この作品は、RMへの国民の健康と医療研究センター（NHMRC）プロジェクトの助成金によってサポートされていました

ここで説明する全ての実験手順は、UNSWオーストラリアの動物実験倫理委員会を遵守し、国民健康と動物実験のための医学研究評議会、オーストラリアの規制に準拠して実施しました。このプロトコルのすべての手順は、関連する動物実験倫理委員会の要求に応じて行われるべきです。 1.アセチルコリンエステラーゼ組織化学染色<ol><li>アセチルコリンエステラーゼの反応液を調製<ol><li> 0.1Mリン酸緩衝液（PB）200mlにアセチルチオコリンヨージド290mgの追加。 900 rpmで磁気攪拌機で混合します。 </li><li>連続的に攪拌しながら、グリシン600mgのを追加します。 </li><li>生成物が完全に溶解するまでゆっくりと連続的に攪拌しながら反応混合物に硫酸銅の420ミリグラムを加えます。 </li></ol></li><li>動物の死体の皮膚を削除切開を行うことで、（組織の共有を介して得られます）灌流ラットまたはマウスの皮膚へ、首の領域から肌を把握し、その足を過ぎてそれを引っ張ります。各筋肉を覆う筋膜が除去されたか、大幅に反応液に筋線維の十分な曝露を確実にするために穿孔のいずれかであることを確認してください。 </li><li>反応混合物に関心の全体または手足を浸し、4℃でO / Nインキュベート。 </li><li>蒸留水で2分間の死体を洗ってください。注：この段階では、筋肉は、青色の着色を示し、運動終板（のMEP）が白い点として見ることができます。 </li><li> 3-5秒間10％の硫化アンモニウム溶液に死体を公開します。 注：筋線維は、急速に褐色に変わり、MEPが黒い斑点ドットとして観察可能になります。反応があまりにも急速に発生し、筋線維が議員と筋線維を区別するために暗すぎる染色されている場合は硫化アンモニウム溶液（ 例えば 5％）のより低い濃度を使用してみてください。反応時間は、取得します議員と筋線維の間の最適なコントラストは、一つのサンプルから他に変化します。したがって、試薬に正確な露光時間を規定することは困難です。コントラストが十分なものとみなされたときの反応は、厳密に監視して停止する必要があります。 </li><li>蒸留水に攪拌しながら、カーカスを2回洗浄し、穏やかにカーカスドライバッチリ。 </li><li>関心のあるすべての筋肉のためのMEPが捕捉されることを保証する写真四肢の両側と内側側面、。 </li></ol> 2.筋肉内注射モーターエンドプレートで<ol><li>マイクロピペットプラーとガラスマイクロピペットを引き出します（プランジャとの傾斜マイクロピペットの使用を推奨します）。解剖顕微鏡の助けを借りて、マイクロピペットの内腔の内径は約0.5mmであるように、ピンセットでマイクロピペットの先端を破ります。 </li><li>使用されているすべての手術器具は、新たにオートクレーブ処理されていることを確認し、目手術領域で無菌です。 </li><li>フルオロゴールド（蒸留水中5％）でマイクロピペットを埋めます。 </li><li>イソフルオラン（O 2中4％）で誘導チャンバ内に動物において麻酔をかけるを誘導します。立ち直り反射を確認し、導入室から取り出す前に、その不在を確認してください。ラットの鼻の上にノーズコーンを固定し、麻酔の維持のためにイソフルオラン（O 2中2％）を提供します。動物の足指をつまむ、ゆっくりペダルの撤退と角膜反射の両方が存在しないことを保証するために、動物の眼に触れます。注：ケタミンおよびxylazilの混合物も使用することができる（80および10mg / kgでそれぞれ、腹腔内送達）。ケタミンは、スケジュール8（「制御」）は、薬物とに接着する必要があります薬の購入、使用、保管、廃棄に関連する必要なプロトコルです。 </li><li>手順の過程で目の乾燥を防ぐために、眼の潤滑剤を適用します。 </li><li> Tを剃りますargeted手足三クロルヘキシジンの交互スクラブと70％のアルコールで剃毛面積を拭くためにガーゼを使用。手術領域に動物を転送します。 </li><li>きれいなアンダーパッドに動物を置き、目標と筋肉（複数可）への良好なアクセスを確保するために適切に配​​置します。 </li><li>離れて下にある筋肉組織からの標的筋肉上の皮膚を持ち上げ、手術用ハサミで皮膚を切開を行うために、歯のグリップをピンセットを使用してください。切開部が完全に興味のある筋肉（複数可）を露出するのに十分な大きさであることを確認してください。筋膜の中断を最小限に抑えがあることを確認します。 </li><li>精神的に筋肉（複数可）にMEP領域の位置と形状を転置するMEPの写真を使用してください。 注：ファインフェルトマーカーは、目的の筋肉（複数可）に写真からMEP地域を再生するために支援することができます。 </li><li>各s 1-2μLの3〜4回の注射、フルオロ金（MEP領域の全長に沿って複数回の注射を行いますhould）十分です。静かに任意の漏出を除去するために、筋肉（複数可）を拭きます。 </li><li>鈍鉗子で互いに近接切開皮膚の両端を持って、外科手術用クリップで傷口を閉じます。傷の全スパンに沿って0.1ミリリットルブピバカイン（水中0.5％）（または他の局所麻酔薬）を潜入。 </li><li>それは完全に麻酔から回復されるまで、麻酔器の電源をオフにして、動物を監視します。 </li><li>筋肉内注射の投与や動物の灌流の間に14日間待ってください。 </li></ol> 3.潅流<ol><li>腹腔内注射によって動物に、ペントバルビトンナトリウム溶液の致死量（150 mgの/ kgの例えば Lethabarb）を管理します。 <ol><li>ペダルの撤退と角膜反射の欠如によって確認されるように、麻酔の深いレベルは、到達するまで密接に動物を監視します。 </li></ol></li><li> perfusio上に配置された解剖ボード上の背中に動物を置きnは沈みます。 </li><li>胸骨の基部を覆っている皮膚を持ち上げるために、歯のグリップをピンセットを使用してください。すぐに胸骨下の強い手術用ハサミで小さな皮膚切開を行い、その後、グリップに胸骨の剣状突起軟骨をピンセットを使用しています。剣状軟骨を保持しながら、胸骨から脇の下に、胸腔の両側に切開を拡大します。 </li><li>心臓を露出させるために、振動板をカットします。 <ol><li>血液の凝固を防ぐために、直接心臓の頂点にヘパリンの0.mlを注入します。 </li><li>左心室へのカニューレの挿入を可能にするために頂部を切断し、その後速やかに止血を用いて適所にカニューレをクランプ。 </li><li>細かいハサミで右心房の切開を行い、すぐに蠕動ポンプを起動します。心房から流出する液体が血液のほぼ自由であり、肝臓の色は淡褐色になるまで、0.1M PBで灌流。そして、ソルと灌流動物の体全体まで、パラホルムアルデヒド（0.1 M PB中4％）の化は、剛性になります。 </li></ol></li></ol> 4.頸髄解剖と組織学のための準備<ol><li>その腹部に動物の死体を置きます。 </li><li>メスで身体の正中線の皮膚をカットし、腸骨のレベルに頭蓋底からそれを反映しています。 </li><li>カットスルーと脊柱の背側面を露出させるために傍脊柱筋を反映しています。 </li><li>アトラス、第二頚椎のセグメント（ すなわち 、C2）の骨棘突起を特定し、対応する基礎となる後根を見つけるために、外科用鉗子やピンセットでそれを削除します。 <ol><li>永久フェルトマーカーでそれを着色し、右C2のルートを特定します。 </li><li> 1によって次の椎骨1つを削除して、色を交互に右後根をマーク（ すなわち 、C2、C4、C6、及びC8は、緑とC3に着色することができる、C5、C7、T1はCOLすることができます青で）論理和。 </li></ol></li><li>静かに脊髄の下端を覆う硬膜を貫通する小手術針（ 例えば 、30 Gゲージ針）を使用します。縦スリットを作るために吻方針を移動させながら上向きに針のベベルで、コードから離れて硬膜を持ち上げます。 <ol><li>硬膜を反映しています。 </li></ol></li><li>新しいメスの刃で1つまたは2つのセグメントブロックに脊髄横断をカットします。 <ol><li>慎重に隣接する二つの根の間のメスの刃途中で各ブロックの左側に小さな基準マークを作る、ブロックごとに、その場での脊髄セグメントのままにしておきます。基準マークは、ブロックの腹側面から見えるようにするように組織を通る十分な深さであることを確認してください。解剖後の組織セグメントの向きを支援するために、動物の解剖学的構造に関していずれか前方または後方に向い、約45°の角度で基準マークを作成します。 </li></ol></li><li>パラホルムアルデヒド（0.1 M PB中4％）の溶液を含む小さな、明確に表示ボトルに、個々のブロックを収集し、RT O / Nで残します。 </li><li>ショ糖（0.1 M PB中30％）の溶液を含むクリーン、明確に表示ボトルにブロックを転送し、少なくとも2日間4℃で維持。 </li><li>クライオ金型内のセグメントを配置し、組織凍結培地でそれらをカバーしました。縦組織学的調製のために、上向きに背側面でブロックを配向させ、低温金型内のブロックを配向させるために基準マークを使用しています。 </li><li> -20℃の低温金型を凍結し、50ミクロンの厚さの切片に低温保持装置で切断しました。注：セクションに直接接着顕微鏡スライド上にマウントし、O / Nを乾燥させるために残すことができます。代替的に、切片を0.1 M PBで満たさ48ウェルプレートに浮遊することができ、その後、スライド上の組織切片を配向する基準マークを使用してマウント。 </li></ol> 5.腰椎共同RD解剖と組織学のための準備<ol><li>それは背中に動物の死体を置きます。 </li><li>動物の腹部に沿って正中切開を行い、内臓を取り除きます。脊柱の腹側面を露出させるために後部腹壁の筋肉を解剖。 </li><li> T13前根が骨を出る短い尾最リブとその隣接するT13の椎骨の位置を確認します。 <ol><li>連続して腹側コードの腹側面のエントリのその時点までのT13腹ルートを追跡するために、微細手術用骨鉗子でT13（ すなわち 、T12、T11と、必要に応じて、T10）に一つずつ二、三の椎骨吻側を削除し、永久フェルトマーカーでマークします。 </li><li>この時点から、色を交互にして着色、L1、L2、L3、L4、L5、L6およびS1用前根のエントリポイントの位置を特定するために同じ手順を繰り返します。 </li></ol></li><li> LUMについては、セクション4.5から4.10に記載したのと同じ手順に従ってください脊髄切開をバー。 </li></ol>

