Name: visualizing astrocyte morphology using lucifer yellow iontophoresis
Uploaded: 2019-09-14T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis at JoVE.com

作者感谢索托女士、余博士和Octeau博士对文本的指导以及评论。NS060677 支持此工作。

这项研究中的动物实验是按照美国国家卫生指南，在实验室动物的护理和使用，并批准校长的动物研究委员会在洛杉矶加州大学。所有实验中都使用了混合性别的成年小鼠（6-8周大）。
1. 解决方案准备
<ol><li>
人工脑脊液（ACSF）溶液
	<ol>
<li>在每个实验前制备新鲜的 ACSF 溶液（135 mM NaCl、5 mM KCl、1 mM MgCl 2、14.7 mM NaHCO3、11mM D-葡萄糖、1.25 mM Na2HPO<su...

<ol>
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J., Murk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+Shapeshifter:+Mechanisms+Allowing+Astrocytes+to+Respond+to+the+Changing+Nervous+System+During+Development,+Injury+and+Disease.">Important Shapeshifter: Mechanisms Allowing Astrocytes to Respond to the Changing Nervous System During Development, Injury and Disease.</a> Frontiers in Cellular Neuroscience. 12, 261 (2018).</li><li>Chai, H., 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=Neural+Circuit-Specialized+Astrocytes:+Transcriptomic,+Proteomic,+Morphological,+and+Functional+Evidence.">Neural Circuit-Specialized Astrocytes: Transcriptomic, Proteomic, Morphological, and Functional Evidence.</a> Neuron. 95 (3), 531-549 (2017).</li><li>Sun, D., Jakobs, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+remodeling+of+astrocytes+in+the+injured+CNS.">Structural remodeling of astrocytes in the injured CNS.</a> Neuroscientist. 18 (6), 567-588 (2012).</li><li>Naskar, S., Chattarji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+Elicits+Contrasting+Effects+on+the+Structure+and+Number+of+Astrocytes+in+the+Amygdala+versus+Hippocampus.">Stress Elicits Contrasting Effects on the Structure and Number of Astrocytes in the Amygdala versus Hippocampus.</a> eNeuro. 6 (1), (2019).</li><li>Sun, D., Lye-Barthel, M., Masland, R. H., Jakobs, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+and+spatial+arrangement+of+astrocytes+in+the+optic+nerve+head+of+the+mouse.">The morphology and spatial arrangement of astrocytes in the optic nerve head of the mouse.</a> Journal of Comparative Neurology. 516 (1), 1-19 (2009).</li><li>Grosche, 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=Versatile+and+simple+approach+to+determine+astrocyte+territories+in+mouse+neocortex+and+hippocampus.">Versatile and simple approach to determine astrocyte territories in mouse neocortex and hippocampus.</a> PLoS ONE. 8 (7), 69143 (2013).</li><li>Kaynig, V., 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=Large-scale+automatic+reconstruction+of+neuronal+processes+from+electron+microscopy+images.">Large-scale automatic reconstruction of neuronal processes from electron microscopy images.</a> Medical Image Analysis. 22 (1), 77-88 (2015).</li><li>Bushong, E. A., Martone, M. E., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasmic+astrocytes+in+CA1+stratum+radiatum+occupy+separate+anatomical+domains.">Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.</a> Journal of Neuroscience. 22 (1), 183-192 (2002).</li><li>Wilhelmsson, U., 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=Redefining+the+concept+of+reactive+astrocytes+as+cells+that+remain+within+their+unique+domains+upon+reaction+to+injury.">Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (46), 17513-17518 (2006).</li><li>Williams, M. 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=Cadherin-9+regulates+synapse-specific+differentiation+in+the+developing+hippocampus.">Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus.</a> Neuron. 71 (4), 640-655 (2011).</li><li>Ogata, K., Kosaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+quantitative+analysis+of+astrocytes+in+the+mouse+hippocampus.">Structural and quantitative analysis of astrocytes in the mouse hippocampus.</a> Neuroscience. 113 (1), 221-233 (2002).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Hubbard, J. A., Hsu, M. S., Seldin, M. M., Binder, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Astrocyte+Water+Channel+Aquaporin-4+in+the+Mouse+Brain.">Expression of the Astrocyte Water Channel Aquaporin-4 in the Mouse Brain.</a> ASN Neuro. 7 (5), (2015).</li><li>Benediktsson, 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=Ballistic+labeling+and+dynamic+imaging+of+astrocytes+in+organotypic+hippocampal+slice+cultures.">Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures.</a> Journal of Neuroscience Methods. 141 (1), 41-53 (2005).</li><li>Fouquet, C., 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=Improving+axial+resolution+in+confocal+microscopy+with+new+high+refractive+index+mounting+media.">Improving axial resolution in confocal microscopy with new high refractive index mounting media.</a> PLoS ONE. 10 (3), 0121096 (2015).</li><li>Luna, G., 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=Astrocyte+structural+reactivity+and+plasticity+in+models+of+retinal+detachment.">Astrocyte structural reactivity and plasticity in models of retinal detachment.</a> Experimental Eye Research. 150, 4-21 (2016).</li><li>Octeau, J. C., 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=An+Optical+Neuron-Astrocyte+Proximity+Assay+at+Synaptic+Distance+Scales.">An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales.</a> Neuron. 98 (1), 49-66 (2018).</li><li>Sosunov, A. 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=Phenotypic+heterogeneity+and+plasticity+of+isocortical+and+hippocampal+astrocytes+in+the+human+brain.">Phenotypic heterogeneity and plasticity of isocortical and hippocampal astrocytes in the human brain.</a> Journal of Neuroscience. 34 (6), 2285-2298 (2014).</li><li>Park, Y. 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=Astrocyte+Specificity+and+Coverage+of+hGFAP-CreERT2+[Tg(GFAP-Cre/ERT2)13Kdmc]+Mouse+Line+in+Various+Brain+Regions.">Astrocyte Specificity and Coverage of hGFAP-CreERT2 [Tg(GFAP-Cre/ERT2)13Kdmc] Mouse Line in Various Brain Regions.</a> Experimental Neurobiology. 27 (6), 508-525 (2018).</li><li>Koeppen, J., 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=Functional+Consequences+of+Synapse+Remodeling+Following+Astrocyte-Specific+Regulation+of+Ephrin-B1+in+the+Adult+Hippocampus.">Functional Consequences of Synapse Remodeling Following Astrocyte-Specific Regulation of Ephrin-B1 in the Adult Hippocampus.</a> Journal of Neuroscience. 38 (25), 5710-5726 (2018).</li><li>Jefferis, G. S., Livet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+and+combinatorial+neuron+labelling.">Sparse and combinatorial neuron labelling.</a> Current Opinion in Neurobiology. 22 (1), 101-110 (2012).</li><li>Lanjakornsiripan, D., 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=Layer-specific+morphological+and+molecular+differences+in+neocortical+astrocytes+and+their+dependence+on+neuronal+layers.">Layer-specific morphological and molecular differences in neocortical astrocytes and their dependence on neuronal layers.</a> Nature Communications. 9 (1), 1623 (2018).</li></ol>

