Name: an improved assay and tools for measuring mechanical nociception in drosophila larvae
Uploaded: 2020-10-29T15:00:00.000+00:00
Duration: 7 min 17 s
Description: Watch this Scientific Journal Video about An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae at JoVE.com

我们感谢托马斯王开发原型冯弗雷丝，帕特里克J.黄改进机械探针检测，布卢明顿果蝇库存控制中心（w1118）和 ppk-Gal4>UAS-mCD8-GFP 飞行股票，和加尔科实验室成员批判性地阅读手稿。这项工作得到了R21NS087360和R35GM126929对MJG的支持。

1. 机械探头结构
<ol><li>使用小型线切割机（图 1C），将每个尼他诺灯丝（图 1B）切成指定长度（图1M-N）垂直于其长轴。灯丝有三种不同的预设直径（图1B）。 注:此处指定的长度是实现所指示的近似压力的指南，使用类似的协议来构建安装。最终，无论灯丝切割的长度和安装孔的深度如何，灯丝都必须在平衡上测量/校准，以获得确切的力/压力值。</li>
	<li>在立体显微镜下检查灯丝的尖端，以确保没有锋利或不规则的边缘保持，因为这些边缘可能会对幼虫的皮肤造成组织损伤并干扰校准。</li>
	<li>使用磨刀石手动平滑机械探头的锋利边缘，直到没有明显的不规则现象持续存在（图 1D）。</li>
	<li>用皮下针在木制冰棒（图1E）末端打一个洞（见 材料表）。将针头至少插入冰棒高度的一半（图 1E）。这创建一个插入尼他诺灯丝的腔室。</li>
	<li>将木胶涂在单根硝基醇长丝（图1F）上，将胶涂层的长丝插入木制冰棒（图1G）的针槽中。允许干燥~5小时。</li>
	<li>将每个机械探头按压到刻度上，直到机械探头弯曲（图 1H-L），从而校准每个机械探头。这是可以用克记录的最大力点。根据灯丝直径（预集）和长度（用户确定），可以生成各种力和压力。</li>
	<li>使用公式 f = ma 将第 1.6 步中记录的质量转换为在米林沃顿 （mN） 中力（力等于质量乘以引力加速度）。f:武力:m:质量:a:引力加速度（9.8米/s2）（图1M）。</li>
	<li>最后，通过将测量的力除以灯丝尖的表面积（图 1M），将计算的力转换为千帕斯卡 （kPa） 中的压力（力/面积）。要计算面积，将不同尼蒂诺长丝的直径从英寸（0.04 英寸、0.06 英寸和 0.08 英寸）转换为厘米。然后，+2（ 其中，r = 硝基醇灯丝半径）确定区域（见 图 1M）。使用不同直径和长度的细丝准备多个探针将生成一套跨越 果蝇 幼虫响应范围的完整集（ 图 1N中显示的示例集）。 注:至少每3-4周检查一次每个机械探头。当压力偏离原测量± 3% 以上时，必须制造新的机械探头。</li>
</ol>2. 幼虫的准备
<ol><li>在25°C孵化器中，在标准食品上提高控制菌株（w1118）幼虫后代或幼虫，其中含有转基因 ppk-Gal4>UAS-mCD8-GFP（ 用于可视化对感官神经元的损伤）。通常，库存通常维持在18°C，但父母和幼虫后代都以25°C的标准玉米粉食品饲养，用于实验。 注:成年苍蝇（5雄10雌，1:2比例）保存在飞瓶中，允许产卵，约24小时。产卵后的时间 （AEL） 从成人被移除开始。</li>
	<li>通过轻轻地将自来水喷入含有幼虫的软蝇食物中，收集大约96小时后的第三个星内幼虫。离开食物的流浪幼虫，或者曾经长过前孢子或后尖刺的幼虫，对于这个检测来说太大/太老了。第二个星内幼虫（~长度小于4毫米）太小。</li>
	<li>倒出软飞食品的内容到一个干净的标准大小的培养皿（100毫米×15毫米）。</li>
	<li>使用钳子，排序中三星，中等大小，幼虫（见 图2A）从较小的（第二星和早期第三星）或更大（晚或徘徊第三星）幼虫。建议用钳子进行温和操作，以避免对幼虫造成任何组织损伤。 注意:使用钳子的转移主要基于水的张力，而不是用钳子的叶片对幼虫施加压力。使用钳子操纵幼虫的替代方案是软漆刷。使用任一工具，用户应练习转移动物，以免造成意外的组织损伤，从而可能使行为测量复杂化。</li>
	<li>将中三星幼虫，使用钳子，转移到一个小的培养皿（30毫米×15毫米），里面装着一小插在室温下用水滋润的苍蝇食物。将幼虫放在小培养皿中，直到进行实验，但不超过20分钟。 注:一般来说，将20-30个幼虫转移到食物塞上，可以提供足够数量的20分钟的行为检测。</li>
</ol>3. 机械节点检测
