著者らは、ビデオで使用されている図面の博士ピーターGerritsに感謝。

注：消毒技術はプロシージャ全体を通して採用する必要があります。手術中に使用されるすべての楽器と材料（綿棒、ガーゼなど）はオートクレーブで滅菌する必要があります。手術用マスク、髪のボンネットと滅菌手袋を着用してください。作業領域と定位固定装置は、徹底的に清掃し、及び70％エタノール溶液で消毒する必要があります。 1。術前の手続き<ol><li>定位固定装置と必要なすべてのマテリアルを設定します。プレ暖かい暖房パッド。 </li><li>そのサポートにカニューレを配置し、それがまっすぐであるかどうか確認して下さい。 </li><li>ガスシステムの電源をONしてください - 周囲の空気と酸素の混合物（総流量の30〜35パーセントは酸素でなければなりません）。 </li><li>ラットを秤量し、麻酔剤を投与する。私たちは、ケタミン（37.5 mg / kg体重）とデクスメデトミジン（0.25 mg / kg）を皮下注射の混合物を使用しています。異なる麻酔プロトコルの場合、Flecknell 4 Hellebrekerを参照してください。S ら。7。 </li><li>ラットが意識を失った後、耳からだけで電気かみそりと目の間にしようと頭の面積を削る。 </li><li>エアチューブの前でその鼻で、加温パッド上にラットを置きます。ラットは、十分な血液の酸素化のレベルを（&lt;90％を削除してはいけません）があることを確認するために酸素濃度計を使用しています。機器の適切な使用については、製造元の指示に従ってください。 </li><li>脱水症状を避けるために、両方の角膜の眼クリーム（Duratears Z、アルコン）を適用します。 </li><li>それは十分に麻酔であることを保証するためにラットの反射神経（尾反射またはつま先-ピンチの反射、などのWalantus ら 8で示した）を確認してください。ラットは強い反射を示すために続ける場合、麻酔の補充が必要になることがあります。 </li><li>動物の前でエアチューブを何つま先-ピンチの反射が表示されていない場合は、定位固定装置にラットを置き、それが両側に同じ読みが表示されるように耳のバーを調整し、再配置鼻のバーでそれを固定すること。ラットが90％以上の血液の酸素化のレベルを示している場合、もう一度確認してください。されていない場合は、鼻に近づけるもたらす、どちらチューブを調整する、または酸素の流量を増加させる。手術中の血液の酸素化レベルと心拍数を監視します。 </li><li>継続的に直腸温度計（優先的に加熱パッドに接続されている）とラットの温度を監視し、手術の最初と最後に値を記録します。加熱パッドを調整するか、38.5に37.5体温を維持するために毛布を使用して℃に</li></ol> 2。手術<ol><li>鎮痛剤を注入する。我々は、カルプロフェンのシングル周術期管理を（4.0〜5.0 mg / kg体重、皮下）を使用している。別の鎮痛剤のプロトコルについては、Hellebrekers ら7を参照してください。 </li><li> tの剃毛面積を清掃彼消毒剤を使って、中心からの髪の周囲に皮膚三回（例えば、クロルヘキシジン0.5％）と局所的に防止するために局所麻酔と血管収縮（のためにリドカイン（20 mg / mlの）とアドレナリン（5 mg / ml）の混合物を注入過度の出血）。 </li><li>耳の裏まで、両眼の間から行く、頭皮の正中線上に約2.5cmの前後切開を加えます。肌をピンチオフすると切開を開いたまま4〜6ブルドッグクランプを使用してください。へらおよび/または綿棒を持つ任意の論理積組織を取り出して、頭蓋骨の表面を露出する面積を掃除。 </li><li>ヘッドが水平になっているかどうかを確認します：まず、ラムダを見つけて、正確にこの場所を介してガイドカニューレを置いて、頭蓋骨に触れる。背腹座標を記録します。次に、頭蓋骨に触れて、正確にブレグマ上のガイドカニューレを挿入して、その背腹座標を記録する。これら2つの座標は同一である必要があります。違いは&gt; 0.3mmである場合、それを修正するために鼻のバーを調整します。 </li><li> M滅菌ハンドドリル（反対側の半球でカニューレの位置の後方半球と他の5 mmのいずれかでカニューレの位置に前方one約5 mm）を使用して頭蓋骨のネジを固定するためのAKE二つの小さな穴。それらがしっかりと固定されるまで完全に頭蓋骨に挿入されることなく、これらの穴に2本の滅菌のネジを置きます。 </li><li>ブレグマで正確に配置されたガイドカニューレで、前後と横方向の座標を記録する。各脳の領域のためのガイドカニューレの配置の正しい場所は、脳地図9-11助けを借りて、ブレグマから加算または減算することによって計算することができます。 </li><li>わずかに頭蓋骨に触れ、その正しい場所にガイドカニューレを置きます。背腹座標を記録します。二国間カニューレを配置するため、他の半球でカニューレの位置次を検索、そして再び背腹座標を記録する。