我感谢Alex L. Kolodkin，因为我在他的实验室里学到了这个填写的准备方案。我也非常感谢杨宏宏的技术援助。本研究得到NRF-2013R1A1A4A01011329（SJ）的支持。

准备<ol><li>通过加入0.5g牛血清白蛋白（BSA）和0.5mL叔辛基苯氧基聚乙氧基乙醇（参见材料表），向具有叔辛基苯氧基聚乙氧基乙醇（PBT）溶液的500ml磷酸盐缓冲盐水（PBS）中制备500mL的1X PBS并搅拌至少30分钟。储存于4°C。使用时比较新鲜，并将溶液储存在干净的瓶子里。 </li><li>通过向6.5mL去离子水中加入2.5mL 16％的多聚甲醛溶液和1mL的10x PBS，制成10mL的4％多聚甲醛。储存于4°C并在一周内使用。 小心:避免皮肤接触多聚甲醛溶液，因为它具有高毒性。 </li><li>通过每1mL DMSO溶解0.1g X-Gal底物制备X-Gal底物（10％w / v X-Gal在二甲基亚砜（DMSO）中）。在-20°C储存在100μL等分试样中。 </li><li>使用10mM磷酸盐缓冲液（pH7.2），150mM NaCl，1mM MgCl 2，3μm制备X-Gal染色溶液MK 4 [FeII（CN） 6 ]，3mM K 3 [FeIII（CN） 6 ]和0.3％叔辛基苯氧基聚乙氧基乙醇。在黑暗中储存于4°C。 </li><li>用去离子水稀释30％的过氧化氢1:10制成3％过氧化氢溶液。 </li><li>通过将1片（10mg）DAB完全溶解在35mL新鲜PBT溶液中来制备3,3&#39;-二氨基联苯胺（DAB）溶液。通过0.2μm注射过滤器过滤，并在-20°C下保存在910μL等分试样中。 小心:将所有DAB废物处理在漂白剂中以摧毁民建联。 </li><li>用苹果汁制作蛋盘。向2L烧瓶中加入35g琼脂和1L去离子水。向1升烧瓶中加入33g蔗糖，2g tegosept和375mL苹果汁。通过高压灭菌30分钟使其熔化。允许两种溶液冷却至约60℃。通过搅拌结合并混合这些溶液。每60毫升培养皿倒入约11毫升的组合溶液。 </li><li>通过混合面包酵母制作酵母糊st进入去离子水（1:0.8比例）并储存在4℃。 </li></ol>胚胎收集（第1天） <ol><li>收集年轻的雌雄和雄蝇（5日以下），并保持1-2天，将它们保持在一个塑料烧杯笼中，里面装有少量的酵母糊（60毫米x 15毫米）。 </li><li>为了最佳收集晚期16期胚胎，在5点左右的时间内建立一个带有苍蝇（&gt; 50）的瓶笼，让它们在25℃下孵育3小时。 </li><li>将苍蝇转移到新鲜的食物瓶中。 </li><li>用盖子关闭蛋盘，并在25℃下在潮湿的培养箱（约70％湿度）中将其倒置过夜。 </li></ol> 3.免疫染色胚胎的制备（第2天） <ol><li>在上午10点20分左右将蛋白质加入1.8毫升PBT溶液中，并用棉签将其从板上松开，同时倾斜。 注意:用cott轻轻地捣碎酵母糊在拭子上，溶解它，以防发现大量的胚胎。开始收集胚胎〜固定前约40分钟（约11:00 AM）。 </li><li>将胚胎从蛋板转移到1.5 mL微量管中，使用1 mL移液管吸头。 </li><li>允许胚胎沉淀并尽可能吸出PBT溶液。用1 mL PBT溶液冲洗胚胎两次。尽可能吸出PBT溶液。 </li><li>向管中注入1 mL 50％的漂白剂（ 即稀释在去离子水中），并通过在室温下（RT）将它们培养在营养器上3分钟来使胚胎脱钙。 