笔者想在美国爱荷华大学的感谢，向我们发送抗体杂交瘤细胞银行的发展研究。这项工作是由以色列科学基金会的资助（29/08号）的支持。还支持从东风集团（德意志研究联合会）的研究资助。

在您开始之前<ol><li>成长所需的飞行文化。保持不拥挤的小瓶（约30苍蝇每毫升50瓶）。让苍蝇奠定只有一个每天在每个小瓶鸡蛋。这将提供幼虫充足的食物供应和之前爬出来的食物，使他们能够达到的最大大小。保持适当的温度小瓶，直到三龄幼虫开始小瓶墙壁上漫步。 </li><li>准备新鲜的股票的磷酸盐缓冲液（PBS）和PBT（0.1％吐温的PBS）。 10毫升的PBS置于冰上。 </li><li>准备1-5毫升的新鲜固定液（4％甲醛在PBT），并保持它在冰上。我们使用瓶装37-38％的甲醛，作为原液。 </li></ol> 1。三龄幼虫的解剖及固定<ol><li>拿起小瓶墙一溜的第三龄幼虫，并将其放置在冰冷PBS 50μL上Sylgard夹层板（Sylgard 184，道康宁公司的有机硅弹性体包在下降组织培养皿）。 </li><li>举行的幼虫，一边背，口附近的钩子，用钳（杜蒙＃5镊子）的罚款，并坚持通过昆虫幼虫的大脑脚（minutien，0.1毫米，不锈钢）。 </li><li>抢与镊子幼虫的后端，轻轻拉和纵向轻轻舒展幼虫。坚持另一种昆虫针后两者之间的气孔。 </li><li>使用弹簧剪刀剪在体壁的水平切口（垂直于前后轴），接近前部和后部的引脚。 </li><li>使用弹簧剪刀剪前幼虫体壁沿背中线后切口。 </li><li>取出内脏（肠，唾液腺，马氏管等）和气管，用细镊子。轻轻洗一次或两次用PBS。 </li><li>用钳子抓住两个体壁瓣，伸展出来和牵制他们每侧两个昆虫针板使用。 </li><li>删除的PBS和添加50μL固定的解决方案（4％甲醛在PBT）。在室温下孵育20分钟。 </li><li>删除的固定液。用PBS洗两次。拉出昆虫针。使用弹簧剪刀剪幼虫的头部和尾部留下一个长方形圆角。转让固定组织用PBS冷冻离心管。 </li><li>一旦足够数量的幼虫是固定的，可以继续直接抗体染色。如果不希望立即染色，固定幼虫应与95％乙醇洗涤3次，每次5分钟，并存储在95％乙醇于-20°C </li></ol> 2。蛹的解剖和固定第I部分<ol><li>出席幼虫固定的小瓶，直到三龄幼虫开始pupariate。检查瓶幼虫已pupariated每隔一小时大关。允许的显着蛹发展在24°C，或24-27小时30-40小时29°C。 </li><li>用细镊子挑20-30日蛹é适当的年龄，并把它们在一个黑暗的瓷器多井解剖菜。要小心，不要损坏利益的组织。 </li><li>从蛹的情况下提取的蛹。开始脱落，鳃盖和继续下去，直到蛹是免费的。投入一个充满与PBT的蛹。继续提取所有的蛹。步骤2.4-2.6应做一次小批量五个蛹。 </li><li>放置在平坦的表面之间的解剖盘井五蛹。用微型清扫刀，切尖的头部和腹部后尖（或者，它可以使用两双镊子撕孔两端的蛹）。 </li><li>在使用罚款钳的地方举行蛹，用1毫升注射器，洗出蛹的内部器官，通过注射前开放PBT。 </li><li>洗净，浸泡在用PBS充满解剖蛹简要移入Eppendorf管放在冰上冷固定液。 </li><li>孵育过夜（ON）在4°C。 </li></ol>第二部分<ol start="8"><li>倒掉固定液和蛹三次，每次5分钟，与PBT洗。保持冰洗蛹。 </li><li>填写与PBT解剖盘的两个井。使用聚乙烯巴斯德吸管转让几个蛹井。 </li><li>一次移动到第二口井的一个蛹。使用完全一致的提示两对锋利的钳子，透明的翅膀蛹角质层脱落：确保蛹以及使用钳子底部，轻轻撕角质层，使用产钳的第二对。一旦角质层被撕裂，把它剥开翼。要小心不要断开从蛹的翅膀。从机翼叶片角质层剥落后，继续剥离角质层翼铰链（位于翼ChOs）。 </li><li>剥离翼角质层后，可以尝试以类似的方式腿角质层剥落。在这个过程中可能会失去很多腿，但尽管产量低，这一步强烈建议，因为它大大提高了腿ChOs的染色。没有进一步的角质层剥落的haltere和腹部ChOs可以可视化。 </li><li> “去皮”蛹放置在一个充满Eppendorf管中，以甲醇和保持在室温。继续所有蛹的表皮剥落，并把它们添加到同一管内。至少有10个很好的去皮蛹应收集每个染色。 </li><li>除去甲醇洗三次，每次5分钟，用95％乙醇。固定蛹可以保持很长一段时间，在95％乙醇于-20°C </li></ol> 3。免疫组化的幼虫和蛹<ol><li>洗与PBT的三次，每次30分钟，在室温下，固定的组织。幼虫可水洗，轻轻摇动旋转板。蛹洗净，无晃动。 </li><li>封闭液（取代了PBT PBT + 5％列印ormal山羊血清）和4对孵化°C。 </li><li>删除封闭液和抗体（封闭液稀释）孵育在4°C </li><li>与PBT清洗四次，在步骤3.1中描述。 </li><li>卸下PBT孵育二次抗体（封闭液稀释）在4°C </li><li> PBT和洗两次一次用PBS在步骤3.1中描述。 </li><li> PBS的安装介质（Dako公司荧光灯安装介质（Dako公司Cytomation，Glostrup，丹麦）和孵化在4°C的替换。 </li></ol> 4。安装幼虫<ol><li>幼虫被安装上了他们的角质层朝下体壁肌肉朝上安装媒体中的显微镜幻灯片。把一个干净的盖玻片上的安装介质中的小水滴，并用它来支付的准备。不适用任何安装幼虫上的压力。 </li></ol> 5。安装蛹<ol><li>准备两个载玻片下降的安装介质，清扫和安装第二。 </li><li>几个蛹放在一个幻灯片。 </li><li>使用弹簧剪刀除去头部和腹部的后部。 </li><li>从腹侧半分开切割的翅膀和腿之间的胸部背侧一半。 </li><li>在安装的解决方案中的第二显微镜幻灯片上放置解剖蛹的身体部位。确保翅膀和腿伸了出来，并尽量减少叠加尽可能组织。 </li><li>将盖玻片上的安装介质下降，并将其放置在样本。并不适用于安装样品的压力。 </li></ol> 6。代表结果第三龄幼虫免疫染色ChOs的例子， 如图1所示。这个例子显示了一个很好的拉伸腹段，在这七个八个ChOs清晰可见。神经元是实验室ELED与MAb22C10（1:20，从发展研究杂交瘤细胞银行获得在美国爱荷华大学）;帽，韧带，帽，附件和韧带附着细胞标记与反αTub85E（1:10，克莱因等。 2010年）。中学抗体荧光染色Cy3标记，或Cy5的标记anti-mouse/rabbit（1:200，杰克逊Immunoresearch实验室）。使用共聚焦显微镜（LSM的510蔡司）查看样品。 HR-35岁的蛹径向腹侧静脉翼ChOs群集如图2所示。 <img alt="图1" src="/files/ftp_upload/3846/3846fig1.jpg" /> 图1六种类型的细胞构成单一CH机关：（一）示意图。帽附着（CA），帽（C）scolopale（），神经（N）的韧带（L）和韧带附着（洛杉矶）。被拉长，聚集在5 ChOs是，LCh5机关跨​​外侧横肌组（LT1-4）。外侧纵肌（LL1），腹纵肌（​​VL1-4）和横向斜肌（LO1），也说明。肌腱细胞是棕色球（Klein等。，2010）所示。 （二）第三龄幼虫腹段在10倍放大倍率观看。目前在每腹节的八个ChOs七是显而易见的：五个横向机关共同组成的机关（pentascolopidial LCh5），一个单一的横向器官（LCh1）和（VChB）腹侧ChOs之一。标记的ChOs（N）的神经元与神经元的标记单克隆抗体22C10（红色）。韧带（L）帽（C）和附着细胞（洛杉矶，加利福尼亚州）标记的抗αTub-85E（蓝色）。此外帽和韧带细胞标记与CHO特定的绿色荧光蛋白记者窝藏调控元件的，DEI轨迹（Nachman 等，未发表资料）。 <img alt="图2" src="/files/ftp_upload/3846/3846fig2.jpg" /> 图2。 </s仲&gt;在桡腹侧静脉的的右翼chordotonal机关，被视为在40X放大。神经元（N）的标记神经元的标记22C10（红B）。韧带（L）和盖帽（三）细胞标记与的反αTub85E抗体（蓝）的CHO特定的GFP报告基因（Nachman 等，未发表资料）。

