此协议是在简多德，托马斯Jessell和安德鲁拉姆斯登的实验室开发的。许多人，包括康拉德巴斯勒，安Calof，托马斯埃德伦德，菲尔汉密尔顿，Domna Karagogeos，沙龙鲁伊斯我Altaba和俊山田决定如何脊髓胶原蛋白的文化植。 Marysia Placzek和马克特西尔儿率先使用在体外植体作为识别轴突导向分子的手段的轴突的生长技术。巴特勒实验室的工作是从3月从美国国立卫生研究院/ NINDS优生优育基金会和R01 NS063999的赠款支持。

第1部分：使用悬滴转染COS细胞聚合制备<ol><li>种子COS - 7（COS）在35mm培养皿中的细胞。当他们到达80％汇合，到使用Lipofectamine2000根据制造商S协议细胞的表达质粒转染1μg。 </li><li>为了准备吊滴，吸转染介质和细胞的转染COS1毫升的1X的磷酸盐缓冲液（PBS）冲洗。治疗与无细胞酶的解离15分钟的中等0.5毫升的细胞。添加OPTI - MEM + 1青霉素/链霉素/谷氨酰胺（P / S / G）+ 10％小牛血清（FBS），停止反应的解决方案1毫升。 </li><li>磨碎的细胞培养皿的表面删除，并转移到一个15毫升锥形管。旋转2分钟沉淀细胞在2000K。删除OPTI - MEM + 1X P / S / G + 10％FBS的100μL上清，重悬沉淀。 </li><li>点上一个35mm培养皿的盖子内的多个20μL滴，反转盘底部顶部的盖子和地点，并在37 ° C培养箱离开几个小时，待细胞总。 </li></ol>第2部分：背侧脊髓外植体的制备<ol><li>从母亲的子宫解剖E11大鼠胚胎，并保持在L15的介质上的冰，直到需要。 </li><li>有了一个削尖的钨针，请从每个胚胎的躯干4-6段部分，从下方前肢芽的区域。用塑料吸管，收集在一个4以及NUNC菜的组织块，置于冰上。 </li><li>当所有的胚胎段已被解剖，用塑料吸管转移到一个解决方案，为1ml L15 + 1mg的dispase在NUNC盘的第二件。在室温下孵育时间不超过5分钟。不要过度的孵化与dispase组织块。 </li><li>在孵化期间，加入热灭活正常山羊血清（HIGS）0.5毫升L1510毫升到培养皿中，漩涡混合。转移到第三NUNC菜的这一解决方案1毫升，并转移到该解决方案的组织块，孵化结束时。置于冰上。注意：如果允许一个小时“休息”冰的组织块，随后的剥离会比较容易。 </li></ol>第3部分：底漆胶原蛋白<ol><li>加入40μl10倍最小的必不可少的中等至360μl胶原蛋白，混合迅速，彻底地轻弹或简要涡旋管。在picofuge迅速降速。该解决方案将是黄色的。，尽可能保持在冰上 。 </li><li>添加足够的0.8M的碳酸氢钠（ 碳酸氢钠3）打开略有橙色的胶原蛋白溶液（见下面的说明） 。再次，混合迅速，彻底地弹和自旋向下picofuge简要。如果解决方案仍然是粉红色混合后，你有太多碳酸氢钠 3，将需要再次开始的胶原蛋白的新鲜管。 </li><li>此时，胶原溶液催芽。它仍将在冰上的液体，但巩固带来至室温时，将在5分钟左右（车削粉红色）。 </li><li>在每一个4孔NUNC菜以及胶原蛋白的现货20μL。使用吸管的笔尖，摊开的胶原蛋白，以形成一个小型的“垫”。让我们在室温胶原。 </li></ol>注：3碳酸氢钠的确切数额将需要每批胶原蛋白的滴定。起点低（11μl），然后添加0.5 -1μl进逐步直到解决方案是一个橙色轻微阴影。 “正 ​​确”的数额通常会变成胶原蛋白粉红色的最小量的碳酸氢钠 3 1ul 。碳酸氢钠3量并不线性比例上升或下降。 第4部分：夹层背侧脊髓外植体和他们的定位与COS在胶原蛋白基质细胞聚集<ol><li>使用新鲜削尖的钨针和一双细镊子（＃5或＃55），取出脊髓周围的中胚层。 </li><li>平行切割的底板与钨针，小心取出脊髓腹侧第五。 </li><li>对开背侧脊髓外植体，确保边缘尽可能干净切。重新修剪的边缘，如果有必要。每个背侧脊髓外植体转移到单独的胶原蛋白，使用移液器装上拉至大约用玻璃毛细管管口垫。 0.5毫米直径。 </li><li>切转染COS细胞约背侧脊髓外植体的外侧边缘的宽度相同的平方的总和。 </li><li>这些广场之一转移到背侧脊髓外植体的胶原蛋白垫，使用口吸管。 </li><li>用口吸管，吸过任何多余的液体，用口吸管。不要过度吸。 </li><li>应用到催芽胶原4μl，垫，使用钨针不直接接触组织的胶原蛋白，在植。通过移动在植体周围的胶原的钨针，东方植，使COS细胞总量的背侧脊髓外植体外侧缘旁。 </li><li>胶原蛋白后，重复的胶原蛋白4μl的应用，以确保外植体完全包裹在胶原。 </li><li>在组织培养罩中，添加OPTI - MEM + 1xP/S/G和30-40小时的文化0.5毫升。 </li><li>修复植0.5毫升4％多聚甲醛在1xPBS 45分钟，冰板。 </li><li>洗两次的1xPBS封闭液+ 0.1％，1％TRITON - X100 HIGS（PBTN）0.5毫升。座在4 ° C的至少几个小时，孵育过夜，是可取的，然后通过免疫组化的过程中。 </li></ol>第5部分：通过免疫组连合轴突的可视化<ol><li> PBTN阻断后，削减的植产钳和一个48孔盘的4孔板。 </li><li>新增的主要抗体200μL，每孔，留在4℃过夜° C。为了形象化连合轴突，使用鼠抗TAG1抗体1:6的解决方案。要确定转染是否成功，还包括对被测试的信号分子，或相关的抗原表位的信号分子已被抗原表位标记的抗体。 </li><li>每个样品洗5 × 1小时PBTN在4 ° C。 </li><li>新增二级抗体200μL，每孔，留在4℃过夜° C。如果反TAG1抗体作为第一抗体，羊抗小鼠IgM二抗耦合FITC或Cy3标记。 如果加上使用光敏二级抗体FITC或Cy3标记，保持箔覆盖从这个角度上的48孔盘。 </li><li>洗净每个样品5 × 1小时PBTN 4 ° C。 </li><li>转移的外植体，以3以及抑郁症的幻灯片，删除多余的液体，Vectashield中型和盖玻片的安装。在轻保险箱商店，4 ° C或-20 ° C </li></ol>第6部分：外植体的代表图片在一个成功的实验，外植体和COS的总将保持彼此相邻，而不是疏远胶原套。会有最小的轴突的繁茂;显着的轴突的繁茂表示，背侧脊髓外植体培养时间过长。将有从外植体生长无滤过泡;出泡发生，如果组织直接接触带有培养基。 使用共聚焦显微镜的信号分子的能力，重新调整轴突评估。通过测量平均角度调整最接近的COS细胞总3的轴突，轴突转岗程度可以量化。如图1所示，COS细胞中表达myc基因标签BMP7（蓝色）重新调整TAG1连合轴突+（绿色），类似的方式，顶板的外植体排斥连合轴突（图BMP7源（图1B ） 1A）。 COS细胞中表达的控制向量3,4连合轴突的方向没有影响。这些植也被标记抗体对转录因子Isl1 / 2（红色），其中装饰运动神经元和背interneurons。这一结果首次显示了BMP家族介导成员顶板chemorepellent 3的活性。 <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> 图1：请<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1853/1853fig1.jpg">点击这里</a>看到图1的放大版本。

