この作品は、NIH（ABRにRLMとR01NS25217とP30NS048154にF32のNS059120）からの助成金によってサポートされていました。

著者は何の競合する金融利益を宣言していません。

ここで説明する方法は、脊髄の最小限の解剖後のゼブラフィッシュ胚の感覚と運動ニューロンの電気的および形態学的特性評価を可能とします。ニューロンは、少なくとも1時間、これらの記録に課される時間制限のために健康なままです。ニューロンは、標準の全細胞構成を使用して記録だけでなく、有核パッチからされています。後者の方法は、イオン電流9の詳細な生?...

<html>ジョージ・ストレジンガー脊椎動物の発生10の遺伝子分析のためのモデル系として、一般的にゼブラフィッシュとして知られ、 ゼブラフィッシュの使用を開拓しました。モデルは、以下を含むいくつかの利点が提供しています：（1）比較的単純で安価な畜産を、 （2）体外受精、最も初期の発達段階から胚への容易なアクセスを可能にします。 （3）透明胚、それらが形成するように、細胞、組織、および器官の直接繰り返し観察を可能にします。 その後数十年にわたり、いくつかの進歩はさらにゼブラフィッシュモデルのパワーを増加させました。具体的には、フォワード遺伝子スクリーニング及び全ゲノム配列決定努力は、14、13、12、11、多くの発生過程に重要な変異および遺伝子の同定に重要な役割を果たし「&gt; 15、16。Gatewayクローニング方法は、転写活性化因子様（TALENs）によって例示さ、トランスジェニックのルーチンアプリケーション17、18に近づくゲノム編集の最近の進歩を可能にし、クラスタ化された定期的interspaced短いパリンドローム反復（CRISPR）-Cas9ヌクレアーゼ、 19、20、21、22に近づく変異の標的に導入を可能と同様に、ノックアウトおよびインをノック。組み合わせ、これらの方法では、ゼブラフィッシュ、特定の行動の根底にある遺伝的メカニズムと、いくつかのヒト疾患の研究のための強力なモデルを作ります23、24、25、26、27。 この作品は、開発に焦点を当てて精神的な規制や神経の発達における電気的活動の役割。焦点は、ゼブラフィッシュモデルはいくつかの利点を提供するために脊髄にあります。まず、胚および幼生の段階でゼブラフィッシュにアクセスすることは比較的容易です。そのため、人は少ないニューロンとシンプルな回路28、29を持っているの発達段階中に脊髄機能を研究することができます。また、ゼブラフィッシュ脊髄は、転写の特性及び特徴的なパターンによって実証されるように、他の脊椎動物と同様ニューロンの多様なセットを持っている30、31、32、33、34、35因子 。 特に、脊髄回路の機能の根底にあるメカニズムを解明することを目指しゼブラフィッシュにおける大多数の研究歩行をサポートするものは、当然のことながら幼虫36、37、38、39、40、41、42、43に焦点を当てています。しかしながら、脊椎機関車のネットワークを形成するニューロンの多くは、初期胚の段階でそれらの分化を開始する、〜9-10時間後に受精（HPF）44、45、46、47、48、49、50、51。