この作品は、健康補助RO1GM - 33205およびLCグリフィスへMH - 067284の国民の協会によっておよびNJホーンスタインのブランダイス大学の夏学部研究奨学金によってサポートされていました。ウッズホール、マサチューセッツ州：このテクニックのための予備実験は、2008年ニューラルシステムと行動のサマーコース（R25 MH059472 NIMH助成金）の一環として、海洋生物学研究所で実施した。

パート1：動物のケアと遺伝的交雑<ol><li> UAS - ChR2と標準のフライのメディア8を含む別のボトルにOK371 - GAL4フライラインを維持する。 </li><li> OK371 - GAL4フライラインとUAS - ChR2行の男性の処女を収集する。 </li><li> 1mMの全トランスレチナール（ATR）と混合標準フライメディアを含むバイアルに雄と雌の両方を置く。 ATR食べ物は〜1分間電子レンジで最初の溶融、通常のフライのメディアによってなされるべきである。一度溶けた、1分間に約30秒間の冷却し、ハエのメディアのすべての10 mlの100％EtOH中100mMのATRの100μlを追加することができます。その後、氷上に置きますバイアル、4で暗いエリアで店舗℃に</li><li>ハエの仲間を聞かせて22から25に暗いエリアで産卵℃に</li><li> 3齢幼虫が見えるようになるまで3-4日待って、その時点で、成人のハエ処分する。 </li></ol>パート2：リグのセットアップ<ol><li>青色LEDへの10倍解剖スコープアイピース（我々の場合で、カールツァイス社、www.zeiss.com、ソーンウッド、ニューヨーク州から）、ヒートシンクの光源（トールラボ、www.thorlabs.com、ニュートン、ニュージャージー州を取り付けます）。ポストとクランプ付きマグネットベースに取り付けます。光源によって生成された電気的ノイズを減らすために電気的に接地されたシールドとヒートシンクをカバーしています。 </li><li>電気生理学リグのエアテーブル上に光源のある場所マグネットベース。 </li><li>回路を制御し、外部電圧源（Powerlab 4月30日、ADInstruments、www.adinstruments.com、コロラドスプリングズ、コロラド州）に制御回路を接続するための光源を接続してください。 </li><li>青色光をアクティブにする回路を制御するために1 - 5Vのパルスを与える。 </li><li>青色光ビームは幼虫の解剖によって占有される領域に焦点を当てされるまで、マグネットベースと光源を調整します。 </li></ol>パート3：解剖<ol><li> sylgardが並ぶ料理の床にカ所0.1ミリメートル虫ピン。 </li><li>ペトリ皿、プラスチックへの食品のメディアや場所から第3齢幼虫を削除します。 </li><li>食べ物を削除するには生理食塩水で幼虫を洗浄します。 </li><li>プルピンの近くに皿を解剖し、½レベルに生理食塩水追加の場所の幼虫。 </li><li>オリエント幼虫は動物の背側表面に沿って実行している2つの銀色のチューブ（気管）を参照できるようにする。 </li><li>気管チューブの間に尾に直接大きなピンを挿入します。縦に体を伸ばして確認してから、幼虫を押しながら、その頭に第二大ピンを配置します。 </li><li>尾の近くに小さな切開を加える、と体の長さをそれを継続する。誤って腹神経および/または体壁の筋肉を切断しないようにハサミの先端がそのよう提起していることを確認します。 </li><li>動物の四隅に置いて4本のピンは、現在開いている中央部です。フィレット動物をするように解剖皿にそれらを設定します。ピンの準備が出教えた。 </li><li>鉗子やハサミを使用して、動物の内臓、気管および脂肪体を除去。 </li><li>生理食塩水で準備をすすぐ。 </li><li>前頭葉（図Aに見られる）と準備の腹側神経節を見つけます。 </li><li>マイクロはさみを使用して、慎重にちょうど前頭葉の後ろの腹側神経節を介してカット。 </li></ol>パート4：筋肉の記録と青色光の刺激<ol><li>電子電極プラー（サッタインスツルメンツ）を使用して10月20日μω電極を引き出します。 </li><li> 3 M KClで引っ張ら電極を埋める。 </li><li>場所は、電気生理学リグ上で解剖顕微鏡下で幼虫の予備校の近くにマイクロマニピュレータと電極ホルダーと操縦電極の先端に電極を埋め。 </li><li>任意の体の壁のセグメント（図A）に筋6（M6）を識別する。 </li><li>慎重にM6に電極を挿入し、迅速な過分極を監視します。筋肉静止膜電位は-30 mVから-70 mVにどこにする必要があります。 </li><li>解剖準備が光ビームの中央になるように青い光を調整します。 </li><li>回路を制御するために20から100ミリ秒の電圧パルスを与える。段階的に回路を制御するために供給される電圧を増加させる。筋細胞（図1B）における興奮性接合部電位を監視。 </li></ol>代表的な結果： 図1Aは、レコーディングのセットアップとフィレット作成の概略を示しています。図1Bは、短い光パルスにより誘発される典型的なEJPを示しています。示すように、EJPの振幅は、支配するM6の両方に知られている2つの運動ニューロンから振幅が​​加算される。より低い光強度のみアクティブに1つのモータユニット（データは示さず）。 <img src="/files/ftp_upload/1133/figure1.png" alt="図1" width="600" height="373" /> 図1：細胞内記録のリグのと青色LED付き）一般的な回路図。脳（BR）は、腹側神経節（VG）のリズミカルな活性を阻害するために削除されます。 ChR2は、GAL4 - UASシステムを使用して運動ニューロンに発現している。 M6筋肉からB）細胞内記録。 40ミリ青色の光パルスは、（127μW/ mm 2の時）確実にM6に大きなシナプス電位（アスタリスク）を呼び起こす。

