我们感谢我们的同事在弗里堡大学生物系，富有成效的讨论。我们感谢布卢明顿联合中心提供飞股票。财政支持这项工作是由瑞士国家科学基金会（PP00P3_123339）和威卢克斯基金会SGS

1。苗种培育<ol><li>保持飞菌株或大众文化的遗传杂交玉米粉培养基上，在25°C下12小时轻12小时黑暗周期中一只苍蝇孵化器配备有光线和定时器。 </li><li>稀释在水中，形成衬垫酵母的流体浆料（10克的背衬酵母稀释用3-4毫升蒸馏水H 2 O）。加一小滴玉米粉食品和覆盖小瓶。让至少一个小时的干燥，以避免成虫坚持酵母膏。将酵母水稀释一小滴表面上的食物可提高产卵。或者可以使用面包酵母膏，20％乙酸（醋酸）（Sigma Aldrich公司，瑞士A6283-100ML）。对于这一点，蘸棉条的尖端的解决方案和玉米粉食品表面上蔓延。 </li><li>将成年苍蝇（至少四天左右，但不是年龄大于10天）在玉米粉食品小瓶。为了获得足够的幼虫重复偏好，投入足够的成年人到每个小瓶试了几次，并允许统计比较。对于遗传杂交，我们通常使用最小的20位女性和5-10男性每小瓶，但一些线路需要更多的女性获得足够的幼虫的后代。 </li><li>允许成人产卵12小时，并将他们转移到新的小瓶。成人可以转移在早晨和傍晚。我们通常将成人七到十天，如果需要采取新的成年人。 </li><li>保持鸡蛋在孵化器每12小时的集合，让增长的幼虫在25°C，12小时光照，12小时黑暗周期和60％的湿度。为了测试为昼夜组件视觉行为举动幼虫持续黑暗（DD）鸡蛋收集后48小时（相当于两光暗周期）。对于这一点，使用一个单独的孵化器不轻，但保持所有其他条件，如温度和湿度相同。要确保传输的小瓶DD孵化器开关前的黑暗阶段。搬家前的小瓶anothER孵化器把小瓶在一个纸箱或小瓶包装用铝箔，以防止光线在传输过程中暴露（包装纸箱或包装有许多工作要做，在“灯相”前孵化器之间的位移）。防止动物，这一点，任何光线照射后，直到实验开始。 </li><li>经过4天（从产蛋84至108小时）收集：早期喂养幼虫（L3）的时间点进行测试（见4。轻偏好测试点。4.4）。 </li></ol> 2。测试设置<ol><li>在一个黑暗的房间，恒定的温度和湿度条件下进行实验。 </li><li>为了保持两个象限培养皿在黑暗中，盖好盖子，用黑胶带和铝箔。为了做到这一点，记下盖周围的中心。 90°，它们之间的分离，也标志着四点在边境的盖子。 ，使其更容易，绘制分在四个象限上的长度为10厘米20厘米见方的一张纸或一台计算机的帮助。切铝箔和黑色带（ 图1A）的 10厘米的正方形。包括两个相对象限胶合广场铝箔，第一层和黑胶带覆盖，要小心，还包括边境的盖子。 在过去的两个略有不同的形式，这样的检测。我们在这里使用的“四分之一板”实验，在培养皿分为四个相等季度10。在替代的“半片”检测培养皿分一半（半暴露于光，在黑暗中一半）1,7,8。直到今天，两个检测之间没有显着的差异已经出版。 </li><li>胶用于标记盖在桌子上的光源下，如一个象限表。马克象限的交点为中心的陪替氏培养皿的周围。使用这些标记作为参考试验板放置的光源下，总是在相同的位置。 </li><li>安装灯，无论是一个简单的白色灯泡（菲利普斯，5W Softtone）或以上的铁支架（Fisher Scientific公司，S47808）的帮助下，在培养皿的LED灯（LED灯，80012白，欧司朗）。调整的方式，均匀照亮整个测试板的高度。我们强烈建议使用LED灯具，因为它们比传统的灯泡发出更具体的波长。此外，发光二极管发出的热量更少。 </li><li>光度计（环境仪表PCE EM882）的帮助下，调整光照强度。为了达到所需的350-760勒克斯的光照强度，移动灯或向下的需要。灯泡插入一个可调节的电源，让更多的灵活性，而不移动的灯（ 图1B）来改变强度。 </li></ol> 3。