この作品は全国多発性硬化症によってサポートされていた社会助成金適応および/ ​​またはDavalos ら 、J Neurosciの方法より転載KAフィギュアや映画へのDDとNIH / NINDS助成NS051470、NS052189とNS066361へRG4595A1 / T。 2008年03月30; 169（1）:1 - 7 2008著作権、エルゼビアの許可を得て。

1。脊椎安定化装置の構築<ol><li>ナリシゲSTS -コンパクト脊髄クランプとナリシゲMA - 6Nヘッド保持アダプタを注文。 </li><li>カスタム設計とその脊柱と尾部がクランプされている間、動物の頭部がサポートされているように整列二つナリシゲの部分を保持するためにステンレス製の底板を作る。デバイス全体は通常低下顕微鏡のステージ上に顕微鏡レンズの下に入る必要があることに留意してください。 </li></ol> 2。動物の手術<ol><li>空気加熱装置とプレヒート顕微鏡室、37でそれを維持℃に</li><li>動物を秤量し、100mgのケタミン、15mgのキシラジンと無菌条件下で0.9％NaCl溶液で希釈した体重1kg当たり2.5mgのアセプロマジンを使用して、麻酔薬のミックスを作る。 </li><li>麻酔ミックス（KXA）を腹腔内注入することにより、動物を麻酔。定期的なつま先の挟み込みは、全体に深い麻酔を確保するために実行する必要があります実験。毎時または必要に応じて半分の投与量で補う。 </li><li>外科手術の実行中に熱のサポートを提供するために、小動物の加熱パッドを使用してください。 </li><li>角膜脱水および損傷を防ぐために目の上に人工涙液軟膏を適用する。 </li><li>トリミング装置とし、完全に切開周辺から髪を削除するには、鋭い刃を持つ最初の動物の背中を剃る。 </li><li>剃毛を取り除くとアルコールパッドで剃毛、皮膚の表面を消毒する。 </li><li>希望する脊髄レベル（胸椎がここに示されている）の皮膚の小さな（〜1.5 cm）の長手方向の切開を行います。 </li><li>慎重に後退による皮下の結合組織を戻す筋肉を公開。 </li><li>慎重に必要なレベルで脊柱から傍脊柱筋を分離。各棘突起の両側から筋肉をデタッチし、デタッチされた筋肉をこすり取る際には、できるだけ少ない切開を行います層の表面からの方法。 </li><li>すぐに吻側と椎弓切除の尾、削除されるラミナを選択し、脊柱の両側の脊髄クランプ用エントリのサイトを準備。慎重にクランプが挿入される4つのポケットを作るために筋肉組織を置換する。 </li><li>脊髄に触れることなく粘膜の下に湾曲した鈍先端はさみの安全な挿入を可能にするストレート歯先端の鉗子と脊柱を持ち上げる。ゆっくり片側、次に反対側にある骨を切断することにより椎弓切除を行います。 </li><li>綿棒を使用するか、少量の出血を制限するために切開隣プレカット小さなgelfoam吸収ゼラチンスポンジの部分を挿入します。必要に応じてさら​​に出血を抑えるために小血管のcauterizerを使用してください。組織を洗浄するために予熱無菌人工脳脊髄液（ACSF）を使用してください。 </li></ol> 3。 in vivoイメージングのための脊柱と準備の安定化<ol><li> PLAC<html>電子脊椎安定化装置のベースプレート上に標高のパッド上に動物とヘッド保持アダプターでヘッドを固定します。 </li><li> STS -椎弓切除に隣接する、事前に用意されたポケットの中の動物（図1）の前後軸に沿ったデバイスの脊髄クランプを置きます。 2個のクランプは〜45 °露出した脊髄（図1）上の水浸レンズを低減するための十分なスペースを可能にするために動物のrostro -尾軸に対しての角度で配置する必要があります。 </li><li>動物の体をイメージング実験（図1）の期間中、標高のパッドを除去した後、空気中に浮遊することができるように尾の基部にSTS -装置の第3のクランプを置きます。 </li><li> ACSFで脊髄のメンテナンスおよびin vivoイメージングのためのこのソリューションでは顕微鏡のレンズの浸漬を容易にするために、露出した脊髄の周りのGelseal（アマシャムバイオサイエンス社）の小さな井戸を構築する。 </li></ol>jove_title&quot;&gt; 4。二光子顕微鏡によるマウス脊髄のin vivo イメージング <ol><li>顕微鏡をカバーする予備加熱チャンバ内に脊椎安定化装置に動物を移し、まっすぐレンズ（図1）の下に椎弓切除を置く低下顕微鏡のステージ上に固定。 </li><li> ACSFのソリューション、それが脊髄クランプまたはGelsealに接触しないことを確認することに慎重に水浸レンズを下ろします。 </li><li>関心のある分野を特定し、それに集中する落射蛍光使用。レーザースキャンモードに切り替え、イメージ化された組織中に存在する蛍光物質のために適切な二光子レーザーの励起波長、dichroicsとバンドパスフィルタを用いた in vivo イメージングで行います。 </li></ol> 5。反復的なイメージングと術後ケア<ol><li>イメージング実験の終了時に、脊椎安定化デバイスからマウスを削除し、慎重に椎弓切除周辺からGelsealを拭いてください。エリアをよく清掃してください。 </li><li>椎弓切除以上の背中の筋肉を復元し、縫合。 </li><li>椎弓切除の皮膚を復元し、縫合、betadineでそれを綿棒。 </li><li> 1ミリリットル栄養と水分補給補助食品として乳酸リンゲル液（バクスターヘルスケア）、ならびに皮下鎮痛治療を（0.1 mg / kgのブプレノルフィン）を提供。 </li><li>防腐処理の腹腔内（マウス1匹あたり0.03ミリリットル、2.27％のエンロフロキサシン注射剤抗菌ソリューションを）管理する。 </li><li>完全な麻酔から回復し、その後個別にそれを収容するまで加熱パッドの上に置いて動物。 </li><li>術後2〜3日の手術と鎮痛治療ごとに8〜12時間後の最初の3〜5日間毎日消毒剤投与を繰り返す。通常の動作と完全な回復を確実にするには、毎日動物を監視します。 </li><li>同じ椎弓切除を介しての再イメージングのために、縫合皮膚と筋肉を再度開き、セクション2で説明した手順を繰り返して - 4。 </li><li>再度見つけるpreviously以前に10を説明するようにマップのように血液の血管を使用して領域を撮像。 </li></ol> 6。代表的な結果すべての動物の手続きは、カリフォルニア、サンフランシスコの大学の制度的動物実験委員会によって設定されたガイドラインの下で行われ、連邦規則に準拠していますれた。脊椎安定化装置の画像と顕微鏡のレンズの下にデバイス上でマウスの位置を示す模式図を図1に示されています。動物の体の下に呼吸運動に十分なスペースを許可することは、脊髄におけるin vivoイメージングで安定が保証されます。それはCX3CR1 GFP / +トランスジェニックマウス18の脊髄に生体内でイメージを作成したとして、 図2は、ミクログリアと血管の間の密接な関係を示し、これでミクログリアが内生的にGFPで標識されています。 図3は、反復の例を示しています。in vivoイメージングでは蛍光脊髄軸索におけるタンパク質（YFP - Hライン3）とミクログリアを（CX3CR1 GFP / +マウスで）発現するマウスで同じ脊髄領域で実行されたとして。 <img alt="図1" src="/files/ftp_upload/2760/2760fig1.jpg" /> 図1二光子顕微鏡を用いたin vivoイメージングのためのマウス脊柱の安定化。（A）カスタムメイドのスチール製ベースプレートをSTS -ナリシゲコンパクト脊髄クランプとMA - 6Nナリシゲをサポートして整列させるために使用されるここに示されているようにアダプターを保持しているヘッド。脊椎安定化デバイス上にKXA麻酔薬で麻酔成体トランスジェニックマウスの（B）適切なポジショニング。インサートは、直ちに吻方と尾側椎弓切除と露出した脊髄組織に脊髄クランプの配置を示します。 &lt;IMG ALT =&quot;図2&quot;SRC =&quot;/ files/ftp_upload/2760/2760fig2.jpg&quot;/&gt; 図2。麻酔したマウスの脊髄における高密度小グリア細胞および血管のin vivoイメージング。の投影が、非整列Z -スタック（A）の血管に近接してCX3CR1 GFP / +マウス（赤、ローダミンデキストランの静脈注射で標識）の脊髄における高密度GFP陽性ミクログリア（緑）。容器の周りに拡張プロセスとし、脊髄実質に向かって血管の壁に接続された単一のミクログリア細胞の（）内のラベル（B）の高倍率画像。スケールバーは、10μmの。 <img alt="図3" src="/files/ftp_upload/2760/2760fig3.jpg" /> 図3。彼らは別の日に移転し、麻酔したマウスの脊髄でイメージを再作成されたのと同じ軸索のセグメントとミクログリアのin vivoイメージングの繰り返し。 （A）YFP -ラボ<html> 0日目と5日のeled軸索。 （B）。それらの周りに同じ血管構造とミクログリア細胞は、日0と1のCX3CR1 GFP / +マウスの脊髄でのin vivoで画像化。スケールバーは、10μmの。 ムービー1。マウス脊髄から生体内で取得した代表的なタイムラプスシーケンス。このシーケンスは、密に蛍光構造を移入組織内で時間をかけて（赤、ローダミンデキストランの静脈注射で標識）を詳細に細かいミクログリアプロセスダイナミクス（緑）と血管との相互作用を示しています。 RAW画像は、バックグラウンドノイズ、明るさやコントラストを補正したとタイムラプスムービーは、画像の位置合わせ、平均または個々の平面のzを選択することなく、連続して取得した画像スタックをzは、投影することにより構築した。血管は、ミクログリアの画像は、z平面の類似の範囲を通過。 Z面の深さ：38μmの。oad/2760/Davalos_Movie_1.movは&quot;&gt;映画を鑑賞するためにはここをクリックしてください。 映画（2）タイムラプスシーケンスは、単一のz -平面のレベルでのイメージング法の生の安定性を実証。 KXA麻酔下で脊髄の安定化デバイス上に配置CX3CR1 GFP / +マウスの脊髄内の同じ単一のz -平面の高速アクイジションでは、より高速な1フレーム/秒のスキャン速度で連続したフレーム間の最小の画像の変位を示していますマウスの呼吸速度。マイナー残留変位は、おそらくビートによるものです。 <a alt="Movie 2" href="http://www.jove.com/files/ftp_upload/2760/Davalos_Movie_2.mov">映画を見て、ここをクリックしてください。</a>

