私たちは、図解のためにジェームス·ペルナに感謝します。この作品は、YZに国立精神衛生研究所からの補助金によって支えられて

承認は、手術およびイメージング調査の開始前にホーム機関から取得する必要がある。この原稿に記載された実験は、カリフォルニア大学サンタクルーズ校施設内動物管理使用委員会からのガイドラインと規則に従って行った。 1。手術<ol><li>すべての手術器具をオートクレーブ徹底的に手術前に70％アルコールでワークスペースを殺菌。 </li><li> KX麻酔溶液（200 mg / kgのケタミンおよび20 mg / kgのキシラジン）の腹腔内（IP）注射によりマウスを麻酔マウスの体重に応じて注：KXの投与量は、健康状態は、歪み、年齢に応じて調整されてもよく、マウスの。最適な用量を決定するために獣医の相談にもお勧めします。 </li><li>マウスの足の指を押し、麻酔の状態を監視するために反射的応答を確認することにより、定期的につま先のピンチテストを実行します。マウスが完全に手術を開始する前に麻酔をかけていることを確認してください。長時間のイメージングセッション中、必要に応じて追加のKXを管理、定期的に、マウスの麻酔状態を確認してください。 </li><li>手術中体温を維持するために、加熱パッドの上にマウスを置く。 </li><li>頭皮を露出させるためにカミソリの刃を使用して、マウスの頭部を剃る。 </li><li>アルコールパッドとベタジン交互で皮膚を拭いて剃毛面積を殺菌。残留髪の切り抜きを削除します。 </li><li>そっとそのため実験中に脱水症状に起因する永久的な損傷を防止し、両眼を潤滑する眼軟膏を適用します。 </li><li>頭皮の正中線に沿ってまっすぐに切開を行い、頭蓋骨の端に向かって横方向に皮膚を移動します。 </li><li>頭蓋骨に付着した結合組織を除去します。 </li></ol> 2。間引き頭蓋骨の準備<ol><li>アイドルマスターに基づいて撮像領域を特定。eotaxic座標注：大血管、ブロックライトの浸透を回避し、画像化された構造をぼかすようにしてください。頭蓋骨は、滅菌生理食塩水で湿っているとき、血管系は最もよく観察される。 </li><li>頭蓋骨の薄い円形領域（0.5〜直径）に高速マイクロドリルを使用してください。むしろ頭蓋骨に対してそれを保持し、押し下げなく、頭蓋骨の表面にドリル平行に移動。外側の緻密骨層と中間海綿骨層の両方になるまでドリルが削除されます（注）。ドリルビットと頭蓋骨の間に長時間の接触を避けるため、過熱による損傷を防ぐため。 </li><li>ドリル薄くなった領域の中心にある顕微刃で内側の緻密骨層を薄くし続ける。均等におおよその厚さの小さな領域（200〜300ミクロン径）に薄くなるまで頭蓋骨に対して押し下げずに頭蓋骨をこすり取る際には、約45度の角度で顕微ブレードを保持するLY、20〜30程度が得られる。による頭蓋骨の自己蛍光を、厚さが二光子顕微鏡下で頭蓋骨の上面と下面との間の距離を走査することによって測定することができる注：20μm未満の頭蓋骨の厚さは、良好な結像品質を提供するが、それはその後の再イメージングセッションで間引き処理を困難にするので、それはお勧めしません。オーバー間伐ないようにするには、頭蓋骨の厚さは、（説明を参照）、手術中に定期的にチェックする必要があります。 </li></ol> 3。固定化<ol><li>丁寧に骨片を除去。 </li><li>オートクレーブ処理された無菌のヘッドプレートの中央開口部の各エッジにシアノアクリレート接着剤の小滴（ 図1参照 ）を配置します。開口部注意の中心に位置して薄くなった地域で頭蓋骨にしっかりとヘッドプレートを保持する：接着剤が薄くなったRを汚染した場合偶然egion、顕微刃で慎重に取り除いてください。 </li><li>優しくヘッドプレートの中央開口部のエッジに頭蓋骨の側面から肌を引き出します。 </li><li>ヘッドプレートはよく頭蓋骨に装着されるまで、約10分待ちます。 </li><li> （ 図1を参照）保持板の2の横方向のブロックにヘッドプレートを配置し、保持板上にマウスを固定化するために、ヘッドプレートの縁の上にネジを締め注：。の間には、皮膚やウィスカーがないことを確認してくださいネジを締める前に、ヘッドプレートとブロック。動物の背中を優しく撫でているときは良いの固定化の準備は解剖顕微鏡下で頭蓋骨の観察可能な動きを示さないはずである。 </li><li> 。未重合接着剤を除去するために生理食塩水でさらさ頭蓋骨をすすぎ注：未重合接着剤は、画像をぼかしや損傷がある目的を顕微鏡としてそれは、残りの接着剤を除去することが重要である&lt;。/李&gt; </ol> 4。イメージング<ol><li> （ 図2参照）、その後のイメージングセッションで画像化された領域を再配置するために使用されているマップ1、のように薄くなった領域と露光され頭蓋骨の血管系の写真を撮る。 </li><li>イメージング顕微鏡下でマウスを置きます。エピ蛍光を使用して10倍の空気目的の下に薄型化エリアの位置を確認し、ビューの中心に最も薄いエリアを移動します。 </li><li>頭蓋骨の上に生理食塩水を一滴を追加し、滅菌水でリンスされた60Xの対物レンズに切り替える。個々の樹状突起棘がはっきりと樹状突起に沿って視覚化された領域を選択します。落射ビューと血管系写真間の血管系を比較することにより、地図上の1対応する領域を特定し、ラベルを付ける。 </li><li>チューン二光子レーザー波長フルオロフォアに記載の方法。例えば、YFPのための920ナノメートル; GFPについて890 nmの;のDsRedとtdTomato 24 1,000 nmの。 </li><li> 2で画像スタックを取得60Xの対物レンズを使用して、z軸に沿って以下の手順。この画像スタックは、約200ミクロン×200ミクロンの領域（512×512ピクセル）をカバーし、（ 図2を参照）、その後のイメージングセッション中に再配置するためのマップ2として使用される。 </li><li> 3倍デジタルズームを使用して、マップ2内の9画像スタックを取得。各画像スタックは、z軸に沿って0.7μmのステップで、70ミクロンX 70ミクロン（512×512ピクセル）のおおよその領域をカバーしています（注）。光毒性を最小限にするために、サンプルで測定した場合、レーザーの強度は40ミリワット以下でなければならない。 </li></ol> 5。復旧<ol><li>イメージングの後、静かに頭蓋骨からヘッドプレートを取り外す。 </li><li>徹底的に頭蓋骨と、残りのすべての接着剤を除去するために、皮膚をきれい注：お肌に残りの接着剤は炎症を引き起こし、頭蓋骨に残っている接着剤は頭蓋骨の浸食となりますしながら、皮膚の治癒が遅くなります新しく成長した骨層にngiogenesis、その後の移転と再結像が困難になる。 </li><li>生理食塩水で数回頭蓋骨と皮膚を洗い流す。 </li><li>無菌手術用縫合糸で頭皮を縫合。 </li><li>別々のケージに加熱パッド上に動物を保管してください。術後疼痛を軽減するためにブプレノルフィンの鎮痛薬（0.1 mg / kg）を皮下投与する。完全に回復した後にホームケージに動物を返します。切開が治癒され、縫合糸が削除されるまで、（少なくとも一日一回チェックしてください）​​密接に動物を監視します。必要に応じてブプレノルフィン鎮痛剤のその後の注射を管理します。 </li></ol> 6。再イメージ<ol><li>繰り返して1.1〜1.8を繰り返します。 </li><li>手順を繰り返し3.1から3.6 </li><li> 1をマッピングするために、血管系のパターンと2をマッピングするために、樹状分岐パターンを比較することで、以前に撮影領域の位置を確認します。 </li><li>再結像は1週間以内に行われた場合、再薄肉の上に新しく成長した骨の薄層を除去するために、ステップ2.3を繰り返す顕微ブレードを使用して祇園。再結像は1週間以上後に行われた場合、ステップ2.2および2.3の両方を繰り返す注：新しく成長した骨層は画質の低下をもたらす元の緻密骨層に比べて少ない縮合型構造からなる。したがって、同じ品質を取得するためには、頭蓋骨以前撮像セッションよりも若干薄くする必要がある。 </li><li>以前は二光子顕微鏡で画像スタックを取っ一致画像スタックを取得する位置及び姿勢を調整する。 </li><li>ステップ4.6のように画像を取得する。 </li><li>繰り返しは、撮影後に5.1から5.5を繰り返します。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-dependent+GCaMP3+reporter+mouse+for+neuronal+imaging+in+vivo.">A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.</a> J Neurosci. 32, 3131-3141 (2012).</li><li>Kuhlman, S. J., Huang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+labeling+and+functional+manipulation+of+specific+neuron+types+in+mouse+brain+by+Cre-activated+viral+gene+expression.">High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.</a> PloS one. 3, (2008).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

