斑马鱼内生应激研究的行为方法
Summary
这份手稿描述了一种测量成年斑马鱼压力行为的简单方法。该方法利用了斑马鱼在压力状态下更喜欢鱼缸的下半部分的固有倾向。我们还描述了将检测与药理学结合的方法。
Abstract
适当地应对有压力的刺激是生物体生存所必需的。对广泛的与压力有关的疾病和精神障碍进行了广泛的研究, 但仍需进一步研究压力的遗传和神经元调节, 以开发更好的疗法。斑马鱼提供了一个强大的遗传模型来研究神经的基础的压力, 因为有大量的突变和转基因线的集合。此外, 药理可以很容易地应用于斑马鱼, 因为大多数药物可以直接添加到水中。我们在这里描述了使用 “新的坦克试验” 作为一种方法来研究斑马鱼的先天应激反应, 并演示如何使用该分析验证潜在的抗焦虑药物。该方法可以很容易地与携带基因突变的斑马鱼系, 或使用转基因方法操作精确神经电路的斑马鱼系结合在一起。该检测方法也可用于其他鱼类模型。总之, 所描述的协议应有助于其他实验室采用这种简单的检测方法。
Introduction
应激反应是潜在有害或厌恶性刺激所导致的行为和生理状态的改变。压力反应在整个动物王国是保守的, 对生物体的生存至关重要.几十年的研究极大地扩大了我们对压力状态背后的一些遗传和神经元机制的了解。今天, 大脑的区域, 如杏仁核和纹状体 2, 和遗传因素,如促肾上腺皮质激素释放激素(crh), 糖皮质激素(gr) 和矿物皮质激素受体(先生) 已经被广泛研究了3,4,5,6。尽管有这些重要发现, 但关于压力的遗传和神经元调节, 仍有许多未知数。因此, 许多与压力有关的疾病缺乏治疗方法。
遗传可修正的模型生物为研究行为的遗传和神经元控制提供了一个有用的工具。特别是鱼类模型的威力非常大: 它们是生成时间短的小生物, 在实验室环境中使用它们很容易, 它们的基因组很容易改变, 作为脊椎动物, 它们不仅具有遗传特性, 而且还具有神经解剖性同源与他们的哺乳动物同行7,8。测量压力的标准检测可以与携带基因突变的斑马鱼系搭配, 也可以与那些可以操纵精确神经元子集的斑马鱼系配对, 并且可以快速有效地评估单个基因或定义神经元的影响。
在行为上, 应激反应可以描述为活动过度活跃或长时间不活动 (类似于 “冷冻”)9, 减少勘探 10, 快速呼吸, 减少食物摄入11, 和对储罐12 底部的首选。例如, 当放入一个不熟悉的鱼缸时, 成年斑马鱼和其他小鱼模型显示出对鱼缸下半部分的最初偏好, 然而, 随着时间的推移, 鱼开始以接近相等的频率12探索顶部和底部的一半。成人治疗已知的减少焦虑的药物会导致鱼类立即探索上半部分10,13.相反, 增加焦虑的药物会导致鱼类对鱼缸的下半部分表现出强烈的偏好,1 2、1 4、1 5.因此, 减少对储罐下半部分的勘探和偏好是简单可靠的应力指标。
与大多数脊椎动物一样, 鱼类的应激反应是由激活下丘脑-垂体-肾间轴 (hpi; 类似于哺乳动物的下丘脑-垂体-肾上腺轴) 14,16.下丘脑神经元表达激素促肾上腺皮质激素释放激素 (CRH) 信号的垂体, 这反过来又释放肾上腺皮质激素释放激素 (促肾上腺皮质激素)。促肾上腺皮质激素然后信号到肾间腺产生和分泌皮质醇, 它有许多下游目标 16, 其中之一是产生低温神经元 3,17的负反馈, 18,19。
在这里, 我们描述了一种评估先天压力行为度量的方法。对于这种行为, 我们详细介绍了使用新的坦克潜水试验12,14的协议。然后, 我们证明, 作为一个例子, 一个已知的抗焦虑药物, 布西酮, 减少压力的行为措施。
Protocol
该协议已获得佛罗里达大西洋大学动物护理和使用机构委员会的批准。 1. 准备工作 指定一个用于进行行为研究的隔离室, 或关闭房间的一部分, 使其被隔离。注: 房间应不受干扰, 交通流量较低, 以避免干扰鱼的正常行为。 将下列材料和设备移入行为室: (一) 相机和镜头, (二) 可连接镜头的红外滤光片, (三) 相机支架, (四) 带有相机采集软件的计算机, (五) 可靠?…
Representative Results
研究斑马鱼的应激为了观察野生斑马鱼在一段时间内的应激行为, 我们在新的储罐试验中测试了来自 AB 菌株24的成年鱼。AB 成年人受上述协议的影响。简单地说, 鱼在行为室的水箱里有1小时的适应期。一个人被放在烧杯里 1 0分钟, 然后轻轻地放在一个不熟悉的水箱 (新的水箱) 里, 里面装满了新鲜的系统水。记录了10分钟的昏迷活动, 并使用商业上…
Discussion
斑马鱼在一个新的鱼缸中表现出强烈的应激反应
在这里, 我们描述了一种简单的行为方法来检查成年斑马鱼的应激反应, 并验证该方法作为一个简单的测量压力的药理学。
这种新型的储罐试验是一种广泛使用的试验, 用于检查斑马鱼和其他种类的 12、14、21、35、<s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了佛罗里达大西洋大学木星生命科学倡议向 ERD 和 ACK 提供的资金支持。这项工作还得到了国家卫生研究院 R21NS105071 (颁发给 ACK 和 ERD) 和 R15MH118625 (颁发给 ERD) 赠款的支持。
Materials
