We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission&#8217;s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the &#8220;Stem Cells Call for Proposals.&#8221; This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by &#8220;Ente Cassa di Risparmio di Firenze&#8221;.

1。軸索のラベリング<ol><li>クライミング繊維は、高分子量デキストランまたは蛍光タンパク質26-29の発現を誘導するプラスミド/ウイルスにコンジュゲート有機染料のいずれかを注入することによって標識することができる。このプロトコルでは、有機色素アレクサフルーアデキストラン488が登山繊維を標識し、小脳皮質（ 図1）でそれらを視覚化する下オリーブに注入される。ここで説明するすべての手順は、厚生労働省のイタリアで承認されている。 </li><li>マイクロピペットプラー上に引いてガラス毛細管を準備します。外径が約30μmになるまでハサミでガラスキャピラリーチューブの先端をトリミング。 </li><li>デキストラン共役系色素の1〜2μlのキャピラリーをロードします。 </li><li>開始する前に、オートクレーブ内のすべての手術器具を滅菌する。処置中に、ガラスビーズ滅菌器を用いて、汚染のリスクを低減する。 </li><li> 9月12日（マウスを麻酔ケタミン（90 mg / kgで）およびキシラジン（10 mg / kg）を腹腔内注射によって週）。動物が完全に尾および/またはつま先のピンチを使用して鎮静されていることを確認。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 </li><li>かみそりの刃や脱毛クリームのいずれかと首上の毛を剃る。エタノール70％と交互にベタジンで2回手術部位に綿棒。 </li><li>定位ホルダーにマウスを置きます。加熱パッドで動物を暖かく保つ。 </li><li>ヘッドがしっかりと保持されるように、ゆっくりと耳の横の骨にearbarsを押してください。ボディと140°の角度をなすように頭を回転させます。所望の配向を穏やかに定位ホルダーにノーズホルダロッドの高さを変化させることによって得ることができる。 </li><li>耳の間の皮膚にリドカインの数滴を適用します。後頭部下の皮膚に約1cmの縦切開を行うために鉗子やカミソリの刃を使用してください。 </li><li>優しく鈍ピンセットで皮下組織や筋肉を分離解剖顕微鏡下でのS。水平筋肉の束を下に押して、大後頭孔を介して硬膜を露出させるために、二つの垂直の筋肉の線維束を離れて保持する。 </li><li>細心の注意を使用して、脳幹を露出するために注射針または非常に鋭いピンセットで硬膜を解剖。硬膜が切断されたときに脳脊髄液の少量のこぼれてもよい。 </li><li>定位マイクロマニピュレータに、垂直からキャピラリー45°のホルダーを回転させます。小脳皮質及び第一頸椎の尾側縁部との間の中間点に、正中線でキャピラリーを配置します。表面から2mmの深さで、ピペットを挿入します。 </li><li> 10〜15分かけて、染料の0.5〜1.5μlの容量を提供します。マイクロインジェクション分配システムは、毛細管内の液体の制御された圧力の配信を可能にする（脈圧= 20 PSI、パルス持続時間= 3〜4ミリ秒、パルス供給= 12ヘルツの周波数）。 </li><li>その後、15分間の場所に毛細血管を残すslowl（2〜3分）、Yはそれを削除します。微妙に筋肉を再調整し、上記の皮膚を縫合。よく全体の手順を通して、いくつかの生理食塩水を適用することにより、水和した筋肉を維持。 </li><li>脱水を避けるために、皮下に0.5ミリリットルの0.9％のNaClを注入する。麻酔から完全に回復するまで加熱ケージに動物を保管してください。 </li><li>皮下手術後2〜3日間、毎日カルプロフェン（5 mg / kgの）を管理。 </li></ol>小脳皮質に2。光学窓注：色素（ 図1を参照）のCFS端末への小脳皮質に下オリーブ核から上2週間以内に輸送される。