AKは、オッペンハイム財団とRHベルックス財団によってサポートされているサポートされています。私たちは、組織学的サポートのための技術的およびK. BuderのためI. Krumbeinに感謝し、TIMM染色に技術的助言のためのJ·ゴールドシュミット（神経生物学、マクデブルク、ドイツのためのライプニッツ研究所）。

全ての動物介入は実験動物の動物実験のための欧州条約および眼と視覚研究における動物の使用に関するARVO声明に従って行われる。全ての実験は、地元の倫理委員会によって承認されています。マウスにおけるON損傷の手順は、他の場所に記載されている9。 1。硝子体内注射マンガン<ol><li>アシスタントの助けを借りて、スキャン前にMRにマンガン2 +注入24時間後に行う。 （滅菌PBS中で420〜450 mg / kg体重）5％抱水クロラール溶液の腹腔内注射により動物を麻酔。追加の局所麻酔のため、前目の穿刺に角膜に液体conjuncain一滴（0.4％塩酸オキシブプロカイン）を適用。目ごとに15ナノモルの Mn 2 +を注入するには、132のLH 2 &lt;中1MのMnCl 2ストック溶液1リットルに希釈することによって、 例えば 、7.5のMnCl 2溶液を調製/サブ&gt; O。 34gの小さなハブリムーバブルニードル（RN針）に接続されている5μLハミルトンシリンジに最終溶液の負荷5μL。 </li><li>右目で始まる場合には、双眼顕微鏡下で、ゆっくりと開いた左側のマウスを置き、左手の親指と人差し指の間に右目を修正。右手で注射器を手に取り、先端に近い針を手に取る。眼球の非外傷性穿刺については、慎重に、それ​​によって強膜血管を温存、角膜輪部の遠位にinfero時空周り約1ミリメートルで硝子体に針を挿入します。 </li><li>ハミルトンシリンジの規模を制御しながら、次の、アシスタントが徐々に2μLの総容量が適用されます。この手順の間、顕微鏡下での最適な針の配置を監視し、液体のレンズやこぼれの穿刺を避ける。液体を最小限に抑えるため、ゆっくりとそれを撤回して、静的にさらに30秒間挿入された針を保つ注射部位からの漏れ。 </li><li>手順では、特別なケアは、目に圧力を避けるために注意すべきである。同様に、眼球を穿刺する過酷なまたは多くの試みを避ける。 の Mn 2 +をONに沿ってのRGCや輸送への取り込みはすでに15ナノモルのMnCl 2で飽和しているので、これは少し不正確注入量によって信号の変動を最小限に抑えることができます。視覚投影の二国間の信号増強のために、左眼用の注入手順を繰り返します。 </li><li>適用さオフロキサシン含有（3 mg / ml）を点眼し、手順は、眼感染症や目の乾燥を防ぐために、一回の後にパンテノール含有軟膏。 MRスキャンを開始するまでの通常の住宅の条件の下でケージにマウスを返します。 </li></ol> MRI用2。動物の準備<ol><li> 2％/ 98％イソフルラン/酸素ガス混合物の投与によりマウスを麻酔。ほぼ水平、ねじられていない位置にマウスのホルダーにマウスをマウントします。インその後、MRスキャナ内部で調整されたMRコイル内へERTこと。適切なシステムによって呼吸と心拍数を監視します。技術的な詳細については、ハーマンら 20を参照してください。 </li><li> MRI、統合された管によってマウスヘッドホルダに接続された蒸発器を介して、最初に1.5％/ 98.5％イソフルラン/酸素ガス混合物の連続注入による供給麻酔中。スキャン中に、記録された重要なパラメータ（ すなわち 、毎分約40回の呼吸の安定した呼吸数を目標）によると、麻酔の濃さ ​​を調整します。加熱装置を使用すると、マウスの腹部部位に位置する温度センサにより測定される、35〜37°Cの間の安定した体表面温度を維持する。技術的な詳細については、ハーマンら 17を参照してください。 </li><li>スキャン後、ホルダーからマウスを解放し、麻酔からの回復を加速する純酸素を供給しています。さらに、使用して、安定した体温を保つ赤色光加熱源。 </li></ol> 3。