本文概述的方法描述了一种在轻度固定脑切片中使用LY染料细胞内电磷化来可视化星形细胞形态的方法。该协议中强调了几个关键因素，有助于成功的LY淋巴磷酸和细胞的形态重建。一个因素是图像的质量和可重复性，这主要取决于小鼠的年龄和灌注的结果。在这项研究中，我们使用6~8周大的C57/BL6N小鼠。成功的灌注（高度依赖于渗透针的正确放置，并注意到由白色的大脑和脑血管中没有血液）是?...

星形细胞是中枢神经系统（CNS）中最丰富的胶质细胞。它们在ion平衡、血流调节、突触形成以及消除和神经递质接受1中发挥作用。星形细胞函数的范围广泛反映在其复杂的形态结构2，3。星形细胞包含几个初级和次要分支，它们分为数千个细枝和传单，直接与突触、树突、斧头、血管和其他胶质细胞相互作用。星形细胞形态因大脑区域而异，这可能暗示它们在神经元回路4中不同地执行其功能的能力。此外，星形细胞已知在发育期间、生理条件和多种疾病状态3、5、6期间改变其形态。
需要一种一致、可重复的方法来准确解决星形细胞形态的复杂性。传统上，免疫性细胞化学已经被用来可视化星形细胞与星形细胞特定或星形细胞富集蛋白质标记。然而，这些方法揭示了蛋白质表达的模式，而不是星形细胞的结构。常用的标记物，如胶质纤维酸性蛋白（GFAP）和S100钙结合蛋白β（S100+），不表达在整个细胞体积中，因此不解决完整的形态7。在星形细胞中普遍存在的荧光蛋白（病毒注射或转基因小鼠报告线）的遗传方法可以识别更细的分支和整体区域。然而，很难区分单个星形细胞，并且分析可能偏向于特定启动子8所针对的星形细胞群。串行截面电子显微镜已被用来揭示星形细胞过程与突触相互作用的详细图片。由于数以千计的星形细胞过程接触突触，目前无法用这种技术9重建整个细胞，尽管随着使用机器学习方法进行数据分析，这种情况预计会发生变化。
在本报告中，我们重点介绍了使用细胞内风泳与Lucifer黄色（LY）染料来描述小鼠星形细胞的过程，以CA1层辐射为例。该方法是基于埃里克·布松和马克·埃利斯曼10、11的开创性过去工作。从轻固定脑切片的星形细胞由其独特的索马形状识别，并充满LY。然后，细胞通过共聚焦显微镜进行成像。我们演示了如何使用LY淋巴磷光来重建单个星形细胞，并对其过程和领地进行详细的形态分析。此外，该方法可与免疫组织化学结合使用，以识别星形细胞和神经元、其他胶质细胞和脑血管之间的空间关系和相互作用。我们认为LY电泳是一个非常合适的工具，用于分析不同大脑区域的形态和小鼠模型的健康或疾病状况7，12，13。

<table border="0" cellpadding="0" cellspacing="0" style="width:995px;" width="994">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">10% Buffered Formalin Phosphate</td>
			<td style="width:192px;">Fisher</td>
			<td style="width:196px;">SF 100-20</td>
			<td style="width:240px;">An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Acrodisc Syringe Filters with Supor Membrane</td>
			<td style="width:192px;">Pall</td>
			<td style="width:196px;">4692</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ag/AgCl ground pellet</td>
			<td>WPI</td>
			<td>EP2</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa Fluor 546 goat anti-chicken IgG (H+L)</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td>A-11040</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa Fluor 647 goat anti-rabbit IgG (H+L)</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td>A27040</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Anti Aquaporin-4 antibody</td>
			<td style="width:192px;">Novus Biologicals</td>
			<td>NBP1-87679</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Anti GFAP antibody</td>
			<td style="width:192px;">Abcam</td>
			<td style="width:196px;">ab4674</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass pipettes with filament</td>
			<td>World precision instruments</td>
			<td style="width:196px;">1B150F-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6NTac mice</td>
			<td>Taconic Stock</td>
			<td style="width:196px;">B6</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>21108</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal laser-scanning microscope</td>
			<td style="width:192px;">Olympus</td>
			<td style="width:196px;">FV1000MPE</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td style="width:192px;">Sigma</td>
			<td>G7528</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disodium Phosphate</td>
			<td style="width:192px;">Sigma</td>
			<td>255793</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrode puller- Model P-97</td>
			<td>Sutter</td>
			<td style="width:196px;">P-97</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fluoromount-G</td>
			<td style="width:192px;">Southern Biotech</td>
			<td style="width:196px;">0100-01</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Heparin sodium injection (1,000 USP per mL)</td>
			<td style="width:192px;">Sagent Pharmaceuticals</td>
			<td>400-10</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris software (Version 7.6.5)</td>
			<td style="width:192px;">Bitplane Inc.</td>
			<td style="width:196px;"></td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isofluorane</td>
			<td style="width:192px;">Henry Schein Animal Health</td>
			<td>29404</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lidocaine Hydrochloride Injectable (2%)</td>
			<td style="width:192px;">Clipper</td>
			<td>1050035</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lucifer Yellow CH dilithium salt</td>
			<td style="width:192px;">Sigma</td>
			<td>L0259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lucifer Yellow CH dipotassium salt</td>
			<td style="width:192px;">Sigma</td>
			<td>L0144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>M8266</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microscope Cover Glass</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td style="width:196px;">24X60-1</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microscope Slides</td>
			<td style="width:192px;">Fisher</td>
			<td style="width:196px;">12-544-2</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Normal Goat Serum</td>
			<td style="width:192px;">Vector Laboratories</td>
			<td>S-1000</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective lens (40x)</td>
			<td>Olympus</td>
			<td style="width:196px;">LUMPLFLN 40XW</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective lens (60x)</td>
			<td>Olympus</td>
			<td>PlanAPO 60X</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PBS tablets, 100 mL</td>
			<td style="width:192px;">VWR</td>
			<td>VWRVE404</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pipette micromanipulator- Model ROE-200</td>
			<td style="width:192px;">Sutter</td>
			<td>MP-285 / ROE-200 / MPC-200</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Potassium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>P3911</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td style="width:192px;">Sigma</td>
			<td>S5761</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td style="width:192px;">Sigma</td>
			<td>S5886</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stimulator- Model Omnical 2010</td>
			<td>World precision instruments</td>
			<td style="width:196px;">Omnical 2010</td>
			<td>A similar alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Triton X 100</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:196px;">T8787</td>
			<td>An identical alternative can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome- Model #3000</td>
			<td style="width:192px;">Pelco</td>
			<td style="width:196px;">100-S</td>
			<td>A similar alternative can be used</td>
		</tr>
	</tbody>
</table>