<ol><li>将三分之一的星状幼虫（使用钳子）放在明亮的场立体显微镜下的黑色或深色乙烯基薄垫上。深色提供对比度，改善幼虫的可视化。最好有一块可自由移动的深色乙烯基，因为它允许用户对齐幼虫而不接触或伤害它。</li>
	<li>将光纤光放在显微镜客观透镜和黑色或深色乙烯基垫之间:这将允许足够的高对比度照明看到幼虫。</li>
	<li>丢弃幼虫，在转移到垫子后不显示正常运动。这些可以干扰正常的无感知行为反应。有关正常运动，请参阅 视频 1。</li>
	<li>用纸巾擦去幼虫周围的任何多余的水，这些水都可能导致幼虫漂浮在乙烯基垫上。</li>
	<li>通过移动深色乙烯基垫来定位幼虫。幼虫的头部/嘴应该指向左边，如果你是右手，反之亦然，如果你是左撇子（图2A-B）。</li>
	<li>将选定的机械探头（通常为 1-2 s）涂抹在幼虫的后背侧，大致位于腹部部分 A8（见图 2B），直到探针弯曲并引出先前测量的压力（图 2C）。重要的是，探头压在幼虫的底体表面，并在探针接触点将幼虫压缩到底垫中。 注:在硝基酚丝尖与多孔角质层表皮之间的接触点，探头低于 2，300 kPa，主要弯曲而不穿透角质层和底层组织。这种探针很少影响幼虫死亡率4。在更高的压力（>5，000 kPa）下，探头会弯曲，偶尔会穿透角质层和底层组织。幼虫的穿刺会损害幼虫的生存4， 如果观察到，这些幼虫通常从行为分析中被丢弃。</li>
	<li>记录每个幼虫的行为反应。如果幼虫在3s内沿着身体轴线显示360°的完整卷，则表示阳性无感知反应（视频2）。其他响应（尝试转动、快速爬行和摆动）对于此检测的目的被视为否定。 注:拉瓦刺激与潜移默化机械刺激（200 kPa）没有引起典型的非感知或滚动反应（视频3）。有些幼虫确实表现出快进或轻触反应，如运动方向的变化。</li>
	<li>丢弃幼虫，准备下一个进行检测，重复步骤 3.1 到 3.7。</li>
	<li>重复步骤 3.1-3.7 直到达到所需的幼虫数量（每个探头使用三到六组 n = 10 幼虫）。 注:当使用低压机械探头（174-462 kPa）时，每个幼虫的检测需要更多时间。这是因为较长的细丝尖更振荡，使得在A8段的中心戳幼虫更加困难。这些探针需要实践。</li>
</ol>4. 结合显微镜评估神经元形态学
<ol><li>将幼虫（基因型 ppk-Gal4>UAS-mCD8-GFP 标记感官神经元）之前用硝醇长丝刺激到科普林罐内的乙醚化室中，里面装着一个 10 mL 烧杯，里面装着一个棉球，里面浸泡着 ~1 mL 的二乙醚。让幼虫在房间里坐~5分钟。 注:我们小组12日公布的先前研究中提供了一个关于乙醚化的详细协议。</li>
	<li>将幼虫轻轻地从乙醚化室冲洗成一个小的培养皿。</li>
	<li>准备好一个显微镜滑梯，两个小盖片（22×22毫米）和一个长盖片（22×54毫米）（见 材料表）。</li>
	<li>在滑梯的两端添加小滴乙醚:油溶液（乙醚与卤碳油溶液的 1:5 比率，见 材料表），然后将小盖片放在小液滴的顶部。这种安排创造了一个小的空间间隙，幼虫可以适合。 注意:将小盖片压在显微镜滑梯上，直到难以滑动。</li>
	<li>在显微镜滑梯中间添加一些以太:油溶液，然后使用钳子将幼虫放置在显微镜滑梯的中心（在小盖片之间）。确保幼虫的前后轴与滑梯的短侧平行，并且正朝上。</li>
	<li>盖住幼虫，将长盖片放在幼虫和两个较小的盖片上。 注意:慷慨地按下长盖片，直到幼虫几乎变平。</li>
	<li>使用粘膜显微镜（见 材料表）的幼虫图像段 A8 使用激光波长 488 （GFP）。 注:立即对幼虫进行成像，因为通过乙醚麻醉会很快消退（约5-10分钟），幼虫会醒来移动，这会使进一步成像复杂化。</li>
	<li>使用 20 倍数光圈 （NA） 0.7 干目标透镜在 1 倍变焦下捕获 1024 x 1024 像素的 Z 堆栈图像，步幅为 1.5μm。</li>
</ol>5. 组织损伤的量化
<ol><li>收集并将 Z 系列堆栈图像（从第 4.8 节）转换为单个 Z 投影（将不同对焦平面拍摄的多个图像平展为单个合成图像）。这可以使用市售软件（例如，奥林巴斯 Fluoview）或任何等效的开源平台执行，例如，斐济/图像 J. 在 TIFF 格式中保存单个 Z 投影。</li>
	<li>打开图像分析程序斐济/图像J。</li>
	<li>单击 "文件"，从菜单栏中单击"打开"，然后从显示的窗口中选择 "打开 "。</li>
	<li>选择以 TIFF 格式保存的存储单个图像投影进行分析。</li>
	<li>单击 "编辑"，从菜单栏中，从显示的窗口中选择 "倒置 "选项。</li>
	<li>单击"图像"，从菜单栏中，然后从显示的窗口选择"调整"，最后选择亮度/对比度选项。</li>
	<li>从工具栏中选择 徒手形状 选项，以测量间隙的区域（如果有）。</li>
	<li>单击 "分析"，从菜单栏中选择 "测量 "选项。这将显示间隙或伤口的区域。</li>
</ol>

