私は 、in vivo細胞内記録と細胞内染色の技法の初期の開発に関するヘルプは、ブラウンとDavid Maxwell ので最初の訓練のためにアンソニーテイラーに感謝。私はcollocalizationマクロのヘルプはM.シルバーに感謝。多くの学者は、誰と私はR.東亜、M.森谷、P.羅、R. Ambalavanarを含むこの技術の開発に提供する洞察力を協力してきました。この手法は、NIHの助成金DE10132、DE15386とRR017971からかなりのサポートと開発されました。

1。動物の準備<ol><li>ペントバルビタールナトリウム（50mg/kg IP）と加熱パッド上の場所でラットを麻酔。動物のバリカンで後部頭蓋骨を覆う皮膚を剃る。麻酔のレベルの手術レベルはつま先を挟まないだけでなく、眼瞼反射欠如である場合撤退の反射や発声の有無をテストすることによって得られていることを保証するために動物を確認してください。麻酔15分ごとのレベルを確認し、ペントバルビタールナトリウム15mg/kgの注射は45分ごとでanesthetisaの外科的レベルを維持する。 </li><li>無菌操作を使用し、腹部と太ももの内側で形成された皺に鼠径部の切開だけで遠位にし、大腿静脈にカニューレ（1ミリメートル直径、クレイアダムス）、および大腿動脈に挿入する。顎下部の正中切開を行う、舌骨下筋を反映している。気管に小さな切開を行い、換気を可能にするカニューレを（直径2mm）を挿入します。動脈カニューレを介してplace.Monitor全身収縮期血圧および拡張期血圧でそれを確保するcannual trachael周り縫合糸をTYE。撤退と眼瞼反射の監視に加えて、今麻酔のレベルを評価するために血圧を監視する。静脈カニューレを介して必要なときに追加の麻酔を管理します。 </li><li>定位フレームにラットを置いて、小脳を露出させる開頭術を行います。齧歯類の人工呼吸器に気管カニューレを取り付けます。加湿空気と100/minの割合で2 cm 3の体積で動物を換気する。 1センチメートルH 2 Oの正側呼気圧力は肺の虚脱を防ぐために維持されるべきである。 15分ごとに無気肺を防ぐために、肺に超インフレにする。その後、温めたミネラルオイル（30℃）で脳の表面を覆う。直接壁と壁間の骨の間の接合部の下にある大静脈洞を避けるように注意してください。 </li><li>電磁振動に結合ロッドにシアノアクリレート接着剤を適用すると顎の正中離開でロッドを取り付けます。どちら/ Dコンピュータの出力または信号発生器から振動子に下顎を使用してコマンド信号を移動します。 </li><li>開頭術に隣接して皮膚の下に電極を接地siliver - 塩化銀を置きます。 </li></ol> 2。電極の準備<ol><li>水平方向の微小電極プラーを使用して石英やアルミノシリケートガラスから微小電極を作製。 </li><li> 0.25M KClおよび0.5Mトリス塩酸緩衝液（pH7.6）または生理食塩水で2％tetramethlyrhodamine（pHは6）に溶解した5〜10％のbiotinamideに微小電極を埋める。電極のテスターと電極のインピーダンスを確認してください。大径の軸索から記録するには60 - 80MΩのインピーダンスを持つ電極を作る、小さな軸索および介在、80 -150MΩのインピーダンスと電極を作る。 </li><li>エレクトロメータのヘッドステージに微小電極を配置。動物の頭の後ろの位置に固定されている小さな望遠鏡（同封のレチクル付き20X）を介して電極を視覚化する。それは電極を定位を高い精度で配置されることができるため、望遠鏡を使用することが重要です。 </li></ol> 3。電気生理学的記録と細胞内染色<ol><li>軟膜の小さな開口部を作成し、電極が脳に触れるとフィードバック信号が生成されるように容量のフィードバックを過度に補償する。これは、脳の表面の正確な位置を可能にします。 </li><li>繰り返される下顎変位を生成し、ステッピングモータと、脳に電極を進める。 </li><li>直流電位の急激な低下によって神経impalementsを認識し、継続的なシナプス活動によってintrasomatic神経impalementsを識別する。静電容量の過補償と電極を賑わっまたはめったに成功した細胞の浸透を生成しないタップ。ため、電極の高インピーダンスから、神経細胞応答はほとんど浸透する前に観察されない。 </li><li>あなたがニューロンを突き刺すと浸透が安定したと判断された後、ランプとホールドと正弦波顎の動きを使って神経細胞の応答を特徴付ける。マップ5ニューロンの受容野を非導電性プローブを用いて頭の周りの皮膚と内口腔をプローブすることにより木の棒など。研究に関連する場合、そのような筋収縮や侵害刺激のような他の機能的に関連刺激に対するニューロンの応答を調べて6一次求心性侵害受容ニューロンの受容体は、侵害刺激に応答する。ペントバルビタールナトリウムのような一般的なanestheseticsは、一次求心性ニューロンの軸索の伝導をブロックするのではなく、シナプス伝達を抑制することはありません。したがって、一次求心性神経細胞軸索の軸索伝導が維持されます。 </li><li> 15 70nA分の総噴射時間のために（DC、1 - 4NA）電流を注入。電流注入時の電極の侵入を監視し、膜電位が- 30mVのより肯定的になれば中止する。</li></ol> 4。組織の処理<ol><li>リンス血管にペントバルビタールの過剰投与（140mg/kg IV）と浸み込ませることにより動物を安楽死させる（0.9％塩化ナトリウム38℃ヘパリンと1ミリリットルの2％のCを含む500台を0.1Mリン酸緩衝液（pH7.4）中4％パラホルムアルデヒドに続いてキシロカイン。 </li><li>それはどちらの正面、矢状または水平面でビブラトームを用いて50 -100μmの時に脳やセクションを削除します。 Secionsは25℃のPBSで自由に浮遊して収集されます。 </li><li> 1から2パーセント正常ヤギ血清でインキュベートすることによりDABのための脳、1％トリトンX - 100、0.01M PBSでの処理アビジンビオチン複合体（1:50エリートステイン）でインキュベートした。 H 2 O 2とニッケル- DABを使用してセクションを反応する。テキサスレッド、4℃で一晩、PBSのアビジン - ビオチン複合体（1:50）で切片をインキュベート℃、その後4℃で一晩PBSTで4％テキサスレッドアビジンDCSでイン​​キュベート。