準備を開始する前に： <ul><li>放浪yhird齢ショウジョウバエの幼虫</li><li> HL3.1（血リンパのような変更）ソリューション</li><li> Sylgard（透明なシリコーンゴム）ブレントとマッケイブ（2008）3で説明した方法を使用して、小さな（35 × 10 mm）のプラスチック製のペトリ皿で準備解剖プレート。 </li><li>短い解剖ピンをカット</li><li>刺激電極のピペット</li><li>シャープ記録ピペット</li></ul> HL3ソリューション： <ol><li> 70のNaCl、5 KClを、4のMgCl 2、10のNaHCO 3、5トレハロース、115スクロース、および5 HEPES、pH 7.2の：解剖と電気生理学的実験の中で、幼虫は（mMで）含まれているHL3.1溶液2に浸漬されています。 </li><li> CaCl 2の濃度は、細胞外Ca 2 +の必要な濃度を提供するためにHL3.1溶液に添加されています。 <ul><li>幼虫の解剖はHL3.1を使用して低Ca 2 +濃度（筋肉の収縮を避けるために）+ 0.25 mMのCaCl 2を 、氷上に置きに実行されます。 </li><li>電気生理学的実験は、通常、HL3.1に記録されます+ 1mMのCaCl 2、しかしこれは、0.4から調整することができます- 1mMのCa 2 +は 、必要に応じて。 </li></ul></li><li> HL3.1ソリューションは、フィルタ使用前に滅菌し、4℃で保存されます˚C. </li></ol>ピペットを刺激し、録音の準備： <ol><li>ヒート= 390、フィラメント= 4、速度= 35、= 200を遅延し、= 0をプル：。レコーディングと刺激ピペットは、次の設定を使用してサターP - 2000レーザーベースのマイクロピペットプラーでプルアップされています</li><li>刺激電極は切断モーターの軸索の幅よりもわずかに大きくしたワイド端で薄肉ホウケイ酸ガラス毛細管から調製される。形の両端は、神経の損傷を最小限に抑えるために、（Narashigaピペット研磨機を使用して）滑らかな仕上げを与えるためにfirepolishedされています。刺激電極は浴溶液（HL3.1 + 1mMのCa 2 +）ので埋められます。 </li><li>記録電極は、シャープピペット（mΩの30〜60）を形成するために引っ張られ、3MのKClで満ちている1.2ミリメートルホウケイ酸ガラス、から調製されています。 </li></ol>パート1：ショウジョウバエの幼虫の解剖<ol><li>三齢放浪幼虫は、以前は次の4を説明するように体壁の筋肉を公開するために解剖されています。 </li><li>解剖はHL3.1で実行されます+ 0.25 mMのCa 2 +は小さなシリコンプレート（35 × 10 mm）に。 HL3.1ソリューションは、幼虫をanaesthetizeするために、解剖用の氷冷でなければならず、解剖前と中に氷上に保持されます。 </li><li>幼虫はHL3.1解剖のソリューションに+ 0.25 mMのカルシウムを添加しても小さい短い解剖ピン（長さは約2 mm）を用いて電気生理学的実験のために変更されたブレントとマッケイブ（2008）によって記載された方法を用いて切開している実験時の顕微鏡対物と電極をヒットする可能性が高い。 </li><li>解剖の後、幼虫の運動軸索が切断されています。これは、静かにCNSを保持していると腹側神経節の後方の筋肉を支配する末梢神経が筋肉（図1）を損傷することなく切断することができますので、少しそれを上げることによって行われます。脳はその後削除されます。 </li><li>解剖幼虫の準備はHL3.1 + 1mMのCa 2 +は 2回洗浄する。 </li></ol><img src="/files/ftp_upload/1109/fig1.png" alt="図1" /> 図1。解剖幼虫のモーターの軸索を切断するために必要な手順を示す回路図。 CNSは、鉗子で持ち上げられ、モータの軸索は脳の基底部で切断されています。 パート2：幼虫の筋肉細胞からの細胞内記録。 <ol><li> HL3.1と準備は、電気生理学的顕微鏡や水没で解剖のプレートを置き、+それが溶液に接触しているので、1.5mMののCa 2 +とバス電極を修正。 </li><li>全ての実験は、サード腹部セグメント（A3）内の筋肉6日に実行されます。筋肉を支配する末梢神経は、吸引電極5を使用して刺激される。 </li><li>細胞内電極が所定の位置にあるときに振動を回避するために、皿に刺激電極を配置します。筋肉6を支配する運動ニューロンへのすぐ近くに刺激電極を置き、カットの神経は、ガラスピペット内にあるまで、静かに吸引を適用する。吸引が適用されるときに神経を伸ばすように注意してください。それはカット神経線維（図2）を引っ張っていないので、それが筋肉との位置に触れていないので、少しピペットを上げる。 <img src="/files/ftp_upload/1109/fig2.png" alt="図2" width="600" height="296" /> 図2。ショウジョウバエの幼虫から一腹部のセグメントの筋肉や神経支配運動ニューロンの回路図。筋肉6時位置に刺激ピペットや切断神経と記録電極の位置が示されている。 </li><li>イントラ携帯電話の録音は、3 MのKClを充填した鋭い微小電極で作られています。第三腹部セグメント（A3）上筋6の中心上に記録電極を配置し、浴溶液のためのゼロを読み取るように入力オフセットを調整します。それは筋肉の表面に触れるまでゆっくりと高光学倍率の下電極とアプローチ筋肉を下げる。筋肉が侵入されていることを確認するためにオシロスコープをご覧ください。静止膜電位は少なくとも-60 mVのでなければ、または動物を使用しないでください。散発的なミニチュア終板電位が表示されるようになるはずです。記録を開始する前に、1分間安定させるためにセルを残す。 </li><li>膜電位の変化は、軸索HS - 2AのヘッドステージとAxoclamp 2Bのアンプで検出し、Clampex V 8.2.0.235（アクソンインスツルメンツ）で記録されます。 Axoclamp 2BはpCLAMPソフトウェアとDigidata 1322Aのインタフェースと連携して動作を実行するコンピュータとのインタフェースです。 </li><li>細胞はmEJP応答を測定するためにあらゆる刺激せずに3分間記録した。トレースは、ミニ解析ソフトウェア（Synaptosoft、V 6.0.3）と決定さmEJP振幅と周波数を用いて分析した。 </li></ol>神経刺激： <ol><li>運動ニューロンの切断端は、全体の筋肉で一貫性のある反応を引き起こすために、両方のモータの軸索に対して十分な強度で方形電圧パルス（0.3秒持続時間）の連続で刺激される。 </li><li>刺激が継続的に持続時間と間隔の回数に応じてパルスを提供するためにプログラムされているマスター- 8パルスジェネレータで生成されます。 </li><li> NMJでのシナプスの回復のための刺激の間に5秒を許可する。 </li><li>刺激の強さを決定する際に、最も低い水準で始まり、いくつかの臨床試験を介して徐々に強度を増加させる。誘発反応の結果、最小の刺激強度は実験で使用する必要があります。 </li><li>適切な刺激強度に達したときに、一つの化合物のEJPと刺激によって誘発one筋肉の収縮があるはずです。 </li><li>各筋肉と平均振幅から少なくとも10誘発電位を記録する。 </li></ol>パート3：代表的な結果<img src="/files/ftp_upload/1109/fig3.png" alt="図3" width="600" height="358" /> 図3。誘発EJPのを示す筋肉6日から代表的な細胞内記録では、分節神経の電気刺激、および散発ミニチュア終板電位、またはmEJPのへの応答です。このようなカントンSのような健康な野生型幼虫の筋肉6のEJPの振幅は、通常は約40 mVのと1-3 mVの間mEJP振幅です。

