我们感谢基督教兰德里热探针的设计，发展批判性阅读的手稿表明热板法，布卢明顿果蝇库存中心飞股票，并Galko实验室成员幼虫热探针法，肖恩·斯威尼丹尼尔·巴布科克。这项工作是由NIH支持以R01 NS069828 MJG和一个国家卫生研究院MARC U型明星训练津贴（T34GM079088休斯敦市中心学者学院大学）研究事业（平均值）为少数获得。

一个典型的提纲准备两个热痛觉检测正常或紫外光敏幼虫的实验步骤如图1所示。 1。幼虫的制备<ol><li>男性和女性的理想基因型的后代幼虫可直接测试。另外，可以建立一种遗传交叉，以获取利益的定义基因型的后代幼虫。成立于含瓶飞食品使用20-30处女的女性和15-20男性十字架。 </li><li>鸡蛋打好后5天内，收获和清洁年初三龄幼虫糊状飞食品舀出，轻轻使劲水通过630微米（孔径）网运行。早期的三龄幼虫（长度3-4毫米，沿前后轴）转让小潮湿的食物垫防止饥饿和dessication的的。如果要测试痛觉过敏，按照后续步骤2.1-2.11。如果幼虫被测试在absenc组织损伤E，直接进行步骤3.1。 </li></ol> 2。醚化和紫外线照射<ol><li>构建醚化室。 <ol><li>削减了1.5毫升的离心管的底部。 </li><li>用热金属锅铲融化离心管切缘。 </li><li>贴上细网融化管表面，并允许降温。醚化室将允许醚烟雾进入，但防止幼虫逃生。 </li></ol></li><li>使用镊子或油漆刷轻轻放置在一个自制的醚化室10月初第三龄幼虫。 </li><li>沿里面的盖子和密切的地方幼虫。 </li></ol>注：接下来的两个步骤，应在通风橱中进行，乙醚是一个潜在的爆炸性的化学品，其烟雾能麻痹一个人，以及幼虫。 <ol start="4"><li>将醚化室含幼虫含有10毫升的烧杯，背着一个罐子内Coplin〜1.5毫升乙醚的棉球浸泡。 </li><li>螺丝帽上的Coplin壶幼虫暴露为2至2.5分钟醚烟雾。请注意，醚化时间较长，可能产生的不利影响幼虫的行为或生存。 </li><li>醚化之后，从Coplin JAR /烧杯中删除醚化室，乙醚烟雾消散在引擎盖几秒钟。你现在可以回到实验室从引擎盖。 </li><li>使用水喷瓶，轻轻冲洗幼虫从醚化室变成一个小型的培养皿。 </li><li>使用镊子和Kimwipe涂抹麻醉幼虫干燥，轻轻贴上他们背侧带固定3“×1”玻璃显微镜幻灯片上双面胶带。 </li><li>将幻灯片上的紫外线照射室的底面和6秒的幼虫暴露在紫外线照射下，在20兆焦耳/厘米2强度。 </li><li>浸泡在水中的幻灯片，轻轻漂浮幼虫的双面scotc的关Ĥ磁带。 </li><li>使用镊子或画笔，轻轻地放置在辐照幼虫1 15×45毫米的一个DRAM玻璃文化小瓶含〜1.0毫升苍蝇的食物，他们可以为可变的时间期限（8 -24小时）恢复25°C或其他所需的热痛觉实验前重新收获的培养温度。 </li></ol> 3。本地热探针法我们提供一个有害的热刺激，个别幼虫体内部分使用定制的专业开发工程（见材料表）生产的热探头。虽然这种探头优化设计功能（〜.07毫米2区的小金属尖，能精确保持设定温度从23°C至65°C）的原则，任何一个小技巧，可以加热的工具到了一段长达20秒的定义温度应该足够了。探头尖端的手背上被用来刺激早期第三龄幼虫，正是升中线在腹段A4（ 见图2）。在这种热刺激反应，幼虫通常会表现出厌恶的退出行为，一个由360度或以上的横向滚动。这种行为是有别于他们的光触摸响应非有害室温金属探测器一般涉及短暂的停顿，在其运动器官的活动13。 热探针法的协议： <ol><li>热探针预先设定的温度所需的设定点。 </li><li>使用画笔或镊子轻轻地转移到一个平面平台（我们通常使用的粘结剂的乙烯切小块）的幼虫，随后将刺激个人幼虫。