Name: subcellular imaging of neuronal calcium handling in vivo
Uploaded: 2023-03-17T12:30:00
Description: Watch this Scientific Journal Video about Subcellular Imaging of Neuronal Calcium Handling In Vivo at JoVE.com

这项工作得到了美国国立卫生研究院（NIH）（R01-NS115947授予F. Hoerndli）的支持。我们还要感谢Attila Stetak博士提供的pAS1质粒。

1. 创造转基因菌株
<ol><li>使用选择的克隆方法8，9，克隆表达载体以包含Pflp-18或Prig-3启动子（用于腹侧神经索中的AVA特异性信号），然后选择Ca2+指示剂，然后是3' UTR（有关更多信息，请参阅讨论）10。质粒及其来源的列表可在补充表1中找到。</li>
	<li>遵循既定的方案，通过显微注射含有...

<ol>
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实施所述方法时的第一个考虑因素涉及为给定的研究问题选择具有理想特性的Ca2+指标。细胞质Ca 2+指示剂通常对Ca 2+具有高亲和力，这些指示剂对Ca 2+的敏感性与动力学（开/关速率）成反比16，17。这意味着需要根据感兴趣的现象优先考虑Ca2+灵敏度或动力学。对于具有相对较高的基础Ca 2+水平的亚细胞区室，例如E...

