This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

注：所有动物的程序都遵循的指导方针，并已批准的机构动物照顾与使用委员会（IACUC）在康涅狄格大学。 注：对于这些实验组成NG2cre 18（JAX＃008533），并诱导NG2creER 16（JAX＃008538）的转基因小鼠杂交到Z / EG 19（JAX＃003920）或gtRosa26：YFP记者20（JAX＃006148）线分别使用到图像NG2细胞和它们的后代。图像成熟少突胶质细胞，PLPDsRed转基因小鼠21人使用。对于一致的存活率，切片可以从小鼠中分离到来自前脑和小脑出生后第10天。 1，切片准备<ol><li>在培养罩，将在6孔板组织培养的插入和加入1 ml片培养基（配方中的试剂部分）。放置在细胞培养孵化器板在37℃下用5％的前夹层的CO 2的至少2个小时。 </li><li>消毒所有工具和组织切碎，用70％的乙醇。 </li><li>泡沫夹层缓冲液（配方中的试剂部）用95％O 2，5％CO 2在冰上至少15分钟清扫开始之前。 </li><li>通过按照批准的动物协议将它们放在冰上5-10分钟麻醉小鼠的幼崽。通过确认该小鼠不向尾部和脚趾夹住响应确定麻醉的适当深度。 </li><li>杀头使用下列动物的协议锋利的剪刀动物。首先使矢状切后外侧切口，用消毒工具快速去除颅骨比前脑和小脑。通过仔细轧制组织的侧切割从大脑和小脑的腹侧表面脑神经。 </li><li>将组织中含有冰冷的氧化夹层缓冲无菌35mm培养皿。 </li><li>使用刀片，切断小脑和前脑。然后做一个正中矢状切救援人员到场唉前脑和小脑，分离半球。 </li><li>切断前脑和小脑的300微米厚的冠状面和矢状切面有手动组织菜刀。注意：A手册组织切碎机可以从解剖到培养孵育快速处理，而不需要琼脂糖嵌入。如前面9所述的vibratome也可以使用。 </li><li>传输分离成片新鲜冒泡冷夹层缓冲冰上。 </li><li>使用小型解剖或重铲分隔各个部分，转让给预孵育6孔板与文化的刀片。 </li><li>使用一次性移液管或P 200枪头文化插入的表面去除多余的夹层缓冲。两到三个前脑或三个小脑切片可以被放置在一个培养插管。 注：对于脑的文化一致的结果，它是理想的取片横跨前三分之一的胼胝体（ 图1A），以便研究二者灰质和白质的区域在相同的切片。每只动物通常会产生6前脑片和6片小脑。 </li><li>将6孔板中培养器并用1ml片介质1天后切片制备，然后每隔一天直到切片固定更换切片平台。 </li><li>根据不同的实验中，使用后的5-7天中培养切片。只使用成为第几天后，透明片，丢弃那些有不均匀的不透明区域。注：片内的不透明区域是由眼可见的，显示为暗细胞在相差一丛。后的技术已经掌握了，死不透明片很少发生，在切片小于1-5％。 </li></ol> NG2细胞分裂和少突胶质细胞分化的2时间推移成像注：要执行的NG2细胞的增殖和分化时间推移成像，我们使用NG2cre：ZEG鼠16这在NG2细胞及其后代（ 图1C-E）的表达绿色荧光蛋白。记者在这条线的表达是足够坚固的切片为至少四周的时间段之后立即获得GFP在切片的实时图像+细胞。最清晰的图像切片，这发生在文化第3-5天的初始变薄期后获得的。 <ol><li>整个实验过程中，保持在细胞培养恒温箱切片在37℃下，只为短暂的时间除去（每片小于15分钟），保持盖子上的6孔板中，同时影像。 </li><li>用配有数码相机的倒置荧光显微镜，在使用10X物镜在第一时间点捕捉图像。注：第一次点将通过实验有所不同，可当准备切片或后一天的任何时间点。 </li><li>在该片段的两种灰质和白质的区域捕获来自多个区域的图像，在时间点之一。注日Ë区域由片内特定地标和通过映射对可用于随后的成像会话的示意性的图像的位置进行成像。有用的地标可包括白质的切片和/或取向的边缘。图像七时五十六每个切片在每个时间点上的位置。 </li><li>在4-6小时的间隔拍摄图像以后，如果检查NG2细胞分裂和少突胶质细胞分化，最好在5-7天。 </li><li>捕获所有区域的最终图像，并立即修复片。加入1 ml固定液（4％PFA中，用0.1M L-赖氨酸的0.01M偏高碘酸钠）对培养插管的底部，然后在切片上添加另一个1毫升。请注意不要直接喷在固定液中的片上，因为它可以从膜分离。修复组织30分钟，并如第4描述进行使用发育阶段特异性标志物免疫组织化学。 </li><li>洗涤切片用0.2M磷酸钠缓冲液3×10米英寸如有必要，存储之前在4℃下的切片在1-3天的0.2M磷酸钠缓冲液中的免疫染色，但最好在24小时内进行染色。用免疫组织化学进行的在第4如下所述来确定已分化为少突胶质细胞（ 图2D-F）的细胞的比例。 </li></ol>的NG2细胞的后代在切片培养的诱导记者转基因小鼠3命运映射注意：要在培养一个特定的时间点跟踪NG2细胞的命运，诱导NG2creER：YFP的转基因小鼠可以使用。 <ol><li>诱导的Cre重组和报告基因的表达通过添加100nM的4OHT溶解于乙醇的培养基中。注：记者阳性细胞应该在〜20-25％的YFP + NG2 + / NG2 +细胞的诱导效率和在该时间点出现在1-2天内将全部细胞在NG2细胞阶段（ 图2A-C）的 </li><li>修复和洗片在不同的时间分秒各种操作后（在章节2.5和2.6中描述）。 </li></ol> 4，免疫组化切片<ol><li>切断附着了用解剖刀刀片的切片的膜。 </li><li>传送片来使用一套镊子的24孔磷酸含板缓冲盐水（PBS）的各个孔中，注意不要拿起膜时打乱片的组合物。 </li><li>传送片以24孔板用封闭含5％正常山羊血清（NGS）和0.1％的Triton-X100的PBS中进行1小时的解决方案。 </li><li>孵育切片的主要抗体的O / N的PBS中5％NGS在4℃。用于鉴定细胞中的NG2细胞阶段中，使用抗体来NG2和PDGFRα。为了识别有区别的少突胶质细胞，利用抗体对结肠腺瘤性结肠息肉病抗原（APC;克隆CC1）。 </li><li>在PBS 3×15分钟第二天洗切片，然后在培养中学antibo模具在PBS中5％NGS 1小时，在室温。在PBS洗3×15分钟片和安装在载玻片上。安装用片和膜对着滑动，使该膜不会成为目标和组织之间。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+sensory+axons+are+mitogenic+for+Schwann+cells.">Evidence that sensory axons are mitogenic for Schwann cells.</a> Nature. 256 (5519), 662-664 (1975).</li><li>Stevens, B., Tanner, S., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+myelination+by+specific+patterns+of+neural+impulses.">Control of myelination by specific patterns of neural impulses.</a> The Journal of Neuroscience. 18 (22), 9303-9311 (1998).</li><li>Stevens, B., Porta, S., Haak, L. L., Gallo, V., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine:+a+neuron-glial+transmitter+promoting+myelination+in+the+CNS+in+response+to+action+potentials.">Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials.</a> Neuron. 36 (5), 855-868 (2002).</li><li>Wake, H., Lee, P. R., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+local+protein+synthesis+and+initial+events+in+myelination+by+action+potentials.">Control of local protein synthesis and initial events in myelination by action potentials.</a> Science. 333 (6049), 1647-1651 (2011).</li><li>Rosenberg, S. S., Kelland, E. E., Tokar, E., De la Torre, A. R., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geometric+and+spatial+constraints+of+the+microenvironment+induce+oligodendrocyte+differentiation.">The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14662-14667 (2008).</li></ol>

