美国国立卫生研究院K08 EB003996

1。纺丝溶液的聚- L -乳酸（PLLA） <ol><li>慢火搅拌溶解0.4克聚乳酸在9 mL氯仿。 </li><li>加入1 mL的二甲基甲酰胺的解决方案，使终浓度为4％聚乳酸（W / V），氯仿:DMF 9:1（V / V）的解决方案。 </li><li>将在聚丙烯或玻璃注射器用钝器23ga金属尖端的解决方案。 </li></ol> 2。纺纱基材的准备1 <ol><li>在氯仿中慢火搅拌85:15的PLGA的8％（W / V）溶液（聚乳酸 - 乙醇酸）。 </li><li>外套干净的玻璃在PLGA的盖玻片表面覆盖每个盖玻片与PLGA解决方案。允许的PLGA干到薄膜（约30分钟）。 </li></ol> 3。静电2 <ol><li>安全PLGA的镀膜玻璃盖玻片集电极与导电碳胶带。对齐纤维，收集器是一个马达驱动车轮。对于随机的纤维，集热器是一个固定板。 </li><li>将注射器尖端20厘米集电极轮泵。泵设置约2毫升/小时，如果使用的是一个轮子，电机300-400 RPM。如果可能的话，一个收藏家的-2千伏直流偏置和15千伏金属尖端应用。在访问一个双极电源的情况下，地面收集器。 </li><li>纤维将喷射注射器尖端和旋转的轮子上收集。继续纺纱，直到获得所需的纤维密度。刷卡用纸巾的金属尖端贴到非导电杆，定期以防止堵塞尖端。 </li></ol> 4。 Moating 1 <ol><li>以下纺纱，"护城河"吹打盖玻片周边各地的PLGA行静电样品。允许的PLGA干。这将保持对齐文化的过程中的纤维。 </li></ol> 5。蛋白质涂层<ol><li>纤维和玻璃控制蛋白质在细胞培养之前需要涂。包括至少一个小时，在蛋白质溶液的基板，然后吸出多余的解决方案，并继续吸气，直到基板干。可能的蛋白质的解决方案包括:PLL（聚- L -赖氨酸）（100微克/毫升），层粘连蛋白（25μg/ mL的）或纤维连接蛋白（50微克/毫升） 。如果使用PLL时，基板应在无菌水冲洗和干燥的两倍以下的初始的涂层。 </li></ol> 6。获取E15大鼠胚胎3 *所有实验均按照美国国立卫生研究院批准，由密歇根州委员会关于使用和爱护动物大学的实验室动物护理和使用指南。 <ol><li>制备方法:检查，如果动物是怀孕。解冻3毫升3毫升胰蛋白酶，FBS和温暖的一个L15瓶。消防波兰巴斯德吸管。所有手术器械消毒30分钟70％的乙醇。 </li><li>准备和热情电镀媒体（ 见表1）。 </li><li>安乐死:执行注射氯胺酮/甲苯​​噻嗪的IP。等待10-15分钟，并检查角膜的缺乏和踏板撤回反射。一旦反射缺席，执行注射苯巴比妥（FatalPlus）的IC。 </li><li>帐篷的腹部皮肤。通过皮肤和肌肉层切暴露腹腔。找到子宫，并在子宫颈癌和卵巢中分离。 </li><li>用剪刀从子宫中取出胚胎，并立即放入L15的温暖。 （以下所有程序完成后在解剖显微镜下和胚胎和解剖线应在温暖的L15淹没在任何时候）。杀头关闭钳周围的下巴和下面的颅骨的枕骨胚胎。 </li></ol> 7。夹层3 <ol><li>胚胎放在一边。保护脊髓一套镊子，而使用另一套剥离四肢和腹部器官。 </li><li>将胚胎和同一套镊子稳定。沿着胚胎的长度剪断，从脖子到尾巴，直到整个线暴露。 </li><li>仔细抓住线结束，并拉向尾部超过本身删除。拉膜和背根神经节（DRG）连接线将免费。 </li><li>如果要执行DRG的外植体或分离的感觉神经元文化，DRG的关闭线剪断，并将它们传送到一个​​温暖的L15的新鲜菜。 <ol><li>对于背根神经节外植体文化，跳到步骤10.2 </li><li>对于分离的感觉神经元文化，跳到步骤9 </li><li>对于运动神经元文化，背根神经节将在步骤7.5中的膜被删除</li></ol></li><li>卸下抓线的高端和剥落下来的脊髓膜，以消除长袜子类似。重复所有胚胎，然后斩成小片（2-3毫米）的线。 </li></ol><pCLASS ="jove_title"&gt; 8。运动神经元（MN）的加工5,6,7 <ol><li>在1000转速为2-3分钟，以沉淀件，倒入一个圆锥形和自旋切碎线和L15。 </li><li>倒掉上清，加入3毫升的胰蛋白酶颗粒线。孵育在37℃水浴20分钟。 </li><li>加入3 mL的胎牛血清的锥形，并在1000转速为2-3分钟，以重新颗粒旋转。 </li><li>倒掉上清，大衣火灾打磨巴斯德吸管与胎牛血清。 </li><li>添加一个巴斯德吸管L15全锥形，然后轻轻磨碎，直到解决的办法是同质性与不可见的团块。避免气泡。 </li><li>准备在L15的Optiprep 9％的解决方案。每两个胚胎（，如果有一个胚胎奇数，四舍五入），准备用3毫升的Optiprep解决方案一管。 </li><li>下降到Optiprep解决方案的同质化解决方案，除以各管均匀。在2000年为15分钟的转速旋转管。 </li><li>仔细收集每管50 mL锥形池前2毫升。在1000 RPM，5分钟的填充锥形冲洗Optiprep细胞L15和自旋。 </li><li>倒掉上清，重悬细胞，少量的电镀媒体（250-500μL）。使用台盼蓝拒识别活细胞计数细胞。跳过步骤10.1 </li></ol> 9。感觉神经元（SN）处理7 <ol><li>倒入成一个锥形管和旋转中删除从脊髓和L15在1000转速为2-3分钟，以沉淀件的背根神经节。 </li><li>倒掉上清，加入3毫升的胰蛋白酶颗粒DRG的。孵育在37℃水浴10分钟。 </li><li>加入3 mL的胎牛血清的锥形，并在1000转速为2-3分钟，以重新颗粒旋转。 </li><li>倒掉上清，大衣火灾打磨巴斯德吸管与胎牛血清。 </li><li>添加一个巴斯德吸管L15全锥形，然后轻轻磨碎，直到解决的办法是同质性与不可见的团块。避免气泡。 </li><li>在5分钟1000转的旋转匀浆。倒掉上清，重悬细胞，少量的锡电镀媒体（250-500μL）。使用台盼蓝拒识别活细胞计数细胞。继续加强10.1 </li></ol> 10。电镀与文化<ol><li>分离SN或MN文化: <ol><li>盖基板与媒体几滴，然后添加适当体积的细胞悬液。应镀游离细胞密度低（25-50个细胞/ mm 2）。允许细胞坚持至少一小时之前，充斥媒体的水井。 </li></ol></li><li>对于背根神经节外植体培养: <ol><li>封面与媒体几滴基板，然后添加DRG的媒体。排列的背根神经节，使它们均匀地分布在整个基板（约4背根神经节，将适合在一个22x22毫米盖玻片）分布。允许的DRG至少4小时坚持之前与媒体充斥井。 </li></ol></li><li>文化可以维持到4天，而媒体的变化。