Name: microfluidics-assisted selective depolarization of axonal mitochondria
Uploaded: 2022-08-04T14:15:00
Description: Watch this Scientific Journal Video about Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria at JoVE.com

这项研究得到了德国研究基金会（HA 7728/2-1和EXC2145项目编号390857198）和马克斯普朗克学会的支持。

所有动物实验均按照上巴伐利亚州政府的相关准则和法规进行。原代神经元由E16.5 C57BL / 6野生型小鼠胚胎制备，遵循先前描述的标准方法6。
1. 微流控装置的组装
<ol><li>在PBS（磷酸盐缓冲盐水）中涂覆一个六孔玻璃底组织培养板，终浓度为20μg/ mL的聚-D-赖氨酸和3.4μg/ mL层粘连蛋白（参见 材料表）。</li>
	<li>将涂层板在室温下在黑暗...

<ol>
	<li>Murali Mahadevan, H., Hashemiaghdam, A., Ashrafi, G., Harbauer, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+neuronal+health:+from+energy+metabolism+to+Parkinson's+disease.">Mitochondria in neuronal health: from energy metabolism to Parkinson's disease.</a> Advanced Biology. 5 (9), 2100663 (2021).</li><li>Dauer, W., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+mechanisms+and+models.">Parkinson's disease: mechanisms and models.</a> Neuron. 39 (6), 889-909 (2003).</li><li>Moratalla, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+vulnerability+of+primate+caudate-putamen+and+striosome-matrix+dopamine+systems+to+the+neurotoxic+effects+of+1-methyl-4-phenyl-1,2,3,6-+tetrahydropyridine.">Differential vulnerability of primate caudate-putamen and striosome-matrix dopamine systems to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine.</a> Proceedings of the National Academy of Sciences. 89 (9), 3859-3863 (1992).</li><li>Cheng, H. -. C., Ulane, C. M., Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+progression+in+Parkinson+disease+and+the+neurobiology+of+axons.">Clinical progression in Parkinson disease and the neurobiology of axons.</a> Annals of Neurology. 67 (6), 715-725 (2010).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2 (8), 599-605 (2005).</li><li>Harbauer, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+mitochondria+transport+Pink1+mRNA+via+synaptojanin+2+to+support+local+mitophagy.">Neuronal mitochondria transport Pink1 mRNA via synaptojanin 2 to support local mitophagy.</a> Neuron. 110 (9), 1516-1531 (2022).</li><li>Ashrafi, G., Schlehe, J. S., LaVoie, M. J., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+of+damaged+mitochondria+occurs+locally+in+distal+neuronal+axons+and+requires+PINK1+and+Parkin.">Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.</a> Journal of Cell Biology. 206 (5), 655-670 (2014).</li><li>Shipman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic+acid+(HEPES)+as+a+tissue+culture+buffer.">Evaluation of 4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic acid (HEPES) as a tissue culture buffer.</a> Proceedings of the Society for Experimental Biology and Medicine. 130 (1), 305-310 (1969).</li><li>Harbauer, A. B., Schneider, A., Wohlleber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondria+by+single-organelle+resolution.">Analysis of mitochondria by single-organelle resolution.</a> Annual Review of Analytical Chemistry. 15, 1-16 (2022).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+mRNA+in+uninjured+and+regenerating+cortical+mammalian+axons.">Axonal mRNA in uninjured and regenerating cortical mammalian axons.</a> The Journal of Neuroscience. 29 (15), 4697-4707 (2009).</li><li>Altman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+TDP-43+condensates+drive+neuromuscular+junction+disruption+through+inhibition+of+local+synthesis+of+nuclear+encoded+mitochondrial+proteins.">Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins.</a> Nature Communications. 12 (1), 1-17 (2021).</li></ol>

本协议描述了一种在微流体装置中播种和培养解离的海马神经元以分别治疗轴突线粒体的方法。本文展示了这种方法与膜敏感染料TMRE和复合物III抑制剂抗霉素A（如前所述7）的实用性，但该方法可以很容易地适应其他线粒体染料或线粒体功能的遗传编码传感器，允许局部的，基于显微镜的读数9。其他神经元细胞类型也可以在微流体室中生长，例如原代皮质神经...

