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Motility Assay for Zebrafish Embryos
斑马鱼胚胎的运动分析   

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Abstract

Analyzing the swimming ability of 2 days post fertilization zebrafish embryos can be a useful technique to study neuromuscular function. Here is a protocol for determining the time it takes for zebrafish embryos to swim a predetermined distance.

Keywords: Muscle(肌肉), Neuromuscular junction(神经肌肉接头), Swimming(游泳), Escape response(逃避反应)

Materials and Reagents

  1. Pipette pump
  2. Glass Pasteur pipette
  3. Overhead transparency sheet
  4. Zebrafish embryo media of choice (Goody et al., 2012)
  5. Embryo ‘poker’ tool (e.g. segment of fishing line super glued in the end of a glass capillary tube, fire polished glass rod)


    Figure 1. Embryo poker tool. This is a useful tool for the positioning and touch stimulus of zebrafish embryos. Fishing line (10 lb, 0.012 inch diameter) is super glued into the end of a glass capillary tube (Sutter Instruments, catalog number: BF100-50-10) with approximately 1 cm of overhang. The glass capillary tube is then wrapped in lab tape.

Equipment

  1. Microscope with high-speed, digital video camera attachment (e.g. Zeiss SteREO Discovery.V12 with Zeiss Axiocam HSm)
  2. Video processing software (e.g. Zeiss Axiovision)
  3. 60 mm Petri dish

Procedure

A. Preparation:

  1. Print the motility wheel (Figure 2) on an overhead transparency sheet.


    Figure 2. Motility wheel. The diameters of these concentric circles provide predetermined distances for zebrafish embryos to swim. The diameters of the circles are, in ascending order: 5 mm, 10 mm, 15 mm, 20 mm.

  2. Place the overhead transparency sheet on the stage of the microscope that will be used to record the videos and adjust the magnification of the microscope such that the edges of the desired concentric circle (e.g. the circle with a 10 mm diameter) are just within the field of view. Do not readjust the magnification setting within a motility assay experiment and use this magnification setting for any subsequent replicates that will be pooled together or compared.
  3. Center a 60 mm Petri dish containing zebrafish embryo media over the concentric circles on the overhead transparency sheet on the microscope stage.
  4. Using a pipette pump and glass Pasteur pipette, transfer one embryo into the Petri dish and use a poker to gently move the embryo into the middle of the concentric circles.


    Figure 3. Microscope set up. A 60 mm Petri dish containing embryo media is placed on top of the motility wheel on top of the microscope stage. An embryo poker tool is used to position a zebrafish embryo in the middle of the motility wheel. Photo credit: University of Maine, Mike Mardosa.

B. Video acquisition:

  1. Begin recording a video when the embryo is stationary and in the center of the concentric circles.
  2. Looking through the eyepieces of the microscope, gently poke the tail of the embryo with a poker. Ensure that you are holding the poker such that your hand does not appear in the video.
  3. When the embryo completely exits the predetermined concentric circle, stop the video recording. A normal 2 day old embryo will exit the circle with a 10 mm diameter in approximately 200 milliseconds on the first poke (Goody et al., 2012).
  4. If the embryo does not completely exit the designated circle, use the poker to reposition the embryo in the center of the circles and repeat steps B1-3. After multiple unsuccessful attempts (~10 attempts), it may be determined that the embryo is incapable of exiting the circle and video recording can be stopped.
  5. Repeat these video acquisition steps until videos of all the desired embryos have been recorded.

C. Video analysis:

  1. ‘Cut’ the videos to only contain the necessary frames, if desired. Save the videos.
  2. For each video, scroll through the frames and determine the first frame in which the entire body of the embryo has exited the predetermined circle. Record that frame number in a spreadsheet. Scroll back to the beginning frames of that same video and determine the last frame in which the embryo is stationary prior to the touch stimulus. Record that frame number in the spreadsheet.
  3. Repeat step C2 for all the videos.
  4. Determine the time lag that occurs between each of the frames (e.g. 9 ms).
  5. Calculate the time it takes an embryo to swim a predetermined distance in milliseconds by subtracting the beginning frame number from the end frame number and multiplying by the time lag value. Record this value in the spreadsheet.
  6. Repeat step C5 for all the motility videos.
  7. After all the biological replicates for a motility experiment have been completed and analyzed, assign the greatest (i.e. slowest) value recorded within a treatment group to all the embryos in that same treatment group that never successfully exited the designated circle.
  8. Calculate the mean and standard deviation or standard error of the mean for each treatment group and statistically compare the values using an unpaired student’s t-test with unequal variance. For an example experimental result using this motility assay, please refer to (Goody et al., 2012).

Acknowledgments

This protocol was adapted from the previous publication: Goody et al. (2012). Development of this protocol was supported by NIH grant RO1 HD052934-01A1 to CAH. MFG would like to thank the University of Maine Graduate School of Biomedical Sciences and Engineering for funding.

References

  1. Goody, M. F., Kelly, M. W., Reynolds, C. J., Khalil, A., Crawford, B. D. and Henry, C. A. (2012). NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy. PLoS Biol 10(10): e1001409.
  2. Westerfield, M. (1993) The Zebrafish Book: A Guide for the Laboratory Use of the Zebrafish (Brachydanio rerio) University of Oregon Press, Eugene.

