Deafferentatedマウス網膜のフラットマウントの準備中のスターバーストアマクリン細胞のパッチクランプ記録
Summary
このプロトコルは、フラットマウント調製物から網膜ニューロンの全細胞パッチクランプ記録を実行する方法を示しています。
Abstract
哺乳動物の網膜は、複数のニューロンタイプの積層組織です。その複雑なシナプスのネットワーク内でどのように処理されるかを視覚信号を理解するために、電気生理学的記録は頻繁に個々のニューロン間の接続を研究するために使用されています。私たちは、GCL（神経節細胞層）およびマウス網膜のINL（内顆粒層）の両方で、遺伝的にマークされたニューロンのパッチクランプ記録のためのフラットマウントの準備を最適化しました。縦と横の両方の接続が前者の構成に保存されているので、フラットマウントで記録INLニューロンを研究するために大きな横方向の成分と網膜回路を可能にすること、スライスよりも好まれています。私たちは、このようなコリン作動スターバーストアマクリン細胞（SACの）として網膜におけるミラー提携ニューロンの応答を比較するには、この手順を使用しています。
Introduction
As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in RhoΔCTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.
Protocol
Representative Results
Discussion
多くのラボでは、フラットマウントの準備15-18でGCLニューロンから記録しているが、私たちの手順は、INLニューロンから記録を可能にします。我々はここ成功したルーチンの録音のために重要であるいくつかのステップを強調する。
網膜の新鮮さと平坦度は、記録ピペットを用いて、それを貫通するために重要です。この点において、パンチニトロセルロース膜?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.
Materials
| Fixed-stage fluorescent microscope with DIC | Olympus | BX51-WI | |
| Micromanipulators | Sutter | MP-225 | |
| Patch clamp amplifier | A-M System | AM2400 | |
| AD converter | National Instrument | NI-USB-6221 | |
| Heater controller | Warner Instrument | TC-324B | |
| Inline heater | Warner Instrument | SC-20 | |
| Peristaltic pump | Rainin | Dynamax | |
| pipette puller | Sutter Instrument | P-1000 | |
| Glass tube with filament | King Precision Glass | Customized | |
| Stimulator | A.M.P.I. | Master-8 | |
| Biocytin | Sigma | B4261 | |
| NaCl | Sigma | S6191 | |
| KCl | Sigma | P5405 | |
| NaHCO3 | Fisher | BP328-1 | |
| Na2HPO4 | Sigma | S0876 | |
| NaH2PO4 | Sigma | S5011 | |
| CaCl2 | Sigma | C5670 | |
| MgSO4 | Sigma | M1880 | |
| D-glucose | Sigma | G6152 | |
| K-gluconate | Sigma | G4500 | |
| ATP-Mg | Sigma | A9187 | |
| Li-GTP | Sigma | G5884 | |
| EGTA | Sigma | E0396 | |
| HEPES | Sigma | H4034 | |
| KOH | Sigma | P5958 | |
| Cs-methanesulfonate | Sigma | C1426 | |
| CsOH | Sigma | 232041 | |
| Syringer filter | Nalgene | 171 | |
| 1 ml syring | Rainin | 17013002 | |
| 10 ul pipette tip | Genesee Scientific | 24-130RL | |
| Streptavidin-488 | ThermoFisher | S-11223 | |
| 10X PBS | Lonza | 17-517Q |
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