著者は、有益なコメントを博士アリステアGarratt（マックスデルブリュックセンター、ベルリンの）感謝します。この作品は、ドイツの研究評議会（DFG）の共同研究センター（SFB665）によってサポートされていました。

1。解剖の手順 注 ：マウスの実験的使用は、実験動物の管理と使用のための公式に承認されたガイドラインに従ってください。 <ol><li>準備する前に、あなたの解剖顕微鏡を設定し、大型と小型のはさみ、大歯鉗子、湾曲鉗子と鉗子（内側研磨のヒントを持っているうちの2つを）解剖デュモン5号の4組を含む解剖に必要な手術器具をレイアウト（詳細については、）特定の試薬や機器の表を参照してください。 100 mmのSylgardコーティングしたシャーレにろ紙のシートを置きます。 、12ウェルプレートの各ウェルに100 mmのペトリ皿および12 ​​mlチューブを冷PBSを注ぎ、氷の上に残す。氷上で2番目の12ウェルプレートと場所の各ウェルに固定バッファのピペット2ミリリットル（PBS、pH7.4中4％パラホルムアルデヒド）。 </li><li>麻酔後、E12.5（膣栓の検出はE0.5として指定された）タイミング妊娠ダムを犠牲に、マウスを置く紙タオル3枚で、最大の腹側で、70％エタノールで腹部を浸す。 </li><li>腹骨盤腔を開き、二国間の子宮角を分離し、氷冷PBSで100 mmディッシュの一つにそれらを転送する。 </li><li>微細な制御下で、小さな、ストレートハサミで子宮壁の縦切開を行い、胎盤から各羊膜嚢を切り取る。 </li><li>離れてピール各胚から羊膜嚢とへその緒を切る。 </li><li>タイピングが要求されるべき場合、小さなはさみを使用して、胚を刎ねるとゲノムDNAの単離のためにヘッドまたはそれらの一部を脇に置く。その後、冷PBSで12ウェルプレートの各ウェルにtorsoの複数形を転送する。 </li><li> PBSを数滴Sylgard皿にろ紙を湿らせ、紙にその背側と胚の胴を置きます。優れた安定性のために、身体から離れて、その尾と四肢をまっすぐ。 </li><li>操作約16倍の倍率で解剖顕微鏡下で、慎重に微細な先端鉗子の2組脊髄上記の胚の皮膚をつまんで、ゆっくりとそれを引き裂く。途中で始まる、尾に向けた最初の進んでして、前方側に向かって途中から再開。 </li><li>乾燥を防ぐためにPBSの二、三滴で時間から時間まで胚を濡らす。 </li><li>それは内部の研磨チップによる微細な鉗子の刃で水平方向のスライド運動によって周囲の軟骨性脊柱からDRGと脊髄を切断胚から削除されたときにそのDRGを防ぐためには、脊髄からオフtearedされています。胚の右側の真ん中に開始、前端にして尾部に向かって、あなたのように働くと。その後、反対側の手順を繰り返します。関連したDRGを裂くように注意してください。 </li><li>胚から完全に緩んで脊髄を切断するために、そのcervicを拾うらは、微細な鉗子を持つ部分とその尾方端に向かって一体に引き出します。 </li><li> 12ウェルプレートのウェルに接続されたDRGと水没孤立脊髄は、固定バッファで満たされ、残りの胚から脊髄の準備を進める。 </li><li>氷上での脊髄少なくとも2時間を修正。 </li></ol> 2。 DRGニューロンのDII標識<ol><li>ラベル付けされる各脊髄の場合は、マイクロピペットプラーで2つのガラス針を準備。 。。。、2 HEAT = 600、VEL = 25、TIME = 175、3 HEAT = 630、VEL = 1 HEAT = 625、VEL = 35、TIME = 175：我々は、サッターモデルP - 97電極プラー（プログラムの設定を使用30、TIME = 150）。 </li><li>微細な制御下でエタノールのDIIの5％（w / v）溶液中の各ガラス針の先端3〜5倍のディップ。エタノールを蒸発させると、針のDII結晶の薄層を作成します。 </li><li>約40倍の倍率で解剖顕微鏡下で働いて、そのvを付けて脊髄を配置スライド上のentralサイドアップ。春のはさみを使用して、後で逆のオープンブックモード（セクションCを参照）で準備のマウントを可能にするfloorplateを切断することによって、全体の長さで腹側にコードを開きます。 </li><li>スライド上で、最大背側に脊髄を置きます。その後、手動で顕微鏡可視化の下でガラス針のDIIに覆われた先端と脊髄の片側に慎重に穴を開けるには毎秒DRGと脊髄にアプローチ。 DIIのほとんど痕跡は、神経細胞のごく少数のラベルを確保するためにピアスDRGに表示されないはず。