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.

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<ol><li>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）10×ストック溶液から哺乳類リンゲル液を調製し、そしてCaCl 2を滴下carbogenationの15分後（95％O 2および5％CO 2）を追加します。最後の1×溶液（mM単位）含まれていますのNaHCO 3、0.8のNa 2 HPO 4、0.1のNaH 2 PO 4、2のCaCl 2、1のMgSO 4および10 D-グルコース120のNaCl、5のKCl、25。 </li><li> SAC振動を特徴づけるために2つの内部のソリューションを使用してください。テスト成体マウスの網膜神経節細胞に長期の全細胞パッチクランプ記録で調製した溶液の有用性とは、使用するまで-20℃で500μlのアリコートで検証バッチを格納します。 <ol><li>膜電位振動を記録するために、（mM単位）が含まれているカリウム系の内部液を使用：125 K-グルコン酸、8のNaCl、4 ATP-Mgを、0.5のLi-GTP、5 EGTA、10 HEPESおよび0.2％ビオシチン（/ワットV）。 KOHでpHを7.3に調整します。 </li><li> （100セシウムメタン、8のNaCl、4 ATP-Mgを、0.5のLi-GTP、5 EGTA、10 HEPESおよび0.2％ビオシチン：興奮性と抑制性シナプス後電流を記録するために、（mM単位）が含まれているセシウムベースの内部液を使用w / v）です。 CsOHを用いてpHを7.3に調整します。 </li></ol></li></ol>レコーディングの日2.準備<ol><li>オープン孔を有するニトロセルロース膜を製造するために、手動で平滑化16 G注射針から作られた、カスタマイズパンチャーを有する膜をパンチし、2清潔なガラスプレート間の膜の平坦化。 whole-のため網膜をマウントし、直径が中央穴0.2ミリメートルを行い、直径1〜1.2ミリメートルである4より大きな穴によってそれを囲みます。 </li><li>電極への内部ソリューションをロードするためのカスタマイズされたバックフィルフィラメントを作るために、真ん中に10μlのピペットチップを溶融し、それが冷えて固まるまで穏やかに溶融部を長くします。使用直前に、それは少し長いパッチピペットよりなるまで、フィラメントをトリミングするきれいなカミソリの刃を使用します。 </li><li>内部のフィラメントとホウケイ酸ガラス管からプログラマブルプラーにパッチピペットを引き出します。実験の日に、マイクロピペットを製造し、すぐにそれらを使用しています。 注：記録SACのために、我々は次のプーラーの設定を使用：熱：484;プル：0;速度：25;時間遅延：1;圧力：400とホウケイ酸チューブの462 ODとIDのランプ値はそれぞれ、1.65ミリメートルと1.0ミリメートルです。ピペットの抵抗は10-14MΩです。 </li><li> 1時間組織の収穫前に、シャキによる内部液のチューブを解凍&gt; 30分間ボルテックス上でそれをngの。 </li></ol> 3.網膜解剖<ol><li>動物はつま先のピンチに応答性を失うまで、酸素の4％イソフルランにより動物を麻酔。頸椎脱臼により動物を生け贄に捧げます。 </li><li>離れてストレート指摘アイリスハサミで視神経ヘッド1〜2ミリメートルで視神経オフ両方の眼球をカット。 </li><li>血液を除去した後、carbogenated哺乳類リンゲル液でそれらを浸漬する清潔なペーパータオル上で眼球をロールバックします。 23 G針で角膜輪部に穴をパンチし、マイクロはさみで角膜縁に沿って切断することにより、眼球を二等分します。細かい鉗子を使用して角膜とレンズを取り外してください。 <ol><li>網膜全体のマウントのために、優しく細かい鉗子で色素上皮オフ網膜全体を剥離し、視神経乳頭に向かってエッジから4 1.5〜2ミリメートルの直交カットを行います。 </li></ol></li><li>皿に打ち抜いニトロセルロース膜を浸し、軽くてその上に網膜をドラッグしますGCLサイドアップ。ホールマウント網膜を調製する際に、中心穴に視神経頭を置きます。別のきれいな皿に網膜を有する膜を転送します。静かにマルタ十字のように見えると穴の上のすべての4辺を築くために細かいペイントブラシで網膜を平らに。 <ol><li>網膜の一部が一度に検討する場合には、色素上皮が付着したままでかみそりの刃で3-4個にステップ3.3から調製された各アイカップをカット。 carbogenated外液中に光から未使用の部分を保護し、12時間以内にそれらを使用しています。 </li></ol></li><li>乾燥ろ紙でニトロセルロース膜をブロットし、鉗子やペイントブラシで硝子体と内境界膜を除去します。 </li><li>すべての網膜のエッジが完全に底部に密封されたガラスカバースリップで記録室にアセンブリを転送する前に膜に結合していることを確認します。