Name: axonal transport of organelles in motor neuron cultures using microfluidic chambers system
Uploaded: 2020-05-05T14:30:00
Description: Watch this Scientific Journal Video about Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System at JoVE.com

这项工作得到了以色列科学基金会（ISF，561/11）和欧洲研究理事会（ERC，309377）的资助。

本议定书对动物的护理和治疗是在特拉维夫大学动物伦理委员会的监督和批准下进行的。
1. MFC 制备
<ol><li>初级模具中的 PDMS 铸件 （图 1）<ol>
<li>按照详细的协议9购买或创建原模（晶圆）。</li>
		<li>在继续涂层步骤之前，使用加压空气清除晶圆平台的任何类型的污垢。晶圆表面应看起来光滑清晰。</li>
		<li>向容器中注入 50 mL 液氮。准...

<ol>
	<li>Misgeld, T., 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=Mitostasis+in+Neurons:+Maintaining+Mitochondria+in+an+Extended+Cellular+Architecture.">Mitostasis in Neurons: Maintaining Mitochondria in an Extended Cellular Architecture.</a> Neuron. 96, 651-666 (2017).</li><li>Devine, M. J., Kittler, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+at+the+neuronal+presynapse+in+health+and+disease.">Mitochondria at the neuronal presynapse in health and disease.</a> Nature Reviews Neuroscience. 19, 63-80 (2018).</li><li>Gibbs, K. L., Kalmar, B., Sleigh, J. N., Greensmith, L., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+axonal+transport+in+murine+motor+and+sensory+neurons.">In vivo imaging of axonal transport in murine motor and sensory neurons.</a> Journal of Neuroscience Methods. 257, 26-33 (2016).</li><li>Mandal, A., Drerup, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+Transport+and+Mitochondrial+Function+in+Neurons.">Axonal Transport and Mitochondrial Function in Neurons.</a> Frontiers in Cellular Neuroscience. 13, 373 (2019).</li><li>Magran&#233;, J., Cortez, C., Gan, W. B., Manfredi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+mitochondrial+transport+and+morphology+are+common+pathological+denominators+in+SOD1+and+TDP43+ALS+mouse+models.">Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models.</a> Human Molecular Genetics. 23, 1413-1424 (2014).</li><li>Anderson, R. G. W., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+of+acidic+intracellular+compartments.">A view of acidic intracellular compartments.</a> Journal of Cell Biology. 106, 539-543 (1988).</li><li>Kiral, F. R., Kohrs, F. E., Jin, E. J., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab+GTPases+and+Membrane+Trafficking+in+Neurodegeneration.">Rab GTPases and Membrane Trafficking in Neurodegeneration.</a> Current Biology. 28, R471-R486 (2018).</li><li>Ya-Cheng Liao, A., 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=RNA+Granules+Hitchhike+on+Lysosomes+for+Long-Distance+Transport,+Using+Annexin+A11+as+a+Molecular+Tether.">RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.</a> Cell. 179, 147-164 (2019).</li><li>Gluska, S., Chein, M., Rotem, N., Ionescu, A., Perlson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+Quantum-Dot+labeled+neurotropic+factors+transport+along+primary+neuronal+axons+in+compartmental+microfluidic+chambers.">Tracking Quantum-Dot labeled neurotropic factors transport along primary neuronal axons in compartmental microfluidic chambers.</a> Methods in Cell Biology. 131, 365-387 (2016).</li><li>Gershoni-Emek, N., 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=Localization+of+RNAi+Machinery+to+Axonal+Branch+Points+and+Growth+Cones+Is+Facilitated+by+Mitochondria+and+Is+Disrupted+in+ALS.">Localization of RNAi Machinery to Axonal Branch Points and Growth Cones Is Facilitated by Mitochondria and Is Disrupted in ALS.</a> Frontiers in Molecular Neuroscience. 11, 311 (2018).</li><li>Neto, E., 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=Compartmentalized+Microfluidic+Platforms:+The+Unrivaled+Breakthrough+of+In+Vitro+Tools+for+Neurobiological+Research.">Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.</a> Journal of Neuroscience. 36 (46), 11573-11584 (2016).</li><li>Ionescu, A., Zahavi, E. E., Gradus, T., Ben-Yaakov, K., Perlson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmental+microfluidic+system+for+studying+muscle-neuron+communication+and+neuromuscular+junction+maintenance.">Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance.</a> European Journal of Cell Biology. 95, 69-88 (2016).</li><li>Zahavi, E. E., 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+compartmentalized+microfluidic+neuromuscular+co-culture+system+reveals+spatial+aspects+of+GDNF+functions.">A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions.</a> Journal of Cell Science. 128, 1241-1252 (2015).</li><li>Altman, T., Geller, D., Kleeblatt, E., Gradus-Perry, T., Perlson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+compartmental+system+underlines+the+contribution+of+mitochondrial+immobility+to+the+ATP+supply+in+the+NMJ.">An in vitro compartmental system underlines the contribution of mitochondrial immobility to the ATP supply in the NMJ.</a> Journal of Cell Science. 132 (23), (2019).</li><li>Schaller, S., 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=Novel+combinatorial+screening+identifies+neurotrophic+factors+for+selective+classes+of+motor+neurons.">Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.</a> Proceedings of the National Academy of Sciences USA. 114, E2486-E2493 (2017).</li><li>Ionescu, A., 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=Targeting+the+Sigma-1+Receptor+via+Pridopidine+Ameliorates+Central+Features+of+ALS+Pathology+in+a+SOD1G93A+Model.">Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model.</a> Cell Death &amp; Disease. 10 (3), 210 (2019).</li><li>Zahavi, E. E., 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+compartmentalized+microfluidic+neuromuscular+coculture+system+reveals+spatial+aspects+of+GDNF+functions.">A compartmentalized microfluidic neuromuscular coculture system reveals spatial aspects of GDNF functions.</a> Journal of Cell Science. 128, 1241-1252 (2015).</li><li>Maimon, 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=Mir126-5p+downregulation+facilitates+axon+degeneration+and+nmj+disruption+via+a+non-cell-autonomous+mechanism+in+ALS.">Mir126-5p downregulation facilitates axon degeneration and nmj disruption via a non-cell-autonomous mechanism in ALS.</a> Journal of Neuroscience. 38, 5478-5494 (2018).</li></ol>

