测量天然产品生物调节特性的平面运动分析
概要
与动物在泉水中的运动相比，单是植物运动用于测量天然产品的刺激和提取特性。
Abstract
描述一种直接、可控的方法，利用非寄生植物， 杜格西亚蒂格里纳，一种自由生活水生扁虫，来研究天然产品的兴奋剂和戒断特性。受益于平面生理学独特方面的实验分析已应用于伤口愈合、再生和肿瘤发生的研究。此外，由于平面学家对各种环境刺激表现出敏感性，并且能够学习和开发有条件的反应，因此它们可用于检查学习和记忆的行为研究。平面学家拥有基本的双边对称性和中枢神经系统，该系统使用神经递质系统，可以用于研究神经肌肉生物调节器的作用。因此，开发了监测平面运动和运动的实验系统，以检查物质成瘾和戒断。由于平面运动提供了一个敏感、易于标准化的动能检测系统来监测刺激作用的潜力，因此平面运动速度（pLmV）测试经过调整，通过确定动物与时间交叉的网格线数，来监测平面动物的刺激和戒断行为。在这里，对该技术及其应用进行演示和说明。
Introduction
所述协议使用平面运动提供一种评估自然物质的生物调节作用的手段。它经过专门调整，以确定这些物质是否作为兴奋剂，以及他们是否与可测量的戒断行为1相关。这种测定，称为平面运动速度（pLmV）测试，首次用于测试已知的药理剂2,2，3。这种基于平面运动性测定的应用自此越来越受欢迎，并已被对天然产品以外的物质感兴趣的不同实验室采用,。对于此测定，将平面放在含有泉水的培养皿中，或放入含有溶解生物调节剂的泉水。由于菜本身被放置在图形纸上，因此动物在容器上移动时与时间交叉的网格线数可用于确定每个条件下的移动速率。光/暗测试，也称为条件位置偏好测试（CPP），是监测平面运动性主题的另一个变化，并评估动物的反应和迁移到黑暗的环境6，7,的速度。也可以使用计算机程序和质量（COM）跟踪中心（8，9，10，11）,9,分析,平面运动的视频跟踪。
将平面图用作此类研究的动物模型与其他动物相比具有若干优势，因为实验者可以轻松控制检测环境。具体来说，在实验前使计划学家挨饿可以防止他们接触其他可能混淆结果的营养或药理剂，而所调查的特定生物调节器只需直接添加到培养水中，即可将特定生物调节剂引入规划者，从而标准化接触。由于平面动物有神经系统和神经递质，让人联想到”高阶”动物，这些动物对神经肌肉刺激的生理和实验反应被认为与其他,,生物体12、13、14、15、16,13,14有生物学上相关。15此外，由于平面学家在实验室中维护成本相对较低，因此为许多研究人员提供了一个可获取的生物模型。
作为一种实验动物，平面动物适合广泛的研究。例如，我们的小组，以及其他研究者使用平面学家来研究肿瘤发生17，18，19。,1917,平面学家还表现出对化学、热、引力、电、照片和磁刺激的一系列反应行为，这些行为构成了其他测定系统的基础。其中一些效应已用于研究这些动物的学习和记忆20，21，22，23，24，25，26，27。20,21,22,23,24,25,26,27目前文献中平面模型的主要用途是平面多能干细胞（称为新细胞）的活动及其在再生中的作用28、29、30。,29,30因此，采用此处描述的模型允许使用其他基于平面的测定进行进一步研究，以提供对天然产品和其他生物调节剂如何影响生物体的更广泛理解。
Protocol
1. 平面畜牧业 使用从生物供应公司购买的平面动物或野生捕获者（如果需要）。该协议中使用的平面学家是供应清单中列出的杜格西亚·蒂格里纳。这个物种也被称为吉拉迪亚蒂格里纳31。其他水生植物物种也是可以接受的2,2，3。注：所述协议适用于从生物供应公司购买的动物。这种检测系统尚未用野生捕获的…
Representative Results
在实验开始之前，应完成运行 pLmV 测定的实验室设置和工作空间的准备。这包括准备习惯容器，如果需要冲洗容器（用于提取实验），在层压网格纸上的培养皿，以及正确放置的相机（图1）。拍摄所有视频后，建议使用通用数据表来标准化调查人员之间的数据收集和演示（图 2 和 图 2 补充）。 摄像机设置应允许对平?…
Discussion
介绍了一种简单易用的平面活动测定，以确定天然产品的兴奋剂和戒断作用。作为行为模型，有必要对评分运动有严格的规程，并明确任何行为的定义，以标准化不同实验者之间的观察。提出的想法演示了如何实现这一点。使用该协议的每个实验室应调整所呈现的信息，以适应所测试的特定产品的效果。建议仔细设置工作空间，以确保参与研究的每个调查员在一致条件下进行测试（<strong class="xfig…
開示
The authors have nothing to disclose.
