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1。ムスカリン性アセチルコリン受容体（mAChR）拮抗薬の皮下投与<ol><li> 1.5mlの反応チューブに滅菌生理食塩水で薬物を溶解することにより無菌条件下でmAChR拮抗薬、（参照、表）の適切な投与量を準備します。以下の拮抗薬を使用した：アトロピン、Methoctramine、4 - DAMP、AFDX - 116、AFDX - 384、MT 7。 </li><li> a1ccインスリン注射器に溶液の50μlのを描き、それぞれのマウスに別々のシリンジを使用してください。また、唯一の対照群の各マウスの生理食塩水を含む注射器を準備。氷の上で注射器に注入する準備が整うまで保管してください。 </li><li>ケタミン - キシラジンカクテルを持つマウスを麻酔する（120 mg / kgのケタミン、8 mg / kgをキシラジン）つま先のピンチに応答がないことを観察することによって適切な麻酔深度を確認してください。 </li><li>適切な鎮静した後、首の尾部領域に頭の吻側領域をカバーする毛を剃る、70％エタノールで離れて残りの髪を拭いてください。 </li><li>場所の実体顕微鏡下でマウス、および注射器の針（ 図1A、B）を軽く引き上げながら、LAL筋肉に針を平行に挿入する。これは、針が結合組織の上に横たわって挿入されると直接LAL筋肉自体を損傷しないようにします。先端がLAL筋肉の尾側面を覆うように針を挿入します。 </li><li>非常にゆっくりとシリンジを引き出しながら、右LAL筋肉上のソリューションを注入し始める、総噴射時間は約1分にする必要があります。適切に注入した場合、解決策は、少なくとも1時間にとどまるが覆っている皮膚に、直径約1 cmである&quot;ドーム&quot;を形成すべきである。一様に注入されたソリューションは、右のLAL筋肉の全体の吻側および尾側の地域をカバーします。 </li><li> 1〜7日間12時間ごとに手順を繰り返します。他のstudies1では、最大21日間の増殖因子を12時間おきに注入するために同じプロトコルを使用していました。異常応答は、最大21日までの日を2倍の麻酔を投与したマウスから観察されなかった。 </li></ol> 2。 LAL筋肉の解剖<ol><li>ペントバルビタールナトリウムの過剰投与（390 mg / kg体重）をマウスを安楽死させる。死は、開胸が行われた後、ハートビートの欠如を評価することによって保証されます。 </li><li>それぞれの足の1ピンと鼻から1ピンで、解剖皿の中で背側にマウスを突き止める。 </li><li>左の肩甲骨周辺の領域への耳にちょうど近位領域から左LALに沿ってカットする小さな春のはさみを使用して、唯一の皮膚を介して最初の切開を加えます。 </li><li>慎重にこの地域で皮膚を取り除くが、これは右のLAL筋肉のための付属のポイントの一つであるとして、右耳に近すぎるカットは避けてください。 </li><li>右と左のLAL筋肉が満たしてヘッドの表面的な面で、正中線に沿って脂質の中央に位置脂肪組織のストリップを見つけます。小さ ​​な春のはさみを使用して、肩（ 図1C）に達するまで左LAL筋の近位端から脂肪組織（左耳に向かって）の右側に1センチメートルカット。 </li><li>一度切開は筋肉の腹側を露出させる小さな鉗子で背面剥離、右LAL筋肉が行われます。慎重にピンセット（ 図1C）で右LAL筋肉を引き上げながら、結合組織や筋膜をトリミング。 </li><li>耳の付け根で右LALの尾方端の周りにカット。に引き渡した右LAL（図1C）を維持、右耳の吻側端に向かって移動し、切断を続行。。 LAL自体を損傷する危険性よりも、このステップでより多くの組織を含める方が良いです。筋肉は、筋肉の上にピペット1X PBSを乾燥し始めた場合。 </li><li>場所は、最大1 × PBS、背側を含む30ミリメートル文化Sylgard皿で右LAL解剖。小さな虫ピンで筋肉の四隅を突き止める。 </li><li>右LAL筋の背側と腹側の表面から小さな鉗子と春のはさみ、クリーンな結合組織を使用する。腹側表面をきれいにするために筋肉を裏返しにして、再度ピン。 2月3日の筋肉は、同じ30ミリメートル皿に置かれるかもしれない。 </li></ol> 3。 LAL NMJsのトリプル免疫染色：1日目<ol><li>免疫染色を開始する前に、すべての必要な試薬を準備する。 1X PBS、2％ウシ血清アルブミンBSA）/ PBS、2％の0.1MグリシンBSA / PBS、2％BSA / PBS、および1X PBS中4％パラホルムアルデヒド（PFA）で0.2％トリトンX100が必要になります。 -20℃の冷凍庫内プレクールメタノール。 </li><li>このプロセスの間に皿の背側にまで固定されたLAL筋肉を残す。 1X PBS溶液を捨て、筋肉をカバーするのに十分な4％（PFA）を追加します。シェーカー上に置いて料理は20分間スピード2または3に設定。特に断りのない限り、料理は、以下のステップのためにシェーカーに配置する必要があります。 </li><li> 4％PFAを破棄し、1X PBSで3回、10&#39;それぞれで筋肉を洗う。 </li><li>最後の1X PBSの洗浄を破棄し、30&#39;のために2％BSA / PBS溶液に0.1Mグリシンを追加。 </li><li> 0.1Mグリシン溶液を捨てるdは、1 × PBSで15&#39;の1:200の濃度でローダミン共役α-ブンガロトキシンを含む追加。 </li><li> α-ブンガロトキシンの液を捨て、それぞれ3回、1 × PBS中で10&#39;の筋肉を洗う。 </li><li>あらかじめ冷却メタノールを添加することにより組織を透過し、正確に5分間-20℃の冷凍庫に皿を置く、ない必要に応じて揺れていない。 </li><li>メタノールを取り外し、10&#39;1X PBSでそれぞれ3回洗浄します。と最後の洗浄およびブロックの組織を捨てる0.2％トリトンX - 100を2％BSA / PBSで1時間。洗浄及びブロッキングのステップは室温で実施されています。 </li><li> 4で、揺れ、一晩筋肉をインキュベートしますブロッキング溶液（参照、表）で希釈した一次抗体のカクテルでC。 </li></ol> 4。 LAL NMJsのトリプル免疫染色：2日目<ol><li>室温で1 × PBSで3回、10&#39;それぞれの筋肉を洗う。 </li><li>室温で1時間（参照、表）のためにブロッキング溶液に二次抗体のカクテルを追加。アレクサ - フルーア647蛍光の光退色を防ぐために、筋肉はこのステップ以降からの光から保護する必要があります。 </li><li> 1X PBSで3回、10&#39;それぞれの筋肉を洗う。 </li><li> 1X PBSの残りの結合組織をきれい、ガラスカバースリップとマウントメディアを使用して、スライド背側LAL筋肉とマウントの外側の境界線をカット。代わりにカバースリップを確保するために明確なマニキュアを使用してください。 </li><li>分析の準備が整うまで-20℃で光とストアからスライドを保護する。 </li></ol> 5。 LAL NMJsの共焦点イメージング<ol><li>高解像度の共焦点画像は、ライカTCS 4D共焦点顕微鏡ライカのプランアポ63Xの油浸対物（1.4NA）で得られる。 </li><li>フルオレセインおよびAlexa 647の信号が同時にそれぞれ、レーザライン488nmの（アルゴン、488 nmの、20 MW）と633 nmの（HeNeレーザ、633nmの、10 mWの）でスキャンした。順次、ローダミンの信号は、レーザーライン561 nmの（DPSS、561、20 MW）でスキャンされます。