注意:这一步不会杀死被破坏的胚胎。 </li><li>让脱钙的胚胎沉淀，尽可能吸出50％的漂白剂，并用1 mL的PBT溶液冲洗胚胎3次。 </li><li>加入0.5mL庚烷，随后在约11:00 AM的室温下向管中加入0.5mL的4％多聚甲醛溶液。 </li><li>孵化在一个养分器上的胚胎在室温下15分钟。 </li><li>取出4％多聚甲醛溶液（底层）。 </li><li>加入0.5 mL 100％甲醇，通过剧烈摇动管30秒，使胚胎变细。 </li><li>除去庚烷（ 即顶层）。 </li><li>加入0.5 mL 100％甲醇，剧烈摇动管10秒。 </li><li>通过点击管子尽可能地吸出甲醇使胚胎沉降。用1 mL 100％甲醇冲洗胚胎，摇动不到10秒。 注意:长期暴露于甲醇会破坏β-Gal活性。 </li><li>取出甲醇并用1 mL PBT溶液冲洗脱皮胚胎3次。 </li></ol> 4.使用LacZ染色对胚胎进行基因分型（第2天） <ol><li>将1mL PBT溶液加入到管中，并将管在37℃的热块或水浴中孵育15分钟。 </li><li>孵育期间，放置X-Gal染色溶液（1 mL / tu在65℃在水浴中，直至变得多云。在37°C水浴中孵育。 </li><li>吸出PBT溶液，并向管中加入1 mL X-Gal染色溶液和20μLX-Gal底物。 </li><li>在37℃下孵育管，使其具有营养作用，直到蓝色沉淀物明显。 注意:第2和第3个蓝色平衡器的孵育时间一般在2-4小时之内。 </li><li>取出X-Gal染色溶液，用1 mL RT PBT溶液冲洗胚胎3次。 </li><li>使用针头或镊子，用光解剖显微镜（1.6X-2.5X目标）手工分选所需基因型的胚胎（ 即未染色的白色胚胎）。 注意:由于一些蓝色平衡剂，如CyO，肌动蛋白-LacZ和TM3，肌动蛋白 -LacZ，携带在肌动蛋白启动子控制下表达细菌β-Gal（LacZ）的转基因构建体，以无处不在的方式染成蓝色的胚胎含有蓝色平衡染色体的一或两个拷贝。因此，未染色的白色胚胎对于所需的致死等位基因是纯合的。 </li></ol> 5.用抗Fasciclin II抗体对胚胎进行免疫染色（第2-3天） <ol><li>使用1 mL移液管吸头收集并转移所需基因型胚胎（ 即未染色的白色胚胎）至0.5 mL微量管。 注意:在这个步骤中，胚胎可以根据形态学标准大致分期，包括角质层的分割模式和头尾的结构9 。在头部退化期间，蛋放置后10至16小时，胚胎经历了最前段9的显着减少。 </li><li>用0.4mL PBT溶液洗涤胚胎一次，并加入0.3mL封闭溶液（5％正常山羊血清和5％DMSO在PBT溶液中）。 </li><li>在室温下将营养物阻断15分钟。 </li><li&gt;加入75μL抗Fasciclin II抗体，并在室温下孵育具有章动作用的管。 </li><li>用0.4mL PBT溶液洗涤胚胎4次，每次洗涤至少20分钟。 </li><li>加入含有山羊抗小鼠HRP抗体（2μg/ mL）的0.3mL封闭溶液（PBT溶液中的5％正常山羊血清），并将管子在室温下孵育过夜。 </li><li>用0.4mL PBT溶液洗涤胚胎6次，每次洗涤至少20分钟。 </li><li>向管中加入0.3mL DAB溶液。 小心:戴上手套，将所有DAB废物/管/尖端放入漂白剂中。 </li><li>加入2μL3％过氧化氢，并在RT中将管子在黑暗中孵化，直到产生所需量的沉淀物。 注意:1D4免疫组化的培养时间一般在0.5-1小时。 </li><li>用0.4mL PBT溶液洗涤胚胎4次。 小心:取出DAB溶液，然后用移液管和二极管清洗前两次洗涤液把它们漂白掉。 </li><li>向管中加入0.2 mL安装的70％甘油/ PBS溶液，并在室温或4°C储存。 注意:保持管远离光线。通过将胚胎置于90％甘油/ PBS溶液10中可获得更清楚的制剂。然而，比在70％甘油/ PBS溶液10中清除的那些更容易解剖在90％甘油/ PBS溶液中清除的胚胎。 </li></ol> 6.分期，分解，安装和成像胚胎（第4天） <ol><li>为了分期，将用70％甘油/ PBS平衡的胚胎转移到载玻片上。 </li><li>收集具有收敛于彼此接触或/并且具有细分为三个带的中肠的腹部节段7（A7），A8和A9 ISN神经的晚期16期胚胎。 注意:1D4抗体染色的胚胎可以基于ISN和ISNb运动轴突的投影图案进行分期。退出CNS，A7，A8和A9 ISN之后晚期-16胚胎的rves会聚到彼此接触，随后发散到远离背部肌肉（ 图1A中的箭头）。然而，在中期16期胚胎中，A7 ISN神经与A8 / A9神经（ 图1B中的方括号）平行延伸。此外，两个半身中的A6 ISNb神经（垂直于腹侧肌）的投影轴与CNS纵向轴突束的后端（箭头和图1C中的一条线）大致对齐。这些形态学标准在不同的基因型之间有所不同。或者，胚胎可以基于肠道形态9,11进行分期。在阶段17之前，中肠具有心形形状，而中肠在阶段17 12细分为三个带。 </li><li> 解剖胚胎。 <ol><li>使用1米L注射器，将每个胚胎移出甘油滴，并将胚胎腹面朝上。在身体长度的1/4处切除前部，并且没有运动轴突的最后部区域。 </li><li>使用针头探针，将终端胚胎移出甘油滴，滚动以将其背面朝上定位，并将其水平放置在野外。 注意:当胚胎移出甘油滴时，与胚胎相关的一点甘油使其粘稠。 </li><li>使用单个非常精细的钨针13沿背侧中线切割胚胎。在胚胎后端做一个小切口，然后继续将背部中线向前切割;相反方向的切割也很好。 </li><li>使用针头探针，将中线切割的胚胎移入甘油滴，将其背面放置，并通过在腹侧（对角线）方向上展开每个体壁（2），将肠道从体壁分离.5X目标）。 注意:确保背部中线完全切割，导致左右两侧体壁之间完全分开。 </li><li>仔细将中线切割的胚胎从甘油滴中移出，然后使用细针探针（2.5X物镜）将身体壁上的两个皮瓣放在玻璃片上。 </li><li>使用非常精细的钨针，通过将其推向侧面（5X物镜）从分离的胚胎中去除内部器官。 注意:另一个类似的解剖程序的更详细的描述可以在网站上找到。 </li></ol></li><li>为了安装，加入2-3μL70％甘油/ PBS溶液清洁，解剖的胚胎，并使用细针探针将其转移到新的载玻片上，将8μL70％甘油/ PBS溶液涂抹在其上中心（2.5X目标）。 </li><li>穿上盖玻片（18毫米x 18毫米）。用常规指甲油（也可以）密封盖玻片的边缘清晰或有色是好的）。 </li><li>根据制造商的说明，在差分干涉对比（DIC）光学显微镜下以高分辨率（20X，40X和63X浸油物镜）拍摄图像。 注意:更好的可视化和表型特征，特别是对于ISNb运动轴突，可以通过使用63X油浸物镜来生产。 </li></ol>