在此协议中，我们描述了一个系统，用于跟踪运动神经元中线粒体和酸性舱的斧头迁移。这种简化的体外平台允许精确控制、监测和操作亚细胞神经元隔间，从而能够对运动神经元局部功能进行实验分析。该协议可用于研究MN疾病，如ALS，专注于了解在疾病10，16,16的斧路运输功能障碍的基本机制。此外，该系统还可用于研究营养因子9<...

MN是具有长斧子的高度极化细胞，在成年人类中达到一米长。这种现象对维护 MN 连接和功能带来了严峻的挑战。因此，MN 依赖于信息、细胞器和材料的适当传输，沿着斧子从细胞体到突触和背部。各种细胞成分，如蛋白质、RNA和细胞器，通过斧子定期穿梭。线粒体是经常在MN中运输的重要细胞器。 线粒体对于MN的正常活动和功能至关重要，负责ATP的提供、钙缓冲和信号过程11、2。2线粒体的斧子运输是一个经过深入研究的过程33，4。4有趣的是，线粒体迁移的缺陷报告涉及几个神经退行性疾病，特别是MN疾病5。酸性隔间是沿着 MN 斧子移动的内在细胞器的另一个例子。酸性隔间包括分裂体、内体、跨Golgi仪器和某些分泌囊泡6。酸性隔间的斧头运输缺陷在一些神经退行性疾病中也被发现，最近的论文强调了它们在MN疾病中的重要性。
为了有效地研究六角运输，经常使用分离体形和体素室的微流体室9,9，10。微流体系统的两大优点，以及分块化和亚克斯的分离，使得它非常适合研究亚细胞过程11。神经元细胞体和斧子之间的空间分离可用于操纵不同神经元隔间（例如，斧子与躯体）的细胞外环境。生物化学、神经元生长/退化和免疫荧光检测都受益于这个平台。MfCs还可以通过与其他细胞类型（如骨骼肌12、13、14）,13,14等神经元进行协管，帮助研究细胞与细胞之间的交流。
在这里，我们描述了一个简单而精确的协议，用于监测运动神经元中的线粒体和酸性隔间传输。通过比较逆行和逆行运动细胞器的相对百分比以及运输速度的分布，进一步展示了该方法的使用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35mm Fluodish &#8211; glass bottom dish</td>
			<td>World Precision Instruments WPI</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>50mm Fluodish &#8211; glass bottom dish</td>
			<td>World Precision Instruments WPI</td>
			<td>FD5040-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor iXon DU-897 EMCCD camera</td>
			<td>Andor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ARA-C (Cytosine &#946;-D-arabinofuranoside)</td>
			<td>Sigma-Aldrich</td>
			<td>C1768</td>
			<td>stock of 2mM in filtered DDW</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X)</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Alomone Labs</td>
			<td>B-250</td>
			<td>Dilute to 10 &#181;g/mL in filtered ddw with 0.01% BSA)</td>
		</tr>
		<tr>
			<td>Biopsy punch 1.25mm</td>
			<td>World Precision Instruments WPI</td>
			<td>504530</td>
			<td>For preperation of large MFC</td>
		</tr>
		<tr>
			<td>Biopsy punch 6mm</td>
			<td>World Precision Instruments WPI</td>
			<td>504533</td>
			<td>For preperation of small MFC</td>
		</tr>
		<tr>
			<td>Biopsy punch 7mm</td>
			<td>World Precision Instruments WPI</td>
			<td>504534</td>
			<td>For preperation of large MFC</td>
		</tr>
		<tr>
			<td>Bitplane Imaris software - version 8.4.1</td>
			<td>Imaris</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#A3311-100G</td>
			<td>5% w/v in ddw</td>
		</tr>
		<tr>
			<td>Chlorotrimetylsilane</td>
			<td>Sigma-Aldrich</td>
			<td>#386529-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>CNTF</td>
			<td>Alomone Labs</td>
			<td>C-240</td>
			<td>Dilute to 10 &#181;g/mL in filtered ddw with 0.01% BSA)</td>
		</tr>
		<tr>
			<td>Density Gradient Medium - Optiprep</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNAse) from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>DN-25</td>
			<td>stock 10mg/mL in neurobasal</td>
		</tr>
