Name: organotypic slice cultures of embryonic ventral midbrain: a system to study dopaminergic neuronal development in vitro
Uploaded: 2012-01-31T12:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro at JoVE.com

我们感谢他们在帮助建立器官切片培养体系和沃尔夫冈夜蛾和批判性阅读的手稿Liviu加布里埃尔Bodea马汀娜Emond和伊莎贝尔Brachmann。我们要感谢弗兰克Costantini的R26记者小鼠和SHH CreER小鼠的克里夫塔宾。这项研究是从科学和北莱茵 - 威斯特法伦州（Programm楚Förderung DER Rückkehr DES wissenschaftlichen Spitzennachwuchses AUS DEM Ausland）的研究部资助的一个研究奖。

本协议修改Daza 等 ，2007年7月 。 1。筹备工作<ol><li>可以准备提前一天<ol><li>准备1X克雷布斯缓冲液（1.5升）： 126 mM氯化钠，氯化钾2.5毫米，1.2毫米的NaH 2 PO 4：H 2 O MgCl 2的 1.2毫米，2.5毫米氯化钙 ，葡萄糖11毫米，25毫米碳酸氢钠3，调整pH至7.4。过滤消毒（0.22微米孔径），并储存在4 ° C </li><li>准备培养液?...

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	<li>Noctor, S. C., Martinez-Cerdeno, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nat. Neurosci. 7 (2), 136-136 (2004).</li><li>Martini, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+selection+of+leading+process+branches+mediates+chemotaxis+during+tangential+neuronal+migration.">Biased selection of leading process branches mediates chemotaxis during tangential neuronal migration.</a> Development. 136 (1), 41-41 (2009).</li><li>Marin, O., Valiente, M., Ge, X., Tsai, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guiding+neuronal+cell+migrations.">Guiding neuronal cell migrations.</a> Cold Spring Harb. Perspect. Biol. 2 (2), a001834-a001834 (2010).</li><li>Ayala, R., Shu, T., Tsai, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trekking+across+the+brain:+the+journey+of+neuronal+migration.">Trekking across the brain: the journey of neuronal migration.</a> Cell. 128 (1), 29-29 (2007).</li><li>Smidt, M. P., Burbach, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+mesodiencephalic+dopaminergic+neuron.">How to make a mesodiencephalic dopaminergic neuron.</a> Nat. Rev. Neurosci. 8 (1), 21-21 (2007).</li><li>Legue, E., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+fate+mapping+using+site-specific+recombinases.">Genetic fate mapping using site-specific recombinases.</a> Methods. Enzymol. 477, 153-153 (2010).</li><li>Daza, R. A., Englund, C., Hevner, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+of+embryonic+brain+tissue.">Organotypic slice culture of embryonic brain tissue.</a> CSH Protoc. , (2007).</li><li>Harfe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+expansion-based+temporal+Shh+gradient+in+specifying+vertebrate+digit+identities.">Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.</a> Cell. 118 (4), 517-517 (2004).</li><li>Srinivas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Dev. Biol. 1, 4-4 (2001).</li><li>Joksimovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+separable+Shh+domains+in+the+midbrain+define+distinct+dopaminergic+progenitor+pools.">Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (45), 19185-19185 (2009).</li><li>Blaess, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal-spatial+changes+in+Sonic+Hedgehog+expression+and+signaling+reveal+different+potentials+of+ventral+mesencephalic+progenitors+to+populate+distinct+ventral+midbrain+nuclei.">Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei.</a> Neural. Dev. 6 (1), 29-29 (2011).</li><li>Hippenmeyer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Biol. 3 (5), e159-e159 (2005).</li></ol>

这里介绍的器官切片培养方法在体外分析发展中国家在胚胎腹侧中脑多巴胺能神经元和它们的迁徙和投影路线系统提供了一个短期的。我们发现，有一些在协议的重要步骤应仔细出席了会议，以获得片，使腹侧中脑多巴胺能神经元的正常发展。最关键的一步是胚胎脑，快速和精确的解剖。相比之下一代成人的大脑切片，这是关键部分的大脑上配有一个冷却系统，使用频率低，与高速结合，?...

