方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量
Summary
After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.
Abstract
Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.
Introduction
突触传递是由含有神经递质的突触小泡的胞吐作用介导的，而这些囊泡必须经过神经末梢内本地吞再循环以维持长期的神经元沟通。由于突触传递在大脑中的重要作用，了解构成突触小泡循环是实现在整个蜂窝通信更好的理解的重要基础的分子机器。在细胞模型系统，肾上腺嗜铬细胞提供了一些最明确的洞察分子机制基础突触囊泡循环。胞吐作用，在神经递质释放的最后步骤，已经极大地研究并通过使用肾上腺嗜铬细胞1,2的检查。事实上，大多数的分子玩家编排的形成，目标，对接和融合分泌颗粒已经确定，由于应用程序股利ERSE技术，嗜铬细胞1。此外，通过提供一个机会以允许参与胞吐作用的蛋白质机械的单泡的分辨率，嗜铬细胞仍然是一个功能强大的模型，以解决囊泡融合3的问题。
细胞贴附电容测量首先在胞吐3中解决单囊泡融合利用。以通过细胞膜导纳测量与小区连接的配置4-7膜片钳技术来检测囊泡一样小〜中直径为60nm已证明的胞吐作用。导纳的定义是多么容易的电路或设备将允许电流流动的措施;它是阻抗的倒数。因此，导纳测量提供的膜电容的理解。这是由小泡膜进入细胞膜的结合来实现;这表明掺入改变表面面积8。每个囊泡融合导致膜电容9,10逐步增加。此外，该导纳测量提供了膜电导和胞吐事件3中 的融合孔导。由于这种技术提供了在胞吐过程中识别单囊泡动力学的独特工具，我们的实验室最近应用这个概念，检测单囊泡11,12的内吞作用。
我们特别感兴趣是网格蛋白介导的内吞作用（CME），它已被认为是一个基本的看家成分在许多细胞13和作为突触小泡胞吞作用在神经端子14,15的主通路。 CME是已知的生物学上重要的，但是，它的动力学仍不能很好地理解，由于在监测单数内吞事件的技术限制。考虑到嗜铬细胞和神经元之间的1胞吐机制的相似性，这是PLausible在嗜铬细胞分裂机制可能会适用于突触小泡内吞作用的神经元。细胞贴附电容测量已用于监测个体的内吞事件并分析裂变动力学，其中大多数方法都未能解决。在我们的细胞附着的录音，在20kHz的正弦波叠加在保持电势，并且输出电流是从两相分离成薄膜电导的一个信道和膜电容，在其它信道锁定放大器16 18。从变化的膜电导和电容，可以计算裂变孔隙，这可能对应于管状薄膜颈部的内化泡连接到前囊夹断质膜的动力学。总的来说，这项技术使我们有机会研究囊泡分裂的调控机制CME的过程中。
Protocol
Representative Results
Discussion
细胞贴附电容测量要求，以顺利取得录音提供高品质的几个关键步骤：1）肾上腺编制可行的，健康的细胞; 2）客运专线涂层的盖玻片; 3）千兆欧密封的形成; 4）该系统的噪声电平;和5）的相位校正。
动物手术，修饰可以潜在地使是调整手术方法，以最适合的灵巧和防止剥离时发生损坏。此外，关于定位，除去肾上腺足够的做法是必要的，这将被用于电生理记录的细胞全部来?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work is supported by a National Science Foundation award (1145581) to LWG.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Poly-D-Lysine | Sigma | P0899 | |
| DMEM | 15066024 | Keep out of UV | |
| Dulbecco’s Modified Eagle Medium | Life Technologies | ||
| Cover Glass | Carolina Biological | 633029 | 12mm |
| Penicillin Streptomycin | Life Technologies | 15140122 | 100mL |
| Insulin-Trans-Sel-X | Life Technologies | 51500056 | Only thaw on ICE! |
| Papain | Worthington | 39S11614 | |
| EPC-7 plus patch amplifier | HEKA | ||
| BNC-2090 data acquisition board | National Instruments | ||
| Igor data acquisition software | Wavemetrics | ||
| P-97 pipette puller | Sutter Instruments | ||
| Microforge | Scientific Instruments | ||
| Borosilicate glass capillaries | Sutter Instruments | B150-110-10 | Outer diameter-1.5 mm |
| Inner diameter 1.10 mm | |||
| Length- 10 cm |
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