我们要感谢Brian Shepherd，Tat Udomritthiruj，Aaron Willey，Ruby Froom，Elise Pitmon和Rose Hedreen，以便早日测试和建立这种方法和室设计。感谢Kelly Tellez和Graham Buchan的阅读和编辑手稿。我们感谢Andrew Seeds和Julie Simpson的开创性工作，并就建议使用亮黄色染料（Sigma）提供建议和支持。这项工作由Mary E. Groff外科和医学研究与教育慈善信托基金，Bronfman科学中心和Hellman研究员计划部分支持。

准备<ol><li> 准备一个将活的果蝇从培养瓶移动到梳妆室的吸气器。吸气器允许有意识的动物转移到行为室，以确保麻醉不会影响随后的行为观察。 <ol><li>使用剪刀切割1.5英尺的管道ID"⅛"，OD¼"。至少1英寸的1毫升一次性微量吸头尖端。在切割尖端上握住1厘米长的网格（开口0.196英寸）。 注意:大多数1毫升尖端具有渐变线，其中尖端位于包装盒中，通常切割到该线。 </li><li>将新鲜的1毫升微量吸头贴在网眼/切口上。观察网格形成两个尖端之间的紧密屏障。新尖端的内部为苍蝇创建了一个保持室。 </li><li>切开新的微量移液器吸头的末端以扩大开口，以允许单个飞行通过。霍尔e将大致匹配梳妆室的开口（直径约1.5-2毫米）。 </li><li>将嵌套的尖端贴合在管道的末端。将尖端推入管中以确保紧密配合，以允许真空压力。 </li><li>在管的另一端，切下200μL微量吸头的尖端，并将狭窄的切割端装入管道。这是吸气器的"嘴"一侧。 </li></ol></li><li>准备粉尘等分试管。称量大约5毫克的亮黄色染料在称重纸上。将灰尘倒入0.6 mL微量离心管中，并紧紧盖住盖子。 注意:我们建议在等分试样中使用丁腈手套，因为一些乳胶手套对染料是可渗透的。 </li><li>对实验中的所有样品重复步骤1.2）（每个室中每次飞行一次）。 </li><li>准备乙醇（EtOH）管:将1 mL 100％乙醇吸入1.5 mL微量离心管中。用于基因型或条件的标记管/ LI&gt; <li>对实验中的所有样品重复步骤1.4）（每个室中每次飞行一次）。盖上并保存EtOH管以完成修饰测定。还包括至少一个"空白"样品，其仅为1mL EtOH而不用于阴性对照。 </li><li>通过拧紧滑动顶板（具有较小的孔），但留下底板（带有较大的孔）以除去灰尘，组装每个梳妆室。 </li><li>将每个必要的梳妆台平放在桌子上，顶面朝下。飞进入面是面朝下的。 </li><li>将5毫克亮黄色染料分批装入每个室中，方法是将管子靠在所需孔上方的表面上。将舱室靠在桌子上，以确保所有的灰尘都通过网格落到顶板上。 </li><li>将底板拧到每个室上，使得圆锥形开口的较宽端面朝外，孔不会靠在井上。保护机器人使其不会滑动并释放染料。 </li><li>将房间翻转并将其平放在桌子上几次，以便灰尘通过网状物落在底板上。 </li><li>将舱室的顶板滑入"打开位置"，使小孔与十五口井对齐，从而将苍蝇引入各个井。 </li></ol>飞尘和美化<ol><li>通过轻轻地吸入吸气器的一端，如同使用吸管一样，将一个飞入吸入器。