Name: ros live cell imaging during neuronal development
Uploaded: 2021-02-09T14:30:00
Description: Watch this Scientific Journal Video about ROS Live Cell Imaging During Neuronal Development at JoVE.com

这项工作得到了国家卫生研究院（Grant R01NS1117701）、国家科学基金会（第1146944-IOS格兰特）、印第安纳创伤脊髓和脑损伤研究基金（格兰特20000289）、普渡研究基金会（格兰特·209911）和普渡大学研究与伙伴关系执行副总裁办公室（格兰特·210362）的支持。我们感谢科里·韦弗博士和海莉·罗德博士建立斑马鱼RGC文化协议。我们感谢海莉·罗德提供图4的数据。我们感谢利亚比亚西和肯尼 · 恩古恩对 Rgc 文化的帮助。我们感谢李将军编辑了文本。我们感谢托比亚斯·迪克博士为含有roGFP2-Orp1的PCS2+载体提供roGFP2-Orp1和邓青博士。图 2 是用 Biorender.com 创建的。

所有动物实验均经过普渡动物护理和使用委员会 （PACUC） 的合乎道德的审查和批准，遵循 NIH 指南，协议2006002050 2020 年 7 月 24 日获得批准。
1. 准备解决方案
<ol><li>E2 介质 （1x）<ol>
<li>通过将 表1 中显示的所有组件组合在一起，准备 100x E2A （500 mL）、500x E2B （100 mL） 和 500x E2C （100 mL） 解决方案。高压灭菌网、E2B 和 E2C 解决方案。存储在4°C。</li>
		<li>对于 1x E2...

<ol>
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有几个关键步骤需要在整个协议中引起注意。我们相信，考虑到这些点将改善实验流。对于初级 RGC 培养，ZFCM（-） 的不育性非常重要，因为这种培养介质不含抗生素，在成像之前或成像期间可能发生污染。为了避免这种情况，我们建议仅在生物安全柜内准备和使用 ZFCM（-），并定期制作新鲜的 ZFCM（-） 介质（步骤 1.5）。此外，层压素库存应保持在-80°C。 解冻的层蛋白应随时储存在 4 °C。不要?...

