著者は、実験室でゼブラフィッシュを確立するために彼らの初期の努力のためにピーター·カストロとマシューDindayに感謝したいと思います。この作品は、健康EUREKAの助成金（＃R01NS079214-01）の国立研究所によって資金を供給された。

1。卵の生産とコレクション<ol><li>ゼブラフィッシュの飼育は、以前8に記載の標準的な手順に従います。簡単に言えば、成人ゼブラフィッシュは、所定の位置に仕切りとタンク繁殖に設定されています。部屋の明かりは、次の朝に来るとき、分周器は繁殖水槽や魚から削除され邪魔されずに、相手の時間の約20〜60分を許可されています。 </li><li>繁殖タンクから卵がストレーナに集められ、卵水で洗浄する。卵は、その後、卵を水でペトリ皿に移しています。未受精卵とデブリはホールピペットを用いて除去される。 </li><li>インキュベーター（28から32℃）に収集卵を含む場所シャーレ。卵が孵化した後、受精後（DPF）を2日程度、ホールピペットで絨毛膜および他の残骸を削除します。 </li><li>希望する曜日受精後（3〜8 DPF）に室温で実験台の上で自由に泳いで幼虫と場所を含むペトリ皿を取り外します。 </li></ol><ol><li>マイクロピペットプラーを使用して、1.2ミリメートルODホウケイ酸ガラスは、愚かなパテのランプの上に敷設して150ミリメートルペトリ皿または空ピペットボックス内の2本の針や店舗へのキャピラリ引っ張る。針は事前にプルアップすることができます。異なる径ガラスキャピラリーは、使用可能なアンプヘッドステージの種類に応じて使用することができます。 </li><li>ナルゲンシリンジフィルター（4 mm）とMicrofilフィラメント（ワーナー精密機器）を添付して1ミリリットルのシリンジを用いて、細胞外記録溶液（2M N​​aCl）で荷物を積み込む微小電極。残りのほとんどまたは全く泡がなくなるまで、針の先端に向かってボーラスを振ったり、タップします。 </li></ol> 3。寒天で固定化<ol><li>卵の水の1.2％低融点寒天の新鮮な溶液を調製します。 〜37℃の水浴セットに置き寒天溶液</li><li>冷凍庫内の記録室及び場所（5〜10分）でカバースリップを準備します。記録室はMATCべき電気生理学リグで使用する時間。私たちは、ワーナー·インスツルメンツ（モデル、RC-26GLP）からロープロファイルのオープンダイヤモンド風呂イメージングチャンバーを使用しています。 </li><li>パスツールピペットまたは転送、場所きれいなペトリ皿プレートの卵の水の小滴中1幼虫のゼブラフィッシュを用いた。この液滴に、麻酔（0.02％tricaine）と麻痺剤（1 mg / mlのα-ブンガロトキシン）を含む溶液の液滴を追加します。 </li><li>運動の損失（5〜10分）のためにゼブラフィッシュを監視します。 </li><li>冷凍庫からカバーガラス/記録チャンバーを取り外します。実体顕微鏡のステージ上でチャンバーを置きます。ピペットを使用して、液滴とカバースリップ/記録室へ転送ソリューション（魚付き）、1.2％低融点アガロースの数mlを混ぜる。 </li><li>ピペットチップまたは微鈍針、魚の背側面をアガロースゲル表面に露出されるように、実体顕微鏡下で位置の幼虫を用いた。次に平らなへら転送を寒天bを用い、硬化（5-10分）にアガロースを許可電気生理学リグ上で設定記録チャンバーにロックします。 </li></ol> 3。細胞外のフィールド録音<ol><li>録音室にゼブラ記録媒体の2-5 mlを加える。薬の変更は、約1ml /分の速度で記録液で灌流チャンバー必要な場合。記録のソリューションも、酸素は必要ありません。 </li><li>三次元マニピュレーターにマウントアンプヘッドステージに微小電極（約1μmの先端径、MΩの2-7）を挿入します。マニピュレーターは、動きと調整の範囲を可能にするために適切な位置にあることを確認します。オフステージの高い、顕微鏡の視野の面に微小電極の先端を持って来て、すぐ上と若干ゼブラフィッシュの頭の前のポイントに低い粗調整を使用しています。 </li><li> &quot;電流クランプ&quot;モードゼロ電極でアンプ付き。 </li><li>それだけでsを触れるように下部電極の先端を粗調整を使用して少し前脳の前のゼブラフィッシュのurface、。 </li><li>それはゼブラフィッシュの皮膚を穿刺するまで微調整の手順を使用すると、電極先端を進める。前脳に数ミクロンを解決してから電極を前進させることができます。 </li><li> Axoscopeソフトウェアを使用して、電流クランプ·モード内の電気的活動を記録します。データは、10kHzのサンプリングレートで取得されています。 </li></ol>

