著者は、原稿の準備の支援についてヴォルテールGungabに感謝。この作品は、からの助成金によって部分的にサポートされていました：H.蘇に国立衛生研究所（T32 GM008440 WLヤングとH.蘇へEJウォーカー、WLヤングにR01 NS27713、NS44155 P01へ）、およびR21 NS070153、とアメリカ人心臓協会（H. suのAHA 10GRNT3130004）。

1。血管から血をクリア<ol><li> 0.25mlの生理食塩水で希釈した10 mg / kgのキシラジンでは100 mg / kgケタミンの腹腔内注射によってマウスを麻酔。 </li><li>一旦麻酔が足のピンチに反応しないことが確認されて、横隔膜を切って、ちょうどxyphoidプロセス下にはさみで胸に切開を行い、胸腔を公開するために、胸郭の背側と腹側のセグメントの間にカットするはさみを使用してください。胸骨と止血剤と隣接胸壁とのセキュアを折り畳むことにより心臓を公開。心臓を露出するためにピンセットで心膜嚢を開きます。 </li><li>流出で鈍22ゲージの針を持つ℃、チューブ37の湯浴でPBSを加えたヘパリン（5単位/ ml）溶液を温めるに接続されている（圧力を100mmHg）灌流ポンプをオンにします。 </li><li>心尖​​付近に、左心室に針を挿入します。すぐに私は血まで、全身の血液の流出を有効にして、フラッシュを継続するために右心房を切開sは、循環から除去。 </li></ol> 2。血管系へのキャスティングエージェントの灌流<ol><li>ミックスフィルタリングMVの化合物を4 mlとMV希釈剤を5ml：Microfil（フローテック株式会社カーバー、MA）製造業者のプロトコルごとにキャスティング溶液を調製します。触媒の450μlを（5％）（MV硬化剤）を追加します。解決策作業時間は20分ですが、適切な粘度を確保するために使用直前に混ぜる。 </li><li> 10mlのシリンジに剤溶液をキャスト撤退、注射器に鈍20ゲージの針を添付し、ソリューションを使用してニードルを埋める。 </li><li>血液を洗い流すために使用されるのと同じ穴から左心室に針を挿入する。心臓に対する止血クランプのある場所に針を固定します。 </li><li>約3ml /分で徐々に左心室に鋳造剤溶液を注入。過剰な圧力がキャスティングエージェントまたは潜在的な血管の破裂の誤った流れにつながる可能性があります。 </li><li>キャストの可視化：を含む成功した灌流の徴候を探します腸と肝臓の血管系におけるING剤、遠位四肢の変色、及び鼻と舌の変色。適切な灌流を達成するために必要に応じてニードルを再調整します。 </li></ol> 3。組織の収集と処理<ol><li> 4℃で一晩、4％パラホルムアルデヒドで脳と場所を削除℃に</li><li>直接室温でますます集中してエタノールにパラホルムアルデヒドから配置することによって、脳を脱水：25％エタノール、1日、50％エタノール、1日、75％エタノール、1日95％エタノール、2日間、および100％エタノール、2日間。 </li><li>室温で2日間、サリチル酸メチルで灌流脳を浸漬することにより、血管周囲の脳組織を明確にする。 </li><li>全体unsectioned明らかに脳の血管系は、解剖顕微鏡（図1）（ライカMZFL III顕微鏡、ライカマイクロシステムズ、バノックバーン、イリノイ州）または明視野顕微鏡（図2）（ライカDM /下サリチル酸メチルに浸漬、脳で画像化することができますLS、ライカマイクロシステムズ）。 </li><li> Reclarification組織が完全にクリアされていない場合に行うことができます。 