对于较长的文化，一半媒体的变化，应做饲料的媒体。饲料媒体电镀媒体减去L -谷氨酰胺的成分相同。 </li></ol> 11。免疫细胞化学1,5,8 <ol><li>修复细胞:吸媒体和30分钟用温水4％PFA（多聚甲醛）取代。然后执行3X 5分钟1X PBS洗。 </li><li>阻断/通透性:准备块/烫发液（1.25％牛血清白蛋白在1X PBS，0.05％TRITON X - 100和2.0％正常山羊血清）。吸PBS和应用块/烫发液，以30分钟的样品。然后执行1X 5分钟1X PBS洗。 </li><li>主要抗体:准备一抗工作液（10％正常山羊血清，0.05％，1.25％的牛血清白蛋白，0.1％NA叠氮Triton X - 100和到10的1X PBS毫升）。添加适量的初级抗体（ 见表2）。封口膜线几盘。地方每个基板上的封口膜的主要抗体溶液200μL珠。反转基板，浮动他们的细胞面朝下放在工作的解决方案的主要抗体过夜（如果房间是空调或特别干燥，水饱和纸件可以添加到菜的一个角落，以防止工作液的蒸发） 。 </li><li>二次抗体:从主要工作的解决方案，可以收集，保存在4 ° C和重用的取出基板和执行2X 5分钟1X PBS洗。应用200μL二次抗体稀释1:200 PBS（也封口膜内衬盘上倒）为4小时。 </li><li>从中学删除，然后进行5分钟2X 1X PBS洗。幻灯片山基板与延长黄金含有的DAPI或其他合适的核反击污点。 </li></ol> 12。代表性的成果: 对齐和随机EL的典型形态ectrospun纤维如图1所示。我们通常会获得百万纯度&gt; 90％的神经元 7,8与本议定书和我们一直保持着DRG的只要一个星期没有任何重大的成纤维细胞生长的文化。图2显示了典型的玻璃和纤维MN文化和免疫后24小时出现。培养三天玻璃和纤维的免疫染色的DRG图3所示。 <img src="/files/ftp_upload/2389/2389fig1.jpg" alt="图1" /> 图1。光纤对准是由集热器的选择操纵。 A）对齐纤维纺到一个旋转的轮子收藏家和B）的随机纤维纺到一个固定的集热器。比例尺= 20微米。 <img src="/files/ftp_upload/2389/2389fig2.jpg" alt="图2" /> 图2代表PLL涂答:经过24小时的文化的运动神经元染色TuJ1（绿色）和DAPI（蓝色））纤维和B）玻璃。比例尺= 20微米。 <img src="/files/ftp_upload/2389/2389fig3.jpg" alt="图3" /> 图3。代表DRG神经丝染色后3天的文化上的PLL涂答:（绿色））玻璃和B）纤维。比例尺= 200微米。 <table border="1"><tbody><tr><td> 库存的解决方案: </td><td> 明尼苏达媒体: </td><td> DRG / SN媒体: </td></tr><tr><td> 10毫克毫升-1白蛋白</td><td> 12.5μL </td><td> - </td></tr><tr><td> 10毫克毫升-1 APO -转</td><td> 50μL </td><td> 40μL </td></tr><tr><td> 50微克毫升-1β-雌二醇</td><td> 2.7μL </td><td> 2.2μL </td></tr><tr><td> 0.1毫克毫升-1素</td><td> 50μL </td><td> - </td></tr><tr><td> 16毫克毫升-1过氧化氢 ​​酶</td><td> 8μL </td><td> - </td></tr><tr><td> 15毫克毫升-1 D -半乳糖</td><td> 50μL </td><td> - </td></tr><tr><td> 50微克毫升-1氢化可的松</td><td> 3.7μL </td><td> 2.9μL </td></tr><tr><td> 0.63毫克毫升-1孕酮</td><td> 0.5μL </td><td> - </td></tr><tr><td> 16毫克毫升-1腐胺</td><td> 50μL </td><td> - </td></tr><tr><td> 50微克毫升-1硒</td><td> 3.4μL </td><td> 4.1μL </td></tr><tr><td> 2.5毫克毫升-1超氧化物歧化酶</td><td> 50μL </td><td> - </td></tr><tr><td> * 500毫微克毫升 -1神经生长因子</td><td> - </td><td> 1毫升</td></tr><tr><td> * 100X PSN </td><td> 0.5毫升</td><td> 0.5毫升</td></tr><tr><td> * B27 </td><td> 1毫升</td><td> 1毫升</td></tr><tr><td> * 2毫米L -谷氨酰胺</td><td> 35μL </td><td> 35μL </td></tr><tr><td> * Neurobasal </td><td>到50 mL </td><td>到50 mL </td></tr></tbody></table> 表1:星号标记（*）组件媒体在使用前。其余的组件，可以准备作为原液，并保存在-20 ° C，直到需要 6,7 。 <table border="1"><tbody><tr><td> 小学（浓度） </td><td> 神经元: </td><td> 神经胶质细胞: </td><td> 成纤维细胞: </td></tr><tr><td>抗β-微管蛋白（TuJ1）（1:1000） </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td>抗神经丝（1:1000） </td><td> ✓ </td><td> X </td><td> X </td></tr><tr><td>抗S - 100（1:25） </td><td> X </td><td> ✓ </td><td> ✓ </td></tr><tr><td> NGFR p75的抗（1:500） </td><td> ✓ </td><td> ✓ </td><td> X </td></tr></tbody></table> 表2。初级抗体的选择是依赖于研究者的目标。上述几种抗体，我们已经在我们的实验室中成功使用的浓度。 S - 100，是特别有用的，以确定雪旺氏细胞和/或污染的成纤维细胞检查，但要注意，它必须使用与NGFR p75的结合，区分神经胶质细胞和fibroblasts4。我们也注意到，NGFR p75的污渍突起很轻，而神经丝或TuJ1是很好的选择，如果我们的目标是可视个别突起。

