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1 記録電極の作製 電極引き手の <ol>ガラス電極を引いて <ol><li>次を使用 <li>プロトコル (材料の表 を参照してください): <ol><li>ライン 1: 熱。510 プル - 速度 30 時間 250;2 行目: 熱 490 プル - 速度 30 250 時間します 。
			注: 時間の単位は 1 単位当たり 0.5 ms に対応します。他のユニットは相対的です。ランプのテストが実行された後、すべてのフィラメントの熱の値を調整する必要があります</li>。
		</ol></li> <li>に確実に ( 図 1 a) 7-10 μ m の範囲で引っ張られた電極内径顕微鏡 (35 倍) を使用します。塵の蓄積を防ぐために密閉容器に毛細血管を格納します</li>。
	</ol></li> <li>火研磨 <ol><li>火ポーランド毛細血管 (1 〜 2 秒の最大熱値の 80-90%) マイクロ フォージを使用して (材料の表 を参照してください)。洗練された電極の最終の内径が 5 μ m ( 図 1 a) 27 を確認します</li>。
	</ol></li> <li>曲げ 注: 2 つのベンドが高倍率の目的の下に筋肉の上に電極を配置するために作られています。
	<ol><li> 図 1 b に示すように装置の使用。マニピュレーターに電極を修正し、それを触れていないフィラメント上の先端の位置</li>
		<li>セット値を最大の 60-70% を熱し、1 〜 2 秒のためマイクロ フォージのペダルを押す (材料の表 を参照)、フィラメントを加熱します。L 字の針を使用すると、電極 ( 図 1 b の拡大) の先端をそっと引き下げます。約 90 o ( 図 1) に曲げることを確認します</li>。
		<li>鉗子を使用して手作業でトーチの炎で、電極を保持し、最初曲げから 7-10 mm の距離で、約 120 o ( 図 1) の角度で 2 つ目のベンド</li>。
	</ol></li></ol> <img alt="Figure 1"class="xfigimg"src="/files/ftp_upload/56493/56493fig1.jpg"/> 図 1。マイクロ ピペット作製の最終手順です。(A) 電極の引っ張ってくると火の研磨後ヒントします。(B.1) 先端曲げのセットアップ。(B.2) ボックスの領域は、右に拡大表示されます。電極とフィラメントは、電極先端がワイヤーの少し上位置とない感動が固定されます。(C.1) 記録電極。(C.2) ボックスの領域は、右に拡大表示されます。約 90 ° の角度であり、最初のベンドと電極の先端間の距離である約 1 mm スケール最初曲げバー = 3 mm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg"target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
 2 準備手順を追加 <ol><li>(mM) の準備 heamolymph のような (HL3) ソリューション: 5 70 塩化ナトリウム、KCl、20 MgCl 2、10 NaHCO 3、5 トレハロース、115 スクロース, 5 HEPES, 1 mM CaCl 2; 7.3 - に pH を調整。7.4. 冷蔵庫でソリューションを維持し、毎週新鮮な。</li>
	<li>が同じ記録方法ガラス刺激 pipets pipets、作る以外にそれらを曲げる必要はありません 。
	注: 刺激電極の作製は、 28 と 27 の詳細で説明されます。最終的な直径 5-7 μ m の範囲にする必要があります火災研磨後</li>。
	<li>注射器に接続されている電極ホルダーに刺激ピペット挿入します</li>。
	<li>小さなシャーレ 35 x 10 mm) シリコーン ゴム エポキシでコーティングから製造解剖板 (材料の表 を参照) 28 の説明に従って 。
	注: シリコーンゴムを使用する前に完全に硬化する必要があります</li>。
	<li>放浪の 3 齢幼虫をピックアップし、前述の 27 , 28 , 29 , 30 で解剖します 。
	注: ボタンを表示するに使用フライひずみ CD8 GFP (材料の表 を参照してください) <ol><li>使用鉗子 (材料の表 を参照してください)、バイアルから 3 rd 幼虫をピックアップします</li>。
		<li>幼虫は、最初の端子を後部に配置することをピンし、別ピン前方 (口フックに近い).</li>
		<li>追加 HL3 ソリューションです</li>。
		<li>はさみを使用して春 (材料の表 を参照) 上のピンから 1 つ下に幼虫の背側にカットを作成します</li>。
		<li>幼虫の左右に 2 の追加ピンを配置すること、幼虫のフィレ肉をピンします</li>。
		<li>勇気と気管鉗子を使用してを削除します</li>。
		<li>春のはさみを使用して腹側神経索外だけ神経をカットします</li>。
