作者感谢理化学研究所的资金。我们也感谢Cagri Yalgin，加Delandre，和周杰伦帕里什讨论遗传和免疫组化协议。

1.Preparation试剂<ol><li>准备钙离子免费HL3.1生理盐水28。 <ol><li> （毫米）：70氯化钠，氯化钾，氯化镁2 20 10 3，碳酸氢钠5 HEPES，115蔗糖，和5海藻糖; pH值7.2。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>设为聚- L -赖氨酸（PLL）盖玻片。 <ol><li> 4.2毫升水溶解100毫克PLL和300μL分装在Eppendorf管中，并冻结在-20 ° C </li><li>涂装前盖玻片，首先解冻等份，把它DDH 2 O10毫升加入20μL的柯达照片- FLO，最终的PLL浓度为0.7毫克/毫升。 </li><li>浸没盖玻片在PLL解决方案的30分钟，捞出晾干，然后用清水冲洗简要水和干燥。重复两次。治疗盖玻片持续约一个月。 </li></ol></li></ol> 2。遗传杂交<ol><li> TØ生成MARCM克隆。下面是一个例子交叉使用泛DA驱动11,27： FRT2A x hsFLP; Gal4的 109（2）80， 无人机，mCD8：：GFP的浴缸，Gal80 FRT2A/SM5-TM6B </li><li> 要生成FLP -克隆 。下面是一个例子交叉使用一类IV专用驱动8： PPK - GAL4 x YW，hsFLP无人机的首次登记税，CD2，Y +一站式FRT - mCD8：绿色荧光蛋白 </li></ol> 3。收集胚胎<ol><li> 果蝇杂交保持在一个收集瓶在25℃，收集胚胎的酵母膏30,31一层薄薄的苹果汁琼脂平板上 29蔓延。 </li></ol> 4。热休克治疗的胚胎<ol><li>反对胚胎已经奠定了苹果汁琼脂平板放置的空盘。密封围绕这两大板块封口膜（图2）。 </li><li>热休克期间，淹没日e在水浴板不放，使用金属的重量。 </li><li>对于MARCM胚胎<ol><li>收集2H胚胎和孵化包围在10厘米培养皿菜浸在25 ° C额外2H组织。 注：调整热休克协议改变了克隆产生的频率。 </li><li>对于数量较少的克隆，着眼于单个孤立的神经元，淹没在上半年的水浴热休克38 ° C。 </li><li>对于数量较大的克隆，热休克45分钟在38℃，在室温下30分钟，然后热休克恢复再额外30分钟。 </li></ol></li><li>收集FLP - 24小时的胚胎和热休克1h后在38 ° C。 </li></ol> 5。筛选克隆的<ol><li>热休克后，取下盖​​子防水安排和苹果汁琼脂平板上放置蘸组织包围在10厘米培养皿菜。文化的胚胎和随后的幼虫在25 ° C，直到漂移ING 第三龄。 注：培养条件下，尤其是营养，已被证明改变DA的树突状乔木形态 32,33 。确保增长的幼虫访问酵母粘贴在任何时候，监测和补充需要粘贴为酵母。 </li><li>这一点在协议开始，轻轻用昆虫钳操纵幼虫。 </li><li>简要轻轻冲洗自来水中的幼虫，然后放到琼脂平板。 注意：此步骤减少从食物或幼虫表面污垢的自体荧光背景。 </li><li>一个强大的荧光解剖显微镜下检查幼虫。确定GFP阳性神经元/体壁细胞的幼虫。 </li></ol> 6， 在体内成像树突<ol><li>一个抑郁症的载玻片放入一个小降80％甘油的幼虫。广场上滑动盖玻片固定幼虫。确保没有空气保持之间的幼虫和coversli第</li><li>轻轻推盖玻片，推出的幼虫，让可视化的神经元的利息。形象mCD8：通过共聚焦显微镜观察GFP标记树突状乔木。 </li></ol> 7。幼虫清扫 开始前的注意事项：DA神经元的树突降低后迅速启动夹层。每一个人幼虫解剖，在不到5分钟，以确保良好的树突形态。 <ol><li>解剖徘徊在Sylgard板描述与以下修改以前34路龄幼虫3： <ol><li> DA神经元轴突总站成像，确保中枢神经系统是完整的和节段性神经不破。开放的幼虫后，首先仔细切开气管的连接体壁，然后轻轻取出整个肠道。 </li><li>如果成像树突单独删除的中枢神经系统，让平坦的安装。 </li></ol></li></ol> 8。 Fixat离子和阻止幼虫鱼片<ol><li>幼虫仍然寄托Sylgard板，固定在4％的PBS PFA上轻轻旋转振动筛在室温下20分钟。 </li><li>卸下固定后的幼虫组织的痕迹（脂肪体，气管等）。 </li><li>洗净PBST（含0.1％Triton X - 100的的PBS）的Sylgard板和筛3X 10分钟。