コー​​ドのもう一方の側に第二針を取る。のみ毎秒DRGを目指しては、個々の軸索の分化を複雑にするかもしれない隣接DRGからラベル付き軸索投射の重複を避けることができます。 </li><li> ° Cでディに染料のために時間を与えるために4℃、周囲温度または一晩で少なくとも6時間暗所での固定バッファとインキュベートして脊髄を返す原形質膜内の軸索に沿ってffuse。脊髄への担保の成長の分析が望まれる場合には、より長い潜伏期間（4時までの二日間° C）を可能にする。 </li></ol> 3。取り付けと顕微鏡分析<ol><li>その背側を持つ単一の脊髄では、PBSのドロップにカバースリップを下に染料拡散、位置に続く。添付のDRGが横方向に正しく向いていると混在しないように注意してください。余分な水分を吸引し、鉗子を用いて反転、オープンブックモードでコードを平らにならす。 </li><li> PBSを用いて顕微鏡スライド上に準備をマウントします。 </li><li>標識した軸索突起の蛍光顕微鏡分析を染色不要なバックグラウンドの上昇を防ぐために、取り付けの日を行ってください。それまでは4℃、暗所でスライドを保持℃を</li></ol> 4。代表的な結果： マウスの脊髄では、求心性のPRを受け取る子宮頸部の8組からojections、胸部の13ペア、60脊髄神経節に合計仙骨DRGの腰椎と4組の5組。いくつかのトレーニングの後、まだ接続されているほとんどのDRGと脊髄は、5分以内で胚から単離することができる。この手順は、E11.5からE13.5マウス胚に添付されたDRGと脊髄の分離に適しています。しかし、最良の結果がE12.5から得られます。ここで説明する標識手順の典型的な結果を図3に示されています。脊髄の全長にわたってDRGの標識は、その後、別の脊椎レベル（ 図4）に軸索の分岐動作を定量化するために使用されることがあります。 <img alt="図1" src="/files/ftp_upload/3667/3667fig1.jpg" /> 図1：Schemeの軸索分岐の二つの主要なモードを描いた。ここに示されるように - - （A）分岐につながる可能性のある成長円錐の活動により分岐するだけでなく、複雑な端末アーバー（B）軸索のシャフト（間質性分岐）における担保の形成。後者のモードでは、6,7皮質と視床皮質軸索を投射するの支配分岐タイプです。 <img alt="図2" src="/files/ftp_upload/3667/3667fig2.jpg" /> 図2胚の脊髄へのDRGニューロンの求心性投射が軸索分岐の両方のタイプが表示されます：。分岐によってDREZ（1）および間質分岐（2）によって待機期間後に得られる娘の支店の副血行路の形態からの軸索最初のブランチを。 <img alt="図3" src="/files/ftp_upload/3667/3667fig3.jpg" /> 図3。胚のマウスのDRGニューロンの単一の軸索軌道の可視化。 （A）付属のDRGと脊髄は、E12.5マウス胚から調製。増加倍率での野生型マウスのDII標識DRGの（スケールバーは1mm。）（BD）背側のビューが表示されます。 Bで毎秒DRGはlabeleですDIIによるD。蛍光画像が反転されて、尾は左にあり、横方向のCとD、で一番下にあります。 Cでの軸索の数が少ないが標識され、より高い倍率でT -のような枝の存在は、脊髄のDREZで識別することができます。 （スケールバーは、250μm以下（B）、100μmの（C）および50μm（D）。） <img alt="図4" src="/files/ftp_upload/3667/3667fig4.jpg" /> 野生型およびC型ナトリウム利尿ペプチド（CNP）欠損E13.5のマウスのT字型の枝の図は、4。定量、単一の吻側または尾ターン。カウント単一の軸索の番号は、各遺伝子型ごとに異なる​​トランクレベルのカッコ内に表示されます。 C -またはR -ターン - 尾または吻側方向にのみ成長、それぞれ。 <img alt="図5" src="/files/ftp_upload/3667/3667fig5.jpg" /> 図5。cGMPのシグナル伝達経路は、脊髄のDREZで感覚軸索分岐をトリガします。の（A）スキームリガンドCNP、受容体グアニル酸シクラーゼNpr2とセリン/胚DRGニューロンにおけるスレオニンキナーゼcGKIαで構成されるcGMPのシグナル伝達経路。 Npr2は、CNPによる刺激によりGTPからcGMPを生成します。 （B）DIIは、野生型およびCNP欠損マウスでは神経細胞をDRGの単一の軸索のトレース。 （スケールバーは25μm。）