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<li>Tosolini, A. P., Mohan, R., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+full+length+of+the+motor+end+plate+regions+in+the+mouse+forelimb+increases+the+uptake+of+fluoro-gold+into+corresponding+spinal+cord+motor+neurons%5BTitle%5D)+AND+%22Front+Neurol%22%5BJournal%5D)">Targeting the full length of the motor end plate regions in the mouse forelimb increases the uptake of fluoro-gold into corresponding spinal cord motor neurons.</a> Front Neurol. 4, 58(2013).</li>
<li>Mohan, R., Tosolini, A. P., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+motor+end+plates+in+the+mouse+hindlimb+gives+access+to+a+greater+number+of+spinal+cord+motor+neurons%3A+an+approach+to+maximize+retrograde+transport%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Targeting the motor end plates in the mouse hindlimb gives access to a greater number of spinal cord motor neurons: an approach to maximize retrograde transport.</a> Neuroscience. 274, 318-330 (2014).</li>
<li>Baumgartner, B. J., Shine, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroprotection+of+spinal+motoneurons+following+targeted+transduction+with+an+adenoviral+vector+carrying+the+gene+for+glial+cell+line-derived+neurotrophic+factor%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Neuroprotection of spinal motoneurons following targeted transduction with an adenoviral vector carrying the gene for glial cell line-derived neurotrophic factor.</a> Exp Neurol. 153 (1), 102-112 (1998).</li>
<li>Gransee, H. M., Zhan, W. -Z., Sieck, G. C., Mantilla, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+delivery+of+TrkB+receptor+to+phrenic+motoneurons+enhances+functional+recovery+of+rhythmic+phrenic+activity+after+cervical+spinal+hemisection%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Targeted delivery of TrkB receptor to phrenic motoneurons enhances functional recovery of rhythmic phrenic activity after cervical spinal hemisection.</a> PLoS ONE. 8 (5), e64755(2013).</li>
</ol>

著者は、彼らが競合する金融利害関係がないことを宣言します。

神経筋条件の実験的治療のための筋肉内ターゲティングおよび対応するα運動ニューロンへの治療導入遺伝子のその後の逆行性伝達は、新戦略ではありません。例えば、この送達方法は、SOD1マウスおよびラット1,9-12ならびにSMA 13とマウスにおいてALS進行の異なる段階での神経筋変性を遅延させるために使用されてきました。有望一方、これらの遺伝子治療のシナリオの有効性...