カバースリップとrehydratに真空グリースを有する膜を固定します外部溶液で網膜を電子。アセンブリの下に気泡をトラップに注意しないこと。 </li><li>正立顕微鏡のステージにチャンバーを設定します。毎分〜3ミリリットルの割合で暖かい（34-35°C）carbogenated外液とチャンバーを灌流。 </li><li>最初の10倍の対物レンズの下で網膜を調べ、その後、微分干渉コントラスト（DIC）および/またはエピ蛍光下GCLとINLニューロンを見るために60倍の水浸レンズを使用しています。 tdTomato発現のSACを視覚化するために、白色を使用する554 nmでの励起フィルターと581 nmでの発光フィルターと組み合わせたLED光源。 </li></ol>フラットマウント網膜から4ホールセルパッチクランプ記録<ol><li>カスタムバックフィルフィラメントにシリンジフィルターを通して内部液をフィルタリングします。 </li><li>ソリューションは&gt;のための銀電極線を覆うまで新鮮に引っ張らマイクロピペットにフィラメントを挿入し、先端付近の内部液を分配5ミリメートル。チューブに接続された10ミリリットルのプラスチックシリンジのプランジャーを押したり引っ張ることによって調整することができ、電極内部が圧力を介して吸入ポール、と電極ホルダーにマイクロピペットを固定します。 </li><li>目的の下でピペットを見つけ、網膜上記〜100μmとそれをダウンさせます。現在フォロアモード（I = 0）の下で、DCが立って直流電圧信号をゼロにオフセットを使用します。それはお風呂にある間に、ピペットを介して固定振幅方形波電流を注入することにより、電流クランプ（I クランプ ）モードでピペット抵抗を測定し、Raccessノブを回して違いを中和します。オームの法則によりピペット抵抗を計算するためにRaccessノブに読書を使用してください。 </li><li>ゆっくりと網膜上記〜10ミクロンに電極をもたらします。電極に正の圧力を適用します。それは網膜に近づくにつれてピペットチップの近くに反射変化を見ます。素早くしかし穏やかGCLにピペットを強制し、正pressuを減らしますすぐに再。 </li><li>ラベルされたニューロンに向かってピペットを移動します。他のニューロン、血管やミュラー細胞のエンドフィートを触れないようにしてください。電極の目詰まりを防止するために必要な場合は、より正の圧力を適用します。 </li><li>ディンプルが表示されるまでの標識ニューロンの正中線近くにピペットチップを置きます。正圧を解放し、原形質膜は、ピペットチップ上に立ち直ることができます。 </li><li>ギガオームシールの形成を助けるために、ピペットに20〜120 pAの負の電流を適用します。ピペットに血漿膜を引っ張るために、必要に応じて穏やかな吸引を適用します。細胞膜を破裂させるためにシール形成した後、5分待ってください。これは、こぼれた内部液を灌流によってクリアすることができます。穏やかな吸引によって、膜を破裂。 </li><li>破水した後、現在のクランプモードにある間、ブリッジバランスに切り替えてRaccessノブを使って調整します。 注：いずれの場合SACのような小細胞については、わずかな調整が必要とされています。 Alternそれぞれ、（-75 mVの周り）塩化物の逆転電位で細胞を保持し、（0 mVの周り）グルタミン酸によってatively、レコード興奮と電圧クランプモード（V クランプ ）における抑制性シナプス後電流。時折、網膜および/またはマイクロマニピュレータードリフトに対応するために、必要に応じてピペットの位置を確認し、調整します。 </li><li>薬理学的治療中と後に、前の膜電位または電流の変化を記録するには、新鮮な上にシナプスブロッカー（ 例えば、CNQX、AP5、ピクロトキシン、ツボクラリン等 ）およびチャネルモジュレーター（ 例えば、フルピルチン、ドーパミン、メクロフェナム酸など）を準備します凍結ストックからの実験の日。 carbogenatedリンゲル液と薬剤を希釈します。灌流を介してバッチ方法でそれらを適用します。 </li><li>記録した後、静かにソーマからピペットを削除します。きれいな皿にチャンバーからアセンブリを転送します。他に網膜をデタッチし、再アタッチパンチホルずにニトロセルロース膜を平坦化定着時網膜平坦性を確保するために、ES。 </li><li> RTで30分間、4％パラホルムアルデヒド中に浸漬することにより、網膜を修正しました。ニトロセルロース膜から網膜を削除し、1×PBSでそれをすすいでください。 4℃で14で、0.4％トリトンX-100を1×PBSで希釈した色素結合ストレプトアビジンでO / N染色によって記録された細胞の形態を明らかにする。退色防止媒体に網膜をマウントし、走査型共焦点顕微鏡を用いて記録された細胞を観察します。 注：揮発性および毒性であるパラホルムアルデヒドを伴う手順は、安全のためのドラフトチャンバー内で行われるべきです。 </li></ol>

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The authors have nothing to disclose.