通过轻轻吹起并将开口朝向目标来存放苍蝇。 </li><li>将一只蝇吸入用于实验的每个孔中。装载井后，滑动顶板，以便在整个舱室装载期间将飞行物捕获在舱室中，并将胶带放置在该井口的开口处，以防止在装载其他井时飞行逃生。如果多个苍蝇被吸入一个单一的好吧，离开这个开放，直到只剩下一只苍蝇。 </li><li>在所有必需的井充满苍蝇之后，翻转室，使得底板向上，使得灰尘有可能通过穿过网状物覆盖苍蝇。 </li><li>通过拧紧螺丝紧紧固定顶板和底板，并旋转室以将苍蝇粉碎4秒。 </li><li>将房间（顶板朝下）撞在桌子上两次，使染料落到另一边。然后，将腔室翻转（顶板朝上）并敲击腔室两次，以确保染料沉淀在底部腔室的地板上，远离保持在顶部腔室中的苍蝇。 </li><li>对于不允许修改的控制蝇，请立即进入协议的步骤3。对于将完成测定的苍蝇，请转到步骤2.7。 </li><li>将房间平放在桌子或台面上，顶板向上放置三十分钟，以便苍蝇修剪。放下小屋呃在噪音和无振动的空间，以保持你的环境恒定，所有的修饰实验（照明水平，湿度和温度）。 RT或25°C带有湿度控制器的桌面孵化器是理想的选择。 </li></ol> 3.样品制备和吸光度分析<ol><li>将顶板向上保持，轻轻拧下底板，将其从室中取出，注意不要弄脏染料。 </li><li>将房间放置在飞行CO 2垫上，气流低，直到苍蝇麻醉。 </li><li>从室中拧下顶板。仔细使用镊子抓住一条腿，并从每个室移动一个除尘飞沫，并将其沉积在含有EtOH的1.5 mL微量离心管中。每个包含一个飞行的15个腔室的传送时间约为3-5分钟。如果/当使用多个室进行惊人的实验时，实验者应该考虑转移时间，以确保实验的有效分期。 </LI&gt; <li>一旦每个微量离心管包含1个飞，关闭盖子并将管反转三次以搅拌并混合染料和乙醇溶液。 </li><li>孵育含有乙醇的管并在室温下飞行5小时，以确保将染料从飞入物中完全除去。 5小时后，短暂地旋转每个管（2秒），以确保染料从苍蝇中清除。 </li><li>将等分试样将50μL实验的1.5mL试管插入96孔板的孔中（如果有的话），注意每个样品在板上的位置。加入200μLEtOH稀释样品5x。稀释避免了重灰尘飞扬或大修饰缺陷的天花板效应。 </li><li>使用平板读数器分析每个样品在397 nm。或者，如果读板器不可用，则在标准分光光度计上测量可见光谱中各个样品和毛坯的吸光度。 </li><li>通过读卡器阅读并记录样品，并将该电子表格与记录仪一起保存d样品在板上。 </li></ol> 4.结果量化<ol><li>编译所有样本的结果，并测量每个基因型或病症的方差。 注意:使用通过单因素方差分析和Bonferroni校正或多次平行比较的其他校正的统计分析将时间0分钟和时间30分钟的染料积累视为单独的条件。 </li><li>使用以下等式计算每个基因型/条件的梳理百分比差异:[（时间0&#39;处的染料积累平均值 - 时间30&#39;处的染料累积平均值）/时间0处的染料累积平均值×100。 </li><li>在条形图中表达每种基因型和条件的百分比，并比较基因型和条件。 </li></ol>