Acknowledgements
作者希望感谢机构促进办公室和莫里斯维尔学院基金会为支持这项工作而提供出版补助金，以及SUNY莫里斯维尔学院科技入门计划（CSTEP）对SUNY莫里斯维尔的本科研究的持续援助和支持。我们还要感谢索菲亚·赫钦斯对所述技术的有益评论。
Materials
| Bottled Water – 1 Gal. | Poland Spring | N/A | Spring water for planarian culture and to prepare solutions |
| Brown Planaria (Dugesia tigrina) | Carolina Biological Supply Company | 132954 | Brown planaria living (other species are acceptable) |
| Flat Paintbrush | Royal Crafter's Choice | 9159 | Flat watercolor paintbrushes for cleaning planarian culture containers |
| Glass Petri Dish – 10 cm | Kimax | N/A | 10 cm diameter (glass) Petri dishes for pLmV assay |
| Glass Petri Dish – 5 cm | Kimax | N/A | 5 cm and Petri dishes for rinsing planarians during withdrawal experiments and for stimulant habituation |
| Grid Paper | Any | N/A | Standard 0.5 cm grid paper for pLmV assay |
| iPEVO Visualizer (software) | iPEVO | https://www.ipevo.com/software/visualizer | Document camera software for video capture and recording |
| Metalware Set with Support Stand and Retort Ring | Any | N/A | Standard chemistry lab ring stand to hold a cell phone camera if used |
| Organic Egg | Any | N/A | Organic egg or beef liver for feeding planarains |
| Polycarbonate Bottle w/ Screw-on Cap – 10 mL | Beckman | N/A | Plastic vials to hold 5 to 10 mL volumes for stimulant habituation |
| Round Storage Container – 10 cm | Ziploc | N/A | 10 cm Round food storage containers for approximately 90 planarians or fewer |
| Round Water Paint Brush | LOEW-Cornell | N/A | Small round watercolor paint brushes (numbers 3 to 6) – soft |
| Transfer Pipette | Any | N/A | Wide bore (5 mL) plastic transfer pipettes to move planarians |
| USB Document Camera | iPEVO | CDVU-06IP | Document camera (or other camera or cell phone camera) |
参考文献
- Moustakas, D. Guarana provides additional stimulation over caffeine alone in the planarian model. PloS One. 10 (4), 0123310 (2015).
- Raffa, R. B., Valdez, J. M. Cocaine withdrawal in Planaria. European Journal of Pharmacology. 430 (1), 143-145 (2001).
- Raffa, R. B., Holland, L. J., Schulingkamp, R. J. Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint. Journal of Pharmacology and Toxicological Methods. 45 (3), 223-226 (2001).
- Thumé, I. S., Frizzo, M. E. Sertraline induces toxicity and behavioral alternations in planarians. Biomedical Research International. 2017, 5792621 (2017).
- Aggarwal, S., et al. Identification of a novel allosteric modulator of the human dopamine transporter. ACS Chemical Neuroscience. 10 (8), 3718-3730 (2019).
- Zhang, C., Tallarida, C. S., Raffa, R. B., Rawls, S. M. Sucrose produces withdrawal and dompamine-sensitive reinforcing effects in planarians. Physiology & Behavior. , 8-13 (2013).
- Zewde, A. M., et al. PLDT (planarian light/dark test): an invertebrate assay to quantify defensive responding and study anxiety-like effects. Journal of Neuroscience Methods. 293, 284-288 (2018).
- Risse, B., Otto, N., Berh, D., Jiang, X., Klämbt, C. FIM Imaging and FIMtrack: two new tools allowing high-throughput and cost effective locomotion analysis. Journal of Visualized Experiments. (94), e52207 (2014).
- Inoue, T., Hoshino, H., Yamashita, T., Shimoyama, S., Agata, K. Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function. Zoological Letters. 1, 7 (2015).
- Hastrom, D., Cochet-Escartin, O., Zhang, S., Khuu, C., Collins, E. M. S. Freshwater planarians as an alternative animal model for neurotoxicology. Toxicological Sciences. 147 (1), 270-285 (2015).
- Risse, B., Berh, D., Otto, N., Klämbt, C., Jiang, X. FIMtrack: an open source tracking and locomotion analysis software for small animals. PLoS One Computational Biology. 13 (5), 100553 (2017).
- Pagán, O. R. Planaria: an animal model that integrates development, regeneration and pharmacology. International Journal of Developmental Biology. 61, 519-529 (2017).
- Palladini, G. A pharmacological study of cocaine activity in planaria. Comparative Biochemistry and Physiology. 115 (1), 41-45 (1996).
- Buttarelli, F. R., Pellicano, C., Pontieri, F. E. Neuropharmacology and behavior in planarians: translation to mammals. Comparative Biochemistry and Physiology Part C. Toxicology & Pharmacology. 147 (4), 399-408 (2008).
- Nishimura, K., et al. Identification of glutamic acid decarboxylase gene and distribution of GABAergeric nervous system in the planarian Dugesia japonica. 神経科学. 153 (4), 1103-1114 (2008).
- Raffa, R. B., Rawls, S. M. . A model for drug action and abuse. , (2008).
