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环境污染物对斑马鱼幼虫的神经行为影响研究

Published: February 05, 2020
doi:
10.3791/60818
, , , ,
1State Key Laboratory of Marine Geology,Tongji University, 2Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering,Tongji University, 3Shanghai Collaborative Innovation Centre for WEEE Recycling,WEEE Research Center of 10 Shanghai Polytechnic University, 4Shanghai Institute of Pollution Control and Ecological Security

Summary

本文提出了利用斑马鱼幼虫模型评价环境污染物神经行为毒性的详细实验方案，包括暴露过程和神经行为指标测试。

Abstract

近年来，越来越多的环境污染物被证明是神经毒性的，特别是在生物的早期发展阶段。斑马鱼幼虫是环境污染物神经行为研究的杰出模型。这里提供了详细的实验方案，用于评估使用斑马鱼幼虫的环境污染物的神经毒性，包括胚胎的采集、暴露过程、神经行为指标、测试过程和数据分析。此外，还讨论了培养环境、暴露过程和实验条件，以确保测定的成功。该方案已应用于精神病药物的开发、环境神经毒性污染物的研究，并可进行优化，进行相应的研究，或有助于力学研究。该协议演示了研究神经行为对斑马鱼幼虫影响的明确操作过程，并可以揭示各种神经毒性物质或污染物的影响。

Introduction

近年来，越来越多的环境污染物被证明为神经毒性1，2，3，4。然而，评估接触环境污染物后体内的神经毒性，不如对内分泌干扰或发育毒性的评估那么容易。此外，及早接触污染物，特别是与环境有关的剂量，在毒性研究5、6、7、8中已引起越来越多的关注。

斑马鱼被确立为一种动物模型，适合在接触环境污染物后的早期发育过程中进行神经毒性研究。斑马鱼是脊椎动物，在受精后比其他物种发育得更快。幼虫不需要喂食，因为幼虫中的营养物质足以维持它们7天的受精后（dpf）9。Larvae从#2 dpf的chorion出来，并发展行为，如游泳和转弯，可以观察到，跟踪，量化和分析自动使用行为工具10，11，12，13开始3-4dpf 14，15，16，17，18。此外，高通量测试也可以通过行为工具实现。因此，斑马鱼幼虫是环境污染物神经行为研究的杰出模型。本文提供了一种利用高通量监测来研究在轻刺激下斑马鱼幼虫的神经行为毒性的协议。

我们的实验室研究了2，2’，4，4′-四溴二苯醚（BDE-47）20，21，6′-羟基/甲氧基-2，2’，4′-四溴二苯醚的神经行为毒性 苯醚（6-OH/MeO-BDE-47）22，使用所提出的协议，使用二苯醚（BDE-209）、铅和商业氯化石蜡23。许多实验室也使用该协议来研究其他污染物对幼虫或成年鱼24、25、26、27的神经行为影响。这种神经行为方案用于帮助提供机械支持，表明低剂量接触双酚A和替代双酚S诱导胚胎斑马鱼过早下丘脑神经发生27。此外，一些研究人员优化了该协议以执行相应的研究。最近的一项研究使用酪蛋白涂层金纳米颗粒（μCas AuNPs）在简单、高通量的斑马鱼模型中消除了淀粉样贝塔（A+）的毒性。研究表明，全身循环中的βCas AuNPs在斑马鱼幼虫血脑屏障和固网内A+42中转移，以非特异性、伴松状的方式引起毒性，行为病理学28支持。

运动、路径角度和社会活动是三种神经行为指标，用于研究斑马鱼幼虫在暴露于污染物后的神经毒性影响。运动是通过幼虫的游泳距离测量的，在接触污染物后可能会损坏。路径角度和社会活动与大脑和中枢神经系统的功能更密切相关。路径角度是指动物运动路径相对于游泳方向30的角度。系统中设置了八个角度等级，从 [-180]-±180°。为了简化比较，根据我们先前的研究21、22，最终结果中的六个类被定义为常规转弯（-10°+0°，0°[10]），平均转弯（-10°+-90°，±10°+90°），以及响应式转弯（-180°~-90°，[90°][90°]）。双鱼社会活动是群体性活动的基本行为;在这里，两个有效幼虫之间的距离为<0.5cm，被定义为社会接触。

这里提出的协议展示了研究对斑马鱼幼虫的神经行为影响的明确过程，并提供了一种揭示各种物质或污染物的神经毒性影响的方法。该协议将有利于研究环境污染物神经毒性的研究人员。

Protocol

该协议符合同济大学动物伦理委员会批准的准则。 1. 斑马鱼胚胎采集 在接触前一天晚上，将两对健康的成年图宾根斑马鱼放入产卵箱，使性别比例保持在1：1。 第二天天亮后30-60分钟将成年鱼移回系统。 将胚胎从产卵箱中取出。 用系统水冲洗胚胎。 将胚胎转移到玻璃培养皿（直径9厘米），并有足够的系统水。 在显微镜下观察胚…

Representative Results

在这里，我们描述了一个协议，用于研究在光刺激下使用斑马鱼幼虫的环境污染物的神经行为效应。在简介中定义了运动、路径角度和社交活动测试。运动和路径角度测试中微板的设置以及软件的图像如下所示。此外，我们自己的研究结果作为例子。两项研究提出了暴露于BDE-47和6-OH/MeO-BDE-47后的运动和路径角度效应。第三项研究介绍了四种商用氯化石蜡对社会行为的影响?…

Discussion

这项工作提供了一个详细的实验方案，以评估使用斑马鱼幼虫的环境污染物的神经毒性。斑马鱼在接触期间经历了从胚胎到幼虫的过程，这意味着对胚胎和幼虫的精心护理至关重要。任何影响胚胎和幼虫发育的东西都会影响最终结果。本文讨论了培养环境、暴露过程和实验条件，以确保整个测定的成功。

在养殖环境中，斑马鱼胚胎和幼虫生活在+28°C的稳定温度下。在这项工?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者感谢国家自然科学基金（21876135和21876136），中国国家重大科技项目（2017ZX07502003-03，2018ZX7701001-22），教育部-上海基金会的资助。儿童环境卫生重点实验室（CEH201807-5）和瑞典研究理事会（第639-2013-6913号）。

Materials

48-well-microplate Corning 3548 Embyros housing
6-well-microplate Corning 3471 Embyros housing
BDE-47 AccuStandard 5436-43-1 Pollutant
DMSO Sigma 67-68-5 Cosolvent
Microscope Olympus SZX 16 Observation instrument
Pipette Eppendorf 3120000267 Transfer solution
Zebrabox Viewpoint ZebraBox Behavior instrument
Zebrafish Shanghai FishBio Co., Ltd. Tubingen Zebrafish supplier
ZebraLab Viewpoint ZebraLab Behavior software
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Tags

Neurobehavioral EffectsEnvironmental PollutantsZebrafish LarvaeHigh Throughput TestingNeurotoxicityZebrafish GenomePsychotropic DrugsEnvironmental ToxicologyLocomotionPath AngleSocial BehaviorHigh Throughput Monitoring SoftwareTracking RotationsProtocol Parameters
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Citazione di questo articolo
Zhang, B., Yang, X., Zhao, J., Xu, T., Yin, D. Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae. J. Vis. Exp. (156), e60818, doi:10.3791/60818 (2020).
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