活性氧物种（ROS）信号调节神经系统的发展和功能1。一个重要的细胞ROS来源是NADPH氧化酶（NOX），这是产生超氧化物和过氧化氢（H 2 O2）2的跨膜蛋白。NOX酶存在于整个中枢神经系统（CNS）中，NOX衍生的ROS有助于神经元发育3、4、5、6。神经干细胞的维持和分化，建立神经元极性，中性生长，突触可塑性已被证明需要足够的ROS 7，8，9，10，11水平。另一方面，NOXs不受控制地生产ROS会导致神经退行性疾病，包括阿尔茨海默氏症、多发性硬化症和创伤性脑损伤12、13、14。因此，生产生理相关的ROS对于维持健康状况至关重要。
基因编码生物传感器的开发极大地促进了细胞ROS的检测。基因编码生物传感器的一个重要优点是 ROS 信号的时间和空间分辨率增加，因为这些传感器可以专门针对不同的位置。对氧化物敏感的GFP（roGFP）是一种类型的ROS生物传感器。roGFP2-Orp1变种通过其Orp1域专门检测H2O2，这是一种来自酵母15，16的谷胱甘肽过氧化物类家庭蛋白。Orp1蛋白的氧化被转移到roGFP2，以改变其构象（图1A）。探测器在405纳米和480纳米附近显示两个兴奋峰，在515纳米处显示一个单个发射峰值。氧化后，兴奋峰周围的荧光强度会发生变化：当405 nm兴奋增加时，480纳米激发会减少。因此，roGFP2-Orp1 是一种比值生物传感器，H2O2-水平通过两个不同波长的荧光增量（图 1B）的兴奋度比率检测到。总的来说，roGFP2-Orp1 是 ROS 成像的多功能工具，可在体内高效使用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62165/62165fig01.jpg"> 图1：roGFP2-Orp1. （A） 氧化剂转移的示意图表示和激发光谱发生在 Orp1 和 roGFP2 之间，以响应 H2O2，导致 roGFP2 的构象变化。（B） roGFP2-Orp1的激发光谱显示两个兴奋峰值在405 nm和480 nm和单个排放峰值在515 nm。H2O2氧化后，405 nm 激发增加，而 480 nm 激发减少。这会导致 H 2O 2存在的比例读出。该数字已从比兰和贝卢索夫（2017）16和摩根等人（2011）25修改。<a href="https://www.jove.com/files/ftp_upload/62165/62165fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
达尼奥雷里奥（斑马鱼）模型系统在应用基因编码生物传感器方面有几个优势。胚胎和幼虫的光学透明度使体内成像具有非侵入性。新的成像工具正在开发中，以实现更高的分辨率和更深的渗透17。此外，还有既定的基因操纵工具（异位mRNA表达，托尔2转基因等）和基因组编辑（塔伦斯，CRISPR/Cas9等），促进转基因动物的生成18。随着斑马鱼胚胎在母体之外发育，该系统进一步允许更容易地获取和操作胚胎。例如，单细胞阶段注射和药物治疗很容易做到。
在这里，我们使用斑马鱼通过注射体外转录的mRNA来暂时表达H2O2特异性生物传感器roGFP2-Orp1。这些胚胎可用于培养神经元的 体外 成像和 体内 成像（图2）。我们描述了从斑马鱼胚胎解剖和电镀视网膜结节细胞（RGCs）的协议，然后评估培养神经元的H2O2水平。然后，我们提出了一种利用共聚焦显微镜对罗格FP2-Orp1表达胚胎和幼虫进行 体内 成像的方法。这种方法不仅能够确定生理H2O2水平，而且还可以确定在不同的发育阶段或条件下发生的潜在变化。总的来说，该系统为检测活细胞和动物中的H2O2 提供了可靠的方法，以研究H2O2 在发育、健康和疾病中的作用。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62165/62165fig03.jpg"> 图2。实验方法大纲。简言之，胚胎采集后，roGFP2-Orp1 mRNA被注射到单细胞阶段斑马鱼胚胎的蛋黄中。发育中的胚胎可用于体外和体内成像的（A）和B。（A） GFP阳性胚胎用于解剖视网膜，用于在34马力下采集RGC。分离的 RGC 镀在 ZFCM （+） 介质中的 PDL/层膜盖上。生长锥成像可以进行RGC延长他们的轴突后，6-24小时的电镀。细胞可以接受不同的治疗，以测量H2O2水平的潜在变化。在这里，我们测量了RGC（红色）生长锥中的H 2 O2水平。（B） GFP阳性胚胎用于体内成像。在理想年龄，胚胎可以麻醉并安装在35毫米玻璃底盘上，用于共焦成像。在这里，胚胎被安装在腹膜成像的通风装置上。示意图显示斑马鱼的视网膜发育。RGC 形成结膜细胞层 （GCL），这是网状网膜的最内层。RGC轴突发展成视神经，穿过中线，形成视基亚西姆。然后，RGC轴突生长，在中脑的光学构造中形成突触。<a href="https://www.jove.com/files/ftp_upload/62165/62165fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35-mm culture dish</td>
			<td>Sarstedt</td>
			<td>83-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillary Tubes</td>
			<td>Sutter/Fisher Scientific</td>
			<td>NC9029378</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes (100 x 15 mm)</td>
			<td>VWR</td>
			<td>25384-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140087</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Mold</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>TE2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscopy</td>
			<td>Zeiss</td>
			<td>710</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium with phenol red</td>
			<td>Gibco/Fisher Scientific</td>
			<td>11-415-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium without phenol red</td>
			<td>Gibco/Fisher Scientific</td>
			<td>21-083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting agarose</td>
			<td>Promega</td>
			<td>V2111</td>
			<td></td>
		</tr>
		<tr>
			<td>mMessage mMachine SP6 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>New England Biolabs</td>
			<td>R0189S</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone/Fisher Scientific</td>
			<td>SH3025601</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma Aldich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylthiourea (PTU)</td>
			<td>Sigma Aldich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>World Precision Instruments</td>
			<td>PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma Aldich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>QiaQUICK PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse slit2</td>
			<td>R&#38;D Systems</td>
			<td>5444-SL-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma Aldich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Steritop 0.22 &#181;m filter</td>
			<td>Millipore</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Ambion</td>
			<td>AM9860</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine Methanesulfonate</td>
			<td>Sigma Aldich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Pipette Puller</td>
			<td>David Kopf Instruments</td>
			<td>700C</td>
			<td></td>
		</tr>
	</tbody>
</table>