<ol>
	<li>Brewster, R., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+specification+of+type+II+sensory+neurons+in+Drosophila.">Origin and specification of type II sensory neurons in Drosophila.</a> Development. 121, 2923-2936 (1995).</li><li>Caldwell, J. C., Miller, M. M., Wing, S., Soll, D. R., Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+larval+locomotion+in+Drosophila+chordotonal+organ+mutants.">Dynamic analysis of larval locomotion in Drosophila chordotonal organ mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 16053-16053 (2003).</li><li>Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeling+the+vibes:+chordotonal+mechanisms+in+insect+hearing.">Feeling the vibes: chordotonal mechanisms in insect hearing.</a> Curr. Opin. Neurobiol. 9, 389-393 (1999).</li><li>Eberl, D. F., Boekhoff-Falk, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Johnston's+organ+in+Drosophila.">Development of Johnston's organ in Drosophila.</a> Int. J. Dev. Biol. 51, 679-687 (2007).</li><li>Eberl, D. F., Hardy, R. W., Kernan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+similar+transduction+mechanisms+for+touch+and+hearing+in+Drosophila.">Genetically similar transduction mechanisms for touch and hearing in Drosophila.</a> J. Neurosci. 20, 5981-5988 (2000).</li><li>Egoz-Matia, N., Nachman, A., Halachmi, N., Toder, M., Klein, Y., Salzberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+cell+adhesion+in+the+Drosophila+wing+is+mediated+by+Delilah,+a+potent+activator+of+&#946;PS+integrin+expression.">Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of &#946;PS integrin expression.</a> Dev Biol. 351, 99-109 (2011).</li><li>Field, L. H., Matheson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chordotonal+organs+of+insects.">Chordotonal organs of insects.</a> Advances in Insect Physiology. 27, 1-228 (1998).</li><li>Gong, Z., Son, W., Chung, Y. D., Kim, J., Shin, D. W., McClung, C. A., Lee, Y., Lee, H. W., Chang, D. J., Kaang, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+interdependent+TRPV+channel+subunits,+inactive+and+Nanchung,+mediate+hearing+in+Drosophila.">Two interdependent TRPV channel subunits, inactive and Nanchung, mediate hearing in Drosophila.</a> J. Neurosci. 24, 9059-9066 (2004).</li><li>Inbal, A., Volk, T., Salzberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+ectodermal+attachment+cells+via+an+EGFR-dependent+mechanism+during+the+organogenesis+of+Drosophila+proprioceptors.">Recruitment of ectodermal attachment cells via an EGFR-dependent mechanism during the organogenesis of Drosophila proprioceptors.</a> Dev. Cell. 7, 241-250 (2004).</li><li>Jarman, A. P., Grau, Y., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=atonal+is+a+proneural+gene+that+directs+chordotonal+organ+formation+in+the+Drosophila+peripheral+nervous+system.">atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system.</a> Cell. 73, 1307-1321 (1993).