小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录
Summary
本协议描述了在少量回收的缓冲液中的氧水平的稳定, 以及在淹没的急性海马切片中记录活动依赖性突触可塑性的方法学方面。
Abstract
尽管自1951年以来, 脑切片的实验已经开始使用, 但问题仍然存在, 这就降低了在执行场电位或胞内记录时对突触传输调制进行稳定和成功分析的可能性。这篇手稿描述的方法学方面, 可能有助于改善实验条件的维护急性脑切片和记录场兴奋性突触后电位在一个商业可用的淹没室与流出-carbogenation 单位。外流 carbogenation 有助于稳定氧气水平的实验, 依靠回收的小缓冲水库, 以提高成本效益的药物实验。此外, 该手稿还有代表性的实验, 考察不同的 carbogenation 模式和刺激范式对突触传递的活动依赖性突触可塑性的影响。
Introduction
在 1951年, 第一次报告的急性脑切片实验进行了1。在 1971年, 在成功的体外录音从梨皮层2,3和发现海马神经元是相互连接横向沿 septotemporal 轴海马4, 其中一个第一个体外海马神经元活动的记录实现了5。神经或 neurostructural 参数的相似性神经元在体内和体外条件仍然是一些辩论的主题6, 但在 1975年, Schwartzkroin7表示, 基神经元的性质是保持在体外和高频刺激 (即,后背) 的传入在海马形成诱导一个长期的促进突触潜能8。神经元活动的电生理记录在体外极大地扩展了活动依赖性突触可塑性的细胞机制的研究910在1973年被极乐发现et al.11在体内实验中用家兔。
研究神经元活动或信号通路的脑切片, 特别是在急性海马切片, 现在是一个标准的工具。然而, 令人惊讶的是,在试管中的实验还没有标准化, 这证明了在急性海马切片的制备和维持方面仍然存在着多种方法。里德et al.(1988)12回顾了在不同类型的切片室中维持急性脑切片的方法学挑战, 以及沐浴培养基、pH 值、温度和氧水平的选择。由于体外切片记录设置的 custom-made 元素, 这些参数在记录室中仍然难以控制。可以找到的出版物, 可能有助于克服一些方法上的挑战, 并描述了新类型的淹没切片室, 如间质 3D microperfusion 系统13, 一个室的层流和氧气增强提供14, 一个具有计算机温度控制的系统15, 以及一个多记录系统16。由于这些房间不易建造, 大多数科学家依赖于商业上可用的切片室。这些室可以安装在显微镜系统, 从而允许的电生理和荧光成像的结合17,18,19。由于这些腔室保持脑切片淹没在人工脑脊液 (aCSF), 高流速的缓冲液的解决方案需要保持, 增加了药物应用的费用。为此, 我们已经采用了一个循环再灌注系统与流出-carbogenation, 提供足够的稳定性, 以长期记录的现场电位在一个浸没的切片室使用相对较小的 aCSF 体积。此外, 我们总结了如何使用这个实验 carbogenation/灌注系统影响的结果, 活动相关的突触可塑性10和如何抑制真核伸长 factor-2 激酶 (eEF2K) 调节突触传输20。
Protocol
这些动物是按照中国上海复旦大学脑科学研究所和医学神经生物学国家重点实验室的既定动物保育标准和程序进行维护的。 1. 溶液制备 注意: 请参见材料表。 准备切片缓冲区 (修改后的 Gey 的解决方案):92 毫米氯化钠, 2.5 毫米氯化钾, 1.25 毫米 NaH2PO4, 30 mm NaHCO3, 25 mm 葡萄糖, 20 mm HEPES, 3 mm Na+-丙酮酸, 10 mm MgSO4, 0.5 mm CaCl2…
Representative Results
在 “协议” 部分, 我们描述了由雄性 C57BL/6 小鼠和雄性大鼠 (5-8 周) 的海马形成 (图 1) 的腹侧和中间部分的急性海马切片的制备。半球在切片机平台上的位置有助于保持它们的稳定, 并消除琼脂或琼脂糖对稳定的需要。灌注系统本身是基于在高转速下运行的蠕动泵, 以提供所需的液体流速。因此, 在泵的标准管迅速老化造成了一个很大的问题, 我们克服了…
Discussion
虽然界面切片室表现出更强大的突触响应25,26,27,28, 淹没腔室为膜片钳记录和荧光提供了额外的便利成像.因此, 我们已经描述了几个方面的现场电位记录的急性海马切片使用商业浸没切片室, 可以很容易地扩展到成像的荧光探针在神经元17,18, <sup class=…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
. 进行、分析、设计了实验并写了手稿。D.X. 和中共协助图准备和进行实验。这项工作得到了自然科学基金 (31320103906) 和111项目 (B16013) 对肺结核
Materials
| Reagents required | |||
| NaCl | Sinopharm Chemical Reagent, China | 10019318 | |
| KCl | Sinopharm Chemical Reagent, China | 10016318 | |
| KH2PO4 | Sinopharm Chemical Reagent, China | 10017618 | |
| MgCl2·6H2O | Sinopharm Chemical Reagent, China | 10012818 | |
| CaCl2 | Sinopharm Chemical Reagent, China | 10005861 | |
| NaHCO3 | Sinopharm Chemical Reagent, China | 10018960 | |
| Glucose | Sinopharm Chemical Reagent, China | 10010518 | |