幼虫应与热探针接触前一水薄膜覆盖。水薄膜覆盖幼虫应该尽可能少，并完全覆盖，幼虫，同时确保未干时，幼虫接触乙烯。 </li><li>对A4在段与尖端的光压力，在大约45°之间的角度探头和表面的幼虫（ 见图2）申请的幼虫轻轻按下探头尖端。压力应引起幼虫表面轻微压痕，通常是足够的，以防止运动。如果幼虫继续移动，适用于稍多的压力，它通常会停止。不记录数据，直到响应或20秒截止实现超越的观点或领域不断探针接触的举动不能从幼虫。 </li><li>继续刺激幼虫展示或达到，直到20秒截止，直到戒断反应，以先到为准。响应幼虫通常先显示一个抬起的头部和尾部的初步行为。这通常是随后撤出至少有360度的滚动行为。只有360度的完整的辊取得为nocifensive行为（HEA初步的d或尾巴加薪是不是）。 </li><li>退缩行为一旦开始释放探头接触和记录延迟或退出的时间。如果没有退缩行为的观察，然后在20秒内的幼虫是一个非应答。进一步的反应可以分为两类。如果在5秒钟内撤出行为，然后幼虫是一个快速响应。如果取款行为是显示5-20秒之间，然后幼虫是一个缓慢的应答（ 见图1巴布科克等人，2009年）1。 </li></ol> 4。全球热板试验热板法测量热痛觉在果蝇幼虫时，整个动物面临着与有害的热刺激。个人中旬第三龄幼虫放置在一个60×15毫米培养皿水80μL滴-原理和实验组照片见图3起来。培养皿中，然后放置到一个坚实的加热块（被称为“热板”）。作为一滴水上升的温度，幼虫表现出了一系列五刻板的行为，我们称之为头部捶打，轧辊，鞭，扣押和瘫痪。作为这些行为一定程度上热板设定温度和水量的功能，幼虫浸入我们在座的，我们已经找到了什么是最佳条件下（95℃热板，80μL下降水），观察它们之间的最小重叠的所有行为。 热板法的协议： <ol><li>放置光纤光导，以确保观看幼虫有足够的高对比度照明。如有必要，使用的测试幼虫。应该是光功率低，被测试的幼虫，以避免环境热。 </li><li>放入热板加热块平坦的表面，并放在热盘上显微镜的基础。热板转到“高”的位置，允许约15分钟的温度稳定，并作出相应调整，表面温度达到95°C </li><li> 80μl蒸馏水的测量，并将在60×15毫米的聚苯乙烯1移液器的Petri菜中的一滴水。 </li><li>使用镊子轻轻一滴水中放入一个干净中旬第三龄幼虫。尝试转移可能的水体积最小的幼虫，使被加热的下降，保持尽可能接近原来的80μL。 </li><li>轻轻的固体加热块放置到培养皿中含有水滴和幼虫对热板。迅速调整菜的位置，所以整个幼虫和看法是一滴水。 见图3和视频1-6。 </li><li>此刻开始定时器被放置在培养皿中的固体加热块加热板和记录每个行为的发病时间。的行为，如果需要的话，也可以是视频录制- 视频1-5为每个行为的有代表性的例子。徕卡软件版本3.7被用来记录电影，录像模式是“连续”，相机的分辨率为300万像素（我们使用0.63x放大计算的图像分辨率为10微米/像素），并分别为44.5帧/秒。 </li></ol> 5。代表结果当地热探针法： 热探针接触后，幼虫通常显示的解除其头部和尾部的初步行为。通常情况下，解除头部被看作第一其次尾巴抬起。几秒钟后，这个初步的行为通常幼虫开始横向滚动，我们称之为“厌恶退缩行为。”后，初步行为或退缩行为显示的时间可能会有所不同温度或GE遗传背景。在我们最初的研究中，我们测量了幼虫参展厌恶撤出探头在不同的设定点温度的百分比，结果发现，48℃，最低气温在所有幼虫响应速度快（&lt;5秒）。在这里，我们报告说，有一个上限，以幼虫热痛觉反应（ 图4A）。 100％的快速反应，观察到探头温度52°C。然而，在54°C和更高，90％或更多的幼虫不能回应，即使经过20小号接触。这些幼虫20小号截止后继续移动。 