</li><li>Jarman, A. P., Sun, Y., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+proneural+gene,+atonal,+in+formation+of+Drosophila+chordotonal+organs+and+photoreceptors.">Role of the proneural gene, atonal, in formation of Drosophila chordotonal organs and photoreceptors.</a> Development. 121, 2019-2030 (1995).</li><li>Kim, J., Chung, Y. D., Park, D. Y., Choi, S., Shin, D. W., Soh, H., Lee, H. W., Son, W., Yim, J., Park, 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=A+TRPV+family+ion+channel+required+for+hearing+in+Drosophila.">A TRPV family ion channel required for hearing in Drosophila.</a> Nature. 424, 81-84 (2003).</li><li>Klein, Y., Halachmi, N., Egoz-Matia, N., Toder, M., Salzberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proprioceptive+and+contractile+systems+in+Drosophila+are+both+patterned+by+the+EGR+family+transcription+factor+Stripe.">The proprioceptive and contractile systems in Drosophila are both patterned by the EGR family transcription factor Stripe.</a> Dev. Biol. 337, 458-470 (2010).</li><li>Matthews, K. A., Miller, D. F., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+implications+of+the+unusual+spatial+distribution+of+a+minor+alpha-tubulin+isotype+in+Drosophila:+a+common+thread+among+chordotonal+ligaments,+developing+muscle,+and+testis+cyst+cells.">Functional implications of the unusual spatial distribution of a minor alpha-tubulin isotype in Drosophila: a common thread among chordotonal ligaments, developing muscle, and testis cyst cells.</a> Dev. Biol. 137, 171-183 (1990).</li><li>Okabe, M., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+induction+of+chordotonal+organ+precursors+in+Drosophila+embryogenesis.">Two-step induction of chordotonal organ precursors in Drosophila embryogenesis.</a> Development. 124, 1045-1053 (1997).</li><li>Rusten, T. E., Cantera, R., Urban, J., Technau, G., Kafatos, F. C., Barrio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spalt+modifies+EGFR-mediated+induction+of+chordotonal+precursors+in+the+embryonic+PNS+of+Drosophila+promoting+the+development+of+oenocytes.">Spalt modifies EGFR-mediated induction of chordotonal precursors in the embryonic PNS of Drosophila promoting the development of oenocytes.</a> Development. 128, 711-722 (2001).</li><li>Todi, S. V., Sharma, Y., Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+molecular+design+of+the+Drosophila+antenna+as+a+flagellar+auditory+organ.">Anatomical and molecular design of the Drosophila antenna as a flagellar auditory organ.</a> Microsc. Res. Tech. 63, 388-399 (2004).</li><li>zur Lage, P., Jarman, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+of+EGFR+and+notch+signalling+in+the+reiterative+recruitment+of+Drosophila+adult+chordotonal+sense+organ+precursors.">Antagonism of EGFR and notch signalling in the reiterative recruitment of Drosophila adult chordotonal sense organ precursors.</a> Development. 126, 3149-3159 (1999).</li></ol>

