Name: live imaging of the mitochondrial glutathione redox state in primary neurons using a ratiometric indicator
Uploaded: 2021-10-20T14:30:00
Description: Watch this Scientific Journal Video about Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator at JoVE.com

这项工作得到了Deutsche Forschungsgemeinschaft（BA 3679/5-1;对于 2289：BA 3679/4-2）。A.K.由ERASMUS +奖学金支持。我们感谢Iris Bünzli-Ehret，Rita Rosner和Andrea Schlicksupp准备了原代神经元。我们感谢Tobias Dick博士提供pLPCX-mito-Grx1-roGFP2。 图4 所示的实验是在海德堡大学尼康成像中心进行的。 图2 是用 BioRender.com 准备的。

所有动物实验均符合国家和机构指南，包括欧洲议会理事会指令2010/63 /EU，并已获得内政部的完全道德批准（海德堡大学动物福利办公室和卡尔斯鲁厄Regierungspraesidium，许可证T14 /21和T13/21）。根据标准程序从新生小鼠或大鼠幼崽中制备原代海马和皮质神经元，并如前所述维持12-14天13。
1. 溶液的制备
<ol><li>成像缓冲液的库存解决方案<ol>
<li>根据 ...

<ol>
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线粒体氧化还原状态的定量和动态测量提供了有关线粒体和细胞生理学的重要信息。有几种荧光化学探针可用于检测活性氧，"氧化还原应激"或"氧化应激"。然而，后一术语没有明确界定，往往缺乏特异性9，17，18。与化学染料相比，Grx1-roGFP2具有几个优点9，19：（i）作为激发比传感?...

几种重要的线粒体酶和信号传导分子受到硫醇氧化还原调节1。此外，线粒体是活性氧的主要细胞来源，并且选择性地容易受到氧化损伤2。因此，线粒体氧化还原电位直接影响生物能量、细胞信号传导、线粒体功能，并最终影响细胞活力3，4。线粒体基质含有大量（1-15 mM）的硫醇二硫化物氧化还原缓冲谷胱甘肽（GSH），以维持氧化还原稳态并建立抗氧化防御5，6。GSH可以共价附着在靶蛋白（S-谷胱甘氨酸化）上以控制其氧化还原状态和活性，并被一系列减少氧化蛋白的解毒酶使用。因此，在研究线粒体功能和病理生理学时，线粒体谷胱甘肽氧化还原电位是一个高度信息性的参数。
roGFP2是GFP的一种变体，通过添加两个表面暴露的半胱氨酸而变得氧化还原敏感，形成人造二硫醇 - 二硫醚对7，8。它在~510nm处具有单个发射峰，在~400 nm和490 nm处具有两个激发峰。重要的是，两个激发峰的相对振幅取决于roGFP2的氧化还原状态（图1），使该蛋白质成为比例传感器。在Grx1-roGFP2传感器中，人戊二醇氧化氢-1（Grx1）已经融合到roGFP29，10的N端。Grx1酶与roGFP2的共价连接为传感器提供了两项重大改进：它使传感器响应特定于GSH / GSSG谷胱甘肽氧化还原对（图1），并且它将GSSG和roGFP2之间的平衡速度提高了至少100，0009倍。因此，Grx1-roGFP2能够对细胞谷胱甘肽氧化还原电位进行特异性和动态成像。
Grx1-roGFP2成像可以在各种显微镜上进行，包括宽视场荧光显微镜，旋转盘共聚焦显微镜和激光扫描共聚焦显微镜。传感器在原代神经元中的表达可以通过各种方法实现，包括脂肪感染11，DNA /磷酸钙共沉淀12，病毒介导的基因转移或使用转基因动物作为细胞源（图2）。本文使用含有1：1比例的AAV1和AAV2衣壳蛋白 13，14 的假型重组腺相关病毒（rAAV）进行实验。使用该载体，通常在感染后 4-5 天达到最大传感器表达，并保持稳定至少 2 周。我们已经成功地将Grx1-roGFP2用于小鼠和大鼠的原代海马和皮质神经元。
在本文中，rAAV介导的线粒体靶向Grx1-roGFP2在原代大鼠海马和皮质神经元中的表达用于评估基础线粒体谷胱甘肽氧化还原状态及其急性扰动。为共聚焦实时成像提供了一个协议，其中包含有关如何（i）优化激光扫描共聚焦显微镜设置，（ii）运行实时成像实验以及（iii）使用FIJI分析数据的详细说明。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamide (DA)</td>
			<td>Sigma-Aldrich</td>
			<td>D3648</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Carl Roth GmbH</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (2.5 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>neoFroxx GmbH</td>
			<td>LC-4522.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2&#183;6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>442611-M</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methyl-D-aspartate (NMDA)</td>
			<td>Sigma-Aldrich</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>neoFroxx GmbH</td>
			<td>LC-5932.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (0.1 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P8574</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Carl Roth GmbH</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine ethyl ester perchlorate (TMRE)</td>
			<td>Sigma-Aldrich</td>
			<td>87917</td>
			<td></td>
		</tr>
		<tr>
			<td>equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>imaging chamber</td>
			<td>Life Imaging Services (Basel, Switzerland)</td>
			<td>10920</td>
			<td>Ludin Chamber Type 3 for &#216;12mm coverslips</td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope, microscope</td>
			<td>Leica</td>
			<td>DMI6000</td>
			<td></td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope, scanning unit</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic pump</td>
			<td>VWR</td>
			<td>PP1080 181-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, camera</td>
			<td>Hamamatsu</td>
			<td>C9100-02 EMCCD</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, incubationsystem</td>
			<td>TokaiHit</td>
			<td>INU-ZILCF-F1</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, microscope</td>
			<td>Nikon</td>
			<td>Ti microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, scanning unit</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td>https://fiji.sc</td>
			<td></td>
		</tr>
		<tr>
			<td>StackReg plugin</td>
			<td></td>
			<td>https://github.com/fiji-BIG/StackReg/blob/master/src/main/java/StackReg_.java</td>
			<td></td>
		</tr>
		<tr>
			<td>TurboReg plugin</td>
			<td></td>
			<td>https://github.com/fiji-BIG/TurboReg/blob/master/src/main/java/TurboReg_.java</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of the mitochondrial glutathione redox state in primary neurons using a ratiometric indicator