<ol>
	<li>Keshishian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545 (1996).</li><li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+development:+insights+from+Drosophila.">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35 (2007).</li><li>Lagow, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+a+hydrophobic+layer+by+a+point+mutation+in+syntaxin+1A+regulates+the+rate+of+synaptic+vesicle+fusion.">Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.</a> PLoS Biol. 5 (4), e72 (2007).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279 (2005).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940 (2003).</li><li>Schroll, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr Biol. 16 (17), 1741 (2006).</li><li>Lin, D. M., Auld, V. J., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+neuronal+cell+ablation+in+the+Drosophila+embryo:+pathfinding+by+follower+growth+cones+in+the+absence+of+pioneers.">Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.</a> Neuron. 14 (4), 707 (1995).</li><li>Ralph Greenspan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The Theory and Practice of Drosophila Genetics, 2nd ed. , (2004).</li></ol>

我々は、開示に関心の競合はありません。

重要なステップは、筋細胞の初期の解剖と入力の両方を含む。最初の解剖中に神経が切断されたり、筋肉が損傷している場合には、実験の残りを継続することは困難である。解剖時に、1つは、背側切開中に可能な限り上方解剖ハサミを角度に非常に慎重でなければならない。筋細胞に入る第二の重要なステップ、中に、1つは、過去の過分極〜30 mVのために監視しなければなりません。 -30 mV?...