そこで、脊髄神経細胞の形態学的および電気的特性が生じ、胚および幼虫期の間でどのように変化するかを理解することOveraのために重要です運動回路の形成と機能のLL理解。 ここで説明する解剖方法は、脊髄の神経細胞からパッチクランプ記録を許可し、正常胚期（〜17から48 HPF）と幼生期（〜3-7日受精後[DPF]）で適用されています。このアプローチは、関心のニューロンへのアクセスを提供するために必要な解剖の量を制限します。プロトコルは、獣医の縫合接着剤におけるゼブラフィッシュ脊髄ニューロンから記録するための他の公表された方法の大部分とは異なる記録チャンバーに胚または幼虫を取り付けるために、かなり微細なタングステンピンよりも、使用されています。 2つの異なるアプローチの可用性は（ すなわち、タングステンピン対縫合のり）電気生理学的解析のためにゼブラフィッシュの胚や幼生を取り付けるための代替オプションを持つ研究者が具体的な実験の目標を達成するために用意されています。 まず、ポップからアクセスして記録するための手順<html>一次感覚ニューロンのピュレーションは、Rohon-ベアード細胞は、記載されています。これらのニューロンの細胞体は、背側脊髄内にあります。 Rohon・ベアード細胞は、多数の脊椎動物種に存在する初期の開発で差別化、および胚タッチレスポンス6、44、47、48の基礎となります。 第二に、脊髄運動ニューロンからアクセスして記録するための手順が詳述されています。ゼブラフィッシュ脊髄運動ニューロンは、神経発生の二つの波の間に生じます。以前生まれの一次運動ニューロンはhemisegment 45、46、49あたりに存在するだけで3-4一次運動ニューロンと、原腸形成の終わり（〜9-16 HPF）で発生します。これとは対照的に、二次運動ニューロンの後に生まれ人口は、より多数で、長期間の間に発生する、〜14 HPFで始まりますEF &quot;&gt; 45、50。中間トランクセグメントにおける二次運動ニューロン起源が主に51 HPF 50によって完成される。二次運動ニューロンは、羊膜46における運動ニューロンの相手であると考えられる。興味深いことに、脊柱上のニューロンは、ドーパミンを介して、運動を制御します胚及び若い幼虫50における幼虫と二次運動ニューロン起源で、51。一次および二次運動ニューロンそれぞれは、いくつかの異なるサブタイプを含む。各一次運動ニューロンのサブタイプは、ステレオタイプその結果、特性の筋肉群を神経支配する末梢軸索を投射軸索の軌跡を識別する。一般に、二次運動ニューロンは、以前に一次運動ニューロンによって確立された軸索の経路をたどる。このように、軸索軌道に対して、一次および二次運動ニューロンを除いて、類似している軸索の厚さと細胞体サイズA一次運動ニューロン45のための大きい再。 第三に、介在ニューロンの数のタイプからの記録のための方法が説明されています。しかしながら、これらの場合に、他の脊髄細胞の除去の限られた量が必要であり、従って脊髄Rohon-ひげ細胞又は運動ニューロンから記録のためのより少ない無傷です。