线粒体氧化还原稳态对神经元的活力和功能很重要。虽然线粒体含有几种氧化还原系统，但高度丰富的硫醇二硫化物氧化还原缓冲液谷胱甘肽被认为是抗氧化防御的核心参与者。因此，测量线粒体谷胱甘肽氧化还原电位提供了有关线粒体氧化还原状态和氧化应激的有用信息。戊二醇1-roGFP2 （Grx1-roGFP2） 是一种基于遗传编码的绿色荧光蛋白 （GFP） 的谷胱甘肽氧化还原电位比率指标，在 400 nm 和 490 nm 处具有两个氧化还原状态敏感的激发峰，在 510 nm 处具有单个发射峰。本文介绍了如何在原代海马和皮质神经元中对线粒体靶向Grx1-roGFP2进行共聚焦实时显微镜检查。它描述了如何评估稳态线粒体谷胱甘肽氧化还原电位（例如，比较疾病状态或长期治疗）以及如何测量急性治疗时的氧化还原变化（以兴奋性毒性药物 N-甲基-D-天冬氨酸（NMDA）为例）。此外，本文还介绍了Grx1-roGFP2和线粒体膜电位指示剂四甲基罗丹明乙酯（TMRE）的共同成像，以证明Grx1-roGPF2如何与其他指标进行多参数分析。该协议详细说明了如何（i）优化共聚焦激光扫描显微镜设置，（ii）应用药物进行刺激，然后用二酰胺和二硫甲状腺素进行传感器校准，以及（iii）使用ImageJ / FIJI分析数据。

生长因子戒断后稳态线粒体氧化还原状态差异的定量分析 为了证明线粒体氧化还原状态稳态差异的定量，在成像前将标准培养基中生长的原代神经元与没有生长因子培养48小时的神经元进行比较。生长因子戒断导致72小时16后凋亡神经元细胞死亡。在48小时后对细胞进行成像，以测试在此之前是否在线粒体氧化还原状态发生变化。在聚L-鸟氨酸包被盖玻片上生长的原...

Watch this Scientific Journal Video about Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator at JoVE.com

