Name: slice it hot: acute adult brain slicing in physiological temperature
Uploaded: 2014-10-30T14:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

このプロトコルで説明されているすべての実験手順はヘブライ大学の動物実験委員会によって承認された。 1.スライスのためのソリューションとツールの準備<ol><li> 表1に記載のイオンを含有する標準生理溶液（SPS）の1リットルを準備します。 <ol><li>脱イオン水（0.055μS/ cmのコンダクタンス）1000mlので満たされた貯蔵ガラス瓶に予め10倍の最終濃度の塩を含むストック溶液を調製する。塩（塩化ナトリウム、塩化カリウム、KH 2 PO 4、そしてMgSO 4）を追加し、完全に溶解するまで撹拌する。 4℃での原液を保管してください。 </li><li>実験日の最終のSPS溶液を作る。磁気撹拌棒を用いて、グルコース（3.6 g）および炭酸水素ナトリウム（2.18グラム）を溶解し、精製水700ml中にストック溶液100mlを追加し、1 Lに精製水を上に追加</li><li> solutiを平衡〜20分間、95％O 2/5％CO 2でそれをガス処理することによってオン。この後、1MのCaCl 2溶液2mlを加える。 </li></ol></li><li>以下のツール（ 図1）を構築。 <ol><li>スライスするための脳と操作するための小さなへらの所望の部分を解剖するための刃でスカルペル、脳を露出するために頭蓋骨を持ち上げるための大きなハサミまたは断頭のために他のツール、頭蓋骨を開くための小さな外科用ハサミ、先端の細い鉗子を準備解剖脳のパーツ。 </li><li>加えて、脳から過剰な液体を除去するためのろ紙の小片を準備、脳のための2つの小さなペトリ皿に脳を接着するために含有する加温した精製水とSPS、およびシアノアクリレート接着剤のために二枚のガラス500mlのビーカー中で解剖するスライシング段階。 </li><li>回復茶にスライス浴からスライスを移動させるためのスライシングおよび広口ガラスパスツールピペット中にスライスを処理するための小さなブラシを準備mber。温かいスライス浴中の細菌増殖を防ぐためのツールを消毒する。さらに、きれいな実験室用手袋を使用しています。 </li></ol></li><li>ギブとエドワーズ15で説明したようにそのような水没スライス回収室を準備します。連続して95％O 2/5％CO 2で、その中にSPSをガスと36℃で保つ。ガス発生がスライスを損傷する可能性が風呂の中に閉じ込め小さな気泡が生じないことを確認してください。 </li><li>暖かい〜マグネチックスターラー又は水浴ヒータプレート上36℃でSPSを300ml。ヒータプレートが使用される場合、イオンの沈殿をもたらし得る過熱からの溶液を、防ぐように注意してください。ケースでは、代わりに古いものを冷却温かい新鮮なSPSを発生します。 </li><li>電気ポットを使用して沸騰させるの精製水〜500ミリリットルを持参して、ビーカーに、スライシング温度より少し暖かい水を得るために、冷たい水で300mlのを兼ね備えています。これは断頭中に精製水を温めて使用してremaiを使用後でスライス浴の生理的温度を維持するためのお湯の寧。 </li><li>腹腔内ペントバルビタール（60 mg / mlの）を0.1ml注入することによってマウスを麻酔し。数分後、動物が感覚の欠如を確認するために、強力なつま先またはテールピンチに応答しないことを確認してください。 </li></ol> 2.脳を解剖<ol><li>頭蓋骨の背面近くに大きなはさみで首を切断することにより素早くマウスを斬首し、頭が過剰な血液を洗い流すために温め、精製水を入れたビーカーに分類してみましょう。 </li><li>おそらく、小さなはさみで首のより多くを除去し、首の筋肉と皮膚の下の頭蓋骨で大後頭孔を公開します。 </li><li>明らかに頭蓋骨の開放を案内するために頭蓋骨の縫合を見ることができるようにするために頭蓋骨の上に肌を引き出します。 </li><li>大後頭孔を通して小さなハサミの下側の先端を挿入し、すぐに向かって、それらを回して開いた頭蓋骨をカット側面。そっと目と前頭頭頂縫合の背後にある場所までの頭頂骨の側縁に沿って切断。その後、（ 図2Aの緑色の破線を参照）頭蓋骨の中心に向かって切り、頭蓋骨の反対側に正中線を横切る。 </li><li> （ 図2（a）の黄色の破線の矢印参照）先端の細い鉗子を使用して、前頭頭頂隅から頭蓋骨を持ち上げ、脳を露出するために起動して側方へ斜めに引き出します。頭蓋骨を持ち上げた時に脳が所定の位置にとどまるように、頭蓋骨が、頭のいずれかの骨や皮膚に付着していないことを確認します。