Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

表1为在本协议中使用的所有材料。 实验获得批准的机构动物护理和使用委员会在坦普尔大学。 1，动物<ol><li>获得野生型幼虫海七鳃鳗（Petromyzon马里努斯 L.），10 - 23厘米的长度-从流喂养密歇根湖，从支流到特拉华河（宾夕法尼亚州），或者流于缅因州（4 7岁）。 </li><li>在实验室中，幼虫保持在50组 - 100动物50加仑淡水坦克在16℃。符合碎石英寸坦克的幼虫在挖洞，水是木炭过滤，空调与空气的石头至少48小时将七鳃鳗在他们之前，去除余氯等杂质。没有必要对喂动物，其中回收自己的碎屑，最多使用的当天。 </li></ol> 2，完全性横断<ol><li>脊髓手术前准备有以下组成的七鳃鳗林格液:110 mM氯化钠，2.1 mM的氯化钾，2.6毫米氯化钙2，1.8毫米氯化镁2和10毫米的Tris缓冲液; pH值7.4。 </li><li>要执行脊髓横断，将幼虫在一个小容器进行麻醉，在冰冷的林格液1％三卡因甲磺酸盐。 </li><li>一旦麻醉的地方在陪替氏培养皿的一半填充的Sylgard（聚硅氧烷）的动物，并使用4个昆虫针（0.15毫米直径），以保持在位置上的动物的手术（一个被放置在动物的口鼻部，一个在尾和两个在第 5 次鳃的水平）。填补了培养皿中几乎达到了林格液的顶部。放菜的幼虫上的冰而进行体视显微镜下手术。 </li><li>要执行脊髓横断背，中线，作一纵切口，用手术刀（＃11）在第 5 次的水平鳃形象化脊髓。最初的皮肤是通过将手术刀刀片的尖端朝上，然后将肌肉组织被切得很平缓，直至脊髓从上面可以看到的开放。时尚的昆虫针两个挂钩，并用它们来保持身体的墙壁打开，而执行脊髓横断。 </li><li>用一把剪刀（Castroviejo＃8）完全横切在第 5 次鳃水平脊髓，垂直插入剪刀脊髓。脊髓已被完全地与一个干净的切割横断。可视化的体视显微镜下脊髓切割端，以确认该脊髓已被完全切断。然后，通过使用一对镊子的（＃5）重新对准脊髓的切断端部的横断后。 </li><li>在进行脊髓横断关闭背侧体壁的伤口有一对细钳子并放置冰上幼虫1小时，以允许伤口吨后Ø干。放一张滤纸冰和幼虫间和七鳃鳗林格液浸泡。低温保存的动物活的同时，它使伤口空气干燥，因此，有助于防止感染。 </li><li>然后转移幼虫小坦克（每罐1幼虫）用冰冷的水24小时。 </li><li>脊髓横断后检查损毁幼虫1天到行为上确认，没有任何动静尾椎受伤的部位。脊髓横断被认为是完整的，如果幼虫能只移动其头部吻端到损伤部位（在第 5 次鳃的水平）。如果是这种情况的幼虫现在可传送到罐（每罐1幼虫）与淡水，在室温（23 ℃），使它们恢复为所选定的时间点。 </li><li>在恢复期间换水，每2至3天。 </li></ol> 3，脑解剖和制备脑原位杂交<ol><li>在受伤后的所需时间点重新麻醉幼虫海七鳃鳗和地点和引脚他们在陪替氏培养皿用林格氏溶液来进行脑解剖如上。 </li><li>解剖大脑了，先作一横切口的鼻孔和松果体器官之间的手术刀。从这里开始利用一对Castroviejo剪刀，同时保持其与钳子切割周围的脑组织。 </li><li>一旦脑露出，通过使用一对镊子取出脉络丛和使用钩子来传播该动物的头部和暴露的脑神经。然后，切断脊髓横向与Castroviejo剪刀。而使用其他钳子切颅神经解剖大脑出了钳子抓住脊髓。 </li><li>解剖后，把大脑的一小块硅胶并保持在适当的位置插入2项等销嗅球和1脊髓。然后，切断后并沿背中线与剪刀的大脑cerebrotectal合缝和偏转翼片横向和管脚他们平与4昆虫针（ 图2）的聚硅氧烷。 </li><li>用脑转移片硅氧烷与含在磷酸缓冲盐溶液4％多聚甲醛（pH 7.4）中而固定，在室温北部沿海地区为3小时，在旋转器上的管。 </li><li>固定后在原位杂交用脑的wholemount如前所述（Swain 等人，1994）。从控制或假手术动物的大脑，应始终平行于从受伤的动物的大脑运行。该wholemounted幼虫脑可以通过原位杂交，因为也由于没有髓鞘10，这使得大脑相当半透明其平而薄的形状并且被研究。 </li></ol>

