NIH（NS - 18205）によって資金を供給。

1。ペア録音のための魚の準備<ol><li>浴溶液、Tricaine（MS222）で浴溶液、ホルムアミド、ニューロンの内部ソリューションと筋肉内部ソリューションと浴溶液を：手順を開始する前に、含まれて5つの主要ソリューションを、準備する。 </li><li>次に、72〜96時間の年齢とTricaineと浴溶液内の場所間の単一の幼虫のゼブラフィッシュを収集する。麻酔薬への曝露の1分以内に、魚はタッチに反応しません。 </li><li>プラスチック製の皿の上に魚を置くと、実体顕微鏡を使用して、ダブルエッジのカミソリの刃で首を切る。 </li><li> sylgardでコーティングし、浴溶液で満たされたガラスの録音室に魚を移す。脊索を通して電解シャープタングステンピンを挿入して、各端に固定します。 </li><li>微細な鉗子のペアのための十分なグリップを提供するピンと魚の吻側端付近の皮膚のフラップを作成します。細かいピンセットで吻側ピンの近くでそれをつかんで覆っている皮膚を取り除きます。ピールゆっくり尾の方向に皮膚を除去する。 </li><li>レコーディングに干渉する可能性のある筋肉の収縮をブロックするために3-5分のためのホルムアミドと浴溶液の3のMLSで魚を扱​​う。通常の浴溶液で5回洗浄する。静かにタングステンピンの側で廃棄を5〜6セグメント上筋の表層の部分を削除します。 </li><li>電気的ノイズを遮蔽するためにファラデーケージに位置する正立顕微鏡に魚を移す。顕微鏡は、長距離40倍目標と4倍まで0.5のズームを作業、固定ステージを装備しています。顕微鏡は、高電力下神経と筋肉の間に準備を移動するために電動XY移動ステージにマウントされています。微分干渉コントラスト光学系の使用は、神経細胞を可視化するのに役立ちます。 </li><li> 0.5 ×ズームの下に、上を覆う筋肉を除去し、脊髄を露出するために広い口径15ミクロンのピペットを使用してください。負圧は、3 ccの使い捨て注射器と筋細胞を介して適用される一つずつ削除されます。この手順は、骨格筋の5〜6背のセグメントごとに繰り返されます。同じワイドボアピペットは、ターゲット速い骨格筋を得るために腹側筋肉の上位2層を除去するために使用されます。 </li><li>これは、ペアの録音の準備を完了し、顕微鏡のズームは、約1.6倍に増加しています。 </li></ol> 2。 CAPモーターニューロンとターゲットの筋細胞のペア録音<ol><li>洗浄セグメントは、CAPの神経細胞を見つけるためにスキャンされます。脊髄の各半セグメントは一つの大きな、10ミクロンの直径、キャップモータニューロンが含まれています。このティアドロップ形の細胞体は、分節境界の尾ほとんど終わり近くに脊髄硬膜の下に表面的に位置​​しています。 CAPのニューロンは、隣接する尾側セグメントでの録音と腹側のターゲットの筋肉のために選ばれている整合性をチェックするためにスキャンされます。 </li><li> 2つのパッチ電極は、記録、ニューロンと筋肉のための秒のいずれかに配置されている。上部電極は、約2ミクロンのタフな脊髄硬膜と先端開口部の浸透のための浅いテーパーを持っています。下部電極は、骨格筋の低抵抗の電圧クランプ記録のための急なテーパーを持っています</li><li>脊髄硬膜を貫通して約30度の角度でニューロンの電極を置き、約1選択されたCAPのニューロンへの吻側半セグメント。約60ミリメートルの水銀の優しい持続陽圧をかけながら軸方向駆動のマニピュレータを用いて電極を進める。硬膜貫通電極の区切りとして、CAPのニューロンは、フォーカスの再調整を必要とする、電極から灌流によって置換されることがあります。 </li><li>電極が接触したときCAPニューロン相馬正圧が解放され、gigaohmシールの直接の形成における通常の結果です。しかし、シールを形成するために失敗するいくつかのケースで、それは穏やかな負圧を適用するために必要となる。シール形成後、電極電位は- 80mVの調整と負圧のアプリケーションでは、細胞膜を破壊している。 CAPのニューロンは、140から180メガオームの入力抵抗と活動電位そのシュート40ミリボルトが特徴です。これは、活動電位が誘発されるまで電流クランプの下に段階的に増加し脱分極ステップを注入することによってテストされています。 </li><li>次に、顕微鏡は、高電力下XY電動トランスレータを使用して、腹側筋肉に再配置されます。速筋細胞は、ターゲットフィールドから選択されます。 </li><li>筋肉の電極は解放されると、gigaohmシールを形成する正の圧力下で標的筋細胞に向かって進んでいる。電極電位は、ナトリウムチャネルを不活性化を-50 mVに調整され、穏やかな吸うの下に細胞膜が破壊される。 100から200メガオームの抵抗と大容量過渡信号のエントリの突然の出現の低下。 </li><li>テストへ活動電位が誘発されるまで、ペアの記録のために、モーターニューロンは、現在の段階的大きい注射による脱分極である。この時点で、終板電流が筋肉に誘発されるべきである。失敗せずにエンドプレートの電流で1ヘルツ結果でmotorneuronの継続的な刺激。刺激周波数が100ヘルツに増加するにつれ、エンドプレート電流は振幅で変動し、刺激の10秒以内に機能的な枯渇を受ける。 </li></ol> 3。代表的な結果一般的に、ニューロンの記録では最大1時間までは安定であるが、筋細胞は、直列抵抗の増加として反映悪化。これが発生すると実験のいずれか終了するか、別の筋細胞では、パッチがクランプされています。すべての記録は一時間以内に終了してください。 <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="図1" /> 図1。motorneuronの活動電位（AP、上）と現在関連付けられている筋肉のエンドプレート（EPC、底部）の録音。 CAPのニューロンのための活動電位は、オーバーシュート40 mVおよびEPCは、1ミリ秒の下に時定数で指数関数時間の経過に沿って300μ秒と崩壊の中で立ち上がるようにしてください。 <table border="1"><tbody><tr><td> の名前 </td><td> レシピ </td></tr><tr><td>浴溶液（mMで） </td><td> 134のNaCl、2.9 KClを、2.1のCaCl 2、1.2のMgCl 2、10グルコース、10のNa - HEPES、pH7.8の</td></tr><tr><td> Tricaineと浴溶液</td><td> 0.02パーセントtricaineを含む浴溶液</td></tr><tr><td>ホルムアミドと浴溶液</td><td> 2Mホルムアミドを含む浴溶液</td></tr><tr><td>ニューロン内部の溶液（mMで） </td><td> 115 K -グルコン酸、15のKCl、2のMgCl 2、10 K-HEPES、5 K - EGTA、4のMg - ATP、pH 7.2の</td></tr><tr><td>筋肉内部溶液（mMで） </td><td> 120のKCl、10 K-HEPES、5 BAPTA、pH7.4の</td></tr></tbody></table> 表1。ソリューション。