钙离子（Ca2+）是许多细胞类型中高度通用的信息载体。在神经元中，Ca2+负责神经递质的活动依赖性释放，细胞骨架运动的调节，代谢过程的微调，以及适当的神经元维持和功能所需的许多其他机制1，2。为了确保有效的细胞内信号传导，神经元必须在其细胞质中保持较低的基础Ca2+水平3。这是通过合作的Ca 2+处理机制实现的，包括将Ca2+摄取到细胞器中，例如内质网（ER）和线粒体。除了在质膜中排列Ca 2+渗透离子通道外，这些过程还导致整个神经元中细胞质Ca2+的异质水平。
休息和神经元激活期间的Ca 2+异质性允许对Ca 2+依赖机制进行多种的，位置特异性的调节1。Ca 2+浓度特异性作用的一个例子是通过肌醇1，4，5-三磷酸（InsP3）受体从ER释放Ca2+。低 Ca2+ 水平与 InsP3 相结合是打开受体的钙渗透性孔所必需的。或者，高Ca2+水平直接或间接抑制受体4。Ca 2+稳态对正常神经元功能的重要性得到了证据的支持，表明细胞内Ca 2+处理和信号传导中断是神经退行性疾病和自然衰老发病机制的早期步骤5，6。具体来说，ER和线粒体的异常Ca2+摄取和释放与阿尔茨海默病，帕金森病和亨廷顿病6，7的神经元功能障碍的发作有关。
研究自然衰老或神经变性过程中的Ca 2+稳态失调需要纵向观察活的，完整的生物体中具有亚细胞分辨率的Ca2+水平，其中天然细胞结构（即突触的排列和离子通道的分布）得以维持。为此，该协议为使用两种现成的基因编码Ca 2+传感器以高空间和时间分辨率记录体内Ca 2+动力学提供了指导。在秀丽隐杆线虫中使用所述方法获得的代表性结果表明，单个神经元的细胞质或线粒体基质中Ca 2+指示剂的表达如何允许快速获取荧光图像（高达50 Hz），这些荧光图像说明了Ca 2+动力学，并具有识别单个脊柱样结构和单个线粒体内Ca 2+水平的额外能力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x/1.40 Oil objective&#160;&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>&#160;UPlanSApo</td>
		</tr>
		<tr>
			<td>10x/0.40 Objective&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>&#160;UPlanSApo</td>
		</tr>
		<tr>
			<td>22 mm x 22 mm Cover glass&#160;</td>
			<td>VWR&#160;</td>
			<td>48366-227&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose SFR&#160;</td>
			<td>VWR&#160;</td>
			<td>J234-100G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam homogenizer</td>
			<td>Andor Technologies</td>
			<td></td>
			<td>Borealis upgrade to CSU-X1</td>
		</tr>
		<tr>
			<td>CleanBench laboratory table&#160;</td>
			<td>TMC&#160;</td>
			<td></td>
			<td>With vibration control</td>
		</tr>
		<tr>
			<td>Disposable culture tubes</td>
			<td>VWR&#160;</td>
			<td>47729-572</td>
			<td>&#160;13 mm x 100 mm</td>
		</tr>
		<tr>
			<td>Environmental chamber</td>
			<td>Thermo Scientific</td>
			<td>3940</td>
			<td>Set to 20 &#176;C</td>
		</tr>
		<tr>
			<td>Filter wheel or slider</td>
			<td>ASI</td>
			<td></td>
			<td>For 25 mm diameter filters</td>
		</tr>
		<tr>
			<td>FJH 185</td>
			<td>Caenorhabditis Genetics Center&#160;</td>
			<td>FJH 185</td>
			<td>Worm strain</td>
		</tr>
		<tr>
			<td>FJH 597</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>FJH 597</td>
			<td>Worm strain</td>
		</tr>
		<tr>
			<td>GFP bandpass emission filter&#160;</td>
			<td>Chroma&#160;</td>
			<td></td>
			<td>525 &#177; 50 nm&#160;(25 mm diameter)</td>
		</tr>
		<tr>
			<td>ILE laser combiner&#160;</td>
			<td>Andor Technologies&#160;</td>
			<td></td>
			<td>4 laser lines&#160;</td>
		</tr>
		<tr>
			<td>ILE solid state 488 nm laser</td>
			<td>Andor Technologies&#160;</td>
			<td></td>
			<td>50 mW</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>Version 1.52a</td>
		</tr>
		<tr>
			<td>IX83 Spinning disk confocal microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>With Yokogawa CSU-X1 spinning disc</td>
		</tr>
		<tr>
			<td>iXon Ultra EMCCD camera&#160;</td>
			<td>Andor Technologies&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low auto-fluorescence immersion oil&#160;</td>
			<td>Olympus&#160;</td>
			<td>Z-81226</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph&#160;</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td>Version 7.10.1&#160;</td>
		</tr>
		<tr>
			<td>Microscope control box</td>
			<td>Olympus</td>
			<td></td>
			<td>IX3-CBH</td>
		</tr>
		<tr>
			<td>Muscimol&#160;</td>
			<td>MP Biomedical&#160;/ Sigma</td>
			<td>02195336-CF&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>pAS1</td>
			<td>AddGene</td>
			<td>194970</td>
			<td>Plasmid</td>
		</tr>
		<tr>
			<td>pBSKS</td>
			<td>Stratagene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCT61</td>
			<td></td>
			<td></td>
			<td>Plasmid available from Hoerndli/Maricq lab upon request</td>
		</tr>
		<tr>
			<td>pJM23</td>
			<td></td>
			<td></td>
			<td>Plasmid available from Hoerndli/Maricq lab upon request</td>
		</tr>
		<tr>
			<td>pKK1&#160;</td>
			<td>AddGene&#160;</td>
			<td>194969</td>
			<td>Plasmid</td>
		</tr>
		<tr>
			<td>Polybead microspheres&#160;</td>
			<td>Polysciences Inc.&#160;</td>
			<td>00876-15</td>
			<td>&#160;0.094 &#181;m</td>
		</tr>
		<tr>
			<td>Stability chamber</td>
			<td>Norlake Scientific</td>
			<td>NSRI241WSW/8H</td>
			<td>Set to 15 &#176;C</td>
		</tr>
		<tr>
			<td>Stage controller</td>
			<td>ASI</td>
			<td></td>
			<td>With filter wheel control</td>
		</tr>
		<tr>
			<td>Standard microscope slide</td>
			<td>Premiere</td>
			<td>9108W-E</td>
			<td>75 mm x 25 mm x 1 mm</td>
		</tr>
		<tr>
			<td>Touch panel controller</td>
			<td>Olympus</td>
			<td></td>
			<td>I3-TPC</td>
		</tr>
		<tr>
			<td>Z-drift corrector&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>IX3-ZDC2</td>
		</tr>
	</tbody>
</table>