<ol>
	<li>Tracey, W. D., Wilson, R. I., Laurent, G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Painless,+a+Drosophila+gene+essential+for+nociception.">Painless, a Drosophila gene essential for nociception.</a> Cell. 113 (2), 261-273 (2003).</li><li>Zhong, L., Hwang, R. Y., Tracey, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pickpocket+is+a+DEG/ENaC+protein+required+for+mechanical+nociception+in+Drosophila+larvae.">Pickpocket is a DEG/ENaC protein required for mechanical nociception in Drosophila larvae.</a> Current Biology. 20 (5), 429-434 (2010).</li><li>Lopez-Bellido, R., Himmel, N. J., Gutstein, H. B., Cox, D. N., Galko, 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=An+assay+for+chemical+nociception+in+Drosophila+larvae.">An assay for chemical nociception in Drosophila larvae.</a> Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences. 374 (1785), 20190282 (2019).</li><li>Lopez-Bellido, R., 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=Growth+factor+signaling+regulates+mechanical+nociception+in+flies+and+vertebrates.">Growth factor signaling regulates mechanical nociception in flies and vertebrates.</a> Journal of Neuroscience. 39 (30), 6012-6030 (2019).</li><li>Hu, 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=Sensory+integration+and+neuromodulatory+feedback+facilitate+Drosophila+mechanonociceptive+behavior.">Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior.</a> Nature Neuroscience. 20 (8), 1085-1095 (2017).</li><li>Kim, S. E., Coste, B., Chadha, A., Cook, B., Patapoutian, 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+role+of+Drosophila+Piezo+in+mechanical+nociception.">The role of Drosophila Piezo in mechanical nociception.</a> Nature. 483 (7388), 209-212 (2012).</li><li>Babcock, D. T., Landry, C., Galko, 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=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Current Biology. 19 (10), 799-806 (2009).</li><li>Babcock, D. T., 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=Hedgehog+signaling+regulates+nociceptive+sensitization.">Hedgehog signaling regulates nociceptive sensitization.</a> Current Biology. 21 (18), 1525-1533 (2011).</li><li>Turner, H. N., Patel, A. A., Cox, D. N., Galko, 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=Injury-induced+cold+sensitization+in+Drosophila+larvae+involves+behavioral+shifts+that+require+the+TRP+channel+Brv1.">Injury-induced cold sensitization in Drosophila larvae involves behavioral shifts that require the TRP channel Brv1.</a> PloS One. 13 (12), 0209577 (2018).</li><li>Im, S. H., 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=Tachykinin+acts+upstream+of+autocrine+Hedgehog+signaling+during+nociceptive+sensitization+in+Drosophila.">Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila.</a> eLife. 4, 10735 (2015).</li><li>Cervero, F., Merskey, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+a+noxious+stimulus.">What is a noxious stimulus.</a> Pain Forum. 5 (3), 157-161 (1996).</li><li>Chattopadhyay, A., Gilstrap, A. V., Galko, 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=Local+and+global+methods+of+assessing+thermal+nociception+in+Drosophila+larvae.">Local and global methods of assessing thermal nociception in Drosophila larvae.</a> Journal of Visualized Experiments: JoVE. (63), e3837 (2012).</li><li>Follansbee, T. L., 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+nociceptive+sensitization+requires+BMP+signaling+via+the+canonical+SMAD+pathway.">Drosophila nociceptive sensitization requires BMP signaling via the canonical SMAD pathway.</a> Journal of Neuroscience. 37 (35), 8524-8533 (2017).</li><li>Im, S. H., Patel, A. A., Cox, D. N., Galko, 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=Drosophila+insulin+receptor+regulates+the+persistence+of+injury-induced+nociceptive+sensitization.">Drosophila insulin receptor regulates the persistence of injury-induced nociceptive sensitization.</a> Disease Models &amp; Mechanisms. 11 (5), (2018).</li><li>Jo, J., 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+caspase+activity+is+required+independently+of+apoptosis+to+produce+active+TNF/Eiger+during+nociceptive+sensitization.">Drosophila caspase activity is required independently of apoptosis to produce active TNF/Eiger during nociceptive sensitization.</a> Cell Death &amp; Disease. 8 (5), 2786 (2017).</li><li>Hwang, R. 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=Nociceptive+neurons+protect+Drosophila+larvae+from+parasitoid+wasps.">Nociceptive neurons protect Drosophila larvae from parasitoid wasps.</a> Current Biology. 17 (24), 2105-2116 (2007).</li><li>Guo, Y., Wang, Y., Wang, Q., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+PPK26+in+Drosophila+larval+mechanical+nociception.">The role of PPK26 in Drosophila larval mechanical nociception.</a> Cell Reports. 9 (4), 1183-1190 (2014).</li><li>Takagi, S., 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=Divergent+connectivity+of+homologous+command-like+neurons+mediates+segment-specific+touch+responses+in+Drosophila.">Divergent connectivity of homologous command-like neurons mediates segment-specific touch responses in Drosophila.</a> Neuron. 96 (6), 1373-1387 (2017).</li></ol>

