テス・J・ラマイヤー氏、ルース・フランク氏、マイケル・A・ムニアック博士に対し、編集とコメントをいただき、2pFLIM買収ソフトウェアを提供するマックス・プランク・フロリダの安田良平博士に感謝します。この作品は、2つのBRAINイニシアチブ賞U01NS094247(H.ZとT.M.)とR01NS104944(H.ZとT.M.)、R01助成金R01NS081071(T.M.)、およびR21グラントR21NS097856(H.Z.)によってサポートされました。すべての賞は、神経障害と脳卒中の国立研究所からされています, 米国.

<ol>
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著者は何も開示していない。

このプロトコルは、頭固定振る舞いマウスにおける神経変調誘発PKA活性を可視化するためにFRET-FLIMセンサtAKARαの使用を示す。このアプリケーションは、得られたFLIM信号が生理的神経調節イベント17に関連していることを実証するために、vitroおよびインビボにおけるtAKARαの広範な試験および特性に基づいている。ここでは、生体内アプリケーションの1つは、運動皮質にお?...

神経変調は、遅いシナプス伝達としても知られており、ストレス、覚醒、注意、移動1、2、3、などの異なる行動状態の間に脳機能を強力に制御します。4.その重要性にもかかわらず、神経調節イベントがいつどこで起こるかの研究はまだ初期段階にある。アセチルコリン、ドーパミン、ノルアドレナリン、セロトニン、および多くの神経ペプチドを含む神経調節剤は、Gタンパク質結合受容体(GPCR)を活性化し、その結果、広範囲のタイムスケールの広いウィンドウを持つ細胞内第2のメッセンジャー経路をトリガーする秒から時間まで。各ニューロモジュレータは、シグナル伝達イベントの明確なセットをトリガするが、cAMP/タンパク質キナーゼA(PKA)経路は、多くのニューロモジュレータ1、5のための共通の下流経路である。cAMP/PKA経路は、神経興奮性、シナプス伝達、可塑性6、7、8、9を調節し、したがって、ニューロンネットワークダイナミクスを調整する。異なるニューロンまたはニューロンタイプが異なるタイプまたはレベルのニューロモジュレーター受容体10を発現するので、同じ細胞外神経調節剤の細胞内効果は異なるニューロン間で不均一であり、したがって、細胞分解能で研究。今日まで, 行動中に生体内の個々のニューロンの神経調節イベントを監視することは困難なまま.
神経変調の時空間ダイナミクスを研究するには、適切な記録モダリティが必要である。微小透析および高速スキャン環状ボルタンメトリーは、神経調節剤の放出を研究するために頻繁に使用されるが、それらは細胞事象11、12を監視する空間分解能を欠いている。集団イメージング13におけるニューロン電気活動のプロキシとして使用されているカルシウムダイナミクスに類似して、PKAイメージングは、細胞分解能でニューロン集団全体の神経調節イベントを読み出すために使用されてもよい。本プロトコルは、動物の行動中に生体内でPKA活動を監視するために改良されたA-キナーゼ活動レポーター(AKAR)の使用について説明する。ここで説明する方法は、生理的神経調節的事象を追跡する時間的分解能を有する細胞内分解能でニューロン集団の同時イメージングを可能にする。
Akarsは、PPKAリン酸化基質ペプチドおよび基質14、15のリン酸化セリンまたはスレオニンに結合するフォークヘッド関連(FHA)ドメインによって連結されたドナーおよび受諾蛍光タンパク質から構成される。PKA経路の活性化に際して、AKARの基質ペプチドがリン酸化される。その結果、FHAドメインはリン酸化基質ペプチドに結合し、それによって2つの蛍光体を近接させ、AKARの閉じた状態と呼ばれる。リン酸化AKARの閉じた状態は、ドナーとインセプター蛍光体との間のフェルスター共鳴エネルギー伝達(FRET)の増加をもたらす。リン酸化ALEVELの割合はPKA活性16のレベルに関連しているので、生体試料中のFRETの量は、PKA活性16、17、18のレベルを定量するために使用することができる。19、20.
