Name: planarian scrunching as a quantitative behavioral readout for noxious stimuli sensing
Uploaded: 2020-07-30T14:30:00
Description: Watch this Scientific Journal Video about Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing at JoVE.com

作者感谢塔潘·高尔先生对手稿的评论。这项工作由NSF职业补助金1555109资助。

1. 定量平面行为分析
<ol><li>实验设置<ol><li>将可调光 LED 面板放在平面上。LED 面板有两个用途：（1） 提供均匀的白色背景和 （2） 用作可调节光源以获得适当的对比度。将 100 mm 培养皿竞技场放在 LED 面板上。 注：为了增加吞吐量，多井板可以用作竞技场23，24，但更大的竞技场有利于自动图像分析。23,</li>
		<li>将相机安装在竞技场上方的环形支架?...

<ol>
	<li>Rink, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+systems+and+regeneration+in+planaria.">Stem cell systems and regeneration in planaria.</a> Development Genes and Evolution. 223, 67-84 (2013).</li><li>Reddien, P. W., Alvarado, A. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+freshwater+planarians+in+environmental+toxicology+studies:+Advantages+and+potential.">The use of freshwater planarians in environmental toxicology studies: Advantages and potential.</a> Ecotoxicology and Environmental Safety. 161, 45-56 (2018).</li><li>Rompolas, P., Azimzadeh, J., Marshall, W. F., King, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+ciliary+assembly+and+function+in+planaria.">Analysis of ciliary assembly and function in planaria.</a> Methods in Enzymology. 525, 245-264 (2013).</li><li>Sabry, Z., 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=Pharmacological+or+genetic+targeting+of+Transient+Receptor+Potential+(TRP)+channels+can+disrupt+the+planarian+escape+response.">Pharmacological or genetic targeting of Transient Receptor Potential (TRP) channels can disrupt the planarian escape response.</a> PLoS One. , 753244 (2019).</li><li>Cochet-Escartin, O., Carter, J. A., Chakraverti-Wuerthwein, M., Sinha, J., Collins, E. M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+Phototactic+Assay+Reveals+Differential+Behavioral+Responses+Based+on+Wavelength.">Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.</a> PLoS One. 9, 114708 (2014).</li><li>Petrus, 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+role+of+TRPA1+in+mechanical+hyperalgesia+is+revealed+by+pharmacological+inhibition.">A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition.</a> Molecular Pain. 3, 40 (2007).</li><li>Takano, 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=Regeneration-dependent+conditional+gene+knockdown+(Readyknock)+in+planarian:+Demonstration+of+requirement+for+Djsnap-25+expression+in+the+brain+for+negative+phototactic+behavior.">Regeneration-dependent conditional gene knockdown (Readyknock) in planarian: Demonstration of requirement for Djsnap-25 expression in the brain for negative phototactic behavior.</a> Development, Growth &amp; Differentiation. 49, 383-394 (2007).</li><li>Nishimura, K., 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=Identification+of+glutamic+acid+decarboxylase+gene+and+distribution+of+GABAergic+nervous+system+in+the+planarian+Dugesia+japonica.">Identification of glutamic acid decarboxylase gene and distribution of GABAergic nervous system in the planarian Dugesia japonica.</a> Neuroscience. 153, 1103-1114 (2008).</li><li>Inoue, T., Yamashita, T., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermosensory+signaling+by+TRPM+is+processed+by+brain+serotonergic+neurons+to+produce+planarian+thermotaxis.">Thermosensory signaling by TRPM is processed by brain serotonergic neurons to produce planarian thermotaxis.</a> Journal of Neuroscience. 34, 15701-15714 (2014).</li></ol>

使用这个协议，我们可以定量地研究物理和化学刺激7，28，29,或基因操纵7,（RNAi）28，2928,29对平面运动的影响。29为了最大限度地提高空间分辨率，最好将摄像机尽可能靠近竞技场，同时确保整个竞技场位于视野中。为了增加吞吐量，可以通过同时记录多个平面图，同时筛选多个平?...