作者没有什么可透露的。

我们使用用尼他诺丝制成的定制机械探针改进了已建立的机械检测1、2、16。 这种金属合金使我们能够使用直径较小的细丝，适合果蝇幼虫的大小。捕鱼线为基础的单鱼已经主宰了飞行机械节点领域至今2，5，6，16。我们的尼他诺灯丝保持其形状和测量压力约约3-5个月（根据我们的经验）。通过改变硝基醇长丝的长度和直径，用户可以产生从次压到几乎完全滚动响应的广泛压力。特别是，用直径较小的尼他诺灯丝制造子保持探针更简单。利用这些探针，我们发现压力，而不是力，会引起更一致的鼻塞行为反应4。我们在这里演示，使用一个成熟的紫外线诱导无感知敏化模型7，10，13，这些灯丝也是研究机械过敏症-合金和高镇痛的有用工具。
先前使用钓鱼线制造的机械探头的研究已经导致行为反应能力2、6、16、17有一定的变异性。有几个因素可以解释这一点。首先，由于压力是重要的变量，因此灯丝尖端的抛光使其四舍五入且没有任何锋利的边缘至关重要。其次，报告压力值而不是仅仅是力对实验的可重复性很重要，因为产生类似力的不同机械探针可以引起不同的压力4。第三，使用有毒探针对每只幼虫只应用一次机械刺激至关重要，因为这种探针在表皮4和感觉神经元水平（图2E）产生依赖剂量的组织损伤。第二次或随后的有毒机械刺激，在组织损伤被诱导后，可能会损害受影响的外周感觉神经元的功能，并引起行为反应的改变。在另一项研究中，幼虫刺激两次与有毒的机械探头大多显示增强的行为反应5，建议发展急性机械敏化（过敏症），这可能是由第一次有毒机械刺激引起的组织损伤。相反，其他作者6报告了混合（增加或减少）行为反应，表明行为反应的改变可能是由于神经元组织的损伤/功能障碍。刺激每个幼虫只有一次消除可能的行为反应差异，无论是由敏感或组织损伤。第四，我们机械地刺激了A8段，这比以前的研究（首选区域A3-A4）2，5，16更后。适用于 A2 或 A8 段的约 3，900 kPa 和 5，300 kPa 之间的探头没有显示任何行为差异4。此外，与 A2-A4 相比，A8 更容易使用产生较低压力（<300 kPa）的机械探针进行刺激，因为该区域的幼虫更薄，因此更容易压缩。其他研究表明，幼虫后端的有毒机械刺激（由刚性昆虫针提供，用钳子固定）大多引起前向运动，而不是逆向或滚动反应18。这种不同的行为反应可能是由于废旧材料（可弯曲的尼他诺灯丝与不可压缩的昆虫针）的特性差异，或向幼虫输送的不同压力（未报告昆虫针的压力值）。
对嗜血杆菌幼虫进行机械结节检测，使该领域发现，不同的机械感官离子通道和神经回路调解机械结节5、6、16、17。然而，与其他感官模式的敏化相比，对机械超敏（合金和高镇痛）的研究滞后——热7、8、10、13、14、冷9和化学3。这种滞后部分可能是由于缺乏合适的机械探头，可以产生跨越俯视到超压的完整响应范围。特别重要的是，特别是对于评估机械合金，是不会引起未受伤幼虫的反向滚动反应的潜移默化探针。我们改进的机械探头的意义在于，它们可以被制造来跨越无害的刺激（低于 174 kPa-200 kPa）或低到高有毒范围（超高 =225 kPa 到 ~5，116 kPa）。在这里，我们演示使用尼蒂诺冯弗雷一样的丝状物，果蝇幼虫开发机械合金和机械高镇痛后紫外线照射。与热敏相比，机械敏化表现出一些差异。与热（热）敏化（高镇痛~8小时和合金过敏症~24小时）相比，机械敏化的发病和峰值都更早（~4小时）7.此外，机械合金和高镇痛是伴随的（均峰值为~4h）。此外，虽然热敏（合金和高镇痛）在以后的时间点7完全解决，机械超敏表现出一个长尾巴，保持略高于基线。德罗索菲拉的冷敏化涉及冷唤起行为9的开关和新的冷唤起行为的出现——这种现象在机械刺激下是不被观察到的。这些在发病、持续时间和观察到的行为上的差异表明，每个感官模式可能由不同的信号通路控制。将此处描述的敏化检测与Drosophila中可用的强大遗传工具相结合，应允许对观测到的机械过敏症（合金和高镇痛）进行精确的基因解剖。

