この作品は、NIHの助成金のEY019312とEY021126（VJK）、ワシントン大学の眼科学部およびVisual科学へEY002687でサポートされていた、と研究によって失明を防ぐために。

全ての実験プロトコルは、実験動物の管理と使用に関する指針に従ったとワシントン大学の制度的動物研究委員会によって承認されました。 1.灌流および試料ホルダーの設定<ol><li>実験の日に新鮮な網膜灌流のための溶液を調製します。蒸留脱イオン水を使用してください。次の三つの溶液のいずれかを使用します。 <ol><li>重炭酸塩含有エイムズ」溶液（1 L）を：エイムスの1ボトル」の準備メディア、そしてNaHCO 3 1.9gの、 </li><li> （mm表示）ロックの溶液を調製する：112.5のNaCl、3.6のKCl、2.4のMgCl 2、1.2のCaCl 2、10.0 HEPES、20.0のNaHCO 3、3.0コハク酸ナトリウム、0.5のNa、グルタミン酸0.02 EDTA、及び10.0グルコース、0.1％のMEMビタミンとアミノ酸酸</li><li> （mm表示）HEPES緩衝リンゲル液を準備します。133.3のNaCl、3.3のKCl、2.0のMgCl 2、1.0のCaCl 2、10.0グルコース、0.01 EDTA、12.0 HEPES、pH値1 MのNaOH〜5.8ミリリットルを7.5に調整し、0.72グラム/ Lリーボビッツ培地のL-15を追加します。任意の灌流媒体と感光体の応答を分離するために、100μMのBaCl 2 - 50μMのDL-AP4、50 - 20を使用してください。 </li></ol></li><li> （mm表示）電極28用の溶液を調製：140.0のNaCl、3.6のKCl、2.4のMgCl 2、1.2のCaCl 2、3 HEPES、0.01 EDTAおよび7.4にpHを調整- NaOHで7.5。電極溶液は、数ヶ月間、室温で保存することができます。 </li><li>試料ホルダーを準備し、テストします。 <ol><li>試料ホルダーの底部のドームの上に黒/グレーの濾紙を接着（ 図1A参照 ）。ドームの平坦な頂部の縁の周りに慎重に二成分5分エポキシ接着剤を広げます。必要に応じて、解剖顕微鏡下でそれを行います。 </li><li>接着剤がほぼ乾燥するまで待ちます（約4分）、フラットなアイテムを使用して、ドームのろ紙を押してください。糊ろ紙実験前少なくとも1日。ザ濾紙は、複数回使用することができるが、記録の月後に交換されるべきです。交換用の紙をインストールする前に、糊残りから慎重にドームをきれいにするために70％エタノールを使用してください。 </li><li> （ 図1AおよびBを参照）、任意の気泡や電極チャネルにねじアダプタで囲まれたスクリューペレット電極を回避しようとしている電極溶液で電極チャネルを埋めます。 4本のネジで試料ホルダーの上下の部分を接続し、電極溶液で灌流ラインを埋めます。 </li><li>マルチメータ（ 図1B）を用いて、各電極対のリード線間の抵抗と電圧を測定します。チャネルは無気泡であり、電極が良好な状態にある場合、抵抗は100Ωkと10 mVの下の電圧以下である必要があります。 </li></ol></li><li>ガラス瓶に灌流培地の700ミリリットル - 400を注ぎます。別の300mlの網膜およびsの解剖において使用するために別個の冷蔵庫の中にそれを引き裂きました。 （ 図1Dを参照）、熱交換器ブロックで灌流チューブをセットし、加熱プレート上で予備加熱されたブロックを配置します。 </li><li> （ 図1Dを参照）接続（エイムズ」やロックのが使用されている場合）2 / O 2ガスをCOおよび灌流チューブ用するを持っているキャップでボトルを囲みます。 37°Cのにメディアでボトルを予熱し、加熱板上または37に設定した水浴中の光刺激装置の上に置きます- 39°Cの17。 <ol><li>プライム重力駆動流を開始するために灌流培地でそれらを充填することにより、灌流ラインが。 <ol><li> LKCシステムでは、その後の実験中にボトル内の灌流液の低下レベルによって影響を受けることなく、流量調節器によって調節される重要な重力流を提供するために、刺激器ユニット17の上にボトルを置きます。 </li><li> Ocuscienceシステムでは、Pを配置溶液の重力ドライブを増加させるために十分に試料ホルダー（および灌流瓶）のレベル以下（ステップ2.8を参照）、ノイズを最小限に抑え、試料ホルダから長い灌流出力線を配置するために、ファラデーケージ内のerfusionボトル。これらの出力線を遮蔽し、ERG信号への電磁ノイズの結合を防止するためにアンプのアースにシールドを接続します。 </li></ol></li></ol></li><li>流量調節器を用いて5ml /分 - 3に灌流を調整します。適切な調節因子でシリンダーから5％CO 2/95％O 2ガスを接続し、エアストーンを通してボトル内のメディアの安定バブリングを確実にするために流量を調整します。 </li></ol> 2.サンプル調製<ol><li>ストレート刃microscissorsは、1つまたは2つの45 Oピンセット、かみそりの刃と濾紙の長方形の一枚など、クリーンでシャープな解剖器具を組み立てます。 </li><li>冷たいperfuの約200mlを注ぎ大きいペトリ皿中のシオン溶液（ドームを含む）を試料ホルダーの底部全体を溶液に浸漬することができるように。ドームの網膜をマウントする場合、このステップは重要となる（ステップ2.6を参照してください）​​。いくつかのソリューションは、カルボゲン（5％CO 2/95％O 2）で飽和するように設計されているが、これは、切開のために必須ではなく、ここに記載した実験で行われていませんでした。 </li><li>典型的な実験では、明/暗サイクル12/12時間で動物を維持し、6のためにそれらを暗適応させる - レコーディングの前に12時間。 （顕微鏡光源の前に、例えば、赤色フィルタを使用）暗赤色灯の下で頸椎脱臼に続いてCO 2吸入により動物を安楽死させると、暗赤色またはIR光の下で、次のすべての手順を実行します。 <ol><li>ピンセットを使用して目を引いて、メディアに入れて。紙の小片（ 例えば、いくつかの定期的なフィルターペーパー）で一度に一つの目を配置したようにします（これはここでは、溶液の外で行われます）ピンセットで目を押さえながら鋸状縁のレベルにほぼ点灯。 </li></ol></li><li> microscissorsで鋸状縁（または目の赤道に近い）に沿って切断し、角膜とレンズを取り外してください。大型シャーレに冷たいメディアにアイカップを置き、他の目で同じ手順を繰り返します。 </li><li>可能な限り無傷のように網膜を維持するために、網膜と強膜の間にはさみを保持することにより、視神経に向かってアイカップの上から小さな切開をカット。グリップ2ピンセットを使用し、網膜をデタッチ互いに離れるピンセットを引くことにより、切開の両側から強膜。 <ol><li>視神経を切断し、遠位表面への物理的な接触の最小量で網膜を分離してみてください。 RPEは、主に解剖プロセスの間に、網膜から自動的に切り離します。これは、実行するよりも、網膜の機械的な妨害を回避することが重要です切開はすぐに一般網膜応答特性に大きな影響を与えることなく、溶液中で少なくとも30分間インキュベートすることができます。 </li></ol></li><li>試料ホルダー（ 図1C）のドームの網膜をマウントします。切開した網膜でペトリ皿内の試料ホルダーの底部を浸します。ドームの上に、上向きに網膜、光受容体の側（分離された網膜の凸面）をスライドさせ、網膜が濾紙上に付着するように試料ホルダーを持ち上げます。他の網膜のための手順を繰り返します。 </li><li>電気網膜間のクロストークと同様にノイズと信号シャントを防ぐために慎重にホルダープレートを乾燥させます。試料ホルダーは、ホルダーの底部と上部の部分の間の溶液が流出することを防止するだけでなく、個々の網膜の光受容体と神経節細胞の辺の電気的分離を助けるために底部のドームの周りにOリングを持っています。 （4本のネジでホルダーの上部を取り付け図1Bを参照）、注射器と針を用いて灌流溶液で灌流チャネルを満たします。 </li><li>熱交換器ブロックの隣に試料ホルダーを移し、試料ホルダ（ 図1D）の入出力灌流ラインを接続します。 ERG増幅器に電極を接続します（上部電極は、アンプに地面/マイナス端子に接続）とアダプタを使用して、試料ホルダーの刺激/コントロールユニットを接続したり、生体内でどのに応じて刺激ユニットに加熱パッドをスライドさせERGシステムが使用されます。 </li></ol> 3.レコーディング<ol><li> in vivoでのシステムのソフトウェアを使用することにより、アンプと刺激の設定を行います。 300 Hzにフィルタリングを1と10 kHzの低域の間の値に取得頻度を設定します。ハイパスフィルタを使用しないでください。必要に応じてフィルタをノッチ60ヘルツ（またはヨーロッパでは50 Hz）を使用してください。 </li><li>光刺激とウィットのないレコードのベースラインH薄暗い刺激（ 例えば 、-35 dB以上0.3 MCDのSM -2の緑色光）は、電極と網膜の試料との間の良好な電気的接続をテストします。網膜の温度と機能が安定した状態に達するように、データ収集を開始する前に20分 - 10を待ちます。 <ol><li>特定の実験によれば、刺激パラメータを選択し、データ収集を開始します。 例えば、千のMCD SMまで0デシベルまでまたは0.1から-40から緑色の光を使用する-2暗所視応答家族のために。 （ 図3を参照して、約1.3のCd SM -2 = 1デシベル） - （30 CDM -2 3）またはプローブフラッシュロッドが背景光によって抑制されなければならない明所録音で約2〜3対数単位明るく点滅を使用してください。 </li><li>しかし、正確な光強度値は、システム·キャリブレーションおよび実験条件に幾分依存する可能性があることを覚えておいてください。経験則として、スケール強度ダウン5による- 10倍の生体内と比較して、 </eM&gt;レコーディング緑色光17を使用して。 （市販アダプタシステムに含まれる）網膜上記開口を有するブラックカバーが試料ホルダに光散乱を減少させ、商業インビボ系の全体野球の両方によって網膜の均質と同じ刺激を容易にするために使用することができます。 注：暗順応網膜から記録のために、典型的な実験の漂白剤、顔料のごくわずかな割合で使用したテスト点滅の強度がするようにRPE主導の再生の欠如は問題ではありません。また、以前にコーン色素再生がまだミュラー細胞の視覚サイクル20を介して、単離された網膜中で行うことができることが示されています。 </li></ol></li><li>実験は数時間に30分まで、典型的持続として、これらの変化は、技術的な問題を示している可能性があるため、実験中ベースラインドリフト、ノイズレベルと応答性を監視します。 注：ノイズの増加または減少D応答振幅は、空気灌流または電極のチャネル内の気泡、低すぎる、または高温、液漏れ/流出または網膜の変位を示すことができます。 </li></ol> 4.クリーニング<ol><li>灌流ラインから試料ホルダーを外し、それを開いて、ろ紙から網膜を洗い流します。電極を取り外し、蒸留水（エタノールを使用していない）でそれらをすすぎます。エタノールおよび/または蒸留水で（灌流チャネルを含む）試料ホルダーを清掃してください。 &gt; 70％エタノールで慎重に同一平面灌流チューブ。 </li><li> （洗剤は使用しないでください）​​蒸留水で灌流ボトルを洗浄します。エタノールはまた、ボトルを洗浄するために使用することができます。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barium+suppresses+slow+PIII+in+perfused+bullfrog+retina.">Barium suppresses slow PIII in perfused bullfrog retina.</a> Vision Res. 19, 1117-1119 (1979).