NIH P40 OD010952、Korein財団、SLCとATKのメイタグフェローシップへマイアミフェローシップ大学によって資金を供給。作者は感謝アメフラシのための国家資源のスタッフを認めるだけでなく、図のための顕微鏡写真を提供したローレンSimonitisとハンナペック、。

1。細胞調製<ol><li> 1日目に、動物の重さと麻酔。 <ol><li> 30グラム、1キロの動物を計量。等張のMgCl 1:1海水の5-10動物ボリュームで麻酔。このような添付airstone付き電動アクアリウムエアーポンプとしてエアレーションと1時間6H 2 0。 </li></ol></li><li>解剖の供給を準備します。 <ol><li>このような生地のピンとしてステンレス鋼ストレートピン、クリーンな解剖トレイを組み立てます。 +ペニシリン/ストレプトマイシン（P / S）、（ 表1、表3を参照ASW）人工海水を含むいくつかのペトリ皿を組み立てます。 </li><li>準備ができて低電力顕微鏡を持っている。きれいな解剖器具などリブ鼻や細かい手術鉗子として、準備、および罰金と大手術ハサミを持っている。使用前に70％isopropolアルコールで楽器をスプレーし、乾燥させます。 </li></ol></li><li>解剖トレイ上の位置動物。 <ol><li>解剖トレイの位置動物腹側を下に。ピン動物体壁とparapodial国境が、背側表面を露出させる、尾と頭を避け、性器溝の縁を通じ</li><li>実行速度の遅い冷たい水道水で背面をすすぐ。性器の溝に沿って、頭の上にスクイーズボトルから70％isopropolアルコールの光のストリームを適用します。 </li></ol></li><li>最初の切開を行います。 <ol><li>シャローを切り取るながら低消費電力顕微鏡を用いた（ 表2を参照）、性器溝が前方シェルマージン由来する点を観察する反射光を、張った状態、リブ·鼻鉗子この原点の左に組織の倍とホールドちょうど良い角度のハサミで性器の溝の右側に体壁の円滑な、平坦な領域を介してすべての道を穴。 </li><li>血リンパは、時にはそれと腹部神経節と胃を運んで、穴から注ぐことが観察されるべきである。 </li></ol></li><li>切開を展開します。 <ol><li>目を拡大し、大きなハサミを使用して腸をニッキング避けるために下刃を上向きに引っ張りながらrhinophores間のポイントに前方に電子切開、。内臓は、切開の外にこぼれることが観察されます。 </li></ol></li><li>神経を取り除きます。 <ol><li>トレイの位置を変更し、頭部領域に顕微鏡再び焦点を合わせる。 </li><li>クリーン鉗子と細かいハサミを使用して、ある時点でのグループとして胸膜ペダルと大脳基底核を除去するために食道の周りにリングに頭部神経節を形成する神経を切断することによって頭部神経節を削除します。 </li><li>食道の腹側に付着した頬神経節を削除します。 </li><li>この神経節から4大神経接続詞その問題を切断することによって腹部神経節を削除します。 </li></ol></li><li>興味のある神経を分離します。 <ol><li>神経節の少なくとも直径に等しい関心の各神経節に付着結合子の長さを残して、離れて頭部神経節をトリム、これは酵素遅らせますオーバー消化次のステップで目的の細胞の。 </li><li>清潔なピンセットでリンスの間を移動する、ASWの2リンス+ P / Sを各ターゲット神経節を置く。 </li><li> PVCの細胞を標的とする場合は、右のペダルhemiganglionに装着右胸膜hemi​​ganglionを残しており、同様に左ペダルhemiganglionに装着左胸膜hemi​​ganglionを残す。 </li><li>不要な神経と受け入れ研究実践によると、動物の残りの部分を破棄します。 </li></ol></li><li>酵素的に神経を消化。 <ol><li>各神経節の場合は、3.75 mgのディスパーゼ、1mgのヒアルロニダーゼ、15ミリリットルのポリプロピレン製コニカルチューブにASW + P / Sの1ml当たり0.3グラムコラゲナーゼタイプXIから成る酵素溶液1mlを準備します。 </li><li>場所は、酵素溶液中の神経をすすぎ、しっかりとキャップを取り付けてください。約23℃（室温）で一晩13-5時間低速でロータリーシェーカー（ 表2参照）にその側にチューブを置きます。 </li></ol></li><li>準備をする培養皿。 <ol><li>顕微解剖（2日目）の前に、組織培養フード内でポリ-D-リジン（PDL）でコーティングされた培養皿を準備します。 〜25分間35mmディッシュの中央に〜0.5ミリリットルPDLを（滅菌水の0.2 mg / ml）で追加します。 </li><li>滅菌水で3回PDLをすすぐ。乾燥することができます。プレートUV-殺菌。 </li></ol></li><li> 2日目は、ニューロンを分離します。 <ol><li> PDLでコーティングされ、それ以外の場合は乾いたシャーレ内にメディアの島を作成するASW + P / Sの約0.5mlの滅菌UVだった35ミリメートル皿の中心を埋める。これは、組織培養フードの外側に、ベンチで行うことができる。一皿はhemiganglion由来する各セルクラスタのために準備されるべきである。 </li><li> SYLGARDコーティングによって作ら準備が直径100mmの解剖皿を持ち、ピンとプローブとして使用するいくつかのサイズの微細な解剖ピンを装備5ミリメートル深い解剖表面を、（ 表3参照）硬化。 ASW + P / S、そして最後Fi付き、その後、70％isopropolアルコールでこの料理をすすぐASWとのそれのll + P / S. </li></ol></li><li>消化された神経の顕微解剖を準備します。 <ol><li>解剖皿に消化胸膜ペダルと頬神経節を含む酵素溶液を注ぐ。 </li><li>反射した光の下で黒の表面に対して顕微鏡のステージ上にお皿を置きます。 </li><li>添付された結合組織を使用して、各胸膜ペダルhemiganglionの背側を突き止める。組織は柔らかく、ストレッチの不寛容になります。 </li></ol></li><li> PVCニューロンを顕微解剖。 <ol><li>きれいな細かい鉗子の2ペアを使用して、プローブとして鉗子の一組で細かい解剖ピンを保持し、胸膜hemi​​ganglionからPVCの細胞を単離する。酵素消化は、削除またはPVCクラスタをカバーする結合組織鞘を離れて壊れ、まだ細胞が互いに付着することになります。 </li><li>解剖の底に細胞クラスタ落下させた後、ペダル胸膜結合18からこれらの細胞を分離するために、プローブを使用ディッシュ。 </li></ol></li><li>培養皿にニューロンを転送します。 <ol><li>静かに気泡を導入しないように注意しながら、その後、培養皿のメディア島にゆっくりとそれを分配する、少し火磨か使い捨てガラスパスツールピペットの先端にクラスタを吸引することによって準備された35ミリ培養皿に細胞クラスタを転送ピペットに。独立した培養皿に他のhemiganglionと場所のPVCクラスタの分離を繰り返します。 </li></ol></li><li> BSCニューロンを顕微解剖して転送。 <ol><li>目的の細胞を倹約するために注意して、頬神経腹側を上にしてピン。頬神経節10の2 BSC細胞クラスターとステップ1.12を繰り返します。 </li><li> BSCクラスタを含む2皿とPVCクラスターを含む2皿：PVCとBSCニューロンの単離ニューロンクラスターを含む4培養皿を生成します。神経節の残りの部分を破棄し、100ミリメートル解剖皿を脇に置きます。 </li></ol></li><li> Dissoc細胞クラスターをイアテ。 <ol><li>低消費電力の顕微鏡のステージ上に細胞を含む培養皿を置き、カバーを取り外します。 </li><li>優しく鉗子で開催された微細な解剖ピンと細胞クラスターに隣接する培養皿の底部をフリックでPVCクラスタを解離する。プラスチックの斑点を掘るしようとしているかのようにフリックは、皿の底のプラスチックにピンの先端を掘っている。プラスチックとの摩擦は、ピンポイントを解放すると、パーカッション波が離れて細胞の近くの塊を壊す媒体を介して産生される。 </li><li> 5-10フリックは、ほぼ完全に細胞を解離するために必要な、しかし、それは細胞が機械的な薄手では破壊されないように、あまりにもフリックではなく、細胞の小さなクラスターを残した方がよいこともできる。 </li><li>カバーやその他の培養皿で繰り返し交換してください。 </li></ol></li><li>細胞培養に保管してください。 <ol><li>に解離細胞とその中心にメディアアイランドを含む培養皿を置きこのような150×25ミリメートルラウンドプラスチックシャーレとして空気の循環を、許可し、17℃にインキュベーターセットにこの料理を置き大型コンテナ</li><li>チャンバ内の湿度の源としての水のオープン皿をインストールします。邪魔されずに一晩にしておきます。細胞はシャーレの中央にポリ-D-リジンコーティングに付着する。 </li></ol></li><li>洪水培養皿。 <ol><li> 3日目に、ゆっくりとASW + P / Sの2.5 mlで料理をあふれさせる反転した細胞培養顕微鏡を用いて細胞を調べ、数える。 17℃のインキュベーターに保管してください。 </li></ol></li></ol> 2。電気生理学方法は、（ 例えばザクマンとネーアー、1995）、多数のテキストに16を説明してきたそのクランプ標準パッチです。このプロトコルは、細胞上の≤100pFの容量、またはセル&lt;プロトコルの3日目と4の間に60μmの径を動作します。プロセスなしで細胞が記録のために最適である。次の特殊consideratイオンが適用されます： <ol><li>大きな細胞はβ= 0.1に設定Axopatch 200Bアンプの構成設定と対応することができる。非常に大きな電流の活性化は、細胞が電圧クランプをエスケープすることがあります。 ASWのソリューションは、（不浸透性N-メチル-D-グルカミンクロライドで置換された、NaClの例フラクション）塩の含有量に置換全細胞電流振幅を低減するために使用することができる。 </li><li>繊維充填ホウケイパッチピペットピペットプラーで引っ張られ、450 mMの塩化カリウムや他の塩からなる細胞内液（ICS）と途中で埋めます（ 表1を参照）が適しており、約0.5〜1 Mオームの抵抗値を持っている必要があります。