<ol>
	<li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury.">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li><li>Davis, G. R., McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey.">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li><li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons.">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li><li>Shifman, M. I., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection.">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li><li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sea+lamprey+UNC5+receptors+cDNA+cloning,+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development.">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li><li>Shifman, M. I., Yumu, l. R. E., Laramore, C., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey.">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li><li>Busch, D. J., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury.">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li><li>Shifman, M. I., Zhang, G., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys.">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li><li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults.">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li><li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+myelin+sheaths:+both+lamprey+and+hagfish+lack+myelin.">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li><li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys.">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li></ol>

The authors declare that they have no competing financial interests.

在这里，我们提出了一个详细的协议来执行一个完整的脊髓横断，后脑部解剖幼虫海七鳃鳗。这个程序允许通过全贴脑原位杂交的方法在脊髓损伤后可识别的脊髓神经元投射之间的基因表达差异分析。在该过程中的关键步骤是一个完整的脊髓横断的正确性能，从而可以通过观察stereomicrocope下脊髓的切割端24小时后，轻轻触摸幼虫的口鼻部具有一对被控制和确认钳子，以查看是否有任何移动尾椎到?...

在哺乳动物脊髓损伤（SCI）是一种破坏性的条件，导致的损伤部位以下功能永久性丧失，因为受伤的轴突不需要通过创伤区再生和重新连接到适当的目标。相反，哺乳动物，七鳃鳗恢复完全性脊髓损伤后运动。1有趣的是，七鳃鳗有一套36脊髓投射神经元是单独识别整个安装，因为他们的大尺寸2,3（ 图1）脑准备。所有这些脊髓投射神经元是由一个高层次的脊髓完全横断金黄地鼠。我们的组和其他的以前的研究已经表明，即使在功能恢复脊髓损伤后某些神经元表现出非常低的再生能力的存在（它们被认为是"坏的蓄热室"），而其他通常通过损伤部位再生的轴突（他们被认为是"GOOD再生"）。2,3这种特性使得七鳃鳗有趣脊椎动物模型来研究在好的和坏的再生脊髓投射神经元，这反过来会导致神经元的企图的固有再生能力的差异之间基因表达的差异再生的轴突在同一外在环境。1 利用该模型，我们以前曾表明，脊髓投射神经元的轴突与导向分子受体如UNC5 4,5和neogenin，6分别介导netrin诱导和RGM的抑制作用，低蓄热能力秀的表情。此外，通过使用这个方法，我们小组还表明，只有在良好的蓄热表明神经丝的表达的恢复的损伤后和在再生过程。最近，布希和摩根7表明，免疫荧光，坏再生器显示的increas突触核蛋白的表达主编受伤后，已经涉及了作者这一事实，即"坏再生"脊髓投射神经元脊髓完全横断5,7,8后慢慢死去。因此，一个完整的脊髓损伤的七鳃鳗的模型已经成为一个非常有用的模型，以了解是什么造就了脊髓神经元投射一个"坏再生"切断后。 进行我们的研究，我们在期望的时间点执行一个完整的脊髓横断手术协议和后脑部解剖损伤后原位杂交执行wholemount。在本方法的文章中，我们提出了一个详细的协议为完全性脊髓损伤手术在七鳃鳗幼体的正常性能，后续维护动物和最终大脑解剖和准备的大脑原位杂交wholemount。一份详细的协议，PErform原位杂交的wholemount在幼虫七鳃鳗的大脑已经被报告过。9另外，该协议用 ​​于脊髓损伤和脑解剖也可用于再加工的大脑用于免疫组化分析或其他组织学方法。

