Name: studying neurobehavioral effects of environmental pollutants on zebrafish larvae
Uploaded: 2020-02-05T15:00:00
Description: Watch this Scientific Journal Video about Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae at JoVE.com

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）。

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
<ol><li>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。</li>
	<li>第二天天亮后30-60分钟将成年鱼移回系统。</li>
	<li>将胚胎从产卵箱中取出。</li>
	<li>用系统水冲洗胚胎。</li>
	<li>将胚胎转移到玻璃培养皿（直径9厘米），并有足够的系统水。</li>
	<li>在显微镜下观察胚...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48-well-microplate</td>
			<td>Corning</td>
			<td>3548</td>
			<td>Embyros housing</td>
		</tr>
		<tr>
			<td>6-well-microplate</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Embyros housing</td>
		</tr>
		<tr>
			<td>BDE-47</td>
			<td>AccuStandard</td>
			<td>5436-43-1</td>
			<td>Pollutant</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>67-68-5</td>
			<td>Cosolvent</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>SZX 16</td>
			<td>Observation instrument</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3120000267</td>
			<td>Transfer solution</td>
		</tr>
		<tr>
			<td>Zebrabox</td>
			<td>Viewpoint</td>
			<td>ZebraBox</td>
			<td>Behavior instrument</td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>Shanghai FishBio Co., Ltd.</td>
			<td>Tubingen</td>
			<td>Zebrafish supplier</td>
		</tr>
		<tr>
			<td>ZebraLab</td>
			<td>Viewpoint</td>
			<td>ZebraLab</td>
			<td>Behavior software</td>
		</tr>
	</tbody>
</table>

studying neurobehavioral effects of environmental pollutants on zebrafish larvae

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。

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?...

Watch this Scientific Journal Video about Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae at JoVE.com

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

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。

环境污染物对斑马鱼幼虫的神经行为影响研究

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

我们的协议为研究这些物质和感兴趣的污染物对斑马鱼幼虫的神经行为影响以及揭示这些制剂的潜在神经毒性提供了一个清晰的过程。该技术可以使用斑马鱼模型中的神经行为的高通量测试进行。该方法可用于评价污染物的等位神经行为效应，以证明潜在的神经毒性，因为斑马鱼基因组与人类具有很高的同源性，因此可以为污染物引起的化学管理和公共卫生问题提供指示。由于斑马鱼具有高通量筛选的优势，该方法为精神药物的发展和环境毒理学研究提供了深入的见解。不要因为研究第一个实验而害怕。如果您遵循协议，您将有一个成功的结果。在进行实验前至少两小时，将室温设定为 28 摄氏度，并在 48 孔微孔的每个孔中添加 800 微升的暴露溶液。然后，使用带改性尖端的一毫升移液器将 200 微升暴露溶液中的一个幼虫转移到微孔板的每个孔中，以准备运动和路径角度实验。为了准备一个六孔微孔板用于社会行为实验，在六孔微孔板的每个井中加入四毫升的暴露溶液，并将两个幼虫在200微升的暴露溶液中转移到每口井中。在开始测试之前，打开高吞吐量监控软件程序并选择跟踪旋转路径角度模块。将 48 孔微孔板转移到记录平台并向下拉盖。单击"文件和生成协议"开始生成新协议，然后输入 48 到位置计数位置。单击参数、协议参数和时间，将实验持续时间设置为 1 小时 10 分钟，将集成周期设置为 60 秒。使用椭圆形形状工具，在左上角周围画一个圆圈。选择圆圈，然后单击"复制"、"右上标记"、"粘贴"和"选择"。使用鼠标将复制的圆圈拖到右上，然后单击"复制"、"右上标记"、"粘贴"和"再次选择"。然后，将复制的圆圈拖到右下角，然后单击"生成"和"清除标记"。系统会自动在盘子的所有其他井上画一个圆圈。接下来，单击"绘制比例"并在屏幕上绘制校准线。输入行的长度，设置单位，然后单击"应用于组"。将动物颜色设置为黑色，将检测阈值设置为 16 到 18，以便检测动物，将非活动速度设置为小速度为小速度，将小到大速度设置为 2.5 厘米/秒。如前所述，将路径角度类从负 180 设置为正 180。要设置光条件，请单击"参数"，"光驾驶"，使用下面的 3 种触发方法之一，并增强刺激以设置光条件。单击"边缘"并设置 10 分钟的暗周期，然后设置三个周期的交替 10 分钟光和暗周期。然后，保存协议。设置所有参数后，请关闭测试室中的照明，让幼虫在系统中适应 10 分钟。在适应期结束时，单击"实验和执行"，然后创建要保存实验文件的文件夹。然后，输入结果名称并单击"背景"和"开始"。在实验结束时，单击"实验"和"停止"。然后将 48 孔微孔板放回光培养箱进行后续实验。由于每组可以包括 16 只动物，因此该系统可用于在单个 48 孔微孔板的不同处理条件下对运动和路径角度进行高通量测试。幼虫的社会活动可以使用六孔板进行测试。在这个具有代表性的实验中，BDE-47的最高浓度组在黑暗时期表现出明显的低活性，而在光时期观察到BDE-47暴露导致的变化。此外，高浓度组6-羟基-BDE-47在受精后五天进行较少的常规和平均转弯。然而，在6-甲氧BDE47接触组中，诱导了反应更多的转弯。斑马鱼的社会活动受到氯化石蜡-70和短链氯化石蜡-52b的刺激，而长链氯化石蜡-52a缩短了幼虫接触的持续时间。重要的是要确保被转移的幼虫的测试应该有一个正常的游泳能力，没有畸形。其他变量，如颜色偏好，浅暗偏好，所有黑暗，斑马鱼的尾巴运动，可以包括回答有关环境污染物的神经行为毒性的其他问题。BDE-47 暴露溶液具有神经毒性，具有内分泌中断潜力，因此一定要避免直接接触皮肤。

Research

JoVE Journal

Environment

Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface

在探索大气蒸发强迫的影响：大气边界层和浅地层的实验整合

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study ...

科研

环境科学

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

对水生生命值评价悬浮泥沙影响的流通系统曝光

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

从个体到群体度评价环境化学物质对蜜蜂发展的影响

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

用于研究铁 (氧) 氢氧化物、微量元素和细菌之间厌氧生物地球化学相互作用的实验柱装置

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

污水灌溉对土壤水力电导率的影响: 耦合场取样与饱和水力电导率的实验室测定

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

研究矿物对有机热液转化影响的实验协议

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

在体外显示砷对皮肤细胞迁移影响的划痕测定

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...