<ol>
	<li>Szebenyi, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+Behaviour+in+Drosophila-Melanogaster.">Cleaning Behaviour in Drosophila-Melanogaster.</a> Animal. Behaviour. 17, (1969).</li><li>Corfas, G., Dudai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+and+dishabituation+of+a+cleaning+reflex+in+normal+and+mutant+Drosophila.">Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila.</a> J Neurosci. 9 (1), 56-62 (1989).</li><li>Yanagawa, A., Guigue, A. M. A., Marion-Poll, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hygienic+grooming+is+induced+by+contact+chemicals+in+Drosophila+melanogaster.">Hygienic grooming is induced by contact chemicals in Drosophila melanogaster.</a> Frontiers in Behavioral Neuroscience. 8, (2014).</li><li>Dawkins, R., Dawkins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+Organization+and+Postural+Facilitation+-+Rules+for+Grooming+in+Flies.">Hierarchical Organization and Postural Facilitation - Rules for Grooming in Flies.</a> Animal Behaviour. 24 (Nov), 739-755 (1976).</li><li>Seeds, A. M., 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=A+suppression+hierarchy+among+competing+motor+programs+drives+sequential+grooming+in+Drosophila.">A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila.</a> Elife. 3, e02951 (2014).</li><li>King, L. B., 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=Neurofibromin+Loss+of+Function+Drives+Excessive+Grooming+in+Drosophila.">Neurofibromin Loss of Function Drives Excessive Grooming in Drosophila.</a> G3-Genes Genomes Genetics. 6 (4), 1083-1093 (2016).</li><li>Tauber, J. M., Vanlandingham, P. A., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+Levels+of+the+Vesicular+Monoamine+Transporter+and+a+Novel+Repetitive+Behavior+in+the+Drosophila+Model+of+Fragile+X+Syndrome.">Elevated Levels of the Vesicular Monoamine Transporter and a Novel Repetitive Behavior in the Drosophila Model of Fragile X Syndrome.</a> Plos One. 6 (11), e27100 (2011).</li><li>Kaur, K., Simon, A. F., Chauhan, V., Chauhan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bisphenol+A+on+Drosophila+melanogaster+behavior--a+new+model+for+the+studies+on+neurodevelopmental+disorders.">Effect of bisphenol A on Drosophila melanogaster behavior--a new model for the studies on neurodevelopmental disorders.</a> Behav Brain Res. 284, 77-84 (2015).</li><li>Chang, H. 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=Overexpression+of+the+Drosophila+vesicular+monoamine+transporter+increases+motor+activity+and+courtship+but+decreases+the+behavioral+response+to+cocaine.">Overexpression of the Drosophila vesicular monoamine transporter increases motor activity and courtship but decreases the behavioral response to cocaine.</a> Molecular Psychiatry. 11 (1), 99-113 (2006).</li><li>Yellman, C., Tao, H., He, B., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+sexually+dimorphic+behavioral+responses+to+biogenic+amines+in+decapitated+Drosophila.">Conserved and sexually dimorphic behavioral responses to biogenic amines in decapitated Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (8), 4131-4136 (1997).</li><li>Feng, G. 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=Cloning+and+functional+characterization+of+a+novel+dopamine+receptor+from+Drosophila+melanogaster.">Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster.</a> Journal of Neuroscience. 16 (12), 3925-3933 (1996).</li><li>Gotzes, F., Balfanz, S., Baumann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Structure+and+Functional-Characterization+of+a+Drosophila+Dopamine-Receptor+with+High+Homology+to+Human+D(1/5).">Primary Structure and Functional-Characterization of a Drosophila Dopamine-Receptor with High Homology to Human D(1/5).</a> Receptors. Receptors &amp; Channels. 2 (2), 131-141 (1994).</li><li>Han, K. A., Millar, N. S., Grotewiel, M. S., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAMB,+a+novel+dopamine+receptor+expressed+specifically+in+Drosophila+mushroom+bodies.">DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies.</a> Neuron. 16 (6), 1127-1135 (1996).</li><li>Sugamori, K. S., Demchyshyn, L. L., Mcconkey, F., Forte, M. A., Niznik, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Primordial+Dopamine+D1-Like+Adenylyl+Cyclase-Linked+Receptor+from+Drosophila-Melanogaster+Displaying+Poor+Affinity+for+Benzazepines.">A Primordial Dopamine D1-Like Adenylyl Cyclase-Linked Receptor from Drosophila-Melanogaster Displaying Poor Affinity for Benzazepines.</a> Febs Letters. 362 (2), 131-138 (1995).</li><li>Pitmon, E., 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=The+D1+family+dopamine+receptor,+DopR,+potentiates+hind+leg+grooming+behavior+in+Drosophila.">The D1 family dopamine receptor, DopR, potentiates hind leg grooming behavior in Drosophila.</a> Genes Brain and Behavior. 15 (3), 327-334 (2016).</li><li>Phillis, R. W., 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=Isolation+of+mutations+affecting+neural+circuitry+required+for+grooming+behavior+in+Drosophila+melanogaster.">Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster.</a> Genetics. 133 (3), 581-592 (1993).</li><li>Hampel, S., Franconville, R., Simpson, J. H., Seeds, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neural+command+circuit+for+grooming+movement+control.">A neural command circuit for grooming movement control.</a> Elife. 4, e08758 (2015).</li><li>Kays, I., Cvetkovska, V., Chen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+analysis+of+single+neurons+to+correlate+synaptic+connectivity+with+grooming+behavior.">Structural and functional analysis of single neurons to correlate synaptic connectivity with grooming behavior.</a> Nature Protocols. 9 (1), 1-10 (2014).</li></ol>

