我们感谢理化学研究所的资金。 P10 - GAL4阿兰文森特（保罗萨巴蒂尔大学，法国图卢兹）是一种礼物。

1。试剂的制备 注:在开始之前进行解剖和免疫组化染色在磁场室和幼虫是使用特殊形状的昆虫引脚牵制。磁性室的建设，编制这些引脚的详细说明， 可以发现在14,15相关文献。简单地说，一个1x1cm方孔切成磁性表贴表，使一个小室的后部盖玻片。双方室是密封的环氧树脂胶，这种胶后室是用70％乙醇使用前清洗几次。准备解剖昆虫引脚弯曲所需要的形状，然后粘到金属选项卡14,15。另外一个金属片，我们从切断黄色提示作出了处理使用一个倒置的平头钢图钉。利用这个磁场室安排允许严密控制OVER引脚定位和组织在夹层延伸。 为了推动在不同子集的DA能神经元调查报告基因的表达，可以使用几种不同的Gal4的线（下野和他的同事 16总结） 。许多这些线路都可以从公共储备中心。在此代表的协议，我们开展免疫这两个DA神经元对比类是共同标记的行:最简单的类，我和最复杂的一流的四（P10 - Gal4的17,18，UAS - mCD8:: Kusabira橙色（KO））。 <ol><li>准备CA +无HL3.1生理盐水<ol><li> （毫米）:70氯化钠，氯化钾，20氯化镁2，碳酸氢钠3，5 HEPES，115蔗糖，5海藻糖; pH值7.2 19 10 5。过滤，消毒，并储存于4 ° C。注意:CA + +免费的解决方案可以防止肌肉收缩，在清扫。 </li></ol></li><li>准备2X PHEM缓冲区<ol><li> （毫米）:130管，60 HEPES，EGTA，20日4硫酸镁4，pH值7.0。过滤，消毒，并储存于4 ° C。 注:这些材料不会解散，直到pH值接近7.0。 </li></ol></li><li>新鲜立即准备固定液前固定。 <ol><li>为了准备在50毫升猎鹰管一25ml的解决方案，第一个混合2G多聚甲醛，100μL1M NaOH溶液和10ml水。 </li><li>在55℃，摇床水浴摇固定液，直到解决方案是明确的。 （总体积约11.5毫升）。 </li><li>在冰酷固定液。 </li><li>添加12.5毫升2X PHEM缓冲区。 </li><li> 1M盐酸调整pH值至7.0。 </li><li>水填充的解决方案，直到卷25毫升。 </li><li>过滤器使用的Whatman纸解决方案。 </li></ol></li></ol> 2。幼虫清扫 开始前的注意事项:微管网络，并特别是那些在感官树突，将迅速击穿后启动夹层。 AChieving在不到五分钟之内立即固定的快速清扫是在本议定书的成功的关键因素。 <ol><li>洗净的幼虫在水中快速移动到使用的头发循环下降。 </li><li>东方的幼虫背侧和腹侧方在玻璃上。注:这个方向是研究腹侧集群神经元。要检查背的集群神经元，颠倒的方向。 </li><li>使用昆虫针的中心针口附近挂钩前结束。将针接近年底，以取得最佳效果。 </li><li>放置在解剖室内HL3.1生理盐水下降。 </li><li>剪下的幼虫后的提示microscissors。注意:此步骤打开光圈microscissors（步骤2.7），将允许访问的幼虫后结束。 </li><li>抓住镊子地区的肠道，现在戳在幼虫后部的光圈。轻轻拉出整个肠道。 </li><li>放置提示一个刀片沿腹中线走向前通过光圈和削减microscissors。 </li><li>利用角落引脚，引脚现在可以自由的角落，后第一，然后前。同时轻轻地伸展开幼虫圆角。 </li></ol> 3。固定，阻断，染色，并安装幼虫鱼片在解剖室内进行开始前的注意事项:所有的固定和染色步骤。在这个过程中，应注意不要敲的昆虫幼虫，因为这可能导致组织损伤引脚。为了防止干燥实验，做特百惠蘸组织包围在一个小容器中所有的染色步骤。 <ol><li>吸的Ca + + HL3.1缓冲区使用一个黄色的提示。立即加入固定液使用另一个pipetteman。 </li><li>轻轻吸管上下钙+ +免费HL3.1缓冲区剩余的痕迹混合固定液在会议厅内。立即吸出，然后添加新鲜固定液进入会议厅。 </li><li>在室温下孵育20分钟。 </li><li> PBST 6X清洗10分钟（0.1％的Triton X - 100的PBS中）。 </li><li>座与5％PBST山羊血清在室温下20分钟。 </li><li>更换封闭液在5％山羊血清PBST稀释的抗体。使用的主要抗体小鼠抗α-微管蛋白（DM1A）和鼠抗CD8（5H10）都稀释1 / 1000。 注:研究者不妨以鼠抗Futsch（22C10）20,21稀释1 / 1000在某些情况下，鼠抗α-微管蛋白（DM1A）（见讨论）。 </li><li>孵育过夜（至少16小时）在4 ° C。 </li><li> 6X PBST清洗10分钟。 </li><li>新增二级抗体溶液稀释于5％山羊血清PBST。使用的抗体的Alexa Fluor 488山羊抗鼠IgG和Cy3标记的驴抗鼠IgG。保持样品的覆盖，以防止荧光团光漂白22。 </li><li>可以在室温下孵育两小时或过夜，4 ° C的室温一小时。 </li><li> 6X PBST清洗10分钟。 </li><li>放置在幻灯片上角质层的一面朝下的幼虫圆角，安装在80％甘油，和密封装入一个"快"与指甲油盖玻片的两侧。 注:为了提高组织的清算，和一个永久性的稳定样品，安装中的DPX如前所述23。 </li></ol> 4。代表性的成果: 荧光染色，共聚焦显微镜下观察。在图1-2不同的分支，在枝蔓乔木有不同的细胞骨架组织。图1显示了一个地区的一类四DA神经元在第一龄幼虫期的乔木。整个乔木标记与mCD8:KO和检测用抗CD8抗体和荧光中学（Cy3标记）。微管蛋白检测抗α-微管蛋白抗体和Fluorescent中学（的Alexa Fluor 488）。