AAKAの初期のバージョンは、主に2色比比イメージング14用に設計されました。脳組織をより深くイメージングする場合、レシオメトリック法は波長依存性光散乱17、18、21による信号歪みに苦しむ。以下に説明するように、蛍光寿命イメージング顕微鏡(FLIM)は、FLIMがドナー蛍光素18、21によって放出される光子のみを測定するので、この問題を排除する。その結果、FRETのFLIM定量は組織深度17の影響を受けない。また、アクセプター蛍化素子の「暗い」(すなわち、低量子収率[QY])変異体を使用することができる。これは第2のセンサーまたは形態マーカー17、19、20の同時イメージングを介して直交神経特性の多重測定を容易にするために色チャネルを解放する。
FLIMイメージングは、蛍光素子が励起状態で過ごす時間、すなわち、蛍光寿命18を定量化する。蛍光素が地上状態に戻ると、励起状態の終わりは、しばしばフォトンの放出と共に起来する。個々の励起分子に対する光子の放出は確率的であるが、集団においては平均蛍光寿命がその特定の蛍光素の特徴である。蛍光色素の純粋な集団が同時に興奮すると、得られた蛍光は単一の指数関数的崩壊に続く。この指数関数的減衰の時間定数は、蛍光タンパク質の平均蛍光寿命に相当し、通常は蛍光タンパク質の1〜4ナノ秒の範囲である。興奮したドナー蛍煙症の地上状態への復帰は、FRETによっても起こりうる。FRETの存在下では、ドナー蛍光の寿命が減少する。非リン酸化AkaRは、比較的長いドナー蛍光寿命を示す。PKAによるリン酸化時に、ドナーとインセプター蛍光体が互いに近づき、FRETが増加するため、センサの寿命が短くなります。したがって、AkaRの集団における蛍光寿命の定量は、PKA活性のレベルを表す。
AAKAの初期のバージョンは、単一細胞分解能での生体内イメージングにうまく使用されていません。これは主に生理的活性化17に対するAKARセンサの低信号振幅によるものです。近年、2光子蛍光寿命撮像顕微鏡(2pFLIM)に利用可能なAKARセンサを系統的に比較することにより、FLIM-AKARと呼ばれるセンサが代替センサを上回ることがわかった。さらに、微小管(tAKARα)、シトゾール(tAKARβ)、アクチン(tAKARδ)、フィラメントアクチン(tAKARε)、膜(tAKARγ)、特定の細胞下の場所でのPKA活性を可視化するために、標的AkaR(tAKARγ)と呼ばれる一連のFLIM-AKAR変異体が開発されました。ポストナプティック密度(tAKAR)。タカーの中で、tAKARαはノルエピネフリンによって引き起こされた信号振幅を2.7倍増加させた。これは、ニューロン中のPKAの大部分が安静状態22,23の微小管に固定されているという知識と一致している。tAKARαは、2pFLIMの既存のAAKAの中で最高のパフォーマーでした。さらに、tAKARαは、複数の神経調節剤によって引き起こされた生理学的に関連するPKA活性を検出し、およびtAKARαの発現は神経機能17を変化させなかった。
最近、tAKARαは、頭部固定振る舞いマウス17におけるPKA活性の可視化に成功した。強制移動は、運動、バレル、および視覚皮質における表面層ニューロン(層1~3、ピアからの約300μmの深さまで)の相馬におけるPKA活性を引き起こしたことが示された。移動誘発PKA活性は、β-アドレナリン受容体およびD1ドーパミン受容体を介したシグナル伝達に部分的に依存していたが、D2ドーパミン受容体拮抗薬の影響を受けなかった。この研究は、2pFLIMを用いて生体内の神経変調イベントを追跡するタカーの能力を示す。
現在のプロトコルでは、強制移動パラダイム中の頭部固定覚醒マウスにおけるPKA活性イメージングの方法全体を6つのステップで説明する。まず、従来の2光子顕微鏡に2pFLIM機能を添加する(図1)。第二に、電動トレッドミルの建設(図2)。第3に、DNAプラスミドの子宮エレクトロポレーション(IUE)、またはアデノ関連ウイルス(AAV)の立体注射によるマウス皮質におけるtAKARαセンサの発現。IUE 24、25およびウイルス粒子26の立体注射のための外科のための優秀なプロトコルは、以前に公開されている。使用した主なパラメータを以下に説明します。前に、頭蓋の窓のインストール。優れたプロトコルは、頭蓋窓手術27、28のために以前に公開されています。標準プロトコルから変更されたいくつかの手順について説明します。第5に、生体内2pFLIMで行う。6番目に、2pFLIM画像の解析(図3および図4)。このアプローチは、他の多くのヘッド固定行動パラダイムおよび脳領域に容易に適用されるべきである。

