这项工作是由来自美国国立卫生研究院（F32 NS059120到RLM和R01NS25217和P30NS048154到ABR）的资助。

作者宣称没有竞争的经济利益。

这里描述的方法允许脊髓的最小解剖后斑马鱼胚胎的感觉和运动神经元的电和形态学特征。神经元至少1小时，强加给这些录音时间限制保持健康。神经元已经使用标准的全细胞构型记录，以及从核贴剂;后者的方法最小化，可以排除离子电流9的详细研究生物物理空间钳的问题。 一个重要的挑战是实现胚胎或幼虫室的坚定执着，以去除皮肤和执行提供访问感兴趣的神经元所需要的有限的解剖。制剂也需要被适当地固定到录音室的全细胞膜片钳方法。即通过使用兽医缝合胶应对这一挑战的方法来连接胚胎或幼虫解剖/记录室这里所描述的，已被用于其它模式生物的解剖性的方法（ 例如，果蝇 ）63。从我们的经验培训他人，我们发现，掌握了最关键的一步是少量胶水的控制和精准投放。这里，胶分配器装置，其允许使用者施加负和正压力，以胶装入或从微量的尖端排出被讨论。使用缝线胶，胚胎和幼虫可被牢固地附接到所述腔室和面向既背朝上或横向。通过这种方式，为各种神经元的不同的接入选项可供选择。另外，在腔室底部有机硅弹性体的层可以比此处指定的1mm时，提供潜在的优点光学甚至更薄。另一种方法，更通常用于连接的准备到记录室中，包括使用的细钨插针1 &lt;SUP&gt;，2，3，4，5。虽然这方法不同，都允许以斑马鱼的脊髓神经元的电接入，从而得到研究人员可以根据目标和实验的挑战，可以选择的选项。 这里所描述的斑马鱼制备允许原位脊髓神经元的电和形态学研究期间分化的他们的早期阶段。通过使用这些方法从脊髓神经元的记录，我们甚至损毁基因6，64，65的识别之前，获得了洞察几个突变的细胞效应。