</li><li>Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+conductance+block+by+barium+in+amphibian+Muller+cells.">Potassium conductance block by barium in amphibian Muller cells.</a> Brain Res. 498, 308-314 (1989).</li><li>Oakley, B., Katz, B. J., Xu, Z., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+buffering+of+extracellular+potassium+by+Muller+(glial)+cells+in+the+toad+retina.">Spatial buffering of extracellular potassium by Muller (glial) cells in the toad retina.</a> Exp. Eye Res. 55, 539-550 (1992).</li><li>Nymark, S., Haldin, C., Tenhu, H., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+measuring+free+drug+concentration:+retinal+tissue+as+a+biosensor.">A new method for measuring free drug concentration: retinal tissue as a biosensor.</a> Invest. Ophthalmol. Vis. Sci. 47, 2583-2588 (2006).</li><li>Walter, P., Luke, C., Sickel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+and+light+responses+in+superfused+bovine+retina.">Antibiotics and light responses in superfused bovine retina.</a> Cell. Mol. Neurobiol. 19, 87-92 (1999).</li><li>Luke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+profile+of+alkylphosphocholines+in+the+model+of+the+isolated+perfused+vertebrate+retina.">The safety profile of alkylphosphocholines in the model of the isolated perfused vertebrate retina.</a> Graefes Arch. Clin. Exp. Ophthalmol. 248, 511-518 (2010).</li><li>Januschowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+toxicity+testing+of+VEGF+Trap-Eye+in+an+isolated+perfused+vertebrate+retina+organ+culture+model.">Electrophysiological toxicity testing of VEGF Trap-Eye in an isolated perfused vertebrate retina organ culture model.</a> Acta Ophthalmol. 92, e305-e311 (2014).</li><li>Kolesnikov, A. V., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transretinal+ERG+recordings+from+mouse+retina:+rod+and+cone+photoresponses.">Transretinal ERG recordings from mouse retina: rod and cone photoresponses.</a> J Vis Exp. , (2012).</li><li>Koskelainen, A., Hemila, S., Donner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+sensitivities+of+short-+and+long-wavelength+sensitive+cone+mechanisms+in+the+frog+retina.">Spectral sensitivities of short- and long-wavelength sensitive cone mechanisms in the frog retina.</a> Acta Physiol. Scand. 152, 115-124 (1994).</li><li>Lyubarsky, A. L., Falsini, B., Pennesi, M. E., Valentini, P., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-+and+midwave-sensitive+cone-driven+retinal+responses+of+the+mouse:+a+possible+phenotype+for+coexpression+of+cone+photopigments.">UV- and midwave-sensitive cone-driven retinal responses of the mouse: a possible phenotype for coexpression of cone photopigments.