<table><tbody><tr><td>Sylgard</td><td>Ellsworth Adhesives</td><td>4019862</td><td>&lt;a href=&quot;http://www.ellsworth.com&quot; target=&quot;_blank&quot;&gt;www.ellsworth.com&lt;/a&gt;</td></tr><tr><td>Minutens Pins</td><td>Fine Science Tools</td><td>26002-10</td><td>&lt;a href=&quot;http://www.finescience.com&quot; target=&quot;_blank&quot;&gt;www.finescience.com&lt;/a&gt;</td></tr><tr><td>Dissecting Dish</td><td>Fisher Scientific</td><td></td><td>&lt;a href=&quot;http://www.fishersci.com&quot; target=&quot;_blank&quot;&gt;www.fishersci.com&lt;/a&gt;</td></tr><tr><td>Neuroprobe Intracellular Amplifier and Head Stage</td><td>A-M Systems</td><td>680100</td><td>&lt;a href=&quot;http://www.a-msystems.com&quot; target=&quot;_blank&quot;&gt;www.a-msystems.com&lt;/a&gt;</td></tr><tr><td>&lt;a href=&quot;http://www.adinstruments.com/products/hardware/corporate/product/ML866/&quot; target=&quot;_blank&quot;&gt;Powerlab 4/30 data acquisition system&lt;/a&gt;</td><td><a href="http://www.adinstruments.com" target="_blank"><img src="/img/new_window.png" /> ADInstruments</a></td><td></td><td>&lt;a href=&quot;http://www.adinstruments.com&quot; target=&quot;_blank&quot;&gt;www.adinstruments.com&lt;/a&gt;</td></tr><tr><td>Grass stimulator</td><td>Grass Technologies</td><td></td><td>&lt;a href=&quot;http://www.grasstechnologies.com&quot; target=&quot;_blank&quot;&gt;www.grasstechnologies.com&lt;/a&gt;</td></tr><tr><td>Desktop Computer</td><td>Dell</td><td></td><td>&lt;a href=&quot;http://www.dell.com&quot; target=&quot;_blank&quot;&gt;www.dell.com&lt;/a&gt;</td></tr><tr><td>Dissecting Scope</td><td>Leica Microsystems</td><td></td><td>&lt;a href=&quot;http://www.leica-microsystems.com&quot; target=&quot;_blank&quot;&gt;www.leica-microsystems.com&lt;/a&gt;</td></tr><tr><td>Light Source</td><td>Dolan-Jenner Industries</td><td>41446-062</td><td>&lt;a href=&quot;http://www.dolan-jenner.com&quot; target=&quot;_blank&quot;&gt;www.dolan-jenner.com&lt;/a&gt;</td></tr><tr><td>Fly Media</td><td></td><td></td><td></td></tr><tr><td>All-Trans-Retinal</td><td>Sigma-Aldrich</td><td>116-31-4</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com&quot; target=&quot;_blank&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>&lt;em&gt;OK-371 Gal4&lt;/em&gt; Flies</td><td>Bloomington Stock center</td><td></td><td></td></tr><tr><td>&lt;em&gt;UAS-ChR2&lt;/em&gt; Flies</td><td>Fiala lab, Griffith lab</td><td></td><td></td></tr><tr><td>LED controller circuit</td><td>Built in Griffith lab</td><td></td><td>&lt;a href=&quot;http://www.ledsupply.com&quot; target=&quot;_blank&quot;&gt;http://www.ledsupply.com&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.futureelectronics.com&quot; target=&quot;_blank&quot;&gt;http://www.futureelectronics.com&lt;/a&gt;&lt;br/&gt;Composed of: &lt;br/&gt;1. 200 mA Buck Puck&lt;br/&gt;2. Blue LED&lt;br/&gt;3. Insulated wire&lt;br/&gt;4. Circuit bread board&lt;br/&gt;</td></tr><tr><td>LED Heat Sink</td><td>Thorlabs Inc.</td><td></td><td>&lt;a href=&quot;http://www.thorlabs.com/&quot; target=&quot;_blank&quot;&gt;http://www.thorlabs.com/&lt;/a&gt;</td></tr><tr><td>Air Table</td><td>TMC</td><td></td><td>&lt;a href=&quot;http://www.techmfg.com/products/accessories/intro3.html&quot; target=&quot;_blank&quot;&gt;http://www.techmfg.com/products/accessories/intro3.html&lt;/a&gt;</td></tr><tr><td>Faraday Cage</td><td>Built in Griffith lab</td><td></td><td></td></tr><tr><td>Leica Leitz M Micro-Manipulator</td><td>Leica Microsystems</td><td>ACS01</td><td>&lt;a href=&quot;http://www.leica-microsystems.com&quot; target=&quot;_blank&quot;&gt;www.leica-microsystems.com&lt;/a&gt;</td></tr><tr><td>Electrode Holder</td><td>Axon Instruments</td><td></td><td>&lt;a href=&quot;http://www.axon.com&quot; target=&quot;_blank&quot;&gt;www.axon.com&lt;/a&gt;</td></tr><tr><td>Borosilicate Glass</td><td>FHC, Inc.</td><td></td><td>&lt;a href=&quot;http://www.fh-co.com/p14-15.pdf&quot; target=&quot;_blank&quot;&gt;www.fh-co.com/p14-15.pdf&lt;/a&gt;</td></tr><tr><td>Electrode Puller</td><td>Sutter Instrument Co.</td><td></td><td>&lt;a href=&quot;http://www.sutter.com&quot; target=&quot;_blank&quot;&gt;www.sutter.com&lt;/a&gt;</td></tr><tr><td>HL 3.1 Saline with 0.8mM Ca2+</td><td></td><td></td><td>Contents (mM):&lt;br/&gt; NaCl:70 KCl:5 CaCl&lt;sub&gt;2&lt;/sub&gt;: 0.8&lt;br/&gt; MgCl&lt;sub&gt;2&lt;/sub&gt;:4Sucrose:115&lt;br/&gt;NaHCO&lt;sub&gt;3&lt;/sub&gt;: 10 Trehalose: 5 HEPES</td></tr><tr><td>Micro-Dissection Tools</td><td>Fine Science Tools</td><td></td><td>&lt;a href=&quot;http://www.finescience.com&quot; target=&quot;_blank&quot;&gt;www.finescience.com&lt;/a&gt;</td></tr></tbody></table>

channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions

市販<em&gt;ショウジョウバエ</em&gt;幼虫の神経筋標本では、シナプスの生理学を研究するための有用なツールであることが実証されています<sup&gt; 1,2,3</sup&gt;。現在のところ、これだけの準備で興奮接合部の電位（EJPsを）呼び起こす可能なことを意味吸引電極の使用を含む。研究と教育研究所の両方で、学生はしばしば困難刺激電極のこのタイプを操縦し、操作している。本研究では、我々は、リモートで吸引電極を使用せず幼虫のNMJでのシナプス電位を刺激する方法を示します。 channelrhodopsin2（ChR2）を発現することにより<sup&gt; 4,5,6</sup&gt;の<em&gt;ショウジョウバエ</em使用して&gt;運動ニューロン<em&gt; GAL4 - UAS</em&gt;システム<sup&gt; 7</sup&gt;、および基本的な電気生理学のリグにマイナーな変更を加える、我々は、青色光のパルスでEJPsを確実に喚起することができた。この手法は、学生のリグの練習時間と資源が限られている神経生理学の教授のラボで特定の使用の可能性があります。

Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

このプロシージャは、神経筋接合部（NMJ）の光パルスでシナプス電位を惹起する青色光活性化藻類チャネルと細胞特異的遺伝子発現のツールを使用しています<em&gt;ショウジョウバエ</em&gt;幼虫。この手法は、安価で研究と教育研究所のシナプス生理学研究のための吸引電極の刺激に代わる使いやすいです。

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 ...

channelrhodopsin2-mediated-stimulation-synaptic-potentials-at

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

13.6K ビュー.  Brandeis. このプロシージャは、神経筋接合部（NMJ）の光パルスでシナプス電位を惹起する青色光活性化藻類チャネルと細胞特異的遺伝子発現のツールを使用していますショウジョウバエ幼虫。この手法は、安価で研究と教育研究所のシナプス生理学研究のための吸引電極の刺激に代わる使いやすいです。

ビデオ: Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

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

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

生物学

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. ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

半無傷でレーザー照射による神経活動のOptogeneticの摂動<em&gt;ショウジョウバエ</emイン·モーション&gt;幼虫

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

神経科学

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 ...

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac.

テトロドトキシン耐性ナトリウム チャネルを用いたショウジョウバエのシナプス接続を調べる

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

シナプスでサイクリング FM 色素: 高カリウムの脱分極、電気とチャネルロドプシン刺激の比較

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

青色および赤色シフトチャネル性肝炎を伴う下コリキュラスニューロンの長距離チャネル性チャネル化補助回路マッピング

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, ...

Synaptic Modulation in Drosophila After Exposure to Prolonged Light

Source: Sugie, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55176">Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light.</a> J. Vis. Exp. (120)
This video demonstrates the rearing of Drosophila melanogaster under specific light conditions to study the role of Bruchpilot proteins in neurotransmitter release and the resulting modulation of synaptic activity.

長時間の光への曝露後のショウジョウバエのシナプス調節

1. Light Exposure Conditions

	From the larva stage until eclosion, keep the flies in a 12 h light/12 h dark cycle (LD) at 25 &#176;C (Figure 1A).
	...

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

要約

さらに動画を探す

この動画の章

ショウジョウバエ幼虫NMJ免疫組織化学

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

半無傷でレーザー照射による神経活動のOptogeneticの摂動

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

テトロドトキシン耐性ナトリウムチャネルを用いたショウジョウバエのシナプス接続を調べる

シナプスでサイクリング FM 色素: 高カリウムの脱分極、電気とチャネルロドプシン刺激の比較

青色および赤色シフトチャネル性肝炎を伴う下コリキュラスニューロンの長距離チャネル性チャネル化補助回路マッピング

長時間の光への曝露後のショウジョウバエのシナプス調節

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

要約

さらに動画を探す

この動画の章

ショウジョウバエ幼虫NMJ免疫組織化学

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

半無傷でレーザー照射による神経活動のOptogeneticの摂動

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

テトロドトキシン耐性ナトリウム チャネルを用いたショウジョウバエのシナプス接続を調べる

シナプスでサイクリング FM 色素: 高カリウムの脱分極、電気とチャネルロドプシン刺激の比較

青色および赤色シフトチャネル性肝炎を伴う下コリキュラスニューロンの長距離チャネル性チャネル化補助回路マッピング

長時間の光への曝露後のショウジョウバエのシナプス調節

テトロドトキシン耐性ナトリウムチャネルを用いたショウジョウバエのシナプス接続を調べる