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笔者宣称他没有竞争的经济利益。

运动轴突指导缺陷的细节通过DAB染色的胚胎的切片制备比通过荧光标记的激光扫描共焦显微镜更快，更准确。因此，固定和1D4染色的胚胎的切片制备最适合于指导提示分子的功能表征。指导线索包括网络，狭缝，信号素（Semas）和水母以及他们的同源受体在蠕虫，苍蝇和脊椎动物中具有进化上保守的作用。在作为狭缝受体的迂回（Robo）家族分子中， 果蝇 Robo在CNS 6,21中首先被确定为调解排?...

胚胎发育期间运动轴突和目标肌肉之间的精确和选择性联系对于果蝇幼虫的正常运动是必不可少的 。每个腹部疾病A2-A7中的30个肌纤维的胚胎图案是由阶段16 1建立的 。在腹侧神经线中产生的36只运动神经元将其轴突延伸到外周以支配特定的目标肌肉2 。通过抗体（小鼠单克隆抗体1D4）3,4免疫组织化学可以观察到运动轴索寻路和靶标识别。在野生型胚胎中的运动轴突投影图案的多个图像可在网络5上获得 。 1D4抗体标记胚胎中枢神经系统（CNS）中线每侧的所有运动轴突和三个纵向轴突束</s， 6 （ 图1C和图2A ）。因此，FasII抗体的免疫组化提供了一个强大的工具，用于识别神经肌肉连接所需的基因，以证明运动轴突指导和靶标识别的分子机制。 在每个腹部A2-A7中，运动轴突突出并选择性地成形为两个主要神经分支，节段神经（SN）和节段间神经（ISN） 2,4和一个小神经分支，横向神经（TN ） 7 。 SN选择性地消弧以产生称为SNa和SNc的两个神经分支，而ISN分裂成称为ISN，ISNb和ISNd 2,4的三个神经分支。其中，ISN，ISNb和SNa运动轴突当晚期16期胚胎用FasII抗体染色时，投影图案最精确地可视化，并被切片（ 图1C和图2A ）。 ISN运动神经元延伸其轴突以支配1,2,3,4,9,10,11,18,19和20,2,4（ 图2A ）的背部肌肉。 ISNb运动神经元支配腹侧肌6,7,12,13,14,28和30,24（ 图2A和2B）。 SNa神经分支突出支配5,8,21,22,23和24,2,4（ 图2A ）的侧肌。由两个运动轴突组成的TN沿着节段边界向内侧投射以支配肌肉25，并且在侧向双极树突状神经元（LBD）中突触周边7 （ 图2A ）。这些目标肌肉神经支配不仅需要在特定选择点的运动轴突的选择性阻塞，而且还需要靶向肌肉识别。此外，在ISN和SNa途径中都发现了一些作为中间靶点的推定的中胚层路标细胞，但不是沿着ISNb途径4 。这可能表明与ISN和SNa运动轴突指导相比，ISNb运动轴索寻路可以以明显的方式进行调节，并且还表明外周运动轴突指导提供了一个有吸引力的实验模型来研究单个指导提示的差异或保守作用分子8 。 这项工作提出了一种标准方法，可视化果蝇中胚胎运动神经元的轴突投影模式。所描述的方案包括如何解剖用1D4a染色的固定胚胎在3,3&#39;-二氨基联苯胺（DAB）中加工用于切片制剂。固定胚胎的扁平制剂的一个关键优点是周边轴突突起和肌肉模式的更好的可视化。此外，这项工作还显示了如何使用LacZ染色法将固定的胚胎基因型分类为所需的突变体胚胎。