<table><tbody><tr><td>Vacuum filter/Storage bottle, 0.22 mm pore</td><td>Corning</td><td>431096</td><td></td></tr><tr><td>Syringe filter 0.2 mm</td><td>Whatman</td><td>6780-2502</td><td></td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A-5040</td><td>Ethyl 3-aminobenzoate methanesulfonate salt&amp;nbsp;</td></tr><tr><td>&amp;alpha;-bugarotoxin</td><td>Tocris</td><td>11032-79-4</td><td></td></tr><tr><td>Tetrodotoxin</td><td>Tocris</td><td>4368-28-9</td><td></td></tr><tr><td>Alexa-549 hydrazine salt</td><td>Molecular Probes</td><td>A-10438</td><td>fluorescent dye</td></tr><tr><td>Spin-X centrifuge tube filter</td><td>Corning</td><td>8161</td><td></td></tr><tr><td>Glass microscope slide</td><td>Fisher</td><td>12-550C</td><td></td></tr><tr><td>Sylgard silicone elastomer kit</td><td>Dow Corning</td><td>184</td><td>silicone elastomer</td></tr><tr><td>Petri dishes</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>30-0038</td><td>inner and outer diameters of 0.78 and 1.0 mm (thin walled glass capillaries)</td></tr><tr><td>Borosilicate glass capillaries</td><td>Drummond Scientific</td><td>1-000-1000-100</td><td>inner and outer diameters of 1.13 and 1.55 mm (thick walled glass capillaries)</td></tr><tr><td>Miniature barbed polypropylene fitting&amp;nbsp;</td><td>Cole-Palmer</td><td>6365-90</td><td></td></tr><tr><td>Vetbond tissue adhesive&amp;nbsp;</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Collagenase XI</td><td>Sigma</td><td>C7657</td><td></td></tr><tr><td>Microelectrode puller</td><td>Sutter Instruments</td><td>&amp;nbsp;Model P-97&amp;nbsp;</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200B</td><td></td></tr><tr><td>Head stage</td><td>Molecular Devices</td><td>CV203BU</td><td></td></tr><tr><td>Motorized micromanipulator&amp;nbsp;&amp;nbsp;</td><td>Sutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Tygon tubing</td><td>Fisher</td><td>14-169-1B</td><td>ID 1/16 IN, OD 1/8 IN and WALL 1/32 IN (flexible laboratory tubing)</td></tr><tr><td>Electrode holder</td><td>Molecular Devices</td><td>1-HC-U</td><td></td></tr><tr><td>Pharmaseal Three-Way Stopcocks&amp;nbsp;</td><td>Baxter</td><td>K75</td><td></td></tr><tr><td>Digitizer</td><td>Axon Instruments</td><td>Digidata 1440A</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Zeiss</td><td>Axioskop2 FS plus</td><td></td></tr><tr><td>40X/0.80W Achroplan objective</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Data acquisition and analysis software&amp;nbsp;</td><td>Axon Instruments</td><td></td><td>PClamp 10 - Clampex and Clampfit&amp;nbsp;</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>Model P-97</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection and Recording Solutions (in mM)&lt;/strong&gt;</td></tr><tr><td>All solutions, except the intracellular, are stable for ~2-3 months when filtered (0.22 mm filter cups) and stored at room temperature (RT).</td><td></td><td></td><td></td></tr><tr><td>The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20 &amp;deg;C) in small aliquots that are individually thawed on the day of use.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Dissection/Ringer&amp;rsquo;s solution</td><td></td><td></td><td>145 NaCl, 3 KCl, 1.8 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 10 HEPES; pH 7.4 (with NaOH)</td></tr><tr><td>Pipette (intracellular) recording solution</td><td></td><td></td><td>135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td></tr><tr><td>Bath (extracellular) recording solution/voltage and current-clamp</td><td></td><td></td><td>125 NaCl, 2 KCl, 10 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 5 HEPES; pH 7.4 (with NaOH).</td></tr><tr><td>Alexa-594 hydrazine salt stock solution.&amp;nbsp;</td><td></td><td></td><td>Prepare a 13.2 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O, aliquot (~100 &amp;micro;l) and store at -20 &amp;deg;C. For use, dilute the stock solutiond 132 fold with pipette solution to a final concentration of 100 mM. After dilution, filter the Alexa-594 containing pipette solution&amp;nbsp; with a centrifuge tube filter.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immobilizing agents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.4% ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td></td><td></td><td>Prepare a 0.4% stock solution in 0.2 M Tris, pH 9 (0.4 g Tricaine/100 mL 0.2 M Tris</td></tr><tr><td colspan="4">Adjust pH to 7 with NaOH and store at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute the stock solution ~25 fold in embryo media</td></tr><tr><td>250 mM &amp;alpha;-bungarotoxin</td><td></td><td></td><td>Prepare a 250 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O (1 mg/500 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,500-fold with extracellular solution to a final concentration of 100 nM.</td></tr><tr><td>1 mM Tetrodotoxin</td><td></td><td></td><td>Prepare a 1 mM stock in ddH2O (1 mg/3 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,000-fold with extracellular solution to a final concentration of 500 nM.</td></tr></tbody></table>

zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons

ゼブラフィッシュは、最初に発達モデルとして導入、他の多くの分野で人気を得ています。急速に発展する生物の大量飼育の容易さは、胚の光学的透明性と組み合わせて、このモデルの初期の魅力的な属性を務めていました。過去20年間、このモデルの成功は、さらに大規模突然変異誘発画面へと遺伝子導入の容易さによって、その従順によって推進されてきました。さらに最近では、遺伝子編集手法は、モデルの力を高めています。 神経発達の研究のために、ゼブラフィッシュ胚及び幼虫は、複数の方法が適用可能なモデルを提供します。ここでは、神経細胞の本質的な特性、電気的興奮の研究を可能にする方法に焦点を当てています。ゼブラフィッシュ脊髄ニューロンの電気生理学的研究のための私たちの準備は、記録室への準備を確保する獣医師の縫合接着剤の使用を含みます。記録のための代替方法ゼブラフィッシュ胚および幼虫から微細なタングステンピン1、2、3、4、5を用いてチャンバに製剤の付着を含みます。最大4幼虫の背側をマウントするために使用されてきたものの、タングステンピンはほとんどの場合、横方向で準備を取り付けるために使用されます。縫合糸の接着剤は両方の向きで胚および幼生をマウントするために使用されています。接着剤を使用して、最小限の解剖は、それによってどんな結果として損傷を回避する、酵素処理を使用せずに脊髄の神経細胞へのアクセスを許可する、実行することができます。しかし、幼虫のために、脊髄の周囲の筋肉組織を除去するために簡単な酵素処理を施す必要があります。ここで説明する方法は、いくつかのdevelopmentaで運動ニューロン、介在ニューロン、及び感覚ニューロンの固有の電気的特性を研究するために使用されていますLステージ6、7、8、9。

我々が正常幼虫DPF 7を介して17個のHPF胚でRohon-ひげニューロンから記録された（ 図5Aおよび5B）。 Rohon-ベアード細胞を記録した場合、調製物は、背側を上に取り付けました。そのような実装は、その表層背側位置と大細胞体のサイズに基づいRohon-ビアード細胞の明確な同定を可能にします。識別は、さらにこれらのニューロンのステレオタイ?...

Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

この原稿は、ゼブラフィッシュの胚および幼生の脊髄神経細胞からの電気生理学的記録のための方法を説明します。準備は、 その場で神経細胞を維持し、多くの場合、最小の切開を必要とします。これらのメソッドは、最初の電気的興奮の買収からの早期の幼虫の段階を経て、脊髄神経細胞の様々な電気生理学的研究が可能になります。

ゼブラフィッシュ<em&gt;その場での</em&gt;脊髄脊髄感覚運動ニューロンからの電気生理学的記録のための準備

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

zebrafish-situ-spinal-cord-preparation-for-electrophysiological

Research

JoVE Journal

Neuroscience

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

11.4K ビュー.  University of Colorado Anschutz Medical Campus (UCAMC). この原稿は、ゼブラフィッシュの胚および幼生の脊髄神経細胞からの電気生理学的記録のための方法を説明します。準備は、 その場で神経細胞を維持し、多くの場合、最小の切開を必要とします。これらのメソッドは、最初の電気的興奮の買収からの早期の幼虫の段階を経て、脊髄神経細胞の様々な電気生理学的研究が可能になります。 

ビデオ: Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

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

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

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

モーターニューロンとターゲットゼブラフィッシュの骨格筋からの対のパッチクランプ記録

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

神経科学

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生きたゼブラフィッシュ胚における運動ニューロン膜電位のバイオセンシング

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

発生生物学

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

胚ゼブラフィッシュマウスナー細胞からパッチクランプ記録

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖やゼブラフィッシュ胚の横方向の取付：脊髄発達の解析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

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 ...

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 ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

ゼブラフィッシュで泳ぐ後側面線の発泡性ニューロンの活動

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

ゼブラフィッシュ幼虫の脊髄再生を研究するための制御された半自動レーザー誘発傷害

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

医学

A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos

Source: Benedetti, L., et al. <a href="https://app.jove.com/v/55297">Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos.</a> J. Vis. Exp. (2017).
This video demonstrates a non-invasive assay to study electrophysiology of motor neurons in zebrafish embryos. Upon measuring tail coiling in the embryos as a result of spontaneous depolarization of the spinal cord motor neurons, a sodium channel blocker is applied to reduce the coiling frequency. Next, the embryos are immobilized in agarose and placed under a fluorescence microscope. The genetically engineered motor neurons express a voltage-sensing biosensor with a donor and an acceptor fluorophore. A decrease in acceptor fluorophore emission indicates a reduction in the spontaneous depolarization of motor neurons due to the sodium channel blocker.

ゼブラフィッシュ胚における運動ニューロンの電気生理学を研究するための非侵襲的アプローチ

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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