记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术
Summary
We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.
Abstract
视网膜是通往视觉系统。要了解视觉信号处理机制，我们研究视网膜神经网络的功能。网络中的视网膜神经元包括许多亚型。超过10种亚型双极细胞，神经节细胞，和无长突细胞已经鉴定了形态学研究。视网膜神经元的多个亚型被认为编码可视信令的显着特征，例如运动和颜色，并形成多个神经通路。然而，在视频信号处理的每个神经元的功能角色尚未完全了解。膜片钳的方法是有用的，解决这一根本问题。在这里，一个协议来记录使用膜片钳记录在暗适应条件鼠标视网膜神经元光诱发突触反应提供。鼠标眼睛的暗适应O / N，和视网膜切片准备使用红外照明和解剖观众在黑暗的房间。红外灯不激活鼠标光感受器，从而保留了它们的光响应。膜片钳是用来记录在视网膜神经元的光诱发反应。在录音表征神经元形态亚型荧光染料注入。此过程使我们能够确定在小鼠视网膜的每个神经元的生理功能。
Introduction
The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms.
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6.
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.
Protocol
Representative Results
Discussion
Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings.
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the …
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| mice (28-60 days old, male) | Jackson laboratory | C57BL/6J strain | |
| Ames' medium powder | Sigma | A1420 | excellent |
| Stereo microscope | Nikon | SMZ745 | excellent |
| dissecting tool_forceps | Dumont | #4, #5, #55 | excellent |
| dissecting tool_scissors | Roboz | RS-5605 | excellent |
| dissecting tool_surgery knife | Surgistar | 7514 | excellent |
| razor blade (for chopper) | EMS | 71970 | excellent |
| chopper | handmade | ||
| infrared viewer | Night Owl Optics | NOBG1 | It shows bright view. Focusing small objects is an issue. |
| infrared pocket scopes | B.E. Meyers | OWL Gen 3 NV pocketscope | excellent view |
| puller | Sutter | P-1000 | excellent. Make consistent size pipettes. |
| dark box | Pelican | dark box | excellent |
| patch clamp system | Scientifica | slice scope 2000 | Excellent setup. Most key components are included in one package. Micromanipulators are excellent. |
| amplifier | Molecular Devices | multiclamp 700B | Excellent and easy control. |
| acquiring software | Molecular Devices | pClamp software | Excellent and easy control. |
| light source (LED) | Cool LED | pE-2 4 channel system | Excellent |
| CCD camera | Q-imaging | Retiga 2000 | Excellent |
| Faraday cage | handmade |
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