<table>
	<tbody>
		<tr>
			<td>0.2 &#956;m cellulose acetate syringe filter</td>
			<td>Nalgene</td>
			<td>190-2520</td>
			<td>Step 3.2.2.</td>
		</tr>
		<tr>
			<td>16x 0.8 NA water-immersion objective</td>
			<td>Nikon</td>
			<td>MRP07220</td>
			<td>Step 5.5.</td>
		</tr>
		<tr>
			<td>3-pin cable</td>
			<td>US digital</td>
			<td>CA-MIC3-SH-NC</td>
			<td>Step 2.5. To connect rotation sensor to the DAQ input of the microscope</td>
		</tr>
		<tr>
			<td>Aluminum bread board</td>
			<td>Thorlabs</td>
			<td>MB1012</td>
			<td>Step 2.5.</td>
		</tr>
		<tr>
			<td>AnimalTracker MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 2.5 and sections 5 - 6. Will be provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Band-pass barrier filter</td>
			<td>Chroma</td>
			<td>ET500-40m</td>
			<td>Step 1.4.</td>
		</tr>
		<tr>
			<td>Cage plate</td>
			<td>Thorlabs</td>
			<td>CP01</td>
			<td>Step 2.4. Used as mount for rotation sensor</td>
		</tr>
		<tr>
			<td>Carbon steel burrs for micro drill, 0.5 mm tip diameter</td>
			<td>FST</td>
			<td>19007-05</td>
			<td>Steps 3.2.3. and 4.4.</td>
		</tr>
		<tr>
			<td>Circular coverslip (5mm diameter)</td>
			<td>VWR</td>
			<td>101413-528</td>
			<td>Step 4.5.</td>
		</tr>
		<tr>
			<td>Custom-made injection needle holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 3.2.4. Technical details provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Dental acrylic</td>
			<td>Yates Motloid</td>
			<td>44114</td>
			<td>Steps 4.3. and 4.5.</td>
		</tr>
		<tr>
			<td>Dental drill; Microtorque ii</td>
			<td>Ram products</td>
			<td>66699</td>
			<td>Steps 3.2.3. and 4.4.</td>
		</tr>
		<tr>
			<td>Dowsil transparent polymer</td>
			<td>The Dow Chemical Company</td>
			<td>3-4680</td>
			<td>Step 4.5. Artificial dura</td>
		</tr>
		<tr>
			<td>Electroporation electrode</td>
			<td>Bex</td>
			<td>LF650P5</td>
			<td>Step 3.1.4.</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bex</td>
			<td>CUY21</td>
			<td>Step 3.1.4.</td>
		</tr>
		<tr>
			<td>Fast green FCF</td>
			<td>Sigma-aldrich</td>
			<td>F7258-25G</td>
			<td>Step 3.1.1.</td>
		</tr>
		<tr>
			<td>FLIMimage MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Section 5. Kindly provided by Dr. Ryohei Yasuda, Max Planck Florida</td>
		</tr>
		<tr>
			<td>FLIMview MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Sections 5. and 6. Will be provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Foam-compatible glue (Gorilla White Glue)</td>
			<td>Gorilla</td>
			<td>5201204</td>
			<td>Step 2.3.</td>
		</tr>
		<tr>
			<td>Headplate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 4.3. Technical details provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Headplate holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 2.6. Technical details provided upon request lead author, used in combination with mounting post bracket and right-angled bracket</td>
		</tr>
		<tr>
			<td>Hydraulic micromanipulator</td>
			<td>Narishige</td>
			<td>MO-10</td>
			<td>Step 3.2.4.</td>
		</tr>
		<tr>
			<td>Krazy glue</td>
			<td>Krazy glue</td>
			<td>KG82648R</td>
			<td>Step 4.3. Cyanoacrylate-based glue</td>
		</tr>
		<tr>
			<td>Low-noise fast photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422PA-40 or H10769PA-40</td>
			<td>Step 1.3.</td>
		</tr>
		<tr>
			<td>MATLAB 2012b</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>Steps 2.6, and sections 5, and 6. Used to run microscope acquisition and data analysis software</td>
		</tr>
		<tr>
			<td>Motor</td>
			<td>Zhengke</td>
			<td>ZGA37RG</td>
			<td>Step 2.4.</td>
		</tr>
		<tr>
			<td>Motor speed controller</td>
			<td>Elenker</td>