2日間サリチル酸メチル、2日間、95％エタノールへの転送の脳、2日間100％のエタノール、およびサリチル酸メチルから。 </li></ol> 4。マイクロCTイメージングまたは組織学的応用 - 組織の準備<ol><li>脳は、直接（図4）明確化した後または、再水和後のマイクロCTを用いて画像化することができます。 </li><li>組織学的分析のために脳組織を準備するには、シリアル化後EtOHを介して脳を再水和する：100％エタノール、1日95％エタノール、1日、75％エタノール、1日、50％エタノール、2日間、25％エタノール、2日間、PBSでと店舗。脳は、標準的な手順を用いてパラフィンに包埋することができます。 </li></ol> 5。代表的な結果血管キャスティングに続いて、cerebrovasculatureは、脳の表面（図1A）に肉眼的に見ることができます。実質内の血管ツリーを可視化し、以下の明確化（図1B）ことができます。詳細な血管構造イメージングと（図3）範囲を解剖し、マイクロCTスキャン（図4）で、明視野顕微鏡（図2）で分析することができます。明視野顕微鏡を使用して、血管系は、脳（図2A）と同様に高い倍率（図2B）で個々の微小血管の切削することなく異なる焦点面で見ることができます。解剖範囲の下で、一つはcerebrovasculatureの三次元構造（図3）のよりよい視覚化を可能にする、1つの焦点面で全体の血管構造を見ることができます。解剖顕微鏡下で撮影された画像は、大血管の形態および全脳の血管系を示すが、明視野顕微鏡（図2B）で撮影した画像に示されている詳細情報が不足していることができる。この手法は、我々の実験BAVMの大人のマウスモデル2で不規則な血管を検出することができました。図5は、マイクロCTスキャン（図5A）および明視野顕微鏡（図5B）によって検出された同じ不規則な船の例を示しています。一緒に、このデータは、我々はtを分析することができることを示しています血管鋳造後の複数のイメージングツールと彼は同じサンプル。 <img alt="図1" src="/files/ftp_upload/2958/2958fig1.jpg" /> 図1。血管キャスティング以下の全マウスの脳。前（A）と溶剤サリチル酸メチルによる（B）明確化の後に。血管は、脳の表面（A）にし、実質、以下の明確化の手順（B）で明確に可視化することができます。 <img alt="図2" src="/files/ftp_upload/2958/2958fig2.jpg" /> 明らかに血管鋳造脳の図2。明視野顕微鏡像。脳は様々な面で、異なる倍率のレベルで画像化することができます。スケールバー200μmの（）と50μmの（B）。 <img alt="図3" src="/files/ftp_upload/2958/2958fig3.jpg" /> 図3。背側から全体の成体マウスの脳のcerebrovasculatureを示す1倍で顕微鏡画像を解剖。 &lt;IMG ALT =&quot;図4&quot;SRC =&quot;/ files/ftp_upload/2958/2958fig4.jpg&quot;/&gt; 図4マイクロCTは背側から図3に示すように、同一の全体の大人のマウスの脳の画像をスキャンする。 <img alt="図5" src="/files/ftp_upload/2958/2958fig5.jpg" /> 図5。脳動静脈奇形を検出するために血管の鋳造法の応用。 A）血管鋳造脳のマイクロCTスキャンでは、腹側のビューから、右半球の拡大、不規則なAVMのような血管の領域を示しています。地域の拡大画像（白いボックス）が右側に表示されます。 B）同じ不規則な血管は、50倍の倍率での明視野顕微鏡下で可視化した。スケールバーは50μm。