正如先前1，在每个温度下的退缩行为的类别，可以绘制和统计比较。鉴于这是一种行为检测，有一些从幼虫，幼虫和个人用户之间的变异。考虑到这一点，我们通常测量3套30％的试验条件下幼虫​​。除了简单的Wi分类我们先前报道的thdrawal延迟，我们在这里报告，也可以测量振幅（卷筒或厌恶退缩行为所花费的时间）（ 见图4）。令人惊讶的是，似乎是逆输入温度和响应的鲁棒性之间的关系，因为较低的温度下出现较多挑起辊（ 图4B）和更多的时间在厌恶撤出花（数据未显示）。这可能表明，接触有害的温度的持续时间可能是鲁棒性的主要因素。 热板实验： 五千篇一律的运动器官的行为被发现后，浸泡在水中的幼虫转移到热板。这些措施说明如下，并显示在视频一起典型的水加热幼虫的运动器官的行为。这些行为观察的平均潜伏期如图5所示一，平均一滴水，每延迟测量温度。这里介绍的最佳实验条件下显示每个不同的行为幼虫的比例介于77至100％（ 图5B），虽然偶尔滚动和鞭打行为被省略，重叠，或以相反的顺序发生。观察到的行为，按时间顺序，介绍如下，可以在观看视频在下面显示的时间： <ol><li> 在热的情况下的正常蠕动运动：运动伴随着搜索头部运动。从6:27看到视频。 </li><li> 头痛击：幼虫移动迅速朝正向或横向运动。这项议案是其在水中的正常运动（ 视频1）类似，但更颠簸摇晃，持续发生。从6:40看到视频。 </li><li> 辊：幼虫滚动横向至少一个完整的360°。从6:50看到视频。这可能发生一个时代的变量有时涉及不完整的轧辊。对于得分的行为，我们只能算完整的360°辊的目的。这种行为是与本地应用程序的热探头看到，最相似的。 </li><li> 鞭：幼虫展品沿前后轴的快速释放收缩运动，使头部很简单的接近尾。从7:11看到视频。经常观察在快速继承或作为滚动的同时鞭打。 </li><li> 劫持：的幼虫延伸出沿前后轴，并表现出高频率全身颤抖的行为，无弯曲。从7:25看到视频。 </li><li> 瘫痪：幼虫停止运动。在一些幼虫，这是永久性的，而其他人则表现出低频脉动运动。从7:36看到视频。 </li></ol>这些数据表明，一滴水和延迟发病的5个字符的温度幼虫/一滴水全球加热后观察acteristic行为是高度相关的。 <img alt="图1。" src="/files/ftp_upload/3837/3837fig1.jpg" /> 图1。纲要的编制和幼虫的检测实验步骤 。化验痛觉与热探头或热板前，来自某只股票或遗传交叉幼虫的粮食收获和清洗。如果痛觉过敏（反对基线痛觉）进行评估，收获后由醚化（接触乙醚），安装，紫外线照射，一段时间恢复对飞食品。收获或回收幼虫，然后用热探头（本地）或热板（全球）检测有害的测试温度。数字是指方法的文字描述步骤（S）所示的部分。 <img alt="图2。" src="/files/ftp_upload/3837/3837fig2.jpg" /> figurE 2。实验设立本地热探针法。热探头热控制单元用于设置和保持探头的温度控制。探头举行前后轴垂直，用来刺激幼虫在45°角的水平。探针接触是在腹段A4，具体如下所示。用户必须保持接触与温和的压力，直到20秒截止或直到滚动行为开始。如果温度被认为有毒，幼虫会显示特点是至少有一个360°辊厌恶的退出行为。轧辊的数量可为单个或多个（ 见图4B）。 <img alt="图3。" src="/files/ftp_upload/3837/3837fig3.jpg" /> 图3。热板法实验设置 。 （一）含有一个60×15毫米培养皿横看卡通中旬第三明星幼虫在80μL一滴水。 （二）卡通顶视图的幼虫放置在中间通过显微镜观察一滴水。 （三）此法工作站的水平视图照片。 （四）幼虫的照片通过显微镜观看。 <img alt="图4。" src="/files/ftp_upload/3837/3837fig4.jpg" /> 图4。量化使用热探针法的行为反应。（一）剧情％的应答者属于每个类别（快，慢，无反应）与温度。 （二）潜伏期厌恶与轧辊的数量增加毒性的四种不同的测试温度（42℃，44°，46°，48°，50°，和52表现出每个幼虫在退缩行为的剧情℃）。 <img alt="图5。" src="/files/ftp_upload/3837/3837fig5.jpg" /> 图5。热板法：延迟时间和温度与BEHavior。（一）每个95的最佳条件下的行为反应延迟和水平均下降温度°C间（热板表面温度）和80μL的水压降（N = 150）。请注意，每个行为是一个特定的时间间隔内观察。一滴水温度测量用的热电偶插入到顶部或底部的下降（N = 10滴为每个位置）和这些测量取平均值。 （二）幼虫的百分比参展每个最佳实验条件下的行为反应。颠簸，抓住和瘫痪几乎100％的时间，同时观察观察分别为77和80％的时间，滚动和鞭打。在较低的表面温度设定点检和瘫痪在200秒内，在更高的设定点，滚动和鞭笞可以跳过或可能发生的同时不遵守。 N = 150。 （三）幼虫的百分比生存以下开始瘫痪行为。幼虫被加热，直至被轧花瘫痪，然后删除恢复到标准的文化条件。模拟处理的幼虫除热暴露同等待遇。形成蛹和可行的成年人进行了量化的7-13天。 N = 120。 <img alt="图6。" src="/files/ftp_upload/3837/3837fig6.jpg" /> 图6。灭活幼虫伤害性感觉神经元增加的行为反应延迟 。情节延迟到每个行为指定的基因型：W 1118 Gal4的109（2）80 = MD-Gal4的/ +无人机的Ork1.Δ - C / +无人机的Ork1.Δ - NC / +，MD- Gal4/UAS-Ork1.Δ-C，和md-Gal4/UAS-Ork1.Δ-NC的。 MD-Gal4的驱动器在所有四个类multidendritic周边感觉神经元无人机调节的转基因表达的UAS-Ork1.Δ-14表达了果蝇开放整流钾离子通道的突触传递所需的修改后的版本， 和 U<html> AS-Ork1.Δ-14数控表达了同样的通道进一步修改后的版本，不干扰突触传递。请注意，只有的幼虫轴承MD-Gal4的驱动程序6和无人机，Ork1.Δ-C的转基因显示为四个五个观察到的行为（捶打，轧辊，扣押，瘫痪）增加延迟。还要注意的是，因为滚动这些动物鞭之前，他们推出的延迟增加。星号表示，学生的t检验P &lt;0.05。 N = 30％基因型幼虫。 补充视频1。 <a href="/files/ftp_upload/3837/MovieS1NormalMovement.avi" target="_blank">点击这里观看视频</a> 。 补充视频（2） <a href="/files/ftp_upload/3837/MovieS2Thrash.avi" target="_blank">点击这里观看视频</a> 。 补充视频3。“目标=”_blank“&gt;点击这里观看视频。 补充视频4。 <a href="/files/ftp_upload/3837/MovieS4Whip.avi" target="_blank">点击这里观看视频</a> 。 补充视频5。 <a href="/files/ftp_upload/3837/MovieS5SeizureParalysis.avi" target="_blank">点击这里观看视频</a> 。 补充视频6。 <a href="/files/ftp_upload/3837/MovieS6All5Behaviors.avi" target="_blank">点击这里观看视频</a> 。