线粒体是神经元中ATP（三磷酸腺苷）的主要供应商。由于神经元健康与线粒体功能密切相关，因此这些细胞器的功能失调与各种神经退行性疾病（包括帕金森病）的发作有关也就不足为奇了1。此外，线粒体中毒已成功用于模拟动物的帕金森病症状2。在动物模型和人类疾病中，神经元的死亡始于远端3，4，这表明轴突线粒体可能更容易受到侮辱。然而，轴突中线粒体的生物学尚不清楚，因为与轴突线粒体的靶向治疗和分析相关的困难，而不会同时干扰细胞体过程。
体外解离神经元培养技术的最新进展现在允许通过微流体装置对轴突和细胞体进行流体分离5。如图1A所示，这些器件具有四个接入井（a/h和c/i），每对（d和f）有两个通道连接。大通道通过一系列450μm长的微通道（e）相互连接。两个腔室之间填充水平的故意差异会产生流体压力梯度（图1B），防止小分子从液位较低的通道扩散到另一侧（图1C，用台盼蓝染料说明）。
我们最近使用微流体装置来研究轴突线粒体自噬的局部翻译要求，即选择性去除受损线粒体6。在本协议中，提出了通过使用线粒体复合物III抑制剂抗霉素A6，7选择性治疗轴突来诱导局部线粒体损伤的不同步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well Glass bottom plate</td>
			<td>Cellvis</td>
			<td>P06.1.5H-N</td>
			<td>Silicone device</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS M5000 widefield microscope</td>
			<td>Thermofischer Scientific</td>
			<td>EVOS M5000</td>
			<td>fully integrated digital widefield microscope</td>
		</tr>
		<tr>
			<td>Hibernate E</td>
			<td>BrainBits</td>
			<td>HE500</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted spinning disk confocal</td>
			<td>Nikon</td>
			<td>TI2-E + CSU-W1</td>
			<td>With incubator chamber</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic devices</td>
			<td>XONA microfluidics</td>
			<td>RD450</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>Sigma</td>
			<td>87917</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidics-assisted selective depolarization of axonal mitochondria

线粒体是神经元中ATP（三磷酸腺苷）的主要供应商。线粒体功能障碍是许多神经退行性疾病的常见表型。鉴于一些轴突的复杂结构和极端的长度，与细胞体对应物相比，轴突中的线粒体可以经历不同的环境也就不足为奇了。有趣的是，轴突线粒体的功能障碍通常先于对细胞体的影响。为了在体 外模拟轴突线粒体功能障碍，微流体装置允许在不影响体细胞线粒体的情况下治疗轴突线粒体。这些腔室中的流体压力梯度阻止了分子逆梯度扩散，从而允许分析线粒体特性以响应轴突内的局部药理学挑战。目前的协议描述了在微流体装置中分离的海马神经元的接种，用膜电位敏感染料染色，用线粒体毒素处理以及随后的显微镜分析。这种研究轴突生物学的通用方法可以应用于许多药理学扰动和成像读数，并且适用于几种神经元亚型。

原代海马神经元在微流体装置中生长7-8天，然后线粒体在两个通道中用膜敏感染料（TMRE）染色25分钟。如图2A所示，这在微凹槽的两侧产生了线粒体的均匀染色，但不足以平衡微槽中间的染色。在轴突侧加入抗霉素A后，体细胞线粒体保留TMRE信号（图2B和视频1），而轴突线粒体的TMRE荧光丢失（图2C和视频1）。</st...

Watch this Scientific Journal Video about Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria at JoVE.com

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

本协议描述了微流体室中神经元线粒体的播种和染色。这些腔室中的流体压力梯度允许选择性处理轴突中的线粒体，以分析其性质以应对药理学挑战而不影响细胞体区室。

微流体辅助轴突线粒体选择性去极化

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many ...