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该协议有几个关键步骤。首先涉及到电纺纤维基板的正常生产。在液体介质中，聚乳酸纤维的PLGA膜和PLGA护城河将单独作为一个单元的玻璃盖玻片。如果省略的PLGA，聚乳酸纤维不会仍然是一个平板 - 他们会卷曲成一个不可用的纠结。因此，PLGA的电影是一个合适的基板文化的过程中保持光纤对准。护城河是添加后纺，以确保纤维的PLGA薄膜牢固地固定和增加结构刚性的电影。护城河也是当基板固定，染色和安装过程中操纵，作为一个有用的处理。 Trituration是第二个关键步骤。应该尽一切努力，以避免形成泡沫。此外，我们已经取得的收益要高得多时，火抛光的吸管是这一步。要火波兰，点燃本生灯和吸管连接一个灯泡。通过吸管尖迅速通过火焰2-3次，同时不断挤压和释放灯泡 - 空气流通过吸管将有助于防止从完全关闭提示。检查吸管尖，以确保孔仍处于打开状态，使用前圆润的边缘出现轻微。 静电是一个非常灵活的过程;电参数进行修改，以产生的各种形态的纤维。 Tan 等 （2005）详细介绍了影响聚乳酸纤维的直径参数的系统研究。王等人 （2009）提出了影响静电纺丝聚乳酸纤维的对齐参数的详细研究。