成功間引き頭蓋骨の準備を取得するには、このプロトコルにはいくつかのステップが重要である。 1）頭蓋骨の厚さ。頭蓋骨は、高密度緻密骨と低密度海綿骨の中間層の2層、サンドイッチ構造を有する。高速マイクロドリルは緻密骨と海綿骨の外側の層を除去するのに適しているが、顕微ブレードは緻密骨の内側の層を薄くするのに最適です。開発中に頭蓋骨の厚さが増加すると剛性ように?...

哺乳類の大脳皮質は、知覚と運動制御から抽象情報処理や認知に、多くの脳機能に参加しています。種々の皮質の機能は、個々のシナプスで情報を通信し、交換する異なるタイプのニューロンから構成されている異なる神経回路上に構築。シナプスの構造および機能は、一貫して経験や病状に応じて変更される。成熟した脳では、シナプス可塑性、機能性神経回路の形成と維持に重要な役割を果たして、両方の強さが変化し、シナプスの追加/削除の形をとる。樹状突起棘は、哺乳類の脳における興奮性シナプスの大多数のシナプス後要素である。一定の売上高および棘の形態学的変化は、シナプス結合1-7において変更が良い指標となると考えられている。 二光子レーザー走査マイクロSCOPYは厚く、不透明な準備と、無傷の脳8でのライブイメージングのために適しています、低光毒性を通じて深い浸透を提供しています。蛍光標識との組み合わせでは、2光子イメージングは​​、生きている脳に覗くと、高い空間分解能と時間分解能を有する個々のシナプスにおける構造的な再編成を追跡する強力なツールを提供しています。様々な方法がライブイメージングのために9-13匹のマウスを作製するために使用されている。ここでは、マウスの皮質におけるシナプス後樹状突起棘の構造的可塑性を調べるためのin vivo二光子イメージングの間引き頭蓋骨の調製を記載する。このアプローチを使用して、我々の最近の研究では、蛍光標識された神経細胞のサブセットおよびin vivo標識技術の急速な発展に伴い、トランスジェニック動物の増加可用性を使用した学習運動技能に応じて、樹状突起棘の変更のダイナミックな絵を描かれているが、ここで説明するのと同様の手順を適用することもできますinvestigaへteの他の細胞型および皮質領域、他の操作と組み合わせ、ならびに疾患モデル16-23で使用した。