光学切片を0.3μmの間隔で収集し、z平面上の画像の数が60から80まで変化させた。ピンホールは、エアリー1（107.97 UM）に調整されます。画像フォーマット1024 × 1024（X、Y）、234.32μmのX 234.32μm及び229.05 nmのピクセルサイズ× 229.05 nmの画像サイズで400Hzの速度の結果で1のズーム倍率、と。 </li><li>ライカTCS - NT取得ソフトウェアは、最大強度の予測にZシリーズの画像を再構成するために使用されます。 </li><li>マルチパネルの画像はAdobe Photoshopを使用して明るさとコントラストに調整されます。 </li><li>典型的なLAL筋肉のために、人は顔の NMJs 途中で約75から100を分析することができるはずです。シナプスの安定性は、事前に、周術期およびシナプス後要素の空間的配置（すなわち、神経、シュワン細胞や筋肉）などの機能、及びシナプス面積の測定（すなわち、シナプスの大きさ）によって決定されます。 </li></ol>代表的な結果： 、正確にニコチンAChRsのシナプス後のクラスター（ 図2、レッド）に並列した単一の筋線維で、成体哺乳類NMJで、単一のモータの軸索は、神経終末の高度に差別化喬木（グリーン図2）を形成細かい枝を詳しく説明。 Perisynaptically、ターミナルシュワン細胞が密接にシナプス前神経末端のすべての支店（;ブルー図2）をカバー。この三者の組織の構造的および機能的完全性は深刻なサブタイプ固有のmAChR阻害剤の毎日のアプリケーションによって摂動されています。 （ 図3）ここで紹介する例では、4 - DAMP、M1、M3、M4とM5 mAChRサブタイプに高い親和性を持つmAChR拮抗薬は、筋肉の表面（ 図3B、全体に多数のNMJsから神経端末の選択的除去を呼び起こすC）。プロセスの拡張子を除いた明るいS100のラベリング（ 図3B&quot;、3C&quot;）によって証明されるように加えて、ターミナルシュワン細胞は8異常に静止です。 Postsynaptically、筋線維は正常であり、nAChRsの失われた（ 図3B&quot;、3C&#39;）はありません。 <img src="/files/ftp_upload/3124/3124fig1.jpg" alt="図1"/> 図1。LAL筋肉の解剖学的位置と組織。吻側（rLAL）、尾側（cLAL）、およびLAL神経（LALn）と対応するエンドプレートのバンドの位置は（、挿入図）が示されている。 LAL筋肉に関しての針の位置と方向は、注射の手順（B）のために示されています。切開ポイントは解剖の手続き（C）として使用されています。 <img src="/files/ftp_upload/3124/3124fig2.jpg" alt="図2"/> 図2。NMJの三者構成。マウスNMJの高倍率焦点ビュー。単一の軸索は、しっかりとターミナルシュワン細胞とそのプロセス（ターミナルシュワン細胞体へ&quot;、アスタリスクポイント）の対象となる微細な端末の枝を（）、詳しく説明する。 Postsynaptically筋線維では、ニコチンAChRsのクラスタは、正確に神経端末の枝に並列したさnalsとターミナルシュワン細胞。 <img src="/files/ftp_upload/3124/3124fig3.jpg" alt="図3"/> 図3。LAL筋は4 - DAMP、mAChR拮抗薬による治療を受けた。車両または4 - DAMPで治療LAL筋肉の低と高倍率共焦点ビュー。溶媒投与筋肉（とは対照的に、A - &#39;&#39;&#39;), 4 - DAMP処理筋肉の欠如の神経終末の数々NMJs（B - B &#39;&#39;&#39;、C - C &#39;&#39;&#39;).図2Bの箱入りの領域は、図2Cにズームされています。