	</ol></li></ol> 3。EJCs の電気録音 <img alt="Figure 2"class="xfigimg"src="/files/ftp_upload/56493/56493fig2.jpg"/> 図 2。レコーディングの設定。サンプル NMJ 落射蛍光機能、高倍率の目的および 2 つのマニピュレーターを搭載した直立の化合物顕微鏡の可動式ステージ上にコーティングされたペトリ皿 (矢印) が置かれたシリコンに固定されています。顕微鏡は防振テーブルに駐留しています。<a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg"target="_blank"> この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol><li>記録 <ol><li>顕微鏡ステージ ( 図 2) で準備とシャーレの場所。お風呂に参照電極を挿入します</li>。
		<li>は、HL3 ソリューションと記録電極をご記入ください。10 倍を目的として (材料の表 を参照)、電極をお風呂に浸すし、筋肉が 6、7 の 2、3、または 4 のマニピュレーターを使用して腹部のセグメント上に配置 (参照 材料表) ( 図 3A.1 と A.2).</li>
		<li>は、60 x に目標を切り替える (材料の表 を参照してください)。エピ蛍光または DIC 光学を使用して関心のある分野に焦点を当てます。シナプスブートン ( 図 3 B.1 3) の上に電極の先端を配置します</li>。
		<li>は、筋肉に非常にやさしく電極を押します。過度の圧力が略記を損傷したり、シナプス活動の増加を誘発します。電極の先端は詰まっている - 場合は、それを置き換えることを確認</li>
		<li>アンプ、A/D ボード、およびコンピューターのスイッチします</li>。
		<li>アンプの電圧クランプ モードを選択します</li>。
		<li>集録ソフトウェアを起動し、選択、&#39; のギャップ フリー &#39; モード</li>。
		<li>コンピューターの画面に mEJCs の外観を観察</li>。
		<li>、MEJCs の振幅は 0.2 - の範囲であることを確認 0.7 na という 
		注: より小さい EJCs を示す記録電極が欠陥を持っていることまたはそのは正しく配置されません</li>。
	</ol></li></ol> <img alt="Figure 3"class="xfigimg"src="/files/ftp_upload/56493/56493fig3.jpg"/> 図 3。シナプス研究の視覚化。ヘミ セグメント x 倍率 (A.1) と 6 と 7 の筋肉を示すボックスの面積を拡大 10 未満の A (A) 明視野イメージ (A.2、矢印マークの記録電極)。スケールバー = 50 μ m。 (B) シナプス研究は、遺伝子にエンコードされた神経細胞マーカー (CD8-GFP) と ショウジョウバエ ラインで可視化エピ蛍光イメージング (B.1) または DIC 光学 (B.2) を使用します。画像は撮影目的と GFP 用フィルター キューブ x 60 (材料の表 を参照してください)。Synaptic ボタンは矢印の付いたマークされ、B.3 に蛍光と DIC イメージのオーバーレイが表示されます。スケール バー = 10 μ m. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg"target="_blank"> この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="2"> <li>刺激 <ol><li>腹部セグメント 2-4 を支配する軸索に近い刺激電極を置くビジュアル コントロールの下で、マニュピュレーターを使用しています</li>。
		<li>電極 ( 図 2) の内側軸索が引っ張られるように注射器のピストンを引いて負圧を適用が電極ホルダーに接続されています</li>。
		<li>刺激をオン。アイソレーション ・ ユニットのノブ (材料の表 を参照) をゼロの電流を設定し、ゆっくり増加、EJCs が表示されるまで (またはしきい値に達するまで).</li>
		<li>現在の刺激閾域における刺激増加約 2 回、EJCs の観測のしきい値と比較して実行します 。
		注: 我々 の経験でこのような刺激の強度は活動電位の障害や活動電位の発火を避けるために最適です。たとえば、0.2 の刺激電流で表示されます EJCs mA、0.4 の電流を使用して、実験を通して mA</li>。
	</ol></li> <li>シール抵抗 <ol><li>に増幅器の電極抵抗スイッチを回して録音 macropatch 電極のシール抵抗を測定 &#34; テストをシール &#34; 位置。GΩ シール抵抗の値が表示されることを確認、&#34; 現在 &#34; ウィンドウ</li>。
		<li>シール抵抗は 0.5 - 2 M の範囲にあることを確認 &#8486;。この範囲外の値を示す電極が正しく研磨または適切に位置しないこと</li>。
