Name: retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes
Uploaded: 2014-05-03T14:00:00
Description: Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

This is supported by 973 MOST grant (Grant No. 2011CB504402, 2012CB947602), National Natural Science Foundation of China (Grant No. 91132724, U1332136) and the 'Hundred Talents Project' of Chinese Academy of Science. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).

1，构建外科器械注：为了确保鱼的过程中和运行后，滴麻醉液乙基3 - 氨基苯甲酸酯（MS-222，或三卡因）在半浓度（0.015％）与1滴/秒的过鱼的嘴的速度仍然活着使用图1中所示的自制滴灌系统。 <ol><li>使一个框， 如图1A（长度为28厘米，宽为10厘米，高度为5厘米），把一个海绵（长度为6厘米，宽度为9厘米，高度为4厘米）的中间，并打开一个差距就?...

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视网膜神经节细胞逆行标记是重要的研究视网膜神经节细胞存活的哺乳动物，但它并没有在斑马鱼中使用。替代的方法，HE染色7和抗体染色8，是不是金标准，用于计数视网膜神经节细胞数，和转基因品系与标记的RGC的所有还没有被构造12,13。在这段视频中，我们介绍一种方法逆行标记视网膜神经节细胞在成年斑马鱼视网膜。虽然大约1％的轴突投射到嗅球或其它区域14?...

由于成年斑马鱼有很强的能力视神经损伤1后再生轴突，一个适当的方法来计算整个视网膜神经节细胞是必不可少的，以评估视网膜神经节细胞的存活和再生2。基于逆行标记视网膜神经节细胞在哺乳动物和金鱼3的方法- 5，我们构建了从成年斑马鱼的顶盖标记视网膜神经节细胞的方法。对于成年斑马鱼，两个关键技术问题应该注意：成年斑马鱼的头骨是非常小的6;他们生活在一个水环境。在这里，我们把头骨与腐蚀剂，最大限度地减少与钻5相关的危险。然后，我们封孔与光固化粘结手术后从而提高动物的生存。 此前，其他一些技术被采用来计算间接的方式视网膜神经节细胞数量。 HE染色的视网膜切片标签的所有类型的细胞中的RGCL 7。在整个Ř抗体标记etina，如胰岛-1，也可以标记无长突细胞8。虽然从视神经残端逆行标记可以标记在视网膜上所有的RGC，它不能被采用在挤压模型，因为它会造成额外损伤视神经。以逆行标签的优势从顶盖，我们已经研究视网膜神经节细胞的存活和再生的视神经损伤。结果表明，几乎所有的视网膜神经节细胞存活和视网膜神经节细胞的90％以上再生的顶盖在第一周的美眉模型9。 为了成功地标记所有视网膜神经节细胞，DiI荧光贴与其他几个商业染料10比较后选择。首先，它是专为在体内组织标记而设计的。其次，它是亲脂性染料，它不能扩散在水中。此外，这种荧光可以持续很长的时间，这使得它非常适合进行视网膜神经节细胞存活的研究。

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">MS222</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:152px;">E10521</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">DiI</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:152px;">N22880</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">lightcuring bond</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Durafill bond</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:152px;">Gluma Etch</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Gluma Etch 35 Gel</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:152px;">Blue LED</td>
			<td style="width:152px;">Shenruo Medical Equipment Co.</td>
			<td style="width:152px;">Power Blue Light Curing Unit</td>
			<td style="width:152px;">China</td>
		</tr>
	</tbody>
</table>

retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes

由于逆行标记视网膜神经节细胞（RGC的）可以从死亡地点隔离视网膜神经节细胞胞体，它已成为黄金标准的视网膜神经节细胞的存活和再生的实验计数视网膜神经节细胞。许多研究在哺乳动物进行了视神经损伤后视网膜神经节细胞研究生存。然而，视网膜神经节细胞在成年斑马鱼的逆行标记尚未报道，尽管一些替代的方法可以在视网膜神经节细胞层（RGCL）计数细胞数。考虑到成年斑马鱼头骨的小尺寸和死亡的钻探头骨后，高风险，我们打开头骨与酸蚀刻的帮助和密封孔与光固化债券，这可能会显著提高患者的生存率。吸收染料5天之后，几乎所有的视网膜神经节细胞被标记。由于这种方法不需要横切视神经，它是不可替代的视网膜神经节细胞存活的研究在成年斑马鱼视神经损伤后。在这里，我们介绍一步该方法步骤，并提供有代表性的成果。

如图4B-D显示，DiI荧光阳性细胞的数目是三分之二DAPI +细胞RGCL的。在正常视网膜中，整个视网膜（ 图4E）的一个蒙太奇图像显示的DiI +视网膜神经节细胞分布在整个视网膜上，但在再生的视网膜（ 图4F），如在中心区域的RGC没有再生到其目标在第一个星期，他们不能被标记。 <img alt="图1...

Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Research

JoVE Journal

Neuroscience

Labeling and Imaging Cells in the Zebrafish Hindbrain

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

逆行神经，大港，并用荧光染料的神经根装载

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...