Name: juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons
Uploaded: 2014-02-25T14:45:00
Description: Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

我们要感谢药用处方。 Huibert Mansvelder和Bert索​​克曼的广泛支持，马塞尔Oberlaender的富有成果的讨论，并提供技术援助，提供神经元追踪，和Brendan Lodder博士。使用ntrode VI用于LabVIEW，由R.布鲁诺（哥伦比亚大学。，NY，USA）慷慨地提供了数据被收购。这项研究是由马克斯·普朗克学会和伯恩斯坦计算神经科学中心，图宾根的支持（由德国联邦教育与研究部（BMBF资助; FKZ：01GQ1002））（RTN），中心Neurogenomics与认知研究（CNCR） ，神经科学校园阿姆斯特丹（NCA），资金CPJdK（NWO-ALW＃822.02.013和ENC-网络＃P3-C3）和阿姆斯特丹VU大学。

1。动物的制备所有实验步骤都进行了按照荷兰法律和评估后，由当地的伦理委员会，在阿姆斯特丹VU大学，荷兰。 <ol><li>麻醉Wistar系大鼠（P25-P45，♂/♀）用异氟烷（在氧气2-3％），随后与氨基甲酸乙酯（20％在0.9％NaCl，1.6〜1.7克/公斤）通过腹膜内注射。通过监测捏撤出，眼睑反射消失，并且触须运动评估麻醉深度。 </li><li>的情况下施用额外剂量的尿烷（初始剂量的10％）...

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该juxtasomal方法允许记录在体内动作电位扣球来自全国各地的行为条件的单台（麻醉，清醒头固定或自由活动）与生物胞素标记的神经元记录为事后的细胞类型分类和/或三维重建的选项。主要的优点是能够获得在细胞外（因此非侵入性）构型的生理参数，还能够与生物胞素16,17,32细胞内标记的神经元。除了 ​​生物胞素标记，这种技术可以用于注射神经元与DNA，RNA，蛋白质，...

神经网络由多种细胞类型，其特点是高度特异性的形态和生理特性1-7。因此，个别细胞类型的网络内执行专门任务的（参见例如Gentet 等人 8和Burgalossi 等 9）。我们才刚刚开始了解整个神经网络的细胞类型特异性的功能现在还是有很多被发现。为此，许多实验室正在实施的实验方法，使从生理参数已取得1,10-15相同的神经元群的形态特征进行分析。在这里，我们展示了juxtasomal标记技术16,17涉及与所记录的神经元与生物胞素的电结合使用传统的补丁移液器在细胞外（因此无创）配置电生理记录。该这种方法的主要优点是，非侵入性的性质确保了单个神经元的动作电位扣球被记录而不改变（ 如透析）的细胞的细胞内含量。其次是电，在juxtasomal方法提供事后细胞识别和重建的选项链接功能（生理），以结构（形态）。通常情况下，形态重构涉及重建的树突和轴突的形态，可以延长至脊柱和/或布顿密度的定量，甚至在用电子显微镜纳米级分辨率重建神经元形态的。该juxtasomal记录技术可以用于各种细胞类型跨越皮质层或子皮质区的范围内的物种的体内录音，虽然大多数研究已应用于该技术在小型啮齿动物如小鼠或大鼠。我们的研究重点是记录和标记的神经元从大鼠初级躯体感觉皮层（S1）和涉及视觉识别记录的神经元18，与精确定位在一个标准化的参考帧组合树突重建逆向工程皮质网络4,19和轴突结构的详细的重建来表征细胞类型特异性地方和远距离投射目标20。 相比其它体内记录技术（细胞内或细胞的全细胞），juxtasomal录音是相对稳定的，因此可以被跨越行为状态应用包括麻醉21,22，镇静剂14，醒头固定23，或什至可自由移动的动物9 。这里，我们表明在聚氨酯麻醉大鼠S1 juxtasomal标签，虽然我们强调该技术的普遍适用性来选择的许多制剂。