没有利益冲突的声明。

在这部影片中所描述的协议提供了一个可视化本体ChOs在幼虫和蛹的阶段。结构和本体ChOs的发展研究迄今已限制主要是胚胎阶段，幼虫的成虫盘。因此，既幼虫和成人ChOs的，更高的发展阶段，许多方面，仍是未知。所描述的协议，结合在果蝇中常见的遗传工具，可以用来确定和研究新的基因和途径，发挥作用，在后ChOs发展阶段。这个协议进行了优化，为CHO可视化，但它可以为其他组织，如6蛹两翼，染色适应。

<table border="1"><tbody><tr><td> 试剂名称/工具 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>杜蒙（或＃55）＃5镊子，biologie尖</td><td>福斯特</td><td> 11252-20或11252-10 （或类似的镊子） </td><td></td></tr><tr><td>奥斯特利茨不锈钢昆虫引脚的，minutiens0.1毫米</td><td> roboz手术仪器有限公司</td><td> RS-6083-10 </td><td></td></tr><tr><td> sylgard 184硅酮弹性体套件</td><td>道康宁公司</td><td> 240.4019862 </td><td></td></tr><tr><td> vannas微型剪刀（3毫米直，7.5厘米，叶片） </td><td> AS Mdeizintechnik有限公司</td><td> 11-590-00 </td><td>具有相同规格的vannas弹簧剪刀，就可以买到从Roboz手术仪器有限公司</td></tr><tr><td>轨道振动筛 - Rotamax-120 </td><tD&gt; Heidolph </td><td> N / A </td><td></td></tr><tr><td> DAKO荧光灯安装中等</td><td> Glostrup，丹麦Dako公司Cytomation </td><td> DK-5302392 </td><td></td></tr><tr><td> X10的PBS配方</td><td colspan="2"> 2克/升的氯化钾，2克/升KH 2 PO 4计 ，80克/升氯化钠， 21.7克/点燃娜2 HPO4.7H 2 </td><td>这一协议的成功的PBS的质量是关键</td></tr><tr><td> PBT的</td><td colspan="2"> X1的PBS +0.1％吐温20 </td><td></td></tr></tbody></table>

visualization of proprioceptors in drosophila larvae and pupae

本体是能够感知的议案，或身体各部位的位置，身体内产生的刺激。在果蝇和其他昆虫的本体是由专门的感觉器官称为chordotonal机关（ChOs）的2。在果蝇中许多其他器官一样，ChOs发展在苍蝇的生命周期的两倍。首先，的幼虫ChOs发展胚胎发育过程中。然后，的成人ChOs开始发展幼虫的成虫盘和继续分化过程中变态。 的发展幼虫ChOs胚胎发育过程中已被广泛10,11,13,15,16研究。每个CHO的核心是一种感官的单位组成的神经元和scolopale细胞。感觉单位之间的两个辅助细胞类型，重视通过专门的表皮的依恋细胞1,9,14角质层被拉长。当一只苍蝇幼虫的动作，相对位移的电子pidermal附着细胞伸展的感觉单位和特定的瞬时受体电位香草（TRPV）的外段在枝蔓8,12的渠道也随之开放。在中枢神经系统，引起信号，然后转移到运动中枢模式发生器电路。 多个ChOs已被形容在成年果蝇7。这些都位于关节附近的成年果蝇的附加物（腿，翅膀和缰绳）和在胸部和腹部。此外，数百ChOs的成人天线共同构成约翰斯顿的器官，换能器声，机械能3,5,17,4。 相反，在胚胎阶段有关的ChOs的发展广泛的知识，很少有人知道这些器官的形态在幼虫期。此外，除了股ChOs的18和约翰斯顿的器官，我们knowled的GE有关的发展和成年果蝇ChOs结构是非常零碎的。 在这里，我们描述了染色和可视化ChOs在三龄幼虫和蛹的方法。这种方法可应用于遗传工具，以更好地描述的形态和了解，在飞行的各种ChOs发展。

Watch this Scientific Journal Video about Visualization of Proprioceptors in Drosophila Larvae and Pupae at JoVE.com

Visualization of Proprioceptors in Drosophila Larvae and Pupae

方法免疫组织化学染色和可视化chordotonal机关在幼虫和蛹<em&gt;果蝇</em&gt;描述。

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 ...

visualization-of-proprioceptors-in-drosophila-larvae-and-pupae

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

11.9K 次观看.  Technion-Israel Institute of Technology. 方法免疫组织化学染色和可视化chordotonal机关在幼虫和蛹果蝇描述。

视频: Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

光偏好的检测，以研究的先天和昼夜调节感光特性<em&gt;果蝇</em&gt;幼虫

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

温度梯度法测定果蝇幼虫的热偏好

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦：了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.