这项工作是支持的加拿大卫生研究院（CIHR），彼得Lougheed医学研究基金会，在Neuroengineering麦吉尔方案的补助金，全宗的RECHERCHE EN桑特魁北克（FRSQ），加拿大创新基金会（CFI ）。塞巴斯蒂安D.朗格卢瓦是硕士培训奖全宗支持DE LA RECHERCHE EN健康魁北克（FRSQ）和卫生研究所（CIHR）加拿大研究所加拿大研究生奖学金硕士奖由弗雷德里克班廷和查尔斯。我们感谢与数字援助杰西卡吨范。

1。胚胎大鼠背脊髓的解剖一般建议置于冰上的L - 15中型和经常改变在清扫菜中保持胚胎的凉爽。这有助于维持组织的完整性。除非另有说明，所有步骤都与两个对杜蒙＃5镊子。为了避免污染，所有的工具和工作表面喷用70％乙醇，并保持封闭夹层介质瓶。菜之间传输的胚胎中，使用切割塑料吸管或穿孔勺。这是至关重要的，不损伤脊髓（刻痕，拉伸）成功地完成了清扫。 制备<ul><li>冷L - 15培养基</li><li> 50毫升的L - 15 + 10％热灭活马血清（HiHS）。置于冰上。 </li></ul>脊髓夹层<ol><li>安乐死E13怀孕上演大鼠（E0 =第一天交配天）与CO 2室根据机构指引。 </li><li>喷雾70％的乙醇，上腹部。捏和拉起降低腹部手术剪钳和切割区域的皮肤。重复通过切断肌肉和腹膜层，以达到腹腔。创建一个V形切口，切割组织沿腹部两侧，胸部。升降机及拉回组织暴露腹腔。 </li><li>子宫是连接三个地点：在较低的中心腹部，双上肢外侧角落。提起子宫的胚胎囊之间的组织敛。剪下的结缔组织切除子宫，并在培养皿中的L - 15在冰充满。 </li><li>接下来的步骤是根据解剖镜下完成。胚外组织和胚胎膜分离胚胎，抢子宫囊之间的组织，与一对镊子。另一对，掐国资委更加透明的一面穿过肤浅膜（黑暗面是胎盘）。轻轻按国资委的胎盘端挤出胚胎。删除所有在培养皿中的L - 15在冰充满的胚胎和地方。 </li><li>将一个胚胎在10厘米用冰冷的L - 15的Petri充满菜。使用microscissors，切去头图1a所示的角度和后部分。 在这些角度切割，将有助于位置放置时，“腹朝下”的胚胎。 </li><li>胚胎的位置“腹正面朝下”（前背向后指向实验者）（图1B）。 </li><li>牢牢把握胚胎从一个侧面，使用钳“牵制”的胚胎（图1C）。不要移动镊子，因为它会撕裂组织。随着一双镊子等，涵盖脱落，皮肤开始从地区之间的“控股”钳（图1D - E）在胚胎的背面。这将会使脊髓，在这一点上，包装膜（脑膜）。 抓斗从两侧的皮肤，而不是从上面的脊髓，以避免刻痕的脊髓。 </li><li>部分分离组织的脊髓（图1F）的右侧。在持物钳的水平开始，封闭钳戳的组织，尽可能接近到脊髓。然后，慢慢地打开镊子撕组织。这应该分离脊髓背根神经节（病种付费），而且还应该破坏腹侧机关。离开。 离开组织增加重量胚胎的前部和后部两端附加了一些组织，从而防止在下一步向上拉。此外，该组织是用来装在后面的步骤中的胚胎。 </li><li>由前年底开始，用勾形的钨针削减脑膜和脊髓沿roofplate（图1G）打开。 </li><li>胚胎旋转180度（后指出，前对实验者的指向）（图1H）。 这允许实验者用同一只手从另一侧的胚胎组织分离。 </li><li>保持到以前分离的方抓住胚胎，并从剩余方使用相同的方法（见1.8）。的组织破坏。完成后，完全分离的脊髓两侧的组织。 另一种方法：彻底分离组织余下的左侧，但离开的前部和后部端连接的右侧上一些组织。有时，这可以帮助脊髓维持在1.12的一步位置。 </li><li>在其一侧脊髓和删除其余的间叶组织和背根节（图1I）。 </li><li>在这一步，脑膜和脊髓是两个“表”到对方apposed组织。牵制较大，脊髓的最前部分，并剥离过短节段脊髓COR的脑膜D（图1J，左）。现在，持有抓住钳跨越两地分居段，平稳，恒定的运动（图1J）脑膜剥离。 不均“剥离”将导致脊髓和/或脑膜层破损。它通常是不可能的，收回部分仍然附着在脑膜脊髓。 </li><li>用塑料吸管，隔离脊髓转移到培养皿中含有的L - 15 + 10％HiHS留在冰上。通常情况下，所有胚胎的脊髓前收集解剖背部分。 </li></ol>脊髓背侧夹层<ol start="15"><li>放置一个脊髓中含有的L - 15 10％HiHS，平趴在一个“开卷”配置一个Petri菜。切去更宽，前部（其中包括后脑的一部分）（图1K）。 </li><li>背侧组织是位于最外侧部分的脊髓（图1L，左）。虽然寄希望于采用了直板的钨针的电源线，使用“L”形的钨针，切出一个地带，是1/5th宽度的一半脊髓。 （图右）1L，。将在15毫升的塑料管，在冰中含有的L - 15 + 10％HiHS背条。 背〜12 E13胚胎神经管段将产生解离电镀后〜3.5-4亿个细胞。可以得到更多的细胞进行脊髓背侧（切割更广泛的背条）粗清扫，但连合神经元的纯度会降低。 </li></ol> 2。连合神经元文化一般建议 应除非另有说明，组织培养罩在无菌条件下进行的所有步骤。使用新鲜培养液和新鲜解冻的补充和试剂 。 在Ca 2 + / Mg 2 +的免费的HBSS，以尽量减少的Ca 2 + / Mg 2 +的依赖性粘附的解离和trituration步骤进行。 制备<ul><li>涂层的盖玻片（使用德国Desag玻璃）或组织培养板（见下文涂层过程）。 </li><li>温暖Neurobasal电镀媒体（见下文），在组织培养皿和在组织培养孵化器的CO 2平衡电镀前至少为1.5 h 。 </li><li> 2.5％胰蛋白酶在37℃水浴</li><li> 2瓶HBSS中的Ca 2 + /镁2 +，在4 ° C时，在37 ° C. </li><li>两个火抛光的玻璃巴斯德吸液管直径通常大小的一半，和一个直径略超过一半。使用消毒的巴斯德吸液管。消防波兰的移液器，使用本生灯（淡蓝色火焰上方）融化尖的直径略有下降。因为这一步是进行组织培养罩外，喷用70％乙醇的组织培养罩下配售的移液器。为更有效的恢复神经元，大衣的含药血清之前trituration一步的离解或（填写与媒体的移液器，并保持30秒）开始前的媒体巴斯德移液器。这将防止细胞粘在trituration巴斯德吸管内。 </li><li> milliQ无菌水洗涤PLL涂层的菜肴或盖玻片</li><li> 12.5％的硫酸镁溶液中的HBSS </li></ul>聚- L -赖氨酸涂层如果使用盖玻片，24小时的酸清洗和消毒前电镀（见Kaech和银行家，2006年）。使用德国Desag盖玻片。 聚- L -赖氨酸盖玻片或塑料组织培养皿大衣： <ul><li>下一个组织文化的引擎盖，覆盖了100微克/毫升的PLL解决方案为1.75-2小时的小圆顶曲面。 </li><li>洗两次澡的milliQ水，至少每洗5分钟（可以在细胞分解步骤如下进行）。 </li><li>在水店，直至使用。不要让PLL涂层表面的干燥。 为了减少盖玻片涂层的PLL浪费，在无菌细菌培养皿中的盖玻片 。 外套和洗涤的液体的圆顶上的盖玻片盖玻片 。 细菌的培养皿中的疏水性，使液体应保持在玻片上 。 电镀前的细胞，组织培养皿中的盖玻片 。 对于塑料组织培养皿上镀的神经元，附着力通常较高，从而突起伸长率可能会减少。 </li></ul>分离和电镀<ol><li>验证背条落户试管底部。删除最巴斯德吸管的L - 15的10％HiHS。快速洗背神经管带，一旦加入3毫升冷（4℃）的HBSS。 </li><li>让背神经管带定居2分钟，然后取出用巴斯德吸管的HBSS。 </li><li>温暖（37℃）的HBSS体积为4.7毫升。 T母鸡添加0.3毫升2.5％胰蛋白酶给予终浓度为0.15％胰蛋白酶。 </li><li>在37℃，7分钟的水浴。通过孵化轻轻搅拌一次中途。 </li><li>加入终浓度为150 U / mL的30μLDNA酶（25万U /毫升）。加入60μl和硫酸镁混合简要终浓度为0.15％，。 对于组织由粗解剖，一个额外的1分钟37 ° C的水浴。 在这个阶段，背神经管段应支离破碎。如果背神经管段还没有开始的片段，它通常是指2.5％的胰蛋白酶股票是旧的，新的股票应解冻，或用冷的HBSS洗没有有效地去除样品HiHS。 </li><li>离心机组织碎片，200克4分钟。 </li><li>巴斯德吸管取出上清液，留在〜50-100μL液体的试管底部。 </li><li>滑动管轻轻地松开颗粒，然后加入5毫升的温暖的HBSS洗细胞。让我们在室温下2分钟定居。 </li><li>在200克离心5分钟。 </li><li>巴斯德吸管取出上清液，留在〜50-100μL液体的试管底部。 </li><li>滑动管，轻轻地松开颗粒和部分悬浮细胞。再加入2毫升温暖的HBSS。 </li><li>使用小（半直径），火抛光玻璃巴​​斯德吸管游离于细胞慢慢地移液和4-6倍。避免气泡，吸液管对管端的液体。不要过度磨碎。 </li><li>用最小的火抛光玻璃巴​​斯德吸管进一步游离于细胞慢慢移液和向下的3-4倍。避免气泡，吸液管对管端的液体。不要过度磨碎。 从粗糙的解剖组织，添加额外的1毫升温暖的HBSS管解离年底 。 