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著者らは、開示することは何もない。

DIIラベリングのための固定組織の使用と組み合わせての準備の容易さと一緒に軸索枝の形成の両方のタイプで構成されるステレオタイプ化された投影パターンは分岐軸索の勉強に有利なモデルをDRGの添付を持つ胚脊髄になります。コー​​ティングされたガラスの針を使用してDIIの微量のアプリケーションが可能に - DRGニューロンの小集団の可視化と、それによって個々の軸索とその分岐パ...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td>実体顕微鏡STEMI DRC </td><td>ツァイス</td><td></td><td></td></tr><tr><td>リン酸緩衝溶液（PBS） </td><td> Biochrom AG </td><td> L182 - 50 </td><td></td></tr><tr><td>パラホルムアルデヒド</td><td>メルク</td><td> 8.18715.1000 </td><td></td></tr><tr><td>標準的な外科はさみ</td><td>ファイン科学ツール</td><td> 14001〜13 </td><td></td></tr><tr><td>歯標準鉗子</td><td>ファイン科学ツール</td><td> 11021〜14 </td><td></td></tr><tr><td>極細アイリスのはさみ</td><td>ファイン科学ツール</td><td> 14088〜10 </td><td></td></tr><tr><td>湾曲鉗子</td><td>ファイン科学ツール</td><td> 11003〜13 </td><td></td></tr><tr><td>デュモン5号ファインチップの鉗子</td><td>ファイン科学ツール</td><td> 11254〜20 </td><td></td></tr><tr><td>デュモン5号鏡面仕上げ鉗子</td><td>ファイン科学ツール</td><td> 11252〜23 </td><td></td></tr><tr><td> Vannas -テュービンゲンバネはさみ</td><td>ファイン科学ツール</td><td> 15008〜08 </td><td></td></tr><tr><td>ろ紙</td><td>フィッシャーサイエンティフィック</td><td> FB59041 </td><td></td></tr><tr><td> Sylgard 184 </td><td>世界のPrecissionインスツルメンツ</td><td> SYLG184 </td><td></td></tr><tr><td> 100 mmのペトリ皿</td><td> Greinerの</td><td> 663102 </td><td></td></tr><tr><td> 12 mlのポリプロピレンチューブ</td><td>カールロート社</td><td> ECO3.1 &lt;/ TD&gt; <td></td></tr><tr><td> 12ウェル培養プレート</td><td>ベクトンディッキンソン</td><td> 35から3043 </td><td></td></tr><tr><td>エタノール</td><td>メルク</td><td> 1.00983.2500 </td><td></td></tr><tr><td>ブラウン/燃えるようなマイクロピペットプラーP - 97 </td><td>サターインストゥルメント（株） </td><td></td><td></td></tr><tr><td>ホウケイ酸ガラス毛細管</td><td>ハーバード装置</td><td> 30から0066 </td><td></td></tr><tr><td> DII（1,1&#39; -ジオクタデシル- 3、3,3&#39;、3&#39; -テトラメチル-過塩素酸塩をindocarbocyanine） </td><td>シグマアルドリッチ</td><td> 468495 </td><td></td></tr><tr><td>顕微鏡はSuperFrost Plusをスライド</td><td>カールロート社</td><td> H867.1 </td><td></td></tr><tr><td>ガラスカバースリップ</td><td>カールロート社</td><td> 1870.2 </td><td></td&gt; </tr></tbody></table>