ために処理するために23℃で蛍光ニッスル染色（NeuroTrace）20分で切片を対比</li><li>ニューロンは、ローダミンで注入した場合、蛍光顕微鏡（例545nm）を直接注入ニューロンを可視化する。 </li></ol> 5。逆行性標識法と組み合わせる方法、免疫細胞化学、共焦点イメージング、定量的局在解析他の方法でこの手法を組み合わせるの成功は、優れた細胞内ラベリングに大きく依存します。 <ol><li>方法は簡単に逆行性神経細胞のラベリングと組み合わせることができますここで可視化した。これを行うには7-10、動物を麻酔し、無菌操作を使用して、このような西洋ワサビペルオキシダーゼ（20％西洋ワサビペルオキシダーゼ。シグマVI、シグマ）などの神経トレーサーを注入し、1％小麦胚芽は、咬筋などのペリフェラルターゲット領域に西洋ワサビペルオキシダーゼ（Sigma）をaggultinated。このニューロントレーサーは、末梢軸索に取り込まと運動ニューロンのsomaの複数形に軸索輸送を介して輸送されます。咬筋の場合は、トレーサーの15μlの滅菌10マイクロリットルのマイクロシリンジを用いて筋肉に注入されます。麻酔から回復し、回復のために自分のケージの中で供されるまで、動物を加熱パッド上に配置して監視されます。 24時間後、動物を麻酔し、生理学的に神経細胞を特徴づけ、細胞内でそれらを染色。その後、コバルトと安定剤や強化などのchromagenとタングステン酸ナトリウムとしてテトラメチルベンジジン（TMB）を使用して、HRPの存在のために上記のように動物やプロセスの組織切片を灌流。 1％ナトリウムの場所のセクションを0.1M PBS（pH6.5）で、15で無水エタノールやアセトンに溶解した0.0007％TMBに溶解し、タングステン酸℃で20分間。て0.3％60分間インキュベーション溶液の100mlあたりH 2 O 2の1.0mlを添加することにより組織切片を反応する。 0.05％ジアミノベンジジン液（pH7.4）、0.02％、コバルト、及び0.01％H 2 10分間0.1M PBS（pH7.4）中のO 2で0.1M PBS（pH6.5）での組織や場所をすすぎます。 37℃説明されているようbiotinamideを注入されたニューロンの可視化のための組織を処理し、標識の感覚ニューロンと運動ニューロンの様々な間の関係を視覚化する。 </li><li>ここに示されているテクニックも容易にimmnocytochemistryと組み合わせることができます。6例として、免疫細胞化学的に正確にラベルされたニューロン内のシナプス（ 図3）を見つけるためにシナプトフィジンのための脳の処理。これを行うには、4℃で2日間C、その後23℃1時間のために抗マウスFITC（1:400）でインキュベーション℃でマウス抗シナプトフィジン抗体（1:10,000）の脳切片をインキュベート</li><li>蛍光マーカーで標識または蛍光イメージングのために処理のニューロンは、共焦点イメージングに最適です。図3は、軸索のブートンを通じて共焦点顕微鏡で得られた光学部の一例です。 （ 図3）。ラベル付けニューロン（ 図4）を介して複数の光のセクションを取得することにより標識されたニューロンのアニメーションを生成する。 </li><li>この方法で標識されたニューロンは、定量的局在の解析に使用することができます。これを行うには、NIHイメージ（http://phy.ucsf.edu/ ° IDL / colocalization.htmで発見）と一緒に公開されているソフトウェアのマクロを使用してください。シナプトフィジン免疫細胞との細胞内のラベルを結合して単一の軸索終末内シナプトフィジンをローカライズする。6 </li></ol> 6。代表的な結果： この方法を使用して取得することができる代表的な結果の概要を図1に示します。この単一の脳幹ニューロンが電気生理学的に明確に見られるように、下顎運動中に記録していた、このニューロンの応答（ 図1左、水色下）が運動中に変調だった。このニューロンは電気生理学的特性評価後にbiotinamideを注入し、続いて視覚化のために処理した。再構築されたニューロン（ 図1中央の、緑色は）本当のことができます解剖学的ランドマーク、この場合には三叉神経運動核が指定（赤枠）のテッド。運動と再構成するときに、神経細胞の応答に基づいて、このニューロンは二筋紡錘求心性ニューロンとして同定することができる。 図2は、顎の変位時のニューロンの生理学的応答の代表的な例を示します。ニューロンの応答は瞬時発火頻度として表されます。神経細胞の応答が密接にこの特定のニューロンは下顎の位置に関連した感覚フィードバックを提供することを示す下顎変位を模倣している。 図3は、ノートでは、シナプスの染色とニッスル染色と組み合わせて細胞内で染色された軸索の高倍率の画像です。軸索神経繊維末端内シナプトフィジン（黄色）の共局在を注意してください。 図4は、シングル、生理学的特徴および細胞内に標識されたニューロンのアニメーションです。 <img src="/files/ftp_upload/2650/2650fig1.jpg" alt="図1"/> 図1の方法の概要。左上：下顎の変位。単一のニューロン（黄色）から中央の細胞内記録（緑）。このニューロンの形態は、細胞内記録と注射後に再建されました。赤いアウトラインは三叉神経運動核の位置を示します。左下：顎運動時に、このニューロンの生理学的な応答を。 <img src="/files/ftp_upload/2650/2650fig2.jpg" alt="図2"/> 図2下顎運動中に生体内で記録された単一の筋肉感覚神経の代表的な生理的反応。顎の変位と神経細胞応答の類似性に注意してください。 <img src="/files/ftp_upload/2650/2650fig3.jpg" alt="図3"/> プロービング筋肉中に応答した細胞内染色の感覚ニューロンのシナプスboutons（赤い腫れ）と図3。ターミナル軸索分岐。シナプトフィジンの後続免疫細胞化学的処理は、軸索ブートン（黄色）内のシナプトフィジンの局在を示しています。グリーンは蛍光ニッスル染色です。 図4。生理反応下顎運動時の生体内で記録された筋紡錘一次求心性ニューロンの軸索のアニメーションは、軸索は、細胞内染色し、可視化のために処理した。 <a href="/files/ftp_upload/2650/collateral4.avi">ここで図4の高解像度のビデオをダウンロード</a> <a href="/files/ftp_upload/2650/medres.mov">ここで図4の中解像度のビデオをダウンロード</a>