<ol>
	<li>Atwood, H. L., Govind, C. K., 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=Differential+ultrastructure+of+synaptic+terminals+on+ventral+longitudinal+abdominal+muscles+in+Drosophila+larvae.">Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae.</a> J. Neurobiol. 24 (8), 1008-1024 (1993).</li><li>Feng, Y., Ueda, A., 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=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li><li>Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., Ramaswami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+of+dynamin+within+individual+Drosophila+synaptic+boutons+relative+to+compartment-specific+markers.">Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.</a> J Neurosci. 16, 5443-5456 (1996).</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=Protocol+for+dissection+of+Drosophila+larvae.">Protocol for dissection of Drosophila larvae.</a> J Vis Exp. , (2008).</li><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster.">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> J Physiol (Lond). 262, 189-214 (1976).</li><li>Katz, L. C., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+and+the+construction+of+cortical+circuits.">Synaptic activity and the construction of cortical circuits.</a> Science. 274, 1133-1138 (1996).</li></ol>

方法はNMJにおいてシナプス機能の変化を検出するために、比較的迅速かつ広範な手段を提供するここで説明する。 in vivoでインタクトな動物を用いて電気生理学的記録を実行し、遺伝的または薬理学的操作を実行する機能は、ショウジョウバエ神経伝達の生理的および遺伝的側面を調査するための理想的な動物モデルにします。 筋細胞は非常に大きいので、いくつかの2?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Small Petri dishes (35 x 10 mm)</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>1008</td>
<td>&nbsp;</td>
<tr>
<td>SYLGARD 182 Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
<td>3097366-1004</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>475002-9902</td>
<td>&nbsp;</td>
<tr>
<td>Light for microscope</td>
<td>&nbsp;</td>
<td>Schott</td>
<td>KLI500</td>
<td>&nbsp;</td>
<tr>
<td>Dissection pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>pClamp 9 software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>PCLAMP 9 STANDARD</td>
<td>&nbsp;</td>
<tr>
<td>Dissection scissors: 3mm Vannas Spring Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-0</td>
<td>&nbsp;</td>
<tr>
<td>Dumont SS Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Thin-walled borosilicate glass capillaries, with filament (1.0 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillaries, with filament (1.2 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B120F-4</td>
<td>&nbsp;</td>
<tr>
<td>Sutter P-2000 Laser Based Micropipette Puller </td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Model P-2000</td>
<td>&nbsp;</td>
<tr>
<td>Pipette polisher</td>
<td>&nbsp;</td>
<td>Narishiga</td>
<td>MF-83</td>
<td>&nbsp;</td>
<tr>
<td>Axon HS-2A head stage</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>Model HS-2A</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Axoclamp 2B amplifier</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>AXOCLAMP 2B</td>
<td>&nbsp;</td>
<tr>
<td>Clampex Software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>v 8.2.0.235</td>
<td>&nbsp;</td>
<tr>
<td>Mini analysis software. v 6.0.3</td>
<td>&nbsp;</td>
<td>Synaptosoft</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Brownlee Precision Amplifier</td>
<td>&nbsp;</td>
<td>Brownlee</td>
<td>Model 410</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Baker</td>
<td>4058-01</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>p-9333 </td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>s6297-1kg</td>
<td>&nbsp;</td>
<tr>
<td>Trelahose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>TO167</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>bp220-212 </td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>h-3375</td>
<td>&nbsp;</td>
<tr>
<td>MgCl-6H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>m2670-1kg </td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>c79-500</td>
<td>&nbsp;</td>
<tr>
<td>Master-8 Pulse Generator</td>
<td>&nbsp;</td>
<td>A.M.P.I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibration table for electrophysiology set up</td>
<td>&nbsp;</td>
<td>Technical manufacturing corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrophysiological methods for recording synaptic potentials from the nmj of drosophila larvae

