Name: collection of frozen rodent brain regions for downstream analyses
Uploaded: 2020-04-23T13:00:00
Description: Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

この作業は、NIH、DA043982、DA046196によってサポートされました。

この研究で使用される動物は、インディアナ大学の制度的動物ケアおよび使用委員会(IACUC)および国立衛生研究所(NIH)ガイドラインによって定められた倫理的および人道的な方法で扱われた。
注:すべてのツールと表面は、任意の作業を開始する前にヌクレアーゼ18を除去するために適切な溶媒で洗浄する必要があります。
1. 脳の保存</...

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この研究は、核酸およびタンパク質の分解を制限しながら、脳の小さな特定の領域を分離する技術を記述する。脳組織への損傷は、生物が死ぬとすぐに起こります。これは部分的に細胞外グルタミン酸の急速な蓄積と21が発生する結果として生じる興奮毒性によるものです。メッセンジャーRNAは、特に分解2222、2323に対して脆弱で?...

この研究は、アレンマウス脳アトラス1などの参考文献を用いて、高品質の核酸またはタンパク質を抽出するための凍結脳領域の解剖をガイドとして示している。この技術では、脳はフラッシュ冷凍され、後で切断および解剖のために-80°Cで保存され、凍結状態で維持される。このプロセスは、研究者が1回のセッションで多数の脳を収穫し、後で複数の脳領域の正確なコレクションのためにそれらを解剖することを可能にする。
多くの場合、遺伝子やタンパク質発現に関する質問に答える際には、関心のある脳領域(ROI)の正確な収集が必要です。薬理学、電気生理学および光遺伝学は野生型または遺伝子組み換えげっ歯類に使用され、観察された行動を支える分子変化を解明するのに役立つ2,2、3、4、,4トランスクリプトームおよびプロテオームの誘発変化の測定はこれらの知見を支持するためにしばしば使用される。定量的逆転写ポリメラーゼ連鎖反応(RT-qPCR)、ウェスタンブロッティング、RNAseq 5、MAPSeq6およびHPLC57などの技術は、堅牢で比較的低コストであり、多くの研究室が小さな脳領域内での分子変化を研究することを可能にする2、4、5、6。4,5,62
,脳領域,,8,9,10,11,12から核酸やタンパク8,9質を抽出して精製する方法はいくつかあります。101112多くの研究室は、収穫時に氷の上で脳を冷やし、切断することによって脳領域を収穫します9,13.このアプローチは、高品質の核酸およびタンパク質をもたらす可能性があるが、組織の微小環境内での分解がこれらの温度で起こり得るため、幾分時間制限がある。これは、一度に多数の動物やROIを解剖しようとするときに特に当てはまるかもしれません。サンプルを凍結しておくと、陰唇標的分子を維持するのに役立ち、比較的純粋なサンプルを収集するために、各セクションの両側のランドマークを慎重に比較する時間を研究者に提供します。レーザーキャプチャは、脳領域10からRNAまたはタンパク質分析のための組織を収集する別の方法です。この手順は、非常に小さく不規則な形状のROIを同定し、単離することができるという機械的解離よりも優れています。しかし、レーザー捕捉は高価な装置および試薬の使用によって制限され、時間がかかり、またサンプル分解の影響を受けやすい場合がある。
凍結組織のマイクロパンチ解剖は新しいものではありません。ミクロス・パルコヴィッツの初期の論文は、基本的なテクニックを詳細14,15,15に詳述している。このプレゼンテーションは、効率を容易にし、必要な機器の費用を削減するためにいくつかの改善と、主に元の作業に従います。例えば、脳の切片は、クライオスタットではなく、凍結された脳ブロックで作られています。これにより、ROI サンプルの収集に必要なセクション数を削減する、より厚いセクションが生成されます。この方法はまた、絶縁箱内のドライアイスに座っている凍結ガラス板のサンプルを解剖する。これは、作業するベンチでサブ凍結段階を生成します。このように解剖されたセクションは操作が容易であり、研究者は目的のROI外の領域からの汚染を制限するために、各セクションの両側を参照と比較することができます。
このプロトコルの利点は、1)脳がプロセス全体を通して凍結状態に保たれ、タンパク質と核酸を保存するのに役立ち、研究者にROIを慎重に収穫する時間を与え、2)必要な試薬は安価であり、ほとんどの分子生物学研究室に見られることです。このプロセスでは、脳全体を脳のマトリックスで0.5~1.0mmに切り離し、氷で継続的に冷やされる凍ったガラス板の上に置きます。アレン・ブレイン・アトラス1または他の脳アトラス16、17,17に見られるランドマークは、関心のある領域を識別するために使用され、冷たいパンチまたはメスのいずれかを使用して解剖されます。1組織が解凍されることは決してないので、この方法で収穫された領域は、下流の分析のための高品質のRNAとタンパク質を提供します。