<ol>
	<li>Clark, K. J., 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=Stressing+zebrafish+for+behavioral+genetics.">Stressing zebrafish for behavioral genetics.</a> Reviews in Neuroscience. 22 (1), 49 (2011).</li><li>Rinkwitz, S., 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=Zebrafish:+an+integrative+system+for+neurogenomics+and+neurosciences.">Zebrafish: an integrative system for neurogenomics and neurosciences.</a> Progress in Neurobiology. 93 (2), 231 (2011).</li><li>Penberthy, W. T., 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=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Frontiers in Bioscience. 7, d1439 (2002).</li><li>Letamendia, A., 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=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS One. 7, e36690 (2012).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nature Genetics. 26 (2), 216 (2000).</li><li>Haffter, P., 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=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral+line.">Mutations affecting development of the zebrafish inner ear and lateral line.</a> Development. 123, 1 (1996).</li><li>Suster, M. L., 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=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Molecular Biology. 561, 41 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Baraban, S. 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=Pentylenetetrazole+induced+changes+in+zebrafish+behavior,+neural+activity+and+c-fos+expression.">Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.</a> Neuroscience. 131 (3), 759 (2005).</li><li>Baraban, S. 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=A+large-scale+mutagenesis+screen+to+identify+seizure-resistant+zebrafish.">A large-scale mutagenesis screen to identify seizure-resistant zebrafish.</a> Epilepsia. 48 (6), 1151 (2007).</li><li>Williams, P., 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=The+use+of+radiotelemetry+to+evaluate+electrographic+seizures+in+rats+with+kainate-induced+epilepsy.">The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy.</a> Journal of Neuroscience Methods. 155 (1), 39 (2006).</li><li>Marsh, E. 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=Interictal+EEG+spikes+identify+the+region+of+electrographic+seizure+onset+in+some,+but+not+all,+pediatric+epilepsy+patients.">Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.</a> Epilepsia. 51 (4), 592 (2010).</li><li>Zhu, 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=Evaluation+and+application+of+modularly+assembled+zinc-finger+nucleases+in+zebrafish.">Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.</a> Development. 138 (20), 4555 (2011).</li></ol>

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ここに提示された細胞外記録法は、脳活動の非常に高感度で迅速な分析を可能にします。これらの録音は、一般的にてんかん患者11と12のげっ歯類モデルにおける異常放電（ すなわち 、発作）の存在を評価するために使用する脳波計（EEG）の監視に類似しています。ここに示されているように外の録音は、薬理学的操作と組み合わせることができます。録音のこれらの...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>低融点アガロース</td><td>フィッシャー·サイエンティフィック</td><td> BP1360-100 </td><td> 1.2％の胚メディアに溶解</td></tr><tr><td>記録媒体</td><td>フィッシャー·サイエンティフィック</td><td> BP3581、P330-3、BP410-1、BP214-500、D16-1、C77-500 </td><td> 1 mMのNaCl、2.9 mMのKCl、10mMのHEPES、1.2mMのMgCl 2、10mMのブドウ糖、2.1 mMのCaCl 2 1N NaOHで約7.3のpH </td></tr><tr><td> Tricaine </td><td>アージェント·ラボ</td><td> MS-222 </td><td> 0.02パーセント</td></tr><tr><td> α-ブンガロトキシン</td><td>バイオサイエンスTocris </td><td> 2133 </td><td> 1 mg / mlの</td></tr><tr><td>ガラスキャピラリーチューブ</td><td>ワーナー·インスツルメンツ</td><td> G120TF-3 </td><td> 2MΩ-7の抵抗にプル</td></tr><tr><td>パッチクランプ増幅器</td><td>ワーナー·インスツルメンツ</td><td> PC-505B </td><td>我々は、現在のクランプモードでワーナー·アンプを使用し、ゲインは2Kに設定2 mVの/ PA、ベッセルフィルタで設定します。匹敵するモデルは、製造元の指示に従って使用することができます。 </td></tr><tr><td>フィルタ/アンプ</td><td>シグナステクノロジー</td><td> FLA-01 </td><td>我々はCygnusプリアンプを使用し、10月20日に設定されたゲイン,1-2Kに設定カットオフ周波数、ノッチフィルターに。匹敵するモデルは、製造元の指示に従って使用することができます。 </td></tr><tr><td>アクソン、A / DボードとAxoscopeソフトウェア</td><td>分子デバイス</td><td>アクソンDigidata 1320A; Axoscope 8.2 </td><td>データは、ギャップのないアクイジションモードを使用してAxoscopeに収集され、10kHzでサンプリング。匹敵するモデルとプログラムは、製造者の指示に従って使用することができます。 </td></tr><tr><td>卵海水</td><td&gt;インスタントオーシャン</td><td></td><td> 3グラムインスタントオーシャン、海塩、10mlの脱イオン水で2 mlの0.1％メチレンブルー</td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