			<td>EK-G00015A1-1</td>
			<td>Step 2.5.</td>
		</tr>
		<tr>
			<td>Motorized micromanipulator</td>
			<td>Sutter</td>
			<td>MP-285</td>
			<td>Step 3.2.4.</td>
		</tr>
		<tr>
			<td>Mounting base</td>
			<td>Thorlabs</td>
			<td>BA1S</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with PH4 and TR2</td>
		</tr>
		<tr>
			<td>Mounting post</td>
			<td>Thorlabs</td>
			<td>P14</td>
			<td>Step 2.6. Used for headplate holder post in combination with PB2</td>
		</tr>
		<tr>
			<td>Mounting post base</td>
			<td>Thorlabs</td>
			<td>PB2</td>
			<td>Step 2.6. Used for headplate holder post in combination with P14</td>
		</tr>
		<tr>
			<td>Mounting post bracket</td>
			<td>Thorlabs</td>
			<td>C1515</td>
			<td>Step 2.6. Used in combination with right-angle bracket and headplate holder</td>
		</tr>
		<tr>
			<td>Optical post</td>
			<td>Thorlabs</td>
			<td>TR2</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and PH4</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>&#925;/&#913;</td>
			<td>&#925;/&#913;</td>
			<td>Step 3.2.2. Protocol: Cold Spring Harbor Protocols 2006, doi: 10.1101/pbd.rec8247</td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>FDS010</td>
			<td>Step 1.2.</td>
		</tr>
		<tr>
			<td>Photon timing counting module</td>
			<td>Becker and Hickl</td>
			<td>SPC-150</td>
			<td>Step 1.1.</td>
		</tr>
		<tr>
			<td>Plasmid: tAKAR&#945; (CAG-tAKAR&#945;-WPRE)</td>
			<td>Addgene</td>
			<td>119913</td>
			<td>Step 3.1.3.</td>
		</tr>
		<tr>
			<td>Post holder</td>
			<td>Thorlabs</td>
			<td>PH4</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and TR2</td>
		</tr>
		<tr>
			<td>Right-angle bracket</td>
			<td>Thorlabs</td>
			<td>AB90</td>
			<td>Step 2.6 Used in combination with mounting post bracket and headplate holder</td>
		</tr>
		<tr>
			<td>Rotation sensor</td>
			<td>US digital</td>
			<td>MA3-A10-250-N</td>
			<td>Step 2.4.</td>
		</tr>
		<tr>
			<td>Rubber mat</td>
			<td>Rubber-Cal</td>
			<td>B01DCR5LUG</td>
			<td>Step 2.1.</td>
		</tr>
		<tr>
			<td>Shaft coupling (1/4 inch x 1/4 inch)</td>
			<td>McMaster</td>
			<td>6208K433</td>
			<td>Steps 2.3. and 2.4.</td>
		</tr>
		<tr>
			<td>ScanImage 3.6</td>
			<td>Svoboda Lab/Vidrio Technology</td>
			<td>N/A</td>
			<td>Steps 5.9. and 6.1.</td>
		</tr>
		<tr>
			<td>Signal splitter</td>
			<td>Becker and Hickl</td>
			<td>HPM-CON-02</td>
			<td>Step 1.3.1.</td>
		</tr>
		<tr>
			<td>Stainless steel axle (diameter 1/4 inch, L = 12 inch)</td>
			<td>McMaster</td>
			<td>1327K66</td>
			<td>Step 2.3.</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment systsem</td>
			<td>David kopf</td>
			<td>1900</td>
			<td>Steps 3.2. and 4.1. modified; Sutter micromanipulator, custom-made injection needle holder, hydraulic micromanipulator</td>
		</tr>
		<tr>
			<td>Two-photon microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Section 5. Built based on Modular in vivo multiphoton microscopy system (MIMMS) from HHMI Janelia Research Campus (https://www.janelia.org/open-science/mimms)</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>14006</td>
			<td>Step 3.2.6.</td>
		</tr>
		<tr>
			<td>Virus: tAKAR&#945; (AAV2/1 hSyn-tAKAR&#945;-WPRE)</td>
			<td>Addgene</td>
			<td>119921</td>
			<td>Step 3.2.2.</td>
		</tr>
		<tr>
			<td>White PE foam roller (8 x 12 inch)</td>
			<td>Fabrication enterprises INC.</td>
			<td>30-2261</td>
			<td>Step 2.1.1.</td>
		</tr>
		<tr>
			<td>White polystyrene fom ball halves</td>
			<td>GrahamSweet</td>
			<td>200mm diameter 2 hollow halves</td>
			<td>Step 2.1.1.</td>
		</tr>
		<tr>
			<td>Zipkicker</td>
			<td>PACER</td>
			<td>PT29</td>
			<td>Step 4.3. Hardening accelerator</td>
		</tr>
	</tbody>
</table>