特定のイオン電流の分離が所望される場合、例えば、この溶液中のK電流、K +の非存在下でのNa +電流は、例えばセシウム+などの他のイオンと交換することができる。 Cl -のより自然なICSが望まれているなら、透過性アニオンとICSの交換も保証されて9。 </li><li>細胞は室温で可能&gt; 1時間にわたる録音で堅牢です。録音は文化の中で24〜48時間に対応した、3日目とプロトコルの4に最適である。 48時間後に細胞表面にあるため糖衣の精緻化にたぶん、ギガオームシールを形成する難しさ、およびプロセス伸長によって引き起こされる空間クランプ問題がある。細胞は&lt;14日で完了することができるような毒性などの非パッチクランプ研究、、のために、便利な、しかしである。 </li><li> ASWと他のソリューション重力供給溶液装置から分配されるだけでなく、細胞内の溶液を、を通して濾過されるべきギガオームシール形成を妨害微粒子を除去するために0.2ミクロンAcrodiskフィルタ（ 表3）注射器に取り付けられた。 </li><li>例えば、D-アスパラギン酸（D-ASP）アゴニストを使用する際に脱感作が生じ、したがってアゴニストのアプリケーションの間で〜1〜2分が推奨されます。逆に、増強は、多くの場合、迅速な繰り返しアプリで観察される例えば、L-グルタミン酸（L-グルタミン酸）などのアゴニストの陽。 </li><li>例えば、L-Gluのようなアゴニスト活性化電流はpicospritzerピペットでアゴニストを適用することによって研究することができる。このピペットは、パッチピペットと同じように引っ張られ、ASWにアゴニストが含まれています。このピペットの先端は、重力供給溶液装置の流出パイプの下方に配置され、流出管（ 図2F）から45°の角度で、アゴニストは速やかに細胞から洗い流さされ、脱感作が最小化されるとき。アゴニストピペットは90°またはパッチピペットへのよりの角度を作成する必要があります。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoafferent+neurons+innervating+tail+of+Aplysia.+I.+Response+properties+and+synaptic+connections.">Mechanoafferent neurons innervating tail of Aplysia. I. Response properties and synaptic connections.</a> J. Neurophysiol. 50 (6), 1522-1542 (1983).</li><li>Wilson, G. F., Richardson, F. C., Fisher, T. E., Olivera, B. M., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+Ca(2+)-sensitive+nonspecific+cation+channel+underlying+prolonged+repetitive+firing+in+Aplysia+neurons.">Identification and characterization of a Ca(2+)-sensitive nonspecific cation channel underlying prolonged repetitive firing in Aplysia neurons.</a> J. Neurosci. 16 (11), 3661-3671 (1996).</li><li>White, B. H., Nick, T. A., Carew, T. J., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+regulates+a+vesicular+class+of+calcium+channels+in+the+bag+cell+neurons+of+Aplysia.">Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of Aplysia.</a> J. Neurophysiol. 80 (5), 2514-2520 (1998).</li><li>Wilson, G. F., Magoski, N. S., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+a+calcium-sensitive+nonspecific+cation+channel+by+closely+associated+protein+kinase+and+phosphatase+activities.">Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities.</a> Proc. Natl. Acad. Sci. U.S.A. 95 (18), 10938-10943 (1998).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J. Vis. Exp. (28), e1355 (2009).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

