This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.

倫理声明：動物を対象とする手順は、ウェイン州立大学の施設内動物管理使用委員会（IACUC）によって承認された。 実験ソリューションの調製<ol><li>実際の実験の前に1週間に解剖ソリューション1日を準備します。なぜなら、より低い温度16でのその強力な緩衝能の網膜解剖のためのHEPES緩衝液を使用してください。 115のNaCl、2.5のKCl、2.5のCaCl 2、1.0のMgCl 2、10 HEPES、28グルコース：（mm）を次のようにすべての化学物質を混ぜる。 NaOHでpHを7.4に調整します。 1週間に冷蔵庫までに解決してください。 </li><li>記録液は、網膜の準備17用に設計された人工脳脊髄液（aCSFの）であるエイムズ」媒体である。実験の日では、チューブ内のエイムズ「粉末（500ミリリットルソリューションの4.4グラム）を秤量。また、 炭酸水素ナトリウム（500ミリリットルソリューションの0.95グラム）を秤量し、Aと混合MES」粉末。 </li><li>実験日によってパッチクランプ記録のための細胞内ピペット溶液を準備します。 111 K-グルコン酸、1.0のCaCl 2、10 HEPES、1.1 EGTA、10のNaCl、1.0のMgCl 2、5 ATP-Mgおよび1.0 GTP-のNaを：（mm）を次のようにすべての化学物質を混ぜる。 KOHでpHを7.2に調整します。従来のシリンジフィルター付きピペット溶液を濾過する。 〜500μlのアリコートを作成し、冷凍庫またはディープフリーザーに保管してください。 </li></ol>実験の日のために2。準備<ol><li> O / N暗順応のために暗い空間に - （雄、8週齢4 C57BL / 6Jまたは類似の背景、）実験前の夜は、ケージにマウスを置く。 </li><li>解剖のための準備： <ol><li>少なくとも10分間、酸素を、ガラスビーカー中 - （200mlの100）の氷の上に置き、気泡液解剖埋める。 </li><li>網膜の準備ストレージ用暗箱の酸素化を開始します。 </li><li>再用グリースレールとプラスチックのカバースリップを準備tinalスライス標本を35mmプラスチックディッシュにそれらの各々を置き。 </li><li>チョッパー（組織スライサー）に新しいカミソリの刃を取り付けます。 </li><li>半分にフィルター膜をカット。これは、網膜スライス用です。 </li><li>解剖顕微鏡の近くに解剖ツールと転送ピペットを合わせます。各ツールは簡単に​​暗闇の中でアクセスできるように作業領域がよく組織されなければならない。 </li></ol></li><li>パッチクランプ記録のための準備： <ol><li>エイムズ&#39;粉末および蒸留水中のNaHCO 3（エイムス4.4グラムおよびNaHCO 3 0.95グラム/ 500ミリリットルのddH 2 O）に溶解する。バブル少なくとも30分間混合酸素（95％O 2および5％CO 2）エイムス「媒体記録温度レベルまで加熱しながら（〜35℃）。まだ吹き込みながらNaHCO 3でpHを7.4に調整します。 </li><li>解剖時の内部ピペット溶液を解凍する。それが解凍された後、細胞内染色のための0.01％のローダミンBを追加します。 </li></ol></li></ol> 3.網膜解剖<ol><li>暗い部屋では、二酸化炭素との二国間気胸を使用して、マウスを安楽死させる。 2の後 - 二酸化炭素を使用しての3分、マウスは意識を失うことになる。 </li><li>マウスは、もは​​やテールピンチに応答しない場合には、すぐに目を脱核、プラスチック皿中の冷たい解剖ソリューションに配置します。動物の死を確実にするために二国間気胸を実行します。すばやく暗箱中皿に目を保​​管。暗闇の中で手続きを行うために赤外線ビューアを使用してください。 注：あるいは、イソフルラン、断頭、または頸椎脱臼は安楽死のために使用することができます。