The authors declare that they have no competing financial interests

髓鞘在中枢神经系统是用于有效的神经元沟通和轴突存活22是必不可少的。 NG2细胞不断产生髓鞘少突胶质细胞到成年期，同时保持常住人口中大部分脑区16,23 - 25。调节这些细胞的分化一定的遗传和分子机制进行了描述，但仍有许多有待发现。器官切片培养物是一种方便的工具来调查这些机制，由于其独特的保持解剖区域，容易操纵的胞外环境中的，健壮的髓鞘形成，并且所有主?...

中枢神经系统Organoytpic切片文化已被证明是研究神经元和神经胶质细胞生物学的semiintact系统1非常有用- 4。这些文化都比较简单采用和保留主要的游离细胞培养的诸多好处，如操控外环境，并易于进行反复长期活细胞成像和电生理记录5 - 9。此外，切片培养维持3维组织细胞结构，区域神经连接性，并且最重要的细胞类型是存在于系统中。这些特性使得这些文化独特而方便的系统研究单个细胞的行为和生理与细胞和环境的相互作用。 NG2细胞为神经胶质细胞在哺乳动物中枢神经系统中的继续增殖和产生微米的人口胚胎和出生后发育过程中10 yelinating少突胶质细胞。它们已被广泛研究中解离的原代细胞培养，以及最近的转基因小鼠系与荧光蛋白的NG2细胞特异性表达的发展促进了体内命运映射和电生理记录在急性切片准备。即使有了这些研究，知之甚少的NG2细胞增殖和少突胶质细胞分化的时间动态。虽然分离的细胞培养物被广泛用于在药理学和遗传操作相对设施，它不适合用于询问这些细胞在不同脑区的功能上的差异特别是当它是需要维持细胞微环境的上下文。切片培养提供了一个简单的替代方案，是适合于药理学操作，并已被用于研究少突胶质细胞髓鞘形成11,12，细胞溶血卵磷脂（LPC），或通过药物治疗15抗体诱发的脱髓鞘13,14，和诱导髓鞘再生后ular响应。 的方法进行调查，并进行实时成像和固定的组织（或postfixation）中描述的NG2细胞增殖和少突胶质细胞分化来自前脑和后脑取器官切片文化分析。这是一个可用于研究单个NG2细胞的细胞命运的除法16后，并发现在生长因子诱导的NG2细胞增殖17与地区和年龄依赖性差异的有效方法。这种相对简单的技术是普及进一步研究细胞的内在和/或环境调节这些神经胶质细胞的生理机能，他们的反应神经元的活动或髓鞘损伤的机制。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Slice Culture Medium</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>11090</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Invitrogen</td>
			<td>14175</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td>25mM</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>1mg/L</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>0.4mM</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Hyclone</td>
			<td>SH30074.03</td>
			<td>25%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>124mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>3.004mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>1.25mM</td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>4.004mM</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>26mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>2.0mM</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>0.075mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents and Supplies</td>
		</tr>
		<tr>
			<td>4-hydroxy tamoxifen (4OHT)</td>
			<td>Sigma-Aldrich</td>
			<td>H7904</td>
			<td>100nM</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>EM Sciences</td>
			<td>19200</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L-6027</td>
			<td>0.1M</td>
		</tr>
		<tr>
			<td>Sodium Metaperiodate</td>
			<td>Sigma-Aldrich</td>
			<td>S-1878</td>
			<td>0.01M</td>
		</tr>
		<tr>
			<td>Rabbit anti NG2 antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>AB5320</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Mouse anti CC1 antibody</td>
			<td>Calbiochem (Millipore)</td>
			<td>OP80</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Mouse anti Ki67 antibody</td>
			<td>Vision Biosystems</td>
			<td>NCL-Ki67-MM1</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Mouse anti NeuN antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Species specific secondary antibodies</td>
			<td>Jackson Immunoresearch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td></td>
			<td></td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture membrane inserts 0.45&#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filters</td>
			<td>Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2 gas mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile six well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting (hippocampal) or weighing spatulas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manual or automatic tissue chopper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm and 60mm sterile Petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Phase Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microcope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

NG2表达细胞（polydendrocytes，少突胶质细胞前体细胞）的第四个主要的神经胶质细胞群在中枢神经系统。在胚胎和出生后发育，他们积极地繁殖，并产生髓鞘少突胶质细胞。这些细胞通常被研究的初级分离培养，共培养神经元，并在固定的组织。使用新的可用的转基因小鼠系切片培养系统可被用于研究细胞增殖少突胶质谱系细胞的分化中的前脑和小脑的灰质和白质的区域。切片培养物从出生后早期小鼠制备的，并保持在培养达1个月。这些片可以在培养期间被多次成像研究细胞行为和交互作用。这种方法允许NG2细胞分裂的可视化，并导致少突胶质细胞分化，同时支持的详细分析步骤区域依赖耳鼻喉科NG2细胞和少突胶质细胞功能的异质性。这是一个功能强大的技术，可用于研究在它十分相似， 在生物体内发现了一个蜂窝环境影响，这些细胞随着时间的内在和外在的信号。

具有代表性的数据的例子如下已使用切片培养从P8 NG2cre两者的前脑和小脑获得：ZEG，NG2creER：YFP和PLPDsRed转基因小鼠品系。 NG2细胞可以被成像在中前脑和小脑切片（ 图1B-D视频1），并且这些细胞的表型天可以固定后进行测定，并与NG2和CC1的抗体（ 图1E）的免疫染色。除了 ​​实时成像，细胞增殖，也可以用免疫组织化学染色对细胞增殖的标记物如Ki67表达（ 图1F）</s...

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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Neuroscience

18.2K 次观看.  University of Connecticut. A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.