Name: quantification of drosophila grooming behavior
Uploaded: 2017-07-19T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Drosophila Grooming Behavior at JoVE.com

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。 <...

<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>

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 ...

Research

JoVE Journal

Neuroscience

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Single Sensillum Recordings in the Insects Drosophila melanogaster and 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

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

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

Methods to Assay Drosophila 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

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

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

A Single-fly Assay for Foraging Behavior in Drosophila

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

Straightforward Assay for Quantification of Social Avoidance 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

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

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

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

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

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

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

Using Linear Agarose Channels to Study Drosophila Larval 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 ...