</a> J. Neurosci. 19, 442-455 (1999).</li><li>Lyubarsky, A. L., Daniele, L. L., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+candelas+to+photoisomerizations+in+the+mouse+eye+by+rhodopsin+bleaching+in+situ+and+the+light-rearing+dependence+of+the+major+components+of+the+mouse+ERG.">From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG.</a> Vision Res. 44, 3235-3251 (2004).</li><li>Azevedo, A. W., Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+protocols+alter+phototransduction:+the+implications+for+retinal+processing+at+visual+threshold.">Experimental protocols alter phototransduction: the implications for retinal processing at visual threshold.</a> J. Neurosci. 31, 3670-3682 (2011).</li><li>Carter-Dawson, L. D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods+and+cones+in+the+mouse+retina.+I.+Structural+analysis+using+light+and+electron+microscopy.">Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy.</a> J. Comp. Neurol. 188, 245-262 (1979).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+vertebrate+photoreceptors.">Adaptation in vertebrate photoreceptors.</a> Physiol. Rev. 81, 117-151 (2001).</li><li>Calvert, P. D., Strissel, K. J., Schiesser, W. E., Pugh, E. N., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-driven+translocation+of+signaling+proteins+in+vertebrate+photoreceptors.">Light-driven translocation of signaling proteins in vertebrate photoreceptors.</a> Trends Cell Biol. 16, 560-568 (2006).</li><li>Schneeweis, D. M., Schnapf, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+photovoltage+of+macaque+cone+photoreceptors:+adaptation,+noise,+and+kinetics.">The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics.</a> J. Neurosci. 19, 1203-1216 (1999).</li><li>Heikkinen, H., Vinberg, F., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesopic+background+lights+enhance+dark-adapted+cone+ERG+flash+responses+in+the+intact+mouse+retina:+a+possible+role+for+gap+junctional+decoupling.">Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.</a> J. Neurophysiol. 105, 2309-2318 (2011).</li><li>Gouras, P., MacKay, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+in+amplitude+of+the+human+cone+electroretinogram+with+light+adaptation.">Growth in amplitude of the human cone electroretinogram with light adaptation.</a> Invest. Ophthalmol. Vis. Sci. 30, 625-630 (1989).</li><li>Peachey, N. S., Goto, Y., al-Ubaidi, M. R., Naash, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+mouse+cone-mediated+electroretinogram+during+light+adaptation.">Properties of the mouse cone-mediated electroretinogram during light adaptation.</a> Neurosci. Lett. 162, 9-11 (1993).</li></ol>

