记录神经元活性的电生理方法-1: 神经元放电特性及电生理平台组成

发布网友 发布时间：2023-05-05 15:29

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热心网友 时间：2023-11-17 00:54

参考文献：
《Guide to Research Techniques in Neuroscience》
《The Axon Guide: A guide to Electrophysiology and Biophysics Laboratory Techniques》

视频：
膜片钳电生理技术_哔哩哔哩

网站：
膜片钳技术|电生理学|电生理学工具-Molecular Devices官网
膜片钳电生理技术 - JoVE

主要是根据电极插入检测样本神经元细胞的位置来划分：

膜片钳有不同的钳制方式，如：

根据需求，不同的电生理记录方法可以分别用于记录体内和体外的神经元细胞的电特性 ( the electrical properties )。(后面将哪种情况下用哪种合适)。

我们首先得知道，电生理研究的是什么？
研究神经元的电生理活动，就研究不同刺激下神经元的放电性质(频率，强弱，时长等)，所以我们必须先了解神经元的电特性。

细节这里不讲(另一篇再整理)，这里有几点需要知道的：

工欲善其事，必先利其器
知道了我们想干嘛，我们先看看看记录神经元电生理活动的设备

The Patch-Clamp Rig

我们先从源头开始讲，那当然是微电极 Microelectrode, ME

These metal electrodes not only provide more stable isolation of single units than micropipettes, but they tend to sample from a larger morphological variety of cells and also help in better localization of electrode tracks to identify where recordings took place in whole brains.

Smaller tips have higher resistances, and they restrict the area from which potentials can be recorded, thus permitting the isolation of the activity of either a fiber or a cell. Large tips and low resistances pick up the activity from a number of neurons and are of limited use in efforts to identify the functional properties of single cells.

Tips with very high resistances are also of little use, as they cannot record neural activity unless they are very close to the cell membrane or actually inside a cell.

The headstage is the central hub that connects the electronic equipment to the tissue preparation. It contains an electrode holder that stabilizes the microelectrode ring recordings and also directly connects the microelectrode to the first stage of amplifier electronics needed to detect the electrical signals. The headstage passes the signal to the main amplifier for the main signal processing. The headstage is carefully positioned by the micromanipulator and is also attached to the microdrive.

Accurately placing a patch electrode onto a 10-20 μm cell requires an optical system that can magnify up to 300- or 400- fold with contrast enhancement (e.g. Nomarski/DIC ( 微分干涉相差显微镜 ), Phase, or Hoffman) and a micromanipulator that stably positions the electrode in 3D space. An inverted microscope(倒置显微镜) is preferable because it allows easier access for electrodes from above the preparation and also provides a larger, more solid platform to bolt the micromanipulator. (更好地为微电极腾出操作的空间) A micromanipulator has the ability to move the electrode in very minute distances along the X, Y, and Z axes. The micromanipulator can then hold that position indefinitely.

A microdrive is used to lower or raise the microelectrode to a specific depth in tissue in very fine steps. It is usually preferable to use remote-controlled microdrive systems to eliminate hand vibration. Thus, the headstage (and consequently the electrode) can be set into place by hand using the micromanipulator and then finely adjusted in and out of tissue using a microdrive for the final approach to the cell.

An instrument that contains the circuitry required to measure electrical currents passing through ion channels or changes in cell membrane potential. The amplifier contains the circuitry necessary to measure current passing through the cell membrane both in magnitude and direction. The amplifier can also measure the cell membrane potential in response to the movement of current.

To initiate current movement, the experimenter can deliver a voltage command (钳制电压) to the cell, and the cell will respond by passing the current necessary to maintain that voltage command. (电压钳，用于测过膜电流。) Conversely, the experimenter may also inject current and then measure the change in membrane potential resulting from that change in current. (电流钳，注入衡定电流测膜电位变化，模拟生理状态下离子流动触发动作电位)

*The current acquired by the amplifier is an analog signal, but in order to perform data analysis needed for high resolution patch-clamp measurements, the analog signal must be converted into a digital one. *

然后既然提到了 带宽 和 采样频率 ，就来说说为什么这两者会影响采样的质量。首先要弄清楚一点， 带宽 是也是表示频率，单位是 Hz，带宽代表的是数据本身的频率，而 采样频率 指的是从数据中抽样的频率。如下图，左边是原始数据的频率，右边是采样数据的频率。很明显可以看出，采样频率越高，数据还原度越高。

然而，带宽只是代表，而并不等于数据本身的频率，因为模拟信号存在着衰减的情况。先来看看从microelectrode 到 Digitizer 的结构。

模拟信号输入路径(Analog-Input-Path)

The analog input path attenuates, amplifies, filters, and/or couples the signal to optimize it in preparation for digitization by the ADC. The ADC samples the conditioned waveform and converts the analog input signal to digital values that represent the analog input waveform. The frequency response of the input path causes an inherent loss of amplitude and phase information.

带宽Bandwidth
It is defined as the frequency at which a sinusoidal input signal is attenuated to 70.7 percent of its original amplitude, which is also known as the -3 dB point. It is defined as the frequency at which a sinusoidal input signal is attenuated to 70.7 percent of its original amplitude, which is also known as the -3 dB point.

Bandwidth describes the analog front end’s ability to get a signal from the outside world to the ADC (analog-to-digital converter) with minimal amplitude loss—from the tip of the probe or test fixture to the input of the ADC. In other words, the bandwidth describes the range of frequencies an oscilloscopecan accurately measure.

(不知道这一块有没有讲清楚，到这里电信号是如何产生，以及如何采集已经基本讲完了，后面就是数据的可视化，屏蔽环境噪音等)

Oscilloscope & Loudspeaker System & Computer & Software

An oscilloscope receives the electrical signal from the amplifier and displays the membrane voltage over time. This is the major source of data output in electrophysiology experiments. They can also be heard by connecting the output of the amplifier to a loudspeaker. Action potentials make a distinctive popping sound, so recording the activity of an active neuron can sound like popcorn popping. Loudspeakers can be helpful when trying to locate a neuron of interest, because different types of neurons have distinctive firing patterns.

Computers have greatly aided electrophysiological studies by automating stimulus delivery and electrical signal recording. Computers can easily manipulate many parameters ring recordings, such as the recording thresholds and stimulus delivery timing. Computers also allow simple real-time data analysis, displaying the results of an experiment, even while the experiment is occurring.

这一篇主要讲了：
1. 主要的电生理技术
2. 神经元电特性
3. 电生理实验的设备

还没讲的有(放到后面吧，看那么多大家也累了)：
1. 什么是电流钳？什么是电压钳？
2. 实现的电路原理 (现在只是简单介绍了一些信号采集的过程)
3. 信号处理 (最原始数据是怎么变成我们拿到手的数据的，这部分我可能能力不足先跳过)
4. 胞外记录，胞内记录，膜片钳的应用
5. 文章中的电生理图怎么看？figure (其实就是拿到手的 data 怎么加工变成文章展示出来的 data)
6. 其他拓展应用，如果结合光遗传