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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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+targeted+recording+of+GFP-expressing+neurons+for+light+responses+and+live-cell+imaging+in+the+mouse+retina.">Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina.</a> Nat Protoc. 5 (7), 1347-1352 (2010).</li></ol>

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への網膜のしっかり取り付けが最も重要であり、タイムリーな再水和（ステップ3.4から3.6）、続いて溶液の過渡吸収することによって達成されます。この短い期間中、通常30秒未満、硝子体が緩んでゼリーのように動作し、剥離することができます。硝子体は、手動で溶液17,19または酵素アクション18,20により除去され、いくつかの公表された方法と比較したときに我々の技術は、より効率的です。別の頻繁に使用される方法は、セルごとに空のパッチピペットを使用してオフに内境界膜を引き裂くことです21,22を記録します。私たちは、網膜のマウント解除を恐れて引き裂き方法を追求しませんでした。しかし、時間に膜から網膜を取り除くことがやや透明でゼリー状の硝子体オフを剥離します。従って、これは、ニトロセルロース膜上の網膜の保持がINLニューロンを記録するために不可欠であるとして、練習を必要とするステップです。良い習慣はよく平坦化し、完全に水和し、ニトロセルロース膜を使用することです。ここで注目すべき点は、記録可能領域との間の見かけ上のトレードオフである（ すなわち、パンチ穴の大きさ）、網膜の平坦性、および膜への付着の堅さ。より大きな記録ウィンドウが好ましいが、より大きなパンチ穴にアタッチする網膜のための少ない膜面積があることを意味します。同様に、小さな穴上に網膜を取り付けることしっかり付着を確実にするが、平坦性が低下することがある、など記録可能な領域です。 硝子体に介して電極を挿入するにはGCLとINL（ステップ4.4）は、我々は電極の詰まりを防止するために正の圧力を加えます。網膜への浸透時にこの圧力の即時の減少はまた、染色のバックグラウンドを減少させるため、発振を維持するために重要です。重要なチェックポイントは、GCLニューロンのためとINLニューロンのための60秒以内、好ましくは30秒未満、浸透とシールを取得するまでの時間を短縮することです。もう一つの重要な点は、ギガオームシールを形成した後、膜破裂する前に5分間の待機期間です。この待機期間は、電極の経路に流出内部液のクリアを確実にし、高カリウム含有量が一時的に隣接するニューロンを脱分極し、振動を乱す可能性があるため、カリウムベースの内部液を用いた場合に特に重要です。最後に、我々のアプローチ（ステップ4.7）の多少型破りな特徴は、細胞に近づく我々、ギガオームシールを形成し、その後、現在のクランプカ月の下で全細胞のアクセスを得ることですデ、私たちの最初の電気生理学的記録で私たちを助けたバージニア・コモンウェルス大学の博士ロリーMcQuiston、から助言を受けました。この方法は、ブレークインの際、迅速な抵抗変化は（オシロスコープを通して見える）電圧変化によって捕捉され、全細胞レベルでの膜電位の急激なスイングから記録されたセルを保護することができます。我々の方法の別の有用な特徴は、正に荷電した膜リン脂質を誘致するのに役立ちますし、シール形成を容易に電極先端に印加される負の電流です。 横網膜回路を保持に関しては、網膜20の水平方向にスライスした準備が示唆されています。この方法は効率的に水平方向に拡大デンドライト記録及び画像化のためにIPLでシナプス結合を公開しつつ、垂直経路が、残念ながら破壊され、したがって、この方法は、限られた目的のために使用することができます。最後に、改良が我々で実装します組織の準備と記録手順は、このようなOFF-SACの7のようなINLニューロンのルーチン目標と録音を可能にします。顕微鏡を増強二光子イメージングとコントラストを組み込むことにより、様々な照明条件下でパッチクランプ記録のために外網状層付近の双極細胞および水平細胞を標的とする現在のフラットマウント調製物で可能と考えられています。

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.

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			<td height="42" style="height:42px;width:195px;">Fixed-stage fluorescent microscope with DIC</td>
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			<td height="21" style="height:21px;width:195px;">Patch clamp amplifier</td>
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			<td height="21" style="height:21px;width:195px;">pipette puller</td>
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			<td height="21" style="height:21px;width:195px;">Glass tube with filament</td>
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			<td height="21" style="height:21px;width:195px;">NaCl</td>
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			<td height="21" style="height:21px;width:195px;">D-glucose</td>
			<td style="width:155px;">Sigma</td>
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			<td height="21" style="height:21px;width:195px;">Li-GTP</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G5884</td>
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			<td height="21" style="height:21px;width:195px;">EGTA</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">E0396</td>
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			<td height="21" style="height:21px;width:195px;">HEPES</td>
			<td style="width:155px;">Sigma</td>
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			<td height="21" style="height:21px;width:195px;">KOH</td>
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			<td height="21" style="height:21px;width:195px;">Streptavidin-488</td>
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			<td height="21" style="height:21px;width:195px;">10X PBS</td>
			<td style="width:155px;">Lonza</td>
			<td style="width:219px;">17-517Q</td>
			<td></td>
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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）を駆動するシナプス電流の振動を明らかにする。抑制性および興奮性シナプス電流は、電圧クランプ状態（ 図1B、下のトレース）の下で、それぞれ、0 mVの及び-75 mVので細胞を保持することによって明らかにされます。 IPL（ 図1C）における典型的なSAC樹状樹枝状分岐と成層のレベルは、内部透析ビオサイチンための事後ストレプトアビジン染色により可視化することができます。リズムと膜電位変動やシナプス後電流の変化の頻度缶パワースペクトル密度を計算することによって定量化すること。薬理学的遮断、またはその欠如は、膜電位振動7の根底にある別のシナプスメカニズムを識別するために使用することができます。 <img alt="図1" src="/files/ftp_upload/54608/54608fig1.jpg" /> 図1.形態とノブのマウス網膜におけるオンとオフスターバーストアマクリン細胞の膜電位。INLでGCLで（A）ON-SACの両方とOFF-SACのは容易に表現tdTomatoのCre-駆動し、記録の対象とすることにより同定しましたDICの下。示されるように、スケールバーは20μmで等しくなります。 （B）上部トレースは示されているようにオンとオフのSACから記録膜電位変化です。下のトレースは、膜電位と不整脈抑制性シナプス後電流（IPSC）の振動を駆動代表リズミカルな興奮性シナプス後電流（EPSC）です。 （Cは）記録された細胞の事後組織学的特徴付けは、IPLでの典型的なSAC樹状形態と層化のレベルを示しています。スケールバーは、示されるように、50ミクロンに等しい。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54608/54608fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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

Nanyang Technological University

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

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

NRCCパッチクランプチップ上で細胞の培養と電気生理学

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

内耳有毛細胞におけるパッチクランプ記録は、単離されたゼブラフィッシュから

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub亜ミトコンドリアの小胞膜のパッチクランプ記録を実行するための&gt; ATPアーゼ小胞の準備とテクニック

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

赤外線神経刺激のメカニズムを調査するための全細胞パッチクランプ

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

胚ゼブラフィッシュマウスナー細胞からパッチクランプ記録

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

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. ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

無傷の神経上皮におけるマウス嗅細胞の穿孔パッチクランプ記録：同定された嗅覚受容体を発現するニューロンの機能解析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

脳スライスにおける全細胞パッチクランプ記録

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...