这项研究是从本科生研究机会计划在UCI（AR和SP）赠款和赠款，从加州阿尔茨海默氏病的倡议和国家卫生部授予机构没有国家的支持。 HD38466，阿尔茨海默氏症研究中心授予没有。 AG16573（JB）

1。皮层组织块冷冻保存 材料的制备 <ol><li>准备无菌水（1:9）的10倍原液稀释1X汉克的缓冲盐溶液（HBSS）。在摄氏4度商店的HBSS。 </li><li>准备10％DMSO冻存液稀释的HBSS（1:9）的二甲基亚砜。冻结介质应该是取得了新的冷冻保存前直到冷冻储存在摄氏4度。 </li><li>钢刀片是用于组织系统斩波。在使用之前，刀片被淹没在70％乙醇为2小时消毒。立即组织处理前，无菌水冲洗3次刀片。避免留在无菌水的剃须刀过多的时间，因为它很容易氧化。 </li><li>一个NALGENE冷冻集装箱装入离心管（2.5毫升），然后带进无菌生物柜。 1毫升的冰鲜冷冻介质添加到每一瓶。冷冻集装箱，然后放置在摄氏4度至少2小时。 </li></ol> 清洗，切菜，和冻结 <ol><li>被冻结的脑组织，脑膜膜和血管的清洁。使用无菌针尖小心地取下冰袋上工作时从组织的碎片。清理组织应轻轻冲洗与冷的HBSS，并转移到一个新的Petri砍菜。 </li><li>冰袋上工作，使用消毒的刀片，迅速 ​​组织切成约1毫米的3块。清理组织工作的一小部分，在更好地控制在切菜过程中的时间结果，产生更均匀的块大小。 </li><li>在50 mL锥形管，慢慢地加入到50毫升的HBSS切碎组织。轻轻冲洗培养皿与HBSS中，以收集任何剩余的组织块。允许组织块下降到试管底部，建立一个松散的组织块颗粒。 </li><li>虽然该组织是解决生物安全柜，把先前冷冻的冷冻集装箱和冷冻管。摘帽所有的离心管，以加速加入组织的过程中。 </li><li>所有的组织块纷纷落户后，轻轻吸出多余的HBSS的，留下一个非常薄的层以上的颗粒的介质。离心气馁，因为它会导致组织块要坚持对方，冻结效率显着减少。 </li><li>使用广泛窍（切割用无菌剪刀尖）的枪头，慢慢地从底部的松散组织块颗粒收集200微升。的材料转移到一个离心管，从底部的沉淀下，再次收集组织。重要的是整个组织分配过程不超过3-4分钟每冷冻集装箱。这将确保该组织是暴露只是一个时间的冻结前的短期内，以二甲基亚砜。组织转移到小瓶后，放置至少4小时，在A - 80℃冷冻的冷冻集装箱。另外，冷冻集装箱可以在一夜之间，在-80℃。重复这个过程中，任何剩余的冷冻集装箱。 </li><li>从冷冻集装箱的冷冻管（S）转移到一个cryobox和长期储存在液氮罐。 </li></ol> 2。解冻和冷冻皮层组织块的培养 材料的制备 <ol><li>一天前解冻的组织样本，聚- L -赖氨酸涂布培养皿/盖玻片准备。皮层神经元在一般情况下，我们准备用500微克/毫升或1毫克/毫升涂菜肴。加入足够的聚- L -赖氨酸溶液（硼酸盐缓冲液中），全面覆盖的菜底部。如果需要盖玻片，确保它们完全淹没下聚- L -赖氨酸的解决方案。孵育至少12小时。在使用之前，彻底的菜肴与自由主义的无菌水冲洗3次，每次5 min。 </li><li>暖的HBSS是用来清洁解冻组织以及组织分解成细胞悬液。取决于解冻组织的需要的HBSS数量会有所不同，但通常为1离心管会稀释在50毫升的HBSS，并将于10毫升的HBSS分离。 </li><li>贝科的改良Eagle中等10％的铁补充牛小牛血清（EBS）和1％的抗生素/ antimycotic（AA）的混合物（DMEM培养液(++))应放置在37℃水浴。 </li><li>电镀液（注(+++))应在使用前新鲜。这种介质是准备加入抗生素/ antimycotic加1％B27和N2补充剂。 </li></ol> 注:媒体名称与穿越（+）追加指示列入媒体的碱基组成的添加剂。在这个文本中，DMEM培养液（+）表示DMEM培养液加10％EBS的加1％，AA，而NB (+++)表示注:PL我们的1％AA加B27的加氮气。 