セントルイスのワシントン大学はXenotec社とのライセンス契約を締結しているし、ex vivoで 、アダプタの販売からロイヤリティを受け取ることができます。

<html>ここではex vivoで ERGアダプタと一緒にin vivoでの ERGシステムのコンポーネントを使用して、2つの単離されたマウス網膜から同時に高品質のex vivoでの ERG記録を得るための重要なステップを示しています。本研究では、同じ溶液（いずれかエイムズ&#39;、ロックのあるいはリンゲル）と動物の両方からの網膜を灌流が、薬物検査のために、別のソリューションの...

電図（ERG）は、光によって誘発網膜の電気的活動を記録するために使用することができ、十分に確立された技術です。 ERG信号は、網膜の抵抗性の細胞外空間に流れる（感光体と双極細胞の軸に沿って）半径方向の電流による電圧変化によって主に生成されます。最初のERG信号はフィッシュアイ1の表面からホルムグレンによって1865年に記録しました。アイントホーフェンとジョリー1908 2は、双極細胞ON、今主に光受容体の活性を反映することが知られている3つの異なるA-と呼ばれる波、B-、およびc波、への光の発症にERG応答を分割し、色素上皮細胞、それぞれ3-8。 ERGは、単離された眼用調製物9から、単離された無傷の網膜（ エキソビボ ）3,10-15間または微小電極を有する特定の網膜層にわたって、（ インビボ ）麻酔した動物またはヒトの目から記録することができます（ローカルERG）4,16。これらの中で、in vivoでの ERGは、現在、網膜機能を評価するために最も広く用いられている方法です。これは、診断目的のために使用することができ、あるいは、動物または患者における網膜疾患の進行を追跡するために非侵襲的な技術です。しかし、in vivoでの ERG記録は、多くの場合、外眼生理学的ノイズ（ 例えば、呼吸や心臓活動）によって汚染された、いくつかの重複する構成要素との複雑な信号を生成します。 ローカルERGは網膜の特定の層を横切って信号を記録するために使用することができるが、それは、最も侵襲性であり、他のERG記録の構成と比較して最も低い信号対雑音比（SNR）を有しています。ローカルERGも技術的に厳しいですし、高価な機器（ 例えば、顕微鏡とマイクロマニピュレータ）が必要です。無傷の、単離された網膜からTransretinal ERG（ex vivoでの ERG）は、インビボで安定した可能ローカルERG方法とHIGとの間の妥協を提供しています動物又はヒト17の無傷の網膜からのH SNRの録音。最近では、この方法は、哺乳動物、霊長類およびヒト網膜18-20に桿体および錐体光受容体の機能を研究するために首尾よく使用されています。また、原因エクスビボ網膜色素上皮が存在しないために、ERG信号の正のC波成分が除去され、顕著な負の遅いPIII成分は、ex vivoでの記録で明らかにされています。遅いPIII成分は網膜21-23におけるミュラーグリア細胞の活性に由来することが示されています。従って、 エキソビボ ERG方法も、無傷網膜におけるミュラー細胞を研究するために使用することができます。いくつかの研究はまた、 エクスビボで ERG記録が網膜24の周りの薬剤の濃度を測定し、薬物25-27の安全性及び有効性を試験するために使用され得ることを示しました。 複数の商業的なインビボ系が入手可能であり、必ずしも大規模な電気生理学的背景を持っていない多くの研究室で使用されます。これとは対照的に、ex vivoでのデバイスは、最近17まで利用されていないと、結果としてごく少数の研究室では、現在、この強力な技術を活用しています。これは、網膜の生理学および病理学についての知識を進めるために、および疾患を失明するための新しい治療法を開発するために、より多くの研究室へのex vivoでの ERG記録を利用できるようにすることが有益であろう。ここでは、単純で手頃な価格のエキソビボ ERG装置17を示し、それは棒状とコーン媒介シグナルを記録するために、いくつかの市販のin vivoでの ERGシステムと組み合わせて使用することができる方法を示す（A及びB波）と機能無傷の野生型マウスの網膜からミュラー細胞（PIIIを遅らせます）。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>In vivo ERG system</td>
			<td>OcuScience</td>
			<td>HMsERG</td>
			<td><a href="http://www.ocuscience.us/id77.html">www.ocuscience.us/id77.html</a></td>
		</tr>
		<tr>
			<td>In vivo ERG system</td>
			<td>LKC Technologies</td>
			<td>UTAS-E 3000</td>
			<td><a href="http://www.lkc.com/products/UTAS/bigshot.html">www.lkc.com/products/UTAS/bigshot.html</a></td>
		</tr>
		<tr>
			<td>Ex vivo adapter</td>
			<td>OcuScience</td>
			<td>Ex VIVO ERG adapter</td>
			<td><a href="http://www.ocuscience.us/id107.html">www.ocuscience.us/id107.html</a></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>North Central Instruments</td>
			<td>Leica M80</td>
			<td><a href="http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;idCategory=27">May use any brand</a></td>
		</tr>
		<tr>
			<td>IR emitter</td>
			<td>Opto Diode Corp.</td>
			<td>OD-50L</td>
			<td><a href="http://www.optodiode.com/">www.optodiode.com</a></td>
		</tr>
		<tr>
			<td>Prowler Night Vision Scopes</td>
			<td>B.E. Meyers Electro Optics</td>
			<td>D4300-I</td>
			<td>Military grade product.</td>
		</tr>
		<tr>