ros live cell imaging during neuronal development

活性氧物种（ROS）是成熟的信号分子，在正常发育、平衡和生理学中非常重要。在不同的ROS中，过氧化氢（H2O2）在细胞信号中的作用最为突出。H2O2在若干物种的开发过程中被牵连。例如，在受精后的头几天，斑马鱼胚胎中检测到H2O2的瞬时增加。此外，消耗一个重要的细胞H2O2源，NADPH氧化酶（NOX），损害神经系统的发展，如分化，轴向生长，和视网膜结节细胞（RGCs）在体内和体外指导。在这里，我们描述了一种在发育过程中使用基因编码的H 2 O2-特异性生物传感器roGFP2-Orp1在培养斑马鱼神经元和整个幼虫中成像细胞内H 2O2水平的方法。这个探针可以在斑马鱼幼虫中短暂或稳定地表达。此外，由于基因表达或体积效应的差异，比例读数降低了检测伪证的可能性。首先，我们演示如何分离和培养从斑马鱼胚胎中衍生的RGC，这些胚胎暂时表达roGFP2-Orp1。然后，我们使用整个幼虫来监测H2O2水平在组织水平。传感器已通过添加 H2O2进行验证。此外，该方法还可用于通过产生具有组织特异性生物传感器表达的转基因动物来测量特定细胞类型和组织的 H2O2水平。由于斑马鱼促进遗传和发育操作，这里展示的方法可以作为测试H2O2在脊椎动物神经元和一般胚胎发育过程中的作用的管道。

养殖斑马鱼RGCs在1d内扩展轴突。图4A显示了H 2 O2-生物传感器的具有代表性的405/480比例图像。细胞体、轴龙和生长锥体在单个神经元中清晰可见。随着时间的推移，这些神经元可以接受不同的治疗，以监测H2O2的变化。我们以前发现，在培养介质中添加 100 μM H2O2可增加比率值，表明可以通过此系统（图 4 ）24</str...

Watch this Scientific Journal Video about ROS Live Cell Imaging During Neuronal Development at JoVE.com

ROS Live Cell Imaging During Neuronal Development

该协议描述了在培养斑马鱼神经元和幼虫中使用基因编码的过氧化氢（H2O2）生物传感器，以评估H2O2 在神经系统发育过程中的生理信号作用。它可以应用于不同的细胞类型，并修改与实验治疗研究活性氧物种（ROS）在一般发展。

