この作品は、財団からの資金援助のおかげで可能にラRECHERCHEMédicale（FRM）、ALSの研究（ALS協会）のミルトンSafenowitzポスドク、NIHの助成NINDS NS05462とNS034382、及びANRグラントHyperMNDを注ぎました。

1。ステップワンプリ麻酔薬：麻酔導入前に10〜15分間、それぞれ流涎および浮腫を防ぐため、アトロピン（0.20 mg / kg）及びmethylprenidsolone（0.05 mg）を皮下に注射する。 2。ステップ2 麻酔導入：ペントバルビタールナトリウム（70 mg / kg）をまたはケタミン/キシラジンの混合物（それぞれ100 mg / kgおよび10 mg / kgの）腹腔内に注入します。マウスがないつま先のピンチ反射が得られないことができるようになるまで下に行きましょう。麻酔が軽すぎると思われる場合は、投与量の1/4で補う。 3。ステップスリー *注：この手術は、端末の手順です。 麻酔の外科平面に達したときは、腹臥位で提起された暖かい毛布の上にマウスを移す。 <ol><li> 100ミリリットル/分の周りの流れに純粋なO 2を提供するマスクを使用してマウスの鼻をカバーしています。 </li><li&gt;はバリカンを使用して、首筋から尾の付け根に背部を剃る。 </li><li>また、右後肢を剃る。 </li><li>仰臥位にマウスを裏返しますが、代わりに酸素マスクを残すために世話をする。 </li></ol> 4。ステップ4 手足に巻回され、作業面の四隅に固定縫合のある場所でマウスを固定します。 5。ステップ5 マウスのコア温度を監視する温度プローブを挿入します。 36℃と38℃の間でコア温度を維持するために、加熱毛布/ランプ電力を調整する 6。気管切開と人工換気<ol><li>鈍はさみを使用して、気管の上にカットして、両側の肌を引き出します。 </li><li>気管を覆う二つの薄い筋（胸骨舌骨）を露出するように鈍い鉗子を用いて、唾液腺を引き裂く。 </li><li> 2 MUSC別、鈍鉗子を用いて気管を明らかにするために彼らの内側に沿って分離レ。 </li><li>デュモン7鉗子を用いて、気管の下に4.0絹縫合糸の2つの長さをスライドさせます。 </li><li> 2軟骨のリングの間で気管内横断カットしてではなく、セクション気管を完全に世話をする。 </li><li>気管ダウン気管チューブを挿入し、その上に糸を結ぶことにより、挿入口の両側に固定します。気管チューブは、マウス人工呼吸器（SAR-830​​/AP、CWE社）とカプノグラフ（μcapstar、CWE社）に接続されています。人工呼吸器は、純粋なO 2のソースに、コンプライアンス袋を通して、接続されています。マウスは人工呼吸と戦っておらず、呼吸終期PCO 2が 4と5％の間で安定しているように、人工呼吸器（呼吸速度100から150拍、呼吸終期の容積170から310μL）のパラメータを調整します。 </li></ol> 7。静脈ラインの配置<ol><li>鈍的剥離技術を使用して、頸静脈を露出首の片側の静脈。このレベルでは、頸静脈は、2つの主要なトランク、前部と後部の顔面静脈に分割されます。 </li><li>これらのトランクのそれぞれについて、1回のセットを次の手順を実行します。 <ol><li>デュモン4または5の鉗子を使用して、その周囲の結合組織から静脈を慎重に分離します。 </li><li>デュモン7鉗子を用いて、静脈の下に6.0絹縫合糸の2つの長さをスライドさせます。静脈の長さに沿って可能な限りそれらを分離。 </li><li>静脈（心臓に近い側）の近位側にある小さな血管クリップを配置し、静脈の遠位側をオフに接続します。 </li><li>非常に微細なアイリスはさみを使用して、完全にセクションに静脈を細心の注意を払っていない取って、静脈内に小さな横切開を行います。 </li><li>アップ血管クリップに、開口部にプレフィル1FRカテーテル（premicath、Vygon）を挿入します。 </li><li> 、デュモン4鉗子で一緒に静脈カテーテルを保持して慎重に血管クリップを削除し、その後いくつかのMORカテーテルをプッシュ静脈へのeはミリメートル。 </li><li>挿入の両側の両方の縫合糸とそれを結ぶことによってカテーテルを固定します。 </li></ol></li><li>カテーテルの一つは、注射器や麻酔の補足投与、必要に応じて（通常は毎年10月30日分）を注入するシリンジポンプに接続されています。 IV用量は、ペントバルビタールナトリウムまたは1250年40μg/ kgの/ケタミン/キシラジン12分のいずれかの6 mg / kgである。他のカテーテルをNaHCO 3（1％）とplasmion（14％）を含む4％グルコース溶液をゆっくり静脈内注入（50μL/ HR）のシリンジポンプに接続されています。 </li></ol> 8。針および縫合糸と首の皮膚を閉じて、腹臥位にマウスを返す 9。後肢の筋肉と神経の解剖<ol><li>はさみを使って、太ももの上からアキレス腱に切り込みを入れる。血管を傷つけないように注意しながら、下層にある筋肉から皮膚を分離。必要に応じて焼灼する。 </li><li&gt;の腿を走って白い線として現れる大腿二頭筋の前縁を識別します。はさみを使って、慎重に膝からのラインに沿って腰の骨へのすべての道を開く。筋肉を区切ります。坐骨神経は現在、大腿二頭筋の下に表示されます。 </li><li>慎重に大腿二頭筋を解剖。出血を防ぐために、必要に応じて/リガチャーを焼灼する。大腿二頭筋は完全に削除されたり、単に坐骨神経と下腿三頭筋の筋肉を露出させるためにリクライニングすることができます。 </li><li>腓腹神経を解剖する。 </li><li> 8/0絹糸を使用し、総腓骨神経の遠位部を縛って、結び目の遠位にカットします。できるだけ腰に近づけて、神経のすべての方法を解剖する。 </li><li>総腓骨、腓腹神経間における脛骨神経を識別します。脛骨神経の別の支店の中では、（以下、脛骨神経とも呼ばれる）深く行く枝からトライセップス·下腿を支配ブランチを識別する。 </li><li> 8/0シルクを使用した枝はトライセップス·下腿を支配そのまま維持しながらスレッド、脛骨神経のすべての支店を結びつける。結び目に対して遠位脛骨神経をカットし、可能な限り神経を解剖する。 </li><li>次の手順に進む一方dessicationを防止するために生理食塩水を吸収させたガーゼで全体の後肢をカバーしています。 </li></ol> 10。椎弓切除術<ol><li>はさみを使って、骨格に沿って切り込みを入れる。下層にある筋肉から皮膚を分離します。 </li><li>皮下の筋肉を分離するために椎骨の両側にある2つの切開を行う。次に、それぞれの側の椎骨の側に取り付ける筋肉の各腱を切った。 </li><li>鈍鉗子と罰金キュレット使用して、明らかに個々の椎骨を特定するために棘突起の周りの脊椎の背側の筋肉の残りの部分を削除します。 </li><li>椎骨T13とL1を識別します。 T13は取り付けリブを持っている最後の椎骨である。脊柱ワット病気カニンガム脊髄脊椎のクランプ（株式会社Stoelting）を使用して固定化する。 T13とL1のそれぞれの側に脊髄クランプを置きます。脊髄を圧迫しますが、縦軸にテンションを少しかけないように注意してください。脊柱はよくピンセットで軽く押し下げて固定されていることを確認してください。 </li><li>細かいrongeursを使用して、それによって脊髄を露出し、T13とL1を介してその後ラミナを脊髄プロセスを削除します。 </li><li>乾燥を防ぐために、生理食塩水を吸収させ、綿またはspongelの小さなビットで露光脊髄をカバーしています。 </li><li>脊髄を取り巻く、背中の上にプラスチック製のお風呂を作ったカスタムを置きます。 4/0絹糸を使ってその場でお風呂を固定します。浴はクイック·キャストシーラント（WPI）とそれを封止することで防水であることを確認します。 </li><li>クイック·キャストが乾燥した後、脊髄を覆っている綿を除去し、ミネラルオイルと一緒にお風呂を埋める。 </li><li>デュモン5鉗子、非常に微細な虹彩のはさみを使って、優しく硬メイトを引っ張るrは脊髄を囲むと、両方の方向に可能な限りそれを開きます。脊髄の両側にある硬膜を折る。 </li><li>それが椎クランプによって中断保つようにマウスがそう嘘をついている上げられたプラットホームを下げます。 </li><li>動物の背中の領域をサポートするために仙骨部の棘突起をクランプするために別の椎クランプを使用しています。 </li><li>第3のクランプは、右後肢と足首を固定するために使用され、膝で90°の角度で屈曲。 </li></ol> 11。アキレス腱解剖<ol><li>手足が膝と足首は90°に曲げて、後肢領域をカバーガーゼを除去し、周囲組織の自由なアキレス腱を解剖。だけでなく、周囲の組織からできるだけ多くの下腿三頭筋を解剖。 </li><li>踵骨に近い足底筋の腱を切断し、その後完全に腱を削除するには、もう一度それをカット。 </li><li>スレッドニードルを使用して、目を通って6/0絹糸を添付電子アキレス腱と腱の周りトリプル結び目を作る。 </li><li>結び目に近い力変換器を配置し、腱の遠位部をカットし、絹糸を使用して力変換器に取り付けます。 </li><li>上腕三頭筋の下腿筋の筋膜の下にステンレス鋼線の2つの長さを挿入します。これらの配線は、筋電図記録用外ACアンプに接続されています。 </li><li> 2バイポーラフック電極上に総腓骨神経と脛骨神経を置きます。 </li><li>陽極は周辺の筋肉に触れると、フック電極の陰極上トライセップス·下腿の神経を置きます。 </li><li>分離ユニットにすべての刺激電極を接続する。 </li><li>脊髄背電位を記録するために外のACアンプに接続されている脊髄の後面には、ボール電極を配置します。背中の筋肉と接触したAg / AgCl参照電極を配置します。 </li></ol> 12。ステップ十二上腕三頭筋の下腿の神経を刺激する最大収縮振幅が観察されるまで、低周波（&lt;1 Hz）での増加強度の50μsecの方形パルスを使用しています。ゆっくり収縮の振幅が最大に達するまで攣縮反応の振幅を監視しながら、筋肉をストレッチする力変換器を移動します。 13。運動ニューロンの細胞内記録この時点から、標準的​​な電気生理学的手法、細胞内電極を準備脊髄ニューロンを貫通し、運動ニューロンとしてそれを識別するために使用されます。 <ol><li>ピペットプラー（P-97マイクロピペットプラー、サッターInstruments）を用いて約1μmの先端にガラスマイクロピペットを引き抜きます。 KClの3M溶液（MΩ電極10から20までの抵抗）で電極を埋める。 </li><li>マイクロポジショナーを使用して、脊髄に細胞内のアンプに接続し、マイクロピペットを、（Axoclamp 2B、アクソン·インスツルメンツ）を駆動する。 eの刺激によって誘発される局所電場電位を監視ACHは神経トライセップス·下腿のモータープールを検索する。 </li><li>微小電極の抵抗を監視しながら、推定上の運動ニューロンを慎重に近づく。膜に押し付けたときに、抵抗が増加します。浸透はいつか細胞内のアンプの &quot;バズ&quot;機能を使用することにより容易に行うことができる。 </li></ol> 14。安楽死手順実験の最後に、動物を断頭続いペントバルビタールの過剰投与（210 mg / kgをIV）によって安楽死させています。

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特別な利害関係は宣言されません。

ここで説明する準備が腰椎運動ニューロンの成体マウス、同時細胞内記録とその軸索によって神経支配筋線維による力の測定には、ことができます最初のものである。 なぜなら動物のサイズが小さいため、この準備のために必要な外科的スキルが身に挑戦することができます。これらのスキルを習得されるただし、一度全体の手術は3時間で行うことができ、動物が録音?...

A correction was made to <a href="http://www.jove.com/video/4312">Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo</a>. There was an error in the name of one author, Marin Manuel. The author's name has been corrected to:
Marin Manuel 
 
instead of: 
 
Manuel Marin

A correction to author list has been made for article <a href="http://www.jove.com/video/4312">Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo</a>.

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

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>硫酸アトロピン</td><td> Aguettant </td><td></td><td></td></tr><tr><td> Methylprenidsolone </td><td>ファイザー</td><td>ソリューションメドロール</td><td></td></tr><tr><td>ペントバルビタールナトリウム</td><td>サノフィ·アベンティス</td><td>ペントバルビタール</td><td></td></tr><tr><td>ケタミン</td><td></td><td></td><td></td></tr><tr><td>キシラジン</td><td></td><td></td><td></td></tr><tr><td>グルコース</td><td></td><td></td><td></td></tr><tr><td>血漿増量剤</td><td>ロジャーBellon </td><td> Plasmagel </td><td></td></tr><tr><td>ブラントはさみ</td><td> FST </td><td> 14079から10 </td><td></td></tr><tr><td>ブラント細かいハサミ</td><td> FST </td><td> 15025から10 </td><td></td></tr><tr><td> Vannas春はさみ</td><td> FST </td><td> 15002から08 </td><td></td></tr><tr><td>ファイン鉗子鋸歯</td><td> FST </td><td> 11370から32 </td><td></td></tr><tr><td>ファイン鉗子鋸歯</td><td> FST </td><td> 11370から31 </td><td></td></tr><tr><td>カニンガム脊髄アダプター</td><td> Stoelting株式会社</td><td></td><td></td></tr><tr><td>クイック·キャストシーラント</td><td> WPI </td><td> ＃KWIK-CAST </td><td></td></tr><tr><td>人工呼吸器</td><td> CWE株式会社</td><td> SAR-830​​/AP </td><td></td></tr><tr><td>カプノグラフ</td><td> CWE株式会社</td><td> μcapstar </td><td></td></tr><tr><td>暖房毛布</td><td>ハーバード装置</td><td> 507221F </td><td></td></tr><tr><td>細胞内のアンプ</td><td>軸索InstrumeNTS </td><td> Axoclamp 2B </td><td></td></tr><tr><td>ピペットプラー</td><td>サターインスツルメンツ</td><td> P-97 </td><td></td></tr><tr><td> KClを</td><td>シグマアルドリッチ</td><td> P9333-500G </td><td></td></tr></tbody></table>

simultaneous intracellular recording of a lumbar motoneuron and the force produced by its motor unit in the adult mouse in vivo

それは独特の特性（1）は、容易に識別可能ターゲット（筋線維）を有するため、非常によく知られた機能を有するもので、中枢神経系のニューロンであるため、脊髄の運動ニューロンは、長い神経機能を研究するための優れたモデル系であった（筋収縮を制御する）、（2）それゆえ、多くの脊髄と降順ネットワークの収束目標、 &quot;最終的な共通経路&quot;の名であること、（3）それが可能鋭い細胞内電極を用いて、それらを貫通するようになり、大きなソーマを持つ。さらに、in vivoで検討したときに、同時に運動ニューロンとその筋肉のターゲットによって開発された力の電気的活動を記録することができる。したがって、同時に、勉強することができるというユニークな立場に実験者を置くin vivoで運動ニューロンの細胞内録音の実行&quot;を参照してモータユニット&quot;（運動ニューロンに与えられた名前は、その軸索のすべてのコンパートメント、およびそれは1を神経支配筋線維）：運動ニューロンに衝突する入力は、運動ニューロンの電気生理学的特性、及び運動ニューロンの生理的機能のこれらのプロパティの影響は、そのモータユニットで発生する力、 すなわち 。準備が麻痺することはできませんので、細胞内記録のための機械的安定性が低下するので、このアプローチは非常にやりがいがあります。したがって、この種の実験は、ネコやラットでは達成されています。それは、正常および遺伝子改変マウスで同様の実験を行うことが可能であった場合は、脊髄運動系の研究は恐るべき飛躍を作ることができます。 技術的な理由のために、マウスの脊髄ネットワークの研究は主に運動ニューロンと脊髄ネットワークが未熟、運動ニューロンは、そのターゲットから分離され、スライスで研究されているin vitro標本で新生児に限られていた、MOtoneuronsは、それらの入力の大部分から分離されています。最近まで、唯一のいくつかのグループは、私たちは成体マウスの生体内で 5,6を運動ニューロンの非常に安定した録音を取得するために許可された新たな準備を発表し、私たちのチームも含め、 生体内2-4に運動ニューロンの細胞内の録音を実行するために管理していた。しかし、これらの録音は、 すなわちこれらの運動ニューロンの力出力を記録する可能性がなく、麻痺した動物で得られた。ここで我々は運動ニューロンの電気生理学的特性とそれらのモータユニットにより開発力の同時録画を得ることができましたこのオリジナルの準備の延長を提示。それは、私たちは彼らの力のプロファイルに基づいて、運動ニューロンの異なるタイプを識別し、それによって、その機能を明らかにすることができますので、これは重要な成果である。遺伝的モデルは、脊髄分節回路7-9を乱し、又はヒトdiseaをreproductingと相まって10,11 SE、我々 はこの手法は、脊髄運動系の研究のために不可欠なツールであることを期待しています。

図1は、貫通後トライセップス·下腿グループから運動ニューロンを同定する方法を示しています。低刺激強度では、唯一の単シナプスEPSP（ 図1A）を観察することができます。高強度で、EPSPが&quot;順行&quot;スパイク（ 図1B）をトリガするのに十分な大きさかもしれません。さらに高い刺激強度では、全か無かの逆行性スパイクは単シナプスEPSP（ 図...

Watch this Scientific Journal Video about Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo at JoVE.com

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

この新しい方法は、単一の成体マウスの運動ニューロンとその筋線維による力の測定を同時に細胞内記録を可能にします。正常および遺伝子改変動物における運動単位の電気的および機械的特性の組み合わせ調査は、神経筋システムの研究のための画期的な製品です。

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 ...

simultaneous-intracellular-recording-lumbar-motoneuron-force-produced

Research

JoVE Journal

Neuroscience

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

14.6K ビュー.  Northwestern University Feinberg School of Medicine. この新しい方法は、単一の成体マウスの運動ニューロンとその筋線維による力の測定を同時に細胞内記録を可能にします。正常および遺伝子改変動物における運動単位の電気的および機械的特性の組み合わせ調査は、神経筋システムの研究のための画期的な製品です。

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神経科学

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

培養皿内の機能モーターユニット：脊髄外植片と筋細胞の共培養

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします<em生体内で&gt;</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

成体マウスの前脳の急性スライスにおける神経芽細胞の移行のタイムラプスイメージング

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（<em&gt; Schistocercaアメリカーナ</em&gt;）嗅覚回路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

へのガイド<em&gt;インビボ</emOptogenetically同定された皮質抑制性介在から&gt;シングル単位記録

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同時2光子<em&gt;インビボ</emマウス脳梁膨大後部皮質におけるシナプス入力とシナプス後ターゲットの&gt;イメージング

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.