脳が頭蓋骨に移動している場合には、頭蓋骨と脳の間の任意の結合組織を除去するための小さなハサミを使用しています。 </li><li>脳が露出しているときに、脳はドライにすることはできません。頭蓋骨と温めで満たされたペトリ皿の中の脳を維持しながら、このようにして、次のステップを実行し、SPSをガス処刑。 </li><li>電子に使用する脳の部分を切り離す小刀やへらを用いて脳の残りの部分からxperiment。小脳を用いた場合には、（ 図2B-Dの赤い線に沿って）中脳と橋を通ってシングルカットにより前脳から切り離し、新鮮含まれている小型のペトリ皿に移動し、SPSはガス処刑。 </li><li>水平面内で小脳をスライスした段階の切断に脳を接着するためのストレートと広い基盤を形成するために、脳幹を削除します。前脳から切り取った面が下を向くように簡単に説明するとろ紙の濡れた片上に小脳を置く。 <ol><li> （ 図2Bの青線で示すように）ベースを形成し、SPSに小脳を返すためにメスで脳幹を切った。カッティングの他の面が必要な場合には、それに応じて小脳ブロック（ 図2C＆Dにおける青い線）をトリム。 </li></ol></li></ol> 3.脳をスライスする<ol><li>これをdと確認することによって、切削ステージを準備RY、ステージの真ん中に瞬間接着剤の小滴を適用する前に。 </li><li>正しい面を上にしてへらを使用して、SPSからの脳ブロックを持ち上げて、ろ紙の小片を用いて脳から余分な液体を除去。次に、単一の移動に伴って、ステージ上で接着剤の上にドロップへらから脳をスライドさせます。 </li><li>乾燥からの脳組織の両側に駆けから糊を防止するために、パスツールピペットを用いてSPSの数滴を適用し、スライスチャンバ内の切断段階を置く。関心（ 例えば、小脳皮質）の領域は、ブレード対向しているので、スライスされる前に、機械的圧力の最小量に供されるように、脳ブロックを揃える</li><li>常に切断チャンバ内にSPSをガス発生ならびに34-37℃に温度を維持しながら、製造者の指示に従って、各スライスを切った。 externを充填することにより、この範囲での切断室の温度を維持するスライサーのアルチャンバー温め精製水で、温度が低すぎると落ちるたびに水を交換する。 注：スライスパラメータは実験的に検討されている組織やニューロンの種類ごとに見つけなければならない。セラミックブレードと小脳ゴルジ細胞とカムデンSMZ 700スライサーの場合、65 Hzから0.05ミリメートル/秒の進行速度で0.75ミリメートルの振動振幅を使用する。小脳核のニューロン高い振幅と周波数（1ミリメートルと90ヘルツ、それぞれ）と遅い前進速度（0.01〜0.02ミリメートル/秒）のために推奨されている。ゴルジおよび小脳核のニューロンの両方のために、スライス厚は300μmであるべきである。 </li><li>スライシング中に、スライスは自分自身にフォールドすることはできません。優しく好ましくはただの実験のために関心のない領域でスライスを触れ、柔らかいブラシでそれらをサポートすることにより、これを防ぐ。ビブラトーム刃に触れることからブラシを防ぐように注意してください。特にセラミックブレードの場合には、均一な光接触ブレードが破損することがあります。 </li><li>広口、ファイアーポリッシュガラスパスツールピペットを使用してリカバリ/保持チャンバーにスライシング室から各スライスを移動します。 </li><li>スライスは、実験を開始する前に、少なくとも1時間室で休むましょう。これが損傷しているか死んで組織および細胞の劣化を可能にし、したがって、クリーナー、スライス面になる。最初の1時間、34-36℃の範囲のチャンバ内のSPSの温度を保つ。スライスはスライスを損傷したり、それらに浮くことができ、気泡によりタッチされていないことを確認してください。 </li></ol> 4.実験<ol><li> 1時間後、回収室温度がRT（17-25℃）まで冷却させ。 注：これは、スライスが細菌の増殖ならびに細胞の代謝を遅くすることにより使用することができる期間を延長するかもしれない。この期間は、脳領域および細胞タイプに依存する。例えば、ゴルジ細胞の特定の種類のスライスに健康であることが判明することができ最後の他のタイプのに対し、わずか6時間後に切断後8時間。 </li><li> 1時間はスライスから渡された後、様々な電気生理学的実験のためのスライスを使用。 </li></ol>

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The authors declare that they have no competing financial interests.

