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?...

<table><tbody><tr><td>Small Petri dishes (35 x 10 mm)</td><td>BD Biosciences</td><td>1008</td><td></td></tr><tr><td>SYLGARD 182 Silicone Elastomer Kit</td><td>Dow Corning</td><td>3097366-1004</td><td></td></tr><tr><td>Dissecting microscope</td><td>Carl Zeiss, Inc.</td><td>475002-9902</td><td></td></tr><tr><td>Light for microscope</td><td>Schott AG</td><td>KLI500</td><td></td></tr><tr><td>Dissection pins</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>pClamp 9 software</td><td>Molecular Devices</td><td>PCLAMP 9 STANDARD</td><td></td></tr><tr><td>Dissection scissors: 3mm Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-0</td><td></td></tr><tr><td>Dumont SS Forceps</td><td>Fine Science Tools</td><td>11200-33</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Thin-walled borosilicate glass capillaries, with filament (1.0 mm, 4 in)</td><td>World Precision Instruments, Inc.</td><td>TW100F-4</td><td></td></tr><tr><td>Borosilicate glass capillaries, with filament (1.2 mm, 4 in)</td><td>World Precision Instruments, Inc.</td><td>1B120F-4</td><td></td></tr><tr><td>Sutter P-2000 Laser Based Micropipette Puller </td><td>Sutter Instrument Co.</td><td>Model P-2000</td><td></td></tr><tr><td>Pipette polisher</td><td>Narishige International</td><td>MF-83</td><td></td></tr><tr><td>Axon HS-2A head stage</td><td>Molecular Devices</td><td>Model HS-2A</td><td></td></tr><tr><td>Micromanipulators</td><td>Sutter Instrument Co.</td><td>MP-85</td><td></td></tr><tr><td>Axoclamp 2B amplifier</td><td>Molecular Devices</td><td>AXOCLAMP 2B</td><td></td></tr><tr><td>Clampex Software</td><td>Molecular Devices</td><td>v 8.2.0.235</td><td></td></tr><tr><td>Mini analysis software. v 6.0.3</td><td>Synaptosoft</td><td></td><td></td></tr><tr><td>Brownlee Precision Amplifier</td><td>Brownlee</td><td>Model 410</td><td></td></tr><tr><td>NaCl</td><td>Baker/VWR</td><td>4058-01</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>p-9333</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>s6297-1kg</td><td></td></tr><tr><td>Trelahose</td><td>Sigma-Aldrich</td><td>TO167</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>bp220-212</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>h-3375</td><td></td></tr><tr><td>MgCl-6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>m2670-1kg</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>c79-500</td><td></td></tr><tr><td>Master-8 Pulse Generator</td><td>A.M.P.I</td><td></td><td></td></tr><tr><td>Vibration table for electrophysiology set up</td><td>Technical Manufacturing Corp.</td><td></td><td></td></tr><tr><td>Faraday Cage</td><td></td><td></td><td></td></tr></tbody></table>

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

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JoVE Journal

Biology

20.9K 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

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

の巨大線維経路からの電気生理学的記録 D.キイロ</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

神経科学

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 ...

生物学

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

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

から電気生理学的記録<em&gt;ショウジョウバエ</em&gt; Labellar味感覚器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

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

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

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

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

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

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

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三令キイロショウジョウバエの中枢神経活動の電気生理学的記録

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

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

Source: Castells-Nobau, A., et al. <a href="https://www.jove.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. ...

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

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の巨大線維経路からの電気生理学的記録 D.キイロ

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

収穫と準備ショウジョウバエ胚

の電気生理学的記録ショウジョウバエ胚

から電気生理学的記録

の記録細胞内の電圧応答のための電気生理学的手法

全細胞電圧クランプ記録のための電気生理学的方法

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

第三令キイロショウジョウバエの中枢神経活動の電気生理学的記録

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