Name: whole cell recording from an organotypic slice preparation of neocortex
Uploaded: 2011-06-03T12:00:00
Description: Watch this Scientific Journal Video about Whole Cell Recording from an Organotypic Slice Preparation of Neocortex at JoVE.com

作者想感谢真弓樱和丽贝卡Foehring优秀的技术援助。此外，我们要感谢博士。罗德里戈安德拉德在实施为我们提供了转基因构造的器官切片文化和基因枪转染协议的博士和珍妮Nerbonne的援助。这项工作是由美国国立卫生研究院授予：NS044163支持NINDS（RCF）。

1。一天的切片前的准备工作我们发现这是更有效的反应釜的手术器械和准备的解决方案前一天的切片。 <ol><li>高压灭菌仪器。 （半无菌条件下进行的手术和切片）。高压灭菌器下面的包，分别在高压灭菌纸包裹： <ul class="none"><li> 手术包：铲，22号手术刀刀片手柄，剪刀，镊子</li><li> 切片包装：3旋钮（刀片控股旋钮和2浴，确保旋钮）和刀片护罩?...

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	<li>Beique, J. C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex.">Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex.</a> Proc Natl Acad Sci U S A. 104, 9870-9875 (2007).</li><li>Buonomano, D. 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M. <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+cultures:+a+technique+has+come+of+age.">Organotypic slice cultures: a technique has come of age.</a> Trends Neurosci. 20, 471-477 (1997).</li><li>G&#228;hwiler, B. H., Thompson, S. M., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Maintenance+of+Organotypic+Slice+Cultures+of+CNS+Tissue.">Preparation and Maintenance of Organotypic Slice Cultures of CNS Tissue.</a> Current Protocols in Neuroscience. , 6.11.1-6.11.11 (2001).</li><li>Guan, D., Lee, J. C., Tkatch, T., Surmeier, D. J., Armstrong, W. E., Foehring, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+biophysical+properties+of+Kv1+channels+in+supragranular+neocortical+pyramidal+neurones.">Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones.</a> J Physiol. 571, 371-389 (2006).</li><li>Guan, D., Lee, J. C. F., Higgs, M., Spain, W. J., Armstrong, W. E., Foehring, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+Kv1+containing+channels+in+neocortical+pyramidal+neurons.">Functional roles of Kv1 containing channels in neocortical pyramidal neurons.</a> J. Neurophysiol. 97, 1931-1940 (2007).</li><li>Guan, D., Tkatch, T., Surmeier, D. J., Armstrong, W. E., Foehring, R. 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我们一直在研究在大鼠大脑皮层（4 9-11）锥体神经元电压门控钾通道的表达和功能的作用。由于缺乏这些渠道的具体药理代理商，我们使用一种遗传方法操纵通道表达（1,14,15,17-19）。我们利用一个器官的文化准备（2,3; 5-8; 12,13,15-22）。Stoppini等（16）的方法修改，以维持细胞的形态和皮质层模式。我们在产后6-17天隔离急性新皮层切片，并保持2-7天文化片。这使得我们研究类似岁的神经元在?...

手术/转/文化： <ol><li>脑切片：坎普登Vibroslice＃MA572世界精密仪器，萨拉索塔，佛罗里达州，美国</li><li>基因枪系统：Bio - Rad公司赫利奥斯＃165-2431（1000阿尔弗雷德诺贝尔驱动器，Bio - Rad公司实验室，大力士，CA 94547） <ul><li>包括：基因枪，稳压器，油管准备站（＃165-2418），注射器试剂盒，TEFZEL管，管材切割机，优化试剂盒（＃165-2424），管材切割机氦软管总成</li><li> Bio - Rad公司氦稳压器（＃165-2413） </li><li>一次性用品HELIOS来自Bio - Rad： <ul><li> 1.6微米的金微载体：＃165-2264 </li><li> TEFZEL管：＃165-2441 </li></ul></li></ul></li><li>孵化器：备考的科学模型3110（热科学：（866）984 - 3766）。 </li></ol>媒体： <ol><li>马血清：Hyclone公司捐助马＃SH 30074。 （Hyclone公司，925西1800南，洛根，UT斯达康84321） </li><li> HMEM（最小的重要媒体加的HBSS和HEPES，没有谷氨酰胺：龙沙BioWhittaker目录＃12 - 137F）：GIBCO / Invitrogen公司（800）955 - 6288，选项1。 </li><li>的HBSS（GIBCO公司汉克斯缓冲液，＃24020-117）​​：GIBCO / Invitrogen公司（800）955 - 6288，选项1。 </li><li> MEM（GIBCO公司最小的必不可少的媒介，＃12360-038），GIBCO / Invitrogen公司（800）955 - 6288，选项1。 </li><li> 250毫升Millipore公司0.2微米的过滤器：＃SC6PU02RE </li><li>塑料转让移液器：费舍尔＃13-711-20。 </li><li> 50毫升微孔steriflip 0.22微米过滤器（＃SCGP00525） </li></ol>从获得的：尔科技大使，1241大道，邮政信箱14989，圣路易斯，密苏里州63132的项目6-8。 录音： <ol><li>吸取玻璃：哈佛GC150TF - 10：哈佛器械，Holliston 10月，84路连山道，马萨诸塞州01746 </li><li>萨特的P - 87水平的电极拉马：萨特仪器公司，一个数字驱动器，诺瓦托，CA 94949 </li><li> Axon仪器Multiclamp 700B放大器：分子器件，公司1311新奥尔良驱动器，桑尼维尔，CA 94089-1136 </li><li> PClamp 10数据采集软件：分子器件，公司1311新奥尔良驱动器，桑尼维尔，CA 94089-1136 </li><li> lectrode位置控制与萨特净资产收益率- 200操控和PC - 200控制器： 萨特仪器公司 ，一个数字，诺瓦托，CA 94949。 </li><li>显微镜：奥林巴斯BX - 50WI直立IR - DIC的光学显微镜</li><li>红外感光摄像头OLY - 150（奥林巴斯）或DAGE - MTI（DAGE - MTI，密歇根大道，01北Roeske市，在46360）。 </li></ol>

whole cell recording from an organotypic slice preparation of neocortex

我们一直在研究大鼠大脑皮层锥体神经元电压门控钾通道的表达和功能的作用。由于缺乏这些渠道的具体药理代理商，我们已经采取了遗传学的方法操纵的渠道表达。我们使用一个器官的文化准备（16），以维持细胞的形态和皮质层模式。我们通常会在产后8-10天隔离急性新皮层切片，并保持3-7天文化片。这使我们能够在我们与急性切片的工作的一个年龄相仿的研究神经元和最大限度地减少了旺盛切片中的兴奋连接的发展。我们从视觉识别的锥体神经元层的II / III或V使用红外线照射（红外）和微分干涉对比显微镜（DIC）在电流或电压钳全细胞膜片钳记录。我们使用基因枪的野生型或突变的钾离子通道的DNA（基因枪）转来操纵表达的渠道，研究其功能。 epifluorescence显微镜与绿色荧光蛋白（GFP）基因共转染后的转染细胞很容易识别。我们比较对相邻的未转染的神经元，在同一层的同一片转染细胞的录音。

Watch this Scientific Journal Video about Whole Cell Recording from an Organotypic Slice Preparation of Neocortex at JoVE.com

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

这是一个协议，准备和保持器官的文化一个新皮层锥体神经元的电气录音的目的的片准备。

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Research

JoVE Journal

Neuroscience

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

科研

神经科学

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

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 ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

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

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 ...