このような運動ニューロン疾患などの神経学的状態から生じる随意運動の制御の喪失とspinal-ならびにデュシェンヌ型筋萎縮症は、罹患者の毎日の生活に高く、長期的な影響を与える衰弱状態です。過去10年間、停止または少なくともこれらの神経筋疾患の有害な影響を遅らせることを目指して研究努力は、世界中の多くの臨床医と科学者のための優先事項となっています。この点で、これらの神経筋疾患を模倣する動物モデルの最近の発生は、これらの条件1-13の発生および進行の根底にある生理学的メカニズムに基本的な洞察を得ることに尽力されています。これらの神経筋疾患の治療は、脊髄への直接アクセスを必要とし、脊髄注射14,15によって達成することができます。遺伝子治療の最近の進歩は、上部の横紋筋肉を標的としていると脊髄1,9-13の前角内に配置され、対応するα運動ニューロンへのシャトル治療遺伝子に下肢。しかし、このかつて有望な戦略は、これらの神経学的状態の結果を改善するために失敗しました。それは、これらの予後不良があり得ることを結論する公正であるが、少なくとも部分的に、これらの保護の遺伝子の低い有効性に起因する、1は、これらの遺伝子送達方法の低い有効性を除外することはできません。 モーターエンドプレート（議員）は、α運動ニューロンから発信大きな末梢運動線維の軸索末端でインデントされた骨格筋線維の専門領域です。一緒に、末梢神経線維終末とのMEPは、神経筋接合部、 すなわち 、シナプスインパルスは、神経伝達物質の順行性遊離、アセチルコリンによってトリガーされる部位を形成します。重要なことは、末梢神経線維とのMEPとの関係は、二DIRECです的な、別のモータが離れてニューロン細胞体16-18から向かってだけでなく、分子や細胞小器官の輸送に関与しているが。これらの解剖学的考慮事項に照らして、MEPが対応する運動ニューロンへの送達および遺伝物質のその後の逆行性輸送のための選択の標的であることが表示されます。この文脈では、運動ニューロンの形質導入の成功は非常にウイルスベクターおよび筋肉の19-20のMEPの筋肉内注射との間の距離に依存することは驚くべきことではありません。しかし、驚くべきことに、実験用ラットとマウスの筋線維のMEPゾーンの正確な位置は、選択の2種が神経筋疾患をモデル化するために、最近まで利用できませんでした。 我々は、ラットおよびマウス21-22でいくつかの前肢の筋肉のためのMEP領域の総合的なマップを生成しています。最近では、我々はMEP rの組織の詳細を示していますマウス後肢23のいくつかの筋肉のためのegion、我々は現在、ラットの後肢上のMEPの特徴を分析しています。私たちの手では、これらの筋肉全体MEPゾーンに向かう逆行性トレーサーの筋肉内注射は、以前に報告されたよりも多くの脊髄セグメントをスパニングしている以上の標識運動ニューロンを生じさせました。ここでは、外部表面上のMEPの位置だけでなく、マウスおよびラットの両方における後肢と前肢の筋肉の深さ全体を明らかにするために、ここ数年にわたって開発されたプロトコルを提示します。

<table><tbody><tr><td>Fluoro-Gold</td><td>Fluorochrome, LLC</td><td>Nil</td><td>Diluted to 5%</td></tr><tr><td>Drummond PCR Micropipets 1-10 &amp;micro;l</td><td>Drummond Scientific</td><td>5-000-1001-X10</td><td>accompanied with plungers</td></tr><tr><td>Acetylthiocholine Iodide</td><td>Sigma Life Science</td><td>A5751-25G</td><td></td></tr><tr><td>Copper(II) Sulfate Anhydrous</td><td>Sigma-Aldrich</td><td>61230-500G-F</td><td></td></tr><tr><td>Tissue-Tek O.C.T Compound</td><td>Sakura Finetek</td><td>25608-930</td><td></td></tr><tr><td>Glycine</td><td>Ajax Finechem</td><td>1083-500G</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine and biotin</td><td>Life Technologies</td><td>D-3312</td><td>Diliuted in distilled water</td></tr><tr><td>Isothesia</td><td>Provet</td><td>ISOF00</td><td>1000 mg/g Isoflurane inhalation vapour</td></tr><tr><td>Autoclip 9 mm Wound Clips</td><td>Texas Scientific Instruments, LLC</td><td>205016</td><td></td></tr><tr><td>Lethabarb Enthanasia Injection</td><td>Virbac (Australia) Pty Ltd.</td><td>LETHA450</td><td></td></tr><tr><td>Formaldehyde Solution</td><td>Ajax Finechem</td><td>A809-2.5L PL</td><td></td></tr><tr><td>SuperFrost Plus glass slides</td><td>Menzel-Glaser</td><td>J1800AMNZ</td><td></td></tr><tr><td>Ammonium Sulphide</td><td>Sigma-Aldrich</td><td>A1952</td><td>Diluted to 10%</td></tr><tr><td>Marcain Spinal 0.5% (Bupivacaine hydrochloride)</td><td>Astrazenca</td><td></td><td>Diluted to 0.25%</td></tr></tbody></table>

intramuscular injections along the motor end plates: a minimally invasive approach to shuttle tracers directly into motor neurons