We demonstrate a method for preparing acute brain slices from mice in physiological instead of ice-cold temperature.
It has been shown11 that the quality of slices obtained in warm conditions is superior when compared with those prepared with cold conditions, provided that the slicer blade has minimal vertical vibration. Slicing in physiological temperature may prevent physiological artifacts caused by the low temperature, such as those related to changes in metabolic processes...

提示された方法の目的は、特に大人や古い動物を使用した場合、 インビトロ電気生理学的実験のために高品質の急性脳スライスを得ることである。 2エレガントな文章にSkredeとWestgaard 1に記載されているように急性脳スライス方法は、現代の神経科学研究の基礎となっており、世界中の無数のバリエーションで使用される。スライスの品質は、スライス当たりの生きているニューロンの数、細胞は電気生理学的および形態学的特性を保持し、ならびに組織の完全でない期間に反映される。また、安定した録画の最大の持続時間は、スライスの品質に依存します。このように、数十年に沿って、元のスライス方法は、さらに多くの場合、Oの切断物の複雑な修飾により、2-10切断後のスライスの回復を強化するために、個々の研究グループによって開発された同様に冷却された生理的なソリューションと動物の心臓内プレ灌流（例えばアスコルビン酸、チオ尿素あるいはH 2 O 2の追加など）ここで、rリカバリソリューション。 最近11が示されているように、スライス時の生理的温度は、ニューロンの健康への冷却よりも有益であると思われる。大人（2-8月）げっ歯類で作業する場合の改善が最も顕著である。劇的な温度変化を回避することにより、そのような可塑13およびイオンチャネル動態13,14のような細胞内の温度依存性プロセス、アーチファクトを防止する。このような変化は、膜電位および細胞内カルシウムシグナル伝達に影響を与えるしきい値をスパイクし、スパイク形状可能性がある。 ここに提示「ホット」急性スライスの調製方法は、小脳、大脳皮質および海馬を含む任意の脳領域から高品質の急性脳スライスを得るための一般的な手順であり、脳幹核16 &lt;/ SUP&gt;だけでなく、嗅球、両方のラット及びマウスで。 注目すべきことに、生理的温度スライス手順は、切断刃がほぼ完全に水平方向に振動し、構造的な欠陥のないことを要求する。このような精度は古いスライサーモデルと達成できない可能性があります。低温は、代謝異常のコストであっても、機械的な損傷に対する組織より耐性にいるようだとして、そのようなケースでは、我々は凍結冷条件のスライス標本を実行することをお勧めします。

<table><tbody><tr><td>Pentobarbital</td><td>CTS</td><td>170066</td><td>Concentration: 60 mg/ml in physiological saline.</td></tr><tr><td>Big scissors&amp;nbsp;</td><td>FST</td><td>14001-16</td><td>Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&amp;nbsp;</td></tr><tr><td>Iris scissors&amp;nbsp;</td><td>Prestige medical</td><td>48,148</td><td>Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5&amp;#8211;2 cm</td></tr><tr><td>Fine tip forceps&amp;nbsp;</td><td>FST</td><td>11254-20</td><td></td></tr><tr><td>Scalpel&amp;nbsp;</td><td>FST</td><td>91003-12</td><td></td></tr><tr><td>Scalpel blade #11</td><td>FST</td><td>10011-00</td><td></td></tr><tr><td>Small spatula&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>2350</td><td></td></tr><tr><td>Filter paper</td><td></td><td></td><td>Any laboratory brand can be used.</td></tr><tr><td>Petri dishes</td><td>Duroplan</td><td>Z231509-1</td><td></td></tr><tr><td>Glass beakers&amp;nbsp;</td><td>SCHOT</td><td>10022846</td><td></td></tr><tr><td>Pasteur pipette&amp;nbsp;</td><td>Maple Leaf Brand</td><td>14672-029</td><td></td></tr><tr><td>Super glue&amp;nbsp;</td><td>LOCTITE</td><td>4091361/1</td><td></td></tr><tr><td>Slicer</td><td>Campden</td><td>7000-smz</td><td></td></tr><tr><td>Ceramic slicing blade</td><td>Campden</td><td>7550-1-C</td><td></td></tr><tr><td>Magnetic heater/stirrer</td><td></td><td></td><td>For heating up the SPS for the procedure</td></tr><tr><td>Electric kettle</td><td></td><td></td><td>For heating up water for temperature control</td></tr><tr><td>Slice recovery chamber + heating unit</td><td>Warner instruments</td><td>&amp;nbsp;BSC-HT +&amp;nbsp; BSC-BUW</td><td>Home-built models may also be used.</td></tr><tr><td>Thermometer</td><td></td><td></td><td>For monitoring SPS temperature during dissection and slicing</td></tr></tbody></table>