如果检查轴突总站，保持Sylgard板的圆角。 （如果染色树突有可能在这一步转移34 0.5毫升管幼虫鱼片） </li><li>在振动筛上一个PBST座在5％的正常驴血清（NDS）幼虫在室温为20分钟。 </li></ol> 9。染色幼虫鱼片<ol><li>删除初级抗体孵育封闭液（5％的NDS / PBST）在4 ° C包围在一个小的特百惠容器过夜蘸组织。 </li><li>管，并从4℃取出，在室温下孵育额外小时。 </li><li> 6X PBST清洗10分钟。新增二级antibody（5％的NDS / PBST），并盖以防止荧光团的照片， 漂白 34 。 </li><li>可以在室温下孵育两小时或过夜，4 ° C的室温下一个小时。 </li><li>在PBST洗幼虫6X 10分钟，并进行安装。 </li></ol> 10。检查的枝蔓乔木安装幼虫鱼片<ol><li>安装每个幼虫圆角平尽可能使用解剖剪刀或手术刀切断头部（包括口挂钩），后（包括气孔）34。 </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，并密封指甲油盖玻片双方一个“快” 装载 34 。 </li><li>一个永久性的安装和更清晰的图像<ol><li>放置到一个PLL盖玻片下降的PBS解剖幼虫鱼片肌肉端时，它会很快坚持盖玻片。 </li><li>安装后删除尽可能多的液体，howeveR不让它完全干燥。 </li><li>以通过乙醇系列盖玻片（附加幼虫圆角）：35％乙醇，50％，70％，95％，终于2X 100％的乙醇，每次10分钟（第二100％的乙醇溶液应经常更换）;最后，在二甲苯洗2 - 3X 10分钟。 </li><li>放在一个干净的幻灯片上的DPX mountant（Distyrene增塑剂，二甲苯 ）的下降，并仔细地躺在上面盖玻片圆角方式。保持在黑暗中，等待一天的DPX设置前成像。 </li></ol></li></ol> 11。轴突总站检查安装幼虫鱼片<ol><li>继续运行节段性神经之间的夹层，固定不变百佳和中枢神经系统的过程中，染色，以便追踪轴突从外周感觉神经元细胞体的VNC。 </li><li>每个幼虫圆角角质层端安装在80％的甘油，在抑郁症的幻灯片。跟踪从DA细胞体的轴突共聚焦显微镜下的VNC的。注：PNS在每个体壁段项目的神经细胞同源段的VNC。 </li></ol> 12。代表性的成果： 代表结果显示在图3-5。图3给出了一类四DA神经元在体内捕获的共聚焦显微镜下，整个乔木。图图4显示了免疫组化标记的Ⅲ类神经枝蔓乔木的一部分收起来，已正确固定保存形态。相关的插图可以不成功的解剖和（抗GFP抗体染色后）固定发生的退化。图5显示了一个IV级在中枢神经系统轴突总站（抗- GFP，绿色）;所有的IV类总站合作与抗CD2（洋红色）染色。 <img alt="图1" src="/files/ftp_upload/3111/3111fig1.jpg" /> 图1协议概述。 a）收集和再热休克胚胎。 B）<html>选择与GFP阳性DA神经元克隆的幼虫，然后在现场幼虫的形象GFP标记的枝蔓乔木。三）解剖幼虫，然后免疫组化染色圆角。 D）山染色圆角，然后形象的树突状乔木和DA神经元克隆的轴突预测。 <img alt="图2" src="/files/ftp_upload/3111/3111fig2.jpg" /> 图2苹果汁琼脂平板热休克的制备。以板（白色箭头，AB），顶部（红色箭头B）添加第二培养皿和密封封口膜，B（蓝色箭头）。 <img alt="图3" src="/files/ftp_upload/3111/3111fig3.jpg" /> 图3 在体内一个mCD8枝蔓乔木形象：GFP标记MARCM的克隆（2.1基因型）代表一类四（v&#39;ada）3龄幼虫神经元。比例尺为50μm。 d/3111/3111fig4.jpg“/&gt; 图4 mCD8：GFP标记的抗GFP抗体染色III类神经元（ddaA），呈现出良好的形态后，成功剥离和固定（绿色）MARCM克隆。插图枝蔓退化（白色箭头）不成功的解剖和固定（洋红色）后发生的。比例尺为25微米。 <img alt="图5" src="/files/ftp_upload/3111/3111fig5.jpg" /> 图5 第三龄幼虫VNC。一个FLP -克隆是一个单一的VI级（VDAB）轴突总站（2.2基因型）检测抗GFP抗体（绿色）。抗CD2标签所有IV类总站（洋红色）。比例尺为25微米。