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利害の衝突は宣言されません。

ここで紹介する方法は、哺乳類NMJsの安定と維持におけるシグナル伝達サブタイプ固有のmAChRの以前に認識されていない役割の調査を可能にします。また、このメソッドは、神経栄養因子および薬理学的作用物質の効果をテストするために有用であろう。例えば、私たちの研究室では、毛様体神経栄養因子（CNTF）は、成体マウスのほぼすべてのLAL神経末端から発芽誘発されることがわかった<a>...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>ケタミン</td><td>ホスピーラ</td><td> NDC0409 - 2051〜05 </td><td>用量：120mg/kg </td></tr><tr><td>キシラジン</td><td>ロイド研究所</td><td> LA33806 </td><td>用量：8mg/kg </td></tr><tr><td>アトロピン</td><td>シグマアルドリッチ</td><td> A0132 </td><td> （&gt; 98％純度）、投与量：0.2mg/kg - 20mg/kg </td></tr><tr><td>アトロピン</td><td>フォイクトグローバルディストリビューション</td><td> AT105 </td><td>医薬品グレード</td></tr><tr><td> Methoctramine </td><td>シグマアルドリッチ</td><td> M105 </td><td>用量：100 - 400M </td></tr><tr><td> 4 - DAMP </td><td>シグマアルドリッチ</td><td> D142 </td><td>投与量：2.5mg/kg </td></tr><tr><td> AFDX - 116 </td><td>バイオサイエンスTocris </td><td> 1105 </td><td> 250M </td></tr><tr><td> AFDX - 384 </td><td>バイオサイエンスTocris </td><td> 1345 </td><td> 50M - 500M </td></tr><tr><td> MT 7 </td><td>国際ペプチド</td><td> PMT - 4340 - S </td><td> 0.1M - 1M </td></tr><tr><td> 1Xリン酸緩衝生理食塩水、pH7.4のバッファ付き</td><td>インビトロジェン</td><td> 10010049 </td><td></td></tr><tr><td>パラホルムアルデヒド</td><td>フィッシャー</td><td> T353 - 500 </td><td> 10g/100mL脱イオン蒸留水に溶解することによって最初の10％溶液を作る; 7.4にpHを調整し、1X PBSで4％を作る</td></tr><tr><td> pentobarbitolナトリウム</td><td> Virbacアニマルヘルス</td><td> NDC - 051311 - 050 - 01 </td><td>用量：390mg/kg </td></tr><tr><td> Sylgard </td><td>ダウコーニング</td><td>パート＃184 </td><td>キットに付属の指示に​​従って、ニーズに応じて複数のサイズの培養皿を（30ミリメートル、60ミリメートル、100ミリメートル）を使用することができます</td></tr><tr><td> 0.1Mグリシン</td><td>シグマアルドリッチ</td><td> G - 7126 </td><td> 2％BSA / PBSの0.185グラムに25mLを追加します。 </td></tr><tr><td> 2％ウシ血清アルブミン（2％BSA） </td><td>シグマアルドリッチ</td><td> A3059 - 100G </td><td> 1X PBS 100mLの中に2グラムBSAを溶解する</td></tr><tr><td> 2％0.2％トリトンX100 BSA / PBS（ブロッキングバッファー） </td><td>シグマアルドリッチ</td><td> T9284 - 100mLの</td><td> 0.2ml/100mL 2％BSA / PBSを溶かす</td></tr><tr><td> α-ブンガロトキシン</td><td>インビトロジェン</td><td> T1175 </td><td> 1:200の濃度で使用</td></tr><tr><td> SMI - 312 </td><td>シュテルンベルガーのモノクローナル抗体</td><td> SMI312 </td><td> 1:1000の濃度で使用</td></tr><tr><td> SV2 </td><td>発達研究ハイブリドーマバンク</td><td> SV2 -上清</td><td> 1:10の濃度で使用</td></tr><tr><td> S100 </td><td>ダコ</td><td> Z0311 </td><td> 1:400の濃度で使用</td></tr><tr><td> FITC -ヤギ抗マウスIgG1 </td><td>ロッシュ</td><td> 03117731001 </td><td> 1:200の濃度で使用しますが、バックグラウンドが高い場合には、1:400を試してみてください</td></tr><tr><td>アレクサ - フルーア647結合ヤギ抗ウサギ</td><td>インビトロジェン</td><td> A21244 </td><td> 1:200の濃度で使用</td></tr><tr><td> Vectashield蛍光マウンティングメディア</td><td>ベクターラボラトリーズ</td><td> H - 1000 </td><td>これはハードセットのメディアではない、あなたは明確なマニキュアでカバーガラスを固定する必要があります。 </td></tr><tr><td>小さな春のはさみ</td><td>ファイン科学ツール</td><td> 15002〜08 </td><td></td></tr><tr><td>解剖の鉗子</td><td>ファイン科学ツール</td><td> 11295〜51 </td><td></td></tr></tbody></table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