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of the rat spinal cord. , (1984).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+recombinations+in+collagen+gels.">Tissue recombinations in collagen gels.</a> Methods in molecular biology. , 325-335 (2008).</li><li>Augsburger, A., Schuchardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24, 127-141 (1999).</li><li>Butler, S. J., Dodd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+BMP+heterodimers+in+roof+plate-mediated+repulsion+of+commissural+axons.">A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons.</a> Neuron. 38, 389-401 (2003).</li><li>Kennedy, T. E., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78, 425-435 (1994).</li><li>Charron, F., Stein, E. <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, 11-23 (2003).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Dodd, J., Morton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+axonal+glycoprotein+expression+on+subsets+of+embryonic+spinal+neurons.">Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons.</a> Neuron. 1, 105-116 (1988).</li><li>Yamauchi, K., Phan, K. D., Butler, 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=BMP+type+I+receptor+complexes+have+distinct+activities+mediating+cell+fate+and+axon+guidance+decisions.">BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions.</a> Development. 135, 1119-1128 (2008).</li></ol>

确定在执行这个实验成功的关键因素，首先，组织不应与太长时间的一个时期dispase治疗，这种治疗会导致组织变得很粘和减少可行性。二，胶原蛋白，必须完全底漆和保持尽可能在冰上。这将成为越来越难以处理，如果它开始设置，即变成粉红色。三，钨针必须始终保持非常尖锐的。

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

在脊椎动物的脊髓后连合轴突一直是非常宝贵的模型系统，在其中找出轴突导向信号。在这里，我们描述了一个在体外实验中，“转岗检测”，已广泛用于研究方向 2连合轴突的外在和内在信号的影响。此法简多德，托马斯Jessell和安德鲁拉姆斯登的实验室中的许多人（详情请参阅确认），这个实验版本用于演示重新定位的关键轴突导向分子的活动，包括BMP在chemorepellent顶板3,4和Netrin1 5和刺猬（SHH）在脊髓中的底板 6 chemoattractive活动。 外植体，包括2-3段脊髓背三分之二是从胚胎一天（五）11大鼠解剖，并在三维胶原凝胶 7中培养。 E11背脊髓植包含新出生的连合神经元，他们的糖蛋白，TAG1 8轴突的表达，可以通过确定的。在30-40小时在培养过程中，连合轴突轨迹是在同一个时间过程相似，在体内看到这些背植详。这轴突的轨迹，可以放置或者测试组织或一个背外植体的外侧边缘接触的候选信号分子表达的COS细胞总的挑战。连合轴突延长追加组织附近的内源性顶板和信号从横向组织异位的影响下成长。在这种情况下，重新调整的程度，连合轴突可以量化的。使用这个实验中，它可能既要研究的一个特定的信号重新调整连合轴突，以及为这个信号直接连合的轨迹9的必要性3,4自给自足。

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

这种检测评估的一种信号分子的能力，这里骨形态发生蛋白7（BMP7），调整连合轴突。一个胚胎脊髓背侧的外植体是培养毗邻COS细胞分泌候选人生长因子的总和。重新调整连合轴突生长外植体内免疫组织化学显示。

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

assaying-ability-diffusible-signaling-molecules-to-reorient-embryonic

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.8K 次观看.  University of Southern California. 这种检测评估的一种信号分子的能力，这里骨形态发生蛋白7（BMP7），调整连合轴突。一个胚胎脊髓背侧的外植体是培养毗邻COS细胞分泌候选人生长因子的总和。重新调整连合轴突生长外植体内免疫组织化学显示。

视频: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

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

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 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 ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons1. These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues2,3. Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FPcm)4-6. We then exemplify the use of this FPcm in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FPcm. Third, the tissue are processed for Western blot analysis of commissural markers.

The floor plate is a crucial structure of the developing spinal cord, providing diffusible signals to progenitors and differentiating neurons. We describe a method to produce floor-plate conditioned medium, to apply it to fresh spinal cord tissue, and to assess the consequences on proteins of interest by biochemistry.

隔离楼板组织条件培养基和生产，以评估楼板释放信号的功能性质的文化

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

生物学

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

摘要

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此视频中的章节

从胚胎脊髓连合神经的解剖和文化

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

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

解剖和斑马鱼的胚胎横向安装:脊髓发展分析

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

隔离楼板组织条件培养基和生产，以评估楼板释放信号的功能性质的文化

脊髓电

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

一种方法来提高背根神经节的对齐和髓鞘

胚胎运动神经元轴突投影模式的可视化

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

摘要

探索更多视频

此视频中的章节

从胚胎脊髓​​连合神经的解剖和文化

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

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

解剖和斑马鱼的胚胎横向安装:脊髓发展分析

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

隔离楼板组织条件培养基和生产，以评估楼板释放信号的功能性质的文化

脊髓电

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

一种方法来提高背根神经节的对齐和髓鞘

胚胎运动神经元轴突投影模式的可视化

从胚胎脊髓连合神经的解剖和文化