著者らは支援を撮影のため手術の撮影機器、D.クォンに対して、L. Belluscioに感謝したいと思い、ビデオのためのK.劉はすべてのバックグラウンド·ミュージックのための支援を、編集、およびK.マクラウド。 KWは学内研究プログラムのNIMH課や遺伝子、認知と精神病プログラムの寛大な支援を認めている。この仕事はNIMH学内研究プログラム（VC、YY SMKW）と学内の臨床および生物学的研究プログラムのNIAAA課（VC、RMC、DML）によってサポートされていました。

後述の実験手順は、メンタルヘルス動物のケアと使用委員会の国立研究所によって承認され、 実験動物の管理と使用のための健康ガイドの国立研究所に合わせてあった。 1。術前準備<ol><li>無菌手術前にホットビーズ滅菌器ですべてのツールをきれいにし、70％エタノールで手術部位を掃除し、きれいなドロップ布を下に置く。滅菌手袋を着用してください。 intraperitonially 0.02ミリリットル/ gであり、与えられた時に作りたての1.2パーセントAVERTIN溶液で動物を麻酔。あるいは、受動スカベンジャー回路付きノーズコーン、誘導のための5％、メンテナンスのために1.5％、イソフルランを通してガスと麻酔。完全鎮静状態を確保するためにテールやつま先ピンチを使用して麻酔レベルをチェックします。 </li><li>無菌の眼軟膏を用いて動物の目をカバーし、作りたてのデキサメタゾンを注入する（0.2 mg / kg）及びカルプロフェン（5 mg / kg）をsubcutaneousl脳の腫れや炎症を防ぐために、11 yである。耳の間の頭蓋骨の上の毛を剃ると、ベタジンスクラブと70％エタノール3交互綿棒で皮膚を消毒する。 </li><li> earbarsと定位手術の段階で動物をマウントし、37℃に設定動物下水循環加熱パッド付き℃、定期的に動物の麻酔レベルをチェックし、必要に応じてオリジナルの麻酔量を補う。 </li><li>エリアを麻痺させるために、頭皮の皮膚の下に0.5％Marcaineの200μlを注入します。皮膚を切開し、頭蓋骨の上に皮弁を取り外します。骨膜を取り外し、きれいな綿棒でその領域を乾燥させます。 </li></ol> 2。慢性頭蓋窓手術<ol><li>穏やかに興味のある脳領域にわたって3〜5ミリメ​​ートルの直径の円を概説する0.5ミリメートルバリで高速歯科用ドリルを使用します。定期的にクリーン綿棒と滅菌0.9％食塩水と離れて明確な骨塵で掘削地点を濡らす。骨、出血した場​​合、gを使用するelfoamはブリードをブロットおよびそれが停止するのを待つように滅菌生理食塩水に浸漬した。 </li><li>最後の骨の層に到達すると、先の細いピンセットで骨の島を持ち上げて外します。ウィンドウは骨縫合を超えた場合に、骨の一番下の棚に硬添付ファイルが発生する可能性があります。骨弁が持ち上げられるように、これらは、優しく除去しなければならない。 </li><li>ロールは、その表面をきれいにし、停止するために、任意の硬膜出血を待つさらさ硬膜の上にそっとゲル発泡体を湿らせた重要なステップは：すべての硬膜出血が停止するまで、先に進まないでください。頭蓋窓の中に閉じ込めになる血液は、通常不透明なウィンドウにつながる。 </li><li>関心領域は、硬膜は、特に厚さは場所の下にある場合、その上に硬膜を除去することが必要な場合があります。このような場合は、優しく、非常に微細鉗子で下PIAから硬膜を分離し、細かい鉗子の別のペアで持ち上げる硬膜の小さな切開を行い、その後、カットエッジをつかみ、そっと硬膜を分割します。硬膜が薄くても強いので、硬膜の無傷のエッジを持つ脳をスライスしないように注意してください。 </li><li>無菌ACSFまたは滅菌生理食塩水で洗い流してください。 </li><li>硬膜またはPIA経由で滅菌ガラスカバースリップ（3-5 mm）を築く重要なステップ：大幅頭蓋骨カーブを囲む場合は、硬膜またはPIAは地域でカバースリップとの密接な接触をすることはないだろうスペースを埋めるためにKwiksil 12接着剤を使用それが画像化されることはありません。 </li><li>エラストマーの接着剤とガラスカバースリップの端をカバーするためにシアノアクリレートゲルを使用しています。 。任意シアノアクリレートゲルが脳表面に触れないように注意してください：シアノアクリレートゲル、またはkrazyglueと歯科用セメント混合物と全露出頭蓋骨重要なステップをカバーしています 。 </li><li>頭蓋骨の反対側の端にあるカスタムメイドの金属頭部固定バーを埋め込む。 </li><li>ケトプロフェン、5 mg / kgを、foのintraperitonial注射後、暖かい回復室に動物を戻すrの疼痛管理。手術後2日以上鎮痛剤を続行します。 </li><li>術後の回復の2週間後には、（誘導の場合は5％、保守のための1.5％）イソフルランで動物を麻酔し、頭部固定用フレームを使用してカスタムメイドの顕微鏡ステージでそれをマウントし、光学的透明度のために頭蓋窓をチェック青色光と照明の下で。脳表面の血管の明瞭さは頭蓋窓の質の高い指標である。血管エッジがはっきりと定義されている場合、ウィンドウが使用可能である可能性が高い。 2週間の術後の回復時間は、手術から回復するために動物のための十分な時間を確保することをお勧めします。頭蓋窓は一般的にクリアして、改善するこの時期に、と硬膜の肥厚や頭蓋骨の再生、ウィンドウの画質を劣化させるまで数ヶ月に付加週間の光学的に透明なままです。 </li></ol> 3。二光子顕微鏡を行動プロトコルおよびレーザー<ol><li>明確なcと動物ranial窓は環境刺激にさらされたり、行動のトレーニングセッションを行い、その後、最大アーク-GFP発現時にその後結像される。行動プロトコルを開始する前に、動物は、ベースラインアーク-GFPの発現レベルでの日々の変動を最小限にするために一貫性のあるホームケージ環境で保たれるべきである。 </li><li>興味のある脳領域に応じて、行動訓練や環境刺激パラダイムを開始します。例えば、動物が日連続で異なるビジュアルな環境にさらされることができ、視覚野10に刺激特異的応答を識別するための視覚的な刺激の後に毎日結像される。 </li><li>ニューロンにおけるアーク-GFPの蛍光は、通常は2時間刺激した後にそのピークレベルに達する。実験のタイムラインは、そのピーク蛍光レベルでのニューロンにおけるアーク-GFP発現の検出を容易にするために最適化することができる。 </li><li>行動訓練セッションまたはenvironmenta後にlの刺激が完了すると、（誘導の場合は5％、保守のための1.5％）イソフルランで動物を麻酔し、二光子顕微鏡でヘッド固定用フレームを使用してカスタムメイドの顕微鏡ステージで動物をマウントします。 </li><li>動物の頭の位置が頭蓋骨に注入された金属棒を使って、ステージに直接接続し、ヘッド固定用フレームに固定されています。イソフルランと酸素が継続的にノーズコーンを介してマウスに供給される。体温は加熱パッドを使用して維持されます。 </li><li>顕微鏡の検出器は、暗い​​部屋で二光子レーザー走査を行うことで、周囲の光から保護されていることを確認します。イメージングのための20倍または25倍（開口数1.05）水​​浸レンズを使用しています。まず、落射蛍光照明の下で、将来像の位置合わせのためのCCDカメラを使って、対象の脳領域の上表面の血管パターンの画像を取得する。 </li><li> 3次元画像スタックを取得するためにスキャン二光子レーザーを起動します。オリンパスFV1000MPEの多光子顕微鏡は、我々のセットアップで使用されています。二光子レーザーの励起波長は920 nmに設定されており、客観から出射されるレーザのパワーが約50 mWで設定されています。放出される蛍光を同時に標本の近くに配置し外部2チャンネルphotomutiplier検出システムを使用して、緑と赤のチャネル（570nmでのダイクロイックミラー、495から540 nmと570から625 nmにおけるバリアフィルタ）で検出される。組織の自家蛍光は、チャンネル13、14の両方に表示され、一方の円弧GFP蛍光は唯一、緑のチャンネルに表示されます。 </li><li>典型的な画像スタックは約320x320x100μmの寸法（幅×長さ×深さ）が0.5μm/ピクセルの水平方向の解像度、と3μm/ピクセルの垂直方向の解像度を持っています。 </li><li>画像スタックを取得した後、そのホームケージに動物を返す。次の行動やイメージングセッションまで動物を乱すことがありません。 desirとして日間にわたる行動やイメージング手順を繰り返しエド。同じ画像の場所に戻って方向付けるために以前に取得した脳表面の血管画像を使用しています。 </li></ol>