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

本文介绍了一种方案，用于使用共聚焦活显微镜确定原代海马和皮质神经元中基底氧化还原状态和氧化还原反应的差异。该协议可以应用于其他细胞类型和显微镜，只需最少的修改。

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

该方法允许研究线粒体氧化还原状态在病理生理学条件下如何受到影响。它也可以用来评估旨在保护线粒体的治疗策略的有效性。这种技术的主要优点是，它允许对线粒体氧化还原状态的动态和动态变化进行器官和特异性评估。该方法可以与附加指标相结合，以同时记录例如线粒体膜电位或除氧化还原状态外的钙浓度。该方案可应用于培养细胞、组织外植体和切片培养物。首先优化扫描共聚焦显微镜设置。为此，请将检测器设置为12位或16位，激活顺序扫描模式并添加第二个序列/跟踪。对于这两个通道，选择指示曝光过度和曝光不足像素的伪彩色查找表，然后选择适合感兴趣对象的物镜。将带有细胞的盖玻片安装到成像室中。加入一毫升成像缓冲液，并将腔室放在显微镜上。使用目镜和透射光聚焦细胞。记录具有不同像素格式和针孔大小的图像。然后记录具有不同激光强度的图像，并相应地调整探测器增益和阈值。最后，记录具有不同扫描速度和不同帧数平均值的图像。对于实时成像，请将延时间隔设置为 30 秒，持续时间设置为 25 分钟。然后安装细胞，将腔室放在显微镜上并如前所述聚焦细胞。切换到扫描模式，并使用实时视图中的 488 纳米通道对焦并定位细胞以进行成像。（可选）要增加每次运行记录的单元格数，请使用多点功能为每个盖玻片成像两到三个视场。开始延时采集并记录五张图像作为两分钟的基线记录。向腔室中加入500微升3X NMDA溶液，以达到30微摩尔的最终浓度，并将另外20张图像记录为10分钟的NMDA响应。接下来，向腔室中加入500微升4X DA溶液，并再记录六张图像。从成像室中吸出缓冲液，并用一毫升DTT溶液代替。再录制 10 张图像后，结束录制并保存图像系列。要导入图像分析软件中的数据，请单击插件，选择生物格式，然后选择生物格式导入程序。在对话框中，选择视图堆栈中的超堆叠，将颜色模式设置为默认值，然后选择自动缩放。通过单击图像，选择类型，然后从选项中选择 32 位，将图像格式更改为 32 位。要将颜色通道拆分为单独的窗口，请单击图像，转到颜色，然后选择拆分通道。调整阈值以选择要分析的线粒体，方法是单击图像，选择调整和阈值。在对话框中，选择默认的红色深色背景和堆栈直方图。当所选像素显示为红色时，单击应用。然后选择将背景像素设置为NAN处理所有图像，并对通道二执行相同的过程。要可视化405到488纳米的比率，请单击过程和图像计算器创建比率图像。在对话框中，选择图像中的第一通道，在图像二中选择操作通道二中的一除法。然后选择创建新窗口并处理所有图像。将比率图像的查找表更改为伪彩色，方法是单击图像，选择查找表，然后触发。为了分析图像，请在比例图像上的单个细胞或线粒体周围绘制ROI。要将投资回报率添加到ROI管理器，请转到分析，单击工具，选择ROI管理器，单击添加并选择全部显示。要测量单个电池的405至488纳米比率，请单击ROI管理器，按Control A选择所有ROI，转到更多并选择多重测量。在对话框中，选择"测量所有切片"和"每个切片一行"。将测量结果导出到电子表格软件后，选择405纳米图像，测量所有ROI的强度，然后再次将测量结果导出到电子表格软件。同样，测量488纳米图像的ROI强度。要保存投资回报率以供将来参考，请按控制条款A选择所有投资回报率，转至更多，然后选择保存。这些来自延时记录的代表性快照显示了NMDA处理之前和之后以及使用DA和DTT进行最大/最小校准后的神经元的比例图像。用30微摩尔NMDA治疗神经元在几分钟内诱导线粒体氧化。对单个荧光通道的分析表明，NMDA诱导的线粒体酸中毒在405和488纳米激发时都会导致GFP荧光下降。在对照实验中，用DA预处理排除了NMDA进一步的线粒体氧化。因此，尽管氧化还原敏感的GFP2荧光强度进行了相当大的淬灭，但405与488的比率没有改变。该实验证实，405至488纳米的比率不受pH值变化的影响。在另一项实验中，并行评估了线粒体膜电位，氧化还原状态和形态。用60微摩尔NMDA治疗神经元导致四甲基罗丹明或TMRE信号的损失和405至488纳米rho GFP比值的增加，随后线粒体的一些延迟四舍五入。当您开始使用此方法时，花时间仔细优化微观设置非常重要。这将有助于在实验过程中保持神经元的健康。始终遵守激光安全规则也非常重要。在现场录制期间添加药物进行篡改时，请确保不要暴露于激光辐射。

Research

JoVE Journal

Neuroscience

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

科研

神经科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

活大鼠皮层神经元线粒体膜电位和活性氧的测定

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

用于可视化神经突在冷冻水合状态下使用低温电子断层扫描准备初级神经元

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Measuring Connectivity in the Primary Visual Pathway in Human Albinism Using Diffusion Tensor Imaging and Tractography

在人类白化病的初级视途径弥散张量成像和测量束成像连通

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...