		<li>シール抵抗録音データ全体を監視し、それを確かめる実験を通じて一定します</li>。
	</ol></li></ol> 4。解析 解析のためのソフトウェアをカスタマイズ <ol><li>分析録音を採用 (材料の表 を参照してください). 
	注: 当研究室は、社内ソフトウェア Quantan 31, EJCs と mEJCs 塊状を記録した ( 図 4) の検出のためにカスタマイズされるを使用します。このソフトウェアは、ガウスのデジタル フィルターが含まれていて、量子多量子イベント ( 図 4 a) の重複ピークの検出が可能します。他のアプローチは 17 で説明します</li>。
</ol> <img alt="Figure 4"class="xfigimg"src="/files/ftp_upload/56493/56493fig4.jpg"/> 図 4。量子解析。 の mEJCs (A) と EJCs (B) Quantan ソフトウェアによって検出します。イベント エリアは緑色でマークされますが、ピークは赤い矢印で示されます。<a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg"target="_blank"> この図の拡大版を表示するのにはここをクリックしてください</a>。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+unit+populations+at+the+Drosophila+larval+neuromuscular+junction.">Quantal unit populations at the Drosophila larval neuromuscular junction.</a> J Neurophysiol. 82 (3), 1497-1511 (1999).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+reduced+calcium+on+quantal+unit+current+and+release+at+the+crayfish+neuromuscular+junction.">The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction.</a> Pflugers Arch. 391 (1), 35-40 (1981).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Ca2++inflow+to+quantal,+phasic+transmitter+release+from+nerve+terminals+of+frog+muscle.">Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.</a> Pflugers Arch. 422 (2), 129-142 (1992).</li><li>Marrero, H. G., Lemos, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Loose-Patch-Clamp method. , (2007).</li><li>Wu, W. H., Cooper, 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=Physiological+recordings+of+high+and+low+output+NMJs+on+the+crayfish+leg+extensor+muscle.">Physiological recordings of high and low output NMJs on the crayfish leg extensor muscle.</a> J Vis Exp. (45), (2010).</li><li>Verstreken, P., Ohyama, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction.">FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.</a> Methods Mol Biol. 440, 349-369 (2008).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Imlach, W., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li><li>Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li><li>Huntwork, S., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li><li>Zhong, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