嗜血杆菌 幼虫在接触不同的有毒刺激时表现出一种典型的逆向滚动行为:热1、机械2和化学3。这种行为显然不同于正常的运动。在这里，我们描述了一个改进的机械检测，可用于评估机械节点和机械敏化。
在最近的一项研究中，我们用尼蒂诺线4制造了冯·弗雷式的灯丝。施加不同力和压力的探针是由形成每个探针的尼蒂诺线的长度和直径变化而形成的。机械探针经过校准，测量的力值（在千禧年，mN）转换为压力（千帕，kPa），基于每个探头4的尖端区域。机械探头的定制制造允许我们生成低于（≤200 kPa）以超高（225 kPa 到 5318 kPa）压力，这在原则上可能有利于研究机械超敏性。使用这些改进的机械冯弗雷样长丝，我们表明，压力4，而不是先前检查的力量2，5，6更一致与逆向行为反应在嗜血杆菌幼虫。这里描述的改进的机械检测也有助于识别一个保存血管内皮生长因子（VEGF）相关的受体酪氨酸激酶信号的通路，调节机械结节在苍蝇和大鼠4。
与热（冷热）和化学感官模式3、7、8、9、10相比，两种超敏模式的机械合金和高镇痛在嗜血杆菌幼虫中研究得相对不足。这可能是由于缺乏具体的机械探头，跨越从无害的刺激到高有毒范围2，5，6。一种通常无害的刺激，在嗜血杆菌幼虫经历组织损伤3、7后引起典型的逆向滚动行为，被称为合金。对典型的有害刺激的夸张滚动反应被称为高过敏症7。有毒刺激被定义为那些引起组织损伤，并可以激活受体11。传染给嗜血杆菌幼虫的有害刺激会损害屏障表皮、外周无知觉神经元3、4、7或两者兼有。
在本文中，我们演示了如何定制和校准适合 嗜血杆菌 幼虫的冯弗雷式机械探针。此外，我们展示了如何使用这些探针来检测 果蝇 幼虫中的机械无知反应。最后，我们进一步证明了这些探针的效用，利用它们来证明在 嗜血杆菌 幼虫组织损伤后，机械过敏，包括合金和高镇痛的存在（见 代表结果）。

<table><tbody><tr><td>Beaker</td><td>Fisher Scientific</td><td>02-540C</td><td>Beaker of 10 ml of capacity. Any similar container will do.</td></tr><tr><td>Black (Arkansas) bench stone</td><td>Dan&amp;rsquo;s Whetstone</td><td>SKU: I200306B24b-HQ-BAB-622-C</td><td>Used to smoothe any irregularities of the nitinol wire tips. https://www.danswhetstone.com/product/special-extra-wide-black-bench-stone-6-x-2-1-2-x-1-2/</td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td>Any equivalent confocal microscope will do</td></tr><tr><td>Coplin Jar</td><td>Fisher Scientific</td><td>08-816</td><td>https://www.fishersci.com/shop/products/fisherbrand-glass-staining-dishes-10-slides-screw-cap/08816#?keyword=08-816</td></tr><tr><td>Diethyl ether</td><td>Fisher Scientific</td><td>E138-500</td><td>For anesthetizing larvae.</td></tr><tr><td>Etherization chamber</td><td></td><td></td><td>This is a homemade customized chamber. Please see details of its construction in our previous published paper12. The purpose of the etherization chamber is allow entry of diethyl ether fumes but prevent larval escape.</td></tr><tr><td>Fiber Optic Light Guide</td><td>Schott AG</td><td>A08575</td><td>Schott Dual Gooseneck 23 inch</td></tr><tr><td>Forceps</td><td>Fine Science Tool</td><td>FS-1670</td><td>For transferring larvae</td></tr><tr><td>Glue</td><td>Aleene&#039;s</td><td>N/A</td><td>Aleene&#039;s&amp;reg; Wood Glue, formerly called (Aleene&#039;s All-Purpose Wood Glue)&lt;br/&gt; https://www.aleenes.com/aleenes-wood-glue</td></tr><tr><td>Graspable holder</td><td>Loew Cornell</td><td>N/A</td><td>Loew-Cornell Simply Art Wood Colored Craft Sticks, 500 pieces.</td></tr><tr><td>Halocarbon oil 700</td><td>Sigma</td><td>H8898-100ML</td><td></td></tr><tr><td>Hypodermic needle 30G 1/2&quot;L</td><td>Fisher Scientific</td><td>NC1471286</td><td>BD Precisionglide&amp;reg; syringe needles, gauge 30, L 1/2 inches. Used to make a hole into the wooden holder for the nitinol wires</td></tr><tr><td>Large Petridish</td><td>Falcon</td><td>351007</td><td>60 mm x 10 mm Polystyrene Petridish</td></tr><tr><td>Microscope (Zeiss) Stemi 2000</td><td>Carl Zeiss, Inc.</td><td>NT55-605</td><td>Any equivalent microscope will do</td></tr><tr><td>Microscope Cover Glass 22x22</td><td>Fisher</td><td>12-545-B</td><td></td></tr><tr><td>Microscope Cover Glass 22x40</td><td>Corning</td><td>2980-224</td><td>Tickness 1 1/2</td></tr><tr><td>Microscope Slides</td><td>Globe Scientific Inc.</td><td>1358Y</td><td></td></tr><tr><td>Mini Diagonal Cutter</td><td>Fisher Scientific</td><td>S43981</td><td>For cutting nitinol filaments</td></tr><tr><td>Nitinol filaments, Diameters: 0.004&amp;rdquo;, 0.006&amp;rdquo;, 0.008&amp;rdquo;</td><td>Mailin Co</td><td>N/A</td><td>Fifteen pieces of each diameter of 12&amp;rdquo; length were ordered.&lt;br/&gt; https://malinco.com/</td></tr><tr><td>Piece of black vinyl</td><td>Office Depot</td><td>N/A</td><td>We use a small piece of vinyl cut from a binder. Dark color provides contrast. A small piece allows orientation of the larva</td></tr><tr><td>Small Petridish</td><td>Falcon</td><td>351008</td><td>35 mm x 10 mm Polystyrene Petridish</td></tr><tr><td>Spatula</td><td>Fisher Scientific</td><td>21-401-10</td><td>Double-Ended Micro-Tapered Stainless Steel Spatula. Used to place the food in the petri dish</td></tr><tr><td>Wipes</td><td>Fisher Scientific</td><td>06-666A</td><td>Kimpes KMTECH, Science Brand. Used to dry larvae of excess moisture.</td></tr><tr><td>&lt;em&gt;W&lt;sup&gt;1118&lt;/sup&gt;&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>3605</td><td>Control strain for behavioral assays</td></tr><tr><td>&lt;em&gt;ppk-Gal4&gt;UAS-mCD8-GFP&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>8749</td><td>Strain for fluorescent labeling of class IV md neurons</td></tr></tbody></table>