板准备<ol><li> 200ml的2.5％琼脂（Sigma-Aldrich公司，瑞士A5093-500G），用双蒸水（Millipore公司）。 </li><li>将培养皿放在桌子上（直径90毫米;格雷纳生物之一，奥地利，4550 Kremsmeinster联系）行允许浇热琼脂糖。 </li><li>琼脂糖微波炉煮，直到解决方案是完全透明和流体。确保液体溶液中不含有气泡。注意：运输仔细的表，因为该解决方案可以非常热！ </li><li>琼脂糖到培养皿中倒入热水，直到整个表面均匀地覆盖有一薄层的溶液，两或三毫米就足够了。要迅速防止琼脂糖凝固涂装前所有板块。让板降温。板在室温下储存，并使用它们只在同一天的准备。 </li></ol> 4。轻偏好测试<ol><li>在实验室的红色光条件下的工作，以防止光线的影响，，由于果蝇是在测试之前不能够感应到红色的波长的光。使用红色灯泡（菲利普斯，PFE712E * 8）安装一盏灯，照亮吨他的工作场所。 </li><li>通过所有的实验中温度保持在25°C。如果需要的话，通过控制室内温度的加热器或冷却装置。 </li><li>采取一些食物持续黑暗下的为期两天的周期（从第1节）饲养的小瓶。由于幼虫通常挖的食品的表面上，采取的最上层（约5毫米深）的帮助下，用抹刀（Fisher Scientific公司，14-373-25A）。传播食品培养皿中盖的外侧，加清水适量，用锅铲轻轻混匀。 </li><li>收集从食物喂养L3幼虫。由于重量轻，将被测试优先选择，只有早期/喂养L3幼虫是至关重要的，因为负趋光性是有保证的。晚/漂泊L3的幼虫或大的幼虫的食物上爬行，想必已经交换了他们的感光特性。可以确认的，因为他们的前气门是开放的，向外侧伸出一个手指状的形式饲喂L3幼虫（负趋光）。后气门s有三个开口，四组大支链的头发。唾液腺延伸到第二腹节11。分段幼虫在显微镜下是方便，而没有白色光应使用。一个红色的灯是必要的，如果分期立体显微镜下，实验前需要照亮幼虫。 </li><li>自来水中简要幼虫洗净，，收集早期喂养三龄幼虫（仍然在红灯）。幼虫转移到试验板之前，用湿的画笔幼虫，并小心地吸收过量的水，用纸巾或滤纸。不要过分干燥的幼虫，因为它可能会损害动物，并影响其行为。 </li><li>使用湿画笔仔细的幼虫转移到板。放置在板中心的一组约30只幼虫。覆盖板的盖子已经准备好与象限板的光源下，准备实验（见秒重刑2）。 </li><li>白色灯打开，并开始计时。让幼虫上自由移动盘5分钟，然后迅速取下盖子，在黑暗和光象限计数的幼虫数。用记号笔标记每个幼虫的位置，可以缓解计数。或者拍摄一张照片的板块，并计算后验。幼虫光偏好不清楚，在墙壁上爬行状幼体或钻入琼脂应被视为中性偏好，刚好被列入总数的幼虫光偏好指数的计算方法。 </li><li>计数后，幼虫和丢弃板更换一个新的测试板接下来的实验中。收集用过的盘子，在高压釜中的塑料袋后的处理和处置。 </li><li>一旦您已执行了足够数量的实验可以测试一个新的基因型。 10-15试验每个基因型是足以进行分析。 </li></ol> 5。数据分析Ş <ol><li>为了方便将数据传送到Excel中的数据表（微软）或者原产（原产实验室）的计算机上进行进一步的统计分析。排列在一列中的动物在黑暗中，在第二列的动物的数量的光象限和第三板（包括“中性”的幼虫）的动物的总数量。 </li><li>计算使用下列公式计算每个实验的黑暗的偏好指数（县）： PREF（黑暗）=（在黑暗的幼虫 - 幼虫数光）/总数幼虫的</li><li>比较统计学上的一组数据组的适当分析。在这里，我们使用Wilcoxon检验统计学比较两组。方差测试一个Tukey多重比较事后检验是可以做到的，如果满足正态分布假设在歧管组比较。 </li><li>使图表清楚地表明线和沿天为一周期的时间点之间的比较。 WE使用Origin软件（原产实验室）测试统计学意义，并产生相应的图表，但任何其他统计软件可以帮助。 </li></ol>