てんかんは、米国では約300万人、最大50万人が世界中に影響を与えます。自発的ないわれのない発作の発生として定義され、てんかんは、脳や遺伝子変異に対する侮辱の結果として取得することができる。動物でのモデル発作への取り組みは、主にげっ歯類で取得侮辱（痙攣薬、刺激や脳損傷）と遺伝子操作（アンチノックダウン、相同組換えや遺伝子組み換え）を利用してきました。ゼブラフィッシュは、げっ歯類ベースのてんかんの研究に貴重な代替手段を提供することができる脊椎動物モデル系1〜3アール。ゼブラフィッシュは脊椎動物遺伝学や開発の研究で広く使用され、哺乳動物への遺伝的類似性の高度を示し、既知のヒト単一遺伝子てんかん変異の約85％のために同族を表現しています。その小さなサイズ（長さ4〜6 mm）のため、ゼブラフィッシュの幼虫を早期に開発し、ARRA中に100μlのできるだけ低い流体ボリュームで維持することができるマルチウェルプレートでyed。試薬は、薬物投与を簡素化し、試験化合物4 の in vivoスクリーニングで急速可能胚が開発した溶液に直接添加することができる。合成オリゴヌクレオチド（モルホリノ）、突然変異誘発、ジンクフィンガーヌクレアーゼとトランスジェニックアプローチが急速に5月7日 、ゼブラフィッシュの遺伝子ノックダウンや変異を生成するために使用することができます。これらのプロパティは、ゼブラフィッシュの研究にてんかんなどの神経疾患の研究では、げっ歯類の上前例のない統計的検出力分析の利点を与える。てんかん研究のための&quot;ゴールドスタンダード&quot;は、中央脳構造（ すなわち 、発作）、効率的に幼虫のゼブラフィッシュの脳活動を記録する方法に由来する異常な放電を監視し、分析することであるため、ここで説明されます。この方法は、従来の細胞外記録技術を適応したものです、無傷のゼブラフィッシュの幼虫の脳活動の長期安定的な監視が可能となります。 S十分な録音が痙攣薬、遺伝子組み換え魚に記録されている自発的な発作の浴アプリケーションによる急性発作のために示されている。

寒天包埋したゼブラフィッシュ幼生の前脳に記録されている電子写真発作のような放電の例を図1に示す。または1mMのピクロトキシン（Bの、8 DPF）、これらの試料中の大振幅マルチスパイクバースト放電が痙攣薬浴のアプリケーション、40mMのピロカルピン（6 DPFにおける）によって誘発された。これらの録音では、固定化され、寒天包埋したゼブラフィッシュを連続最大90分まで?...

Watch this Scientific Journal Video about Forebrain Electrophysiological Recording in Larval Zebrafish at JoVE.com

Forebrain Electrophysiological Recording in Larval Zebrafish

ゼブラフィッシュ幼生前脳における細胞外電場電位を記録するためのシンプルな方法が記載されている。方法は、堅牢を提供<em生体内で&gt;</em&gt;発作のような活動の読み出し。この手法は、てんかん関連遺伝子または痙攣薬の投与により誘発される発作を運んで遺伝的に修飾されたゼブラフィッシュの幼虫と一緒に使用することができます。

ゼブラフィッシュ幼生における前脳電気生理学的記録

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

forebrain-electrophysiological-recording-in-larval-zebrafish

Research

JoVE Journal

Neuroscience

17.0K ビュー.  University of California, San Francisco. ゼブラフィッシュ幼生前脳における細胞外電場電位を記録するためのシンプルな方法が記載されている。方法は、堅牢を提供発作のような活動の読み出し。この手法は、てんかん関連遺伝子または痙攣薬の投与により誘発される発作を運んで遺伝的に修飾されたゼブラフィッシュの幼虫と一緒に使用することができます。

ビデオ: Forebrain Electrophysiological Recording in Larval Zebrafish

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

神経科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

無傷アダルトゼブラフィッシュの脳における電気生理学的記録

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

神経電位電極の体系的電気化学的および電気生理学的評価方法

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

幼虫のゼブラフィッシュにおける脊髄切断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同時電気生理学的記録およびげっ歯類の脳における阻害剤のマイクロ注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

からの電気生理学的記録<em&gt;ショウジョウバエ</em&gt;低揮発性の臭気物質に対するトリコイドセンシラ

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

分離およびゼブラフィッシュ稚魚の脳から神経細胞、大食細胞、ミクログリアの RNA の抽出

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...