解離技術はグリアや他の未確認の細胞の小さな数字が点在50〜100孤立したニューロンを含む感覚ニューロンの文化を生み出す、ここで説明。プロトコルの中で最も重要なステップは、神経、酵素溶液中に残存する時間であり、そして、個々の細胞にクラスターを分解する消化細胞塊の解離をフリック。酵素消化（ステップ1.8）が使用可能温度で最適化する必要があります。 23°遅い振とうしな...

海洋opistobranch軟体動物、 アメフラシは 、何十年もの間、有用な神経生物学的モデルとなっています。これは最高の馴化と古典的条件7,8のモデルとして知られています。このモデルでは学習と記憶に関する研究は、彼がArvidカールソンとポールグリーンガード10と共有賞で、エリックR.カンデル2000年に生理学·医学ノーベル賞を受賞した。この無脊椎動物の神経系の要素が付加左神経と筋肉との動物から解剖されている削減の準備からの電気録音を、関与する研究は、 アメフラシの個々の神経細胞の役割を解明に役立っています。しかしアメフラシでの学習を構成する精密な分子メカニズムの識別は、多くの場合、個々のドナー動物から1つずつ取得し別の技術、感覚ニューロンと運動ニューロンの長期共培養を採用し、培養皿21でシナプスを形成させ。 我々は、他の1,3,6、14、15、16は、名目上均質なニューロンのクラスタの解離短期間培養物を作るためにニューロンは、このモデルで対象とすることができる同定ならびに長期の実験では、その耐久性の容易さを利用しているている私たちは、パッチクランプ構成で電圧クランプの下にイオン電流を勉強。多くのアメフラシニューロンは長続き実験操作のための時間を可能にするためにパッチクランプを繰り返しラウンドに立ち上がる。技術は、腹部の神経節の神経分泌袋細胞、解離我々はここで説明する胸膜と頬神経節の感覚ニューロンとしてニューロンのためではなく、非常に大規模なニューロン&gt;60μmの径などのL7またはR2などに便利です腹部神経節。我々は他の場所に記載感覚ニューロン共培養とは異なり、私たちの文化でアメフラシ血清を使用しない。この手順を使用して得られたほとんどのニューロンはtに対してプロセスがなくてもなります彼は全体のセル電圧記録を促進する文化の中で最初の48時間、しかし、その後、新芽や栄養素、および/または成長因子の不足から、死ぬ前におよそ14日間軸索と他のプロセスを詳しく説明します。 この手法は、頬や胸膜神経の生理的に文書化された地域から皿当たり50〜100のニューロンの初代培養を生成します。このプロトコルは、多数の実験は、動物ごとに複製が必要な実験で単一細胞生理学の側面を研究する研究者にとって有用です。それは一致した治療群と対照培養物から研究その利益を可能にして、左右hemigangliaに標的細胞の解剖学的分離に起因する文化のペアを生成します。 プロトコルは、頬神経の頬Sクラスター（BSC）ニューロン、および胸膜神経節の胸膜ventrocaudal（PVC）のニューロンを対象としています。これらの細胞は、全体セル電圧の記録および表示ローバスに適したサイズであるTグルタミン酸応答。議論の方法論は、 アメフラシ神経系の中で最も神経に適しています。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Artificial seawater ASW</td>
 <td>Sigma-Aldrich</td>
 <td>assorted</td>
 <td>(mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 15 HEPES-NaOH, pH 7.6</td>
 </tr>
 <tr>
 <td>Intracellular solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>(mM): 450 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4</td>
 </tr>
 <tr>
 <td>Poly-D-lysine</td>
 <td>Sigma</td>
 <td>P6407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>penicillin/streptomycin added to ASW at 1:100</td>
 <td>Lonzo Walkersville, Inc.</td>
 <td>17-603E</td>
 <td>5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin</td>
 </tr>
 <tr>
 <td>Neutral dispase II</td>
 <td>Roche Diagnostics</td>
 <td>10165859001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hyaluronidase</td>
 <td>Sigma-Aldrich</td>
 <td>H4272</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>collagenase type XI</td>
 <td>Sigma-Aldrich</td>
 <td>C9407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamate (L-Glu)</td>
 <td>Sigma-Aldrich</td>
 <td>49601-100G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Aspartate (D-Asp)</td>
 <td>Sigma-Aldrich</td>
 <td>11200-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>N-methyl-D-aspartate (NMDA)</td>
 <td>Biomol</td>
 <td>100002-268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Asp</td>
 <td>Sigma</td>
 <td>A6683-25G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)</td>
 <td>Sigma</td>
 <td>A6816-5MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glu R antagonists</td>
 <td>various</td>
 <td>various</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>kynurenate</td>
 <td>Sigma-Aldrich</td>
 <td>61250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>APV</td>
 <td>Sigma-Aldrich</td>
 <td>A5282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-propanol</td>
 <td>VWRSP</td>
 <td>BDH1133</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloriding solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O</td>
 </tr>
 <tr>
 <td>Sylgard silicone 2-part polymer</td>
 <td>World Precision Instruments (WPI)</td>
 <td>SYL184</td>
 <td>Provides pin-out surface for small dissection dishes</td>
 </tr>
 <tr>
 <td>0-40x zoom magnification microscope for dissections</td>
 <td>Wild</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Techniquip 150 Watts Fiber Optic Illuminator</td>
 <td>Microoptics of Florida</td>
 <td>TQ FOI-150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RotoMix 50800 orbital mixer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) &amp; 40x objectives</td>
 <td>SR Research Ltd.</td>
 <td>Eyelink II</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tektronix digital oscilloscope</td>
 <td>SR Research Ltd.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pClamp 10 data acquisition and analysis software</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PC with Windows XP or higher operating system</td>