<ol>
	<li>Sawin-McCormack, E. P., Sokolowski, M. B., Campos, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+genetic+analysis+of+Drosophila+melanogaster+photobehavior+during+larval+development.">Characterization and genetic analysis of Drosophila melanogaster photobehavior during larval development.</a> J. Neurogenet. 10, 119-135 (1995).</li><li>Sprecher, S. G., Pichaud, F., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+and+larval+photoreceptors+use+different+mechanisms+to+specify+the+same+Rhodopsin+fates.">Adult and larval photoreceptors use different mechanisms to specify the same Rhodopsin fates.</a> Genes Dev. 21, 2182-2195 (2007).</li><li>Sprecher, S. G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons.">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454, 533-537 (2008).</li><li>Xiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-avoidance-mediating+photoreceptors+tile+the+Drosophila+larval+body+wall.">Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.</a> Nature. 468, 921-926 (2010).</li><li>Diaz, N. N., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptors:+unconventional+ways+of+seeing.">Photoreceptors: unconventional ways of seeing.</a> Curr. Biol. 21, R25-R27 (2011).</li><li>Emery, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+CRY+is+a+deep+brain+circadian+photoreceptor.">Drosophila CRY is a deep brain circadian photoreceptor.</a> Neuron. 26, 493-504 (2000).</li><li>Mazzoni, E. O., Desplan, C., Blau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+pacemaker+neurons+transmit+and+modulate+visual+information+to+control+a+rapid+behavioral+response.">Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response.</a> Neuron. 45, 293-300 (2005).</li><li>Keene, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+visual+pathways+mediate+Drosophila+larval+light+avoidance+and+circadian+clock+entrainment.">Distinct visual pathways mediate Drosophila larval light avoidance and circadian clock entrainment.</a> J. Neurosci. 31, 6527-6534 (2011).</li><li>Gong, Z. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+Pairs+of+Neurons+in+the+Central+Brain+Control+Drosophila+Innate+Light+Preference.">Two Pairs of Neurons in the Central Brain Control Drosophila Innate Light Preference.</a> Science. 330, 499-502 (2010).</li><li>Lilly, M., Carlson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=smellblind:+a+gene+required+for+Drosophila+olfaction.">smellblind: a gene required for Drosophila olfaction.</a> Genetics. 124, 293-302 (1990).</li><li>Bodenstein, D., Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+postembryonic+development+of+Drosophila.">The postembryonic development of Drosophila.</a> Biology of Drosophila. , 275-367 (1950).</li><li>Pittendrigh, 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=Circadian+systems:+Entrainment.">Circadian systems: Entrainment.</a> Biological Rhythms. 4 Handbook of Behavioral Neurobiology, 95-124 (1981).</li><li>Collins, B., Kane, E. A., Reeves, D. C., Akabas, M. H., Blau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balance+of+Activity+between+LN(v)s+and+Glutamatergic+Dorsal+Clock+Neurons+Promotes+Robust+Circadian+Rhythms+in+Drosophila.">Balance of Activity between LN(v)s and Glutamatergic Dorsal Clock Neurons Promotes Robust Circadian Rhythms in Drosophila.</a> Neuron. 74, 706-718 (2012).</li><li>Keene, A. C., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+photobehavior+in+fruit+fly+larvae.">Seeing the light: photobehavior in fruit fly larvae.</a> Trends Neurosci. 35, 104-110 (2012).</li><li>von Essen, A. M., Pauls, D., Thum, A. S., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+visual+classical+conditioning+in+Drosophila+larvae.">Capacity of visual classical conditioning in Drosophila larvae.</a> Behav. Neurosci. 125, 921-929 (2011).</li></ol>

