transretinal ERG的录音，从小鼠视网膜杆和锥光反应
Summary
我们描述一个transretinal视网膜电图（ERG）录音获得完整的小鼠视网膜的杆状和锥状光反应相对简单的方法。这种做法从感光细胞的突触传递块孤立的光反应，并记录他们使用场电极置于整个平板式的孤立视网膜。
Abstract
有两个不同的类脊椎动物的视网膜成像感光杆和视锥细胞。棒是能够探测到单光子的光，而视锥细胞在瞬息万变的明亮的光线条件下连续运行。光杆和锥感光细胞的外节的视觉色素的吸收触发一个光传导级联反应，最终导致封闭的环核苷酸门控通道的细胞膜和细胞极化。现有的和潜在的这种膜的光致变化可以注册为光响应，无论是古典吸电极记录技术1,2或transretinal药物阻断突触后反应元件3-5孤立视网膜电图记录，从图（ERG）。后一种方法允许从小鼠感光细胞的药物访问长久的录音和获得稳定的光反应FR特别有用OM的稀缺和脆弱的鼠标锥。在视锥细胞的情况下，进行这样的试验，无论是在暗适应条件和以下强烈的光照漂白基本上所有的视觉色素，监察锥感光恢复的过程中，在暗适应6,7。在这部影片中，我们将展示如何进行暗适应小鼠视网膜棒和M / L，锥的驱动transretinal录音。使用野生型小鼠（C57BL / 6）视网膜棒的录音将进行。为简单起见，锥录音将获得杆转导的转基因α-亚基基因敲除（Tα – / – ）小鼠缺乏信号8杆。
Protocol
1。使电极玻璃电极的准备。重达120毫克，琼脂和混合在10毫升蒸馏水（最终琼脂浓度为1.2％）。在热水浴融化的琼脂溶液。填补玻璃毛细管（我们使用以下尺寸的Word Presision仪器TW100-4毛细血管长度为100毫米，外径/ ID = 1/0.75毫米，内部容积为44μL）琼脂解决方案，使用塑料注射器。巩固在室温下10分钟的琼脂。因此，最多可以填写200毛细血管（琼脂混合物的其余部分可以存放在-4°C和重新使?…
Discussion
杆和锥驱动transretinal ERG的录音上面介绍的方法，正在成为一个调查，在野生型和转基因动物的鼠标感光功能强大的工具。除了基本的光响应特性的简单特征，这个简单的技术提供了很大的反响稳定性，在近乎完整的视网膜准备进行长期的实验。无论是暗适应的棒最大响应幅度和野生型小鼠光敏性至少1小时内是稳定的，从开始录音，暗适应锥最大振幅和光敏性通常不下降30-40分钟后超过10％录音。?…
Declarações
The authors have nothing to disclose.
Acknowledgements
从研究职业发展奖防盲支持，国立卫生研究院资助EY19312 EY19543（VJK），以及不受限制地从研究的拨款，以防止盲目性和EY02687（华盛顿大学眼科及视觉科学系）。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
| DL-AP-4 | TOCRIS bioscience | 0101 | ||
| All other reagents | Sigma-Aldrich | |||
| Glass capillaries | World Precision Instruments | TW100-4 | For making electrodes | |
| Filter paper | HARG | Millipore | HABG01300 | |
| Photometer | UDT Instruments | S350 | For light calibration | |
| Radiometric silicon sensor | UDT Instruments | 221 | For light calibration | |
| Anti-vibration table | Technical Manufacturing Corporation | TMC 78-239-02R, TMC63-26012-01 | To minimize mechanical noise | |
| Air compressor Panther P 15TC | Werther International | P 15TC | Connected to anti-vibration table | |
| Stereomicroscope | LEICA | MZ9.5 | For mouse eye dissection | |
| Infrared image converters | B.E.Meyers | ProwlerTM | Bound to stereomicroscope | |
| Differential amplifier DP-311 |
Warner Instruments | DP-311 | ||
| Axon Digidata 1440A Digitizer | Molecular Devices | 1440A | ||
| Dual Channel 8-pole Filter | KROHN-HITE Corporation | 3382 | ||
| Ceramic resistor | TE Connectivity | CGS SBCHE618RJ | For reheating the perfusion solution | |
| Thermocouple | T | Physitemp Instruments | IT-18 | |
| Temperature monitor | Omega | DPi32 | Connected to thermocouple | |
| LED 505 nm | TT Electronics/ Optek Technology | Digi-key P/N 365-1185-ND | To apply test flashes/bleaching light | |
| Cautery pen | Bovie | AA25 | For marking the dorsal part of the mouse eyeball | |
| pCLAMP 10 Electrophysiology Data Acquisition and Analysis Software | Molecular Devices |
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Citar este artigo
Kolesnikov, A. V., Kefalov, V. J. Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses. J. Vis. Exp. (61), e3424, doi:10.3791/3424 (2012).