作者声明没有利益冲突。

梳理分析相对简单，但我们会提醒实验者特别注意以下问题。在引入苍蝇和染料之后，通过拧紧顶板和底板上的螺丝来保持紧密的密封，对于可重现的结果至关重要。亮黄色染料非常细，松散的接头将会损失室内边缘的染料。每个井的染料含量的不规则性可能容易导致梳理量化，因为不均匀的粉尘会增加梳理事件的差异和假阳性率。此外，我们鼓励实验者在30分钟的梳理期间，意识到他们选择保?...

果蝇黑腹果蝇 （ D. melanogaster ）是一种强大的天生行为，涉及到多个独立运动程序的协调1 。果蝇清除身体的灰尘，微生物和其他可能抑制正常生理功能如视力和飞行的病原体，或导致显着的免疫挑战。在感测和响应机械2和免疫激活3时 ，苍蝇重复地将腿部在一起或在目标身体区域上摩擦，直到其足够清洁并且梳理进行到身体的另一部分。苍蝇执行在不同的较量疏导运动在很大程度上发生在刻板图案1,4。优先考虑信号，行为层次变得明显。电路和活动模式已被确定为支持ofa模型，首先发现层次顶部的修饰程序，并抑制随后修饰的身体区域的平行信号5 。最重要的是给头，然后是腹部，翅膀，最后是胸部5 。 黑腹果蝇的修饰程序是研究神经回路，调节分子信号和神经递质的理想系统。例如，神经纤维瘤功能6的损害， 果蝇脆弱的X型精神发育迟滞蛋白（ dfmr1 ） 7的损失和双酚A（BPA） 8的暴露都会导致过度的梳理和类似于神经纤维瘤病的离散人类症状的脆弱X综合征和自闭症谱系障碍和注意缺陷多动障碍（ADHD）的方面。修饰行为也可以是习惯在突变菌株2上差异化，将该运动程序提供给行为可塑性的研究。可以用果蝇建模的神经学现象的广度需要一种新颖的比较方法来测量苍蝇自身修养的能力。 囊泡单胺转运体和多巴胺和身体中的其它生物胺的相对丰度的联合作用已被证明介导果蝇梳理行为9,10。八聚胺和多巴胺刺激斩首苍蝇中可比的后腿修饰活性，而八胺的前驱酪胺也触发了较少的梳理7 。四多巴胺受体已经在果蝇 11，12，13，14被确定</sup&gt;。通过使用本方案中描述的修饰测定方法，我们确定了I型家族多巴胺受体DopR（ DopR，dDA1，dumb ）在后腿修饰行为中的作用15 。 疏导可以通过查看清洁度通过该动物可以用标记染料或荧光灰尘5,16喷粉整个身体后充分培训的程度来间接定量。残留在身体上的剩余灰尘可以用作总体行为的相对标记。经过足够的时间培养后，尘土飞逝，可能会在修饰行为中表现出特定的赤字。随着梳理调查变得更加广泛，方案已经将诸如斩首加入颈部连接神经10的措施 ，刷毛的触觉刺激以引起修复反应2 ，和录像的行为15 。修饰的直接观察可以通过使用视觉观察和手动记录特定事件疏导4的频率和持续时间来容易地研究。 我们设计了一个十五分钟的梳妆室，可以用3D打印机或激光切割机构造，蓝图设计可用于复制15 。该设计使用两个连接的中心板，其具有通过网格分开的开口和分开的两个另外的滑动顶部和底部平板，从中分别装载飞行物和/或染料。经过粉尘飞行时间后，我们将其沉积在乙醇中以溶解染料，并测量该溶液在染料波长处的吸光度。平板读数器可用于多个平行样品，或者单次读取分光光度计可用于单个样品。这种方法最大限度地减少了由处理引起的误差低成本测试法以更小，更具成本效益的规模运行。这种方法是由Julie Simpson和Andrew Seeds率先推出的方法衍生和修改的，他们使用带加热元件的较大梳妆室进行温度敏感电路操作5 。以下方案展示了全身梳理的量化，并显示了用于定量各个身体部位的染料积累的替代方法。我们还提供了WT和DopR突变体之间的样本比较数据，以及用于计算修饰行为的简单性能指标的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High-Flex Tygon PVC Clear Tubing</td>
			<td>McMaster-Carr</td>
			<td>5229K54</td>
			<td>ID 1/8&#34;, OD 1/4&#34;, used with micropipettor tips and mesh to construct mouth aspirators</td>
		</tr>
		<tr>
			<td>Micropipette tips (1ml and 200ul)</td>
			<td>Genesee Scientific</td>
			<td>24-165, 24-150R</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Screen, 2&#34; x 2.6&#34;</td>
			<td>McMaster-Carr</td>
			<td>9318T44</td>
			<td>Used to construct grooming chamber and mouth aspirators</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Yellow Dye</td>
			<td>Sigma-Aldrich</td>
			<td>201375-25G</td>
			<td>we recommend use of nitrile gloves while handling this product</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher Scientific</td>