			<td>Red filter</td>
			<td>Rosco Laboratories</td>
			<td>Roscolux #27 Medium Red</td>
			<td>May be used instead of IR system</td>
		</tr>
		<tr>
			<td>Red head light</td>
			<td>OcuScience</td>
			<td>ERGX011</td>
			<td><a href="http://www.ocuscience.us/catalog/i29.html">www.ocuscience.us/catalog/i29.html</a></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>WPI, Inc.</td>
			<td>500086</td>
			<td><a href="http://www.wpiinc.com/">www.wpiinc.com/</a></td>
		</tr>
		<tr>
			<td>Dumont tweezers #5</td>
			<td>WPI, Inc.</td>
			<td>14101</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com</a></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Metler Toledo</td>
			<td>AB54-S/FACT</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>pH meter and electrode</td>
			<td>Beckman Coulter</td>
			<td>pHI 350</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>60129</td>
			<td><a href="http://www.sigmaaldrich.com/">May use any brand</a></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>63020</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>1.0 M solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>B0750</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris Bioscience</td>
			<td>101</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Succinic acid disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>224731</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>L-Glutamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G2834</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Leibovitz culture medium L-15</td>
			<td>Sigma-Aldrich</td>
			<td>L4386</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>MEM vitamins</td>
			<td>Sigma-Aldrich</td>
			<td>M6895</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M5550</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>UN3156</td>
			<td>5% CO2</td>
		</tr>
	</tbody>
</table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

我々は、実験プロトコル上述の異なる標準灌流液（ 図2）を用いて、 図1に示しに従って、暗順応の野生型（WT）C57BL / 6マウスの網膜からフラッシュ応答を記録しました。応答波形と速度だけでなく、桿体の感度はエイムスの類似しているように見えた」とロックのメディア（ 図2AおよびB）。一方、HEPES緩衝リンガー液（重炭酸塩なしまたは5?...

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

9.1K ビュー.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

ビデオ: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神経科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

全体の開発、 ショウジョウバエの脳のex vivoで培養

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

ex vivoでマウス神経上皮における単一細胞分裂のライブイメージング

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

皮膚における単一神経終末のin vivo二光子顕微鏡で

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

ポリリボソーム画によって中枢神経系Translatomeの生体尋問中

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

無傷の鋤鼻器官と副嗅球のex vivoでの準備

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同時2光子<em&gt;インビボ</emマウス脳梁膨大後部皮質におけるシナプス入力とシナプス後ターゲットの&gt;イメージング

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

脳スライスにおける恐怖回路のex vivo光遺伝学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...