隆抗体，能够识别的果蝇蛋白Bruchpilot（BRP），CAST1/ERC家庭成员的活动区（Wagh 等人 ，2006年），是一个重要组成部分。 BRP是直接形成活动区的T -酒吧，并负责有效聚类的Ca 2 +通道下方的T -酒吧密度（富凯等人，2009年） 。 BRP突变体减少的Ca 2 +通道密度，郁闷诱发囊泡释放，并改变短期的可塑性（ 基特尔等，2006） 。活跃区的改建已发现在果蝇的疾病模型。例如，使用的NC82抗体的免疫荧光显示，活跃的区域密度下降在肌萎缩性脊髓侧索硬化症和皮特霍普金斯综合征（Ratnaparkhi等，2008; Zweier等，2009）。模型。因此，活跃的区域，或其他疾病的果蝇幼虫模型，突触蛋白的评价可能会提供一个宝贵的初步线索，一个突触缺陷的存在。 准备整个安装解剖果蝇幼虫的免疫分析NMJ需要一些技巧，但可以稍加练习，大多数科学家所完成。是提供多个幼虫在同一解剖盘解剖和免疫染色方法，限制各基因型之间的环境差异，并提供足够的动物重现性和统计分析的信心。

Watch this Scientific Journal Video about Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction at JoVE.com

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

神经肌肉接头（NMJ）<em&gt;果蝇</em&gt;是一个重要的模型系统，为研究正常的突触功能以及某些神经系统疾病中发现的突触功能的扰动。我们提出了一个解剖的协议<em&gt;果蝇</em&gt;幼虫电机系统和免疫活性区蛋白内NMJ。

在活动区域​​的解剖和影像果蝇神经肌肉交界处

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Research

JoVE Journal

Neuroscience

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Single Drosophila Ommatidium Dissection and Imaging

Single Drosophila Ommatidium Dissection and Imaging

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

科研

神经科学

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

为什么量化事项：表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Measuring Neuromuscular Junction Functionality

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

两种算法的高通量和多参数定量<em&gt;果蝇</em&gt;神经肌肉接头形态

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

果蝇成腿的解剖和免疫组织化学，以检测已识别运动神经元的神经肌肉接头处的变化

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

在活动区域​​的解剖和影像果蝇神经肌肉交界处

在活动区域的解剖和影像果蝇神经肌肉交界处