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<td>PLLA</td>
<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
<tr>
<td>L15</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>Albumin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2289 
</td>
<td>&nbsp;</td>
<tr>
<td>Apo-transferrin</td>
<td>&nbsp;</td>
<td>Akron </td>
<td>AK8227</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC 23009-0010</td>
<td>&nbsp;</td>
<tr>
<td>Galactose</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>G0625</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7556</td>
<td>&nbsp;</td>
<tr>
<td>Putrescine </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5780</td>
<td>&nbsp;</td>
<tr>
<td>Selenium </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S9133</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-estradiol</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>E 1132 </td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>H0396</td>
<td>&nbsp;</td>
<tr>
<td>Catalase </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C40 </td>
<td>&nbsp;</td>
<tr>
<td>Superoxide dismutase</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S4636</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>PSN</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15640 </td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1680149</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1689149</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Optiprep</td>
<td>&nbsp;</td>
<td>Accurate </td>
<td>2011</td>
<td>&nbsp;</td>
<tr>
<td>PLL</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4832 </td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>F 4759 </td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Anti-TuJ1</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556321</td>
<td>&nbsp;</td>
<tr>
<td>Anti-neurofilament</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB1987</td>
<td>&nbsp;</td>
<tr>
<td>Anti-S100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2532</td>
<td>&nbsp;</td>
<tr>
<td>Anti-NGFR p75</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N3908</td>
<td>&nbsp;</td>
<tr>
<td>Normal goat serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G6767</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8032</td>
<td>&nbsp;</td>
<tr>
<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
<td>&nbsp;</td>
<tr>
<td>Prolong Gold +DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36931</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the culture of primary motor and sensory neurons in defined media on electrospun poly-l-lactide nanofiber scaffolds

静电是微纳米尺度的纤维生产的技术。纤维可与不同高度完全是随机的对齐，对齐度，静电。此外，纤维可纺的各种材料，包括可生物降解的聚合物，如聚- L -乳酸（PLLA）。这些特点使电纺纤维适合在组织工程中的各种脚手架应用。我们的重点是使用对齐神经再生的电纺纤维。我们曾表明，对齐静电聚乳酸纤维直接的初级感觉和运动神经元在体外的生长。我们维持生物材料尽可能接近体内发现真正的神经元模型进行评估时，一个主要的细胞培养系统的使用是必不可少的的。在这里，我们描述了在我们的实验室中使用electrospin纤维支架和文化背根神经节外植体，以及分离的感觉和运动神经元上的静电支架，技术。然而，静电和/或很容易适应这里介绍的养殖技术在其他应用程序使用。

Watch this Scientific Journal Video about The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds at JoVE.com

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

不结盟纤维直接的神经元在体外的生长，是一个潜在的神经再生的支架组成部分。我们描述了一个准备静电纤维基材和主大鼠E15感觉（DRG）和运动神经元无血清培养的过程。用免疫细胞化学神经元的可视化也包括在内。

初级运动和感觉神经元上的静电聚- L -乳酸纳米纤维支架中定义的媒体文化

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

the-culture-primary-motor-sensory-neurons-defined-media-on

Research

JoVE Journal

Neuroscience

19.4K 次观看.  University of Michigan. 不结盟纤维直接的神经元在体外的生长，是一个潜在的神经再生的支架组成部分。我们描述了一个准备静电纤维基材和主大鼠E15感觉（DRG）和运动神经元无血清培养的过程。用免疫细胞化学神经元的可视化也包括在内。

视频: The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

体外刺激对龟脑神经的运动测量

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...