安楽死の方法は、国立衛生研究所（NIH）および/または施設の動物実験委員会のガイドラインに従ってください。 </li><li>暗い部屋では、解剖顕微鏡と赤外線照明を調整します。顕微鏡下で、10cmのプラスチックシャーレWにその皿から目を移す冷却された解剖ソリューション番目。プラスチック皿の底に置かれ、チューブを介して連続的に気泡酸素。それが低すぎないように流量を調節し、それは解剖を乱すのに十分高くないことを確認してください。 </li><li>角膜および水晶体除去： <ol><li>マイクロメスで角膜の上に切開を行います。眼球のもう一方の端にある視神経を保持することは動くから目を防ぐことができます。外科ハサミを使ってカットを拡張します。その周りにいくつかの強膜で角膜を切り取る。 </li><li>細かい鉗子でレンズをつかみ、ゆっくりとレンズを引き出します。硝子レンズを外しすぎて粘着性である場合は、ハサミで硝子体をカット。 </li><li>アイカップが行われると、そっと小さなトランスファーピペットとアイカップに冷たい、酸素解剖ソリューションを注ぐ。 注：切開が角膜に行われた後、眼球を素早く収縮。これは、プロ中の目を変形として、眼の形状を維持することが重要であるcedureは、網膜を損傷し、網膜剥離の原因になります。 </li></ol></li><li>網膜の背側と腹側の識別： 注：緑、紫外線（UV）の不均一な分布は、特定の網膜の領域を識別し、18が円錐ので光応答記録のために重要である。 <ol><li>視神経乳頭近傍を通過網膜アイカップ全体に行くの行を特定します。この行は、主に視神経の腹側を通過する。他の19の腹側であるが、視神経を含む側面は、背側である。赤外線視聴者の下で明確にこのランドマークを参照してください。 </li><li>実験の目的に応じて、不要な側のカットを作る。これは、アイカップから網膜を単離した後に有用であろう。 </li></ol></li><li>硝子体除去： <ol><li>必要に応じて、15分間、ヒアルロニダーゼ（0.5mg / ml）を有するアイカップをインキュベートする。 </li><li>極細ピンセットで硝子体を除去します。硝子体は主であるボトムと目をカップの外縁に取り付ける。そっと触れたり、網膜を突っついせずにそれを削除します。硝子体がしっかりアイカップに添付さかもしれませんが、アイカップから網膜剥離を避けるために、静かに取り除く。全く緊張が感じられなくなるまで引っ張って続けます。 注：この手順は、マウスの網膜を切開するために特に重要である。しかしながら、そのようなラットまたはサンショウウオなどの他の種の網膜は問題ではない。 </li></ol></li><li>アイカップから網膜を分離します。強膜をつかみ、静かに鉗子の裏側を使用して網膜をはがす。 注：ホールマウント網膜の準備が孤立した網膜から作ることができる。縁で三から四カットを作成し、網膜を平らに、またはいくつかの部分に網膜をカット。 </li><li>網膜トリムと網膜（ ステップ3.5）の不必要な半分を捨てる。同じプラスチック皿の中で、二つに残りの網膜をカット。網膜の各部分については、作るために縁を断つ網膜のスラブ、および折りたたみエッジをトリム。 </li><li>大ホールピペット（〜2ミリリットル）を使用して、ガラス板上に網膜のスラブを転送します。小さなピペットで過剰の溶液を吸い取り、スラブを平らに濾紙を使用し、迅速に組織の上にフィルター膜の断片を置き。 <ol><li>小さなトランスファーピペットを使用して、フィルター膜上に溶液を解剖の低下を置く。液が膜に広がるまで〜15秒待ってください。この時間の間に、網膜組織は、フィルター膜に付着するであろう。 </li><li>フィルターの下や周りに、より冷たい解剖ソリューションを注ぐ。フィルター膜と網膜組織がフロートします。鉗子でフィルター膜をつかみ、スライシング室に配置します。組織上の解剖ソリューションを注ぎ、そして暗箱で準備を格納する。 注：ここで説明する手順は非常に重要です。これは、網膜組織は、フィルタMEMにこだわっていることを確認するために迅速にこれらのステップを実行することが重要ですブレーンと乾燥しない。 </li></ol></li><li>チョッパーを用いて、スライスに網膜組織を切断する。約200μmの厚さは、パッチクランプ研究のために可能である。 </li><li>スライスチャンバ内のグリースレールにプラスチック製のカバースリップを置きます。プラスチックカバースリップ上グリースレールの上に網膜スライスを転送します。横断面が見えるようにスライスを90°回転させます。押し下げてカバースリップ上にフィルタ膜が、その後、グリースをろ紙の側面を覆う。 <ol><li>スライスはプラスチック製カバースリップ上に固定化された後、カバースリップをつかみ、35ミリメートルのプラスチックの皿に移す。網膜の準備に冷たい解剖ソリューションを注ぎ、準備を水没。継続的に酸素化されるべきである暗箱で各皿を、保管してください。 注：すべての手順は、フィルター膜が変形しないように慎重に行う必要がある。そうでない場合は、網膜のスライスが簡単にフィルター膜から離れる。 </li></ol></li></ol><ol><li>録音の準備： <ol><li>首相エイムズ」媒体との灌流チューブが。チューブを充填するとき、すべての気泡が通過できるように。 </li><li>プラーでパッチクランプ記録ピペットを作る。ピペットチップが埋め戻されるまで1分間ピペットの裏側でピペット溶液、ディップピペットを充填する。次に、マイクロピペットフィラーを使用してピペットの〜1月3日を記入してください。湿ったピペットボックスの各ピペットを保管してください。 </li><li>パッチクランプ記録のための機器の電源を入れ、コンピュータ、増幅器、CCDカメラ、顕微鏡を含む。 </li></ol></li><li>室内光を遮断してください。赤外線ビューアを使用して顕微鏡ステージ室への網膜スライス標本を置きます。それが固定化された後、連続灌流を開始する。 33から37℃での灌流温度を設定します。 </li><li> CCDカメラでスライス面を表示します。ターゲットCEターゲットの場所に焦点を当てるLLタイプが存在する。 （ すなわち、それは滑らかに見える表面と良好な細胞形状を持つべき）記録パッチクランプのための健康そうなセルを選択します。 </li><li>ピペットホルダーに記録ピペットを置きます。スライス標本にピペットを進める。それはスライス標本（上〜2ミリメートル）に近い場合、顕微鏡でピペットの先端を見つける。ピペットチップは、顕微鏡下で可視になると、標的細胞に向かって下に先端を移動させる。 </li><li>アンプを設定します。 0 MV / 0 PA（ゼロ）でピペットを調整し、〜10Hzで〜5 mVのの連続電気パルスを開始します。ピペット抵抗をチェックします。理想的な抵抗はほとんどの網膜神経細胞のために、5〜10MΩです。 </li><li>先端から吹き出す開始します。マウスピースを使用するか、またはピペットは、浴溶液に浸漬する際に正圧を適用するために、注射器を使用しています。先端は、標的細胞の表面上にあるまで、連続的に内部液を吹き飛ばす。 <ol><li>正圧が目の上の小さなディンプルを行うとE細胞表面、少し先端を進め、吹き出し停止。増加するはずであるピペット抵抗を、確認してください。それが継続的に増加している場合、それを残して、それが&gt; 1GΩ（ギガシール）に達するまで抵抗を監視します。抵抗が自然に増加しない場合は、ギガシールになるまで、穏やかに負圧を加える。 </li></ol></li><li>ギガシールが達成された後、mVのを-70する保持電位を変更する。その後、断続的に、ピペットチップの内部に膜を破裂する負圧を適用する。全細胞構成が行われると、ピペット抵抗は500MΩと1GΩ、容量性電流が見られるの間とすることができる。時々自発性シナプス後電流を観察することができる。 </li><li> -80から+ 40mVまでのIV関係を記録。電位依存性チャネルの異なる種類の細胞型に依存して活性化される。 </li><li>レコード·光誘発シナプス電流または電圧。 </li></ol>