<html>乔治·斯特雷伊辛格率先使用斑马鱼 ，俗称斑马鱼作为脊椎动物发育10的遗传分析模型系统。该模型提供了几个优点，包括:（1）相对简单和便宜畜牧业; （2）外部受精，从而允许方便地从最早的发育阶段的胚胎;和（3）的透明胚胎，从而允许细胞，组织和器官的直接和重复观察，因为它们形成。 在随后的几十年里，进行了若干改进进一步增加了斑马鱼模型的力量。特别是，正向遗传学屏幕和全基因组测序工作发挥了重要作用的基因突变和基因关键的标识许多发育过程11，12，13，14，"&gt; 15，16。Gateway克隆方法已经允许转基因的常规应用方法17,18。在基因组编辑的最新进展，通过转录激活状（TALEN的）中列举和群集规则间隔开的短回文重复序列（CRISPR）-Cas9核酸酶，允许有针对性地引入突变，以及敲除和敲入接近19，20，21，22，组合，这些方法使斑马鱼为底层的具体行为和一些人类疾病的遗传机制的研究提供强大的模型23，24，25，26，27。 今年工作重点放在发展心理调节和电活动的神经发育中的作用。重点是脊髓，为此，斑马鱼模型提供了几个优点。首先，它是相对容易的，在胚胎和幼体阶段访问斑马鱼;因此，可以在具有较少的神经元和较简单的电路28，29的发育阶段研究脊髓功能。此外，斑马鱼脊髓具有一组不同的神经元，类似于其他脊椎动物中，因子30，31，32，33，34，35所证明由转录的特性和特征图案。 旨在揭示背后脊髓电路的功能的机制的多数在斑马鱼的研究中，尤其是支持运动的，是可以理解集中于幼虫阶段36，37，38，39，40，41，42，43。然而，许多形成脊柱机车网络中的神经元的启动它们的分化在早期胚胎阶段，〜9-10小时后受精（HPF）44，45，46，47，48，49，50，51。鉴于此，了解如何脊髓神经元的形态学和电学性质发生和胚胎和幼虫阶段之间的变化是一个重要overa的运动电路形成和功能LL理解。 这里描述的解剖方法允许从脊髓神经元膜片钳记录，并在胚胎阶段（〜17-48 HPF）和幼体阶段（〜3-7天受精后[DPF]）得到成功应用。该方法限制了解剖，以提供对感兴趣的神经元所需要的量。所述协议从多数的其它公开的方法的不同之从在兽医缝合胶斑马鱼脊髓神经元的记录使用的，而不是一个细钨针，胚胎或幼虫附着到记录室。的两种不同的方法的可用性（ 即，缝合胶相对于钨针）用于安装所述斑马鱼的胚胎或幼虫用于电生理分析为研究人员提供替代方案，以实现其特定的实验目标。 首先，从弹出访问和记录过程<html>初级感觉神经元的ulation，Rohon-胡子细胞，进行了描述。这些神经元的细胞体位于背侧脊髓内。 Rohon-胡子细胞存在于许多脊椎动物物种，在开发早期分化，和背后胚胎触摸响应6，44，47，48。 第二，用于访问和从脊髓运动神经元的记录程序的详细。期间神经发生的两个波斑马鱼脊髓运动神经元引起的。在出生较早初级运动神经元出现在原肠胚形成（〜9-16 HPF）的端部，只有3-4本每hemisegment 45,46，49初级运动神经元。相反，次级运动神经元的后出生人口是比较多，而且在长时间内产生，起始于14〜HPFEF"&gt; 45,50。在中间躯干段二级运动神经元起源主要是由51 HPF 50完成。二级运动神经元都被认为是运动神经元的羊膜对方46。有趣的是，脊椎神经元，经由多巴胺，调节运动在胚胎的幼虫和次级运动神经元起源和年轻幼虫50,51。初级和次级的运动神经元的每一个包括若干不同的亚型。每个初级运动神经元亚型的项目的外围轴突其支配的特性的肌肉群，从而导致刻板，识别轴突轨迹。通常，次级运动神经元遵循先前由初级运动神经元建立的轴索途径。因此，相对于轴突轨迹，原发性和继发性运动神经元是相似的，不同之处在于厚度轴突胞体和尺寸的重新更大初级运动神经元45。 三，讨论从几个类型的interneurons的记录方法。然而，在这些情况下，需要去除其它脊髓细胞的有限量的，因此，脊髓比从Rohon-胡子细胞或运动神经元的录音少完好无损。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vacuum filter/Storage bottle, 0.22 mm pore</td>
			<td>Corning</td>
			<td>431096</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter 0.2 mm</td>
			<td>Whatman</td>
			<td>6780-2502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma</td>
			<td>A-5040</td>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt&#160;</td>
		</tr>
		<tr>
			<td>a-bugarotoxin</td>
			<td>Tocris</td>
			<td>11032-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>4368-28-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-549 hydrazine salt</td>
			<td>Molecular Probes</td>
			<td>A-10438</td>
			<td>fluorescent dye</td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter</td>
			<td>Corning</td>
			<td>8161</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Fisher</td>
			<td>12-550C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>184</td>
			<td>silicone elastomer</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>30-0038</td>
			<td>inner and outer diameters of 0.78 and 1.0 mm (thin walled glass capillaries)</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Drummond Scientific</td>
			<td>1-000-1000-100</td>
			<td>inner and outer diameters of 1.13 and 1.55 mm (thick walled glass capillaries)</td>
		</tr>
		<tr>
			<td>Miniature barbed polypropylene fitting&#160;</td>
			<td>Cole-Palmer</td>
			<td>6365-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive&#160;</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>Sigma</td>
			<td>C7657</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter Instruments</td>
			<td>&#160;Model P-97&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr>
			<td>Head stage</td>
			<td>Molecular Devices</td>