主要支流为微管蛋白是积极的，有些单薄的偏科是微管蛋白阴性。电影1是通过类似的I类DA神经元染色的连续切片。图2显示了一个地区的一类四DA神经元在第一龄幼虫期的乔木Futsch和CD8抗体染色。主要支流Futsch阳性，一些薄的偏科Futsch阴性。图3。在幼虫体壁的气管显示一个复杂的微管组织。电影2和3显示了通过体壁肌肉和气管染色的连续切片。 <img alt="图1" src="/files/ftp_upload/3662/3662fig1.jpg" /> 图1。图1显示了一个地区的一类四DA神经元在第一龄幼虫期的乔木。整个乔木标有mCD8::正和检测抗- CD8抗体和荧光中学（Cy3标记）。微管蛋白检测抗-＆-肾小管ñ抗体和荧光二（Alexa的488）。连续共聚焦Z -节（0.5微米），小组交流C&#39; - C"，单从面板C.分支机构（黄色箭头）或无（紫色箭头）微管突出的例子抗体染色。红色箭头突出微管在底层上皮细胞。 <img alt="图2" src="/files/ftp_upload/3662/3662fig2.jpg" /> 图2。图2显示了一个类似的一类四DA神经元在第一龄幼虫期的乔木Futsch和CD8抗体染色地区。整个乔木标有mCD8::正和检测抗- CD8抗体和荧光中学（Cy3标记）。使用反Futsch抗体和荧光中学（488 Alexa的）检测Futsch。主要支流Futsch阳性，一些薄的偏科Futsch阴性。 （黄色箭头）或无（紫色箭头）Futsch分行的例子是突出的。 图3。气管染色使用以上所述的抗微管蛋白的协议。幼虫三龄和解剖如前面所述23,24。电影3显示了从一个正方形标志着领域扩大连续切片。 电影1。连续切片追踪整个I类神经树突状乔木微管蛋白染色。整个乔木与mCD8标记:正（洋红色），检测抗- CD8抗体和荧光中学（Cy3标记）。微管蛋白（绿色）是检测抗α-微管蛋白抗体和荧光中学（的Alexa Fluor 488）。规模:视频图像的一侧对应到46.88μm <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">，点击这里查看</a>电影节<a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">。</a> 。 电影2追查BOD微管蛋白染色的连续切片 。Ÿ壁肌肉的第三龄幼虫。微管蛋白是检测抗α-微管蛋白的抗体和荧光中学（的Alexa Fluor 488）。规模:视频图像的一侧对应到46.88μm <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">，点击这里查看</a>电影节<a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">。</a> 。 电影（3）跟踪一个标方在Figure.3第三龄幼虫体壁气管，微管蛋白染色的连续切片。微管蛋白是检测抗α-微管蛋白的抗体和荧光中学（的Alexa Fluor 488）。规模:视频图像的一侧对应到46.88μm <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">，点击这里查看</a>电影节<a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">。</a> 。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+microtubule+growth,++TIPs,+and+kinesin-2+are+required+for+uniform+microtubule+polarity+in+dendrites.">Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites.</a> Curr. Biol. 20, 2169-2177 (2010).</li><li>Pawson, C., Eaton, B. A., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formin-dependent+synaptic+growth:+evidence+that+Dlar+signals+via+Diaphanous+to+modulate+synaptic+actin+and+dynamic+pioneer+microtubules.">Formin-dependent synaptic growth: evidence that Dlar signals via Diaphanous to modulate synaptic actin and dynamic pioneer microtubules.</a> J. Neurosci. 28, 11111-11123 (2008).</li><li>Williams, D. W., Tyrer, M., Shepherd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+and+tau+reporters+disrupt+central+projections+of+sensory+neurons+in+Drosophila.">Tau and tau reporters disrupt central projections of sensory neurons in Drosophila.</a> J. Comp. Neurol. 428, 630-640 (2000).</li></ol>