slice it hot: acute adult brain slicing in physiological temperature

ここでは、急性脳スライスを調製するためのプロトコルを提示する。この手順は、主に、結果の品質を決定する電気生理学的パッチクランプ実験のための重要な要素である。これは、成熟した動物からの高度髄脳構造を扱う場合は特に、手順を切削中に冷却工程を省略すると、健康なスライスおよび細胞を得るのに有益であることが示されている。上昇した温度のみに依存推測することができ、神経の健康をサポートすることにより、正確なメカニズムは、それはそれを理にかなっていても、可能な限り、スライシングが行われる温度は、温度関連のアーティファクトを防止するために、生理学的条件に近づけるべきである。この方法の別の重要な利点は、手順の単純さと、したがって、短い準備時間である。実証の方法では、成体マウスが使用されているが、同じ手順が若いマウスならびにラットに適用することができる。また、以下のパッチCLアンプ実験は、水平小脳切片上で実行されるが、同じ手順は、他の面と同様に、脳の他の後方の領域で使用することができる。

記載の方法で調製したスライスは、様々な電気および光遺伝学的実験のために使用することができる。 図3A及び図3Cでは、微分干渉（DIC）光学系の下で見、それぞれ水平小脳スライス冠状大脳皮質切片の代表的な例を示している。小脳スライスでは、小脳ニューロンのいくつかのタイプを容易に目標と電気生理学的記録を可能にする、それらの位置及び細胞体形状に?...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

スライスこれホット：生理的温度における急性成人の脳スライス

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

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Neuroscience

26.3K ビュー.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

ビデオ: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

神経科学

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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 ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

ATP または急性脳スライスにおけるセロトニンに対するミクログリアのプロセスの魅力の二光子励起イメージング

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

成人人間の脳からの短期自由浮遊スライス培養

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

成虫マウスおよび老化マウスからの海馬スライスにおける低酸素症の最小化

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

急性マウス脳スライスによる、海馬ネットワーク活動の調査

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

軽度外傷性脳損傷の覚醒閉鎖頭部損傷モデルを用いたシナプス可塑性変化の評価

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

スライスこれホット：生理的温度における急性成人の脳スライス

要約

さらに動画を探す

この動画の章

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

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

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

ATP または急性脳スライスにおけるセロトニンに対するミクログリアのプロセスの魅力の二光子励起イメージング

成人人間の脳からの短期自由浮遊スライス培養

成虫マウスおよび老化マウスからの海馬スライスにおける低酸素症の最小化

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

急性マウス脳スライスによる、海馬ネットワーク活動の調査

軽度外傷性脳損傷の覚醒閉鎖頭部損傷モデルを用いたシナプス可塑性変化の評価

スライスこれホット：生理的温度における急性成人の脳スライス

要約

さらに動画を探す

この動画の章

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

黒質ドーパミンニューロンの細胞体樹状突起のドメインからの亜細胞パッチクランプ記録

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

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

ATP または急性脳スライスにおけるセロトニンに対するミクログリアのプロセスの魅力の二光子励起イメージング

成人人間の脳からの短期自由浮遊スライス培養

成虫マウスおよび老化マウスからの海馬スライスにおける低酸素症の最小化

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

急性マウス脳スライスによる、海馬ネットワーク活動の調査

軽度外傷性脳損傷の覚醒閉鎖頭部損傷モデルを用いたシナプス可塑性変化の評価

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