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 ...

organotypic-slice-cultures-to-study-oligodendrocyte-dynamics

Research

JoVE Journal

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.

视频: Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

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

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

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 ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...