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特別な利害関係は宣言されません。

ここで説明するin vivoイメージング法で生きている動物で、複数の日にわたってニューロンの同じセットでアーク遺伝子発現変化の反復検査が可能になります。それは様々な行動の経験に応じて、個々のニューロンにおける神経可塑性関連分子動力学に関する情報を取得するための効率的かつ汎用性の高い方法です。そのようなin situハイブリダイゼーションおよび免疫染?...

<table border="1"><tbody><tr><td> 機器の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> FV1000多光子レーザー走査顕微鏡</td><td>オリンポス</td><td> FV1000MPE </td><td>イメージング</td></tr><tr><td>解剖顕微鏡</td><td> Omano </td><td> 555V107 </td><td>手術</td></tr><tr><td>マウスに対する定位手術の段階</td><td>ハーバード装置</td><td> 726335 </td><td>手術</td></tr><tr><td> 20Xまたは25X水浸対物レンズ</td><td>オリンポス</td><td> XLPL25XWMP </td><td>イメージング</td></tr><tr><td>ヘッド固定用フレーム付き顕微鏡ステージ</td><td>カスタムメード</td><td> N / A </td><td>イメージング</td></tr><tr><td>ファイン鉗子</td><td>ファイン科学ツール</td><td> 11251から20 </td><td>手術</td></tr><tr><td>歯科用ドリルバリ</td><td>ファイン科学ツール</td><td> 19007から05 </td><td>手術</td></tr><tr><td> CCDカメラ</td><td> QImaging </td><td> QICAM 12ビット</td><td>イメージング</td></tr></tbody></table>

in vivo two-photon imaging of experience-dependent molecular changes in cortical neurons

経験に応じて変更することが脳の能力は、健康な脳機能に不可欠であり、この過程で異常が脳障害の1,2の様に貢献しています。より良い脳の回路は、動物の経験に反応するメカニズムを理解するために生きている動物で、長期間にわたってニューロンの指定されたセットの中で経験依存分子の変化を監視する機能が必要です。経験と関連した神経活動は神経細胞1,2における遺伝子発現の変化を引き起こすことが知られているが、そのような変化を検出する方法のほとんどは、複数日にわたって同じニューロンの繰り返し観測を許可していないか、または個々のニューロン3を観察するのに十分な解像度を持っていない、4。ここでは、以上の個々の皮質ニューロンにおける経験依存性の遺伝子発現の変化を追跡するために遺伝的にコードされた蛍光レポーターを用いた in vivo二光子顕微鏡に組み合わせた方法を記載する<html>日々の経験のコース。 定評のある経験依存性遺伝子の一つが活性調節細胞骨格関連タンパク質（ARC）は5,6です。アークの転写は急速であり、非常に激化神経活動3により誘導され、そのタンパク質産物は、グルタミン酸受容体との長期的なシナプス可塑性7のエンドサイトーシスを調節する。アークの発現は広く3特定の行動に関与する神経回路をマッピングするための分子マーカーとして使用されてきた。これらの研究の大部分では、アーク式は固定の脳切片におけるin situハイブリダイゼーションや免疫組織化学によって検出した。これらのメソッドはArcの発現が調べていなかったアーク発現の細胞パターン日間反復または一味違った体験の複数のエピソードに変更される可能性がありますどのように、行動の経験の後、興奮性ニューロンのサブセットに局在していたことが明らかになっていますが。 ntentが&quot;&gt; 生体内では 2光子顕微鏡は、生きている脳8,9における経験依存的細胞の変化を調べるための強力な方法を提供しています。2光子顕微鏡による生きた神経細胞におけるアーク式の検査を有効にするには、我々は、以前に生成されたノックをGFPレポーターが内因性アークプロモーター10の制御下に置かれているマウス系統インチこのプロトコルでは、生きた動物におけるニューロンのアンサンブルにおける経験依存アーク-GFPの発現パターンを追跡するための手術の準備と撮像法について説明していますこの方法では、慢性頭蓋窓は最初の利息の皮質領域にわたってアーク-GFPマウスに移植した。これらの動物は、その後、繰り返し数日間にわたって所望の行動パラダイム後の2光子顕微鏡で撮像した。このメソッドは、帳簿動物に一般的に適用することができる経験依存性分子の変化の他の蛍光レポーター4。

<html>このプロトコルは、生きた動物の個々の皮質ニューロンにおける経験依存的分子の変化を追跡する方法について説明します。慢性頭蓋ウィンドウが最初に遺伝子発現の蛍光レポーターを運ぶマウスへの関心の皮質領域上に作成されます。二光子顕微鏡は、その後、個々のニューロンにおける行動的に誘発され分子の変化を観察して、複数の日にわたってニューロンの同じセットでこのよ?...

Watch this Scientific Journal Video about In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons at JoVE.com

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

ニューロンにおける経験依存的分子変化は行動の課題に応じて適応する脳の能力のために不可欠である。アン<em生体内で&gt;</em&gt; 2光子イメージング法は、遺伝的にコードされた記者を通じて、個々の皮質ニューロンにおけるそのような分子の変化の追跡を可能にする、ここで説明されます。

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 ...

in-vivo-two-photon-imaging-experience-dependent-molecular-changes

Research

JoVE Journal

Neuroscience

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

21.5K ビュー.  National Institute of Mental Health. ニューロンにおける経験依存的分子変化は行動の課題に応じて適応する脳の能力のために不可欠である。アン 2光子イメージング法は、遺伝的にコードされた記者を通じて、個々の皮質ニューロンにおけるそのような分子の変化の追跡を可能にする、ここで説明されます。

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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

慢性二光子用薄型頭蓋骨のウィンドウテクニック in vivoで</em神経炎症のモデルのマウスミクログリアの>イメージング

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

神経科学

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

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 significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

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

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

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 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 ...

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 ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

ラット皮質ニューロンにおける繊維状アクチン（F-アクチン）涙点の定量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脳スライスにおける神経樹状突起における 2 光子カルシウム イメージング

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

経瞳孔二光子マウス網膜のin vivoイメージング

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...