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我々は、開示することは何もない。

方法は2つの光子顕微鏡を用いて麻酔したマウスの脊髄における人口密度の高い蛍光細胞構造の安定及びin vivoでの反復的なイメージングを可能にするここで説明する。実現性はカスタムメイドの脊椎安定化装置や呼吸器誘発性運動のアーチファクトを減少させる麻酔療法の結果です。脊椎安定化装置は、マウス本体の下に呼吸スペースを可能にし、市販の脊髄クランプとヘッド取付部?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ローダミンBデキストラン</td><td>インビトロジェン</td><td> D1841 </td><td>で希釈した70kDaの、 ACSF（3％w / v）の</td></tr><tr><td>ケタミン塩酸</td><td> Bionichepharma </td><td> NDC番号：67457-001-10 </td><td> 50mg/ml、注射</td></tr><tr><td> Anased </td><td>ロイド研究所</td><td> NADA番号：139から236 </td><td>キシラジン注射液、 20mg/ml </td></tr><tr><td>アセプロマジン</td><td> Vedco </td><td> NADA番号：117から531 </td><td> 10mg/ml、注射</td></tr><tr><td>人工涙液 軟膏</td><td>フェニックス 製薬</td><td> NDC番号：57319-760 - 25 </td><td>潤滑油</td></tr><tr><td> Betadine </td><td>フィッシャー</td><td> 19-061617 </td><td></td></tr><tr><td>マクファーソン、ウェストコット はさみ</td><td>世界の精密 楽器</td><td> 555500S </td><td>湾曲した、鈍チップ はさみ</td></tr><tr><td>ストレートピンセット</td><td>世界の精密 楽器</td><td> 555047FT </td><td>歯先端の鉗子</td></tr><tr><td>小血管は焼灼する</td><td>ファイン科学ツール</td><td> 18000〜00 </td><td></td></tr><tr><td> Gelfoam </td><td>ファルマシア、ファイザー社</td><td>ミキサーミルMM400 </td><td></td></tr><tr><td>コンパクト脊髄 クランプ</td><td>ナリシゲ</td><td> STS - </td><td></td></tr><tr><td>頭部保持アダプタ</td><td>ナリシゲ</td><td> MA - 6N </td><td></td></tr><tr><td> Gelseal </td><td>アマシャム バイオサイエンス（株） </td><td> 80-6421-43 </td><td></td></tr><tr><td>乳酸リンゲル</td><td>バクスターヘルスケア&lt;/ TD&gt; <td> 2B8609 </td><td></td></tr><tr><td> Buprenex </td><td> Reckitキットベンキーザー ティカルズ社</td><td> NDC番号：12496 - 6757から1 </td><td>ブプレノルフィン、 注射可能な</td></tr><tr><td> Baytril </td><td>バイエル</td><td> NADA 140から913 </td><td>エンロフロキサシン、 抗菌性注射剤 2.27パーセント（20ミリリットル） </td></tr><tr><td>加熱パッド - 大</td><td>ファイン科学ツール</td><td> 21060〜10 </td><td></td></tr></tbody></table>