両方の座標は（または&lt;0.3 mmの異なる）同じにする必要があります。 </li><li>手のドリラーで、滅菌鉛筆で頭蓋骨にカニューレの位置をマークすると、ガイドカニューレの助けを借りて、サイズと正しい場所を確認し、バリの穴を作成します。穴が行われると、静かにカニューレの遮るもののない挿入を可能にする髄膜を開ける滅菌針を使用してください。 </li><li>最初の穴にカニューレを配置し、それが最後の腹側の座標に到達するまで、慎重に下ろします。歯科用セメントを準備し、寛大にカニューレを固定するためにカニューレと、1つまたは両方のネジの周りにそれを適用します。セメントが完全に乾くまで待ちます。その後、慎重に背腹バーを上方向に回して、カニューレのサポートを削除します。 </li><li>サポートへの2番目のカニューレを置き、他の半球のカニューレの位置に移動します。穴にカニューレを置き、前の手順を繰り返します。ネジとセメントとカニューレの大きな面を覆う、そしてセメントが乾燥する前に、皮膚からの剰余金を削除する。 </li><li>注入暖かい（〜37 ° C）補水を確保するために滅菌生理食塩水（〜10 ml / kgを、SC）。 </li><li>セメントが完全に乾いた後、カニューレのサポートを削除し、閉塞を防ぐために各カニューレに無菌のピンを配置。 </li><li>滅菌生理食塩水で創傷面積を清掃し、フロントと傷の背中を縫合。 </li><li>その鼻の前にガスチューブを交換し、定位固定装置から動物を取り外します。酸素飽和度のレベルと体温を監視し続ける。 </li><li>デクスメデトミジンと注射麻酔薬が使用されている場合は、その拮抗薬のatipamezoleを注入（0.25 mg / kgの、SC）と動物が目覚める（約5分）まで待ちます。 </li><li>回復のケージにラットを置きます。低体温を避けるために、ビバリウムの部屋にそれを返す前に、少なくとも1時間のために動物を観察できる場所で28℃でまたは加熱パッド上にインキュベーターにケージを置きます。 </li></ol> 3。手術後のケア<ol><li>中<html>手術後の最初の4日間、体重と実験室の日誌や&quot;動物福祉の日記&quot;における動物の状態に関する他の観察の日々の記録を保つことによって、ラットの回復を監視する。 </li><li>病気の明白な徴候を示す動物、傷の感染、体重や福利削減の他の徴候の損失は、特別なケアを受ける必要があります：手術後の痛みを最小限に抑えるために鎮痛薬の余分線量を、例えば、粉の食物と水の混合液を標準固形飼料に加えて、水分補給をサポートするために、ラットの食欲、および/または生理食塩水の皮下注射を刺激する。 </li><li>ラットではこれらの介入後に改善、または体重の損失が表示されない場合は犠牲が麻酔薬の過剰摂取（人道的エンドポイント）を持つ動物、（手術前の体重に比べて）&gt; 15％です。 </li><li>ラットは、通常、行動実験の開始前に少なくとも7日間で回復する必要があります。 </li></ol> 4。代表的な結果jove_content&quot;特に酸素補給と血中酸素濃度、心拍数と体温の継続的なモニタリングに関して、私たちの外科手術への様々な変更かどうかを判断するには&gt;、強化された動物の生存率と手術後に全身状態を改善し、我々は比較我々は標準プロトコルで動作24のコホート（20匹ずつ）の非生存率はこれらの変更を実装した後に手術を受けた動物の20コホートの非生存率（20​​ラットそれぞれで構成される）。図1aに示すように、非生存率が大幅に変更されたプロトコル（P &lt;0.05、Mann - WhitneyのU検定、二つの両側）で運転されたコホートのサンプルで減少した。また、図1b、手術後の体重減少のに表示されている修正されたプロトコルで操作したラットのもかなりのような標準的な手順（術後1日目で運転ラットのそれに比べて減少した：P &lt;0.05、術後2日目：P &lt;0.01、術後3日目：P = 0.17、スチューデントt検定）。 <img alt="図1" src="/files/ftp_upload/3528/3528fig1.jpg" /> 図1非生存率や手術後の体重減少に対する外科的改変の影響。標準プロトコルで動作ラットのそれと比較して変更されたプロトコルで動作ラットの（A.）非生存率。非生存率は、（中央値±四分位範囲）手術を生存しなかったラット20匹、のコホートごとに、ラットの割合として算出した。 * P &lt;0.05、Mann - WhitneyのU検定両側二つ（変更プロトコルと標準プロトコルのための24のコホートにn = 20のコホート）。最初の（POD1）、秒（POD2）とサード（POD3）術後日中（B.）体重減少（手術前の重量の割合として平均± SEM）。 * P &lt;0.05、** P &lt;0.01、スチューデントt -検定（N = 60あたりのグループ）。