- Hall, F., Morita, M., Best, J. B. neoplastic transformation in the planarian: I cocarcinogenesis and histopathology. The Journal of Experimental Zoology. 240 (2), 211-227 (1986).
- Voura, E. B., et al. Planarians as models of cadmium-induced neoplasia provide measurable benchmarks for mechanistic studies. Ecotoxicology and Environmental Safety. 142, 544-554 (2017).
- Van Roten, A., et al. A carcinogenic trigger to study the function of tumor suppressor genes in Schmedtea mediterranea. Disease Models and Mechanisms. 11 (9), 032573 (2018).
- Mason, P. R. Chemo-klino-kinesis in planarian food location. Animal Behaviour. 23 (2), 460-469 (1975).
- Van Huizen, A. V., et al. Weak magnetic fields alter stem cell-mediated growth. Science Advances. 5 (1), 7201 (2019).
- Brown, H. M., Ogden, T. E. The electrical response of the planarian ocellus. Journal of General Physiology. 51 (2), 255-260 (1968).
- Inoue, T., Yamashita, T., Agata, K. Thermosensory signaling by TRPM is processed by brain serotonergic neurons to produce planarian thermotaxis. The Journal of Neuroscience. 34 (47), 15701-15714 (2014).
- Byrne, T. Effects of ethanol on negative phototaxis and motility in brown planarians (Dugesia tigrina). Neuroscience Letters. 685, 102-108 (2018).
- de Sousa, N., et al. Transcriptomic analysis of planarians under simulated microgravity or 8g demonstrates that alteration of gravity induces genomic and cellular alterations that could facilitate tumoral transformation. International Journal of Molecular Sciences. 20 (3), 720 (2019).
- Best, J. B., Rubinstein, I. Maze learning and associated behavior in planaria. Journal of Comparative and Physiological Psychology. 55, 560-566 (1962).
- Shomrat, T., Levin, M. An automated training paradigm reveals long-term memory in planarians and its persistence through head regeneration. The Journal Experimental Biology. 216, 3799-3810 (2013).
- Robarts-Galbraith, R. H., Newmark, P. A. On the organ trail: insights into organ regeneration in the planarian. Current Opinion in Genetics & Development. 32, 37-46 (2015).
- Ivancovic, M., et al. Model systems for regeneration: planarians. Development. 146 (17), 167684 (2019).
- Herath, S., Lobo, D. Cross-inhibition of Turing patterns explains the self-organized regulatory mechanism of planarian fission. Journal of Theoretical Biology. 485, 110042 (2019).
- Itoh, M. T., Shinozawa, T., Sumi, Y. Circadian rhythms of melatonin-synthesizing enzyme activities and melatonin levels in planarians. Brain Research. 830 (1), 165-173 (1999).
- Itoh, M. T., Igarashi, J. Circadian rhythm of serotonin levels in planarians. Neuroreports. 11 (3), 473-476 (2000).
- Hinrichsen, R. D., et al. Photosensitivity and motility in planarian Schmedtea mediterranea vary diurnally. Chronobiology International. 36 (12), 1789-1793 (2019).
- Raffa, R. B., Desai, P. Description and quantification of cocaine withdrawal signs in planaria. Brain Research. 1032 (1-2), 200-202 (2005).
- Pagán, O. R., et al. A cembranoid from tobacco prevents the expression of induced withdrawal behavior in planarian worms. European Journal of Pharmacology. 615 (1-3), 118-124 (2009).
- Rawls, S. M., Patil, T., Yuvasheva, E., Raffa, R. B. First evidence that drugs of abuse produce behavioral sensitization and cross-sensitization in planarians. Behavioural Pharmacology. 21 (4), 301-313 (2010).
- Venturini, G., et al. A pharmacological study of dopaminergic receptors in planaria. Neuropharmacology. 28 (12), 1377-1382 (1989).
- Ouyang, K., et al. Behavioral effects of Spenda, Equal and sucrose: Clues from planarians on sweeteners. Neuroscience Letters. 636, 213-217 (2017).
- Pagán, O. R., Montgomery, E., Deats, S., Bach, D., Baker, D. Evidence of nicotine-induced, curare-sensitive, behavior in planarians. Neurochemical Research. 40 (10), 2087-2090 (2015).
- Shibata, N., Agata, K. RNA interference in planarians: feeding and injection of synthetic dsRNA. Methods in Molecular Biology. 1774, 455-466 (2018).
- Pagán, O. R., et al. Reversal of cocaine-induced planarian behavior by parthenolide and related sesquiterpene lactones. Pharmacology Biochemistry and Behavior. 89 (2), 160-170 (2008).
- Vouga, A., et al. Stereochemistry and neuropharmacology of a ‘bath salt’ cathinone: S-enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates. Neuropharmacology. 91, 109-116 (2015).
- Chan, J. D., Marchant, J. S. Pharmacological and functional genetic assays to manipulate regeneration of the planarian Dugesia japonica. Journal of Visualized Experiments. (54), e3058 (2011).