 <td>PC Solutions</td>
 <td>&nbsp;</td>
 <td>Thinkserver with solid state hard drives (80GB) and low noise monitors</td>
 </tr>
 <tr>
 <td>Flaming/Brown P87 micropipette puller</td>
 <td>Sutter Instruments, Novato, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon instruments electrode holder assembly for Axopatch 200B preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>CV203BU</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digidata 1200 A/D converter</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Picospritzer, powered by N2 adjustable for pressure and duration</td>
 <td>Parker Hannifin, Cleveland, OH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TMC Micro-G Vibration isolation table</td>
 <td>Ametek</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday cage</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>custom manufacture</td>
 </tr>
 <tr>
 <td>Burleigh Piezoelectric Clamshell Micromanipulators</td>
 <td>Burleigh Instruments; Thorlabs</td>
 <td>&nbsp;</td>
 <td>presently PCS-5000; -6000 series + mounts</td>
 </tr>
 <tr>
 <td>Narishige M-152 manual manipulators (for perfusion system and picospritzer)</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filament pipette glass,1.5 mm OD, 0.84 mm ID -</td>
 <td>WPI</td>
 <td>1B150-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3 inch length</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ag/AgCl half cell</td>
 <td>WPI</td>
 <td>EP4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Angled Scissors</td>
 <td>Fine Science Tools</td>
 <td>15006-09</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumostar Fine forceps</td>
 <td>Fine Science Tools</td>
 <td>11295-00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>35 mm falcon tissue culture dishes</td>
 <td>VWRSP</td>
 <td>25382-064</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>falcon 150 x 25 mm tissue culture dishes; 1013</td>
 <td>VWRSP</td>
 <td>1013</td>
 <td>also can be made into small dissection dishes with sylgard</td>
 </tr>
 <tr>
 <td>sylgard</td>
 <td>WPI</td>
 <td>SYL184</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>animal dissection tray</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>For 6-bore gravity-fed perfusion system</td>
 </tr>
 <tr>
 <td>Aluminum clips with screw hole ends</td>
 <td>hardware store</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>23 gauge needles (manually file off points)</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Polyethylene tubing 0.022"ID x 0.042"OD; 427411</td>
 <td>Becton-Dickinson</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>H-7 pipette stand/holder for microcap perfusion array</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>one-way valves</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Drummond Microcaps 1 &mu;l</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>18 gauge needles for suction (filed off points)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethylene tubing</td>
 <td>Cole Parmer</td>
 <td>4.27436E+11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>fine dissection pins</td>
 <td>Fine Science Tools</td>
 <td>26002-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>capillary tubes</td>
 <td>Kimble 71900-100</td>
 <td>&nbsp;</td>
 <td>fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS</td>
 </tr>
 <tr>
 <td>modeling clay</td>
 <td>craft store</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dish holder for microscope stage with isolated ground bath</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Custom manufacture</td>
 </tr>
 <tr>
 <td>pasteur pipettes</td>
 <td>VWRSP</td>
 <td>14672-412</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pipette bulbs</td>
 <td>VWRSP</td>
 <td>53283-911</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>acrodisk syringe filters</td>
 <td>VWRSP</td>
 <td>28144-040</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>thick-walled 1.5 mm diameter borosilicate filament glass</td>
 <td>WPI</td>
 <td>1B150F-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>High purity nitrogen cylinder and bifurcating regulator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Tables 1-3. Lists of Reagents, Materials, and Equipment.

isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents

海洋腹足類軟体動物アメフラシカリフォルニは、学習と記憶の研究において特に重要で、神経系機能のモデルとして由緒ある歴史を持っています。このような研究のための一般的な準備は、感覚と運動ニューロンを最小限解剖動物、または個々の感覚と運動ニューロンの技術的に精巧な神経細胞の共培養にそのまま残されているものです。あまり一般的では公称均質ニューロンの小さなクラスターは短期培養における単一細胞に解離されている孤立したニューロンの準備です。そのような単離された細胞は、パッチクランプ技術、およびこれらのコンダクタンスの目標変調を用いたイオン電流の生物物理学的特徴付けに有用である。このような培養物を調製するためのプロトコルが記載されている。プロトコルは胸膜と頬神経の容易に識別グルタミン酸感覚ニューロンを活用して、それらの解離と最小限のメンテナンスを説明I血清を含まない数日間のn文化。

このプロトコルにおいて対象となる神経節内の感覚ニューロンの位置は、BSC及びPVCニューロンは、 図1に示されている。 BSCのニューロンは頬神経節の腹側に2対称の楕円形のクラスター、そのまま神経節（ 図1A）で頬塊から離れて対向する面に配置されています。 PVCニューロンは中心軸（ 図1B）に向かって胸膜神経節の背面の周りをラップする二国間の?...

Watch this Scientific Journal Video about Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents at JoVE.com

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

我々は、海洋アメフラシの神経系の解剖を説明<em&gt;アメフラシ</em&gt;麻酔の後、短期·組織培養のための神経細胞、およびパッチクランプ法を介した単一細胞イオン電流の録音の分離。

の感覚ニューロンの分離<em&gt;アメフラシカリフォルニ</em&gt;グルタミン酸作動電流のパッチクランプ記録のために

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

isolation-sensory-neurons-aplysia-californica-for-patch-clamp

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Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

12.2K ビュー.  University of Miami. 我々は、海洋アメフラシの神経系の解剖を説明アメフラシ麻酔の後、短期·組織培養のための神経細胞、およびパッチクランプ法を介した単一細胞イオン電流の録音の分離。

ビデオ: Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

の電極の作製と注入アメフラシカリフォルニ無傷、自由に振る舞う動物における多チャンネル神経と筋肉の録音のための

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

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 ...

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Sf9細胞でとアメフラシ感覚ニューロンにおけるPKC転座のライブイメージング

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

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  ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

体外における筋モータープールのために運動ニューロンの識別<em&gt;アメフラシカリフォルニ</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

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 ...

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.