什么都没有透露。

光偏好测试幼虫的先天优势，感光特性。该法是很容易的建立，允许多次重复，以较低的成本和光传感和处理提供了有价值的信息。实验范式允许多少人喜欢浅色或深色的相对快速定量。这种偏好可以显示作为原油的百分比，或者偏好指数（县）。 PREF被表示为动物的差异，优选的光，优选黑暗除以动物的总的动物。 光偏好测试中的一个关键点是实验的时间。在这里，我们测...

在这里，我们描述了一个基于行为的检测暗（或光）幼虫偏好。幼虫的反应与一个强大和刻板避光反应在觅食阶段（L1至L3月初）1。幼虫避光行为的测定法的目的是评估和比较亮或暗的幼虫自由移动，在涂有琼脂的陪替氏培养皿中的一组的偏好。这种行为的检测不仅提供信息的敏感性，一体化和时间的视觉系统可塑性，它进一步提供了光敏感性及其过程控制的昼夜系统的提示。 果蝇幼虫眼（也被称为Bowlig的器官; BO），光感的主要器官。每只眼睛是由12个光感受器（PR），8 PRS表达绿色，：敏感rhodopsin6（RH6）和四个永久居民表达了对蓝色敏感rhodopsin5（RH5）2,3。此外PRS，ALSØIV级的multidendritic神经元，涵盖幼虫体壁，已经确定以应对4,5有害光线强度。它也被称为位于中央的幼虫脑起搏器的神经元表达光敏蛋白隐花色素（喊），作为时钟固有的蓝色光传感器大脑内的6,7。有趣的野生型动物photophobicity显示一昼夜成分在不同时间点的过程中，白天和夜间的时候，用这个方法测试。对L3幼虫觅食表现出较强的photophobicity在黎明和photophobicity的黄昏时进行测试时光黑暗的偏好7。有趣的是，只有RH5-PRS都需要避免轻，而RH6​​-PRS是可有可无的。两者RH5-PR和RH6-PRS参与重置分子钟光8。惊魂途径，必须协调与其他光感的途径，以协调在一个适当的行为输出一天的过程中。乙酰胆碱在PRS光回避行为，以及夹带的分子时钟起着至关重要的作用。从PRS昼夜起搏器神经元阻断神经递质乙酰胆碱减少光黑暗偏好测定8避光反应。采用相同的检测，两个对称的神经元对最近发现的第三龄幼虫果蝇 9开灯偏好。这两对神经元功能在后期幼体阶段，当动物留下的食物大概是找到一个合适的的pupariation网站。然而，视觉通路如何相互作用，一昼夜地控制幼虫的视觉行为的问题在很大程度上仍然未回答。光偏好测定允许昼夜时间点之间的比较，飞线和昼夜状态下不同光质。该法是很容易准备，价格低廉，一直是有用的，我以前Ñ​​几个实验室的描述和研究光派生行为在幼虫。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Sigma-Aldrich</td>
 <td>A5093-500G</td>
 <td>2.5%; Sigma-Aldrich, 9471 Buchs, Switzerland </td>
 </tr>
 <tr>
 <td>Petri dishes</td>
 <td>Greiner Bio-One GmbH</td>
 <td>633180</td>
 <td>90-mm diameter; Greiner Bio-One GmbH, 4550 Kremsmeinster, Austria</td>
 </tr>
 <tr>
 <td>LEDs Lamp</td>
 <td>OSARAM </td>
 <td>80012 White</td>
 <td>LED lamp, 80012 White</td>
 </tr>
 <tr>
 <td>Environment Meter</td>
 <td>PCE</td>
 <td>PCE EM882</td>
 <td>Lux, Temp, RH%</td>
 </tr>
 <tr>
 <td>Thermostatic cabinet</td>
 <td>Aqua Lytic (Liebherr)</td>
 <td>ET636-6</td>
 <td></td>
 </tr>
 <tr>
 <td>Light timer</td>
 <td>Timer T</td>
 <td>6185.104</td>
 <td>230V/50HZ (check specifications for your country)</td>
 </tr>
 <tr>
 <td>Universal thermostat</td>
 <td>Conrad</td>
 <td>UT200</td>
 <td></td>
 </tr>
 <tr>
 <td>Humidifier</td>
 <td>Boneco</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Balck tape</td>
 <td>Tesa</td>
 <td></td>
 <td>5 cm </td>
 </tr>
 <tr>
 <td>Glue</td>
 <td>Uhu</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>lncubator lamp</td>
 <td>Phillips</td>
 <td>Softtone</td>
 <td>5W</td>
 </tr>
 <tr>
 <td>Timer clock</td>
 <td>Ziliss</td>
 <td></td>
 <td>Ziliss, Switzerland</td>
 </tr>
 <tr>
 <td>Excel Software</td>
 <td>Microsoft</td>
 <td></td>
 <td>Excel</td>
 </tr>
 <tr>
 <td>Origin Software 8.5</td>
 <td>OriginLab</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Backer Yeast</td>
 <td>Migros Switzerland</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Iron support stand 17X28CM</td>
 <td>Fisher Scientific</td>
 <td>S47808</td>
 <td></td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Sigma Aldrich</td>
 <td>A6283-100ML</td>
 <td>20% acetic acid dilluted in H2O</td>
 </tr>
 <tr>
 <td>Red light lamp</td>
 <td>Phillips</td>
 <td>PFE712E*8C</td>
 <td></td>
 </tr>
 <tr>
 <td>Spatula </td>
 <td>Fisher Scientific</td>
 <td>14-373-25A</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>EA</td>
 <td>EA PS 2042-06B</td>
 <td>Optional</td>
 </tr>
 <tr>
 <td>Aluminium foil</td>
 <td>Prix Coop</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heater</td>
 <td>GOON</td>
 <td>NSB200C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microwave Oven</td>
 <td>Intertronic</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Standard corn meal fly food</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Destilled water </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