			<td>12-812</td>
			<td>set to &#34;touch&#34;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carolina Biological Supply</td>
			<td>86-1282</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR International</td>
			<td>10025-726</td>
			<td></td>
		</tr>
		<tr>
			<td>0.65 ml microcentrifuge tubes</td>
			<td>VWR International</td>
			<td>20170-293</td>
			<td>tubes can be reused with successive assays</td>
		</tr>
		<tr>
			<td>UV 96 well plate</td>
			<td>Corning</td>
			<td>26014017</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy HTX Platereader</td>
			<td>BioTek</td>
			<td>need to download catalog to access product number</td>
			<td>http://www.biotek.com/products/microplate_detection/synergy_htx_multimode_microplate_reader.html?tab=overview</td>
		</tr>
		<tr>
			<td>Gen5 Microplate Reader and Imager Software</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>https://www.microsoftstore.com/store/msusa/en_US/pdp/Excel-2016/productID.323021400?tduid=(65d098c0e83b86c952bdff5b0719c83f)(256380)(2459594)(SRi0yYDlqd0-LI..ql4M2LoZBEhcBljvIA)()</td>
		</tr>
		<tr>
			<td>Drosophila Incubator</td>
			<td>Tritech</td>
			<td>DT2-CIRC-TK</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34; acrylic plastic</td>
			<td>McMaster-Carr</td>
			<td>8473K341</td>
			<td></td>
		</tr>
		<tr>
			<td>8-32 nuts</td>
			<td>McMaster-Carr</td>
			<td>90257A009</td>
			<td></td>
		</tr>
		<tr>
			<td>8-32 x 1&#34; hex cap screws</td>
			<td>McMaster-Carr</td>
			<td>92185A199</td>
			<td>the bottom plate needs to be tapped for this size screw</td>
		</tr>
		<tr>
			<td>8-32 x 1/2&#34; hex cap screws</td>
			<td>McMaster-Carr</td>
			<td>92185A194</td>
			<td>the second plate from the top needs to be tapped</td>
		</tr>
		<tr>
			<td>2-56 3/8&#34; flat head phillips machine screws</td>
			<td>McMaster-Carr</td>
			<td>91500A088</td>
			<td>these hold the two middle plates together</td>
		</tr>
		<tr>
			<td>0.175&#34; ID, 1/4&#34; OD, 0.34&#34; aluminum pipe</td>
			<td>McMaster-Carr</td>
			<td>92510A044</td>
			<td>Manufactured in-house; product listed is approximately the same dimensions and should work for size 8 screws.&#160; These act as sheaths for the 1&#34; screws and set the hex cap up slightly from the surface of the top plate</td>
		</tr>
	</tbody>
</table>