<table>
	<tbody>
		<tr>
			<td>0.5 mm Mouse coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 505C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>0.5 mm Rat coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 705C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>1.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Dot Scientific</td>
			<td>229443</td>
			<td>For storing frozen ROIs</td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-03</td>
			<td></td>
		</tr>
		<tr>
			<td>4-12% NuPage gel</td>
			<td>Invitrogen</td>
			<td>NPO323BOX</td>
			<td>protein gradient gel</td>
		</tr>
		<tr>
			<td>Bioanalyzer System</td>
			<td>Agilent</td>
			<td>2100</td>
			<td>RNA analysis system</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Millipore Sigma</td>
			<td> 
			D8938</td>
			<td>Glass tissue homogenizer</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Lite</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>Model 180</td>
			<td>Cool lamp</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>LabRepCo</td>
			<td>11074010</td>
			<td></td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>ThermoFisher</td>
			<td>78440</td>
			<td>protease inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Low profile blades</td>
			<td>Sakura Finetek USA Inc.</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-actin antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>JLA20</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td>2000C</td>
			<td>Used in initial RNA purity analysis</td>
		</tr>
		<tr>
			<td>No. 15 surgical blade</td>
			<td>Surgical Design Inc</td>
			<td>17467673</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking buffer</td>
			<td>LiCor Biosciences</td>
			<td>927-40000</td>
			<td>Western blocking reagent</td>
		</tr>
		<tr>
			<td>Omni Tissue Master 125</td>
			<td>VWR</td>
			<td>10046-866</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>rabbit anti-KCC2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>94725S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>RNA Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>73034</td>
			<td>Used to extract RNA from small tissue samples</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Excelta Corp.</td>
			<td>16050103</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard razor blades</td>
			<td>American Line</td>
			<td>66-0362</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596026</td>
			<td>Used to extract RNA from tissue</td>
		</tr>
	</tbody>
</table>

collection of frozen rodent brain regions for downstream analyses

神経生物学の理解が進むにつれて、分子分析は内側前頭前野(mPFC)や側坐核などの小さな脳領域でしばしば行われます。本研究の課題は、検討する微小環境を維持しながら、正しい領域を解剖することです。このホワイト ペーパーでは、ほとんどのラボですぐに利用できるリソースを使用した、シンプルで低コストの方法について説明します。この方法は、プロセス全体を通して組織を凍結させることによって核酸およびタンパク質を保存する。脳は、脳マトリックスを使用して0.5〜1.0mmのセクションに切断され、凍結ガラス板の上に配置されます。各セクション内のランドマークは、アレンマウス脳アトラスなどの参照と比較され、領域は冷たいメスや生検パンチを使用して解剖されます。その後、使用するまで組織を-80°Cで保存する。ラットとマウスのmPFCを通じて、側坐核、側側海馬および腹側海馬および他の領域は、qRT-PCRおよび西洋アッセイを用いて正常に分析された。この方法は、明確なランドマークによって識別することができる脳領域に限定されています。

この方法を検証するために、成体CD1野生型雄マウスおよびRNAおよびタンパク質から内側前頭前野を採取し、抽出し、特徴付けた。RNAを毛細管電気泳動法で解析した。分解されたRNAは、28Sおよび18Sリボソームバンドの強度の損失を示し、また25〜200ヌクレオチド間のスミアとして分解産物を示す(図5A、サンプル1)。高品質のRNAは、低分子量領域においてシグナルがほとんど...

Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Collection of Frozen Rodent Brain Regions for Downstream Analyses