| NaH2PO4 | Sinopharm Chemical Reagent, China | 20040718 | |
| HEPES | Sigma | H3375 | |
| Sodium pyruvate | Sigma | A4043 | |
| MgSO4 | Sinopharm Chemical Reagent, China | 20025118 | |
| NaOH | Sinopharm Chemical Reagent, China | 10019718 | |
| Tools and materials for dissection | |||
| Decapitators | Harvard apparatus | 55-0012 | for rat decapitation |
| Bandage Scissors | SCHREIBER | 12-4227 | for mouse decapitation |
| double-edge blade | Flying Eagle, China | 74-C | |
| IRIS Scissors | RWD, China | S12003-09 | |
| Bone Rongeurs | RWD, China | S22002-14 | |
| Spoon | Hammacher | HSN 152-13 | |
| dental cement spatula | Hammacher | HSN 016-15 | |
| dental double end excavator | Blacksmith Surgical, USA | BS-415-017 | |
| Vibrating Microtome | Leica, Germany | VT1200S | |
| surgical blade | RWD, China | S31023-02 | |
| surgical holder | RWD, China | S32007-14 | |
| Electrophysiology equipment and materials | |||
| Vertical Pipette Puller | Narishige, Japan | PC-10 | |
| Vibration isolation table | Meirits, Japan | ADZ-A0806 | |
| submerged type recording chamber | Warner Instruments | RC-26GLP | |
| thermostatic water bath | Zhongcheng Yiqi，China | HH-1 | |
| 4 Axis Micromanipulator | Sutter, USA | MP-285, MP-225 | |
| Platinum Wire | World Precision Instruments | PTP406 | |
| Amplifier | Molecular Devices, USA | Multiclamp 700B | |
| Data Acquisition System | Molecular Devices, USA | Digidata 1440A | |
| Anaysis software | Molecular Devices, USA | Clampex 10.2 | |
| Fluorescence Microscope | Nikon, Japan | FN1 | |
| LED light source | Lumen Dynamics Group, Canada | X-cite 120LED | |
| micropipettes | Harvard apparatus | GC150TF | extracelluar recording |
| borosilicate micropipettes | Sutter, USA | BF150-86 | patch clamp |
| tungsten electrode | A-M Systems, USA | 575500 | |
| peristaltic pump | Longer, China | BT00-300T | |
| tubes for peristaltic pump | ISMATEC, Wertheim, Germany | SC0309 | 1x inflow, ID: 1.02mm |
| tubes for peristaltic pump | ISMATEC, Wertheim, Germany | SC0319 | 2x tubes for outflow, ID: 2.79 mm |
| CCD camera | PCO, Germany | pco.edge sCMOS | |
| lens cleaning paper | Kodak | ||
| 50 ml conical centrifuge tube | Thermo scientific | 339652 | |
| Prechamber | Warner Instruments | BSC-PC | |
| Inline heater | Warner Instruments | SF-28 | |
| Temperature Controller | Warner Instruments | TC-324B |
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Citar este artículo
Weng, W., Li, D., Peng, C., Behnisch, T. Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System. J. Vis. Exp. (131), e55936, doi:10.3791/55936 (2018).