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W., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+representation+of+the+glomerular+map+in+the+Drosophila+protocerebrum.">Spatial representation of the glomerular map in the Drosophila protocerebrum.</a> Cell. 109, 229-241 (2002).</li><li>Shimono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidendritic+sensory+neurons+in+the+adult+Drosophila+abdomen:+origins,+dendritic+morphology,+and+segment-+and+age-dependent+programmed+cell+death.">Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death.</a> Neural Dev. 4, 37-37 (2009).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18, 377-402 (2004).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <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 Protocols. , (2000).</li><li>Kaczynski, T. J., Gunawardena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Embryonic+Nervous+System+in+Whole-mount+Drosophila+Embryos.">Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos.</a> J. Vis. Exp. (46), e2150-e2150 (2010).</li><li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures.">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> J Vis Exp. , (2009).</li><li>Medina, P. M., Swick, L. L., Andersen, R., Blalock, Z., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+forward+genetic+screen+for+identifying+mutations+affecting+larval+neuronal+dendrite+development+in+Drosophila+melanogaster.">A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster.</a> Genetics. 172, 2325-2335 (2006).</li><li>Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+and+AMPK+maintain+epithelial+cell+polarity+under+energetic+stress.">LKB1 and AMPK maintain epithelial cell polarity under energetic stress.</a> J Cell Biol. 177, 387-392 (2007).</li><li>Brent, J., Werner, K., McCabe, B. D. <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+NMJ+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. 25, e1108-e1108 (2009).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

没有利益冲突的声明。

果蝇幼虫DA神经元模型提供了一个优良的遗传系统调查机制，控制神经元的形态和电路形成。 MARCM是一般用于标签和产生突变的DA神经元克隆。 MARCM，我们使用一个泛神经（ 如Gal4 的 C155）或DA神经元特异性司机。使用泛神经的驱动程序，它可以直接使用公共储备中心广泛使用的几个股票。然而，使用DA神经元的特定的驱动程序可以是有利的，因为标记克隆不会产生在中?...