in vivo imaging of the mouse spinal cord using two-photon microscopy

in vivoで遺伝的に特定の細胞型2-3に蛍光タンパク質を発現するように設計されているマウスでは、二光子顕微鏡1を用いたイメージングでは、大幅にin vivoで 4-7 の多くの組織で生理的および病理学的プロセスの知識を広げている。中枢神経系（CNS）の研究では、小説などの下でニューロン、アストロサイト、ミクログリア、などの細胞の挙動について、しばしば予想外の所見の茄多を生産している脳内のin vivoイメージングの幅広い応用がなされている生理学的または病理学的条件8月17日 。しかし、主に技術的な合併症は、生きているマウスの脊髄の研究ではin vivoイメージングの実装を制限してきた。特に、肺と心臓への脊髄の解剖学的接近は、イメージング生体脊髄やりがいのある仕事になる重要な動きのアーティファクトを生成します。 </P&gt; 我々は、脊柱を安定させる呼吸器誘発性の動きを減らし、それによってin vivoで画像にマウス脊髄をする二光子顕微鏡の使用を容易にすることによって脊髄のイメージングの固有の限界を克服する新しい方法を開発した。これは、呼吸器誘発性運動の有意な減少をもたらす、深い麻酔の方法でカスタマイズされた脊椎安定化装置を組み合わせることによって実現されます。このビデオプロトコルは、組織の損傷を維持して最小限に出血が長期間にわたって安定した生理的条件下で維持することができる生きている脊髄の小さな領域を公開する方法を示しています。高分解能in vivo で詳細にミクログリアと血管の間の密接な関係を取得した代表的な生のイメージ。タイムラプスシーケンスは、生きたマウスの脊髄でのミクログリアのプロセスの動的な挙動を示しています。また、同じz -フレームの連続スキャンが示すのこのメソッドは画像の位置合わせ後の取得を必要としない画像および/またはタイムラプスムービーのスタックを生成するために達成することができる卓越した安定性。最後に、我々は生体内で進行中の生理学的または病理学的プロセスの縦断的研究を可能にする、このメソッドは、後でタイムポイントでの脊髄の同じ領域を再検討し、再イメージングするために使用できる方法を示します。

Watch this Scientific Journal Video about In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy at JoVE.com

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

マウス脊柱を安定させ、繰り返し実行するために低侵襲プロトコル<em&gt; in vivoで</em二光子顕微鏡を使用して&gt;脊髄の画像が記載されている。このメソッドは、呼吸器誘発性の動きを最小限にしてもアライメントや後処理他を必要としない生の画像データを生成するために脊椎安定化装置と麻酔薬の処方を組み合わせたもの。

二光子顕微鏡を用いたマウス脊髄の in vivoイメージングで

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

in-vivo-imaging-of-the-mouse-spinal-cord-using-two-photon-microscopy

Research

JoVE Journal

Neuroscience

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

23.7K ビュー.  University of California, San Francisco. マウス脊柱を安定させ、繰り返し実行するために低侵襲プロトコル in vivoで脊髄の画像が記載されている。このメソッドは、呼吸器誘発性の動きを最小限にしてもアライメントや後処理他を必要としない生の画像データを生成するために脊椎安定化装置と麻酔薬の処方を組み合わせたもの。

ビデオ: In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

皮質ニューロンにおける経験依存性分子の変化の in vivo二光子イメージングにおける

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

神経科学

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

皮膚における単一神経終末のin vivo二光子顕微鏡で

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

二光子<em&gt; in vivoで</em&gt;間引き頭蓋骨の準備を使用して、マウス皮質における樹状突起棘のイメージング

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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

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

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

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

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

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

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

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

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

マウス脊髄における細胞ダイナミクスの二光子イメージング

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

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

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

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

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

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

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

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

CLARITY / CUBIC技術を用いたマウスの脊髄にセロトニン繊維のイメージング

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニック マウス

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

生体内での2光子蛍光寿命イメージング顕微鏡を用いて頭固定振る舞いマウスにおけるタンパク質キナーゼの活動を可視化する

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...