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実験動物を使用するための実験手順は、カリフォルニア大学サンフランシスコ校（UCSF）の動物実験使用の委員会によって承認された。

ここでは、さまざまなモダリティで撮像される成体マウスのcerebrovasculatureの鋳造のための方法を報告する。疾患モデルBAVMにこのメソッドのアプリケーションは、不規則な血管の3次元解析が可能になります。潜在的な将来の研究では、マイクロCT画像の定量化、組織の組織学的分析、および血管疾患の進行の診断が含まれています。 一般的に発生する問題と提案脳血管系の完全な血流が常に満たされるわけで。左心房や大動脈を破裂させずに遠位血管に到達するのに十分な圧力を有することは重要な問題です。これを克服するために、約100mmHgの一貫性のある圧力を適用する。また、大動脈にエージェントをキャストの適切な流出を確実にするために針位置を調整する。キャスティングエージェントは、脳の血管に到達できるように頸動脈が制限されていないことが重要です。あたりの改善小血管と相関する重要な手順融合は、以下のとおりです。血管から血液の（1）適切なクリアリング、鋳造剤の（2）ろ過、および鋳造剤注入時（3）針の配置。ケアは、左心房と肺にエージェントを鋳造の注入を避けるために注意すべきである。他の研究者が成功し、追加の手順を使用せずに、マウス3とラット4 cerebrovasculatureを灌流している。 hemotoxylinは93％の血管内腔の血流5を示したとMicrofil灌流血管の手動分析の対比。常に破棄または完全にそれはすぐに重合し、そしてツールは複数の被験者のために使用することはできないとして鋳造剤と接触する任意のツールをきれいに。周囲のマウスの脳組織の明確化が困難な場合があります。サリチル酸メチルの浸漬の後、組織は、依然として血管のイメージングが困難、不透明表示されることがあります。適切な脱水と明確化を確保することがこの問題を解決する必要があります。アルコールとメチルの中にロッカーの脳組織を配置するalicylateの治療は、組織の明確化を向上させることができます。 技術の欠点この手法の限界は、（1）それはすぐに触媒を添加した後、それはすぐに濃くなるので、それを混合した後、鋳造剤溶液を注入することが必要であること、（2）キャストのエージェントは、容易に大きなコンダクタンスの船を埋め、そうではありません確実にこのような細動脈（抵抗血管）と毛細血管（栄養血管）などの小型船を、塗りつぶし、およびいくつかのグループは、マイクロCTイメージング1,6のためにMicrofilを好むものの、（3）それは、マイクロCTイメージングに最適なX線造影ではありません。しかし、適切な針の配置、船の位置と灌流圧を含む上記の提案の提案、以下、小血管の完全なキャスティングを実現することができます。様々な解析ツールへの適応性は、様々で、このエージェントは動脈と静脈の循環を含めて、脳の構造を分析するための貴重なツールとなります視点。 技術のアプリケーションこの方法の利点は、その広範な応用の可能性があります。キャストは動脈、静脈、および本体内のすべての組織の毛細血管を含むすべての船舶を、いっぱいになる、様々な種の複数の臓器の血管構造を解析するため。以前、他のグループは、ヒト、サル9、羊10、ラット4,11,12、およびマウスの3,13の7大と小型の8隻は、この血流を使用している。我々は現在、健康と病気の状態2の両方で、マウスの脳内微小血管の構造を解析する上でこの技法を使用しての証拠を示しています。さらに、様々な臓器の血管の可視化を向上させるためにさまざまなサイズと異なる色の血管を灌流するために、様々な粘度とMicrofil解決方法があります。例えば、赤は淡い脳実質に容易に可視化すること、および黄色のコントラストもでできます。暗赤色心筋。また、材料が放射線不透過性であるため、血管系は、マイクロCTは、3D回転可能な画像を得るためにスキャンで撮像することができます。組織はまた、撮像された後パラフィン切片と組織学的分析に使用することができます。 結論我々はここにcerebrovasculatureの形態学的構造を研究するために分析の様々な技法を用いて適応することができます成体マウスの脳の血管の血管鋳型を作るためにプロトコルを示す。我々はまた、動脈から静脈循環へシャント血その拡大、不規則な形状の容器に代表される、BAVMの疾患の状態のモデルにこの方法の応用のための証拠を提供している。利用可能な血管の鋳造方法には、さまざまなものがあります。我々は血管鋳型のためにここに提供するシンプルなプロトコルは、様々な画像診断法を用いて成体マウスの脳の血管を調べるためのツールと​​して使用することができます。