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

利害の衝突は宣言されません。

我々は、高頻度刺激時に小胞放出とリサイクルの過程を研究するために主に（温とブレーム、2005）ゼブラフィッシュペアの録音を使用している。このプロセスは、重要なシナプス成分に起因する点変異する危険にさらされている通常のシナプス伝達を特徴と運動性の突然変異体で中断されます。たとえば、シナプス後受容体の凝集体（小野ら 、2001、2002、2004）とシナプス前トランスミッタの合成（Wang ら 、2008）非協調水泳で結果を混乱させる突然変異。ペアの録音は、このように行動、遺伝学と生理学をつなぐ、機能的な欠陥の原因を特定できる情報を提供しています。それは、現在、さらに賢明なプロモーターを使用してCAPのモーターニューロンの一過性発現によりシナプス伝達の根底にあるプロセスを解明することができるはずです。この方法では、ドミナントネガティブまたは変異遺伝子は、特にこれらのニューロンに発現しており、両方の行動や電気生理学的影響は決定することができます。全体のセル構成で提供される付加的な利点は、潜在的にそのようなクロストリジウム毒素やカルシウムのバッファとして小胞のリサイクルを、変えることができる薬剤とCAPの相馬の透析が可能になります。細胞体と筋肉の間の短い距離のために、拡散は、レコーディングの時間枠内でよく発生する必要があります。この準備の可能性は盗聴されるようになっています。この年齢で魚の透明性とシナプスの表面的な場所でも、光のツールの使用（; Fetchoと東島、2004 Fetchoとオマリー、1997）が容易になります。我々は、FM色素などSynaptophluorin（Li ら 、2003）などの遺伝的指標を用いて生きている魚のエンドとエキソサイトーシスの測定で非常に成功している。さらに、蛍光共役毒素は生きた魚（アイバニーズ-タロンら 、2004）のイメージングのためのシナプスタンパク質を標識するために使用することができます。この準備のシンプルさを考えるとそれは、今後の大きな可能性を秘めています。