特定的试剂和设备表<table border="1"><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> DMEM培养液</td><td> Sigma - Aldrich公司</td><td> D6429 </td><td></td></tr><tr><td>葡萄糖30％ </td><td> Sigma - Aldrich公司</td><td> G7528 - 250 </td><td></td></tr><tr><td>马血清</td><td> Invitrogen公司</td><td> 26050-088 </td><td></td></tr><tr><td> DMEM（4,5克/ L，GLC，与L -谷氨酰胺，娜PYR，碳酸氢钠） </td><td> Sigma - Aldrich公司</td><td> D6429 - 500 </td><td></td></tr><tr><td>青霉素/链霉素100X </td><td> Sigma - Aldrich公司</td><td> P4333 - 20 </td><td></td></tr><tr><td> L -抗坏血酸</td><td> Sigma - Aldrich公司</td><td> A4403 </td><td> 200mm的股票和存储准备在-20 ° C </td></tr><tr><td>超纯LMP琼脂糖</td><td> Invitrogen公司</td><td> 15517-022 </td><td></td></tr><tr><td> Millicel插入</td><td> Millipore公司</td><td> PICMORG50 </td><td></td></tr><tr><td> μ盘35毫米，低</td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Vibratome </td><td> MICROM </td><td>航模650V </td><td></td></tr><tr><td>刀片</td><td>普莱诺有限公司</td><td> 121-6 </td><td></td></tr><tr><td> Histoacryl胶</td><td> BRAU9381104 </td><td>布劳恩蛇牌</td><td></td></tr><tr><td>穿孔勺直径 直径15毫米</td><td>精细科学工具</td><td> 10370 -18 </td><td></td></tr><tr><td>钳5 Dumoxel </td><td>精细科学工具</td><td> 11252 - 30 </td><td></td></tr></table> immunostainings使用的抗体： <table border="1"><tr><tD&gt; 抗体的名称 </td><td> 公司 </td><td > 目录编号 </td><td> 评论（可选） </td></tr><tr><td>兔抗酪氨酸羟化酶</td><td> Millipore公司</td><td> AB152 </td><td>稀释1:500 </td></tr><tr><td>小鼠抗酪氨酸羟化酶</td><td> Millipore公司</td><td> MAB318 </td><td>稀释1:500 </td></tr><tr><td>小鼠抗BrdU </td><td> BD Pharmingen公司</td><td> 555627 </td><td>稀释1:200 </td></tr><tr><td>兔抗裂解的caspase 3 </td><td>细胞信号技术</td><td> 9661 </td><td>稀释1:200 </td></tr><tr><td>驴抗兔IgG - Alexa的488 </td><td> Invitrogen公司</td><td> A21206 </td><td>稀释1:500 </td></tr><tr><td>驴抗鼠IgG - Cy3标记</td><td>杰克逊ImmunoResearch </td><td> 715-165-150 </td><td>稀释1:200 </td></tr></table>

organotypic slice cultures of embryonic ventral midbrain: a system to study dopaminergic neuronal development in vitro

鼠标是一个很好的的模式生物研究哺乳动物大脑的发展，由于大量的分子和基因数据。然而，发展中国家的小鼠大脑是不容易操纵和体内成像合适，因为小鼠胚胎是交通不便，不透明。器官型胚胎的大脑切片文化，因此被广泛用于研究在体外培养的小鼠大脑发育 。体外操纵或允许使用的转基因小鼠基因表达的修改，所以可以用荧光蛋白标记的神经元或神经胶质细胞的亚群。标记细胞的行为，然后可以使用时间推移成像观察。已定时成像研究细胞行为背后晚1-2萌芽阶段大脑皮层的发展，特别是成功。胚胎器官的大脑区域以外的前脑切片在文化系统较差establisHED。因此，时移成像数据描述了神经细胞迁移的财富，是仅限于前脑3,4。它仍然是不知道，是否背的大脑中发现的原则腹脑区的真实。在腹侧的大脑，神经元是神经元集群，而不是层组织，他们往往要经过复杂的迁徙轨迹，以达到他们的的最终位置。腹脑不仅是一个腹侧大脑发育良好的模型系统，也包含如多巴胺能神经元疾病过程中的有关神经元的人口。虽然功能和多巴胺能神经元变性一直在成人和脑老化的非常详细的调查，很少有人知道这些神经元的行为在其分化和迁移阶段5。我们在这里描述从胚胎一天（五）12.5鼠腹侧中脑切片培养的一代。这些切片邪教URES有可能适合监测在体外几天的多巴胺能神经元发展。我们强调在胚胎发育的早期阶段产生的脑切片的关键步骤， 并讨论保持在体外培养的多巴胺能神经元的正常发展的必要条件。我们也可以从时间的推移成像实验结果。在这些实验中，被打成腹侧中脑前体（包括多巴胺的前体）和他们的后裔在镶嵌的方式，使用一个基础的Cre / loxP位诱导的命运映射系统 6 。

Watch this Scientific Journal Video about Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro at JoVE.com

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

描述一个方法来生成从E12.5小鼠胚胎脑器官切片。可用于器官切片文化观察多巴胺能神经元或其他腹侧中脑的神经元的行为。

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Research

JoVE Journal

Neuroscience

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

In ovo Expression of MicroRNA in Ventral Chick Midbrain

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

接口3D工程神经细胞微电极阵列：一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...