このような腓腹筋と前脛骨筋などのげっ歯類の後肢の筋肉が、、頻繁に哺乳類NMJsの形成と維持に不可欠なシグナルの in vivo薬理試験で使用されます。しかし、皮下または筋肉内投与後にこれらの筋肉への薬物の浸透は、しばしば不完全または不均一であり、多くのNMJsは影響を受けませんができます。このようなミニポンプなどの機器との全身投与は、時空間の効果を向上させることができるが、このアプローチの侵襲的な性質は、交絡炎症応答および/または直接筋肉の損傷を引き起こす可能性があります。それは時間のかかるシリアルセクショニングと豊富な免疫染色​​を必要とするためまた、後肢の筋肉におけるNMJsの完全な分析は困難です。 マウスLALは首の背部に表面的にある筋肉の薄い、平らなシートです。それは、関数が耳介を移動することが速筋筋肉です。それは頭蓋骨の正中線に由来し、それぞれの耳介の軟骨部に横方向に拡張する吻側および尾側部分が含まれています。筋肉は、それが終了する茎乳突孔を次のように尾側に投影する顔面神経の枝によって供給される。我々と他の人がNMJsと筋肉での薬物の in vivo効果で短期と長期の両方の調査のための利点を提供する便利な準備であることがLAL発見した。最初に、その表面的な場所は軽い麻酔下での薬剤の複数のローカルアプリケーションを容易にします。第二に、その薄さ（筋線維の2-3層）は、筋肉内のほぼすべてのNMJsの可視化と分析を可能にします。第三に、その神経支配のパターンと一緒に無傷の神経でそれを解剖しやすさは、 試験管 9,5 の補足的な電気生理学的解析を可能にします。おそらく最後の、そして最も重要な、小さなappliedボリューム（〜50μlのは）簡単に、全体の筋肉の表面を覆って薬剤へのすべてのNMJsの制服と長期の暴露を提供し、体系的なアプローチ1,8が不要になります。

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

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

我々は、挙筋オーリス筋（LAL）若い成体マウスの筋肉にムスカリン性シグナル伝達の阻害剤の反復投与のためとwholemountsにおける神経筋接合部のその後の免疫染色（NMJs）するための手順を説明します。 LAL筋肉は明らかにするためのユニークな利点があります<em&gt; in vivoで</emNMJs上&gt;薬理作用。

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

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

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K ビュー.  Arcadia University. 我々は、挙筋オーリス筋（LAL）若い成体マウスの筋肉にムスカリン性シグナル伝達の阻害剤の反復投与のためとwholemountsにおける神経筋接合部のその後の免疫染色（NMJs）するための手順を説明します。 LAL筋肉は明らかにするためのユニークな利点があります in vivoで薬理作用。

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

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

ラットの薬物自己投与時の超音波発声の評価

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

神経科学

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

腸神経系ではα-シヌクレインの介在物の強制経口および画像解析を用いたロテノンの経口投与

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

慢性ニコチン投与によるマウスノックインで蛍光標識ニコチン性受容体のスペクトル共焦点イメージング

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

ガスクロマトグラフィーによるオペラントエタノール自己投与とエタノール判別中エタノールのマイクロダイアリシス

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

重度の脊髄損傷後のフィブリンおよび成長因子カクテル神経幹細胞の生存と分化の促進

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

カニ手足の固有感覚と緊張受容体：学生実験室の練習

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

管理のリアルタイム fMRI ニューロフィード バック トレーニングのプロトコル

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

気体の麻酔薬は、キセノンと亜酸化窒素の健康なボランティアの管理中に記録脳波電磁

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

オーリスの挙筋哺乳類の神経筋伝達電圧クランプ条件下での試験のための準備

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

マウスの神経幹細胞と脳における5-ヒドロキシメチルシトシンの検出

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...