visualizing astrocyte morphology using lucifer yellow iontophoresis

星形细胞是神经回路的重要组成部分。它们将整个中枢神经系统 （CNS） 分片，并参与各种功能，包括神经递质清除、组子调节、突触调节、神经元代谢支持和血流调节。星形细胞是一个复杂的细胞，具有索马，几个主要分支，和许多精细的过程，接触神经皮内不同的细胞元素。为了评估星形细胞的形态，有必要有一个可靠和可重复的方法来可视化其结构。我们报告一种可靠的方案，在成年小鼠的轻度固定脑组织中使用荧光路西法黄色（LY）染料对星形细胞进行细胞内电泳。该方法具有几个特征，可用于表征星形细胞形态。它允许对单个星形细胞进行三维重建，这有利于对其结构的不同方面进行形态分析。免疫组织化学与LY电泳也可用于了解星形细胞与神经系统不同成分的相互作用，并评估标记的星形细胞中蛋白质的表达。该协议可以在各种中枢神经系统疾病小鼠模型中实现，用光显微镜严格检查星形细胞形态。LY风泳提供了一种评估星形细胞结构的实验方法，特别是在损伤或疾病的情况下，这些细胞被建议经历显著的形态变化。

这项研究中报告的数据来自每个实验中4只小鼠的7-12个细胞。在适当情况下，在图形面板中报告平均数据。
为了评估星形细胞形态，我们使用LY染料进行细胞内风泳，以填充CA1层辐射中的星形细胞，如图1总结。图2描绘了一个代表性的星形细胞及其精细的形态结构。使用 488 nm 激光线（步长 0.3 μm 和 3.0±3.5 倍数字变焦）在共聚焦激光扫描显微...

Watch this Scientific Journal Video about Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis at JoVE.com