<table border="0" cellpadding="0" cellspacing="0" width="1253">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ketamine</td>
			<td style="width:181px;">Bioniche Pharma</td>
			<td style="width:108px;">67457-034-10</td>
			<td style="width:775px;">Mixed with xylazine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Xylazine</td>
			<td style="width:181px;">Lloyd laboratories</td>
			<td style="width:108px;">139-236</td>
			<td>Mixed with ketamine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saline</td>
			<td style="width:181px;">Hospira</td>
			<td style="width:108px;">0409-7983-09</td>
			<td>0.9% NaCl for injection and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Razor blades</td>
			<td style="width:181px;">Electron microscopy sciences</td>
			<td style="width:108px;">72000</td>
			<td>Double-edge stainless steel razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Alcohol pads</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">06-669-62</td>
			<td>Sterile alcohol prep pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Eye ointment</td>
			<td style="width:181px;">Henry Schein</td>
			<td style="width:108px;">102-9470</td>
			<td>Petrolatum ophthalmic ointment sterile ocular lubricant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">High-speed micro drill</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">18000-17</td>
			<td>The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Micro drill steel burrs</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">19007-14</td>
			<td>1.4 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Microsurgical blade</td>
			<td style="width:181px;">Surgistar</td>
			<td style="width:108px;">6961</td>
			<td>The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Cyanoacrylate glue</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">NC9062131</td>
			<td>Fix the head plate onto the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Suture</td>
			<td style="width:181px;">Havard Apparatus</td>
			<td style="width:108px;">510461</td>
			<td>Non-absorbale, sterile silk suture, 6-0 monofilament</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Dissecting microscope</td>
			<td>Olympus</td>
			<td style="width:108px;"></td>
			<td>SZ61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Two-photon microscope</td>
			<td style="width:181px;">Prairie Technologies</td>
			<td style="width:108px;"></td>
			<td style="width:775px;">Ultima IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">10X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 0.30, air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">60X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 1.1, IR permeable, water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ti-sapphire laser</td>
			<td style="width:181px;">Spectra-Physics</td>
			<td style="width:108px;"></td>
			<td>Mai Tai HP</td>
		</tr>
	</tbody>
</table>

two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

哺乳類の大脳皮質では、神経細胞がシナプスで非常に複雑なネットワークや情報交換を形成している。シナプス強度、ならびにシナプスの追加/削除の変化は、神経可塑性の構造的基礎を提供する、経験依存的な様式で起こる。大脳皮質で最も興奮性シナプスのシナプス後構成要素として、樹状突起棘はシナプスの良いプロキシであると考えられている。マウス遺伝学および蛍光標識技術、個々のニューロンのシナプスの構造およびそれらの撮影の利点は、無傷の脳内で標識することができる。ここでは、 生体内での経時的蛍光標識後シナプス樹状突起棘に追従する二光子レーザー走査顕微鏡を用いて、経頭蓋撮影プロトコルを導入する。このプロトコルは、完全な頭蓋骨を保持し、髄膜および皮質の曝露によって引き起こされる炎症作用を回避間引き頭蓋骨の準備を利用する。そのため、画像はSUの直後に取得することが可能rgeryが実行されます。実験手順は、数時間から数年の範囲の様々な時間間隔で繰り返し行うことができる。この調製物の適用はまた、生理学的および病理学的条件下で、異なる皮質領域、層、ならびに他の細胞型を調査するために拡張することができる。

YFP-Hラインマウス25で、黄色蛍光タンパク質は、皮質の表層への先端樹状突起を投影V層錐体細胞のサブセットで表現しています。間引き頭蓋骨の製剤を介して、蛍光標識された樹状セグメントは、繰り返し時間から数ヶ月の範囲の、種々の撮像間隔で二光子顕微鏡で画像化することができる。ここでは、個々の棘だけでなく、糸状仮足は明らかに樹状突起に沿って可視化することが?...

Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

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-vivo-imaging-dendritic-spines-mouse-cortex-using-thinned

Research

JoVE Journal

Neuroscience

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

17.8K ビュー.  University of California, Santa Cruz. 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.

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

間伐-頭蓋骨の準備を使用してマウス視覚野の慢性的イメージング

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

内在性NADH蛍光の二光子イメージングによるマウス大脳皮質におけるMicroregional低酸素の検出

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

マウス脳の長期イメージングのためのポリッシュと強化間引き頭蓋骨ウィンドウ

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

ための間引き頭蓋骨皮質ウィンドウ·テクニック<emインビボで&gt;</em&gt;光干渉断層イメージング

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

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 ...

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 ...

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 ...