我们感谢犹他大学的斑马鱼设施畜牧业。 RID是由美国国立卫生研究院R56NS053897支持，以及LKB是由霍华德休斯医学研究所医学 - 到 - 毕业倡议支持的博士前受训者。

斑马鱼是按照标准程序提出和繁殖;实验已获犹他州实验动物管理和使用委员会的大学。 1，准备手术的板<ol><li>使手术板块下使用60毫米培养皿和的Sylgard 184有机硅弹性体套件，下列制造商的说明。填写菜不超过半满以上，并允许聚合。店铺覆盖在室温下。 </li></ol> 2制备的微量移液器<ol><li>通过加热和使用作出显微注射针相同的设置拉薄壁硼硅毛细管中微量拉马制造微量。 </li><li>在解剖镜下，折断微量的尖端直径用钳子约200微米。 </li><li>锥破碎边缘与microgrinder最初至35°，接着在25℃的第二坡口。确保针尖锋利，SMO超视距。店内完成斜面微量在少量粘土的培养皿中。 </li></ol>斑马鱼幼虫3。准备<ol><li>前7天手术，成立了男性和女性的斑马鱼的交配坦克。 </li><li>收集胚胎第二天早上，3小时后，指示灯会亮起，以确保最大收益。如果使用的是转基因记者一行如玻璃转化温度（elevl3：EGFP）knu3，排序受精胚胎100/100毫米的钢板在25毫升的E3在28.5°C。如果使用野生型，排序在28.5℃，受精胚胎25/100毫米的钢板在25毫升的E3 </li><li>如果使用的是记者一行，筛选胚胎的荧光表达在48 HPF。允许鉴定胚胎在25/100毫米的钢板的密度成熟在25毫升的E3在28.5°C。 </li><li>当幼虫在5 DPF，覆盖的Sylgard与E2 + 10 mg / L的硫酸庆大霉素（GS）+三卡因准备手术板块。 <ol><li>通过加入25ml E2 + GS到100 mm的Petri显示准备恢复盘小时。 </li><li>用胶带在一起3拭子准备手术刀。这将形成有三个凹槽的三角形的工具。 </li><li>通过绑扎它到凹槽中的一个安装在该拭子一个微量制备。 </li></ol></li><li>如果重复使用微量，冲洗至干净为止与E2 + GS用1ml注射器和27号针头安装在拭子之前。 </li></ol> 4。手术<ol><li>麻醉1盘幼体的时间（25鱼）与三卡因。鱼被充分麻醉，当他们不再表现出触摸响应。重要的是鱼完全手术前麻醉，否则当手术刀触及他们，他们会抽搐。解剖显微镜下进行手术。 </li><li>幼虫转移手术板块。 <ol><li>在最大放大倍率，旋转一个幼虫的时间，以便它是否位于其侧背面最接近手拿着手术刀。 </li><li>位置钳，使他们安息在的Sylgard，角度对幼虫的宽度。 </li><li>支撑对钳子的臂中的一个的玻璃手术刀，在肛门孔的水平切入幼虫的背侧侧面，即一定不要切断超出脊索的腹边缘。扭动手术刀切断脊髓。 </li><li>重复剩余的幼虫。 注意：如果一个幼虫渗出，也不会从手术中恢复。立即从手术取出钢板的幼虫，并通过三卡因过量安乐死吧。 </li></ol></li><li>一旦完成手术对一批幼虫，转移受伤的动物恢复盘。这是为了支持麻醉结算。 <ol><li>注意：收集受伤幼虫转移时，请务必将其收集头部或尾部第一：不要通过弯曲应力幼虫损伤部位。 注意：用于外科手术的所有装置可以重复使用，包括微量。 </li></ol></li></ol> 5。恢复<ol><li>风帆ansfer受伤幼虫从恢复盘到100mm板在25/plate的密度填充25毫升E2 + GS。允许在28.5℃培养箱中恢复。 </li><li>检查板每天，去除病，死动物。不要更改媒体，直到Coleps（淡水原生动物）是在媒体上看到。当改变媒体，请不要将鱼转移到一个新的板块;相反，去除尽可能多的媒体尽可能和洪水同板块新媒体。必要时重复，减少Coleps人口。 </li><li>每天用少量的粉状鱼苗喂食物。 注：现场食物（ 如草履虫或轮虫）不能被输送到受伤的幼虫，他们已经恢复运动之后。否则，活食拓殖会损伤部位，并杀死幼虫。 </li></ol>