まばらにラベルされたCFSは、以下のように行うことができる永久頭蓋窓の下で可視化することができる。 <ol><li>すべての手術器具を滅菌する。 </li><li>前述したように、マウスを麻酔。動物をチェックするためにつま先のピンチを使用し、完全に鎮静されている。 </li><li> SKを拭く、頭の上に髪を剃る70％アルコールおよびベタジンの交互スワイプで中や眼軟膏を適用します。定位フレームにマウスを置き、しっかりと頭を保持するために耳のバーを配置します。 </li><li>皮下頭蓋窓サイトで脳と可能な炎症の腫れを防ぐために、デキサメタゾン（0.2 mg / kg）をし、カルプロフェン（5 mg / kgの）を管理。 </li><li>耳の間から目の上に、頭蓋骨の上の皮膚にリドカインの低下を適用し、フラップをカット。メスでそれをこする前に骨膜上に再びリドカイン溶液を適用します。 </li><li>小脳上記の頭蓋骨の薄い半円形の領域に歯科用ドリルを使用してください。頭蓋骨の深さは半円の周囲全体にわたってほぼ一定であるように、ウィンドウは、通常、中枢及び裏面に向かって縫合糸配置されている。この非常に厚い領域上のラムダ縫合および反復的な掘削に近すぎるウィンドウを配置すると、浮腫の脳と形成の過熱の原因となります。 </li><li>止血の適用出血例で頭蓋骨にスポンジ。 </li><li>カミソリの刃やピンセットの助けを借りて、2等分にカバーガラスをカットします。カバーガラスは、骨の島よりも大きめにピンセットで骨のフラップを削除する前に、同じような形状をしていることを確認してください。いない場合は、カバーガラスの形状を変更、または別のものを選択してください。骨をオフに描きながらピンセットで硬膜に手を触れないでください。慎重に硬膜上にカバーガラスを置く。注：硬膜上にガラスの全面の付着が長期的な頭蓋のウィンドウの予言されている。 </li><li>繰り返しコットンスティックで生理食塩水を用いてリンスして乾燥さガラスの下に気泡を除去。また、これはガラスを配置した後、小さな毛細血管からこぼれするかもしれないいくつかの残留血液を取り除くのに役立ちます。アクリル接着剤と歯科用セメント30の混合物で窓をシール。 </li><li>完全に麻酔から回復するまで、回復室での加熱パッド上に動物を保管してください。再しないでください完全に回復するまで、ホームケージにマウスを回す。彼らは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人のままにしないでください。 </li><li>皮下手術後2〜3日間、毎日カルプロフェン（5 mg / kgの）を管理。 </li></ol> 3。 インビボ多光子レーザー軸索切断<ol><li>第1の撮像セッションを実行する前に、手術から2〜7日待ちます。第ガルバノミラーの対によってスキャンした後、水浸20Xによって試料上に集束されるサファイアレーザ光源（120 fsの幅のパルス、90MHzの繰り返し率）：ここで使用される特注の二光子顕微鏡のTiが設けられている目的（NA 0.95、WD 2ミリメートル）。クローズドループピエゾステージは、客観的に400程度までの軸方向変位を実行します。光電子増倍管は、蛍光シグナルを収集します。 </li><li>頭蓋窓がobjectiと平行にかぶさるように、動物を麻酔し、定位ホルダーにヘッドの位置決め焦点面を見る。 </li><li> TPFは頭蓋窓の下皮質を画像化して探る。最も明るいものの中で破損する軸索を選択してください。 </li><li> Z-スタックを取得する標的軸索の3次元再構成を得るために、（通常は視野は512×512ピクセル、2μmのZ軸ステップで、100×100μm2である。）画像化されたボリュームの中では、焦点面に主に並列にある軸索の枝に病変部位を選択します。この配向は、急速に成功した解剖を予測する形態学的変化を、得点することができます。 </li><li> CFの末端部分の選択された点用量、高エネルギーを照射。これは、LabVIEWソフトウェアを介して行われます。（〜4ラインスキャンを行うためのガルバノミラーの位置を設定し、画像化（対物レンズの後に測定≈300 MW、）に使用される電力よりも5〜10倍以上のレーザーパワーを上げる - 10μm）を、損傷部位での400のためのレーザシャッタ開 - 600ミリ秒。