<table border="1"><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> SZX16荧光解剖镜下（GFPHQ过滤） </td><td>奥林巴斯</td><td> SZX16 </td><td></td></tr><tr><td>活昆虫钳</td><td>福斯特</td><td> 26030-10 </td><td></td></tr><tr><td> 26毫米x 76毫米抑郁症的载玻片</td><td> Toshinriko有限公司</td><td> T8 - R004 </td><td></td></tr><tr><td> Sylgard 184（或Silpot 184） </td><td>道康宁</td><td> 3097358-1004 </td><td></td></tr><tr><td>聚- L -赖氨酸</td><td>西格玛</td><td> P - 1524 </td><td>该产品已被证明最有效的</td></tr><tr><td> DPX安装培养​​基</td><td>西格玛</td><td> 44581 </td><td></td></tr><tr><td>兔抗GFP </td><td> Invitrogen公司</td><td> A - 11122 </td><td>稀释1:500 </td></tr><tr><td>大鼠抗- CD8 </td><td> Caltag </td><td> 5H10 </td><td>稀释1:200 </td></tr><tr><td>小鼠抗CD2 </td><td>抗体ð serotec </td><td> MCA443R </td><td>稀释1:700 </td></tr><tr><td>鼠抗- Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td>稀释1:10 </td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

神经系统的发育需要的神经元​​的地位和身份，准确的神经元类的特定的树突状发展和轴突布线，正确规范。最近的树突状树枝状（DA） 的果蝇幼虫周围神经系统（PNS）的感觉神经元已成为强大的遗传模型，在其中阐明神经元分化的一般和特定类的机制。有四个主要的DA神经元类（I - IV）1 。他们被命名为了增加枝蔓乔木的复杂性，并在其分化2-10的基因控制类特定的差异。 DA感官系统是一个实际的模型研究的树突状形态 11-13的控制背后的分子机制，因为：1）它可以利用强大的基因在果蝇中的可用工具，2）的优势，DA神经元枝蔓乔木展开在下方的光骑士尺寸只有2AR幼虫角质层很容易地可视化体内的高分辨率，3）类特定的树突形态的多样性有利于比较分析，发现控制简单与高度分支的树突树形成的关键要素，以及4）树突状乔木的定型不同的DA能神经元的形状有利于形态的统计分析。 DA神经元活动的修改输出的一个幼虫的运动中枢模式发生器 14-16 。不同的DA神经元类有不同的感觉方式，和它们的激活会引起不同的行为反应14,16-20。此外，不同类别发送到果蝇幼虫中枢神经系统的腹 ​​神经索（VNC） 的 21轴索预测刻板。这些预测终止两个DA神经元感觉方式和位置在体壁的树突状领域7,22地形意见书23。因此，DA的轴突预测的检查可用于澄清的内在机制， 地形测绘7,22,23，以及一个简单的电路调节幼虫运动14-17的接线。 我们在座的生成和分析遗传马赛克24标记的DA通过MARCM（马赛克Repressible细胞标记分析）1,10,25和FLP - 22,26,27技术（在图1总结）神经元的实用指南。

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

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

感觉神经元的树突分枝<em&gt;果蝇</em&gt;幼虫周围神经系统是有用的模型，以澄清一般和神经元类特定的神经细胞分化的机制。我们提出了一个实用指南生成和分析树突状树枝状的神经元遗传马赛克。

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 ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Research

JoVE Journal

Neuroscience

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

15.2K 次观看.  RIKEN Brain Science Institute. 感觉神经元的树突分枝果蝇幼虫周围神经系统是有用的模型，以澄清一般和神经元类特定的神经细胞分化的机制。我们提出了一个实用指南生成和分析树突状树枝状的神经元遗传马赛克。

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

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

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

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

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during 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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

去除<em&gt;果蝇</em从幼虫鱼片的感官神经元和表皮细胞的免疫荧光分析&gt;肌肉组织

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...