NINDS、米国心臓協会、老化研究のためのアメリカ連合、ダイムの月によって資金を供給

1。準備開始する前に、必要な手術器具、解剖や文化、メディア、および7日齢のラットまたはマウスの仔を組み立てる。プロトコルは、マウスやラットの準備のための同じです。 （ⅰ）材料および機器<ul><li>営業ハサミ（ストレート長さ6&quot;、猫＃RS6818、Roboz、ゲイサーズバーグ、MD） </li><li> Microは、ハサミ、解剖（長さ4&#39;&quot;、猫＃RS5910を。Roboz、ゲイサーズバーグ、MDは） </li><li>炭素鋼デュモンピンセット（猫＃RS - 5045.Roboz、ゲイサーズバーグ、MD） </li><li>改質ガラス&quot;転送&quot;ピペット（削除されたパスツールピペットの下部とエッジのスムージング） </li><li> ACLARプラスチックフィルムが正方形で（ハネウェル、＃812071、ポッツビル、ペンシルバニア州）&quot;× 2で&quot;2にカット</li><li>の3でのスポンジ4（ケンドール、猫＃3157、マンスフィールド、マサチューセッツ州）カバー</li><li> 30ミリメートルMillicell膜インサート（0.4μmの、ミリポア、ベッドフォード、MA） </li><li>両刃のカミソリの刃を持つMcllwainのティッシュチョッパー（マッキルウェーン組織チョッパー、ビブラトーム社、セントルイス、MO） </li><li> CCDカメラと画像解析ソフトウェアとの倒立顕微鏡</li></ul> （ⅱ）解剖媒体（DM、pH7.3に1リットル） <table border="0"><tbody><tr><td>ハンクス平衡塩類</td><td> 1バイアル（95.2 g/1L）（シグマ：H2387） </td></tr><tr><td> NHCO 3（4.2 mM）を</td><td> 350mgを</td></tr><tr><td> HEPES（10mM）を</td><td> 2.83グラム</td></tr><tr><td>グルコース（33.3 mM）を</td><td> 6.0グラム</td></tr><tr><td>ペニシリン/ストレプトマイシン（100倍） </td><td> 10mLの</td></tr><tr><td> BSA（0.3％） </td><td> 300mgの</td></tr><tr><td>し、MgSO 4 · 7H 2 O </td><td> 1.44グラム</td></tr></tbody></table> （ⅲ）培養培地（400 mL）に<table border="0"><tbody><tr><td> MEM（Invitrogen社11575〜032） </td><td> 200 mLの</td></tr><tr><td> ***ハンクのバランス塩（インビトロジェン24020〜117） </td><td> 100mLの</td></tr><tr><td>ウマ血清（Invitrogen社26050〜070） </td><td> 100mLの</td></tr><tr><td> HEPES緩衝液（1 M、インビトロジェン15630〜080） </td><td> 5 mLの</td></tr><tr><td>ペニシリン/ストレプトマイシン（100倍） </td><td> 4 mLの</td></tr><tr><td colspan="2"> ***培養液を加える前に、500mLのハンクのバランス塩に12.8グラムのブドウ糖を追加</td></tr></tbody></table> （IV）動物 7日齢ラットまたはマウスの出生後の仔、実験ごとに一般的には1リットル。 2。文化の日に<ol><li>すべての解剖ツールとACLARフィルムは開始前に30分間70％エタノールに浸漬する必要があります。解剖フード、ブレード、および組織のチョッパーの表面をエタノールで消毒する必要があります。 </li><li>ウェルあたり1mLの培養液を追加し、各ウェルのミリポア透過膜を挿入し、いくつかの（3-4）6ウェル組織培養プレートを準備します。場所、6ウェルのスライスを転送する準備が整うまで、37℃インキュベーターでプレート。 </li><li> DMと氷の上で場所を持ついくつかの（4-6）60 mmのプレートを準備します。 </li><li>急速にはさみで首を切ると70％ETOHに浸し、そして迅速に頭蓋骨のsaggital切開と脳を公開。慎重に冷たいDMですぐに脳と場所を削除します。二つの半球に分離して2-3秒のカバーのスポンジに各半球内側を下に置きます。 </li><li>そっとティッシュチョッパーでACLARフィルムの正方形と場所の上に半球を持ち上げ、配置、350μmのセクションでcoronally脳をカット。終了したら、そっと冷たいDMの区分け半球と水没してAlcarの正方形を取る。 </li><li>解剖顕微鏡を用いて、海馬切片を分離。ラットの場合は、尾側海馬への吻側またがる5つのセクションがあるはず、とマウスは3-4のセクションがあるはずです。 </li><li> 37℃組織培養インキュベーターから細胞膜を挿入した6ウェルプレートを取り出してください。ガラス転移のピペットを使用して透過性の膜の上に個別に海馬切片を移す。あまりにも多くのDMがメンブレン上に放出されている場合は、パスツールピペットでDM過剰を取り除く。それぞれの膜に5つの海馬スライスを置きます。条件ごとに少なくとも3ウェル、または15のスライスを許可する。 </li><li>毒性の実験を開始する前に12-14日のために5％CO加湿インキュベーター中37℃で培養プレートをインキュベートする。培養液は週に2回変更することができます。 </li></ol> 3。スライス培養における海馬神経損傷を誘発し、定量化<ol><li>培養中の12-14日後、培養培地に5μg/ mLのヨウ化プロピジウム（PI）を追加します。このステップでは、基底細胞死の可視化と定量化が可能になります。 </li><li>翌日、PIを定量化各海馬スライスのCA1サブリージョン（および/ ​​またはCA3や歯状必要に応じて）の蛍光と取得は、基底細胞の死を（時間と呼ばれる= 0時間、またはT 0）を表すPI蛍光測定値を意味する。画像取得と定量化のために、我々は、オープンラボのソフトウェア（即興、英国）を使用しますが、他の画像取得と解析ソフトウェアは、蛍光強度を定量化するために使用することができます。 </li><li>すぐにT 0 PI蛍光の画像取得の後、ご入金方法（我々は神経毒性2,5を誘導するために一時間に10μMNMDA、酸素-グルコース欠乏、またはLPSを使用している）ことによって神経損傷を誘発する。毒性刺激後、5μg/ mLのPIを含む新しい培地と培地を交換して一晩のスライスを維持する。 </li><li> 3日目に、PI 24における実験条件の蛍光、またはT 24を意味し定量化する。 </li><li>この画像取得後、直ちに培地を変更し、10μMNMDAと5μg/ mLのPIと最大の神経細胞死を達成するために一晩インキュベートするスライスを追加します。 </li><li> 4日目に、T maxとして定義されている最大の神経細胞死のPIの蛍光を、意味し定量化する。次のようにパーセントの細胞死を計算します（T 24 - T 0）/（T MAX - T 0）。 </li></ol> 4。代表的な結果<img src="/files/ftp_upload/2106/2106fig1.jpg" alt="図1" /> 図1（）海馬と、その後の実験的な傷害と画像取得の世代のためのタイムライン。 （B）basally海馬スライスの代表画像（T 0）、24時間、10μMNMDA（T 24）に1時間暴露後の、そして最大の神経傷害を得るためにNMDAさらに24時間または10μM（T max）の後に。