ラインスキャンを配向CF面に垂直な軸索を完全に離断する。明るいスポット（ 例えば 、静脈瘤）を照射すると、通常軸索を解剖するのに役立ちます。レーザー軸索切断用の波長は、イメージング（920 nm）を使用したのと同じである。 </li><li>通常照射領域における軸索の蛍光は、一過性に起因する光退色に減少する。アブレーションがうまく実行されたことを確認するために数分照射後の同じ領域のスタックを取得する。領域は膨潤を開始し、軸索がビーズ様構造を形成する場合、ニューロンは、正常に解剖された。この場合は、数時間（分、数十から24時間まで）で軸索の先端部分は退化し、完全に消えてしまいます。 </li><li>残念ながら、完全な軸索切断を生成するために必要なエネルギー量は常に予測できません。照射の結果のみが光退色している​​場合、蛍光が急速に拡散を染色するために回収されるべきであり、照射が有効に再び繰り返されるべきであるLY軸索を解剖。また、ケース内の腫れの兆候は5内に表示されない-の増加、新規スキャンで再試行して、（。同様にレーザーdendrotomyについて説明したものに、アレグラMascaro ら 23を参照）、20分、または腫脹は回復が続いている時間および/またはレーザパワーを滞留。 </li><li> インビボイメージング2光子レーザの軸索切断に追従日での最終的な軸索 ​​リモデリングをモニタリング（ 図2および図3）。血管パターンは、次の日で解剖軸索を取得するために基準パターンとして使用される。 </li><li>皮下カルプロフェン（5 mg / kg）を、実験の全期間ごとに撮像セッションを管理します。 </li></ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+motility+and+its+modulation+by+activity+are+branch-type+specific+in+the+intact+adult+cerebellum.">Axonal motility and its modulation by activity are branch-type specific in the intact adult cerebellum.</a> Neuron. 56, 472-487 (2007).</li><li>Shinoda, Y., Sugihara, I., Wu, H. S., Sugiuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entire+trajectory+of+single+climbing+and+mossy+fibers+in+the+cerebellar+nuclei+and+cortex.">The entire trajectory of single climbing and mossy fibers in the cerebellar nuclei and cortex.</a> Prog Brain Res. 124, 173-186 (2000).</li><li>Strata, P., Tempia, F., Zagrebelsky, M., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+trophic+interactions+between+climbing+fibres+and+Purkinje+cells+in+the+rat+cerebellum.">Reciprocal trophic interactions between climbing fibres and Purkinje cells in the rat cerebellum.</a> Prog Brain Res. 114, 263-282 (1997).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+craniotomy+surgery+procedure+for+chronic+brain+imaging.">A craniotomy surgery procedure for chronic brain imaging.</a> J Vis Exp. (12), (2008).</li></ol>