<ol>
	<li>Houweling, D. A., B&#228;r, P. R., Gispen, W. H., Joosten, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+bridging+the+lesion+and+the+role+of+neurotrophic+factors+in+repair.">Spinal cord injury: bridging the lesion and the role of neurotrophic factors in repair.</a> Progress in brain research. 117, 455-471 (1998).</li><li>Mikami, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+dendritic+cells+in+injured+adult+spinal+cord+results+in+activation+of+endogenous+neural+stem/progenitor+cells+leading+to+de+novo+neurogenesis+and+functional+recovery.">Implantation of dendritic cells in injured adult spinal cord results in activation of endogenous neural stem/progenitor cells leading to de novo neurogenesis and functional recovery.</a> Journal of neuroscience research. 76 (4), 453-465 (2004).</li><li>Chernoff, E. A. G., Sato, K., Corn, A., Karcavich, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+regeneration:+intrinsic+properties+and+emerging+mechanisms.">Spinal cord regeneration: intrinsic properties and emerging mechanisms.</a> Seminars in Cell &amp; Developmental Biology. 13 (5), 361-368 (2002).</li><li>Kuscha, V., Barreiro-Iglesias, A., Becker, C. G., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+tyrosine+hydroxylase+and+serotonergic+systems+in+the+regenerating+spinal+cord+of+adult+zebrafish.">Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish.</a> The Journal of comparative neurology. 520 (5), 933-951 (2012).</li><li>Becker, C. G., Lieberoth, B. C., Morellini, F., Feldner, J., Becker, T., Schachner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1.1+is+involved+in+spinal+cord+regeneration+in+adult+zebrafish.">L1.1 is involved in spinal cord regeneration in adult zebrafish.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 24 (36), 7837-7842 (2004).</li><li>Hui, S. P., Dutta, A., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+after+crush+injury+in+adult+zebrafish+spinal+cord.">Cellular response after crush injury in adult zebrafish spinal cord.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 239 (11), 2962-2979 (2010).</li><li>Goldshmit, Y., Sztal, T. E., Jusuf, P. R., Hall, T. E., Nguyen-Chi, M., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf-dependent+glial+cell+bridges+facilitate+spinal+cord+regeneration+in+zebrafish.">Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 32 (22), 7477-7492 (2012).</li><li>Reimer, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+regeneration+in+adult+zebrafish.">Motor neuron regeneration in adult zebrafish.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 28 (34), 8510-8516 (2008).</li><li>Hale, M. E., Ritter, D. A., Fetcho, 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=A+confocal+study+of+spinal+interneurons+in+living+larval+zebrafish.">A confocal study of spinal interneurons in living larval zebrafish.</a> The Journal of comparative neurology. 437 (1), 1-16 (2001).</li><li>Bhatt, D. H., Otto, S. J., Depoister, B., Fetcho, 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=Cyclic+AMP-induced+repair+of+zebrafish+spinal+circuits.">Cyclic AMP-induced repair of zebrafish spinal circuits.</a> Science. 305 (5681), 254-258 (2004).</li><li>McClenahan, P., Troup, M., Scott, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fin-tail+coordination+during+escape+and+predatory+behavior+in+larval+zebrafish.">Fin-tail coordination during escape and predatory behavior in larval zebrafish.</a> PloS one. 7 (2), (2012).</li><li>Kim, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repressor+activity+of+Headless/Tcf3+is+essential+for+vertebrate+head+formation.">Repressor activity of Headless/Tcf3 is essential for vertebrate head formation.</a> Nature. 407 (6806), 913-916 (2000).</li></ol>

作者什么都没有透露。

当最初学习这个技术，我们建议尝试在单个会话不超过50-100个断面。掌握这一技术后，我们能够横切高达每300小时的胚胎;然而，吞吐量这个级别需要每周练习了几个月。我们还建议实行带记者一行和验证完整的横断直到不完全性脊髓横断伤的发生率降低到1％以下。 在成年斑马鱼脊髓横断是一个行之有效的和强大的技术研究轴突再生和神经损伤后。通过将这种分析成幼虫生物...

重大创伤人类脊髓常常导致永久性瘫痪和下面损伤水平感觉丧失，由于无法再生的轴突或重新开始神经发生1,2。相反，哺乳动物，但是，anamniotes包括蝾螈和斑马鱼（ 斑马鱼 ），即使完全脊髓横断3,4展现强劲复苏。 成年斑马鱼是一种行之有效的模式，研究恢复过程脊髓损伤5-7。下面的完整脊髓横断，感觉和机车功能重建是在成年斑马鱼6周后，受伤8观察。为了检查体内的再生过程中，我们转向透明斑马鱼幼鱼9。 在这里，我们提出了一个方法来样带5天后受精（DPF）的斑马鱼幼虫USI脊髓纳克斜面显微注射移液管的手术刀，从哈特改性等人 10此方法支持高吞吐量，低死亡率，和可再现性。通过练习，幼虫300 /小时，可横切，和6个月以上个断面，其中包括超过3,600动物，98.75％±0.72％存活，直到后7天损伤（DPI）。我们的数据显示感官和运动的快速恢复，以及：1 dpi时，所有的运动者受伤的鱼只由胸鳍运动驱动。然而，幼虫开始响应由2 dpi至钨针尾触摸到横断，重新建立C-弯曲的移动由3 DPI，并通过5 dpi 11个图显示掠夺游泳。使用抗乙酰化微管蛋白抗体染色，我们已经证实，轴突是从损伤部位在1 dpi的缺席，但拥有5 DPI越过损伤部位。我们相信，该协议将提供宝贵的技术轴突再生和神经在脊髓损伤后的研究。 </p&gt;