我们什么都没有透露。

要理解复杂的细胞形态如何实现它重要的是要能够准确地检测微管组织。在这里，我们描述了一个强大的免疫组化标签方法检测树突状树枝状感觉神经元的树突的微管组织。除了染色的感觉神经元，这种方法实现了强大的气管，肌肉和其他体壁组织的免疫组化染色。 我们使用此协议，研究开发的DA能神经元的感官树突微管组织。这些树突是非常敏感，受伤或应力25，?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录 数</th><th>评论 （可选） </th></tr><tr><td>镊子</td><td>杜蒙</td><td> 11251-20 </td><td></td></tr><tr><td> Microscissors </td><td>福斯特</td><td> 15000-08 </td><td></td></tr><tr><td>鼠抗α-微管蛋白（克隆:DM1A） </td><td>西格玛</td><td> T9026 </td><td>稀释1 / 1000 </td></tr><tr><td>鼠抗Futsch（克隆:22C10） 上清</td><td>发展 研究 杂交瘤细胞银行</td><td> 22C10 </td><td>稀释1 / 1000 </td></tr><tr><td>鼠抗- CD8（克隆:5H10） </td><td> Caltag </td><td> MCD0800 </td><td>稀释1 / 1000 </td></tr><tr><td>的Alexa Fluor 488抗鼠IgG </td><td> Invitrogen公司</td><td> A - 11001 </td><td>稀释1 / 500 </td></tr><tr><td> Cy3标记的抗鼠IgG </td><td>杰克逊Immunoresearch </td><td> 712-166-150 </td><td>稀释1 / 200 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

要了解如何实现复杂的细胞形态的差异，重要的是要准确地按照微管组织。 果蝇幼虫体壁含有几种类型的细胞模型来研究细胞和组织的形态发生。例如气管是用于检查1，管形态发生和感觉神经元的果蝇幼虫一般的澄清和神经树突状分化2-5和变性 6级的具体机制已成为一个主要系统的树突状树枝状（ DA） 。 之间的神经元类，树突分支的形状可以显著不同，甚至在单个神经元的不同分支 7,8中。 DA能神经元的遗传研究表明，差的细胞骨架的组织可以在树突分支的形状4,9-11形态差异的基础。我们提供了一个强大的免疫标记法到ssay 在体内 DA的感觉神经元枝蔓乔木微管组织（图1，2，电影1）。该协议说明了第一龄幼虫的解剖和免疫组化，一个阶段，积极的感觉神经元树突生长和分支组织时发生12,13。 除了染色的感觉神经元，这种方法实现了强大的标签（电影2，3）微管在肌肉组织，气管（图3，电影3），和其他的体壁组织。这是宝贵的希望分析在体壁的微管组织调查机制时，在原地调查控制的组织和细胞的形状。

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

要理解的复杂的细胞形状，如神经细胞树突，如何在发展过程中取得的，重要的是要能够准确地检测微管组织。在这里，我们描述了一个强大的免疫组化标记法研究微管组织的感觉神经元树突树突分枝，气管，肌肉，及其他<em&gt;果蝇</em&gt;幼虫体壁组织。

免疫组化标记在感觉神经元树突的微管，气管，并在肌肉果蝇幼虫体壁

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

Research

JoVE Journal

Neuroscience

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

12.3K 次观看.  RIKEN Brain Science Institute. 要理解的复杂的细胞形状，如神经细胞树突，如何在发展过程中取得的，重要的是要能够准确地检测微管组织。在这里，我们描述了一个强大的免疫组化标记法研究微管组织的感觉神经元树突树突分枝，气管，肌肉，及其他果蝇幼虫体壁组织。

视频: Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

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

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

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

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

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 ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...