We have nothing to disclose.

このプロトコルは、蛍光色素で下オリーブのニューロンを標識する方法を示しています。続いて、小脳皮質上の頭蓋窓を実行するための方法が記載されている。この手法は、オリーブ小脳ニューロン、登山繊維の末端部分に光アクセスを提供します。残念ながら、両方のラベリングと開頭手術の結果も、熟練したオペレータの手の中に極めて低い（3匹のマウスのうち、通常は1標識し、3頭側?...

機械的損傷、毒性傷害または神経変性疾患から生じる軸索切断は、通常、細胞体10-13から取り外された軸索の遠位部分の変性が続く。いくつかの例外を除い2,7,14,15で、成体動物のCNS ​​における切断軸索は通常、再成長プログラム16をアクティブにすることはできません。 リトルは、細胞および細胞内レベルでの変性のイベントをリアルタイムでダイナミクスについてはほとんど知られていない。神経損傷を制限し、神経細胞の再生を促進するための新たな戦略の開発を単独で負傷した神経細胞が退化して再生成するメカニズムを明らかにし、最初のステップとして、必要とします。この研究は、最も直接的に生体内での単一ニューロンのダイナミクスを監視することで対処されている。一光子蛍光イメージング技術は、可視光の強い散乱によって制限されるが、二光子励起は、リチウムの深い皮質層に到達する細胞内分解能3,4,17でマウスを見る。蛍光タンパク質を選択的に18-20ニューロンの亜集団で発現させたトランスジェニックマウスを利用して、TPF顕微鏡21,22は、 インビボでの開発の際のシナプスの可塑性および軸索伸長の探査に適用されている。単独で損傷を受けたニューロンのT彼の能力に損傷が特に関心のある軸索を対象と傷害のモデルと2光子イメージングによる生体モニタリングにカップリングすることによって調べることができる後の再成長。フェムト秒パルスの多光子吸収は、単一の樹状突起または単一の棘5,23を破壊するために使用されている。また、この損傷パラダイムは連絡デンドライト6を中断させることなく、単一の軸索の枝を切ることができます。一度損傷し、小脳登山繊維（CFS）、その軸索を再生するために、特定のニューロン集団を許可する機能を解剖するのコンテキスト内で有用なモデルSiがあるNCE彼らも成体動物24,25における損傷後の顕著な塑性特性を保持する。最近でのCFの長期イメージングは、これらの軸索は、レーザー軸索切断6に従う日で再成長させることが可能であることを示した。 このプロトコルは、順行性トレースを通じてオリーブ小脳の神経細胞とその軸索伸長にラベルを付ける方法を説明します。近隣のニューロンが蛍光標識されると、それらは頭蓋窓の下で数週間または数ヶ月間の任意の時点で繰り返し監視することができる。 生体内でのレーザー軸索切断により、単一の軸索の分岐を分析する手順は、次に説明する。 ここで紹介するテクニックは、in vivoでの軸索リモデリングのメカニズムに新たな洞察を提供し、神経変性を制限し、軸索再生を促進するための治療戦略の開発を助けるかもしれない。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument Inc</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0720</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser, 120 fs width pulses,&#160; 90 MHz repetition rate</td>
			<td>Coherent</td>
			<td>Chameleon&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spongostan, haemostatic sponge&#160;</td>
			<td>Ferrosan</td>
			<td>MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometric mirrors&#160;</td>
			<td>GSI Lumonics</td>
			<td>VM500+</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier modules&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>H7710-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW System Design Software</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Voren, 1 mg/ml (dexamethasone-21- isonicotinate)</td>
			<td>Boheringer Ingelheim</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rymadil (carprofen)&#160;</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine clorohydrate 2%</td>
			<td>ATI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 dextran</td>
			<td>Life Technologies</td>
			<td>D22910</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser nanosurgery of cerebellar axons in vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

このプロトコルは、単一のニューロンに軸索のラベリング、in vivoイメージングやレーザー軸索切断を実行する方法を説明した。実験のタイムラインを図1に示す。 アレクサフルーア488デキストランで標識し、in vivoでの二光子顕微鏡法による頭蓋窓の下で可視化したCFの一例を図2に報告されている。6,27、以前に報告?...

Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

Laser Nanosurgery of Cerebellar Axons In Vivo

レーザーnanodissectionに結合された2つの光子イメージングは​​、細胞内の分解能で中枢神経系の変性と再生のプロセスを研究するための有用なツールである。このプロトコルは、ラベル画像、およびin vivoでの小脳皮質内の単一の登山繊維を分析する方法を示しています。

小脳軸索のレーザーNanosurgery<em&gt;インビボ</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

laser-nanosurgery-of-cerebellar-axons-in-vivo

Research

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

8.1K ビュー.  University of Florence. レーザーnanodissectionに結合された2つの光子イメージングは​​、細胞内の分解能で中枢神経系の変性と再生のプロセスを研究するための有用なツールである。このプロトコルは、ラベル画像、およびin vivoでの小脳皮質内の単一の登山繊維を分析する方法を示しています。 

ビデオ: Laser Nanosurgery of Cerebellar Axons In Vivo

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

神経科学

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Cの生体レーザー軸索切断にエレガンス

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脳顆粒ニューロンの遺伝子操作<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;ニューロンの形態と移行を研究するために、

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

共焦点肉眼検査を使用した生後小脳顆粒細胞の移動のex vivoイメージング

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

神経細胞死とマウス プライマリ小脳顆粒ニューロンの変性をモデリング

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

生体内でマルチ モーダル イメージングとマウス レーザー誘発脈絡膜新生血管モデルの解析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

その場で有髄軸索のスペクトル反射型顕微鏡

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人小脳スライスにおける長期うつ病誘導の評価

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...