<table><tbody><tr><td>Tricaine methane sulfonate</td><td>Spectrum</td><td>TR108</td><td>Benzocaine saturated solution in PBS for sacrifice</td></tr><tr><td>Scalpel #3</td><td>Fine Science Tools (FST)</td><td>10003-12</td><td></td></tr><tr><td>Blades for scalpel: #11</td><td>Fine Science Tools&amp;nbsp;</td><td>10011-00</td><td></td></tr><tr><td>Castroviejo scissors #8</td><td>Fine Science Tools&amp;nbsp;</td><td>15002-08</td><td></td></tr><tr><td>Forceps #4 &amp;amp; #5</td><td>Dumont, Switzerland</td><td>Roboz RS4955</td><td>#4 for dissection of Spinal cord; #5 for stripping menninges</td></tr><tr><td>Dissecting Microscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Sylgard</td><td>Dow Corning Co.</td><td>184</td><td></td></tr><tr><td>Insect pins 0.15, 0.20 mm</td><td>Austerlitz</td><td>No catalogue #</td><td>0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td></tr><tr><td>7 ml HDPE Scintillation Tubes with Caps</td><td>Fisher Scientific</td><td>03-337-1</td><td></td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Science (EMS)</td><td>19210</td><td>Dilute to 4% in PBS</td></tr></tbody></table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

经过完全性脊髓损伤，海七鳃鳗在第一瘫痪横断以下的水平。然而，它们恢复运动数周之后，与此相伴的propriospinal轴突和从脑干脊髓伸出的轴突的短距离再生（几个毫米）。其中36个大型识别脊髓投射神经元，有些是很好的再生等是坏的再生。这些神经元可最容易在wholemount CNS制剂来鉴定。为了了解在有利于神经元内在机制或在脊椎动物中枢神经系统损伤后抑制轴突再生，我们确定好坏再生器之间的差异的基因表达，以及如何表达由脊髓横断影响。本文阐述了在淡水舱的技术外壳幼虫和近期改造成海七鳃鳗，产下的显微视觉完全性脊髓横断，并准备BR泉和脊髓wholemounts用于原位杂交。简要地，将动物保持在16 &#160; °C和麻醉的1％苯佐卡因的七鳃鳗铃声。脊髓经由背侧入横断与虹膜切除术剪刀和该动物被允许在淡水罐在23℃下进行恢复。用于原位杂交，将动物再次麻醉，并在大脑和脊髓经由背侧的方法除去。

作为可使用这种方法时得到的结果的一个例子，wholemounted大脑的表示控制的可识别的脊髓投射神经元和2周neogenin转录物的表达的代表性图像受损后幼虫海七鳃鳗示于图2。的读者可以参考先前的研究报告6完全脊髓横断和识别脊髓投射海七鳃鳗的神经元的全部细节再生能力后neogenin的表达之间的关系。简要地说，结果表明，在受伤后有变化，在识别脊髓投射神经元的neogenin受体的...

Watch this Scientific Journal Video about Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey at JoVE.com

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

七鳃鳗完整的脊髓损伤后恢复运动。然而，一些脊髓投射神经元是很好的再生和其他人都没有。本文说明了技术的外壳海七鳃鳗幼体（最近转化成人），产生完全性脊髓横断和原位杂交准备wholemount大脑和脊髓的。

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

complete-spinal-cord-injury-brain-dissection-protocol-for-subsequent

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

9.8K 次观看.  University of Edinburgh. 七鳃鳗完整的脊髓损伤后恢复运动。然而，一些脊髓投射神经元是很好的再生和其他人都没有。本文说明了技术的外壳海七鳃鳗幼体（最近转化成人），产生完全性脊髓横断和原位杂交准备wholemount大脑和脊髓的。 

视频: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

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 ...

科研

神经科学

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

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 ...

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 ...

生物学

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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 ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

受控半自动激光诱导损伤研究斑马鱼幼虫脊髓再生

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

完全性脊髓损伤和脑解剖协议后续Wholemount

摘要

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此视频中的章节

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

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

解剖和斑马鱼的胚胎横向安装:脊髓发展分析

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

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

一个

斑马鱼

立体定向激光捕获创伤性脑区显微解剖

受控半自动激光诱导损伤研究斑马鱼幼虫脊髓再生

完全性脊髓损伤和脑解剖协议后续Wholemount

摘要

探索更多视频

此视频中的章节

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

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

解剖和斑马鱼的胚胎横向安装:脊髓发展分析

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

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

一个

斑马鱼

立体定向激光捕获创伤性脑区显微解剖

受控半自动激光诱导损伤研究斑马鱼幼虫脊髓再生

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生