この手順は、安価で一般的に入手可能なツールを使用して高品質のタンパク質およびRNAを得るために離散的な凍結脳領域のコレクションを記述する。

下流解析のための凍結げっ歯類の脳領域の収集

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

この手順は、安価で一般的に入手可能なツールを使用して高品質のタンパク質およびRNAを得るために離散的な凍結脳領域のコレクションを記述する。脳領域は解剖によって収穫から凍結されるため、標的分子は保存され、研究者は慎重に解剖し、関心のある領域を保存する時間があります。関心のある地域を特定するのは、最初は困難です。一般的な脳のランドマークを使用して、必要な領域に関連してセクションを出現し、後退すると、識別が明らかになります。安楽死させた成人CD-1野生型マウスから脳を取り除いた後、フラッシュは液体窒素またはイセオペンタンのいずれかで組織を60秒間凍結する。ドライアイスで予冷し、マイナス80度で保管してください。組織を解剖する24時間前に、解凍した冷凍庫パックの積み重ねにきれいな脳マトリックスを置く。そして、カミソリスロットの底とパックの上部の間に約半センチメートルを残すことを確認するために、2つの冷凍パックの間のマトリックスに側面を挟みます。断熱箱に氷を充填して、上から約5センチメートルまで、解剖用の冷凍ガラス板を設置します。その後、上にドライアイスの2.5センチメートルの層を置きます。そして、黒いプラスチックシートでそれをカバーしています。プラスチックの上にガラス板を置きます。そして、プレートの境界の周りにドライアイス。冷凍庫から凍結した脳マトリックスを取り出し、脳を上に挿入します。箱の中の温度に10分間平衡させる。この間、蓋を開けておきます。冷たい鉗子を使用して、矢状の正弦と横の正弦がブロックの垂直な溝と一致するように、マトリックス内の脳の位置を調整します。脳が所定の位置に入ったら、冷やしたカミソリの刃を中心近くに置き、約1ミリメートルを組織に押し込みます。次に、脳の両端に1つの冷たいブレードを置き、マトリックスにずっと押し込みます。その後、ロストラルの端に冷やしたブレードを追加し、一度に1つずつスロットに入れ、1ミリメートル静かにティッシュに押し込みます。1ミリ間隔でブレードを追加し続け、尾端に向かって作業します。すべてのブレードが所定の位置にある場合は、指、手のひら、または鈍い物体で上を押し下げ、それらを左右にゆっくりと揺らし、組織を通してそれらを移動させます。ブレードがスロットの底に達したら、ブレードのグループの両側をつかみ、前後に揺らすことによってマトリックスからそれらを解放します。ブレードのグループを解放した後、それらをガラス板の上にrostral側に置き、さらに容易に分離するためにサンプルを凍結するためにスタックの隣または上にドライアイスを置きます。次に、鋭いエッジを持つスタックを下に置き、親指と指の間でスタックをシフトしてブレードを分離します。ガラス板のセクションをロストラルからカウダルに並べ、指の間で曲げるか、2番目の冷たいブレードで分離することによって、ブレードから組織を分離します。時には、組織はブレードの両側に付着し、尾骨を尾翼方向に維持するために注意を払う必要があります。解剖を開始する前に、アレンマウス脳アトラスまたは別の参照を開き、関心のある地域を識別するために必要なランドマークを見つけます。チルド鉗子またはブレードを使用してセクションを反転させます。また、セクション全体で関心領域が一貫していることを確認します。きれいなメスやパンチでセクションにカットし、金属を優しくしっかりとティッシュに押し込みます。カットを行うために前後にそれを揺らす。目的の領域を収穫した後、ラベル付きの事前冷蔵1.5ミリメートルチューブに入れ、マイナス80度で保存します。組織を処理するには、RNA抽出用のタンパク質抽出またはグアニジニウム含有溶媒に冷たいRIPAバッファーを追加し、すぐにガラスダウンまたは機械的ホモジナイザーで均質化します。この方法を検証するために、成体CD-1野生型雄マウスから内側前頭前野を採取し、RNAおよびタンパク質を特徴付けた。毛細管電気泳動で解析すると、分解されたRNAは、28Sおよび18Sリボソームバンドの強度の損失、ならびに25〜200ヌクレオチド間のスミアを示す。高品質のRNAは、低分子量領域ではほとんどない信号を示す明確なリボソームバンドを示しています。凍結解剖法を用いて得られたRNAを、採取したばかりの組織から調製したRNAと比較した。どちらの方法も、高い完全性の数と強いリボソームバンディングを持つRNAを生成します。この方法を使用して後で分析するために組織の微小環境を保存できることを確認するために、数週間にわたって保存されていた解剖された内側前頭前野からRNAを抽出した。すべてのサンプルは、8以上の明確なリボソームバンディングとRNA完全性番号を持つ高品質のRNAを生成しました。凍結解剖試料のタンパク質の完全性を確認するために、タンパク質を回収し、高分子量標的KCC2に対する抗体を用いてニトロセルロースおよびプローブに移し、より低分子量のプロテインアクチンを用いた。すべてのケースでバンドは鋭く、識別可能な内訳製品なしで明確でした。これは、セクションの微小環境を維持し、脳アトラス画像との比較のためにセクション形態を維持するので、プロセス全体を通して組織を凍結し続けることが重要です。この方法で収集された対象領域は、マイナス80°Cで数ヶ月間保存することができ、RT-qPCR、RNA-Seq、ウェスタンブロット分析、およびHPLCを含む多くの下流アプリケーションで利用することができます。この方法は、これらの薬物が脳の発達にどのような影響を与えるかをよりよく理解するために、THCおよび他のカンナビノイドにさらされた周産周産げ歯類の辺縁系内の分子変化を探求するために使用されてきた。

collection-of-frozen-rodent-brain-regions-for-downstream-analyses

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

齧歯類の脳における同時fMRIと電気生理学

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

神経科学

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

げっ歯類からの神経細胞とヒト以外の霊長類の脳スライスのDiOLISTIC標識

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

トータルRNAの単離およびダウンストリーム次世代シーケンシングと遺伝子発現プロファイリングのための離散マウスの脳領域のマイクロダイセクションのための非レーザーキャプチャー顕微鏡のアプローチ

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

網膜切片の形態学的解析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

大人の齧歯類の脳の従来の従来にない地域から神経幹細胞を増殖させる

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

齧歯類の中枢神経系の生体光遺伝学的刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

聴覚弁別学習した後、マウス脳領域の高分解能定量的シナプスプロテオームプロファイリング

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

パーソナライズされた齧歯動物の脳でのマイクロインジェクション針

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

外傷によって影響を受ける脳のレーザーキャプチャーマイクロダイ セクション定位アトラス誘導レーザー

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...