an improved assay and tools for measuring mechanical nociception in drosophila larvae

对嗜血杆菌中机械结节的公开检测导致了行为的可变评估。在这里，我们制造，用于果蝇幼虫，定制金属镍钛合金（尼蒂诺）长丝。这些机械探针类似于脊椎动物用来测量机械节点的冯·弗雷灯丝。在这里，我们演示了如何制造和校准这些机械探头，以及如何从潜移默化（无害或非有害范围）产生完全的行为剂量响应到超负（低至高有毒范围）刺激。为了证明探针的效用，我们调查了果蝇幼虫组织损伤引起的过敏症。机械合金（对通常无害的机械刺激的过敏）和高镇痛（对有毒机械刺激反应过度）尚未在嗜血杆菌幼虫中建立。使用通常无害的机械探针或通常引起逆向行为的探针，我们发现果蝇幼虫在组织损伤后会产生机械过敏（合金和高过敏症）。因此，我们在这里说明的机械探针和测定可能是解剖机械超敏的基本分子/遗传机制的重要工具。

我们开发了定制的机械探针，使用硝基醇长丝（图1A，N），以引起机械唤起的行为，并产生一个完整的行为剂量反应曲线，使用无害和有毒的机械探头不同强度（图2D），证明这些探头可用于研究基线（在没有伤害的情况下）机械节点。
我们的行为测定结果确定，探针施加压力低于200千帕（约1.57 mN）（图1M），当应用于 嗜血杆菌 幼虫，不会引起逆向滚动反应（图2D 和 视频3）。不出所料，这些潜移默化或非有毒的机械探头（175千帕或200千帕）不会引起可见的神经元组织损伤（图2E）。因为它们不会诱发损伤，因此此类探针可用于评估机械合金（对通常非有害的机械刺激的过敏）。相反，超常或有毒的探针（从462千帕到5，116千帕），以剂量依赖的方式引起增强的行为反应（图2D），更高的压力引起更强的行为反应。不出所料，超高的机械压力也诱发剂量依赖组织损伤的外周感官神经元本身（图2E）。从每组四个幼虫中测出的组织损伤面积（μm2 ±标准偏差）为:2，051.03 ± 703.81（462 kPa），5，102 .29 ± 1，004.67 （2，283 kPa） 和 12，238.83 ± 3，724.11 （5，116 kPa）。因此，大于或等于462 kPa（约63 mN）的压力，它唤起逆向滚动反应（在幼虫的25%或更多），并导致可见的神经元组织损伤（图2E），可能适合研究机械性高镇痛（对通常有毒的机械刺激过敏）。无感知机械探头（≥462 kPa）总是诱发组织损伤（n = 10，经过定性评估），但并不总是引起逆向滚动响应。
为了评估机械超敏（合金和高镇痛），我们使用了一个成熟的嗜血杆菌幼虫模型的无知觉敏化，使用紫外线（UV）照射诱导组织损伤7，12。这一分析有助于剖析热无知觉超敏8、9、10、13、14、15的遗传和细胞机制。为了确定紫外线治疗是否导致机械合金失调，中三星对照（w1118）幼虫是模拟照射或紫外线照射（15-20mJ/cm2）（图3A）。然后，幼虫在2小时、4小时、8小时、16小时和24小时处理后用通常的潜移默化机械探针（200千帕，1.57米N）进行行为测试。大约20%的幼虫在紫外线治疗后最早在2小时后做出反应，50%的幼虫在4小时反应，相比之下，6.6%和8.3%的模拟紫外线照射动物（图3B）。这表明紫外线引起的组织损伤导致机械合金在4小时后照射。在较晚的时间点（8小时、16小时和24小时）时，紫外线处理幼虫的行为反应在16%-20%的响应者范围内（平均值为n=3-6组，每组10幼虫），与模拟辐照对照组（在3%-6%的响应者范围内）相比略有增加（但不具有统计学意义）， 平均值为n=3-6组，每组10幼虫）（图3B）。
为了研究机械性高镇痛，使用了超压（462 kPa，3.63 mN），通常诱发约20%的幼虫（图2D）的逆向滚动反应，并导致神经元组织损伤（图2E）。我们应用了 462 kPa 探针到幼虫的侧，无论有没有紫外线引起的组织损伤（图 3A ）。我们发现，紫外线治疗后在4小时、8小时和16小时探测的幼虫在逆向滚动反应中显著增加，其中4小时是行为超敏的峰值（60%响应）:模拟紫外线照射动物表现出27%的逆向反应（图3C）。与机械合金尼亚类似，紫外线处理动物在8小时、16小时和24小时的行为反应（在36%-42%范围内）在统计学上与未处理的幼虫没有区别（在20%-26%之间）。与明星阶段的中间第三阶段相比，星期后期第三阶段的Larvae确实显示出基线行为反应的轻微下降。我们假设这可能是由幼虫的大小增加（图2A）或覆盖身体的层盖的厚度增加。这一事实可以解释为什么在后期开发阶段，紫外线处理不会引起更大的机械敏化，如观察到的4小时后紫外线处理。