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+regulating+dendritic+outgrowth,+branching,+and+routing+in+Drosophila.">Genes regulating dendritic outgrowth, branching, and routing in Drosophila.</a> Genes Dev. 13, 2549-2561 (1999).</li><li>Walters, E. T., Illich, P. A., Weeks, J. C., Lewin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+responses+of+larval+Manduca+sexta+and+their+sensitization+by+noxious+stimuli+in+the+laboratory+and+field.">Defensive responses of larval Manduca sexta and their sensitization by noxious stimuli in the laboratory and field.</a> J. Exp. Biol. 204, 457-469 (2001).</li><li>Woolf, C. J., Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptors--noxious+stimulus+detectors.">Nociceptors--noxious stimulus detectors.</a> Neuron. 55, 353-364 (2007).</li><li>Hwang, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+neurons+protect+Drosophila+larvae+from+parasitoid+wasps.">Nociceptive neurons protect Drosophila larvae from parasitoid wasps.</a> Curr. Biol. 17, 2105-2116 (2007).</li><li>Xiang, Y. <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>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Sweeney, N. T., Li, W., Gao, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+single+neurons+in+vivo+reveals+specific+roles+of+flamingo+in+neuronal+morphogenesis.">Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis.</a> Dev. Biol. 247, 76-88 (2002).</li><li>Kernan, M., Cowan, D., Zuker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+mechanosensory+transduction:+mechanoreception-defective+mutations+of+Drosophila.">Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila.</a> Neuron. 12, 1195-1206 (1994).</li><li>Nitabach, M. N., Blau, J., Holmes, 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=Electrical+silencing+of+Drosophila+pacemaker+neurons+stops+the+free-running+circadian+clock.">Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock.</a> Cell. 109, 485-495 (2002).</li></ol>

