Name: biosensing motor neuron membrane potential in live zebrafish embryos
Uploaded: 2017-06-26T13:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos at JoVE.com

The authors would like to thank Simona Rodighiero for her priceless support with the FRET imaging analysis.

pHuC_Mermaid质粒生成注意:美人鱼是通过配对肠绒氨酸杆菌的电压感应域（VSD）开发的生物传感器（现在 Ciona robusta ） 5含有FRET配对荧光团Umi-Kinoko Green（mUKG:供体）的磷酸酶（Ci-VSP）的电压传感器和橙色发射荧光蛋白Kusabira Orange（mKOk:受体）的单体版本。对于该生物传感器，通过膜去极化诱导的VSD结构域的构象变化增加了供体和受体荧光蛋白的接近度，从而增加它们之间的能量转移（增加FRET比率） 3 。 VSD保证生物传感器在质膜上的有效定位。生物传感器（在脊髓中，信号在中间神经元和运动神经元中均可检测到）的神经元表达是通过克隆美人鱼操作en阅读框（ORF）在斑马鱼泛神经启动子HuC 6下 。 <ol><li> 使用T3通用引物和Mermaid Sma I引物（5&#39;TATCCCGGGATTCGACGGTTCAGATTTTA）通过聚合酶链反应（PCR）从pCS4 +美人鱼质粒3与Pfu（校正）DNA聚合酶通过聚合酶链反应（PCR）扩增Mermaid ORF，以在上游插入Sma I限制性位点的美人鱼ORF。 <ol><li>使用以下PCR混合物:0.5μLPfu DNA聚合酶，7.5μL特异性10×缓冲液，1μL10mM dNTPs，2.5μL二甲基亚砜（DMSO），0.5μL（50ng）质粒制备物和1 μL每个引物的20μM储备溶液，总体积为50μL。 </li><li>在95℃下将PCR混合物加热15秒，在50℃下加注15秒，并在72℃下延长4分钟，总共35个循环。 </li></ol></li><li>凝胶纯化具体钝PCR产物使用商业凝胶纯化试剂盒，遵循制造商的方案。 </li><li>根据制造商的方案将DNA片段克隆到pCMV-SC钝化载体中。 </li><li> 将Mermaid阳性质粒（命名为pCMV-SC_Mermaid）与Sma I限制酶线性化，用于以下插入HuC启动子。 <ol><li>设置限制反应如下:0.5μL（10U） SmaI限制酶，2μL试剂盒10x缓冲液和5μg总体积为20μL的质粒DNA。将反应在25℃下孵育1小时。 </li></ol></li><li>使用商业凝胶纯化试剂盒按照制造商的方案对消化的质粒进行凝胶纯化。 </li><li> 使用一对HuC特异性引物（HuCprom-forw1_SalI:5&#39;-GTAGTCGACCAGACTTGTCAAAAGGGTCCA和HuCpro），以Pfu DNA聚合酶和斑马鱼基因组DNA为模板，PCR扩增HuC启动子（pHuC）m-rev1:5&#39;-TCCATTCTTGACGTACAAAGATG），跨越ATG上游的3,150-bp区域。 <ol><li>使用以下方案设置PCR混合物:将0.5μLPfu DNA聚合酶，7.5μL特异性10×缓冲液，1μL10mM dNTP，2.5μLDMSO，200ng基因组DNA和1μL20μM每个引物的储备溶液总体积为50μL。 </li><li>在95℃下进行2分钟的初始步骤后，将PCR混合物在95℃加热15秒，在50℃下加热15秒，并在72℃下延长4分钟，总共35个循环。 </li></ol></li><li>按照制造商的方案，使用商业凝胶纯化试剂盒纯化特异性钝性PCR产物。 </li><li>使用1μLT4 DNA连接酶和1μL10μL总体积的特异性10X缓冲液，将等摩尔量的纯化的pCMV-SC_Mermaid（步骤1.5）和pHuC DNA（步骤1.7）。将反应温育16小时4°C。 </li><li>根据制造商的说明书，用5μL连接反应（步骤1.8）转化感受态细胞的等分试样。 </li><li> 选择pHuC阳性克隆（pHuC_Mermaid），启动子通过SalⅠ -EcoVR双重消化（ Sal I限制性位点插入到启动子片段的上游，而Eco RV限制性位点位于pCMV-SC质粒的多聚腺苷酸化区PolyA的下游）。 <ol><li>设置限制反应如下:将0.5μL（10U）的Sal I和Eco RV限制酶，2μLkit10X缓冲液和1μg的pHuC_Mermaid DNA，总体积为20μL。将反应在37℃下孵育1小时。将反应运行到琼脂糖凝胶上。 </li></ol></li></ol>胚胎显微注射<ol><li>转移受精的例如使用塑料移液器从野生型（AB株）或Sod1-G93R成年斑马鱼4获得的gs至10mm培养皿。 </li><li>在冷（4℃）的鱼水中冲洗胚胎，并立即用200微克的pHuC_Mermaid质粒使用微量注射器（有关显微注射程序的全面和详细描述，参见参考文献7和8）将其微量注射到卵黄中。 </li><li>使用塑料移液器将胚胎转移到培养皿中，并将它们在28℃的鱼水中孵育，直到达到所需的发育阶段（受精后20-24小时），进行以下分析。 </li></ol> 3.自发尾线卷绕分析注意:评估20-24 hpf胚胎中有或没有药物利鲁唑的自发尾丝卷曲行为。 <ol><li>将胚胎转移到装有含有0.2％DMS​​O（瑞利唑载体）的鱼水的90mm圆形培养皿中，并使用two具有尖锐技巧的珠宝商的镊子。孵育胚胎5分钟。 </li><li>在使用安装在立体显微镜上的数码相机进行1分钟的录像时，可以在RT检测尾盘。以30帧/秒的时间分辨率获取时间序列。 </li><li>通过计算每个时间单位的弯曲数（对侧和同侧）的数量来计算自发尾盘的频率。 </li><li> 为了评估药物利鲁唑的效果，轻轻地使用塑料巴斯德移液器将胚胎转移到装有含有5μM利鲁珠的鱼的新的90mm培养皿中。 <ol><li>孵育胚胎5分钟，然后记录1分钟的视频并执行如上所述的行为分析。 </li></ol></li></ol>美人鱼生物传感器可视化在生物胚胎中的成像设置:同时检测供体和受体信号<ol><li>将20 - 24 hpf胚胎装入1％低熔点琼脂糖的鱼水中37°C在一个35毫米玻璃底的成像盘。定位在他们身边的胚胎。等待琼脂糖在室温下固化。 </li><li>将成像盘转移到安装在倒置显微镜上的反相共聚焦阶段。通过用488nm氩激光线激发mUKG并记录其在495和525nm之间的发射，用20X物镜（0.7数值孔径NA）来识别表达生物传感器的运动神经元。 </li><li>对于FRET测量，使用488nm激光线激发FRET对的供体mUKG。同时使用在双向模式下以8,000Hz工作的谐振扫描器，由供体（495和525nm）发射的荧光和由mKOk受体发射的荧光（550和650nm之间的FRET通道）发射的荧光。如果可用，请使用473 nm激光。 注意:供体的激发效率将稍微降低（85％而不是93％，488 nm），但受体的交叉激发将也可以减少（从478 nm激光线到488 nm到17％的17％）。 </li><li>为了减少光毒性和荧光团漂白，通过降低激光线的功率（在采集软件的光束路径窗口）来最小化样品的照度。 </li><li>优化激励以匹配在实验开始时设置的增益和偏移参数，并在整个会话期间保持不变。要设置偏移量，请将图像的颜色更改为强度值（通过使用Q查找表），然后在关闭激光扫描时，转动偏移旋钮（智能偏移），使背景像素稍微强度高于零。使用相同的查找表，扫描时切换激光，转动增益旋钮（智能增益）以最大化信噪比，小心避免饱和像素。 </li><li>使用打开的针孔（2个通风单元），获取16位图像，以提供足够的动态范围进行定量e分析。避免平均以提高采集速度并减少光漂白。 </li><li>在软件采集窗口中，从下拉菜单中选择512 x 64像素（像素大小:605 nm）的图像字段大小。 </li><li>从采集模式窗口中，从下拉菜单中选择xyt （时间流逝，单个xy平面），通过获取单个xy平面记录胚胎脊髓神经元电压的变化，设置采集参数，每30个记录一个图像ms 1分钟</li><li>为了评估利鲁唑给药对相同神经元膜去极化的影响，在加入含有5μM利鲁珠的鱼水后5分钟获得一个新的数据。 </li><li>对于FRET分析，使用ImageJ宏Biosensor_FRET（表示单链FRET生物传感器。 </li><li>评估t 1处的每个神经元的基底膜FRET比例为（（FRET平均值 - FRET背景）/（供体平均值 - 供体背景）），其中FRET和Don或平均强度是在获取的每个通道的细胞周围绘制的相同感兴趣区域（ROI）中计算的平均荧光强度，FRET和供体背景是在没有荧光样品的视场的ROI中计算的平均荧光强度。 注意:有关插件使用的详细分步说明， <a href="http://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/biosensor-fret">请</a>访问<a href="http://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/biosensor-fret">www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/biosensor-fret</a>网站。 </li><li>使用图形软件比较不同实验范例之间的去极化的频率，幅度和持续时间。使用不配对的学生t检验比较两组，并将P &lt;0.05时的平均值视为统计学差异。 </li></ol>