除了在干细胞和再生研究,1、2、3,2方面的受欢迎程度外，淡水平面学家长期以来一直用于行为研究4、5，5利用它们相对大的尺寸（几毫米长）、易于和低廉的实验室维护成本，以及广泛的可观察行为。计算机视觉和自动跟踪对平面行为研究的,,引入6、7、8、9、10、11，,7使行为表型的定量分化得以实现。8,9,1011动物行为是神经元功能的直接读出。由于平面神经系统的中等大小和复杂性，但与脊椎动物大脑,12、13、14,13共享保存的关键元素，研究平面体行为可以提供对神经元作用的保守机制的洞察，这些机制在更复杂的生物体中可能很难直接探寻。14因此，平面是比较神经生物学研究,8，12，15，16，17，18，19，20，21,12,15,16,1718,19,20的宝贵模型。此外，水生环境允许快速和容易接触化学品，以研究其对大脑功能在再生和成人平面，使他们成为一个受欢迎的系统神经毒理学22，23，24，25，26。22,23,24,25,26
平面主义者有三个不同的步态，称为滑翔、近位和皱。每个步态在特定情况下表现出来：滑翔是默认的步态，当硅体功能受损7，27,27时，出现坐相看，而皱逸是一种逃生步态——独立于西莉亚功能——以响应某些有害刺激7。我们已经表明，擦伤是一种特定的反应，由某些化学或物理线索的感觉引起，包括极端温度或pH值，机械损伤，或特定的化学诱导剂，因此不是一般的应激反应7,7，28，29。,29
由于其特异性和陈规定型参数，可以很容易地量化使用这个协议，皱起是一个强大的行为表型，使研究人员能够进行机械研究，解剖感官通路和行为25，28的神经元控制。此外，在神经毒理学研究22、24、25、30中，24,25皱力已被证明是测定对神经系统发育和功能的不良化学影响一个敏感终点30。由于几种不同的感官通路似乎汇合通过各种机制诱导皱起，皱起不同于其他平面行为，因为各种，但特定的刺激可以用来解剖不同的神经元电路，并研究如何集成不同的信号，以产生皱起表型。
重要的是，物种差异是存在的，其中一种化学物质可能在一个平面物种中引起皱起，但在另一种物种中引起不同的行为反应。例如，我们发现，阿南达米德诱导在平面物种杜格西亚贾波尼卡，但诱导在施密特亚地中海28的异位体。此示例强调了能够可靠地区分不同步态的重要性，因为它们是不同分子机制的表型表现。然而，使用定性观察数据很难区分与异形体分，因为两个步态都是肌肉驱动的，并且有质量相似性,7，28。因此，为了区分步态，有必要进行西莉亚成像或定量行为研究，允许基于特征参数7，28,进行区分。由于Cilia成像在实验中具有挑战性，需要像高放大倍数复合显微镜和高速照相机,7、28等专门7设备，因此研究人员不像定量行为分析那样广泛地获得这种成像。
在这里，我们提出了一个协议：（1）使用各种物理（有害温度，截肢，近UV光）和化学（甲基同位素（AITC），辛纳尔醛）刺激的诱导和（2）使用自由可用的软件对平面行为进行定量分析。通过量化四个参数（体长振荡的频率、相对速度、最大振幅和身体伸长和收缩的不对称度）7，可以区分滑翔、近位等文献中报告的其他行为状态，如蛇形运动15 或表皮15。此外，虽然在7种不同植物物种中保存，但每种物种都有其特有的频率和速度;因此，一旦确定一个物种的滑翔和皱逸速度，单靠速度就可以作为一种手段来区分滑翔和近体29。该协议假定没有事先接受计算图像分析或行为研究的培训，因此也可以在本科教学实验室环境中应用于平面行为实验。补充材料中提供了促进协议适应的示例数据。

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	<tbody>
		<tr>
			<td>Allyl isothiocyanate, 95% (AITC)</td>
			<td>Sigma-Aldrich</td>
			<td>377430-5G</td>
			<td>CAUTION:&#160; Flammable and acutely toxic; handle in a fume hood with appropriate PPE.</td>
		</tr>
		<tr>
			<td>Camera lens, 2/3 25mm F/1.4&#160;</td>
			<td>Tamron</td>
			<td>23FM25SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates, 6 well, tissue culture treated</td>
			<td>Genesee Scientific&#160;</td>
			<td>25-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL polypropylene, sterile</td>
			<td>MedSupply Partners</td>
			<td>62-1019-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cinnamaldehyde, &#62;95%</td>
			<td>Sigma-Aldrich</td>
			<td>W228613-100G-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimmable A4 LED Tracer Light Box</td>
			<td>Amazon</td>
			<td>B07HD631RP</td>
			<td></td>
		</tr>
		<tr>
			<td>Flea3 USB3 camera</td>
			<td>FLIR</td>
			<td>FL3-U3-13E4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat resistant gloves</td>
			<td>Fisher Scientific</td>
			<td>11-394-298</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>HP88854200</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean Sea Salt, prepared in deionized water</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td>Prepare in deionized water at 0.5 g/L.</td>
		</tr>
		<tr>
			<td>Montj&#252;ic salts, prepared in Milli-Q water</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td>Prepare in milli-Q water at 1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, 1.2 mM NaHCO3; adjust pH to 7.0 with HCl.</td>
		</tr>
		<tr>
			<td>Petri dishes, 100 mm x 20 mm, sterile polystyrene</td>
			<td>Simport</td>
			<td>D210-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, 20-200 &#956;L range</td>
			<td>Rainin</td>
			<td>17008652</td>
			<td></td>
		</tr>
		<tr>
			<td>PYREX 150 mL beaker</td>
			<td>Sigma-Aldrich</td>
			<td>CLS1000150</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade, 0.22 mm</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Roscolux color filter:&#160; Golden Amber</td>
			<td>Rosco</td>
			<td>R21</td>
			<td>Alternatively purchase the Roscolux Designer Color Selector (Musson Theatrical product #SBLUX0306) which includes all 3 color filters together.</td>
		</tr>
		<tr>
			<td>Roscolux color filter:&#160; Medium Red</td>
			<td>Rosco</td>
			<td>R27</td>
			<td></td>
		</tr>
		<tr>
			<td>Roscolux color filter:&#160; Storaro Red</td>
			<td>Rosco</td>
			<td>R2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Samco transfer pipette, 62 &#181;L large aperture</td>
			<td>Thermo Fisher</td>
			<td>691TS</td>
			<td></td>
		</tr>
		<tr>
			<td>Support stand&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-947-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>VWR</td>
			<td>89095-600</td>
			<td></td>
		</tr>
		<tr>
			<td>UV laser pointer</td>
			<td>Amazon</td>
			<td>B082DGS86R</td>
			<td>This is a Class II laser (405nm &#177;10nm) with output power &#60;5 mW.</td>
		</tr>
	</tbody>
</table>