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

星形细胞是形态复杂的细胞，其多重过程和茂密的区域就是例证。为了分析它们的精细形态，我们提出了一种可靠的方案，用于在轻度固定组织中执行细胞内路西法黄色淋病。

使用路西法黄电磷学可视化星形形态

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

这种方法可用于促进非常精细的星细胞过程的可视化，用于评估疾病和稳定状态期间突触的星细胞神经元相互作用。此技术也可以应用于任何小鼠模型、细胞群体或大脑区域。要准备具有适当电阻的锐电极，请用微管拉拔器上的灯丝拉拔硅酸盐玻璃单筒电极。将拉拔的电极存放在封闭的容器中，以防止灰尘进入尖端，并保持电极从箱底升高，以防止尖端断裂。要用荧光笔黄色染料溶液填充电极，请将电极放在垂直位置，尖端朝下，将一到两个微升荧光笔黄色溶液移液器插入电极背面。等待 5 到 10 分钟，让溶液通过毛细管作用移动到尖端，然后轻轻地将填充电极固定到连接到操纵器的电极支架中，电极支架的银线与电极内的荧光素黄色接触。对于染料喷射测试，轻轻地将一个轻轻固定的大脑切片放入一个玻璃底部盘，在室温下装满0.1摩尔PBS，用带尼龙弦的铂琴将切片固定到位。将电极连接到电压源，将接地电极放入包含脑片的浴缸中。将共体显微镜的目标移动到感兴趣的大脑区域，并使用 10X 水浸透镜将电极降入溶液中，将电极移动到视场中心。在亮场照明下，使用 40X 水浸透镜仔细检查电极，以确保电极清晰无碎屑或气泡。使用 488 纳米激光切换到共声激光扫描显微镜。以 12 伏打开刺激器以测试染料喷射。较大的荧光染料云应可见在电极的尖端。然后，在亮场下缓慢地将电极朝向切片，停在表面上方。要用离子磷填充感兴趣的星形细胞，请使用红外差分干扰对比度来识别直径约 10 微米的细长椭圆形索玛塔的星形细胞，在切片表面以下 40 到 50 微米。这是海马的地层半径，正下方是金字塔细胞层。当我们通过组织移动时，我们可以看到血管、神经元、星细胞和其他细胞类型。选择星细胞后，将细胞移动到视场中心，然后缓慢地将电极尖端降低到切片中，在组织中导航，直到电极与细胞体位于同一平原上。一旦星形细胞的细胞体清晰可见并勾勒出轮廓，缓慢而轻轻地向前推进电极，移动电极，直到尖端刺穿细胞的索马。缓慢地上下移动目标的焦点，以注意电极是否位于索马内。电极尖端进入电池内后，以 0.5 至 1 伏打开刺激器，以连续将电流喷射到电池中。使用共体显微镜观看细胞膜，增加数字变焦以可视化细胞的细节，并确保电极的尖端在细胞内可见。等待约 15 分钟，直到更精细的分支和过程出现定义，然后关闭电压，轻轻地从电池中退出电极尖端。要对填充的细胞进行成像，请等待 15 到 20 分钟，让细胞返回到原始形式，然后调整共合显微镜上的设置，以确保在 40 倍目标下出现更精细的分支和过程。设置步长为 0.3 微米的 Z 堆栈，在成像时移动目标，直到没有来自单元的信号，然后将该步骤设置为顶部。然后向下移动目标，对焦穿过单元格，直到没有信号，然后将该步骤设置为底部。成像完成后，检查电极有无染料弹出。如果出现大型染料云，电极可用于下一片。否则，更换电极。通过生成最大强度投影，可以观察到其域中星细胞结构的详细视图。四 X 级放大到星细胞显示一个主要分支、几个次要分支的最大强度投影，以及周围分支和传单的分布。此处显示 CA1 星细胞的原始图像。在这些后续图像中重建由星形细胞所包围的细胞体、主要分支、过程和领土体积。重建后，对索马、整个细胞和领土的体积进行量化。并且统计了主要分支机构的数量。作为标记中间星细胞纤维丝的细胞骨骼蛋白在细胞索马、主要分支和一些二级星细胞分支中表达，但不是在更精细的分支和工艺中。在这项具有代表性的分析中，没有发现由胶质纤维酸蛋白标记的原枝数和由荧光素黄色标记的原枝数量有显著差异。此外，由胶质纤维酸蛋白标记的星细胞的细胞面积和体积明显小于该区域，体积以荧光素黄色可视化，表明胶质纤维酸蛋白是标记主要分支的可靠标记，但对确定细胞的整体面积或体积没有用。从电极尖端释放的染料量取决于电极电阻，电极电阻必须足够高，才能使稳定的染料流被喷射。未来的研究可以通过超分辨率光显微镜或免疫组织化学分析星细胞结构中特定蛋白质的表达来研究星细胞结构的不同成分。

Research

JoVE Journal

Neuroscience

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

科研

神经科学

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Analyzing Dendritic Morphology in Columns and Layers

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

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 ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

应用 ImageJ 对免疫组化制备组织显微照片小胶质细胞形态学的定量研究

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...