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動物実験は、実験動物の管理と使用に関するガイドに記載されているguidlinesと規則に従って（NIH出版番号86から23まで、1985年改訂）とメリーランド州の動物実験委員会の大学で行われた。

ここに示した方法は、単一ニューロンの機能に重要な洞察を提供し、どのように個々のニューロンの応答は、神経回路に貢献する強力な手法です。9この知識は、感覚運動機能を理解する上での基本です。この手法の最大の強みは、生理学、形態とシナプス形態と分布を含むニューロンに関する多数のパラメータの決定を可能にされているです。そのようなような神経回路のような逆?...

<table border="1"><tbody><tr><th>試薬や機器の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>電磁バイブレータ</td><td>リン動的システム</td><td> V101 </td><td></td></tr><tr><td>信号発生器</td><td>フィードバックシステム</td><td> PFG605 </td><td>台形の出力信号を作り出すことができる</td></tr><tr><td>電極のガラス</td><td>サターインスツルメンツ</td><td> AF100 - 68から10 </td><td>フィラメントと</td></tr><tr><td>電極プラー</td><td>サターインスツルメンツ</td><td>モデルP - 2000やP - 80 </td><td></td></tr><tr><td> biotinamide </td><td>ベクターラボラトリーズ</td><td> SP - 1120 </td><td> 4℃で保存</td></tr><tr><td>テキサスレッドアビジンDCS </td><td>ベクターラボラトリーズ</td><td> - 2016 </td><td></td></tr><tr><td> tetramethlyrhodamine </td><td>分子プローブ</td><td> D - 3308 </td><td> 3000分子量、リジン修正可能な</td></tr><tr><td>マウス抗シナプトフィジン抗体</td><td>ケミコン</td><td> MAB5258 </td><td></td></tr><tr><td>蛍光ニッスル染色</td><td> Neurotrace、分子プローブ</td><td> N - 21480 </td><td></td></tr><tr><td>電極のテスター</td><td>ウィンストンエレクトロニクス</td><td> BL - 1000 - B </td><td>電極のインピーダンスを測定する</td></tr><tr><td>電位計</td><td>アクソンインスツルメンツ</td><td> Axoprobe 1A、Axoclamp 2B </td><td></td></tr></tbody></table>