			<td>CV203BU</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized micromanipulator&#160;&#160;</td>
			<td>Sutter Instruments</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Fisher</td>
			<td>14-169-1B</td>
			<td>ID 1/16 IN, OD 1/8 IN and WALL 1/32 IN (flexible laboratory tubing)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td>Molecular Devices</td>
			<td>1-HC-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmaseal Three-Way Stopcocks&#160;</td>
			<td>Baxter</td>
			<td>K75</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Axon Instruments</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td>Zeiss</td>
			<td>Axioskop2 FS plus</td>
			<td></td>
		</tr>
		<tr>
			<td>40x/0.80w Achroplan objective</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition and analysis software&#160;</td>
			<td>Axon Instruments</td>
			<td colspan="2">PClamp 10 - Clampex and Clampfit&#160;</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="2">Dissection and Recording Solutions(in mM)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">All solutions, except the intracellular, are stable for ~ 2 - 3 months when filtered (0.22 mm filter cups) and stored at room temperature (RT).</td>
		</tr>
		<tr>
			<td colspan="4">The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20&#176;C) in small aliquots that are individually thawed on the day of use.&#160;</td>
		</tr>
		<tr>
			<td colspan="2" rowspan="1">Dissection/Ringer&#8217;s solution</td>
			<td></td>
			<td colspan="2">145 NaCl, 3 KCl, 1.8 CaCl2.2H2O, 10 HEPES; pH 7.4 (with NaOH)</td>
		</tr>
		<tr>
			<td colspan="2">Pipette (intracellular) recording solution</td>
			<td></td>
			<td colspan="2">135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td>
		</tr>
		<tr>
			<td colspan="2">Bath (extracellular) recording solution/voltage and current-clamp</td>
			<td></td>
			<td colspan="2">125 NaCl, 2 KCl, 10 CaCl2.2H2O, 5 HEPES; pH 7.4 (with NaOH).</td>
		</tr>
		<tr>
			<td colspan="2">Alexa-594 hydrazine salt stock solution.&#160;</td>
			<td></td>
			<td colspan="2">Prepare a 13.2 mM stock in ddH2O, aliquot (~ 100 &#181;l) and store at -20&#176;C. For use, dilute the stock solutiond 132 fold with pipette solution to a final concentration of 100 mM. After dilution, filter the Alexa-594 containing pipette solution&#160; with a centrifuge tube filter.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immobilizing agents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">0.4 % ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td></td>
			<td colspan="2">Prepare a 0.4% stock solution in 0.2M Tris, pH9 (0.4 g Tricaine/100 ml 0.2 M Tris</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">Adjust pH to 7 with NaOH and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute the stock solution ~ 25 fold in embryo media</td>
		</tr>
		<tr>
			<td>250 &#956;M &#945;-bungarotoxin</td>
			<td></td>
			<td colspan="2">Prepare a 250 &#956;M stock in ddH2O (1 mg/500 &#956;l), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute 2,500-fold with extracellular solution to a final concentration of 100 nM.</td>
		</tr>
		<tr>
			<td>1 mM Tetrodotoxin</td>
			<td></td>
			<td colspan="2">Prepare a 1 mM stock in ddH2O (1 mg/3 ml), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute 2,000-fold with extracellular solution to a final concentration of 500 nM.</td>
		</tr>
	</tbody>
</table>

zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons

斑马鱼，首先介绍了作为一个发展模式，在许多领域都得到了普及。饲养迅速发展生物体的大数自如，与胚胎光学透明度结合，担任该模型的初始吸引力属性。在过去的二十年里，这种模式的成功得到了进一步的顺从大规模诱变筛选，并通过易于转基因的推动。最近，基因编辑方法扩展了模型的力量。 对于神经发育研究，斑马鱼胚胎和幼虫提供到可应用多种方法的模型。在这里，我们专注于让神经元，电兴奋性的本质属性的研究方法。我们对斑马鱼的脊髓神经元的电生理研究编制涉及使用兽医缝合的胶水，以确保准备录音室。记录替代方法从斑马鱼胚胎和幼虫涉及使用细钨针1，2，3，4，5的制备腔室的连接。钨销是最经常用于安装在横向取向的制备中，虽然它已被用于安装幼虫背的一面朝上4。缝合胶水已被用于安装在两个方向的胚胎和幼虫。使用胶水，一个最小的夹层可以执行，允许访问脊髓神经元而不使用酶处理的，从而避免了任何所得的损坏。然而，对于幼虫，有必要施加一个简短酶处理以除去脊髓周围的肌肉组织。这里所描述的方法已被用来在几个developmenta研究运动神经元的interneurons和感觉神经元的内在电性能升级6，7，8，9。