<table border="0" cellpadding="0" cellspacing="0" style="width:733px;" width="733">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">60mm petri dish</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">82050-544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">100mm petri dish</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">89038-968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">NC9644388</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">borosilicate capillary tubing: OD 1.00mm ID 0.78mm</td>
			<td style="width:179px;">Warner Instruments Inc.</td>
			<td style="width:119px;">64-0778</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">forceps</td>
			<td style="width:179px;">Fine Scientific Tools Inc.</td>
			<td style="width:119px;">11252-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">disssection microscope</td>
			<td style="width:179px;">Nikon</td>
			<td style="width:119px;">SMZ6454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">microgrinder</td>
			<td style="width:179px;">Narishige</td>
			<td style="width:119px;">EG-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Gentamycin Sulfate</td>
			<td style="width:179px;">Amresco Inc.</td>
			<td style="width:119px;">0304-5G</td>
			<td>dissolve in water 10mg/ml, store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tricaine</td>
			<td style="width:179px;">Acros Organics</td>
			<td align="right" style="width:119px;">118000100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">cotton tipped applicator, wood, 6-inch</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">1ml syringe</td>
			<td style="width:179px;">BD</td>
			<td align="right" style="width:119px;">309625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">27 ga. needle</td>
			<td style="width:179px;">BD</td>
			<td align="right" style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fry food</td>
			<td style="width:179px;">Argent Labs</td>
			<td style="width:119px;">F-ARGE-PTL-CN</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">micropipette puller</td>
			<td style="width:179px;">Sutter Instrument Co.</td>
			<td style="width:119px;">Model P-97</td>
			<td>Box Filament FB330B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">20x E2 (1L)</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">17.5g NaCl</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S671-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">0.75g KCl</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">P217-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">2.90g CaCl2&#183;2H2O</td>
			<td style="width:179px;">Sigma&#160;&#160;&#160;</td>
			<td style="width:119px;">C7902-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">4.90g MgSO4&#183;7H2O</td>
			<td style="width:179px;">Merck</td>
			<td style="width:119px;">MX0070-1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">0.41g KH2PO4</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">P285-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">0.12g Na2HPO4</td>
			<td style="width:179px;">Sigma&#160;&#160;&#160;</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">500x NaCO3 (10ml)</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td>make fresh, discard extra</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">0.35g NaCO3</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">1x E2 (1L)</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td>store at RT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">50ml 20x E2</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:269px;">2ml fresh 500x NaCO3</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spinal cord transection in the larval zebrafish

脊髓损伤是由于缺乏低于损伤水平轴突再生，以及为无法重新开始脊髓神经发生下列哺乳动物失败的感觉和运动恢复。然而，一些anamniotes包括即使在脊髓完全切断了斑马鱼表现出感觉和功能恢复。成年斑马鱼是一个既定的模式生物再生研究脊髓损伤后，用感觉和运动恢复6周后受伤。要利用体内分析的再生过程中的透明斑马鱼幼虫可用以及遗传工具的成人无法访问，我们用斑马鱼幼虫脊髓横断后，研究再生。在这里，我们展示了一种可重复和可核查的横切幼虫脊髓。横断后，我们的数据表明感觉恢复在开始后2天伤（DPI），机智h时的C-弯曲运动检测的3 DPI和恢复自由游泳5 DPI的。因此，我们提出了斑马鱼幼虫作为一个配套工具的成年斑马鱼来进行恢复脊髓损伤后的研究。

以降低周围的损伤部位的组织损伤的严重程度，微量的适当斜角是至关重要的。 图1A示出一个正确的斜面的尖端。使用尖太宽（ 图1B）往往导致更高的死亡率，由于切口的背主动脉的可能性增加，同时一个提示，太窄（ 图1C）的倾向一目了然从皮肤而不是切割组织。 实施本技术，但有利的是使用一个记者线如玻璃转化温度（elevl3?...

Watch this Scientific Journal Video about Spinal Cord Transection in the Larval Zebrafish at JoVE.com

Spinal Cord Transection in the Larval Zebrafish

脊髓横断后，成年斑马鱼具有功能恢复六个周后，损伤。要利用幼虫的透明度，恢复快的，我们提出了横断幼虫脊髓的方法。横断后，我们通过后3天观察伤感觉恢复在开始后2天伤，和C-弯曲运动。

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

spinal-cord-transection-in-the-larval-zebrafish

Research

JoVE Journal

Neuroscience

11.8K 次观看.  University of Utah. 脊髓横断后，成年斑马鱼具有功能恢复六个周后，损伤。要利用幼虫的透明度，恢复快的，我们提出了横断幼虫脊髓的方法。横断后，我们通过后3天观察伤感觉恢复在开始后2天伤，和C-弯曲运动。 

视频: Spinal Cord Transection in the Larval Zebrafish

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

斑马鱼幼虫的前脑的电生理记录

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

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 ...

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

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

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

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

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 ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...