| Camera | We use Point Grey Grasshopper3 USB camera with lens from Edmund Optics. | ||
| Infrared filter | Edmund Optics | ||
| Video Acquisition Program | Use programs such as Virtualdub or FlyCapture because the acquisition framerate can be set. | ||
| Infrared LED lights | |||
| Assay tank | Aquaneering | Part number ZT180 | Size: M3 1.8 liter |
| Stand and clamp, or standard tripod for camera | |||
| 250mL beaker | |||
| Tracking software | We use Ethovision XT 13 from Noldus Information Technology | ||
| Buspirone chloride | Sigma-Aldrich | B7148 | |
| Randomized trial generator | We use the RANDBETWEEN function in Microsoft Excel |
References
- McEwen, B. S. Stress, adaptation, and disease. Allostasis and allostatic load. Annals of the New York Academy of Sciences. 840, 33-44 (1998).
- Tovote, P., Fadok, J. P., Lüthi, A. Neuronal circuits for fear and anxiety. Nature Reviews Neuroscience. 16 (6), 317-331 (2015).
- Facchinello, N., et al. Nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor. Scientific Reports. 7 (4371), (2017).
- Ziv, L., et al. An affective disorder in zebrafish with mutation of the glucocorticoid receptor. Molecular Psychiatry. , (2013).
- Grone, B. P., Maruska, K. P. Divergent evolution of two corticotropin-releasing hormone (CRH) genes in teleost fishes. Frontiers in Neuroscience. , (2015).
- Fuller, P. J., Lim-Tio, S. S., Brennan, F. E. Specificity in mineralocorticoid versus glucocorticoid action. Kidney International. , (2000).
- Zhdanova, I. V. Sleep and its regulation in zebrafish. Reviews in the Neurosciences. 22 (1), 27-36 (2011).
- Patton, E. E., Zon, L. I. The art and design of genetic screens: zebrafish. Nature Reviews Genetics. , (2001).
- Duboué, E. R. E. R., Hong, E., Eldred, K. C. K. C., Halpern, M. E. M. E. Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish. Current Biology. 27 (14), 2154-2162 (2017).
- Facchin, L., Duboue, E. R., Halpern, M. E. Disruption of Epithalamic Left-Right Asymmetry Increases Anxiety in Zebrafish. Journal of Neuroscience. 35 (48), 15847-15859 (2015).
- Øverli, &. #. 2. 1. 6. ;., Sørensen, C., Nilsson, G. E. Behavioral indicators of stress-coping style in rainbow trout: Do males and females react differently to novelty. Physiology and Behavior. , (2006).