dii-labeling of drg neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord

ここでは、親油性トレーサー1,1&#39; -ジオクタデシル- 3、3,3&#39;、過塩素酸塩（DII）1 3&#39; - tetramethylindocarbocyanineを使用して拡散染色により胚脊髄にDRGニューロンの小グループの軌跡をラベル付けする手法を提案。遺伝子が変異されたマウス系統のものと野生型の軸索経路の比較では、神経系の配線に不可欠な機構である軸索分岐の制御の候補タンパク質の機能的役割をテストできます。軸索分岐は、それによって情報の並列処理のための物理的な基礎を提供する、複数のターゲットに接続する個々のニューロンが可能になります。軸索成長の中間ターゲット領域での影響は、終末分枝と区別されることがあります。また、軸索枝形成の異なるモードが成長円錐の活動（分割または遅延ブラジャーからかどうかの分岐の結果に応じて分類することができるnching）またはプロセスにおける軸索のシャフトから担保の出芽からは、間質分岐2（ 図1）と呼ばれる。 DRGからのニューロンの中央の突起軸索分岐の両方のタイプを研究するために有用な実験系を提供する：それらの求心性軸索は10〜13胚日の間に脊髄の後根のエントリーゾーン（DREZ）に到達（E10 - E13）、それらT -またはY字型の分岐部の紋切り型のパターンが表示されます。得られた2つの娘の軸索は、脊髄の背側縁に、それぞれ、吻側または尾側方向に進むと待機期間の担保は、特定の中継ニューロンに灰白質（間質分岐）とプロジェクトを貫通して、これらの幹の軸索から発芽した後にのみ彼らはさらに樹枝状になる脊髄のラミナ（端子分岐）3。 DIIの追跡機能は、PRにあるように見えた脊髄の後根のエントリーゾーンで成長円錐を明らかにしたその分岐を示唆分割のocessが成長円錐自体4（ 図2）の分割によって引き起こされる、しかし、他のオプションはよく5として議論されている。 このビデオでは、接続されているDRGを残しE12.5マウスの脊髄を分析する方法を第一を示しています。 DIIの試料微量の以下の固定は、キャピラリーチューブから引っ張ってガラスの針を使用して、DRGに適用されます。インキュベーションステップの後、標識された脊髄は、蛍光顕微鏡を使用して、個々の軸索を分析するために反転のオープンブックの準備としてマウントされます。

Watch this Scientific Journal Video about DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord at JoVE.com

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

齧歯類の脊髄への感覚求心性のステレオタイプの予測は、単一の軸索のトレースを通して軸索分岐を研究するために簡単にアクセス可能な実験系を提供する。

マウス胚の脊髄の全載標本での分岐軸索勉強するDRGニューロンのDII -標識

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

dii-labeling-drg-neurons-to-study-axonal-branching-whole-mount

Research

JoVE Journal

Neuroscience

24.8K ビュー.  Max Delbrück Center for Molecular Medicine. 齧歯類の脊髄への感覚求心性のステレオタイプの予測は、単一の軸索のトレースを通して軸索分岐を研究するために簡単にアクセス可能な実験系を提供する。

ビデオ: DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

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

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

ホールマウントの胎児神経系の可視化ショウジョウバエ胚

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人の全体のマウントの準備ショウジョウバエ</emジャイアント繊維染料注入のための>腹神経索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

アン<em&gt;エクスビボ</em&gt;レーザー誘起脊髄損傷モデルがリアルタイムで軸索変性のメカニズムを評価するために

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

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

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

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 ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胎児運動ニューロンの軸索突起パターンの視覚化<em&gt;ショウジョウバエ</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

マウスにおける線込みおよび脊髄窓移植

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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 ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

ラットおよびマウス脳への神経幹細胞の動脈内デリバリー:脳虚血への応用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

脊髄刺激に対する運動ニューロン応答の理解

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

著者スポットライト:脊髄刺激の進歩 - パッチクランプ電気生理学による運動ニューロンの細胞応答の探索 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

脊髄刺激時の運動ニューロンにおける全細胞パッチクランプ記録のための脊髄スライスの ex vivo 調製

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...