从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核
Summary
我们提供了用于诱导的长时程增强，在海马CA1区和核富集级分的从片的tetanized区域的随后分离的详细协议。这种方法可以被用来确定活性依赖的核蛋白质导入在学习和记忆的细胞模型。
Abstract
学活性依赖的蛋白表达，细胞内转位，或磷酸化是必不可少的理解突触可塑性的潜在的细胞机制。长时程增强（LTP）和诱导的急性海马长时程抑制（LTD）被广泛地接受为学习和记忆的细胞模型。有迹象表明，使用活细胞成像和免疫组化的方法来可视化活动相关的蛋白质动力学大量的研究。然而，这些方法依赖于抗体的免疫细胞化学在单个神经元的荧光标记蛋白或过表达的适宜性。蛋白质的免疫印迹是一种替代方法提供的结果，独立确认。在制备由个别tetanized海马切片的亚细胞级分的第一限制因素是材料的低量。二，处理程序是至关重要的，因为升甚至很短，小的操作艾尔文片可能会引起一定的激活信号级联。在这里，我们描述，以便获得足够纯度的核浓缩部分的足够量的从大鼠脑急性海马切片的CA1区的优化工作流程。作为一个代表性的例子中，我们表明，突触核蛋白质的信使的ERK1 / 2磷酸化形式雅各积极进入细胞核后，诱导LTP，并且可以从CA1区神经元核浓缩部分进行检测。
Introduction
突触N-甲基-D-天冬氨酸受体（NMDA受体）发挥在突触可塑性和细胞存活信号而激活突触外NMDA受体的可触发的神经变性和细胞死亡的关键作用。这些变化依赖于严密控制/受规管活动相关的基因表达，因此需要激活突触或树突和细胞核7之间不断的沟通。在MAP激酶ERK1 / 2的突触NMDA受体的信号的下游效应，并参与NMDA受体活化诱导的基因表达，而经由突触外NMDA受体信号没有或ERK1 / 2的活性8,11的抑制作用。
有迹象表明，已经显示出远端树突和细胞核之间穿梭蛋白的数量。许多这些蛋白质含有核定位信号，并正在积极沿着在动力蛋白和importin依赖性微管运输到核6,9。 InterestinglY，一些使者只是过境到细胞核针对特定突触的刺激。例如，环AMP应答元件结合蛋白2（CREB2）的逆行运输，通过化学股份有限公司但不LTP 12诱导。本地化的NMDA受体依赖的突触刺激驱动CREB调节转录共激活因子（CRTC1）进入细胞核，易位过程，其涉及在长期海马可塑性4。它最近的研究表明，该蛋白信使雅各布转位到后两个核，突触和突触外NMDA受体的激活和调节CREB依赖性基因转录的5。信号的突触和突触外原点编码雅各的翻译后修饰。突触活动引起雅各ERK1 / 2磷酸化依赖于在位置180（pJacobS 180）的一个关键丝氨酸这是一个必要的后续易位初级海马文化的核心。此外，我n个急性海马pJacobS 180转位到谢弗抵押LTP后核CA1区神经元而不是公司咯 。 pS180雅各导致的可塑性相关的基因表达增加，这基因表达反馈到突触功能。形成鲜明对比，雅各布该易位至细胞核后突触外NMDA受体活化不磷酸化在Ser180和可能具有不同的蛋白复合物有关在核中引起“CREB切断”和突触触头10回缩。
对突触核蛋白质的信使核进口的大多数已发表的研究中分离的神经细胞原代培养工作已经完成。因此，这将是有趣的，如果这样的调查结果可以复制使用海马生理更多的相关条件下神经元的连接和功能更好的保存。在这里，我们提出了一个优化的协议，以评估LTP-DE通过免疫印迹侧基的蛋白质信使核转。这个方法也适合于分析中的粗核级分的蛋白质的活性依赖的磷酸化。具体来说，目前的协议包括制备急性海马CA1区片，感应和记录的LTP。接着，CA1区用显微镜解剖，分离出刺激的区域。我们结合和修饰赵和他的同事17引入的更改提供CellLytic核抽提试剂盒的协议核隔离。优化过程包括解剖海马CA1区的低渗缓冲液裂解使细胞肿胀，核释放。细胞裂解和细胞核形态可以通过显微镜检查来确定。铀浓缩是一个短离心步骤来实现的。免疫印迹分析用抗体的NeuN和NSE2，核或细胞质级分的特异性标记物，表明该方法可以被用作快速和可重复的协议来隔离这些亚细胞组分，并研究样蛋白的磷酸化非常不稳定的翻译后修饰。此外，此方法是有利的，对于小的组织样本来自海马切片的CA1区解剖区域导出，并且可以组合使用，以海马切片的免疫组化分析。
Protocol
Representative Results
Discussion
在上述协议中所述的步骤提供指导如何准备从青年或成年大鼠海马急性切片，诱导和记录LTP，迅速解剖切片的刺激区域，并准备核浓缩部分的研究活动相关的蛋白质动力学。这种方法源于对彼此独立地使用几种不同的方法组合。我们优化的工作流程，并为初学者建立自己的实验后，LTP的诱导，研究蛋白质的亚细胞再分配提供足够的细节。作为一个例子，我们证明了LTP的晚期形式诱导S180的核积累磷…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by the DFG (SFB 779 TPB8, Kr1879/3-1 MRK), DIP grant (MRK), EU FP7 MC-ITN NPlast (MRK), Center for Behavioral Brain Sciences (CBBS, Sahsen-Anhalt), (AK and SB), MM is a recipient of European Molecular Biology Organization (EMBO) Long-Term Fellowship (EMBO ALTF 884-2011) and Marie-Curie IEF.