<table border="1"><tbody><tr><td> の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> ノート </td></tr><tr><td> Microfil </td><td> FlowTech、（株） </td><td> MV130 </td><td> 4ヶ月の貯蔵寿命</td></tr><tr><td>メチルサリチル酸</td><td>フィッシャーサイエンティフィック</td><td> O3695 - 500 </td><td>組織を明確にする</td></tr></tbody></table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

血管イメージングは​​、脳動静脈奇形（BAVMs）などの脳血管疾患の臨床診断と管理に不可欠です。動物モデルは、脳血管疾患の病因論と潜在的な治療法を研究するために必要です。大動物用のイメージング血管系は比較的簡単です。しかし、マウスの脳の疾患モデルの血管撮像法を開発することにより、遺伝子組み換えマウスのラインのコストと可用性のが望ましい。イメージングは​​、マウス脳血管ツリーが課題です。人間と大きな動物、大血管での脈管構造学を評価するためのゴールドスタンダードでは（コンダクタンス）のレベルは、X線カテーテルコントラストベースの血管造影、小型げっ歯類には適していない方法です。 この記事では、我々は多くのdを使用して分析することができます動脈、静脈、毛細血管を含む、全体の血管床の耐久性のあるスケルトンを生成する脳血管キャスティングの手法を提案ifferentモダリティ。マウスcerebrovasculatureの微小血管の完全な鋳造が困難になる可能性がありますが、これらの課題は、このステップバイステップのプロトコールで対処されています。血管鋳造材料の心臓内の血流を介して、身体の全ての船舶は、キャストされています。脳を除去し、有機溶剤サリチル酸メチルを用いて明らかにすることができる。脳血管の三次元画像は、任意の従来の明視野顕微鏡や解剖顕微鏡で簡単にかつ安価に視覚化することができます。キャストcerebrovasculatureもイメージングおよびマイクロコンピュータ断層撮影（マイクロCT）1を用いて定量することができる。さらに、撮像された後、鋳物の脳は、組織学的分析のためのパラフィンに埋め込むことができます。 他の技術と比較して、この血管鋳造法の利点は、CTが原因で放射線不透過性の特性にスキャン、明視野顕微鏡分析など、さまざまな分析ツール、へその広範な適応です。材料だけでなく、組織学的および免疫組織化学的分析のteristic。組織のこの効率的な使用は、動物の使用量を節約し、コストを削減することができます。我々は最近、顕微鏡レベル2で成人BAVMのマウスモデルにおける不規則な血管を可視化し、マイクロCTスキャンによって撮像された不正な形式の船舶の追加のイメージを提供するために、この方法の応用を実証している。この方法は欠点を持っていると分析のすべてのタイプの理想的ではないかもしれないが、それは容易に学習し、広く体全体の血管の血管鋳造に適用することができる、シンプルで実用的なテクニックです。

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

この記事では、我々は実行しやすいとイメージに成体マウスの脳の血管ツリーを利用することができる脳血管キャスティングのためのシンプルで実用的なテクニックを紹介。

3Dイメージングと形態素解析のための成体マウスの脳血管キャスティング

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

cerebrovascular-casting-adult-mouse-for-3d-imaging-morphological

Research

JoVE Journal

Neuroscience

21.5K ビュー.  University of California, San Francisco. この記事では、我々は実行しやすいとイメージに成体マウスの脳の血管ツリーを利用することができる脳血管キャスティングのためのシンプルで実用的なテクニックを紹介。

ビデオ: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

認知に関する情動の損なう効果の脳イメージング研究

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

神経科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

成体マウスの前脳の急性スライスにおける神経芽細胞の移行のタイムラプスイメージング

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

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

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

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

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

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

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

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

NF-κB依存性神経新生の調節および分析のための方法

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

並列の単核 RNA 配列のため大人の脊髄核の分離

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

刺激特異的皮質視覚誘発潜在的形態パターン

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

マウスコクレアの特定周波数領域におけるリボンシナプスの形態学的および機能的評価

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神経クラッシュ損傷のラットモデルにおける機能的評価のための3D運動学的解析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

大人のマウスにおける人工内耳表面製剤

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...