著者が明らかに何もありません。

ショウジョウバエでは、シナプス伝達の研究に有利なモデル生物を表します。いくつかの録画設定は、シナプス電位、2 つの電極電圧クランプ33,34, 焦点の macropatch とシナプス電流の録音の細胞内の録音を含む、幼虫 NMJ で使用されています。ここで説明したシナプス電流の録音。後者の手法では、可視化研究でシナプス伝達の正確な定量をこ...

ショウジョウバエは、シナプス伝達を制御する分子機構を研究するエクセレント モデル システムです。ショウジョウバエの神経筋システムは、グルタミン酸とグルタミン酸作動性のリリースの保存の機能を勉強したがってショウジョウバエの神経筋接合部 (NMJ) を使用できます。以来 1 月および 1 月の研究1、3 齢幼虫は興奮性接合電位 (EJPs) または (EJCs) の流れを監視することによって誘発と自発的なシナプス伝達を研究に広く使用されています。EJPs はよく鋭いガラス微小電極を細胞内に記録され、彼らは特定の筋線維のシナプスを作るすべてのボタンを含む全体の NMJ の活動を反映しています。
対照的に、神経やシナプス下肢近くマイクロ ピペット チップを配置することで先んじて、リリースのサイトの限られた数のアクティビティを記録できます。この手法がカッツと Miledi2、採用された当初と焦点細胞外記録が正常にカエル3,4、5マウス6を含むいくつかの NMJ 製剤で採用されています。,7,8甲殻類9,10、11,12,13,14,15,16, ショウジョウバエ17,18,19,20,21,22,23。このアプローチは、記録電極24,25macropatch に最適化した Dudel によってさらに開発されました。Dudel の実装では、この手法の方が、緩いパッチ クランプ法26を一致しました。
ショウジョウバエ幼虫 NMJ、明確なシナプス研究と遺伝的コード化神経細胞蛍光タグ (材料の表を参照) と形質転換線がすぐに利用できます。これらの利点は、EJCs と選択したシナプスブートン20,21,22mEJCs を記録することができました。ここでは、このテクニックの詳細をについて説明します。

<table><tbody><tr><td>Sutter P-97</td><td>Sutter instrument</td><td>P-97</td><td>Microelectrode puller</td></tr><tr><td>Narishige MF-830</td><td>Narishige</td><td>MF-830</td><td>Microforge</td></tr><tr><td>WPI MF200</td><td>WPI</td><td>MF200</td><td>Microforge</td></tr><tr><td>Glass capilaries</td><td>WPI</td><td>B150-86-10</td><td>Glass capilaries</td></tr><tr><td>Microtorch 1WG61</td><td>Grainer</td><td>1WG61</td><td>Microtorch</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>SYLGARD 184</td><td>Silicone for dissection plates preparation</td></tr><tr><td>Dissection pins</td><td>Amazon</td><td>B00J5PMPJA</td><td>Pins for larvae positioning</td></tr><tr><td>Tweezers</td><td>WPIINC</td><td>500342</td><td>Tweezers for placing pins, removing the guts and tracheas.</td></tr><tr><td>Scissors</td><td>WPIINC</td><td>501778</td><td>Scissors for cutting the cuticula of the larvae and nerves.</td></tr><tr><td>Olympus BX61WI</td><td>Olympus</td><td>BX61WI</td><td>Upright microscope</td></tr><tr><td>Olympus Lumplan FL N 60x</td><td>Olympus</td><td>UPLFLN 60X</td><td>Microscope objective 60X</td></tr><tr><td>Olympus UPlan FL N 10x</td><td>Olympus</td><td>Uplanfl N 10X</td><td>Microscope objective 10X</td></tr><tr><td>Narishige Micromanipulator</td><td>Narishige</td><td>MHW-3</td><td>Three-axis Water Hydraulic Micromanipulator</td></tr><tr><td>npi Electronic GmbH ELC-03XS</td><td>npi Electronic GmbH</td><td>ELC-03XS</td><td>Electrophysiological amplifier</td></tr><tr><td>A.M.P.I Master 8</td><td>A.M.P.I.</td><td>Master 8</td><td>Electrical stimulator</td></tr><tr><td>A.M.P.I Iso-Flex</td><td>A.M.P.I.</td><td>Iso-Flex</td><td>Stimulus isolator</td></tr><tr><td>TMC antivibration table</td><td>TMC</td><td>63-9090</td><td>Antivibration table</td></tr><tr><td>TMC Faraday cage</td><td>TMC</td><td>81-333-90</td><td>Faraday cage</td></tr><tr><td>Digidata 1322A</td><td>Axon Instruments</td><td>Digidata 1322A</td><td>Digidata</td></tr><tr><td>Computer</td><td>Dell</td><td>Dell Dimension 5150</td><td>Computer with Win XP OS</td></tr><tr><td>Electrode holder</td><td>WPI</td><td>MEH3SW</td><td>Electrode holder</td></tr><tr><td>Optical filter</td><td>Omega optical</td><td>XF 115-2</td><td>Filter cube for Green Fluorescent Protein (GFP) detection</td></tr><tr><td>pCLAMP 8</td><td>Axon Instruments</td><td>8.0.0.81</td><td>Software for signal recording</td></tr><tr><td>Quantan</td><td>In-house software</td><td>-</td><td>Software for signal processing</td></tr><tr><td>Canton-S (Wildtype)</td><td>Bloomington Stock Center</td><td>64349</td><td>Control fly line</td></tr><tr><td>cpx SH1</td><td>Generous Gift of J.T. Littleton</td><td>-</td><td>Complexin knock-out fly line with increased spontaneous exocytosis</td></tr><tr><td>CD8-GFP</td><td>Bloomington Stock Center</td><td>5137</td><td>Fly line with neuronal fluorescent (GFP) Tag</td></tr></tbody></table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