subcellular imaging of neuronal calcium handling in vivo

钙（Ca 2+）成像已主要用于检查神经元活动，但越来越清楚的是，亚细胞Ca2+处理是细胞内信号传导的关键组成部分。体内亚细胞Ca2+动力学的可视化，其中神经元可以在其天然的完整回路中研究，已被证明在复杂的神经系统中具有技术挑战性。线虫秀丽隐杆线虫的透明度和相对简单的神经系统能够实现荧光标签和指示剂的细胞特异性表达和体内可视化。其中包括已被修饰用于细胞质以及各种亚细胞区室（例如线粒体）的荧光指示剂。该协议能够以亚细胞分辨率在体内进行非比率Ca 2 +成像，从而可以分析Ca2 +动力学，直至单个树突棘和线粒体的水平。在这里，使用具有不同Ca 2 +亲和力的两个可用的遗传编码指示剂来证明使用该协议测量单对兴奋性中间神经元（AVA）中细胞质或线粒体基质内的相对Ca2 +水平。结合秀丽隐杆线虫中可能的遗传操作和纵向观察，该成像方案可能有助于回答有关Ca2 +处理如何调节神经元功能和可塑性的问题。

这两种方案能够以高空间分辨率快速获取体内单个神经突的亚细胞区域和细胞器内的差异Ca2+水平。第一个方案允许以高时间和空间分辨率测量细胞质Ca2+。这里使用谷氨酸能AVA命令中间神经元15中GCaMP6f的细胞特异性表达来证明这一点，其神经突贯穿腹侧神经索的整个长度。GCaMP6f荧光（50 Hz）的快速采集能够检测Ca 2+流入的方向传播（...

Watch this Scientific Journal Video about Subcellular Imaging of Neuronal Calcium Handling In Vivo at JoVE.com