<ol>
	<li>Werblin, F. S., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+retina+of+the+mudpuppy,+Necturus+maculosus+II.+Intracellular+recording.">Organization of the retina of the mudpuppy, Necturus maculosus II. Intracellular recording.</a> J Neurophysiol. 32 (3), 339-355 (1969).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annu Rev Physiol. 46, 455-472 (1984).</li><li>Lukasiewicz, P., Werblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+slowly+inactivating+potassium+current+truncates+spike+activity+in+ganglion+cells+of+the+tiger+salamander+retina.">A slowly inactivating potassium current truncates spike activity in ganglion cells of the tiger salamander retina.</a> J Neurosci. 8 (12), 4470-4481 (1988).</li><li>Kaneko, A., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+L-glutamate+on+the+anomalous+rectifier+potassium+current+in+horizontal+cells+of+Carassius+auratus+retina.">Effects of L-glutamate on the anomalous rectifier potassium current in horizontal cells of Carassius auratus retina.</a> J Physiol. 358, 169-182 (1985).</li><li>Kamermans, M., Werblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA-mediated+positive+autofeedback+loop+controls+horizontal+cell+kinetics+in+tiger+salamander+retina.">GABA-mediated positive autofeedback loop controls horizontal cell kinetics in tiger salamander retina.</a> J Neurosci. 12 (7), 2451-2463 (1992).</li><li>Cook, P. B., McReynolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+inhibition+in+the+inner+retina+is+important+for+spatial+tuning+of+ganglion+cells.">Lateral inhibition in the inner retina is important for spatial tuning of ganglion cells.</a> Nat Neurosci. 1 (8), 714-719 (1998).</li><li>Pang, J. J., Gao, F., Wu , J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-evoked+current+responses+in+rod+bipolar+cells,+cone+depolarizing+bipolar+cells+and+AII+amacrine+cells+in+dark-adapted+mouse+retina.">Light-evoked current responses in rod bipolar cells, cone depolarizing bipolar cells and AII amacrine cells in dark-adapted mouse retina.</a> J Physiol. 558 (Pt. 558 (Pt 3), 897-912 (2004).</li><li>Euler, T., Masland , R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-evoked+responses+of+bipolar+cells+in+a+mammalian+retina).">Light-evoked responses of bipolar cells in a mammalian retina).</a> J Neurophysiol. 83 (4), 1817-1829 (2000).</li><li>Berntson, A., Taylor , W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+characteristics+and+receptive+field+widths+of+on-bipolar+cells+in+the+mouse+retina.">Response characteristics and receptive field widths of on-bipolar cells in the mouse retina.</a> J Physiol. 524 Pt. 524 (Pt 3), 879-889 (2000).</li><li>Sterling, P., Smith, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+for+a+binary+synapse.">Design for a binary synapse.</a> Neuron. 41 (3), 313-315 (2004).</li><li>Borghuis, B. G., Marvin, J. S., Looger, L. L., Demb, J. 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+imaging+of+nonlinear+glutamate+release+dynamics+at+bipolar+cell+synapses+in+the+mouse+retina.">Two-photon imaging of nonlinear glutamate release dynamics at bipolar cell synapses in the mouse retina.</a> J Neurosci. 33 (27), 10972-10985 (2013).</li><li>Dowling, J. E., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+retinal+dystrophy+in+the+rat.">Inherited retinal dystrophy in the rat.</a> J Cell Biol. 14, 73-109 (1962).</li><li>Eggers, E. D., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(A),+GABA(C)+and+glycine+receptor-mediated+inhibition+differentially+affects+light-evoked+signalling+from+mouse+retinal+rod+bipolar+cells.">GABA(A), GABA(C) and glycine receptor-mediated inhibition differentially affects light-evoked signalling from mouse retinal rod bipolar cells.</a> J Physiol. 572 (Pt 1), 215-225 (2006).</li><li>Ichinose, T., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mode+of+retinal+presynaptic+inhibition+switches+with+light+intensity).">The mode of retinal presynaptic inhibition switches with light intensity).</a> J Neurosci. 32 (13), 4360-4371 (2012).</li><li>Ichinose, T., Fyk-Kolodziej, B., Cohn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+ON+cone+bipolar+cell+subtypes+in+temporal+coding+in+the+mouse+retina.">Roles of ON cone bipolar cell subtypes in temporal coding in the mouse retina.</a> J Neurosci. 34 (26), 8761-8771 (2014).</li><li>Baicu, S. C., Taylor, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base+buffering+in+organ+preservation+solutions+as+a+function+of+temperature:+new+parameters+for+comparing+buffer+capacity+and+efficiency.">Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency.</a> Cryobiology. 45 (1), 33-48 (2002).</li><li>Ames, A., Nesbett, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+retina+as+an+experimental+model+of+the+central+nervous+system.">In vitro retina as an experimental model of the central nervous system.</a> J Neurochem. 37 (4), 867-877 (1981).</li><li>Haverkamp, S., 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+primordial,+blue-cone+color+system+of+the+mouse+retina.">The primordial, blue-cone color system of the mouse retina.</a> J Neurosci. 25 (22), 5438-5445 (2005).</li><li>Wei, W., Elstrott, J., Feller, M. 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><li>Farre, C. G., M Bruggemann, A., Fertig, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+screening+-+automated+patch+clamp+on+the+rise.">Ion channel screening - automated patch clamp on the rise.</a> Drug discovery today. Technologies. 5 (1), e1-e34 (2008).</li></ol>