<ol>
	<li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , (1959).</li><li>Richardson, C. A., Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+and+post-operative+analgesia+following+experimental+surgery+in+laboratory+rodents:+are+we+making+progress?.">Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.</a> Altern. Lab Anim. 33, 119-127 (2005).</li><li>Stokes, E. L., Flecknell, P. A., Richardson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reported+analgesic+and+anaesthetic+administration+to+rodents+undergoing+experimental+surgical+procedures.">Reported analgesic and anaesthetic administration to rodents undergoing experimental surgical procedures.</a> Lab Anim. 43, 149-154 (2009).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia - A Practical Introduction for Research Workers and Technicians. , (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIRECTIVE+2010/63/EU+OF+THE+EUROPEAN+PARLIAMENT+AND+OF+THE+COUNCIL+of+22+September+2010+on+the+protection+of+animals+used+for+scientific+purposes.">DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes.</a> Official Journal of the European Union. , (2010).</li><li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+Stereotaxic+Surgery+in+Rodents.">Survivable Stereotaxic Surgery in Rodents.</a> J. Vis. Exp. (20), e880-e880 (2008).</li><li>Hellebrekers, L. J., Booij, L. H. D. J., Flecknell, P. A., Van Zutphen, L. F. M., Baumans, V., Beynen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia,+analgesia+and+euthanasia.">Anaesthesia, analgesia and euthanasia.</a> Principles of Laboratory Animal Science. , 277-311 (2001).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J. Vis. Exp. (6), e236-e236 (2007).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1986).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2007).</li><li>Swanson, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain Maps: Structure of the Rat Brain. , (1992).</li></ol>

著者らは、開示することは何もない。

このビデオプレゼンテーションの主な目的は、定位手術の基本原則と行動の神経科学を​​理解することです。すでに定位手術を行っている研究者はまた、このビデオの恩恵を受けると、自分の研究室で使用するための手続きの改良のいくつかを検討するかもしれない。手術手技1-3を最適化する方法についてますます知識、人間と獣医学4,12、国民意識に関する動物福祉の問?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td></tr><tr><td>アルコール70パーセント</td><td> VWR </td></tr><tr><td> Antisedan（atipamezole） </td><td>オリオン</td></tr><tr><td>硫酸アトロピン0,5 </td><td> Pharmachemie BV </td></tr><tr><td>ブルドッグ止血クランプ</td><td> Aesculap </td></tr><tr><td>カニューレ</td><td>コンポーネントサプライ株式会社</td></tr><tr><td>クロルヘキシジン0.5パーセント</td><td> AppepPharma </td></tr><tr><td>粉体のクリーニング</td><td> Alconox </td></tr><tr><td>コットンスティック</td><td>ハルトマンBV </td></tr><tr><td> Dexdomitor（デクスメデトミジン） </td><td>オリオン</td></tr><tr><td>ドリル</td><td>ドレメル8000 </td></tr><tr><td> Duratears Z </td><td>アルコン</td></tr><tr><td> Durogrip Naaldvoerder逆130ミリメートル</td><td> Aesculap </td></tr><tr><td> Durotip Fijne schaar ret.sp / ST 110ミリメートル</td><td> Aesculap </td></tr><tr><td>ガーゼ</td><td> Medicomp（5x5の） </td></tr><tr><td>暖房パッド</td><td>ハーバード装置</td></tr><tr><td>昆虫ピン（スタイレット） </td><td> Entosphinx（チェコ共和国） </td></tr><tr><td>ケタミン10％（ケタミン） </td><td> Alfasan </td></tr><tr><td>マイクロホールステッドストレート先端</td><td> Aesculap - 獣医</td></tr><tr><td> Molinea </td><td>ハルトマンBV </td></tr><tr><td>塩化ナトリウム0.9％ </td><td>バクスター</td></tr><tr><td>針（25G） </td><td>テルモ</td></tr><tr><td>酸素濃度計</td><td> EDANインスツルメンツ株式会社</td></tr><tr><td>ペントバルビタール</td><td> UMCGの薬局</td></tr><tr><td> Rimadyl </td><td>ファイザー</td></tr><tr><td>手術用メスの刃23号</td><td>スワンモートン</td></tr><tr><td> ScalpelholderのNR。 4 133ミリメートル</td><td> Aesculap </td></tr><tr><td>スクリュードライバー</td><td>従来のハードウェアストア</td></tr><tr><td>迅速なシンプレックス（歯科用セメント） </td><td> Kemdent </td></tr><tr><td>頭蓋骨のネジ</td><td>コンポーネントサプライ株式会社</td></tr><tr><td>へら</td><td> VWR </td></tr><tr><td> Spongestan特別</td><td>ジョンソン＆ジョンソン</td></tr><tr><td> Stereotacts </td><td> Kopfは楽器</td></tr><tr><td>手術用鉗子100ミリメートル</td><td> Aesculap </td></tr><tr><td>縫合糸材料Safil 5 / 0 </td><td> Aesculap </td></tr><tr><td>注射器10ミリリットル</td><td> Omnifix </td></tr><tr><td>注射器1ミリリットル</td><td>テルモ</td></tr><tr><td>シリンジ5ミリリットル</td><td> Omnifix </td></tr><tr><td>キシロカイン（リドカイン/アドレナリン） </td><td>アストラゼネカ</td></tr></tbody></table>