线粒体对神经元健康至关重要。在许多神经退行性疾病中，轴突线粒体是第一个遭受痛苦的疾病，但尚不清楚是什么让轴突线粒体如此特别。本协议中使用的腔室中的流体压力梯度允许对线粒体轴突进行选择性处理。这使得细胞体过程不受干扰，并使我们能够专注于轴突生物学。我们在这里展示了线粒体用膜电位敏感染料的去极化，但这种方法可以应用于许多不同的神经元细胞类型和荧光读数。首先，将编码的六个孔板放入无菌罩中，并用无菌双蒸水洗涤两次。让板在倾斜位置干燥三到五分钟。通过真空抽吸或移液去除多余的水分。然后将微流体室浸泡在80%乙醇中。再次，在倾斜位置干燥微流体室三到五分钟，然后用移液器吸头除去多余的乙醇。完全干燥后，将微流体室放在孔和板的中心。轻轻敲击微流体室的边界和中间的微槽部分，以正确连接到玻璃板上。首先，在1.5毫升反应管中收集所需数量的解离海马神经元。在室温下以1000倍G离心管四分钟。弃去上清液，并将沉淀重悬于八微升B-27神经基础培养基中。然后将细胞溶液分配到微流体室的通道入口中。点击板的背面以协助流过通道。使用移液器在通道出口处吸出任何剩余的细胞悬液。将微流体室与细胞在37摄氏度和5%二氧化碳下孵育15至20分钟。接下来，用50微升B-27神经基础培养基填充顶部轴突孔，并点击板的背面以辅助流动。用 150 微升 B-27 神经基础培养基填充体侧的每个孔，在顶部形成一个张力气泡。还要用100微升的B-27神经基础培养基填充轴突侧的两个孔。体外培养七到八天后，确保轴突已通过微凹槽生长，并延伸到轴突隔室。通过去除 B-27 神经基础培养基并加入 100 微升预热的成像培养基到轴突和体细胞通道的顶部孔中来清洗装置。让培养基流向较低的孔。从下孔中取出培养基，从顶部孔中取出任何剩余的培养基，然后用新鲜培养基重复，在洗涤结束时使孔空。然后将100微升的5纳摩尔TMRE稀释液加入体细胞和轴突顶孔中。让培养基流过两个隔室，直到所有孔中的体积相等。用含有TMRE的培养基填充。在体细胞隔室中选择一个感兴趣的区域，并跟随它穿过微凹槽进入轴突隔室。确保在体细胞和轴突隔室中成像的微凹槽彼此匹配。更改文件名后，使用561纳米激发和625纳米发射波长获取红色荧光图像。通过检查体细胞侧是否存在张力气泡来确保体细胞和轴突腔之间的体积差异。然后从轴突侧的两个孔中取出成像介质，通道内的培养基除外。为了诱导线粒体去极化，将160微升在成像培养基中稀释的20微摩尔抗霉素A添加到顶部轴突孔中。让它流过通道，直到两个轴突孔中的体积相等。重复成像，通过识别微凹槽，确保成像的位置与基线采集期间相同。使用该协议，原代海马神经元在微流体装置中生长，然后用膜敏感染料TMRE进行线粒体染色。在微槽的两侧观察到线粒体的均匀染色，但在中间没有。在轴突侧加入抗霉素A后，体细胞线粒体保留TMRE信号，而轴突线粒体的荧光丢失 在组装设备之前，请确保孔中或设备上没有水分。否则，腔室将不会密封。使用这种方法，我们可以研究轴突线粒体功能丧失和局部功能的影响，以及它对逆行信号传导和全局细胞存活的影响。长期以来，人们一直认为线粒体生物发生只发生在细胞体内。这种方法使我们能够揭示轴突线粒体损伤需要对短蛋白PINK1进行局部翻译。

Research

JoVE Journal

Neuroscience

Mechanical Manipulation of Neurons to Control Axonal Development

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

科研

神经科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Subtype-selective Electroporation of Cortical Interneurons

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

表征分子马达的组成对运动轴突货物用“货物映射”分析

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

使用串行块面部扫描电子显微镜脑线粒体分析

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...