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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
<td>DPM1B</td>
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<td>0.002 x 3 inch tungsten rod</td>
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<td>A-M Systems Inc</td>
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<td>Sylgard 184 Elastomer</td>
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<td>Dow Corning Corp.</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

幼虫ゼブラフィッシュは、脊髄運動ニューロンとターゲットの筋肉からの同時パッチクランプ記録を可能にした最初の脊椎動物のモデルシステムを表す。これは、両方の細胞型と視覚的形態と位置に基づいて、単一のセグメントCAPモータニューロンを区別する能力へのアクセシビリティの直接の帰結です。このビデオでは、CAPモータニューロンと標的筋細胞と同様に各セルのタイプから録音するための方法論を識別するために使用される微細な方法を示しています。キャップ運動ニューロン型の識別は、染料の充填によって、またはそのような活動電位波形と、セルの入力抵抗のような生物物理的特徴によって確認されています。一時間に複数の異なるターゲットの筋細胞から長期的な録音を可能にするため日常的に最後のモーターニューロン記録。モーターニューロンの発火パターンの制御は、神経筋接合部におけるシナプス伝達の周波数依存性の測定を可能にします。大規模量子サイズと全体のセル電圧のクランプが提供する低ノイズのおかげで、単一のイベントのすべては筋肉で解決することができます。この機能は、リリースのプロパティ、小胞のリサイクルだけでなく、シナプス抑制や促進などの基本的なシナプスの特性の調査を可能にします。 in vivoでの準備日食直前の神経筋モデル系でこのことによってもたらされる利点は、モータニューロンは通常、細胞外電極で刺激し、筋肉が全細胞パッチクランプを行うには大きすぎるしている特徴と研究。ゼブラフィッシュの準備は、トランスジェニック系統、モルホリノノックダウン、薬理学的介入とin vivoイメージングを含めたアプローチの広い範囲で電気生理学的解析を組み合わせることに従順です。これらのアプローチは、ゼブラフィッシュの突然変異系統から提供された神経筋疾患のモデルが増えていると相まって、人間の神経筋疾患の新しい理解のためにドアを開きます。

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

幼虫ゼブラフィッシュは、脊髄運動ニューロンとターゲット骨格筋からの同時パッチクランプ記録を可能にした最初の脊椎動物のモデルシステムを表す。このビデオでは、分節キャップ運動ニューロンと標的筋細胞と同様に各セルのタイプから録音するための方法論を識別するために使用される微細な方法を示しています。

モーターニューロンとターゲットゼブラフィッシュの骨格筋からの対のパッチクランプ記録

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.1K ビュー.  Oregon Health and Sciences University. 幼虫ゼブラフィッシュは、脊髄運動ニューロンとターゲット骨格筋からの同時パッチクランプ記録を可能にした最初の脊椎動物のモデルシステムを表す。このビデオでは、分節キャップ運動ニューロンと標的筋細胞と同様に各セルのタイプから録音するための方法論を識別するために使用される微細な方法を示しています。

ビデオ: Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

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

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

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

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

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

神経科学

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

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

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

内耳有毛細胞におけるパッチクランプ記録は、単離されたゼブラフィッシュから

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

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

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

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

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

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

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

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

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

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

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

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 ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

器官型海馬スライスにおけるペア全細胞記録

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神経筋接合部：測定シナプスサイズ、断片化および共焦点蛍光顕微鏡を用いてシナプスタンパク質の密度の変化

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

急性脳スライスにおけるニューロンマイクロ回路の電気生理学的および形態学的特徴は、ペアパッチクランプ記録を使用して、

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

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 ...