当游离的细胞，它是没有必要的分解所有的细胞团块和聚合。停止吹打和向下或改变一个直径较小的巴斯德吸管，如果您没有看到进一步吹打细胞聚集规模进一步减少 。 </li><li>让任何剩余的组织碎片，1分钟，在管内定居。这是没有必要的细胞转移到新的试管。 </li><li>取20μL细胞悬液，加入5μL台盼蓝。一个血球计数细胞。 神经元应≥95％可行的台盼蓝拒。 </li><li>板的神经元在Neurobasal电镀媒体（见下面的食谱） 。 <ul><li>建议电镀密度为获得分离出神经元（低密度文化，以避免结块或相互重叠的神经元）： </li><li> 120 000 - 180 000个细胞/孔为6孔板</li><li> 60 000 - 75 000个细胞/孔12孔板</li></ul></li><li> 16-18小时后，改变媒体Neurobasal生长介质（见下面的食谱） 不要使用真空泵吸从培养皿中的媒体变化的媒体时，轻轻地用吸管 。 这就避免了撞出的神经元。 </li></ol>代表性的成果： 电镀后4小时，应坚持以聚- L -赖氨酸（PLL）涂层表面的神经元。在相衬照明，坚持胞体通常是相对平坦的，椭圆形（图2a）。没有很好地坚持细胞出现的领域，移动时，菜是非常轻轻地放在一边挖略有。许多因素可能阻碍细胞粘附（见讨论）。 大多数神经元在体外培养 30小时后，已经扩展了一个可见的生长锥（图2C，D）一个轴突。如果穷人的轴突生长观察，验证，Neurobasal生长介质已与新鲜培养基和补充。在这些条件下至少有6天的神经元保持健康。此程序已被证明是可靠的产量连合神经元高度浓缩的准备工作，与〜90％的神经元表达DCC（荫等人，2009年）。脊髓背侧地带，准备细胞悬液的宽度会影响文化的纯洁性，具有更大的纯度时使用更薄条。示例应用程序是显示在图3（免疫）。荫等人的文章。 （2009年），为更多的例子。 <img src="/files/ftp_upload/1773/1773fig1tmb.jpg" border="0" alt="图1" /> 图1。脊髓清扫步骤示意图。 D =背，V =腹。 <a href="https://www.jove.com/files/ftp_upload/1773/1773fig1.jpg">点击这里查看大图。</a> <img src="/files/ftp_upload/1773/1773fig2.jpg" alt="图2" /> 图2代表孤立连合的结果PLL的镀膜玻璃盖玻片上镀的神经元。 A，B）电镀后4小时，神经一直坚持到表面。酒吧= 20微米。 C，D）电镀后的30小时，大多数神经元轴突与一个可见的生长锥延长。酒吧= 20微米。 <img src="/files/ftp_upload/1773/1773fig3.jpg" alt="图3" /> 图3。一个连合神经细胞免疫染色为γ-微管蛋白（绿色），与F - actin的鬼笔环肽（F - actin的，红色）和细胞核的DAPI（蓝色）标记。 配方和意见 Neurobasal电镀媒体<ul><li> Neurobasal </li><li> 10％热灭活胎牛血清（HiFBS） </li><li> 2毫米L -谷氨酰胺（200毫米原液） 可选：青霉素/链霉素抗生素（使用正常浓度的一半） </li></ul> Neurobasal生长介质<ul><li> Neurobasal </li><li> B27（1 / 50稀释股票） </li><li> 2毫米L -谷氨酰胺（200毫米原液） </li><li>可选：青霉素/链霉素抗生素（使用正常浓度的一半） 一旦媒体，它可以被保存在4 ° C为两个星期。电镀前至少1.5小时，在组织培养箱内培养的细胞，在培养皿中进行组织培养的地方媒体和地方媒体的温度和pH值达到平衡。 </li></ul> Neurobasal Neurobasal培养基瓶已被打开后，它可以保留一个月，在4 ° C黑暗环境中。 Neurobasal已超过一个月打开，否则细胞存活率会降低处置。 热灭活胎牛血清（HiFBS）或马血清（HiHS） 热灭活胎牛血清或HS，热量在56 ° C在一个30分钟的水浴。摇动一瓶大约每隔10分钟左右。 （使用同样大小的瓶子装满水的准确性，一个放置温度计一瓶水，到56℃达到。开始在这一点上的时间。）热灭活胎牛血清可能需要离心清除沉淀物，并可以分装和重新冻结在-20 ° C L -谷氨酰胺始终解冻每个实验的L -谷氨酰胺的新鲜等分。 B27 可以保持在20 ° C的长期储存，或4 ° C的长达一个月的B27的等分。