このビデオでは、我々は、ショウジョウバエの幼虫の神経筋接合部（NMJ）でシナプス伝達を記録するための電気生理学的方法を説明します。幼虫の神経筋システムは、シナプスの生理学と神経伝達の研究のためのモデルのシナプスであり、遺伝学を定義し、実験的操作にアクセスできる状態になっている貴重な研究ツールです。幼虫は、体壁筋肉組織、中枢神経系、および末梢神経を公開するために解剖することができます。ショウジョウバエとその神経支配のパターンの筋肉が十分に特徴付けされていると筋肉が細胞内記録のために簡単にアクセスできます。 A7 - 個々の筋肉は8腹部セグメント、セグメントA2に繰り返されるパターンに配置された30の筋肉と各内での位置や向きによって識別することができます。解剖ショウジョウバエの幼虫は薄いと個々の筋肉と運動ニューロンの軸索の束であると透視可視化することができる<sup&gt; 1</sup&gt;。トランスジェニックコンストラクトは、視覚的に識別するためのまたは特定の組織で遺伝子産物を操作するための標的細胞を標識するために使用することができます。幼虫では、興奮性接合部電位（EJPs）はシナプスでの運動ニューロンからのグルタミン酸の小胞の放出に反応して生成されます。解剖幼虫では、EJPは、細胞内電極で筋肉に記録することができます。活動電位を人為的に、腹側神経節に後方カット穏やかな吸引によってガラスのピペットに引き込まれ、電極で刺激されている運動ニューロンに誘発することができます。これらの運動ニューロンは、刺激の異なる焼成しきい値を持つものがあり、それらを同時に起動したとき、彼らは筋肉の応答を生成します。 NMJシナプスを介して伝送される信号は、運動ニューロン支配するその筋肉に記録することができます。 EJPsとミニチュア興奮性接合部電位（mEJPs）は膜電位の変化として見られている。電気生理学的応答は、修正された最小限の血リンパのような溶液中で室温で記録されます<sup&gt; 2</sup&gt;（HL3）5mMのMgを含む<sup&gt; 2 +</sup&gt;と1.5mMのCaの<sup&gt; 2 +</sup&gt;。誘発EJPsの振幅の変化は、シナプス機能と構造の違いを示すことができます。デジタル化された記録はEJPの振幅、mEJPの周波数と振幅、および量子コンテンツを分析している。

Watch this Scientific Journal Video about Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae at JoVE.com

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

ここでは、ショウジョウバエの幼虫の神経筋接合部でシナプス伝達を測定するための電気生理学的方法を説明します。誘発放出は、運動ニューロンの軸索を刺激することによって人工的に開始され、NMJの透過は、筋肉に誘発シナプス後応答によって測定することができる。

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. ...

electrophysiological-methods-for-recording-synaptic-potentials-from

Research

JoVE Journal

Biology

20.8K ビュー.  Columbia University College of Physicians and Surgeons. ここでは、ショウジョウバエの幼虫の神経筋接合部でシナプス伝達を測定するための電気生理学的方法を説明します。誘発放出は、運動ニューロンの軸索を刺激することによって人工的に開始され、NMJの透過は、筋肉に誘発シナプス後応答によって測定することができる。

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

3齢幼虫ショウジョウバエの幼虫から成虫ディスクの解剖

生物学

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

がくからパッチクランプの静電容量の録音のための方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ショウジョウバエ幼虫NMJの解剖

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

ショウジョウバエ幼虫NMJ免疫組織化学

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 ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

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. 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.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

収穫と準備ショウジョウバエ</em電気生理学的記録とその他の手続のための>胚

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

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

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

ザリガニ開閉筋肉のNMJにおいて歴史的なビューと生理学のデモンストレーション

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...