（ 图5A和5B），我们已经成功地从Rohon胡子神经元记录在17个HPF胚胎到7 DPF幼虫。当Rohon-胡子细胞记录，将制备物固定背的一面朝上。这种安装允许基于它们的表面背的位置和大胞体大小Rohon-胡子细胞的明确识别。识别另外由这些神经元的超极化的定型静息膜电位（ 图5， 小图表 ）6，54证实。此外，由于初级感觉神经元，Rohon-胡子神经元缺乏突触输入。因此，在没有电刺激的，在电流钳模式（ 图5B"）记录，而应该发生在膜电位没有变化。由于从Rohon-胡子细胞的初始记录在斑马鱼中进行&lt;SUP类= "外部参照"&gt; 6，各种转基因品系（ 例如，TG（islet2b:GFP），TG（NGN:GFP），且Tg（isletss:GFP））已经产生，在这些神经元表达荧光报告，进一步促进他们识别55，56，57。 <img alt="图5" src="/files/ftp_upload/55507/55507fig5.jpg" /> 图5:在1从Rohon-胡子神经元全细胞电压和电流钳记录和2 DPF胚胎和7 DPF幼虫。 （A）的向外电压钳记录和内向电流从Rohon-胡子神经元在1-（细黑线）得到的2-（粗黑线）和7-DPF（灰线）的胚胎/幼虫。保持电位是-80毫伏和电流由一个去极化步骤，以20毫伏引出。 （B）单动作电位是通过短暂（1ms）中的电流引起的注射（〜0.35 NA）至1-（细黑线）的Rohon-胡子神经元，2-（粗黑线）和7-DPF（灰线）的胚胎/幼虫。 （B"）在不存在的电刺激的，在膜电位没有变化，如自发突触后去极化，发生在Rohon-胡子神经元。插图表总结搁从Rohon-胡子神经元记录的1-（N = 21）和2-（N = 9）的dpf胚胎和7-（N = 7）单丝旦幼虫膜电势的值。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55507/55507fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 转基因株系允许其他脊髓神经元亚型的明确识别也可用。在这些当中，MNX1转基因品系的Tg（MNX1:GFP）表达在脊髓运动神经元的一个子集的绿色荧光蛋白（GFP）及其规范（〜14-16 HPF）后不久&lt;SUP类="外部参照"&gt; 58,59。由于每个hemisegment（ 图6A）内的初级运动神经元的刻板的定位，与在转基因MNX1 GFP表达在一起，所以能够识别各种初级运动神经元亚型（ 图6B和6C）。包括在记录电极溶液中的荧光染料允许轴突轨迹的可视化，提供运动神经元的身份附加的确认，因为一些的interneurons也表达在TG（MNX1:GFP）GFP线。可替代地，另一种转基因品系，其允许运动神经元的鉴定是ET2线60。 <img alt="图6" src="/files/ftp_upload/55507/55507fig6.jpg" /> 图6:全细胞电压和电流钳从1运动神经元的dpf斑马鱼安柏瑞录音操作系统。 （A）一种卡通描绘存在于所述斑马鱼脊髓初级运动神经元亚型的特定形态特征。初级运动神经元可以通过体细胞的一个段内的位置来识别（ 即，喙[的RoP]，内侧[MIP]，或尾鳍[CAP]）45。此外，每个亚型延伸经由不同路径的轴突到外围。将合并使用的Tg（MNX1:GFP）的线和染料标记揭示的定型轴突在心轴和运动神经元亚型的记录期间的标识。使用这里介绍的方法，能够以相同的hemisegment内从三个不同的初级运动神经元亚型顺序记录。 （B）电压钳记录中示出从的RoP，MIP和CAP，都在一个单一hemisegment获得的。电压步骤到20毫伏是用来从-80mV的保持电位激发电流。 （C）在电流钳ř从的RoP，MIP和CAP，短暂（1毫秒，0.4〜NA）ecordings电流注射的施加以触发一个动作电位。膜电位在〜-65举行毫伏。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55507/55507fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 初级和次级的运动神经元之间的主要区别是出生早期神经元的胞体较大。然而，次级运动神经元亚型是不是由体细胞大小或位置识别。用于从特定辅运动神经元的录音中，两个转基因品系，TG（GATA2:GFP）和TG（islet1:GFP），已被用于识别次级运动神经元腹侧与背侧和突出轴突，分别为55，61。然而，第三次级运动神经元亚型存在于斑马鱼脊髓，用AXONS该项目背部和腹部62。因此，染料可以被用于填充次级运动神经元记录期间识别的形态的基础上（ 图7A）8,9上亚型。通常情况下，从二次运动神经元，电压钳（ 图7B中 ， 星号 ）或电流钳记录（ 图7C和图7C"， 箭头 ）中自发的或突触事件被记录。 <img alt="图7" src="/files/ftp_upload/55507/55507fig7.jpg" /> 图7:全细胞电压和电流钳从2次级运动神经元的dpf胚胎录音。 （A）在TG（GATA2:GFP）线，两个不同的二次运动神经元亚型表达GFP 62。在左hemisegment，腹侧辅助运动神经元（ 星号和箭头指示胞体和轴突，分别地）。在邻近hemisegment，右侧（尾侧），有一个腹侧/背次级运动神经元（ 星号表示体细胞; 箭头指示两个轴突，一个突出腹侧[底部箭头]以及其他背侧[顶箭头]）。这些神经元标记与记录时的红色荧光染料。为了鉴定腹侧/背次级运动神经元，这是至关重要的，以确保解剖不会在相邻的尾hemisegment除去肌肉，从而损坏或去除所述背轴突。在记录之后，因为它是从制剂（ 右顶部星号 ）拉离所述神经元细胞体保持附着在微量。当使用染料记录期间填充神经元，染料常常泄漏而电极是在浴，产生了红色荧光背景在脊髓和脊索（ 底部星号在延髓[左] hemisegment &lt;可见<html> / em&gt;的）。从腹侧和腹侧/背次级运动神经元得到的（B）的电压钳记录。电压的步骤（至-30，-10，+ 10，+ 30，50，70，90，和110毫伏）引起向外和向内的电流。松开动作电位/去极化可以是存在于所述记录（ 星号 ）。 （C）在从二次运动神经元，短暂（1毫秒）增加振幅的电流注射的电流钳记录施加到神经元触发动作电位（ 星号 ）。 （C"）由〜0.4-NA电流注射的在次级运动神经元触发单个动作电位的例子被示出。在此阶段，自发动作电位也观察到（C和C"， 箭头 ）。 （D）长时间（100毫秒）电流注射的（〜0.35 NA）引发动作电位的重复放电。膜电位在〜-65举行毫伏。负载/ 55507 / 55507fig7large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

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

这手稿描述了从斑马鱼胚胎和幼虫的脊髓神经元的电生理记录的方法。制备维持神经元在原地 ，往往涉及到最小的清扫。这些方法允许对多种脊髓神经元的电生理学研究中，从通过早期幼体阶段的初始电兴奋采集。

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 ...

zebrafish-situ-spinal-cord-preparation-for-electrophysiological

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

11.4K 次观看.  University of Colorado Anschutz Medical Campus (UCAMC). 这手稿描述了从斑马鱼胚胎和幼虫的脊髓神经元的电生理记录的方法。制备维持神经元在原地 ，往往涉及到最小的清扫。这些方法允许对多种脊髓神经元的电生理学研究中，从通过早期幼体阶段的初始电兴奋采集。 

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

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 Transection in the Larval Zebrafish

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 to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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

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

脊髓神经元从新生小鼠中分离和培养

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

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 ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

从产前Islmn:GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...