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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
		</tr>
		<tr>
			<td>a-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&#160;</td>
			<td>Cole-Palmer</td>
			<td>6365-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive&#160;</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>&#160;Model P-97&#160;</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&#160;&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Axon Instruments</td>
			<td colspan="2">PClamp 10 - Clampex and Clampfit&#160;</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="2">Dissection and Recording Solutions(in mM)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">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>
		</tr>
		<tr>
			<td colspan="4">The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20&#176;C) in small aliquots that are individually thawed on the day of use.&#160;</td>
		</tr>
		<tr>
			<td colspan="2" rowspan="1">Dissection/Ringer&#8217;s solution</td>
			<td></td>
			<td colspan="2">145 NaCl, 3 KCl, 1.8 CaCl2.2H2O, 10 HEPES; pH 7.4 (with NaOH)</td>
		</tr>
		<tr>
			<td colspan="2">Pipette (intracellular) recording solution</td>
			<td></td>
			<td colspan="2">135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td>
		</tr>
		<tr>
			<td colspan="2">Bath (extracellular) recording solution/voltage and current-clamp</td>
			<td></td>
			<td colspan="2">125 NaCl, 2 KCl, 10 CaCl2.2H2O, 5 HEPES; pH 7.4 (with NaOH).</td>
		</tr>
		<tr>
			<td colspan="2">Alexa-594 hydrazine salt stock solution.&#160;</td>
			<td></td>
			<td colspan="2">Prepare a 13.2 mM stock in ddH2O, aliquot (~ 100 &#181;l) and store at -20&#176;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&#160; with a centrifuge tube filter.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immobilizing agents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">0.4 % ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td></td>
			<td colspan="2">Prepare a 0.4% stock solution in 0.2M Tris, pH9 (0.4 g Tricaine/100 ml 0.2 M Tris</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">Adjust pH to 7 with NaOH and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">For use, dilute the stock solution ~ 25 fold in embryo media</td>
		</tr>
		<tr>
			<td>250 &#956;M &#945;-bungarotoxin</td>
			<td></td>
			<td colspan="2">Prepare a 250 &#956;M stock in ddH2O (1 mg/500 &#956;l), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">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 colspan="2">Prepare a 1 mM stock in ddH2O (1 mg/3 ml), prepare 100 &#181;l aliquots, and sotre at -20&#176;C.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

縦断のために脊髄商工を移植するための手順<em&gt;インビボ</emマウス脊髄の&gt;イメージング

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

前根刺激のための斜め脊髄スライスの調製

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

成体マウスの脊髄組織からの初代混合グリア培養の調製

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

新生児マウスからの脊髄ニューロンの単離と培養

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニック マウス

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

リズミカルにアクティブな準備体外新生児齧歯動物脳幹-脊髄損傷で薄くスライス

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

出生前イスルmnからのオキュロモータ、トロクレア、脊髄運動ニューロンの単離と培養 :GFPトランスジェニックマウス

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...