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）。

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没有利益冲突的声明。

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 ...

whole-cell-recording-from-an-organotypic-slice-preparation

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JoVE Journal

Neuroscience

14.1K 次观看.  University of Tennessee Health Science Center. 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。