脊髄運動ニューロンの完全性に影響を及ぼす疾患は、最も衰弱性の神経学的状態の間です。過去数十年にわたり、これらの神経筋障害のいくつかの動物モデルの開発は、これらの条件の進行を遅延または逆転を狙っ異なる治療シナリオを科学コミュニティを提供しています。ニューロンの逆行性機構を利用することにより、これらのアプローチの一つは、脊髄運動ニューロンに対応するシャトル治療遺伝子のために、骨格筋を標的とするようになっています。一度有望が、このような遺伝子送達アプローチの成功は、これまでに得示した形質導入運動ニューロンのサブ最適数によって妨げられてきました。運動終板（のMEP）は、運動ニューロンα脊髄へ直接シナプス接触している骨格筋で高度に専門領域です。この点において、それは、これまで、努力は逆行することに注意することが重要です運動ニューロンへの転送遺伝子は、対象となる筋肉のMEP領域の位置を参照することなく行われました。ここでは、単純なプロトコル1）骨格筋の表面上のMEPの正確な位置を明らかにし、2）運動ニューロンへの筋肉内送達および逆行性トレーサーのその後の最適な逆行性輸送を案内するために、この情報を使用することが記載されています。我々は、欧州議会議員のターゲティングを介して脊髄運動ニューロンへの治療遺伝子の逆行性輸送を調査へのさらなる研究において、これらのトレース実験からの結果を利用したいと考えています。

アセチルコリンエステラーゼ組織化学的染色は、筋肉の幅にわたってモータのエンドプレートの位置を明らかにした。 図1は、全体のラットの前肢で行わかかる染色の結果を示します。これは、硫化アンモニウム溶液の濃度を最適化するために提案されている（ 例えば 、5〜7％の代わりに10％）だけでなく、試料を溶液中に浸漬する時間としては、筋肉繊維上の非特異的バ...

Watch this Scientific Journal Video about Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons at JoVE.com

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

モーターエンドプレートに沿って筋肉内注射：直接運動ニューロンへのシャトルトレーサーへの低侵襲アプローチ

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

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Research

JoVE Journal

Neuroscience

12.6K ビュー.  University of New South Wales. The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

ビデオ: Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

皮下ムスカリン拮抗薬の管理およびマウスにおける挙筋オーリスの筋筋のトリプル免疫染色

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

神経科学

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

マルチカラーで神経回路の地形を明らかにし

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

腰椎運動ニューロンおよび成体マウスでそのモーターユニットによる力の同時細胞内記録<emインビボ&gt;</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

コレラ毒素とビオチン化デキストランアミン二重標識が続く仮性狂犬病ウイルスとペリフェラルターゲットからトレース経シナプス

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

ミニ浸透圧の移植は、病態生理学的状態で脳の再編成を検討するためにポンプ冊子トレーサーの配達

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

脳の外植片にレーザー誘導神経トレース

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

マウスにおける横隔膜の運動ニューロンの逆行性神経解剖学的トレーシング

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

軸索輸送の In Vivo イメージングのためのツールキットの拡張

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Lumbar Intrathecal Injection of Gene Therapy Vectors for Central Nervous System Targeting in Mice and Rats

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This approach bypasses the blood-brain barrier to directly access the cerebrospinal fluid and preferentially target cells within the central nervous system. Multiple published preclinical studies employing the lumbar intrathecal injection route have contributed to the development of gene therapy clinical trials; however, the described protocols are variable and dispersed across multiple resources. Here, a comprehensive set of protocols for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats for preclinical gene therapy studies is presented. With proper training, this injection technique can be performed quickly and reliably. In addition to detailing the injection protocol at each developmental stage, associated parameters, such as injection volume, that can influence study outcomes are discussed. To demonstrate the application of lumbar intrathecal injections for targeting the central nervous system, the expression of adeno-associated virus serotype 9 in the brain, spinal cord, and peripheral tissues is presented following a successful or unsuccessful injection.

A lumbar intrathecal injection represents a translationally relevant route of administration for delivering gene therapy to the central nervous system. This comprehensive standardized protocol for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats aims to guide researchers in adopting this technique for preclinical gene therapy studies.

マウスおよびラットにおける中枢神経系を標的とした遺伝子治療ベクターの腰椎髄腔内注射

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This ...

Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

<meta charset="utf8"></head><body>ヒト間葉系幹細胞の脊髄腔内注射とラット脳への移動の追跡

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

ヒト間葉系幹細胞の脊髄腔内注射とラット脳への移動の追跡

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain ...