我々は、技術的な支援をBakhos A. Tannous、博士のrAAV-EGFPベクターを用いて私たちを提供するためのマサチューセッツ総合病院、チャールスタウン、マサチューセッツ州の神経科学センターのベクター開発·生産担当ディレクター、ジョン·むちで打つことに感謝します。この作品は、神経疾患や脳卒中の国立研究所からの助成金によってR01 NS050408を（JS）にサポートされていました。

記載外科手術はコロンビア大学の施設内動物のケアと使用委員会（IACUC）によって承認されています。 1。機器およびウイルス粒子懸濁液の調製<ol><li>装置を清掃し、消毒、手術器具と椎骨L1を固定するために使用されるVノッチスパイクを滅菌します。 </li><li>プルとベベルガラスピペット。我々は、40μmの先端径を持ち、20°の角度で傾斜がついていピペットを使用しています。ガラスピペットを滅菌する。 </li><li>定位フレームを設定し、マニピュレータ上にマイクロシリンジインジェクタを取り付けるとコントローラにインジェクタを接続します。 </li><li>コンプレッションフィッティングキットを使用してマイクロシリンジにガラスピペットのいずれかを添付してください。 </li><li>マイクロシリンジからプランジャを取り外し、ミネラルオイルを注射器を埋める。オイルレッドO（1 - （2,5 - ジメチル-4 - （2,5 - dimethylphenylazo）フェニルアゾ）-2 - ナフトール）が、そのvを高めるために鉱物油に添加してもよいisibility。プランジ​​ャーを挿入し、先端に至るまで押し込みます。慎重に気泡を作成することは避けてください。 </li><li>バイオセーフティキャビネット内でウイルス粒子の懸濁液を準備します。氷の上で冷凍ウイルスを解凍し、使用直前に、滅菌した所望の粒子濃度にリン酸緩衝生理食塩水で希釈。 </li><li>インジェクタ上のホルダーにマイクロシリンジを挿入します。 </li><li>小さ ​​なプラスチック製のシート上にウイルス懸濁液のピペットを5μl、 例えばパラフィルム。ドロップにガラスピペットの先端を下げ、マイクロシリンジを埋めるためにプランジャーを引く。目詰まりを防ぐためのピペット先端に小さな気泡を作成します。 </li></ol> 2。椎弓切除術<ol><li>ベンチや消毒剤で加熱パッドを拭くことによって手術領域を準備します。 </li><li>マウスを麻酔。我々は、（誘導中に3％、メンテナンス時の2〜3％）とイソフルラン吸入麻酔を使用します。 </li><li>時の乾燥から目を保護するために、それぞれの目の上に置いて潤滑剤操作。 </li><li>腰痛からのマウスの首にファーを剃ると、クロルヘキシジンまたはポビドンヨード、70％エタノールなどの局所消毒のワイプを交互に皮膚の消毒。外科用ドレープで無菌的に調製現場を隔離し、ブピバカイン（0.25％、生理食塩水で1:10に希釈）を用いて切開部位に浸潤する。 </li><li>正中線に沿って肋骨の尾側（2-3 cm）で皮膚を切開し、背骨を覆う筋膜を分離します。 </li><li>脊髄は脊柱より生後発育中に、以前の成長が止まるので、脊髄分節L4は第1腰椎（L1）の下に位置しています。椎骨L1はリブの最後のペアを保持している椎骨尾に位置しています。その背面に付いている小さな脊髄筋肉と靭帯を除去することによって椎骨L1を識別し、公開します。 </li><li>少しアドソン鉗子と椎骨L1を持ち上げたままにします。背portioを削除するには、専用の椎弓切除鉗子を使用n個の椎骨（背骨やラミナ）と脊髄を露出するの。脊髄の損傷を防ぐ。 </li><li>定位フレーム内の加熱プレート上にマウスを移す。その後の動作中にマウスの温度を監視します。 </li></ol> 3。注射<ol><li> Vノッチスパイクで椎骨L1を固定してください。椎骨は、呼吸中に動かないようにスパイクが脊椎を安定させなければなりません。 </li><li>近いので、ピペットの先端が椎弓切除部位を超えていることをマイクロシリンジを持参してください。あなたはピペットを出るウイルス懸濁液が表示されるまでプランジャーを押し下げます。滅菌綿棒で液滴を削除します。 </li><li>露出した脊髄の吻側、最も部分にピペットチップを置きます。センター後正中溝上記ピペット、その後500μmで横方向に先端を移動。コー​​ドの表面にチップと穿刺硬膜を下げるか、あなたはunbeveledガラスピペットを使って作業しているときは、dは穿刺に面取りされたスチール製のカニューレを使用浦。脊髄にガラスピペット300μmの先端を下げます。 </li><li> 200 NL / minの速度でウイルス懸濁液の1μlを注入します。 </li><li>注射の終了時に、ゆっくりと後退させるピペット前に少なくとも2分待ってください。 </li><li>脊髄分節L4にウイルスベクターの完全な分配を達成するために露出した脊髄の尾最部でステップ3.3から3.5を繰り返します。 2注射部位は、対象地域の組織の損傷を回避するために吻側およびL4セグメントの尾側に位置しています。 </li></ol> 4。創縫合<ol><li> Vノッチクランプから椎骨L1を離すと定位フレームからマウスを削除します。 </li><li> 5.0ビクリルと筋膜を縫合。閉鎖筋膜は椎弓切除部位のためのカバーを提供しています。 </li><li>ナイロン縫合糸または外科用ステープルで皮膚を閉じます。 </li></ol> 5。術後処置<ol><li>柔らかい、nonparticularな寝具を使用して回復ケージにマウスを移す。トンの上に置きます快適な呼吸のために彼がサイド。それは通院、完全に警戒され、飲酒を開始するまで、動物を監視します。 </li><li>我々は、カルプロフェンの毎日の皮下注射（5.0 mg / kg）を使って72時間術後の鎮痛を提供します。 </li><li>実験が完了するまで、最初の3日間毎日検査で術後の回復、隔日または週3日を監視します。 </li><li>創傷治癒が完了したときに、7〜10日間運転後の皮膚の縫合糸またはステープルを外します。 </li><li>実験終了後の動物を安楽死させる。 </li></ol>

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著者らは、彼らが競合する経済的利益を持っていないことを宣言します。

定位ベクターの注入は、神経変性11の予防または治療のために傷害9,10、または遺伝子治療から再生中に6,7またはoptogenetic郭清8、軸索ガイダンスを広げる経シナプスウイルスに基づいて、このような神経ネットワークのマッピングなどのアプリケーションに脊髄ニューロンを標的にできます12。ウイルスベクターは、体性感覚、運動と自律神経?...

マウスにおける条件遺伝子操作の高度な技術は、中枢神経系におけるシナプス経路と機能の接続の探査への多面的なアプローチを可能にします。導入遺伝子は、エストロゲン受容体1の変異型リガンド結合ドメインを認識するリプレッサーまたは遺伝子の転写活性化因子、またはタモキシフェンとして機能するように設計することができるテトラサイクリン制御トランスに作用するようドキシサイクリンなどの小分子エフェクターによって調節することができる。不可逆的な導入遺伝子の改変は、一般的デオキシリボ核酸（DNA）リコンビナーゼによって達成される。 CRE（原因組換え）とFLP（フリッパーゼ組換え酵素）が切除、反転またはそれぞれ1のloxP（クロスxの軌跡上、P1）またはFRT（フリッパーゼ認識対象）サイトが隣接してDNA断片の転移を触媒する。アプリケーションでは、遺伝子の活性化またはサイレンシングおよび誘導リボ核酸（RNA）干渉を含む<sup&gt; 2。例えば、β-ガラクトシダーゼまたはアルカリホスファターゼなどの蛍光または酵素レポーターの条件式は、ニューロンにラベルを付け、それらの局所組織と接続3を調べるために使用することができます。北米（の大規模な変異導入プロジェクト<a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ）、ヨーロッパ（ <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucommは</a> ）で、マウス胚性幹細胞クローンのライブラリーを製造しています最終的には全体のマウスゲノムをカバーする条件付き遺伝子標的とトラップ。これらのクローンから生成されたマウスは、選択的な遺伝子操作（たとえば、ニューロンの特定の集団へ特異的プロモーターまたは遺伝子座の下でのDNAリコンビナーゼを発現するマウス系統の拡大数と交配することができる<a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index 。PHP</a> ）。 <p class= &quot;jove_content&quot;&gt;興味のあるニューロン集団に特異的なプロモーターが知られていないか、領域内のすべてのニューロンによって表現されていない場合には、神経細胞や関心のある特定の領域の異なる集団に遺伝子操作を制限することは、単独で標的遺伝子によって達成されない場合があります興味のある。脊髄では、実験的なデザインは、1つまたは2つの頭尾方向のセグメントへの遺伝子操作の空間的制約が必要な場合があります。 CreまたはFLPを発現するウイルスベクターの定位注入フロックス化またはflrted対立、いわゆるDNA断片がloxPまたはFRT部位に隣接されているマウスの脊髄の領域に遺伝子組換えを制限することができます。マウスを表現リコンビナーゼで動物を交配に起因する構成的DNA再構成とは異なり、この戦略はまた、遺伝子の活性化やサイレン以上時間的な制御を提供します。コー​​ドするウイルスベクターは、フロックス化または導入遺伝子はcorrespoを発現するマウスで遺伝子操作の逆のオプションを提供しています浮気ニューロン特異的プロモーターの下流にリコンビナーゼをnding。ニューロンへの親和性を持ついくつかの組換えベクターは、4利用可能です。大容量（ガット）、アデノウイルス、アデノ随伴ウイルス、単純ヘルペスウイルスやレンチウイルスは、一般的に神経親和性のベクトルを使用されています。研究課題に適したウイルスを選択すると、実験デザインの重要な部分です。導入遺伝子のサイズ、配送ルート、グリア細胞とは対照的に、神経細胞への感染の特異性、感染症の効能は、炎症性および毒性の副作用が4を考慮する必要があります。 ここでは、脊髄後角へのウイルスベクターを、私たちは痛みの神経生物学上の我々の研究での条件付き遺伝子調節のために採用している手法の定位注射について説明します。後角には侵害受容性ニューロンを含む一次体性感覚ニューロンからの求心性入力を受け取る。投射ニューロンからそれを伝える前に、ローカルの介在ニューロンは情報を処理する脳5〜背角。我々は、構成的に活性サイトメガロウイルスプロモーター下に強化緑色蛍光タンパク質（EGFP）を発現する神経向性組換えアデノ随伴ウイルス（rAAVの）と脊髄分節レベルL4で後角ニューロンの感染を示しています。

<table border="1"><tbody><tr><td> 材料名 </td><td> 会社 </td><td> カタログ番号 </td><td></td></tr><tr><td>脊髄ベースプレート</td><td>デビッドKopfはインスツルメンツ</td><td> 912 </td><td></td></tr><tr><td>小動物の定位器</td><td>デビッドKopfはインスツルメンツ</td><td> 900 </td><td></td></tr><tr><td>マウスガス麻酔ヘッドホルダー</td><td>デビッドKopfはインスツルメンツ</td><td> 923-B </td><td></td></tr><tr><td>調整可能なベース·マウント</td><td>デビッドKopfはインスツルメンツ</td><td> 982 </td><td></td></tr><tr><td> Vノッチスパイク</td><td>デビッドKopfはインスツルメンツ</td><td> 987 </td><td></td></tr><tr><td>小動物温度制御システム</td><td>デビッドKopfはインスツルメンツ</td><td> TCAT-2LV </td><td></td></tr><tr><td>アドソン鉗子</td><td>ファイン科学ツール</td><td> 11006から12 </td><td></td></tr><tr><td>椎弓切除鉗子</td><td>ファイン科学ツール</td><td> 11223から20 </td><td></td></tr><tr><td> SYS-Micro4コントローラとUltraMicroPump（1） </td><td>ワールド·精密機器</td><td> UMP3-1 </td><td></td></tr><tr><td>マイクロシリンジ、65RN </td><td>ハミルトン</td><td> 7633から01 </td><td></td></tr><tr><td> RNコンプレッションフィッティング、1ミリメートル</td><td>ハミルトン</td><td> 55750から01 </td><td></td></tr><tr><td>ホウケイ酸ガラスキャピラリー</td><td>ワールド·精密機器</td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td>ナリシゲ</td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

ウイルスベクターの実質内注射は、神経細胞や中枢神経系の特定領域の異なる集団の中の条件遺伝子操作を可能にします。我々は、マウスの脊髄後角における標的遺伝子発現またはサイレンシングを可能に定位注入技術を実証する。手術は短時間です。それは動物と脊椎の健常運動性の迅速なリカバリを提供する、単一の椎骨の椎弓切除術を必要とします。傾斜のガラスカニューレとシリンジの低速および使用に小さなベクター懸濁液量の制御された注射は組織病変を最小限に抑えます。ベクトルの局所免疫応答は、採用ウイルス固有の性質に依存し、我々の経験では、組換えアデノ随伴ウイルスを使用した場合、マイナーと短命です。このような強化された緑色蛍光タンパク質などのレポーター遺伝子は、ベクターの監視空間分布を容易にし、有効性や携帯仕様トランスフェクションのficity。

反対側、腹角および後根神経節の背角を温存注入後角（ 図1）の神経細胞で成功したトランスフェクションの利回り堅牢な遺伝子発現を、。 <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="図1" /> 図1。後角ニューロンのトランスフェクション。L4脊髄の左後角における蛍光レポーターEGFP（緑色）の（）式は、2週間のrAAV-EGFPの定位注入後?...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

ウイルスベクターは、標的遺伝子操作が可能になります。我々は、脊髄後角へのウイルスベクターの定位注射、一次体性感覚求心性および中枢神経系の神経細胞間のシナプスの接触の著名なサイトを使って、条件付き遺伝子発現やマウス脊髄におけるアブレーションのための方法を示しています。

マウス脊髄における条件遺伝子操作のためのウイルスベクターの定位注射

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

Research

JoVE Journal

Neuroscience

30.2K ビュー.  Centre National de la Recherche Scientifique (CNRS). ウイルスベクターは、標的遺伝子操作が可能になります。我々は、脊髄後角へのウイルスベクターの定位注射、一次体性感覚求心性および中枢神経系の神経細胞間のシナプスの接触の著名なサイトを使って、条件付き遺伝子発現やマウス脊髄におけるアブレーションのための方法を示しています。

ビデオ: Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髄電気生理学II：細胞外吸引電極の作製

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

神経科学

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

によって胚および成体神経幹細胞の拡大<em&gt;子宮内</em&gt;エレクトロポレーションまたはウイルス定位注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

永続かつ広範な神経伝達のための新生児マウス脳の脳室内ウイルス注射

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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

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

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

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

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

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

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

マウス脊髄における細胞ダイナミクスの二光子イメージング

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生児マウス脳の線条体細胞の遺伝的操作の定位手術

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

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 ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

レンチ ウイルスのベクトルの直噴ハイライト ラット脊髄における複数の運動経路

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

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 ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

生体 内 カルシウムイメージングのための定位ウイルス注入および勾配指数レンズ移植

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...