解冻和培养 <ol><li>从液氮罐取出离心管，并迅速解冻在37 ° C直到水浴中只有一小冰粒是观察里面的小瓶内容。 </li><li>轻轻转移的内容，以温暖的HBSS 50毫升的小瓶，在一个锥形管。使用宽口尖轻轻驱逐任何剩余的组织块的离心管中卡住。反转管的3-4倍，并允许组织在底部慢慢收集。组织应该迅速解决，但如果块太小，这可能不会发生光离心（〜200 ×克）建议。 </li><li>吸干多余的HBSS。 </li><li>添加10毫升温暖的HBSS组织颗粒和300μL0.25％胰蛋白酶和50μLDNAase。在37℃水浴孵育5分钟，然后在轨道摇床设置为80 rpm和37 ° C的一个额外的5分钟。 </li><li>这一时期后，将进入生物安全柜的组织块，并用10 mL的吸管仔细搅动的组织块，直到形成阴天细胞悬液。停用温暖的DMEM培养液中添加10毫升的胰蛋白酶（+）的细胞悬液。 </li><li> 1200 × 克 5分钟离心分离细胞。吸弃上清，在10毫升的温暖的DMEM重悬细胞沉淀（+ +）和量化使用血球细胞数。 </li><li>聚- L -赖氨酸涂层菜肴板细胞。通常，一个60毫米的菜将被镀与1 × 10 6细胞。 </li><li>允许细胞组织培养箱培养约1小时的菜/盖玻片的重视。建议在一个板块监测的附件的进展，每10分钟，直到有效附件。然后，轻轻地更换新鲜的DMEM (++).中期</li><li> 24小时后，更换的DMEM（+）用温水注(+++).部分介质的变化（50％）进行每5天用新鲜的NB (+++).健康的文化通常24小时后，进程的分化和增长的迹象。文化在这些条件下，执行部分介质的变化，每4-5天，可维持长时间的时间可达4-6周。 </li><li>对于生成的神经前体细胞（筹备），类似的协议，离心后，没有EBS的DMEM培养液的异常。细胞是镀金的T - 25的DMEM/F12（1:3），B27与表皮生长因子（20毫微克/毫升）的补充，让neuropheres形成烧瓶。要区分的NPC，神经球镀金层粘连蛋白（10微克/毫升）的DMEM/F12（1:3）培养基用N2涂盖玻片。 </li></ol> 3。代表性的成果健康的文化会表现出显着的生长和分化，后5天，解冻后，通常会在其增长稳定10天（图1）。这些文化Immuncocytochemical染色揭示众多的星形胶质细胞（图2A）和神经元对人类和大鼠原代神经元的文化（图2B）。此协议也适合对NPC的一代自由浮动的神经球（图3A），区分条件下，高品质（图3B，C）的混合培养的结果。 <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="图1" /> 图1。冷冻人脑皮层组织细胞培养 ，冷冻的人脑皮层组织块接种于多聚赖氨酸涂布盖玻片解冻，生长10天。白天5细胞扩张明显，达到10天汇合。比例尺:100微米。 <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="图2" /> 图2。细胞培养免疫细胞化学染色（A）培养染色标记的胶质神经胶质纤维酸性蛋白（GFAP，厚箭头）。 （二）发现神经元与神经元的标记β-微管蛋白III（TIII，细箭头）。 （三）人类和鼠文化展示丰富的神经胶质细胞和神经元后的10天中文化。一抗:鼠抗GFAP（1:1000）和兔抗TIII（1:1000）。二抗:抗鼠Alexa的594（1:500）和抗兔Alexa的488（1:500）。核染色:Hoestch蓝色（1:1000）。比例尺:50微米。 <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="图3" /> 图3。代神经球。（一）冰冻组织处理，如上所述，并允许传播神经球。 （二）神经球被镀上盖玻片与众不同的条件下生长10天，导致细胞从神经球迁移。 （三）这两种神经元和星形胶质细胞是目前在区分神经干扩大边缘。核counterstaining，如在图2中使用的小学和中学的抗体。