<ol>
	<li>Charron, F., Stein, E., Jeong, J., McMahon, A. P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. , 113-1111 (2003).</li><li>Fabre, P., Shimogori, T., Charron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+ipsilateral+retinal+ganglion+cell+axons+at+the+optic+chiasm+requires+the+Shh+Receptor+Boc.">Segregation of ipsilateral retinal ganglion cell axons at the optic chiasm requires the Shh Receptor Boc.</a> Journal of Neuroscience. 30, 266-275 (2010).</li><li>Helms, A. W., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitors+of+dorsal+commissural+interneurons+are+defined+by+MATH1+expression.">Progenitors of dorsal commissural interneurons are defined by MATH1 expression.</a> Development. 125, 919-928 (1998).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1, 2406-2415 (2006).</li><li>Okada, A., Charron, F., Morin, S., Shin, D. S., Wong, K., Fabre, P. J., Tessier-Lavigne, M., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boc+is+a+receptor+for+sonic+hedgehog+in+the+guidance+of+commissural+axons.">Boc is a receptor for sonic hedgehog in the guidance of commissural axons.</a> Nature. 444, 369-373 (2006).</li><li>Yam, P. T., Langlois, S. D., Morin, S., Charron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+guides+axons+through+a+noncanonical,+Src-family-kinase-dependent+signaling+pathway.">Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway.</a> Neuron. 62, 349-362 (2009).</li></ol>