		<tr>
			<td>Dow Corning High-vacuum silicone grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554-1EA</td>
			<td>For epoxy mold preperation</td>
		</tr>
		<tr>
			<td>DPBS 10X</td>
			<td>Thermo Fisher</td>
			<td>#14200-067</td>
			<td>dilute 1:10 in ddw</td>
		</tr>
		<tr>
			<td>Dumont fine forceps #55 0.05 &#215; 0.02 mm</td>
			<td>F.S.T</td>
			<td>1125520</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy Hardener</td>
			<td>Trias Chem S.R.L</td>
			<td>IPE 743</td>
			<td>For epoxy mold preperation</td>
		</tr>
		<tr>
			<td>Epoxy Resin</td>
			<td>Trias Chem S.R.L</td>
			<td>RP 026UV</td>
			<td>For epoxy mold preperation</td>
		</tr>
		<tr>
			<td>FIJI software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Alomone Labs</td>
			<td>G-240</td>
			<td>Dilute to 10 &#181;g/mL in filtered ddw with 0.01% BSA)</td>
		</tr>
		<tr>
			<td>Glutamax 100X</td>
			<td>Thermo Fisher</td>
			<td>#35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>HB9:GFP mice strain</td>
			<td>Jackson Laboratories</td>
			<td>005029</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS 10X</td>
			<td>Thermo Fisher</td>
			<td>#14185-045</td>
			<td>Dilute 1:10 in ddw with addition of 1% P/S and filter</td>
		</tr>
		<tr>
			<td>iQ software</td>
			<td>Andor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors, curved, 10 cm</td>
			<td>AS Medizintechnik</td>
			<td>11-441-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors, straight, 9 cm</td>
			<td>AS Medizintechnik</td>
			<td>11-440-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>#L-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Thermo Fisher</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Red</td>
			<td>Thermo Fisher</td>
			<td>L7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitotracker Deep-Red FM</td>
			<td>Thermo Fisher</td>
			<td>M22426</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti micorscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S) Solution</td>
			<td>Biological Industries</td>
			<td>03-031-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithin (PLO)</td>
			<td>Sigma-Aldrich</td>
			<td>#P8638</td>
			<td>Dilute 1:1000 in flitered 1X PBS</td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>DOW Corning Corporation</td>
			<td>#3097358-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td>stock 25 mg/mL in 1XPBS</td>
		</tr>
		<tr>
			<td>Vannas spring microdissection scissors, 3 mm blade</td>
			<td>F.S.T</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Yokogawa CSU X-1</td>
			<td>Yokogawa</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

axonal transport of organelles in motor neuron cultures using microfluidic chambers system

运动神经元 （MNs） 是具有长斧子的高度偏振细胞。Axonal 传输是 MN 健康的关键机制，有助于神经元的生长、发育和生存。我们描述了一种使用微流体室 （MfCs） 来跟踪 MN 斧子中荧光标记的细胞器的六角传输的详细方法。该方法速度快，价格相对便宜，便于在空间和时间中监测细胞内线索。我们描述了一个分步协议：1） 制造聚二甲基硅氧烷（PDMS）MFCs;2） 在MFC中电内脊髓外泄和MN分离培养的电镀;3） 线粒体和酸性隔间的标签，然后进行实时共聚焦想象;4） 手动和半自动六角运输分析。最后，我们证明了HB9：：GFP腹腔脊髓外植性强子的线粒体和酸性隔间在运输上的差异，以证明系统的有效性。总之，该协议为研究各种六角组件的斧头传输提供了一个有效的工具，以及 MFC 使用的简化手册，以帮助发现空间实验的可能性。

按照所述协议，小鼠胚胎HB9：：GFP脊髓外植在MFC中培养（图4A）。ex植物生长了7天，当斧子完全进入远端隔间。线粒体深红色和Lysotracker红色染料被添加到远端和近端隔间，以标记线粒体和酸性隔间（图4C）。对远端凹槽中的Axons进行了成像，对电影进行了分析如下：首先，我们使用kymograph分析（?...

Watch this Scientific Journal Video about Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System at JoVE.com