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The authors have nothing to disclose.

这里提出的方案允许我们探讨斑马鱼胚胎脊髓运动神经元的电性质与自发卷取行为之间的关联，最早的定型运动活动，出现在17 hpf的胚胎发育，并持续到24 hpf 10 。 我们的方法为研究人员提供了研究完整胚胎神经系统的工具，充分保护了开发功能网络中细胞间相互作用的复杂性。斑马鱼胚胎的体内成像通过将其浸入低熔点琼脂糖中简单地固定来进行。?...

体内全身成分分析允许科学家以最可靠的方式研究细胞行为。当细胞细胞相互作用（接触和非接触依赖性）受到严重影响时，尤其是在神经系统中，膜电压变化驱动可激活细胞之间的通讯。理解由这些电信号编码的信息是了解神经系统在生理和疾病状态下工作的方式的关键。 为了研究大多数非侵入性生理条件下的细胞电学性质，最近开发出几种遗传编码电压指标1 。与前几代光电压传感器（主要是电压敏感染料） 2相反，GEVIs允许完整神经系统的体内分析，以及它们的表达可以限于特定的细胞类型或种群。 斑马鱼胚胎是体内 "底物"，可以利用GEVIs的巨大潜力。事实上，由于其光学透明度及其简化且进化上保守的神经系统，斑马鱼模型允许直接识别和操纵网络中的每个蜂窝组件。事实上，使用基于FRET的GEVI美人鱼3导致在肌萎缩性侧索硬化（ALS）斑马鱼模型中鉴定脊髓运动神经元行为的先兆症状变化4 。 以下体内协议描述了如何以神经元特异性方式监测表达美人鱼的完整斑马鱼胚胎中脊髓运动神经元的电学性质。此外，它展示了药理学诱导的chan具有这种电性质的ges可能与胚胎自发盘旋的频率的变化相关联，这种变化的运动活动表征斑马鱼在非常早期的发育阶段的运动行为。