Subcellular Imaging of Neuronal Calcium Handling In Vivo

目前的方法描述了一种非比率方法，使用现成的遗传编码钙指示剂在秀丽隐杆线虫中进行高分辨率、亚区室钙成像。

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

该技术可用于促进我们对神经元中钙处理的理解，以及区室钙如何调节对体内神经元功能重要的不同机制。该技术的主要优点是，它可以在秀丽隐杆线虫的生理条件下进行更高分辨率的快速体内成像，可以与其他荧光工具结合使用。这种技术可以在许多不同的背景下提供调节神经元钙信号传导的机制。例如，在衰老、神经元损伤和神经变性期间。演示该程序的将是研究技术员Ennis Deihl。Kaz Knight，博士候选人，Rachel Doser，我实验室的博士后研究员。首先克隆含有启动子的表达载体，然后是钙指示剂和三种选择的素数UTR。通过将含有钙指示剂转基因中的救援 DNA Lin-15 plus 的 DNA 混合物显微注射到一天大的成年秀丽隐杆线虫的性腺中，创建转基因秀丽隐杆线虫菌株。将注射的亲本蠕虫保持在20摄氏度，直到F1后代成年。通过筛查多外阴表型缺失来选择转基因成虫。这表明含有钙指示剂的额外染色体阵列的表达。克隆传代没有多外阴表型的成年F1后代。对具有钙指示剂最佳表达的后续几代转基因菌株进行实验。使用能够进行长期延时成像的显微镜。在低放大倍率物镜下定位蠕虫，然后切换到高放大倍率物镜以定位神经元。使用能够以超过 50 FPS 的速度快速采集图像的超灵敏相机获取图像。接下来，对钙指示剂使用标准排放过滤器。添加 Z 漂移校正器，用于采集超过 10 秒的图像流。在13×100毫米的玻璃培养管中，通过将分子级琼脂溶解在M9中并微波几秒钟来制备三毫升10%琼脂。要制作琼脂垫，首先，通过添加两层实验室胶带来准备两张载玻片。然后将显微镜载玻片放在两个载玻片之间。切下 1000 微升移液器吸头的尖端，并用它来将一小滴琼脂移液到盖玻片的中心。通过在琼脂顶部按下另一张幻灯片来压平琼脂。冷却后，使用10毫升管的开口将琼脂切成小圆盘，然后去除周围的琼脂。接下来，通过将麝香酚粉末溶解在M9中来制备蠕虫滚动溶液，以产生30毫摩尔的原液。用聚苯乙烯珠以一比一的比例稀释原液，制成滚动溶液。为了定位蠕虫进行成像，首先，将1.6微升的滚动溶液放在琼脂垫的中心。然后使用首选的蠕虫镐，将所需年龄的蠕虫转移到琼脂垫上的滚动溶液中。等待大约五分钟，让麝香酚减少蠕虫运动，然后将 22 x 22 毫米的盖玻片放在琼脂垫的顶部。对于腹侧神经索中的神经突成像，通过轻轻滑动盖玻片来滚动蠕虫。要将蠕虫安装在显微镜上，请将一滴浸油滴在盖玻片上。使用明场中的低放大倍率物镜找到蠕虫。然后切换到100倍物镜，并使用GCaMP或mito GCaMP的488纳米成像激光器的照明来定位AVA神经突。对于体内GCaMP成像，在采集设置中调整成像激光，直到罗勒GCaMP荧光处于相机动态范围的中间范围内，并将曝光时间设置为20毫秒。使用 GCaMP 荧光以 100 倍放大倍率定位 AVA 神经突后，设置 Z 漂移校正器，并启动连续自动对焦。成像前根据需要手动校正焦平面。以 100 倍放大倍率获取图像流。使用该协议，以高时间和空间分辨率测量细胞质钙。GCaMP6F在谷氨酸能AVA命令中间神经元中的细胞特异性表达揭示了钙内流的定向增殖。GCaMP6F荧光的亚细胞定量显示，钙内流的开始在仅10微米外的神经突的一部分中延迟。此外，沿着AVA神经突发现的树突状脊柱状结构在GCaMP6F荧光中显示出闪光，该闪光与神经突活化无关，并且不会导致钙流入的传播。此外，使用具有AVA特异性表达线粒体基质局部钙指示剂mito GCaMP的蠕虫菌株测量单个神经突中单个线粒体中的相对钙水平。结果显示，相对大量的钙同步摄取到线粒体的一个子集中。具体来说，钙对一些线粒体的摄取是同步的，而邻近的线粒体似乎没有吸收钙。同样，线粒体亚群显示出少量钙的快速摄取和释放。这似乎也是同步的，但仅适用于线粒体的一个子集。速度和精细操作对于定位动物和保持神经元功能至关重要。动物健康也至关重要。即使是一点点饥饿也是有问题的。最难学习的部分是动物的快速定向，用于共聚焦显微镜而不会损坏。我的建议是大量的练习和良好的控制。该协议可以应用于从神经元，其他神经元或秀丽隐杆线虫中神经元以外的细胞中的其他亚细胞位置释放钙的实验。由于秀丽隐杆线虫中的这种方法使动物完好无损，因此可以解决有关完整神经系统的单个神经元体内亚细胞钙处理调节的问题。

Research

JoVE Journal

Neuroscience

Subcellular Imaging of Neuronal Calcium Handling In Vivo

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

科研

神经科学

Super-resolution Imaging of Neuronal Dense-core Vesicles

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

在永久性幼虫特有的神经重构的C语言活体成像线虫

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...