综合起来，我们的结果表明， 果蝇 幼虫在紫外线诱发的组织损伤后，会发展成机械合金和机械性高镇痛。机械合金和高镇痛的高峰期相同，紫外线治疗后4小时:然而，机械性高镇痛有一个更明显的时间尾巴，因为它返回基线比机械合金更慢。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61911/61911fig01.jpg"> 图1:开发一种类似冯·弗雷的工具，以评估 嗜血杆菌 幼虫的机械节点。 （A） 用于研究 嗜血杆菌 幼虫中机械探针的图片。（B） 硝基醇丝及其相对直径显示为相对尺度。（C） 用于切割硝基醇丝的对角线切割机的图片。（D） 用锋利的石头平滑切割的尼他诺灯丝的锋利边缘。（E） 用于在探头的木冰棒手柄上打孔的皮下针。针尖需要达到手柄杆的至少一半高度，才能插入安全的灯丝。（F-G）通过用插入孔粘入木制冰棒手柄，将硝基醇长丝粘附。（H-L）通过按压机械探头来校准它们。（M） 不同机械探头产生的力值（mN）和压力（在 kPa 中）。用于构造探头的每个硝基醇长丝的长度 （P1-P10:P:探针）详细为厘米（厘米）。（N） 一套完整的机械探头的图片，从174千帕到5，116千帕不等。 <a href="https://www.jove.com/files/ftp_upload/61911/61911fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61911/61911fig02v2.jpg"> 图2:机械节点检测:冯·弗雷样的灯丝产生逆向滚动行为的剂量反应曲线，并导致感觉神经元组织损伤。 （A） 果蝇 幼虫不同阶段（第二和第三星）的图片。比例尺栏: 2 毫米 （B） 第三星 果蝇 幼虫的果体视图的卡通。红点表示应用机械探头的腹部段。T: 胸科部分:答:腹部部分。其他解剖地标被标记。（C）检测的卡通:机械探针应用于幼虫的，直到它弯曲到下面的表面，然后保持 2s 。如果压力足够高，这会在释放时引起反向滚动响应。（D） 行为剂量反应:每个蓝点代表幼虫的百分比，这些幼虫对10种动物体内的机械刺激做出逆向滚动反应。小提琴情节的百分比的逆向滚动行为诱导不同的机械探头。kPa:千帕。框图表示中位数（绿色）、胡须（红色）表示第 10 和第 90 个百分位数。（E） 组织损伤: 第三个星内幼虫 （基因型 ppk-Gal4>UAS-mCD8-GFP标记无感知感官神经元） 在背段 A8 与指示的压力进行探测。然后检查荧光标记的配对 ddaC IV 类感官神经元（穿过正名中线）（见第 4 节和第 5 节）。白色区域（红色星号）表示间隙或组织损伤。比例:100μm。在面板 B 中，幼虫显示在正向视图中，而 C 显示的是横向视图。机械探针压在幼虫的鼻角膜侧面，在探头尖端和周围区域的接触点产生凹陷状口袋。向心室侧弯曲的实心黑线是口袋的顶部，而破折号的灰色横向线表示侧面和口袋底部。 <a href="https://www.jove.com/files/ftp_upload/61911/61911fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61911/61911fig03v2.jpg"> 图3:紫外线损伤后的机械超敏。 （A） 实验设计的示意图，以测试敏感性。中三星被模拟处理（非紫外线）或紫外线照射。然后在模拟治疗或辐照后，在不同的时间点（2小时、4小时、8小时、16小时和24小时）进行机械节点检测。 （B） 机械合金:在模拟处理或紫外线照射后，在指示的时间点用通常的潜移默化或非有害的机械刺激（200 kPa，1.57 mN）进行探测后，表现出逆向滚动的幼虫百分比。（C） 机械性高镇痛:在模拟治疗或紫外线照射后，在指示的时间点用通常超常或有毒的机械刺激（462 kPa，3.63 mN）进行探测后，表现出逆向滚动的幼虫百分比。错误条表示平均值 +/- SEM.用于统计分析的双尾未修制 t测试:*p < 0.05，**p < 0.01:ns:不重要。每个红点，在B和C面板中，代表10个幼虫的平均比例，n = 3-6套每个时间点/条件。 <a href="https://www.jove.com/files/ftp_upload/61911/61911fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
视频1:嗜血杆菌幼虫的正常运动。<a href="https://cloudflare.jove.com/files/ftp_upload/61911/Video_1_regresolu.mp4" target="_blank">请点击这里下载此视频。</a>
视频2:嗜血杆菌幼虫的有害机械刺激。<a href="https://cloudflare.jove.com/files/ftp_upload/61911/Video_2_regresol.mp4" target="_blank">请点击这里下载此视频。</a>
视频3:对嗜血杆菌幼虫进行下垂机械刺激。<a href="https://cloudflare.jove.com/files/ftp_upload/61911/Video_3_Subthreshold_short.mp4" target="_blank">请点击这里下载此视频。</a>