<table><tbody><tr><td>Low Melting Point Agarose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9414</td><td></td></tr><tr><td>DMSO&amp;nbsp;</td><td>Sigma-Aldrich</td><td>W387520</td><td></td></tr><tr><td>Riluzole</td><td>Sigma-Aldrich</td><td>R116</td><td></td></tr><tr><td>Pfu Ultra HQ DNA polymerase&amp;nbsp;</td><td>Agilent Technologies - Stratagene Products Division</td><td>600389</td><td></td></tr><tr><td>T3 Universal primer&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up system</td><td>Promega</td><td>A9280</td><td></td></tr><tr><td>Universal SmaI primer&amp;nbsp;</td><td>Eurofins</td><td></td><td></td></tr><tr><td>StrataClone Mammalian Expression Vector System / pCMV-SC blunt vector&amp;nbsp;</td><td>Agilent Technologies - Stratagene Products Division&amp;nbsp;</td><td>240228</td><td></td></tr><tr><td>SmaI&amp;nbsp;</td><td>New England Biolabs</td><td>R0141S</td><td></td></tr><tr><td>T4 DNA ligase</td><td>Promega</td><td>M1801</td><td></td></tr><tr><td>SalI</td><td>New England Biolabs</td><td>R0138S</td><td></td></tr><tr><td>EcoRV</td><td>New England Biolabs</td><td>R0195S</td><td></td></tr><tr><td>35 mm, glass-bottomed imaging dish&amp;nbsp;</td><td>Ibidi</td><td>81151</td><td></td></tr><tr><td>forceps</td><td>Sigma-Aldrich</td><td>F6521</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica Microsystems</td><td>M10 F</td><td></td></tr><tr><td>Digital camera</td><td>Leica Microsystems</td><td>DFC 310 FX</td><td></td></tr><tr><td>Leica Application Suite 4.7.1 software&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>QuickTime Player, v10.4</td><td>Apple</td><td></td><td></td></tr><tr><td>Confocal microscope (inverted)</td><td>Leica Microsystems</td><td>TCS SP5</td><td></td></tr><tr><td>Microinjector&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>Femtojet</td><td></td></tr><tr><td>ImageJ macro Biosensor_FRET&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>GraphPad Prism 6.0c&amp;nbsp;</td><td>GraphPad Software, Inc</td><td></td><td></td></tr></tbody></table>

biosensing motor neuron membrane potential in live zebrafish embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

携带在pHuC泛神经元启动子控制下的基于FRET的美人鱼生物传感器编码序列的表达载体，其驱动仅在神经系统中的蛋白质的合成，通过显微注射递送到单细胞受精卵中为了获得瞬时转基因胚胎（ 图1 ， 左图）。掌握显微注射技术后，美人鱼阳性胚胎的百分比接近100％。其中，只有在质膜上仅表达适量的美人鱼生物传感器的胚胎被考虑用于FRET分析/图...

Watch this Scientific Journal Video about Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos at JoVE.com

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

biosensing-motor-neuron-membrane-potential-in-live-zebrafish-embryos

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Anatomy and Physiology

Developmental Biology

Unit 1

The Nervous System and Nervous Tissue

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6.6K 次观看.  Università degli Studi di Milano. Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

视频: Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

科研

神经科学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

发育生物学

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

斑马鱼游泳期间后横向线阿弗伦特神经元的活动

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) in response to an action potential (AP). Since SVs are recycled locally at the presynaptic terminal, coordination of SV exocytosis and endocytosis is important for sustained synaptic transmission. A pH-sensitive green fluorescent protein, called pHluorin, provides a powerful tool to monitor SV exo/endocytosis by targeting it to the SV lumen. However, tracking AP-driven SV recycling with the pHluorin-based probes is still largely limited to in vitro culture preparations because the introduction of genetically encoded probes and subsequent optical imaging is technically challenging in general for in vivo animal models or tissue preparations. Zebrafish is a model system offering valuable features, including ease of genetic manipulation, optical clarity, and rapid external development. We recently generated a transgenic zebrafish that highly expresses a pHluorin-labeled probe at motor neuron terminals and developed a protocol to monitor AP-driven SV exo/endocytosis at the neuromuscular junction (NMJ), a well-established synapse model that forms in vivo. In this article, we show how to prepare larval zebrafish NMJ preparation suitable for pHluorin imaging. We also show that the preparation allows time-lapse imaging under conventional upright epifluorescence microscope, providing a cost-effective platform for analyzing NMJ function.

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

解剖斑马鱼幼鱼神经肌肉接头突触囊泡循环的实时成像

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) ...

A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos

Source: Benedetti, L., et al. <a href="https://app.jove.com/v/55297">Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos.</a> J. Vis. Exp. (2017).
This video demonstrates a non-invasive assay to study electrophysiology of motor neurons in zebrafish embryos. Upon measuring tail coiling in the embryos as a result of spontaneous depolarization of the spinal cord motor neurons, a sodium channel blocker is applied to reduce the coiling frequency. Next, the embryos are immobilized in agarose and placed under a fluorescence microscope. The genetically engineered motor neurons express a voltage-sensing biosensor with a donor and an acceptor fluorophore. A decrease in acceptor fluorophore emission indicates a reduction in the spontaneous depolarization of motor neurons due to the sodium channel blocker.

一种研究斑马鱼胚胎中运动神经元电生理学的无创方法

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...