- Levin, E. D., Bencan, Z., Cerutti, D. T. Anxiolytic effects of nicotine in zebrafish. Physiology & behavior. 90 (1), 54-58 (2007).
- Bencan, Z., Sledge, D., Levin, E. D. Buspirone, chlordiazepoxide and diazepam effects in a zebrafish model of anxiety. Pharmacology Biochemistry and Behavior. 94 (1), 75-80 (2009).
- Cachat, J., et al. Measuring behavioral and endocrine responses to novelty stress in adult zebrafish. Nature Protocols. 5 (11), 1786-1799 (2010).
- Mathuru, A. S., et al. Chondroitin fragments are odorants that trigger fear behavior in fish. Current Biology. , (2012).
- Alsop, D., Vijayan, M. The zebrafish stress axis: Molecular fallout from the teleost-specific genome duplication event. General and Comparative Endocrinology. , (2009).
- Evans, A. N., Liu, Y., MacGregor, R., Huang, V., Aguilera, G. Regulation of Hypothalamic Corticotropin-Releasing Hormone Transcription by Elevated Glucocorticoids. Molecular Endocrinology. , (2013).
- Fenoglio, K. A., Brunson, K. L., Avishai-Eliner, S., Chen, Y., Baram, T. Z. Region-specific onset of handling-induced changes in corticotropin- releasing factor and glucocorticoid receptor expression. Endocrinology. , (2004).
- Liposits, Z., et al. Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF). Histochemistry. , (1987).
- Facchin, L., Duboué, E. R., Halpern, M. E. Disruption of epithalamic left-right asymmetry increases anxiety in Zebrafish. Journal of Neuroscience. 35 (48), (2015).
- Chin, J. S., et al. Convergence on reduced stress behavior in the Mexican blind cavefish. Developmental Biology. , (2018).
- Wong, K., et al. Analyzing habituation responses to novelty in zebrafish (Danio rerio). Behavioural Brain Research. 208 (2), 450-457 (2010).
- Matsunaga, W., Watanabe, E. Habituation of medaka (Oryzias latipes) demonstrated by open-field testing. Behavioural Processes. 85 (2), 142-150 (2010).
- Walker, C. Chapter 3 Haploid Screens and Gamma-Ray Mutagenesis. Methods in Cell Biology. , (1998).
- Rihel, J., et al. Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science. 327, 348-351 (2010).
- Peal, D. S., Peterson, R. T., Milan, D. Small molecule screening in zebrafish. Journal of Cardiovascular Translational Research. , (2010).
- Murphey, R., Zon, L. Small molecule screening in the zebrafish. Methods. 39 (3), 255-261 (2006).
- Gammans, R. E., et al. Use of buspirone in patients with generalized anxiety disorder and coexisting depressive symptoms. A meta-analysis of eight randomized, controlled studies. Neuropsychobiology. 25 (4), 193-201 (1992).
- Maaswinkel, H., Zhu, L., Weng, W. The immediate and the delayed effects of buspirone on zebrafish (Danio rerio) in an open field test: A 3-D approach. Behavioural Brain Research. , (2012).
- Gebauer, D. L., et al. Effects of anxiolytics in zebrafish: Similarities and differences between benzodiazepines, buspirone and ethanol. Pharmacology Biochemistry and Behavior. , (2011).
- Maximino, C., et al. Fingerprinting of psychoactive drugs in zebrafish anxiety-like behaviors. PLoS ONE. , (2014).
- Horváth, J., Barkóczi, B., Müller, G., Szegedi, V. Anxious and nonanxious mice show similar hippocampal sensory evoked oscillations under urethane anesthesia: Difference in the effect of buspirone. Neural Plasticity. , (2015).
- Costall, B., Kelly, M. E., Naylor, R. J., Onaivi, E. S. Actions of buspirone in a putative model of anxiety in the mouse. Pharm Pharmacol. 40 (7), 494-500 (1988).
- Barros, M., Mello, E. L., Huston, J. P., Tomaz, C. Behavioral effects of buspirone in the marmoset employing a predator confrontation test of fear and anxiety. Pharmacology Biochemistry and Behavior. , (2001).