Watch this Scientific Journal Video about An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae at JoVE.com

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

该协议的目的是展示如何对 嗜血杆菌 幼虫的机械结节进行改进的检测。我们在这里使用测定来证明机械过敏（合金和高过敏症）存在于 嗜血杆菌 幼虫中。

测量 果蝇 拉瓦的机械结节的改进的检测和工具

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

an-improved-assay-tools-for-measuring-mechanical-nociception

Research

JoVE Journal

Behavior

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

4.7K 次观看.  The University of Texas MD Anderson Cancer Center. 该协议的目的是展示如何对 嗜血杆菌 幼虫的机械结节进行改进的检测。我们在这里使用测定来证明机械过敏（合金和高过敏症）存在于 嗜血杆菌 幼虫中。

视频: An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

科研

生物学

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

生物工程

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 ...

神经科学

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

免疫与感染

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

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

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

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

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

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

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

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 Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Local Heat Probe Assay: A Method to Assess Nociception of High Temperatures in Drosophila Larvae

Source: Chattopadhyay, A., et al. <a href="https://www.jove.com/video/3837/" target="_blank">Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae</a>. J. Vis. Exp. (2012).
Drosophila larvae have dedicated sensory neurons&#8211;nociceptors&#8211;whose response to potentially harmful thermal stimuli triggers aversive&#8211;or so-called &#34;nocifensive&#34;&#8211;behaviors in the animal. This video describes a method to assess thermal nociception by recording larval behavior in response to local application of a heat probe onto the cuticle. This method is, therefore, called the local heat probe assay.

局部热探针测定:一种评估果蝇幼虫对高温伤害感受的方法

Source: Chattopadhyay, A., et al. Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae. J. Vis. Exp. (2012).
Drosophila larvae ...

测量果蝇拉瓦的机械结节的改进的检测和工具

摘要

探索更多视频

此视频中的章节

高分辨率测量的气味驱动在果蝇幼虫的行为

使用微流体芯片实时成像和损伤研究的响应

局部和全局的方法评估热痛觉

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

一个行为分析对机械感觉MARCM为基础的克隆

去除

新的分析冷痛觉在

一个简单的神经元机械损伤方法学研究

局部热探针测定:一种评估果蝇幼虫对高温伤害感受的方法

测量 果蝇 拉瓦的机械结节的改进的检测和工具

摘要

探索更多视频

此视频中的章节

高分辨率测量的气味驱动在果蝇幼虫的行为

使用微流体芯片实时成像和损伤研究的响应

局部和全局的方法评估热痛觉

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

一个行为分析对机械感觉MARCM为基础的克隆

去除

新的分析冷痛觉在

一个简单的神经元机械损伤方法学研究

局部热探针测定:一种评估果蝇幼虫对高温伤害感受的方法

测量果蝇拉瓦的机械结节的改进的检测和工具