quantification of drosophila grooming behavior

果蝇的美容行为是一个复杂的多步运动计划，需要协调的前肢和后肢的运动。在这里，我们提出了一个整理测定方案和新颖的室设计，可以成本效益可扩展，可以进行小型或大规模的果蝇梳理研究。苍蝇在整个身体上撒上了黄色染料，并给予时间从室内的身体上除去染料。然后将苍蝇沉积在一定体积的乙醇中以溶解染料。测量和记录用于修饰的和未移植的动物的染料 - 乙醇样品的相对光谱吸光度。该方案产生个体苍蝇染料积累的定量数据，可以容易地平均和比较样品。这允许实验设计容易地评估突变动物研究或电路操作的修饰能力。这个有效的过程既是通用又可扩展的。我们显示wWT动物和突变体动物之间的协议和比较数据为果蝇 I型多巴胺受体（DopR）的扫流。

修整测定产生定量数据，以在设定的测量时间（30分钟）后，基于剩余在苍蝇体上的累积染料的相对剩余量来评估行为性能。 图1中突出显示了滑动梳理室设计的示例图像和测定的主要步骤。苍蝇通过在染料存在下涡旋聚集大量的染料从立即粉尘（ 图2d，2e ）。除尘蝇可以在分析后保留一定范围的染料积累（ 图2f，2g ）。因此，溶解在...

Watch this Scientific Journal Video about Quantification of Drosophila Grooming Behavior at JoVE.com

Quantification of Drosophila Grooming Behavior

该协议描述了果蝇中可扩展的个体梳理分析技术，产生强大的定量数据来测量梳理行为。该方法是基于在一段时间内比较未修饰动物与修饰动物的身体上染料积累的差异。

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

quantification-of-drosophila-grooming-behavior

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.0K 次观看.  Williams College. 该协议描述了果蝇中可扩展的个体梳理分析技术，产生强大的定量数据来测量梳理行为。该方法是基于在一段时间内比较未修饰动物与修饰动物的身体上染料积累的差异。 

视频: Quantification of Drosophila Grooming Behavior

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

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 ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

染色昆虫行为检测:麻醉的交配行为<em&gt;果蝇</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

果蝇美容行为的量化

摘要

探索更多视频

此视频中的章节

在昆虫单感器记录果蝇按蚊

从查明的神经元和取食行为的钙信号的同时记录果蝇

对检测方法果蝇行为

Optogenetic刺激的逃避行为

单苍蝇检测方法的觅食行为的

简单的检测方法的社交回避的量化

染色昆虫行为检测:麻醉的交配行为

在气味引导行为的高分辨率定量

为什么量化事项:表型的表征在

用线性琼脂糖渠道研究