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

斧头迁移是运动神经元健康的重要机制。在该协议中，我们提供了一种使用微流体室跟踪运动神经元斧头酸性隔间和线粒体的斧头传输的详细方法。

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

斧质性运输对神经元健康和生存能力至关重要。我们的协议描述了一种用于监测和分析斧弓传输的优雅方法。并可适用于各种神经元疾病模型。使用微流体室可实现精确的空间时间控制，这对研究斧头生物学至关重要。斧质传播异常与多种神经退行性疾病（如ALS）有关。微流体平台支持研究基本机制以及测试疗法，以修复斧头传输缺陷。微流体室平台可以很容易地适应与癌症研究的不同方面，包括各种神经亚型的斧头生长和退化。这种方法很复杂，通常教动手。视觉演示将增加任何有兴趣研究斧弓运输的科学家的可访问性。首先在主模具中铸造 PDMS。使用加压空气清除晶圆平台上的任何污垢，确保晶圆在继续前看起来光滑和清晰。接下来，将液氮填充容器，用 23 量针准备 10 毫升注射器。在化学烟气罩中，将含有晶圆的晶圆放在可密封的容器中，并在注射器中填充每个含有板的晶圆两毫升液氮。打开氯三甲基硅烷瓶。用注射器刺穿橡胶盖，将氮气注入瓶子中。在不拔出针头的情况下，将瓶子倒置，并针对每个含有板的晶圆降低两毫升氯三甲基硅烷。将氯三甲基硅烷分散在容器中，但不得直接在含晶片上。关闭容器。每片晶圆孵育五分钟。接下来在 50 毫升管中重 47.05 克 PDMS 基座，以 16 比 1 基比加添加 PDMS 固化剂，总重量为 50 克。在低速旋转器上混合 PDMS 10 分钟，然后倒入每个包含板的晶圆中，达到所需的高度。将板放在真空干燥器内两个小时。这将去除滞留在PDMS内的空气，并形成一个清晰的均匀模具。之后在烤箱中孵育盘子三小时或在70摄氏度下过夜。创建微流体室或 MFC。用手术刀切割并去除板中的 PDMS 模具，确保不要因为模具易碎而强制模具。按照文本手稿中的说明打孔和切割腔室，具体取决于实验设置。对于脊髓外植培养，在大型 MFC 的端侧打两个 7 毫米井，确保它们与通道边缘重叠。在近端，在通道中间打一个七毫米井，与最小的重叠，以便有足够的空间留给外植。在近角通道的两个边缘打两个额外的一毫米孔。将 PDMS 切成单个腔室。然后将 MFC 朝上，用 20 测量针在打孔 7 毫米井上的三个小外层洞穴雕刻。对于分离的运动神经元培养，在小型 MFC 的两个通道的边缘打四个六毫米的孔，将 PDMS 切割成单个腔室。要对 MFC 进行消毒，请将 50 厘米长的胶带在长长的长磁带上铺在长凳上。然后按胶带上的腔室的两个面，然后将其拉回来。将清洁室放在新板中，并在分析等级中孵育70%乙醇，在轨道摇床上孵育10分钟。将乙醇处理，并在组织培养罩或70摄氏度的烤箱中干燥室。然后将腔室放在组织培养级玻璃底沟的中心，并在边缘施加轻微力，将 PDMS 与菜底结合。在70摄氏度下孵育10分钟。然后按压室，加强依从性。在紫外线下孵育碟子10分钟，然后继续编码MFC。每毫升PL二节增加1.5毫微克。确保巴解组织通过这些渠道运行。直接在通道入口处移液涂层介质。在光学显微镜下检查 MFC，检查气泡。如果气泡阻塞了微槽，请将 MFC 放在真空干燥器中两分钟。从 MFC 通道中清除多余的空气。然后与巴解组织一夜之间孵育。用层压代替巴解组织，再孵育 MFC 过夜。电镀前，用培养介质清洗层压素。解剖一只 ICR E12.5 怀孕的小鼠，将胚胎与羊膜囊分离。将胚胎放入HBSS硅盘中。取下头部和尾巴，翻转到他的肚子上。轻轻地从胚胎上剥下肤浅的皮肤，露出脊髓。使用柔和的钳子将脊髓从头部分离到头部和尾部。取出脑膜，然后取下后角，将脊髓切成一毫米厚的横向部分。从 MFC 的近侧隔间中处置所有介质。拿起一个脊髓外植与移液器，在总体积为四微升。并注射它尽可能接近洞穴。通过横向出口从近井中抽出多余的液体。再重复上一步两次，并确保外植嵌入近向通道中。慢慢添加150微升的SCEX介质，到近井。电镀后一天。更换近侧隔间中的 SCEX 介质，并将丰富的 SCEX 介质添加到近侧隔间。在近井和近井之间保持每口井至少 15 微升的体积梯度。四到六天后，轴子应穿过到外侧隔间，并准备好进行轴向传输成像。要标记线粒体和酸性隔间，准备新的SCEX介质与100纳米摩尔米托跟踪深红色调频，和100纳米摩尔LysoTracker红色。并将其添加到微流体室。在37摄氏度下孵育30至60分钟。然后用温暖的SCEX介质洗三次。继续对斧弓传输进行实时成像。以三秒间隔获取 100 个时间间隔图像系列。每部电影总共五分钟。在微流体室中七天后。小鼠胚胎HB9 GFP脊髓外植。我们沾满了米托跟踪器深红色和利索跟踪红色染料。给线粒体和酸性隔间贴上标签。对义沟中的轴孔进行了成像，并采用Kymograph分析来确定一般运动分布。这揭示了逆行方向的偏差，仅在酸性城市隔间，但不是在线粒体运输。粒子密度也得到量化，与HB9 GFP脊髓外子轴突中的酸性隔间相比，线粒体颗粒数量更高。接下来，使用半自动化软件进行单粒子传输分析，然后使用内部代码进行。这一分析表明，酸性成分和线粒体具有相似的粒子速度。但是，只有酸性隔间表现出对逆行运动的偏向。隔离胚胎脊髓可能很难在第一。多次练习这个程序，确保使用正确的胚胎年龄和适当的解剖工具。微流体室的使用改变了我们研究斧头生物学的方式。它允许我们可视化局部的斧神群过程，我们曾经假设这些过程只发生在细胞体中。

Research

JoVE Journal

Neuroscience

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

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

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

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 ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

神经肌肉接头处：测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

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 ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...