Summary

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

Published: October 13, 2016
doi:

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

倫理的承認 – ベイラー医科大学の制度的動物のケアと使用委員会によって承認されたような動物を対象とする手順は、研究用動物のための国立衛生研究所のガイドラインの規則や規制に従って行いました。 1.外部および内部ソリューション網膜解剖の間およびその後の電気生理学的記録で外液として、哺乳動物のリンゲル液を使用してください。記録の日に(カルシウムなし)1…

Representative Results

deafferentatedマウス網膜からのオンとオフ型のSACの代表的な録音は、図1に示されている。GCLとINLの両方におけるコリン作動性細胞が確実にtdTomato蛍光により同定し、DIC( 図1A)の下で全細胞パッチクランプ記録のために標的とすることができますそれらの膜電位(上部トレース)および(下部のトレース、 図1B)を駆動するシナプ?…

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

References

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Cite This Article
Tu, H., Hsu, C., Chen, Y., Chen, C. Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina. J. Vis. Exp. (116), e54608, doi:10.3791/54608 (2016).

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