Name: electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo
Uploaded: 2013-05-29T13:00:00
Description: Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

<p class="jove_content"我们感谢博士尤瓦的戈特利布-德罗尔电插图。这项工作是由渠务署授予国家在以色列的心理生物学研究所和萨克森 - 以色列研究合作计划和补助AK从以色列科学基金会，以色列卫生部和优秀传统遗产中心的支持生物医学科学合作。</p&gt;

1。后脑电穿孔 1.1处理蛋<ol><li>将鸡蛋在培养箱（37-38.5°C）水平。胚胎电孵育65-70小时后，当他们到达16-17（HH）阶段（25-30体节）。 </li><li>从培养箱中取出鸡蛋，它们保持在水平位置。 </li></ol> 1.2准备工作<ol><li>上拉玻璃毛细管（直径0.5 mm）。 </li><li>与L形的弯曲的黄金Genetrodes电极（直径3 mm）连接到脉冲发生器的适配器保持。电参数包括25伏，5个脉冲数，45?...

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OVO中电是一个可行的，可靠的，有效的工具来检查细胞规范和轴突指导在鸡胚神经系统的发展20。在这个协议中，我们描述了一个模式把小鸡后脑电E2.75使用增强子元件，​​使有条件的标签特定的interneurons。这一战略的结合换位系统PiggyBac转介导外源基因插入到鸡基因组，这使跟踪的的轴突航线，预测和突触网站在胚胎发育晚期。 以前的方法标签轴突/把小鸡?...

后脑是一个关键的中继枢纽神经系统之间的通信中枢和周围神经系统神经网络通过递增和递减。它调节的基本功能，包括呼吸，意识，听觉和运动协调1-3。在早期胚胎发育过程中，脊椎动物的后脑瞬时细分成重复菱沿其前后轴（AP），不同的神经细胞类型的形成，并产生多个脑干中心4。后脑也被划分成基底和鼻翼板，在指定离散神经祖细胞变得和区分不同的DV地点3,5,6沿背腹轴（DV）。怎样早期AP和DV特定的神经元模式的脑干功能设计电路的设立在很大程度上是未知。 为了获得这个基本知识的问题，工具都需要以特定亚群的神经元在早期后脑贴上标签，以跟踪它们的轴突轨迹和连接更高级的阶段。我们先前已经利用特异性增强子元件，和一个CRE / LoxP位条件的表达背脊髓在早期鸡胚7-9的轴突轨迹跟踪系统。在当前的手稿中，我们有针对性的后脑和标记后期胚胎后脑的interneurons，轴突和其突触目标，使用修改后的电战略和PiggyBac转 - 介导DNA换位的升级实验范式。我们的新战略，可以在一侧的后脑和他们的的轴突预测和突触位点不同的胚胎阶段的跟踪标记不同的神经元亚型，从2至12天电。基于这种方法，我们贴上了最背的分组后脑的interneurons（dA1/Atoh1 + </s&gt;细胞），并揭示了两个对侧上升轴突投射图案，分别来自不同AP的位置和拉长了一个独特的索。 DA1轴突被发现在听觉核，中脑，小脑10多层的项目，并形成突触。 小鸡电的结合，遗传神经元和先进得多的发展阶段分析预测网站跟踪研究在大脑中形成的神经网络，并阐明控制电路形成的分子机制，提供一个独特的平台。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		 </tr>
		 <tr>
			<td>L-shaped gold Genetrodes 3 mm electrodes</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0162 </td>
		 </tr>
		 <tr>
			<td>pulse generator, ECM 830</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0002</td>
		 </tr>
		 <tr>
			<td>OCT (Optimal Cutting Temperature) Compound</td>
			<td>Tissue-Tek
				Sakura</td>
			<td>4583 O.C.T. Compound </td>
		 </tr>
		 <tr>
			<td>Nail Polish</td>
			<td>From Any Commercial Supplier </td>
			<td></td>
		 </tr>
		</tbody>
 </table>

electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

鸡胚神经管电具有许多优点，如快速，高效的成神经细胞的外源基因表达。在这篇稿件中，我们提供了一种方法，展示了独特的电穿孔DNA禽流后脑E2.75为了明确标示神经祖细胞的一个子集，以及如何按照他们的轴突预测和突触的目标非常先进的发展阶段， E14.5。我们利用新的遗传工具，包括特异性增强子元件，​​酶Cre /液氧 - 基于质粒和PiggyBac转介导的DNA换位后脑细胞的一种亚型（最背的分组的interneurons，DA1）系统来驱动GFP表达。随后在早期和晚期胚胎阶段，在不同的脑干区域的轴突轨迹和DA1轴突的目标。这种策略有助于先进的技术，针对感兴趣的细胞在胚胎后脑TRA庆安电路形成多个发展阶段。

这个协议最近发现轴突的模式和投影网站的DA1群的interneurons在小鸡后脑10。要具体标明这些轴突，增强子元件（Atoh1的），先前已经被作为具体的脊柱DI1神经元8,12,13其特征在于，确认表达在后脑DA1单元10。该元件上游克隆到Cre重组和合作电在E2.75随着一个CRE依赖细胞质GFP记者质粒（pCAGG LoxP位停止LoxP位cGFP的图1BI）。由平坦的安装准备分析hindbrains 48小时以下的电?...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

神经网络是建立在胚胎大脑发育神经生物学的一个基本问题。在这里，我们结合电穿孔技术与新的遗传工具，如酶Cre / LOX-质粒和PiggyBac转介导的DNA换位系统在禽流后脑背中间贴上标签，并跟踪他们的轴突预测和突触在不同发育阶段的目标。

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

小鼠神经嵴干细胞的培养鸡胚提取物的生产

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

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

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...