のMRIプロトコル<ol><li>プロトコルは、35ミリメートル×38ミリメートル直径の有効視野に専用の、SNR効率、小動物コイル（直線偏光リッツコイル）を装備した3テスラスキャナのために検証されます。送受信モードでコイルを動作させてください。 </li><li>その最終的な位置にある動物と、周波数分析器を用いてコイルの曲との一致を調整します。手動で画像の均一性および品質を最適化するために、送信機基準電圧とシム電流を調整する。 </li><li>計画のための矢状および横表示で0.5ミリメートル×0.5ミリメートル×2ミリメートルの解像度で、T 1加重2D東証画像を取得する。計画MRスキャンを使用して、冠状測定方向にMEMR画像を取得、左右方向に沿って位相エンコードを動物の頭部に平行になるように回転される。取得時間を最小限にするために調整ビューの長方形のフィールドを使用実際のヘッドの大きさ。位相エンコード方向の画93.8％長方形のフィールドを使用して、ベースマトリックス256、視野14.08ミリメートル×50.65ミリメートル×54ミリメートル、および0.11 mmの128スライス：次のパラメータを使用して、甘やかされて育った3D点滅シーケンス（VIBE 3D）を採用スライス解像度のスライス厚は61％に設定する。 </li><li> 、0.21ミリメートル×0.21ミリメートル×0.18ミリメートル（補間0.09ミリメートル×0.1ミリメートル×0.1ミリメートル）、エコー時間T E = 6.51ミリ秒の有効分解能を提供し、512×480×128で、最終的なイメージを作成する面内補間をアクティブにする繰り返し時間 t r = 16ミリ秒、帯域幅= 160 Hzの/ PX、フリップ角は= 22°。約30分の合計取得時間（T A）を達成するために2つの平均値と3繰り返しを適用します。 </li></ol> 4。のMRIデータ解析<ol><li>ソフトウェアsyngo fastViewを使用してデータを分析します。定量的な信号増強のため、定義された領域Oを選択Fの2次元平面のMRIの録音への関心と強化された構造（SI MEMRI）、組織のバックグラウンド（のSI backgr）とノイズ（のSD N）の標準偏差の信号強度（SI）を決定する。必要に応じて、LGNとSC構造のための神経解剖学的向きを容易にするために、マウス脳アトラスを使用しています。 ：式を用いて、コントラスト対雑音比（CNR）を算出</li><li> CNR =（SI MEMRI - SI backgr）/ SD N </li><li>各サンプルの平均のCNR計算のための3つの連続した​​画像を定量化する。両側性に注入された動物では、独立して各半球を分析。 </li><li>水平冠状および矢状画像の描写のために、オリジナルの3D MRIデータセットから多断面再構成を計算します。これらの処理された画像は、定量分析にはお勧めできません。レチノの生気に満ちた3D再構成（最大強度の予測、MIPの）を作成するには- 視蓋投影は、血管造影後処理ソフトウェアモジュールを使用しています。 </li></ol> 5。 の Mn 2 +オートメタログラフィー（TIMM染色） <ol><li> MEMRI以下の Mn 2 +をトレース脳構造のTIMM染色のために、15〜150ナノモルの Mn 2 + ivit 24時間前画像への投与量を注入する。 </li><li> MRスキャンした後、PBS中で30ミリリットルの氷冷0.325パーセントのNa 2 S（pH7.4）で動物を灌流。凍結切片に網膜凍結サンプルを分析。 </li><li>クライオトーム上で15μmの厚さの連続した​​、赤道部分をカット。 </li><li> Angenstein ら 22によると、固定剤および凍結保護がない状態でTIMM染色21を実行します。 </li></ol> 6。統計解析事後分散分析に続いて、単一の比較のためのスチューデントのt検定を用いた統計分析を実行。データは平均±標準誤差として提示されている。個々のN番号は各実験ごとに個別に与えられた。 （; P≤0.01、**、P≤0.001、*** P≤0.05、*）0.05≤Pに達した結果は統計的に有意であると考えている。