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?...

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 Rho&#916;CTA 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.

<table><tbody><tr><td>Fixed-stage fluorescent microscope with DIC</td><td>Olympus</td><td>BX51-WI</td><td></td></tr><tr><td>Micromanipulators</td><td>Sutter</td><td>MP-225</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>A-M System</td><td>AM2400</td><td></td></tr><tr><td>AD converter</td><td>National Instrument</td><td>NI-USB-6221</td><td></td></tr><tr><td>Heater controller</td><td>Warner Instrument</td><td>TC-324B</td><td></td></tr><tr><td>Inline heater</td><td>Warner Instrument</td><td>SC-20</td><td></td></tr><tr><td>Peristaltic pump</td><td>Rainin</td><td>Dynamax</td><td></td></tr><tr><td>pipette puller</td><td>Sutter Instrument</td><td>P-1000</td><td></td></tr><tr><td>Glass tube with filament</td><td>King Precision Glass</td><td>Customized</td><td></td></tr><tr><td>Stimulator</td><td>A.M.P.I.</td><td>Master-8</td><td></td></tr><tr><td>Biocytin</td><td>Sigma</td><td>B4261</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S6191</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>BP328-1</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S0876</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S5011</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C5670</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M1880</td><td></td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G6152</td><td></td></tr><tr><td>K-gluconate</td><td>Sigma</td><td>G4500</td><td></td></tr><tr><td>ATP-Mg</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>Li-GTP</td><td>Sigma</td><td>G5884</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E0396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>KOH</td><td>Sigma</td><td>P5958</td><td></td></tr><tr><td>Cs-methanesulfonate</td><td>Sigma</td><td>C1426</td><td></td></tr><tr><td>CsOH</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>Syringer filter</td><td>Nalgene</td><td>171</td><td></td></tr><tr><td>1 ml syring</td><td>Rainin</td><td>17013002</td><td></td></tr><tr><td>10 &amp;mu;l pipette tip</td><td>Genesee Scientific</td><td>24-130RL</td><td></td></tr><tr><td>Streptavidin-488</td><td>ThermoFisher</td><td>S-11223</td><td></td></tr><tr><td>10x PBS</td><td>Lonza</td><td>17-517Q</td><td></td></tr></tbody></table>

patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

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。

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?...

Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

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。

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

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

patch-clamp-recording-starburst-amacrine-cells-flat-mount-preparation

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.1K ビュー.  Baylor College of Medicine. 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。 

ビデオ: Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

神経科学

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

生物学

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

パッチクランプキャパシタンス測定とCa 2 +</sup網膜スライスにおける単一神経終末で>イメージング

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

マウス網膜からERG Transretinal·レコーディング：ロッドとコーンの光応答

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

無傷の網膜におけるGFP発現細胞集団の電気生理学的特性

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

マウスの網膜神経節細胞におけるシナプスと人工コンダクタンスによるダイナミッククランプの実装

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

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.

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

マウス分裂網膜における双極性および水平神経細胞の研究

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

著者スポットライト:Split Retina技術を使用してアクセスを強化し、実験を加速する 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

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

要約

さらに動画を探す

この動画の章

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

パッチクランプキャパシタンス測定とCa^{2 +イメージング}

マウス網膜からERG Transretinal·レコーディング：ロッドとコーンの光応答

無傷の網膜におけるGFP発現細胞集団の電気生理学的特性

マウスの網膜神経節細胞におけるシナプスと人工コンダクタンスによるダイナミッククランプの実装

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

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

要約

さらに動画を探す

この動画の章

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

パッチクランプキャパシタンス測定とCa 2 +イメージング

マウス網膜からERG Transretinal·レコーディング：ロッドとコーンの光応答

無傷の網膜におけるGFP発現細胞集団の電気生理学的特性

マウスの網膜神経節細胞におけるシナプスと人工コンダクタンスによるダイナミッククランプの実装

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

パッチクランプキャパシタンス測定とCa^{2 +イメージング}