Name: dissection and culture of commissural neurons from embryonic spinal cord
Uploaded: 2010-05-25T17:15:00
Description: Watch this Scientific Journal Video about Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord at JoVE.com

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?...

<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 ...

Research

JoVE Journal

Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

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

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 ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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

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

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

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

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

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

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

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

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 ...

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