<ol>
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W., 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=Impact+of+repeated+topical-loaded+manganese-enhanced+MRI+on+the+mouse+visual+system.">Impact of repeated topical-loaded manganese-enhanced MRI on the mouse visual system.</a> Invest Ophthalmol Vis Sci. 53, 4699-4709 (2012).</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。

視覚系のMEMRIはナイーブおよび病理学的条件下での機能性を評価するための従来の神経生物学の手法を拡張します。離れて孤立した中枢神経系の線維トラクトの整合性にユニークな洞察を提供するから、MEMRIを簡単に視覚に指定されたパラダイムの即時の影響を調査するために、行動試験、 例えば 、検眼、視覚ベースの水作業を補充することができます。また、電気生理学的および...

その有利な神経解剖学的構造に基づいて、げっ歯類の視覚系は、神経保護1又はプロ再生効果2,3を媒介するために、薬理学的化合物およびそれらの能力を評価するためにユニークな可能性を提供しています。また、最近では、シナプス前足場タンパク質ファゴット4を欠損したマウスで例示したように、マウスの突然変異体の機能的および神経解剖学的特性の研究を可能にします。さらに、補助的なツールの広いスペクトルが電図および行動試験、および固有信号の光イメージングによる皮質再配列の決定により、 例えば 、網膜神経節細胞（RGC）とのRGC軸索番号だけでなく、RGC活性の特色に追加与える。レーザー顕微鏡の最新技術開発が視神経（ON）と脳のホールマウント標本の深い組織蛍光イメージングによってRGC再生の現場での可視化を可能にする。このhistolog中iCalのアプローチは、光シート蛍光顕微鏡と組み合わせて、テトラヒドロフランに基づく組織のクリアをONに求心路遮断および視索5に再入力する単繊維の解像度を可能にします。このような技術は、解像度と成長パターンの決定に優れているかもしれませんが、彼らは、特に長期的な再生過程を評価することが望まれている個々の成長の節目、の繰り返しと縦の分析を有効にしないでください。 造影MRIは、マウスおよびラット6,7における網膜視蓋投影の最小侵襲的可視化のために採用されています。これは、網膜細胞の常磁性イオン（ 例えば 、Mnが2 +）の直接眼内送達によって達成することができる。カルシウムアナログとしてはMn 2 +が電位依存性カルシウムチャネルを経由して、RGC細胞体に組み込まれ、積極的に無傷で、ONと視神経管の軸索細胞骨格に沿って搬送。それは脳核内に蓄積しながら、それが10,11発生することがありますが、視覚的投影、外側膝状核（LGN）と上丘（SC） すなわち 、一次視覚野への経シナプス伝達は、無視できる程度の8,9が表示されます。 MR配列決定の下、常磁性の Mn 2 +は、T 1スピン-格子緩和時間12を短くすることにより、主にMRコントラストを増強する。例えばMn 2 +、MRI（MEMRI）が正常に傷害13,14後の軸索再生および変性の評価を含めたラットの様々な神経解剖学的および機能的研究に適用されている強化され、レチノ-視蓋投影15の正確な解剖学的マッピング薬物治療16後の軸索輸送特性だけでなく、決定。 の Mn 2 +の取り込み及び輸送神経細胞だけでなく、改善されたMRIプロトコルの投与量、毒性、および動力学における近年の改良により、トランスジェニックの研究への適用を拡大してきた一般的に臨床現場で使用される17 3テスラスキャナを用いて、マウス9。 ここでは、マウスの網膜視蓋投影のインビボ画像化に適した長手MEMRIプロトコルを提示し、ナイーブおよび種々の神経変性条件下での Mn 2 +依存性のシグナル増強を評価することにより、その適用性を例示する。我々のプロトコルは、一般的に、専用の動物のスキャナよりもアクセスすることが適度な3 Tの磁場でMRデータ収集上の特定の重点を置きます。ナイーブマウスでは、腸管固有の信号強度が、実質的にかつ再現性硝子（ivit） の Mn 2 +アプリケーションの後に増加になることができる方法を示しています。定量的には、視覚投影に沿っての Mn 2 +伝播が（3と26カ月齢のマウスの間で測定される）正常な老化プロセスとは独立して発生し、増加は暗闇に視覚刺激と適応に難治性である。対照的に、マンガン<s&gt; 2 +上視床および中脳センターの濃縮は、自発的アポトーシスRGC死からおよび変性19日苦しんでノックアウトマウス（P50 KO）挫滅18急性だけでなく、NFKB1で以下に減少する。このように、従来の組織学的分析による膨張で、個々の動物の縦MEMRI分析は、神経変性プロセスのユニークな反応速度のプロファイリングを可能にします。これは、薬理学的または遺伝的な介入に関連した神経保護および軸索再生の研究のために有用であることを証明する必要があります。

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			<td height="21" style="height:21px;width:363px;">Manganese (II) chloride solution 1M</td>
			<td style="width:269px;">Sigma Aldrich, Taufkirchen, Germany</td>
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			<td>0.4% oxybuprocaine hydrochloride</td>
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			<td>Dr. Mann Pharma, Berlin, Germany</td>
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			<td>420-450 mg/kg body weight</td>
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			<td>Actavis, Munich, Germany</td>
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			<td style="width:269px;">Hamilton Company, Reno, NV, USA</td>
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			<td>SYR 5 &#181;l, 75 RN, no NDL</td>
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			<td height="21" style="height:21px;">34 G&#160; needle (34/35/pst4/tapN)</td>
			<td style="width:269px;">Hamilton Company, Reno, NV, USA</td>
			<td style="width:119px;">207434/00</td>
			<td>removable needle RN, 34 gauge, lenght 38.1 mm, point style 4</td>
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			<td style="width:269px;">Zeiss, Oberkochen, Germany</td>
			<td style="width:119px;"></td>
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			<td colspan="2">Siemens Medical Solutions, Erlangen, Germany</td>
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			<td>Doty Scientific Inc., Columbia, SC, USA</td>
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			<td>tissue embedding for cryo-sections</td>
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			<td>Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">208043</td>
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			<td height="21" style="height:21px;width:363px;">gum arabic</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
			<td style="width:119px;">4159</td>
			<td rowspan="5">for TIMM staining</td>
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			<td height="21" style="height:21px;width:363px;">Hydroquinone (C6H6O2)</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
			<td style="width:119px;">3586</td>
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			<td height="21" style="height:21px;width:363px;">Citric acid (C6H8O7)</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
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			<td height="21" style="height:21px;width:363px;">Tri-sodium citrate dihydrate (C6H5Na3O7 x 2H2O)</td>
			<td style="width:269px;">Merck, Darmstadt, Germany</td>
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in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice

齧歯類の視覚システムは、視床と中脳の中心を入力する視神経を形成する網膜神経節細胞とその軸索を包含し、視覚野のシナプス後予測。その明確な解剖学的構造、便利なアクセス可能性に基づいて、ニューロンの生存、軸索再生、およびシナプス可塑性の研究のために好ま構造となっています。 MRイメージングにおける最近の進歩は、マンガン媒介コントラスト強調（MEMRI）を使用して、この突起のレチノ-視蓋部分のin vivo可視化を可能にした。ここでは、（200ミクロン）3の解像度は一般的な3テスラスキャナを用いて達成することが可能なマウスにおける視覚投影、MEMRIを説明するためのプロトコルを提供する。私たちは、15ナノモルのMnCl 2の単回投与量の硝子体内注射は24時間以内で完全な投影の飽和向上につながる方法を示しています。網膜を除いて、信号強度の変化が独立しているその一致視覚刺激または生理的老化の凹み。私たちは、縦方向に、この手法をさらに適用する軸索急性視神経損傷を受けて変性症はMn 2 +輸送によるパラダイム病変部位で完全に逮捕を監視します。逆に、活性の Mn 2 +輸送は、生存率、数、および軸索線維の電気的活性に定量的に比例する。このような分析のために、我々は視覚的な突起を含む感覚の自発的な萎縮を表示するトランスジェニックマウスモデル（NF-κB活性P50 KO）での視覚的なパスに沿っての Mn 2 +輸送動態を例示している。これらのマウスでは、MEMRIは減少したが、このようにしてNF-κBの突然変異により、構造および/ ​​または機能障害の徴候を明らかにし、野生型マウスと比較しての Mn 2 +輸送を遅延しないことを示します。 要約すると、MEMRIは便利in vivoアッセイを橋渡しし、characterizatiための死後の組織学を投稿神経線維の完全性と活動の上。これは、縦方向の軸索変性と再生に関する研究、本物または誘導性表現型変異マウスの調査のために非常に有用である。

正確に視覚投影の活力と機能性を評価するために、このイメージング技術の能力は、硝子体とのRGCによるその取り込みに非毒性の Mn 2 +投与量の正確なアプリケーションに依存している。この主要な仮定は、層の特定の Mn 2 +取り込みがオートメタログラフィー（TIMM染色）21によって実証されている図1にテストされています。網膜切片を対?...

Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

このビデオでは、ナイーブマウス視覚投影の造影MRイメージングのための急性視神経挫滅損傷および慢性視神経変性に関連視神経変性の繰り返しと縦のin vivo試験では、臨床3 Tスキャナを使用して、方法を示しているノックアウトマウス（P50 KO）。

マウスにおける造影MRIによる視神経線維の整合性in vivoイメージング

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

in-vivo-imaging-optic-nerve-fiber-integrity-contrast-enhanced-mri

Research

JoVE Journal

Neuroscience

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

13.2K ビュー.  Jena University Hospital. このビデオでは、ナイーブマウス視覚投影の造影MRイメージングのための急性視神経挫滅損傷および慢性視神経変性に関連視神経変性の繰り返しと縦のin vivo試験では、臨床3 Tスキャナを使用して、方法を示しているノックアウトマウス（P50 KO）。 

ビデオ: In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

哺乳類の中枢神経系における視神経切断後の実験操作のための方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

神経科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高解像度<em&gt;インビボ</em&gt; 3T磁気共鳴画像を用いたヒト海馬サブフィールドのためのマニュアルセグメンテーションプロトコル

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

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 ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

実験視神経の脱髄のラットモデルにおける視覚誘発電位の記録

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

ヒト皮質下の高分解能構造磁気共鳴イメージング<I&gt;インビボ</I&gt;と死後

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

エレクトロポレーションによる齧歯類坐骨神経内のシュワン細胞へのインビボ遺伝子導入で

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.