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
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			<td height="25" style="height:25px;width:224px;">SM-6 control system</td>
			<td style="width:275px;">Luigs &#38; Neumann</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:224px;">LN- Mini 23 XYZ</td>
			<td style="width:275px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LN- Mini 55 Manipulatorblock X2</td>
			<td style="width:275px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Lynx-8 amplifier</td>
			<td style="width:275px;">Neuralynx</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Axoclamp-2B amplifier</td>
			<td style="width:275px;">Axon Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Osada model EXL-M40</td>
			<td style="width:275px;">Osada, inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Piezoelectric device</td>
			<td style="width:275px;">Physik Instrumente</td>
			<td style="width:119px;">PL140.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Labview</td>
			<td>National Instruments, Austin, TX, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ntrode Virtual Instrument&#160;</td>
			<td>R. Bruno, Columbia Univ., NY, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">(Labview acq. software)</td>
			<td style="width:275px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:224px;">Sugi absorbent swabs</td>
			<td style="width:275px;">Kettenbach</td>
			<td style="width:119px;">30601</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Cytochrome C from equine heart</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">C2506</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Catalase from bovine liver</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">C9322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">DAB</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">D5637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">H2O2</td>
			<td style="width:275px;">Boom</td>
			<td style="width:119px;">7047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Vectastain standard ABC-kit</td>
			<td style="width:275px;">Vector</td>
			<td style="width:119px;">PK6100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Triton X100</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Urethane</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">U2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Isoflurane</td>
			<td style="width:275px;">Pharmachemie</td>
			<td style="width:119px;">45.112.110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Lidocaine</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">L5647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Simplex rapid dental cement</td>
			<td style="width:275px;">Kemdent</td>
			<td>ACR308/ACR924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Biocytin</td>
			<td style="width:275px;">Molekula</td>
			<td style="width:119px;">36219518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">PFA</td>
			<td style="width:275px;">Merck Millipore</td>
			<td style="width:119px;">8187151000&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Trizma base</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">T4661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Mowiol 4-88</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:119px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Analytical grade glycerol</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:119px;">49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">HEPES</td>
			<td style="width:275px;">Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">NaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">31434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">KCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">60130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">CaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">22,350-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">MgCl2</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:119px;">63072</td>
			<td></td>
		</tr>
	</tbody>
</table>

juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons

大脑皮质的特征是多层次和许多不同的细胞类型，它们共同作为网络负责许多高级认知功能，包括决策，感官引导行为或内存。为了理解如何这样复杂的神经元网络执行这样的任务，一个关键的第一步是确定在网络中的单个细胞类型的函数（或电活性），优选当动物正在执行一个相关的认知任务。另外，为了确定单个神经元网络和形态学的架构，以允许反向工程皮质网络的解剖结构是同样重要的。可今天的技术突破允许录制清醒细胞活性，动物行为与事后识别记录神经元的有价值的选择。在这里，我们展示了juxtasomal生物胞素标记技术，它涉及到记录动作电位人在细胞外（或宽松的补丁）配置扣球使用传统的补丁移液器。该juxtasomal记录配置是横跨行为条件相对稳定且适用，包括麻醉，镇静，清醒头固定，并且即使在可自由移动的动物。因此，这种方法允许在动物的行为来重建单个神经元，最终，整个皮质微电路的连接细胞类型特异性动作电位扣球。在这个视频稿件，我们将展示如何在juxtasomal配置单个神经元可在氨基甲酸乙酯麻醉大鼠的事后鉴定和形态重构被贴上生物胞素。

详细的知识对单个神经元的三维结构对于阐明神经元网络的组织原则至关重要。我们的方法包括一个管道，以实现高品质的生物胞素标记从体内制备，从而使事后神经元分类和树突和单个神经元，在高分辨率的轴突架构的详细重建。根据juxtasomal标签的质量，神经元恢复与不同的DAB-强度，从微弱到可以非常精确地对应于记录位置19,26位置激烈的DAB信号。在我们的实验室，一个?...

Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

要了解神经网络的结构，单个神经元的功能和形态特征是必要的。在这里，我们证明juxtasomal生物胞素标记，其允许电生理记录在胞外结构，但保持对胞内标记的神经元为事后重建的树突和轴突体系结构的能力。

Juxtasomal生物胞素标记来研究单个皮层神经元的结构与功能的关系

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

免疫组化和多标签与同一宿主物种抗体研究成人神经发生

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...