<table><tbody><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>t-Octylphenoxypolyethoxyethanol</td></tr><tr><td>16% Paraformaldehyde Solution</td><td>Ted Pella</td><td>18505</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma-Aldrich</td><td>30435</td><td></td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma-Aldrich</td><td>71500</td><td></td></tr><tr><td>X-Gal Substrate</td><td>US Biological</td><td>X1000</td><td>X-Gal (5-Bromo-4-chloro-3-indolyl-b-D-galactoside galactopyranoside)</td></tr><tr><td>Dimethyl Sulfxide</td><td>Sigma-Aldrich</td><td>D4540</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Potassium hexacyanoferrate(II) trihydrate</td><td>Sigma-Aldrich</td><td>P9387</td><td></td></tr><tr><td>Potassium hexacyanoferrate(III)</td><td>Sigma-Aldrich</td><td>244023</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma-Aldrich</td><td>216763</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine Tetrahydrochloride</td><td>Sigma-Aldrich</td><td>D5905</td><td></td></tr><tr><td>Agar</td><td>US Biological</td><td>A0930</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3</td><td></td></tr><tr><td>Tegosept (Methy 4-Hydroxybenzoate)</td><td>Sigma-Aldrich</td><td>H5501</td><td></td></tr><tr><td>Culture Dish (60 mm)</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>Tricon Beaker</td><td>Simport</td><td>B700-100</td><td>This is used to make a plastic beaker cage for embryo collection.</td></tr><tr><td>Yeast</td><td>Societe Industrielle Lesaffre</td><td>Saf Instant Yeast Red</td><td></td></tr><tr><td>Cotton Swab (Wooden Single Tip Cotton PK100)</td><td>VWR</td><td>14220-263</td><td></td></tr><tr><td>Eppendorf Tube (1.5 ml)</td><td>Sarstedt</td><td>#72.690</td><td></td></tr><tr><td>Bleach</td><td>The Clorox Company</td><td>Clorox</td><td></td></tr><tr><td>Heptane</td><td>Sigma-Aldrich</td><td>246654</td><td></td></tr><tr><td>Methanol</td><td>J.T. Baker</td><td>UN1230</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Life Technologies</td><td>16210-064</td><td></td></tr><tr><td>Anti-FasciculinII Antibody</td><td>Developmental Studies Hybridoma Bank</td><td>1D4 anti-Fasciclin II</td><td></td></tr><tr><td>Goat Anti-mouse-HRP Antibody</td><td>Jackson Immunoresearch</td><td>115-006-068</td><td>AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Goat Anti-Mouse IgG+IgM (H+L)&lt;br/&gt; (min X Hu, Bov, Hrs Sr Prot</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G9012</td><td></td></tr><tr><td>Slide Glass</td><td>Duran Group</td><td>235501403</td><td></td></tr><tr><td>Coverslip</td><td>Duran Group</td><td>235503104</td><td>18 x 18 mm</td></tr><tr><td>1 ml Syringe</td><td>Becton Dickinson Medical(s)</td><td>301321</td><td></td></tr><tr><td>Tungsten Needle</td><td>Ted Pella</td><td>#27-11</td><td>Tungsten Wire, &amp;oslash;0.13mm/6.1m (&amp;oslash;.005&quot;/20 ft.)</td></tr><tr><td>Nutator (Mini twister)</td><td>Korean Science</td><td>KO.VS-96TWS</td><td>Alternatively, BD Clay Adams Brand Nutator (BD 421125)</td></tr></tbody></table>

visualization of the axonal projection pattern of embryonic motor neurons in drosophila

功能性神经肌肉回路的建立依赖于发育中的运动轴突和目标肌肉之间的精确连接。运动神经元通过响应大量来自周围细胞外环境的轴突指导线索，延长生长锥体以沿着特定途径导航。生长锥目标识别也在神经肌肉特异性中起关键作用。这项工作提出了一个标准的免疫组织化学方案，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。该方案包括几个关键步骤，包括基因分型程序，以排序所需的突变体胚胎;免疫染色程序，用fasciclin II（FasII）抗体标记胚胎;和解剖程序，从固定胚胎产生鱼片制剂。周边的运动轴突突出物和肌肉图案在圆形胚胎的扁平制剂中比在wh中更好地可视化油籽胚胎。因此，用FasII抗体染色的固定胚胎的切片制备为表征运动轴突寻路和靶标识别所需的基因提供了有力的工具，也可以应用于功能丧失功能遗传屏障。

神经发育期间运动轴突和目标肌肉之间的精确连接取决于在特定选择点的选择性轴突 - 轴突排斥和目标识别。在果蝇中 ，运动轴突之间的选择性排斥部分由第1类和第2类信号素（Semas）（包括Sema-1a，Sema-2a和Sema-2b）8,14,15,16,17,18的组合作用。因此， Sema-1a功能的丧失常常导致ISNb轴突在特定选择点处失效，导致其表现出异常浓厚的形态（ 图2C中的<...

Watch this Scientific Journal Video about Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila at JoVE.com

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

这项工作详细描述了一个标准的免疫组织化学方法，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。用FasII抗体染色的固定胚胎的切片制备提供了一个强大的工具，用于表征神经发育期间运动轴索寻路和靶标识别所需的基因。

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

visualization-axonal-projection-pattern-embryonic-motor-neurons

Research

JoVE Journal

Neuroscience

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

7.7K 次观看.  Chonbuk National University. 这项工作详细描述了一个标准的免疫组织化学方法，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。用FasII抗体染色的固定胚胎的切片制备提供了一个强大的工具，用于表征神经发育期间运动轴索寻路和靶标识别所需的基因。 

视频: Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

科研

神经科学

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

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 ...

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 ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

发育生物学

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

制备<em&gt;果蝇</em&gt;中央神经元<em在原位</em&gt;膜片钳

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

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 ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

通过成人角质层可视化果蝇腿运动神经元轴突

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

胚胎运动神经元轴突投影模式的可视化

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