<ol>
	<li>Robbins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+brain+tissue.">Cryopreservation of human brain tissue.</a> Exp Neurol. 107, 208-213 (1990).</li><li>Ware, C. B., Nelson, A. M., Blau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-rate+freezing+of+human+ES+cells.">Controlled-rate freezing of human ES cells.</a> Biotechniques. 38, 879-880 (2005).</li><li>Paynter, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practical+issues+for+cryopreservation+of+nerve+cells.">Principles and practical issues for cryopreservation of nerve cells.</a> Brain Res Bull. 75, 1-14 (2008).</li><li>Thirumala, S., Gimble, J. M., Devireddy, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+stromal+vascular+fraction+of+adipose+tissue+in+a+serum-free+freezing+medium.">Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.</a> J Tissue Eng Regen Med. 4, 224-232 (2010).</li></ol>

冷冻保存提供了一个宝贵的脑组织样本，以供将来使用银行的机会。在这里，我们描述了一个简单而有效的的协议，同时生成神经元丰富的文化和神经前体细胞从冷冻的脑组织块。这种经济的过程避免了传统的冷冻保存技术，利用更昂贵的速率控制冰柜的成本。该协议允许可行的解冻后的神经元文化的一代提供一个快速而有效的的手段，以冻结组织块。整个冻结过程中可以采取低至20分钟。此外，使用这种方法组织块的初级神经元文化也可以被解冻后产生自由浮动的神经球生长的NPC。在这方面，在我们的冷冻介质血清缺乏确保细胞是未分化状态保存。与众不同的条件下，神经球生产从冰冻组织显示扩张和分化率从新鲜组织生成的神经球非常媲美。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995-073</td>
<td>High gluclose 1X</td>
</tr>
<tr>
<td>Bovine calf serum supplemented</td>
<td>&nbsp;</td>
<td>HYCLONE</td>
<td>SH30072.03</td>
<td>For culture medium </td>
</tr>
<tr>
<td>Neurobasal-A Medium (1X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>1088-022</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>N2 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>Trypsin (10X)</td>
<td>&nbsp;</td>
<td>Cellgro </td>
<td>25-054CI </td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Deoxyribonuclease 1 from bovine pancreas (DNase)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4527-30KU</td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) (10X)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14065</td>
<td>Cleaning tissue, washes, and freezing medium</td>
</tr>
<tr>
<td>Poly-L-Lysine- 500mg</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2636</td>
<td>Substrate for adhesion of neuronal cells</td>
</tr>
<tr>
<td>antibiotic-antimycotic (100X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>To prevent contamination</td>
</tr>
<tr>
<td>Large orifice pipette Tips (1-200 ul)</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>02-681-141</td>
<td>To prevent shear stress to cells</td>
</tr>
<tr>
<td>Graduated pipette tips (101-1000 ul)</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1111-2721</td>
<td>&nbsp;</td>
<tr>
<td>21 G1 precision guide needles</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>305165</td>
<td>To clean tissue</td>
</tr>
<tr>
<td>10 ml pipette </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1071-0810</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>926-9-04</td>
<td>&nbsp;</td>
<tr>
<td>Single edge razor blade</td>
<td>&nbsp;</td>
<td>Smith Brand</td>
<td>67-0238</td>
<td>To chop tissue</td>
</tr>
<tr>
<td>60 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>For general culture</td>
</tr>
<tr>
<td>100 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0010</td>
<td>For cleaning tissue</td>
</tr>
<tr>
<td>Cryogenic box </td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5026-1010</td>
<td>&nbsp;</td>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5100-0001</td>
<td>"Mr. Frosty"</td>
</tr>
<tr>
<td>2.0 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5012-0020</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>D128-500</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Ethanol 200 proof</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>For sterilizing razor blade</td>
</tr>		</tbody>
		</table>
		</div>

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

在这项研究中，我们勾勒出成功皮质的脑组织块冷冻保存和解冻生成神经元文化的高度浓缩的标准化协议。本议定书的冷冻介质使用的是10％的二甲基亚砜（DMSO）在汉克的缓冲盐溶液（HBSS）稀释。皮层组织块转移到含有冻结介质的离心管中，并慢慢地冻结在-1 ° C /分钟的速度控制冷冻集装箱。始终产生神经丰富的文化展出文化和神经网络的显着扩大，在第5天，10天之内迅速神经炎增长冰冻组织块解冻后的处理和分解。的星形胶质细胞标记神经胶质纤维酸性蛋白（GFAP）和神经元的标记β-微管蛋白Ⅲ类，免疫细胞化学染色显示培养的神经元和星形胶质细胞的数量高。组织块解离后的神经前体细胞培养新一代导致在迅速扩大的与众不同的条件下产生大量的神经元和星形胶质细胞的神经球，。这个简单的冷冻保存的协议，允许快速，高效，廉价的保存皮质的脑组织块，其中赠款为神经元，星形胶质细胞和神经元前体细胞培养后一代增加了灵活性。

Watch this Scientific Journal Video about Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures at JoVE.com

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

在这里，我们描述了一个高效的冷冻保存的方法和解冻皮质的脑组织块生成神经元文化的高度浓缩。这个简单的协议提供了神经元，星形胶质细胞和神经元前体细胞培养后一代的灵活性。

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

cryopreservation-cortical-tissue-blocks-for-generation-highly

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.6K 次观看.  University of California, Irvine. 在这里，我们描述了一个高效的冷冻保存的方法和解冻皮质的脑组织块生成神经元文化的高度浓缩。这个简单的协议提供了神经元，星形胶质细胞和神经元前体细胞培养后一代的灵活性。

视频: Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

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. ...

科研

神经科学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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 ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

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

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...