我们什么都没有透露。

这里描述的检测可用于定性和定量评估有害的热刺激的反应不同基因型的幼虫。热探针法的一个主要特点是，刺激只在一个单一的轨迹。这大概只有一小部分，IV类神经元段由探头接触的可能立即11个相邻节段的射击。由于局部刺激，模仿的热探针法检测定位到一个特定区域，如手接触热炉身体有害刺激的感官体验。热探针法的缺点是，它有一些用户到用户的变化，可能可以归因于三个因素：一）与用户应用的探头幼虫的压力，二）探针的精确位置对幼虫相对底层的痛觉神经元，，三）精确昂乐在探头接触表面的幼虫。 我们以前报道分类幼虫的定量策略到无反应，反应缓慢，快速反应撤军延迟到一个给定的温度1。在这里，我们报告幼虫的反应，以更高的温度。有趣的是，我们发现幼虫热痛反应，有一个上限，这个上限之间52和54°C。这可能表明，幼虫不具备瞬时受体电位（TRP）通道门能够在温度超过52°C。另外，它可以建议使用的神经细胞或肌肉开始或进行损坏电机的响应之前，他们甚至可以运作厌恶撤出。我们还报告分析不同的撤军幅度响应为响应“鲁棒性”指标使用的轧辊。天真，希望，这些参数会随着温度的升高或刺激时间增加。令人惊讶的是，我们发现，这种情况并非如此。幼虫在低端的有毒的范围（42℃）的温度较长时间的刺激表现出更多的面包和花费更多的时间滚动比幼虫探讨在较高的温度（48-52℃）。这表明，在有毒的温度窗口，它主要是时间曝光，决定响应的幅度。由于幼虫暴露于高度有毒的温度（48-52°）平均响应速度非常快，他们没有表现出许多轧辊的幼虫暴露的时间长，温度减少有害。响应振幅分析报告增加了另一个维度沿不同的基因型或环境操作可以比较定量。 热板法的一个主要特点是，它涉及一个全球性的接触到有毒热。正因为如此，它更像是坐在比触摸热炉加热釜的动物。虽然这是不明确时，幼虫可能会遇到在野外的全球有害的刺激，在实验室对这一全球性曝光的行为反应是较局部刺激后观察到的复杂。热板法的强度，还指出，其他3是因为接触幼虫是不是该协议的一个组成部分，它有小的用户到用户的变异。唯一实质性的差异，似乎是在确定每个行为开始时，这可以反复观看/熟悉的最小化。一个有趣的差异之间的化验是令人厌恶的行为开始时的温度。这些都是远低于热探头在热板法。幼虫接触与热探针（头部和尾部加注）展出了初步的行为可能是一个关联的观察〜27°C的热平台的头部捶打é检测。这是可能的响应，这反映了“不适”比“痛”。我们没有观察到因素的相关鞭打，扣押，即使在高温和麻痹（高达48°C）温度在热探针法，它可能是发射的身体从多个地区的感觉神经元的临界质量需要对这些行为带来的。有趣的是，癫痫和瘫痪的行为观察温度（〜34 - 37℃）以下的观察表明，全球刺激可能涉及神经元的反应是不够的总结，与当地的行为触发探头与热的痛觉阈值的底热探针的应用。这些温度实际上是为有毒的幼虫认为是支持的观察，幼虫开始瘫痪的行为，并随后获准恢复对飞食品，在大多数情况下生存（图5C）。进一步支持的论点，热PL吃检测读出伤害性反应，是一个事实，即阻断已知的伤害性感觉神经元的突触传递，大部分观测到的行为（图6）增加了延迟。更高温度鞭打行为，有没有延迟增加的观察表明，其他感觉神经元不表达MD-Gal4的可能需要这种行为。 总之，这两个实验涉及个别幼虫暴露定义温度 - 一个小的金属探测器，在当地的下降在全球试验中迅速加热水浸泡法和热尖有害的热刺激。不同的遗传背景和/或暴露于不同环境条件（例如加号或减号组织损伤）的果蝇幼虫的行为反应，可以使用这些实验研究和量化。最终，从这些实验结果将有助于我们更好地了解基因网络控制NOCiception在果蝇和其他相关物种。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>热棒</td><td>亲开发工程</td><td>自定义建立在需求</td><td>可应要求提供联系信息</td></tr><tr><td>干浴孵化器</td><td> Fisher Scientific则</td><td> 11-718 </td><td> 1固体加热块加热块和1个16毫米井</td></tr><tr><td>徕卡DFC290 12v/400mA 彩色摄像机</td><td>徕卡公司</td><td> 12730080 </td><td>任何同等 相机会做。 </td></tr><tr><td>徕卡MZ6显微镜</td><td>徕卡公司</td><td>为MZ6变焦身体的一部分（光学载波）是10445614 </td><td></td></tr><tr><td>肖特王牌Modulamp单位</td><td>肖特北美公司</td><td> A20500 </td><td></td></tr><tr><td>肖特双鹅颈23英寸光纤光导</td><td>肖特北美公司</td><td>肖特A08575 </td><td></td></tr><tr><td>温度控制装置</td><td> TSCI公司。 </td><td>客户定制</td><td>详情可应要求提供</td></tr><tr><td>蔡司STEMI 2000显微镜</td><td>蔡司</td><td> NT55-605 </td><td>任何同等 显微镜会做。 </td></tr><tr><td>镊子</td><td>福斯特</td><td> FS-1670 </td><td></td></tr><tr><td> 1毫米网</td><td>杰纳西科学</td><td> 57-101 </td><td></td></tr><tr><td>画笔</td><td>迪克布利克艺术材料</td><td> 06762-1002 </td><td></td></tr><tr><td>紫外线交联剂</td><td> Fisher Scientific则</td><td> 1199289 </td><td></td></tr><tr><td> coplin罐</td><td> Fisher Scientific则</td><td> 08-816 </td><td></td></tr><tr><td> 10毫升烧杯</td><td> Fisher Scientific则</td><td> 02-540C </td><td></td></tr><tr><td>乙醚</td><td> Fisher Scientific则</td><td> E138-500 </td><td></td></tr><tr><td> 35×10毫米聚苯乙烯培养皿</td><td>鹘</td><td> 351008 </td><td>我们没有测试 替代的菜肴。 </td></tr><tr><td>玻璃载玻片</td><td>康宁</td><td> 26003 </td><td></td></tr><tr><td>热电偶</td><td> Omega工程公司</td><td> HH802U </td><td></td></tr><tr><td>一块乙烯</td><td>欧迪办公</td><td> 480009 </td><td></td></tr><tr><td>离心管</td><td>丹维尔科学公司</td><td>的C-2170 </td><td></td></tr></tbody></table>