Materials
| Table 1. Equipment | |||
| CED 1401 AD/DA converter | Cambridge Electronics Design, UK | ||
| Axopatch 200B amplifier | Axon，USA | ||
| Axon Digidata acquisition system | Axon，USA | ||
| Clampex 10.0 Data acquisition and analysis software | Axon，USA | ||
| Leica microscope | Leica TCS SP2，Germany | ||
| P-97 Standard microelectrode puller | Sutter，USA | ||
| Stereomicroscope | Leica | Leica S4E, Germany | |
| Vibrotome | Leica VT1000S, Germany | ||
| Isolated pulse stimulator | Model2100, A-M systmes, USA | ||
| Centrifuge | Thermo Scientific, HERAEUS, FRSCO17 | ||
| SDS-PAGE system | BIORAD | ||
| Cooler | JULABO | ||
| Bright field Microscope | NIKON ECLIPSE TS100 | ||
| Cell culture incubator | Thermo Electron Corporation, HERAEUS | ||
| Micromanipulator | Luigs & Neumann, SM-5, Germany | ||
| Blotting chamber and electric power supplier | Hoefer Scientific Instruments, San Francisco | ||
| Cooler | Julabo, F12 | ||
| Centrifuge | Thermo Scientific, Heraeus, FRESCO 17 | ||
| Submerged type Recording Chamber | custom made | ||
| U-shape and submerged type incubator | custom made | ||
| Small surgical scissors | |||
| Scalpel | |||
| Thin spatula | |||
| Plastic Pasteur pipette | |||
| Plastic culture dish | |||
| Bunsen beaker | |||
| Syringe | |||
| Table 2. Reagents | |||
| Name of Reagent | Company | Catalog Number | Comments/Description |
| NaCl | ROTH | Art.-Nr.3957.1 | ≥99.5%, p.a.,ACS,ISO |
| KCl | ROTH | Art.-Nr.6781.1 | ≥99.5%, p.a.,ACS,ISO |
| CaCl2·2H2O | MERCK | 1.02382.0500 | pro analysi |
| MgSO4·2H2O | MERCK | 5886.05 | pro analysi |
| MgCl2·6H2O | AppliChem | CAS-NO: 7791-18-6; EC-NO:2320946 | for Molecularbiology |
| KH2PO4 | MERCK | 12034.025 | for Molecularbiology |
| Na2HPO4·2H2O | MERCK | 1.06574.1000 | extra pure |
| Glucose·H2O | ROTH | Art.-Nr.6887.1 | for Molecularbiology |
| Hepes | ROTH | Art.-Nr.9105.4 | PUFFERAN, ≥99.5%, p.a. |
| NaHCO3 | MERCK | 1.06329.1000 | pro analysi |
| Protease inhibitor Coctail | ROCHE | ||
| Phosphostop | ROCHE | ||
| Bicuculline | Tocris bioscience | ||
| Isofluran | Baxter | ||
| Table 4. Antibodies | |||
| Primary antibodies | Company | Catalog Number | Species |
| pJac-s180 | Biogenes/ purified | rabbit (dil. 1:100) | |
| NeuN | Milipore | MAB377 | mouse (dil. 1:1,000) |
| NSE | Cell Signaling | D20H2 | rabbit (dil. 1:1,000) |
| beta-actin | Sigma | A-5441 | mouse (dil. 1:5,000) |
| Secondary antibodies | Species | ||
| IgG HRP Conjugated | DAKO | goat anti – mouse (1:5,000) | |
| IgG HRP Conjugated | NEB | goat anti – rabbit (1:5,000) |
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