planarian scrunching as a quantitative behavioral readout for noxious stimuli sensing

淡水平面学家通常平稳地滑过其腹侧的 cili 推进。然而，某些环境条件会诱发肌肉驱动的运动形式：peristalsis 或皱当。虽然近体病由阴性缺陷导致，但皱毛与硅酸功能无关，是对某些刺激（包括截肢、有害温度、极端 pH 和乙醇）的特定反应。因此，这两个肌肉驱动的步态是机械上不同的。然而，它们可能难以从质量上区分。在这里，我们提供了一个使用各种物理和化学刺激诱导擦伤的协议。我们详细介绍了皱起的定量特征，它可以用来区分它与近位和滑翔，使用免费可用的软件。由于皱当是一种普遍的平面步态，尽管具有特定物种的特征差异，因此，在适当考虑时，此协议可以广泛适用于所有植物学家物种。为了证明这一点，我们比较了行为研究中使用的两种最流行的平面物种， 杜格 西亚·贾波尼卡和施密特亚· 梅迪特拉内亚，对同一组物理和化学刺激的反应。此外，scrunching 的特异性允许将该协议与 RNA 干扰和/或药理暴露结合使用，以解剖所涉及的分子靶点和神经元回路，从而有可能为诺西感知和神经肌肉交流的重要方面提供机械洞察力。

外视近UV感知在S.地中海平面图是TRPA1依赖，并已被建议链接到H2O2版本17。由于H2O2暴露诱导TRPA1依赖性皱在S.地中海和D.japonica平面28，在第2.1.4节的步骤可用于测试近UV光暴露是否诱导在这两个物种的皱当。当D.japonica平面师在暴露在接近紫外线的光线下时，会皱着一下（10/10），而S.地中海平面图师...

Watch this Scientific Journal Video about Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing at JoVE.com

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

淡水平面学家表现出三个步态（滑翔、近位和皱），这些步态通过定量行为分析是可区分的。我们描述了一种利用各种有害刺激诱导擦伤的方法，其定量，以及从异形体和滑翔的区别。利用基因敲击，我们证明了皱当作为定量表型读出的特异性。