physiological, morphological and neurochemical characterization of neurons modulated by movement

個々の神経細胞と神経回路におけるそれらの機能の役割は、感覚と運動機能の神経機構を理解する上での基本です。動物が運動中に静的1,2または記録細胞外神経活動のときに感覚運動メカニズムのほとんどの研究は、神経細胞のいずれかの検査に依存しています。3,4これらの研究は、感覚運動機能のための基本的な背景を提供しているが、彼 ​​らはどちらかの機能的な情報を評価しないこれは、運動中に発生した場合、または完全にニューロンの解剖学、生理学、神経化学的表現型を特徴づけるために彼らの能力は限られています。技術は生体の運動の中に個々のニューロンの大規模な特性評価を可能にするここに示されています。この手法は、一次求心性神経細胞を研究するだけでなく、運動ニューロンと感覚運動の介在を特徴づけるためにだけ使用できます。最初に単一ニューロンの応答は、ニューロンの受容野の決定に続いて下顎骨の様々な動きの間に電気生理学的手法を用いて記録されます。神経トレーサーは、細胞内ニューロンに注入され、脳は、ニューロンが光、電子または共焦点顕微鏡（図1）可視化できるように処理されます。神経形態はニューロン（図2,3）の生理的応答と相関させることができるように特徴づけられる神経細胞の詳細な形態は、再構築されます。この通信ではこの手法の実装を成功させるための重要なキーの詳細とヒントが提供されています。貴重な追加情報は、他の技術とこの手法を組み合わせることにより、研究対象のニューロンのために決定することができます。逆行性神経標識は、標識されたニューロンのシナプスなるとニューロンを決定するために使用することができる、従って神経回路の詳細な測定を可能にします。免疫細胞は標識されたニューロン内の神経伝達物質を調べ、設定されたラベル付きニューロンのシナプスニューロンの化学的表現型を判定するために、このメソッドと組み合わせることができます。標識されたニューロンは、超微細構造の特徴とラベルされたニューロンの超小型回路を決定するために電子顕微鏡のために処理することができます。全体的にこのテクニックは、徹底的にこのように感覚運動機能におけるニューロンの役割にかなりの洞察を可能にする生体運動の中に神経細胞を特徴づけるために強力な方法です。

Watch this Scientific Journal Video about Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement at JoVE.com

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

テクニックは運動中に哺乳類の神経細胞の生体内生理的反応の定量化と神経形態、神経化学的表現型とシナプス超小型回路とニューロンの生理学を相互に関連付ける説明されています。

運動によって変調された神経細胞の生理的、形態学的および神経化学的キャラクタリゼーション

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

physiological-morphological-neurochemical-characterization-neurons

Research

JoVE Journal

Neuroscience

13.7K ビュー.  University of Maryland. テクニックは運動中に哺乳類の神経細胞の生体内生理的反応の定量化と神経形態、神経化学的表現型とシナプス超小型回路とニューロンの生理学を相互に関連付ける説明されています。

ビデオ: Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

メモリの眼球運動のモニタリング

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

神経科学

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

脳皮質機能および変更された重力状態の間、神経認知パフォーマンスとの間のコヒーレンス

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病ラットの熱侵害受容閾値の迅速定量

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

吸引ピペット技術を用いた嗅覚受容ニューロンから記録匂い物質誘発性応答

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

免疫沈降およびアシル-ビオチン所（阿部）による培養海馬神経細胞におけるタンパク質パルミトイル化の検出

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

電子プローブX線マイクロアナリシスによるニューロンにおける総カルシウムの測定

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

誘導された神経細胞への成人の脳の周皮細胞由来細胞の系統の再プログラミング

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

急性脳スライスにおけるニューロンマイクロ回路の電気生理学的および形態学的特徴は、ペアパッチクランプ記録を使用して、

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

横隔運動ニューロンによってダイヤフラム神経支配の機能的および形態学的評価

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

神経化学的マーカーのためのビオシチンで満たされたと加工セクションの免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...