ショウジョウバエ神経筋接合部 (NMJ) はグルタミン酸作動性シナプス伝達を研究するエクセレント モデル システムです。ショウジョウバエ幼虫 NMJ で可視化研究からシナプス電流の焦点の macropatch 録音の手法について述べる。この方法で高倍率、長距離水浸対物レンズ、差動干渉の対照 (DIC) の光学、蛍光顕微鏡と同様に、マイクロ ピペット、記録のカスタマイズ加工を必要添付ファイル。記録電極は選択したシナプスブートン DIC 光学、エピ蛍光、あるいは両方に可視化の上に配置されます。この方法の利点は、リリースのサイトの限られた数のシナプスの活動を監視できます。記録電極は直径数ミクロン、および電極縁外に配置されるリリース サイト記録電流に大きな影響はないです。記録された性シナプス電流高速キネティックがあるし、容易に解決することができます。これらの利点、または非同期の自発的なシナプス活性の増強と突然変異のはえ系統の研究のため特に重要です。

焦点距離 macropatch の録音は、選択したシナプス ボタン (図 5) からシナプス アクティビティの監視を有効にします。シナプスブートン (図 5 a, サイト 1) の上に電極を配置すると、記録された mEJCs (図 5サイト 1) は騒音レベルを大幅に上回る振幅があるし、上昇の段階 (サブ ミリ秒の範囲) でシャープし?...

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

ショウジョウバエ幼虫神経筋接合部のシナプス電流の焦点 Macropatch 録音

ショウジョウバエ第 3 齢幼虫神経筋接合部シナプス ボタンの可視化から塊状性シナプス電流を記録できます。この手法は、単一のシナプスブートンのアクティビティを監視できます。

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

6.0K ビュー.  Wayne State University. ショウジョウバエ第 3 齢幼虫神経筋接合部シナプス ボタンの可視化から塊状性シナプス電流を記録できます。この手法は、単一のシナプスブートンのアクティビティを監視できます。

ビデオ: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

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

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

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.  ...

神経科学

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

で、アクティブなゾーンの解剖とイメージングショウジョウバエ神経筋接合部

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

ショウジョウバエの幼虫のNMJからシナプス電位を記録するための電気生理学的方法

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

生物学

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

シナプス電位のChannelrhodopsin2仲介刺激でショウジョウバエ神経筋接合部

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

解離性とC. elegansの神経筋接合部から録音する

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

の調製<em&gt;ショウジョウバエ</emための&gt;中央ニューロン<em&gt;その場で</em&gt;パッチクランプ

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

の電気生理学的記録ショウジョウバエ胚

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

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 ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

ショウジョウバエ幼虫の神経筋接合部の透過型電子顕微鏡法のためのサンプル調製プロセスの最適化

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

ショウジョウバエ神経筋接合部(NMJ)の定量化:シナプスの形態と機能を評価する方法

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...