简介

分析2天后受精斑马鱼胚胎的游泳能力可以是一个有用的技术,研究神经肌肉功能。 这里是一个协议,确定斑马鱼胚胎游动预定距离所需的时间。

关键字:肌肉, 神经肌肉接头, 游泳, 逃避反应

材料和试剂

  1. 移液泵
  2. 玻璃巴斯德吸液管
  3. 顶部透明度标题
  4. 选择斑马鱼胚胎培养基(Goody等人,2012)
  5. Embryo"扑克"工具(例如线条超级粘在玻璃毛细管末端,火抛光玻璃棒)


    图1. Embryo扑克工具。 这是斑马鱼胚胎定位和触摸刺激的有用工具。 将钓鱼线(10磅,0.012英寸直径)超级粘合到具有约1cm悬垂的玻璃毛细管(Sutter Instruments,目录号:BF100-50-10)的末端。 然后将玻璃毛细管包裹在实验室带中。

设备

  1. 具有高速数字摄像机附件的显微镜(例如,具有Zeiss Axiocam HSm的Zeiss SteREO Discovery.V12)
  2. 视频处理软件(例如 Zeiss Axiovision)
  3. 60 mm培养皿

程序

A.制备:

  1. 在高架透明板上打印活动轮(图2)。


    图2.运动轮。 这些同心圆的直径提供斑马鱼胚胎游泳的预定距离。圆的直径按照升序:5mm,10mm,15mm,20mm。

  2. 放置在显微镜的舞台上,将用于记录视频和调整显微镜的放大倍率,使所需的同心圆的边缘(例如圆的10毫米直径)正好在视野内。不要在运动性测定实验中重新调整放大倍率设置,并对任何后续重复使用此放大倍数设置,这些重复将合并在一起或进行比较。
  3. 将包含斑马鱼胚胎培养基的60 mm培养皿置于显微镜载物台顶部透明片上的同心圆上。
  4. 使用移液管泵和玻璃巴斯德吸管,将一个胚胎转移到培养皿中,并使用扑克将胚胎轻轻地移动到同心圆的中间。


    图3.显微镜设置。 将含有胚胎培养基的60mm培养皿放置在显微镜载物台顶部的运动轮的顶部。 胚胎扑克工具用于将斑马鱼胚胎定位在运动轮的中间。 照片来源:缅因大学,Mike Mardosa

B.视频采集:

  1. 当胚胎静止并位于同心圆的中心时,开始录制视频。
  2. 看着显微镜的目镜,用扑克轻轻地戳着胚胎的尾巴。 确保你握着扑克,让你的手不出现在视频中。
  3. 当胚胎完全退出预定的同心圆时,停止视频记录。 正常的2天龄胚胎将在第一次捅时在大约200毫秒内离开具有10mm直径的圆圈(Goody等人,2012)。
  4. 如果胚胎没有完全退出指定的圆,使用扑克将胚胎重新定位在圆的中心,并重复步骤B1-3。 在多次尝试失败(〜10次尝试)之后,可以确定胚胎不能退出圆圈并且可以停止视频记录。
  5. 重复这些视频采集步骤,直到所有所需的胚胎的视频已经记录

C.视频分析:

  1. '剪切'视频只包含必要的帧。 保存视频。
  2. 对于每个视频,滚动通过帧并且确定胚胎的整个身体已经离开预定圆的第一帧。 在电子表格中记录框架编号。 回滚到该相同视频的开始帧,并确定胚胎在触摸刺激之前静止的最后帧。 在电子表格中记录框架编号。
  3. 对所有视频重复步骤C2。
  4. 确定在每个帧之间发生的时滞(例如 9 ms)。
  5. 通过从结束帧编号减去开始帧编号并乘以时滞值,计算胚胎以毫秒为单位游动预定距离所花费的时间。 在电子表格中记录此值。
  6. 对所有运动视频重复步骤C5
  7. 在完成和分析了用于动力实验的所有生物学复制品之后,将在处理组内记录的最大(即最慢)值分配给同一处理组中从未成功退出指定圈。
  8. 计算每个治疗组的平均值和标准偏差或平均值的标准误差,并使用具有不等方差的不成对的student's t检验统计学比较值。对于使用这种运动性测定的实验结果的实例,参考(Goody 。,2012)。

致谢

该协议改编自先前的出版物:Goody等人(2012)。该协议的开发由NIH授予RO1HD052934-01A1至CAH支持。 MFG感谢缅因大学生物医学科学与工程研究生院的资助。

参考文献

  1. Goody,M.F.,Kelly,M.W.,Reynolds,C.J.,Khalil,A.,Crawford,B.D.and Henry,C.A。(2012)。 NAD +生物合成改善了肌肉营养不良的斑马鱼模型。

    em> 10(10):e1001409。
  2. Westerfield,M。(1993)The Zebrafish Book:A Guide for the Laboratory Use of the Zebrafish(Brachydanio rerio) University of Oregon Press,Eugene。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Goody, M. F. and Henry, C. A. (2013). Motility Assay for Zebrafish Embryos. Bio-protocol 3(11): e787. DOI: 10.21769/BioProtoc.787.
  2. Goody, M. F., Kelly, M. W., Reynolds, C. J., Khalil, A., Crawford, B. D. and Henry, C. A. (2012). NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy. PLoS Biol 10(10): e1001409.
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