我们已经描述了一个方法来剖析和文化连合从胚胎大鼠脊髓神经元。这个程序已经在我们的实验室经常使用可靠准备的神经元的轴突导向的研究细胞和分子机制。细胞生物学和免疫组织化学实验，解剖一窝产生足够的神经元。当需要更多的细胞，如在许多生物化学实验，解剖两个窝可能是必需的。对于一个受过训练的人，可以进行清扫和分解〜20胚胎在不到4个小时。更长的时间范围内将导致一个?...

胚胎大鼠背脊髓的解剖（参见表我） <ul><li> E13怀孕-上演大鼠</li><li>乙醇70％ </li><li>手术剪刀，精细的科学工具</li><li>杜蒙＃5，精细的科学工具钳， </li><li>培养皿</li><li> L - 15培养基</li><li>解剖镜下，徕卡</li><li>塑料转移移液器</li><li> Microscissors，精细的科学工具</li><li>钨针和针持有人（一个勾形针，一个L形状针，直针），精细的科学工具</li><li>热灭活马血清（HiHS） </li><li> 15毫升的塑料管</li></ul>连合神经元文化（参见表我） <ul><li>组织培养孵化器（37 ° C，5％CO 2，湿度控制） </li><li>德国Desag盖玻片和/或塑料组织培养皿</li><li>聚- L -赖氨酸，西格玛</li><li>无菌milliQ H 2 O </li><li> Neurobasal，Invitrogen公司</li><li>热灭活胎牛血清（HiFBS） </li><li> L -谷氨酰胺</li><li> B27，Invitrogen公司</li><li>青霉素/链霉素抗生素</li><li> 37 °彗星水浴</li><li>无菌巴斯德滴管</li><li>本生气体刻录机</li><li> HBSS中，钙2 + /镁2 +自由，Invitrogen公司</li><li> DNA酶，沃辛顿</li><li> 硫酸镁 </li><li>离心分离</li><li> Hemacytometer </li><li>台盼蓝溶液</li></ul>

dissection and culture of commissural neurons from embryonic spinal cord

连合神经元已被广泛用于研究的内在机制，在胚胎脊髓发展的轴突指导。这些神经元的胞体位于脊髓背侧，其轴突遵循胚胎发育过程中的定型的轨迹。最初连合轴突项目的腹部朝向floorplate。越过中线后，这些轴突转向对大脑前部和项目。上述每个步骤是受几个指导线索的行动。连合神经元高度浓缩的文化，非常适合解决许多轴突寻路机制，包括车削实验，免疫组织化学和生物化学实验。在这里，我们描述的方法来剖析和文化连合从E13大鼠脊髓背侧神经元。首先，是孤立的脊髓和背条被解剖了。胰蛋白酶和机械破碎成细胞悬液，然后背的组织是分离的。神经元被镀上聚- L -赖氨酸镀膜玻璃盖玻片或组织培养皿。 30小时后<em&gt;在体外</em&gt;，大多数神经元轴突延长。文化的纯度（阴<em&gt;等。</em&gt; 2009），通常在90％以上，可评估，LH2连合神经元标记的DCC和TAG1（赫尔姆斯和约翰逊，1998年）免疫标记。这种神经元的准备是一个有用的工具<em&gt;在体外</em&gt;连合在轴突的生长和脊髓发展的指导细胞和分子机制的研究。

Watch this Scientific Journal Video about Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord at JoVE.com

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

这个视频演示的方法来剖析和文化连合从E13大鼠脊髓背侧神经元。是有用的游离连合神经元轴突的生长和指导，研究细胞和分子机制。

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 ...

dissection-culture-commissural-neurons-from-embryonic-spinal

Research

JoVE Journal

Neuroscience

16.7K 次观看.  Institut de Recherches Cliniques de Montréal. 这个视频演示的方法来剖析和文化连合从E13大鼠脊髓背侧神经元。是有用的游离连合神经元轴突的生长和指导，研究细胞和分子机制。

视频: Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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 ...

科研

神经科学

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

小鸡Statoacoustic神经节和脊髓植在胶原凝胶夹层和文化突起生长实验

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

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 ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

从胚胎脊髓​​连合神经的解剖和文化

从胚胎脊髓连合神经的解剖和文化