local and global methods of assessing thermal nociception in drosophila larvae

在这篇文章中，我们展示了实验，以研究在果蝇幼虫的热痛觉。一个实验涉及到空间受限的（本地）1,2热感受器的刺激，而第二个涉及批发（全球）激活大多数或所有3个这样的神经元。总之，这些技术让果蝇伤害性感觉神经元的行为功能的可视化和量化。 果蝇幼虫是一个既定的模型系统研究热痛觉潜在的有害的进化保守的跨物种1,2温度，感官反应。这些研究对果蝇的优点是相对简单的神经系统和复杂的基因技术，可以用来解剖的基础生物学4-6在果蝇的分子基础，在所有的多细胞动物的反应作者e以有毒热刺激，一般涉及“nocifensive”提出的刺激7厌恶撤出。这种刺激是免费的神经末梢感受器，通过检测的有机体反应的幅度取决于接收8伤害性刺激感受器的数量。在果蝇中，它是IV级树突状树枝状检测，除了他们最近发现的作用作为感光10 9有害的热和机械刺激感觉神经元。这些神经元，这已经非常好，发育水平的研究，arborize在表皮屏障表，并与几乎所有11,12表皮细胞接触。每个IV类神经元的项目，他们可能会连接到二阶神经元轴突进入中枢神经系统的11腹神经索项目的大脑。 在基准条件下，伤害性感觉神经NS将不会触发，直至达到一个相对高门槛。这里所描述的实验，让研究者量化基线的行为反应，据推测，致敏，随之而来的下列组织损伤。每个检测有害的热刺激引起不同但相关的运动器官的行为反应，并允许研究人员在果蝇幼虫各方面的热痛觉可视化和量化。检测可应用于幼虫所需的基因型或不同环境条件下，可能影响痛觉提出幼虫。由于热痛觉跨物种保守，在果蝇的遗传解剖，从收集到的结果可能会告知在其他物种的热伤害性的认识，包括脊椎动物。

Watch this Scientific Journal Video about Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae at JoVE.com

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae | Neuroscience | Video

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

在这篇文章中，我们展示了实验研究热​​痛觉<em&gt;果蝇</em&gt;幼虫。一个实验涉及到空间受限的（本地）的热感受器的刺激<sup&gt; 1,2</sup&gt;，而第二个涉及的大部分批发（全球）激活或所有这些神经元<sup&gt; 3</sup&gt;。总之，这些技术允许的行为功能的可视化和量化<em&gt;果蝇</em&gt;伤害性感觉神经元。

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 ...

local-global-methods-assessing-thermal-nociception-drosophila

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Biology

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Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Human Biology

Sensory Systems

SCIENTISTS IN ACTION

15.7K 次观看.  The University of Texas MD Anderson Cancer Center. 在这篇文章中，我们展示了实验研究热​​痛觉果蝇幼虫。一个实验涉及到空间受限的（本地）的热感受器的刺激 1,2，而第二个涉及的大部分批发（全球）激活或所有这些神经元 3。总之，这些技术允许的行为功能的可视化和量化果蝇伤害性感觉神经元。

视频: Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

在人类婴儿的伤害性电生理测量和分析

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

科研

神经科学

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 ...

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 ...

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 ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

测量 果蝇 光感受器中光色素水平的电生理学方法

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...