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。

microfluidics-assisted-selective-depolarization-of-axonal-mitochondria

Research

JoVE Journal

Neuroscience

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

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

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 injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

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 ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

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

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

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

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using 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

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using &quot;Cargo Mapping&quot; Analysis

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

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

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

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

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, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

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

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

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

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

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 ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...