The authors have nothing to disclose.

Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings. 
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the ...

The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms. 
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6. 
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mice (28-60 days old, male)</td>
			<td style="width:145px;">Jackson laboratory</td>
			<td style="width:203px;">C57BL/6J strain</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; medium powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:203px;">A1420</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:145px;">Nikon</td>
			<td style="width:203px;">SMZ745</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:203px;">#4, #5, #55</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_scissors</td>
			<td style="width:145px;">Roboz</td>
			<td style="width:203px;">RS-5605</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_surgery knife</td>
			<td style="width:145px;">Surgistar</td>
			<td style="width:203px;">7514</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">razor blade (for chopper)</td>
			<td style="width:145px;">EMS</td>
			<td style="width:203px;">71970</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chopper</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">infrared viewer</td>
			<td style="width:145px;">Night Owl Optics</td>
			<td style="width:203px;">NOBG1</td>
			<td>It shows bright view.&#160; Focusing small objects is an issue.</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">infrared pocket scopes</td>
			<td style="width:145px;">B.E. Meyers</td>
			<td style="width:203px;">OWL Gen 3 NV pocketscope</td>
			<td>excellent view</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puller</td>
			<td style="width:145px;">Sutter</td>
			<td style="width:203px;">P-1000</td>
			<td>excellent.&#160; Make consistent size pipettes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dark box</td>
			<td style="width:145px;">Pelican</td>
			<td style="width:203px;">dark box</td>
			<td>excellent</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">patch clamp system</td>
			<td style="width:145px;">Scientifica</td>
			<td style="width:203px;">slice scope 2000</td>
			<td style="width:323px;">Excellent setup.&#160; Most key components are included in one package.&#160; Micromanipulators are excellent.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amplifier</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">multiclamp 700B</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acquiring software</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">pClamp software</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:216px;">light source (LED)</td>
			<td style="width:145px;">Cool LED</td>
			<td style="width:203px;">pE-2 4 channel system</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD camera</td>
			<td style="width:145px;">Q-imaging</td>
			<td style="width:203px;">Retiga 2000</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques

網膜は視覚系へのゲートウェイである。視覚信号処理メカニズムを理解するために、網膜の神経回路網の機能を調査する。ネットワーク内の網膜神経細胞は、多数のサブタイプが含まれる。双極細胞、神経節細胞、およびアマクリン細胞の10以上のサブタイプは、形態学的研究によって確認されている。網膜ニューロンの複数のサブタイプは、そのような動きや色などの視覚的シグナルの明確な特徴をコードする、複数の神経経路を形成すると考えられる。しかし、視覚的な信号処理における各ニューロンの機能的役割は十分に理解されていない。パッチクランプ法は、この根本的な問題に対処するのに便利です。ここでは、暗順応状態でパッチクランプ記録を用いて、マウスの網膜神経細胞の光誘発性シナプス応答を記録するためのプロトコルが提供される。マウスの眼には、O / N暗順応であり、網膜スライス標本は、赤外線照明と視聴者を使用して、暗い部屋で解剖されている。赤外光にはありませんマウスの光受容体を活性化し、したがって、それらの光反応性を保持します。パッチクランプは、網膜神経細胞の光誘発性の応答を記録するために使用される。蛍光色素は、ニューロンの形態学的サブタイプを特徴付けるための録音中に注入される。この手順では、マウスの網膜に各ニューロンの生理的機能を決定することが可能になります。

代表的なスライス標本は、スライス標本が平坦面に神経節細胞の光受容体を示す、真っ直ぐな位置にある。 図1に示されず、濾紙からの剥離される。スライスが傾いている場合、製剤の唯一の部分は、それが困難なパッチクランプのための適切な細胞を同定することを可能にする焦点である。記録のために、それは通常、光沢のある表面を有して良い相馬、さえ丸い形状、目に見...

Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

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

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. ...

recording-light-evoked-postsynaptic-responses-neurons-dark-adapted

Research

JoVE Journal

Neuroscience

8.3K ビュー.  Wayne State University School of Medicine. 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.

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

空気相Electroolfactogram記録を用いたマウス嗅覚神経細胞の分析応答

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

神経科学

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 ...

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 ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

遺伝的にコードされたカルシウムセンサーを発現する鋤鼻器のスライス標本のイメージング神経細胞応答

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 ...

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

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

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 ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

スライス パッチ ・ クランプの技術学習によって誘発される可塑性を分析するため

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...