- Heinen-Kay, J. L., et al. Predicting multifarious behavioural divergence in the wild. Animal Behaviour. 121, 3-10 (2016).
- Thompson, R. R. J., Paul, E. S., Radford, A. N., Purser, J., Mendl, M. Routine handling methods affect behaviour of three-spined sticklebacks in a novel test of anxiety. Behavioural Brain Research. 306, 26-35 (2016).
- Hamilton, T. J., et al. Establishing zebrafish as a model to study the anxiolytic effects of scopolamine. Scientific Reports. , (2017).
- York, R. A., Fernald, R. D. The Repeated Evolution of Behavior. Frontiers in Ecology and Evolution. 4, 143 (2017).
- Jakka, N. M., Rao, T. G., Rao, J. V. Locomotor behavioral response of mosquitofish (Gambusia affinis) to subacute mercury stress monitored by video tracking system. Drug and Chemical Toxicology. , (2007).
- Hu, C. K., Brunet, A. The African turquoise killifish: A research organism to study vertebrate aging and diapause. Aging Cell. , (2018).
- Maximino, C., et al. Measuring anxiety in zebrafish: A critical review. Behavioural Brain Research. 214 (2), 157-171 (2010).
- Maximino, C., Marques de Brito, T., Dias, C. A. G., Gouveia, A., Morato, S. Scototaxis as anxiety-like behavior in fish. Nature protocols. 5 (2), 209-216 (2010).
- Godwin, J., Sawyer, S., Perrin, F., Oxendine, S., Kezios, Z. Adapting the Open Field Test to assess anxiety related behavior in zebrafish. Zebrafish Protocols for Neurobehavioral Research. , 181-189 (2012).
- Agetsuma, M., et al. The habenula is crucial for experience-dependent modification of fear responses in zebrafish. Nature Neuroscience. 13 (11), 1354-1356 (2010).
- Valente, A., Huang, K. H., Portugues, R., Engert, F. Ontogeny of classical and operant learning behaviors in zebrafish. Learning and Memory. , (2012).
- Baker, M. R., Goodman, A. C., Santo, J. B., Wong, R. Y. Repeatability and reliability of exploratory behavior in proactive and reactive zebrafish, Danio rerio. Scientific Reports. , (2018).
- Friedrich, R. W., Genoud, C., Wanner, A. A. Analyzing the structure and function of neuronal circuits in zebrafish. Frontiers in Neural Circuits. , 7 (2013).
- Friedrich, R. W., Jacobson, G. A., Zhu, P. Circuit Neuroscience in Zebrafish. Current Biology. 20 (8), (2010).
- Marquart, G. D., et al. A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies. Frontiers in Neural Circuits. , (2015).
- Randlett, O., et al. Whole-brain activity mapping onto a zebrafish brain atlas. Nature Methods. 12 (11), 1039-1046 (2015).
- Gupta, T., et al. Morphometric analysis and neuroanatomical mapping of the zebrafish brain. Methods. 1046 (18), 30011-30012 (2018).
- Marquart, G. D., et al. High-precision registration between zebrafish brain atlases using symmetric diffeomorphic normalization. GigaScience. , (2017).
- Ronneberger, O., et al. ViBE-Z: A framework for 3D virtual colocalization analysis in zebrafish larval brains. Nature Methods. , (2012).
- Subedi, A., et al. Adoption of the Q transcriptional regulatory system for zebrafish transgenesis. Methods. 66 (3), 433-440 (2014).
- Scheer, N., Campos-Ortega, J. A. Use of the Gal4-UAS technique for targeted gene expression in the zebrafish. Mechanisms of Development. 80 (2), 153-158 (1999).
- Chatterjee, D., Tran, S., Shams, S., Gerlai, R. A Simple Method for Immunohistochemical Staining of Zebrafish Brain Sections for c-fos Protein Expression. Zebrafish. , (2015).
Cite This Article
Chin, J. S., Albert, L. T., Loomis, C. L., Keene, A. C., Duboué, E. R. Behavioral Approaches to Studying Innate Stress in Zebrafish. J. Vis. Exp. (147), e59092, doi:10.3791/59092 (2019).