visualizing protein kinase a activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy

神経変調は、脳機能に強力な制御を行使します。.神経調節システムの機能不全は、神経学的および精神的障害をもたらす。その重要性にもかかわらず、細胞分解能を持つ神経調節イベントを追跡する技術が出現し始めたばかりです。ドーパミン、ノルエピネフリン、アセチルコリン、セロトニンなどの神経調節因子は、それぞれのGタンパク質結合受容体を介して細胞内シグナル伝達イベントを引き起こし、神経興奮性、シナプス伝達、およびその他の神経学的刺激性を調節する。ニューロンネットワークにおける情報処理を調節する。上記の神経調節剤は、cAMP/タンパク質キナーゼA(PKA)経路に収束する。そこで、単細胞分解能を有する生体内PKAイメージングは、神経電気活動に対するカルシウムイメージングに類似した方法で神経調節イベントの読み出しとして開発された。本明細書では、頭部固定振る舞うマウスの皮質における個々のニューロンのレベルでPKA活性を可視化する方法が提示される。これを行うには、Förster共鳴エネルギー伝達(FRET)に基づく改良されたA-キナーゼ活性レポーター(AKAR)がtAKARαと呼ばれる使用されます。この遺伝的にコードされたPKAセンサは、DNAプラスミドの子宮エレクトロポレーション(IUE)、またはアデノ関連ウイルス(AAV)の立体注射を介して運動皮質に導入される。FRETの変化は、光散乱脳組織におけるFRET信号を定量するための比量的FRET測定に対する利点を提供する2光子蛍光寿命イメージング顕微鏡(2pFLIM)を使用して画像化されます。強制移動中のPKA活動を研究するために、tAKARαは、目を覚ます、頭固定マウスの皮質の上の慢性頭蓋窓を通して画像化され、速度制御された電動トレッドミルで実行または休息します。このイメージングアプローチは、対応する行動誘発PKA活動を研究し、生体内イメージングのための他のFLIMベースのセンサに適用されます。

FRET-FLIMセンサは、神経変調に関与するcAMP/PKA経路を含む多くの異なるシグナル伝達経路の可視化を可能にします。現在のプロトコルは、最近開発されたtAKARαセンサを2pFLIMと組み合わせて利用して、頭部固定振る舞いマウスにおけるPKA活動を可視化する。図 1に示すように、既存の 2 フォトン 顕微鏡のほとんどは、3 ~ 4 つのコンポーネントを追加?...

Watch this Scientific Journal Video about Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy at JoVE.com

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

タンパク質キナーゼAの活動を可視化する手順を提示し、マウスの頭部固定、振る舞いをする。改良されたA-キナーゼ活性レポーターtAKARαは皮質ニューロンで発現され、頭蓋の窓を通してイメージングのためにアクセス可能にされる。2光子蛍光寿命イメージング顕微鏡は、強制移動中の生体内でのPKA活性を可視化するために使用されます。

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

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

我々の方法は、ターゲットAキナーゼ活性レポーターα、またはtAKARと呼ばれる新しいプロテインキナーゼAセンサーを利用する

visualizing-protein-kinase-activity-head-fixed-behaving-mice-using

Research

JoVE Journal

Neuroscience

8.3K ビュー.  Oregon Health & Science University. 我々の方法は、ターゲットAキナーゼ活性レポーターα、またはtAKARと呼ばれる新しいプロテインキナーゼAセンサーを利用する

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

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 ...

神経科学

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 ...

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 ...

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 ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

2光子ホログラフィック顕微鏡を用いた神経活動の評価と操作

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

インビボ 大きな頭蓋窓を用いたマウスからの広視野および2光子カルシウムイメージング

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

ゼブラフィッシュの前脳における行動追跡と神経活動パターン

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

著者スポットライト:成体ゼブラフィッシュの脳研究の進歩 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

ゼブラフィッシュ成魚の行動性における前脳活動の2光子カルシウムイメージング

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used ...