平面性皱毛作为有毒刺激感的定量行为读出

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

该协议允许研究人员通过将定量行为分析与分子和化学扰动相结合，阐明平面行为和这一行为背后的秒机制。这种技术很容易获得所有技能水平，因为它不需要先进的仪器或专门的软件，以获得定量行为读数的自由移动平面。皱当可用于研究平面体中的异常，并用作检测的敏感终点，破坏由异种生物和疾病引起的神经系统功能。首先，在平面上放置可调光 LED 面板，该面板将提供均匀的白色背景，并可用作可调光源以获得适当的对比度。在 LED 面板上放置一个 100 毫米的培养皿竞技场。将摄像机安装在竞技场上方的环形支架上，并根据需要调整其位置、高度和对焦，使整个竞技场以视野为中心并处于焦点位置。在竞技场上填充大约 25 毫升的适当曝光介质，以达到最大音量，打开 LED 面板并关闭可能对录制质量产生负面影响的所有其他光源。使用转移管道将平面在竞技场中心放置。在测试化学溶液时，将平面水与平面水进行尽可能少的转移，使化学的浓度没有显著变化。开始以本机斐济格式将数据记录为图像序列。对于滑翔实验，记录一到两分钟的滑翔行为，用于擦伤或近视实验，记录足够长的时间，以捕获至少三个连续的直线上发生的振荡。实验完成后，终止录制。如果平面达到竞技场的边界而不能满足终止标准，则移液器将平面器移回竞技场的中心。打开斐济实验的原始图像序列，将其转换为八位，并使用图像堆栈底部的箭头工具或滑块平移图像序列。要提取时间段和感兴趣的区域，请使用矩形工具绘制包含平面图的完整路径的感兴趣区域。右键单击图像堆栈并选择重复，选中重复堆栈的框。输入兴趣序列的第一帧和最后一帧，然后单击"确定"。对于滑翔实验，提取滑翔期，其中平面体移动至少两倍的身体长度，以进行皱擦或永久实验。提取一个实例，当平面体在直线上经历至少三个连续和完整的身体振荡。对重复的图像堆栈应用阈值，将图像二进制化，并从背景中提取平面图，调整滑动条，使整个平面图以红色突出显示。精确值取决于成像质量。保留深色背景框、堆叠直方图，并且不未选中重置范围。滚动浏览图像堆栈以确保良好的阈值范围，然后单击"应用"。在将堆栈转换为二进制窗口中，将方法设置为默认值，将背景设置为浅色。取消选中此窗口中的所有框，然后单击"确定"。将显示一个双化图像，显示白色背景上的黑色平面图。确保整个平面图在图像序列的所有帧中可见。通过单击分析和设置测量值来设置测量值。选中区域、质量中心、堆栈位置和拟合椭圆的框，然后单击"确定"。选择打开的图像堆栈并选择分析，并分析粒子。在分析粒子窗口中，选择"显示"和"蒙版"以打开一个新堆栈，显示使用所选参数检测到的所有对象。设置大小过滤器，通过输入平面图的近似区域来消除不必要的噪音。然后选中显示结果和清除结果的框，然后单击"确定"。使用面板底部的滑块平移蒙版图像堆栈，以确保没有平面图的杂色或帧。在结果窗口中，使用文件保存数据，并将 CSV 扩展名另存到文件名中，以将数据另存为逗号分隔值。保存图像堆栈的数据后，关闭相应的图像堆栈结果和蒙版窗口。为了通过有害温度引起擦伤，在热盘上将玻璃烧杯中的平面水加热到 65 摄氏度。将平面放在竞技场的中心，等待，直到它直立方向，并开始滑翔，然后开始录制。使用P 200移液器缓慢而一致地移液100微升的预热平面水，后咽到平面的尾端，以诱导皱。停止录制，一旦擦伤停止。将平面放在回收容器中，如果进行更多实验，则将介质与新鲜室温平面水交换。要通过截肢来诱导擦伤，请将一名平面者转移到竞技场的中心，然后等待，直到平面者直立方向并开始滑翔。然后开始录音，用干净的剃刀刀片截肢。确保切割位置在实验中保持一致。前片停止皱起后停止录制。取出这两块，将它们放在一个单独的容器中，并允许它们再生七天。截肢的平面师一旦再生，就可以重新融入家庭容器。该协议用于测试是否接近紫外线照射，诱导在S.mediterranea和D.japonica平面，而D.japonica平面，当暴露在接近紫外线光，S.mediterranea平面图要么显示尾部变薄或没有反应。D.japonica平面体至少表现出三条连续的直线皱子的皱擦参数的量化，揭示了该物种的特征皱擦参数在小鼠中与250微摩尔甲醛未知TRPA的一种角膜的对比暴露，导致皱毛和S.mediterranea。而D.japonica平面显示蛇一样和振荡运动的混合物被滑翔或有力的头部转弯打断。对至少连续三次振荡的样本进行定量，对四个参数中的三个值产生显著较低的值，而预期在 D.japonica 中，该参数的皱痕值显著降低，表明观察到的振荡运动没有擦伤。RNAi 确认了在 S.mediterranea 中对辛纳尔醛暴露的擦伤和反应的特异性。在180秒内暴露在平面水中，所有控制RNAi平面者皱着。与 Smtrpa 相比， 没有一个 Rnai 平面师证明 S. mediterranea 在肉桂醛中擦伤需要 Smtrpa 一个。必须保持一致，如何操纵动物，以减少噪音，并确保行为测量的可重复性。可以扩展此项目以包括体形分析，从而识别和量化此处未捕获的其他平面行为。

Research

JoVE Journal

Biology

Computer-Generated Animal Model Stimuli

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

科研

生物学

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

A Quantitative Fitness Analysis Workflow

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

FRET Imaging in Three-dimensional Hydrogels

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...