light preference assay to study innate and circadian regulated photobehavior in drosophila larvae

光作为环境信号来控制各级动物行为。 果蝇幼虫的神经系统是作为一个独特的模型，回答基本问题，如何快速昼夜行为之间的光信息处理和共享。 果蝇幼虫显示刻板的避税行为，当暴露在光线下。探讨可比简单的明暗偏好测试可应用于光依赖行为。参与传感和处理视觉输入的神经通路在脊椎动物和节肢动物部分重叠与处理光的昼夜信息。迷人的光传感系统和昼夜系统如何互动，以保持输出协调行为的问题，仍然在很大程度上未被果蝇是影响生物模型来处理这些问题，由于在大脑中的神经元数量少，遗传工具的可用性。神经manipul的BULLETIN。呈现明暗偏好测定允许视觉行为包括昼夜的控制趋光性了一系列的调查。

继上述协议，我们测试了两种不同的昼夜倍CT0和CT12明暗偏好早期幼虫第三体育场野生型广东-S飞。成人12小时光照，12小时黑暗饲养，并留下12小时产卵。幼虫生长的前两天，在相同的光线暗的政权。因为我们想在测试昼夜调制恒定的条件下（自由运行的生物钟），幼虫，然后转移到不断的黑暗未来三天，直到进行测试（ 图3A）。 这里，我们用350勒克司，因为它已...

Watch this Scientific Journal Video about Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae at JoVE.com

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

在这里，我们描述了果蝇幼虫光暗偏好测试。此法提供了先天和昼夜节律调节的光传感和处理感光特性的信息。

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 ...

light-preference-assay-to-study-innate-circadian-regulated

Research

JoVE Journal

Neuroscience

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

12.8K 次观看.  University of Fribourg. 在这里，我们描述了果蝇幼虫光暗偏好测试。此法提供了先天和昼夜节律调节的光传感和处理感光特性的信息。

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

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

含量自发活动，研究昼夜节律和睡眠参数果蝇</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

测量小鼠使用车轮运行活动的昼夜和急性对光反应

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

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

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 ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

口味偏好测定成人<em&gt;果蝇</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

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 ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...