<ol>
	<li>Stoppin, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-173 (1991).</li><li>McCullough, L., Wu, L., Haughey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Function+of+the+PGE2+EP2+Receptor+in+Cerebral+Ischemia.">Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia.</a> J Neurosci. 24 (1), 257-257 (2004).</li><li>Czapiga, M., Colton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+microglia+in+organotypic+slice+cultures.">Function of microglia in organotypic slice cultures.</a> J Neurosci Res. 56, 644-644 (1999).</li><li>Newell, D. W., Barth, A., Papermaster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+non-glutamate+receptor+mediated+toxicity+caused+by+oxygen+and+glucose+deprivation+in+organotypic+hippocampal+cultures.">Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures.</a> J Neurosci. 15, 7702-7702 (1995).</li><li>Wu, L., Wang, Q., Liang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+Effects+of+prostaglandin+recetor+signaling+on+neuronal+survival.">Divergent Effects of prostaglandin recetor signaling on neuronal survival.</a> Neuroscience letter. 421, 253-253 (2007).</li><li>Hailer, N. P., Jarhult, J. D., Nitsch, R., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+in+vitro:+Analysis+of+morphology+and+adhesion+molecule+expression+in+organotypic+hippocampal+slice+cultures.">Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures.</a> Glia. 18, 319-319 (1996).</li></ol>

利害の衝突は宣言されません。

実験を複製するときに再現性と一貫性のある結果を確保するために実行するには、2つの重要なポイントがあります。最初に、実験前に12-14日のために海馬スライスの年齢を聞かせすることが重要です。スライスの分離により、大幅なミクログリアの応答がある、とミクログリアをin vitroで 3,6 で 10日目で発生安静分岐し状態に戻ると、しばらくの間有効なまま。第二に、P...

the organotypic hippocampal slice culture model for examining neuronal injury

器官海馬スライス培養では、海馬の基本的なアーキテクチャとコンポジションが相対的に1を保存されている神経細胞傷害のメカニズムを調べるためにin vitro法です。器官培養系は、神経細胞のアストロサイトやミクログリアの効果の検査は可能ですが、ex vivoでの準備として、血流、または末梢炎症性細胞の動員の影響には対応していません。そのために、この培養法は、頻繁に海馬の錐体ニューロンに興奮毒性と低酸素障害を調べるために使われるだけでなく、炎症反応を調べるために使用されています。ここで我々は、出生後の齧歯類の脳から海馬スライス培養を生成する神経細胞傷害を誘発する有毒な刺激を投与し、海馬神経細胞死を測定すると定量化するための方法を説明します。

Watch this Scientific Journal Video about The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury at JoVE.com

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

organoptypic海馬スライス培養モデルは、<em&gt; in vitroで</em&gt;モデルは、パラダイムの様々な神経損傷を調べるために使用。この記事では、スライス培養を生成し、神経損傷を定量化するための方法を説明します。

神経損傷を調べるための器官型海馬スライス培養モデル

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

the-organotypic-hippocampal-slice-culture-model-for-examining

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JoVE Journal

Neuroscience

19.0K ビュー.  Stanford University School of Medicine. organoptypic海馬スライス培養モデルは、 in vitroでモデルは、パラダイムの様々な神経損傷を調べるために使用。この記事では、スライス培養を生成し、神経損傷を定量化するための方法を説明します。

ビデオ: The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

末梢神経突起伸長のリアルタイムイメージングのためのGFP発現マウス胚の器官型スライス培養

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

神経科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海馬切片培養

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

出生後の脳における神経細胞の移動の高分解能タイムラプスイメージングのための器官型スライスアッセイ

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚腹側中脳の器官型スライス培養：ドーパミン作動性ニューロンの発達を研究するための体制 in vitroで</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

器官切片培養におけるプルキンエ細胞の樹状突起の形態分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

器官型海馬スライスにおけるペア全細胞記録

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Entorhino海馬器官型スライス培養における神経血管リモデリングの解析

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度初代海馬ニューロンの文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

行動するマウスにおける海馬シータ振動の光同調

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...