神经元发育过程中的 ROS 活细胞成像

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

我们的协议描述了在培养斑马鱼神经元和幼虫中使用过氧化氢生物传感器。该协议将帮助研究人员剖析活性氧物种在正常生理学和病理生理学中的作用。这项技术的主要优点是实时检测活体动物和培养细胞中的过氧化氢。这种方法可以应用于其他动物，如啮齿动物模型系统。要准备盖片，请使用储存在 100% 乙醇中的酸洗盖片。使用钳子从存储容器中取出一个盖片，并点燃它以去除残留的乙醇。空气完全干燥盖片，将其以一个角度放置在35毫米培养盘内。准备聚-D-莱辛或PDL，或工作解决方案。将 PDL 涂抹到每个盖片的中心，避免溶液扩散到边缘，并在室温下孵化 PDL 20 至 30 分钟。确保 PDL 不会干涸。用 0.5 毫升无菌水清洗 PDL，让盘子完全干燥。准备层压素工作解决方案。并将其应用到每个盖片的中心，确保避免溶液扩散到边缘。在潮湿的孵化器中将板在 37 摄氏度下孵化两到六个小时，避免层压素溶液干燥。要在板视网膜结节细胞或RGC中进行胚胎解剖，准备并标记四个35毫米组织培养皿，并填充4毫升70%乙醇。E2媒体一。E2媒体二。和 E2 媒体三，解剖当天。把盘子放在冰箱里。当斑马鱼胚胎受精后34小时，将涂有层蛋白的培养皿从孵化器中取出。用 0.5 毫升的 1X PBS 洗盖滑三次。最后洗完后，在每个培养皿中加入四毫升 ZFCM®介质。避免干燥盘子。取回准备好的冷藏培养皿，使其与室温平衡。用 15 微升 ZFCM®介质填充 4 到 6 个 PCR 管。从孵化器中取出斑马鱼胚胎，将胚胎浸入含有70%乙醇的35毫米组织培养皿中，5-10秒内进行灭菌。使用移液器的转移，将胚胎转移到E2介质一盘，含有无菌E2介质，以洗去多余的乙醇。然后，将胚胎转移到E2介质两道菜中，用锋利的钳子去除它们的胆汁。最后，将胚胎移植到E2介质三道菜进行解剖。使用一对细钳，位置和持有胚胎前蛋黄与钳子之一。并删除尾部后部的蛋黄囊与其他钳子。用钳子抓住脖子，摘下头部，让大脑和眼睛暴露在 E2 介质中。用细钳尖，轻轻地从头部滚动眼睛，同时用第二个钳子按住颅面组织。将四只眼睛转移到先前准备的含有 ZFCM® 的管子上下三次，用 p20 移液器约 45 次分离细胞。将 ZFCM® 与分离的细胞转移到盖片的中心。将培养物保持在22摄氏度的多苯乙烯泡沫架上，以吸收振动。在成像当天，检查显微镜下的细胞，以验证 RGC 轴突的生长。对于活细胞成像，将盖片从培养皿转移到活细胞成像室。使用配备 DIC 目标 OG-590 长通红色滤镜和 EMCCD 摄像头的倒置显微镜。在成像之前，用 ZFCM-后用 10X 目标定位单元替换 ZFCM®介质，使用油浸入目标以 60 倍的放大倍数获取图像。使用额外的 1.5 倍放大倍数。首先获取 DIC 图像。然后，使用适当的滤镜集对 roGFP2-Orp1 进行映像。拍摄完第一组图像后，与包含不同治疗解决方案的媒体交换介质。应每 30 分钟更换一次成像媒体，以避免 pH 和渗透性变化。对于体内成像，将 22 至 24 小时受精后胚胎保存在 E3 介质中，其中含有 0.003% 无甲基蓝苯乙烯硫酸酯。每天交换介质并去除死胚胎。在理想年龄，麻醉和安装胚胎在1%的糖在35毫米玻璃底培养皿。胚胎可以按多面、通风或横向方向，具体取决于成像的兴趣区域。凝固后，用E3介质填充菜肴，不加甲基蓝，但用0.016%三卡因填充。设置显微镜进行成像。使用倒置激光扫描共聚焦显微镜对安装在糖滴底部的胚胎进行成像。兴奋 roGFP2-Orp1，并获取与所需的排放过滤器相应的图像。通过胚胎所需的部分获得五微米部分厚度的 z 堆栈。成像后，用细钳从Agarose中取出胚胎，并把它们保存在孵化器和甲基蓝色自由介质中，用苯乙烯硫烯直到预期年龄。此处显示了过氧化氢生物传感器和养殖斑马鱼 RGC 扩展轴突的代表性图像。细胞体、轴龙和生长锥体在单个神经元中清晰可见。在培养介质中添加过氧化氢可增加比率值，表明该系统可以检测到实时变化。过氧化氢水平的量化显示在面板中，B.To 确定整个斑马鱼胚胎的过氧化氢水平，mRNA在一个细胞阶段被注射，导致所有组织表达roGFP2-Orp1生物传感器。斑马鱼幼虫的头部区域显示在两个不同的时间点，重点是网状腺中的过氧化氢水平。测量了斑马鱼胚胎中受精后两天和受精后五天过氧化氢的基础水平。在施肥后的两天，比例值明显低于施肥后的五天。此外，每种动物的视网膜过氧化氢含量都有不同程度的增加。使用细钳去除眼睛和眼睛与移液器的柔和分离是这个程序中最关键的步骤。此协议之后可以进行药物治疗，以调查 ROS 信号中涉及的因素。

Research

JoVE Journal

Neuroscience

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Analyzing Dendritic Morphology in Columns and Layers

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

在行为和刺激反应期间为整个大脑成像准备成人德罗索菲拉褪黑麦片加斯特

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...