rodent stereotaxic surgery and animal welfare outcome improvements for behavioral neuroscience

特定の脳領域へのカニューレの移植のための定位手術は何十年も目を覚まし、行動を動物に局所的に操作する神経伝達物質やシグナル伝達経路の効果を調べるために非常に成功した実験的な手法となっている。さらに、電気生理学的刺激と記録の研究のための電極の定位注入は、動物の振る舞い​​における神経可塑性と脳のネットワークの私達の現在の理解に役立っている。げっ歯類1-4、動物福祉の問題に関する国民意識と厳格な法律（例えば、実験動物5の使用に関する2010年欧州連合（EU）の指令）の手術手技の最適化について、増え続ける知識は特に関連して、これらの外科的処置を絞り込むために私たちを促しただけでなく、手術中の酸素補給のための新しい手順や血液の酸素と心拍数のレベルの連続監視を実装する手術後のケアのための標準化プロトコルを導入する。我々の観察では、これらの変更が増加生存率と手術後の動物の一般状態（例えば、以下の体重減少とより積極的な動物）の改善をもたらしたことを示している。このビデオプレゼンテーションでは、私たちのいくつかの変更に特別な注意と定位手術のこのタイプに関係する一般的な手順が表示されます。我々は、ラットではこれらの外科的処置を説明しますが、それは定位固定装置6の特別なアダプタを使用して、マウスまたは他の小実験動物に手術のこのタイプを実行することも可能です。

Watch this Scientific Journal Video about Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience at JoVE.com

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

げっ歯類での定位手術は動物の行動を、目を覚ましに薬物の標的投与や電気刺激やレコーディングが可能になります。このビデオプレゼンテーションでは、この長年の手順その成功生存率向上と縮小手術後の体重減少に、最近の手続き型の改良を紹介します。

行動神経科学のための齧歯類定位手術と動物福祉のアウトカムの改善

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

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JoVE Journal

Neuroscience

38.9K ビュー.  University of Groningen. げっ歯類での定位手術は動物の行動を、目を覚ましに薬物の標的投与や電気刺激やレコーディングが可能になります。このビデオプレゼンテーションでは、この長年の手順その成功生存率向上と縮小手術後の体重減少に、最近の手続き型の改良を紹介します。

ビデオ: Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

齧歯類モデルで中枢神経系の疾患を治療するための全身および局所薬物送達

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

神経科学

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

齧歯類のために全体の動物灌流固定

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

大人のラットの特定の脳領域の興奮毒性病変に対する定位手術

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

鳴禽類には聴覚フィードバックを操作するための軽量、ヘッドフォンベースのシステム

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

齧歯類の中枢神経系の生体光遺伝学的刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

マウス海馬にオリゴマーアミロイドベータの定位注入

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

非拘束EEG Radiotelemetry：硬膜外とディープ脳内定位EEG電極配置

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

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, ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

検出技術その場で革新的な RNA 側坐齧歯動物の核内ドーパミン D1-アルファ受容体の同定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

子豚を自由に移動するから非侵襲的脳波

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...