この研究は、国立精神衛生研究所、国立衛生研究所の学内研究プログラムの部門（DIRP）によって賄われていた。

<ol>
	<li>Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activity+in+early+neuronal+development.">Electrical activity in early neuronal development.</a> Nature. 444, 707-712 (2006).</li><li>Beggs, J. M., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+avalanches+in+neocortical+circuits.">Neuronal avalanches in neocortical circuits.</a> J. Neurosci. 23, 11167-11177 (2003).</li><li>Beggs, J. 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利害の衝突は宣言されません。

1。技術的な問題： <ol><li> 無菌テクニック。多国間環境協定と文化の準備の準備はすべて無菌条件下で層流フード内で実行されます。神経活動に影響を与える抗生物質は、準備および文化プロセス中にいつでも使用されていません。 </li><li> MEA上のMEA。組織の生存上のプラズマ/トロンビン凝固や組織の付着は、プラズマ/トロンビン凝固及び大気への組織暴露の時に必要な時間の間で注意深くバランスを取る必要。大気中に長時間暴露は、組織の変性をトリガしながら短い凝固時間は、MEAからセクションの早期の剥離をリスク。トロンビン溶液の強さは、凝固プロセスの速度を決定するので、それは成功したMEAの表面に、健全な文化を取り付けるための非常に重要なパラメータです。我々は、1000単位（; 1 NIHのユニット= 0.324 ± 0.073 mgの1,000個ロット）を使用して最良の結果を得る。重要なことは、培養中にプラズマ破裂を促進する空間的に不均質凝固におけるプラズマ/トロンビン溶液の結果の不完全混合。これらのプラズマ&quot;の穴は&quot;深刻な文化の健康に影響を与えると微小電極から文化を切り離し、それによって電気生理学的記録の品質を損なう。 MEA /組織の組み立て時の冷却板での作業凝固を遅らせ、プラズマ/トロンビンソリューションおよび組織切片の適切な配置の均質な混合が可能になります。 同様に、凝固のわずか5分後に水没文化に培養チャンバーに、単一液滴の培地を添加することにより、大幅に表面張力に起因する組織の剥離の危険性を低減します。成功する文化がMEAに平らにし、わずかに不完全な組織- MEAの接触または組織の変性（例えば、図1D）のいずれかの主要な徴候なしに、数週間のための健全な成長を示す展開されます。 </li><li> 組織の解剖。マイクロナイフは非常に私たちの解剖のプロセスを改善している。我々は約2 mm幅の&quot;ブレード&quot;を使用してペンチを分割し、メスホルダーでそれらを保持しているから、我々は、ダブルエッジのカミソリの刃（100050〜00 - - 壊れやすいメス刃ファイン科学のツール）を使用してください。それによって大幅に起因する文化の健康を改善する総合的な組織を引っ張るために機械的ストレスを低減するブレードの滑らかな、縦の企業の動きを使用して冠状スライスから解剖組織サンプル。 </li><li>組織の冷却。準備中にスライスし、組織切片の適切な冷却は、文化の成功に不可欠です。我々は、金属板の下側取り付けられたペルチェ素子で構成されたカスタムメイドのコールドプレートを使用してください。ペルチェ素子によって発生する熱は、冷水の血流を介して削除されます。これは、大幅に準備時間を短縮し、準備の各段階で冷却を標準化する（DOLD研究所エンジニアリング131プランテーション博士セガン、TX 78155;（830）560〜1471））。 </li><li> インキュベーターの条件。カスタムメイドのインキュベーター高精度の内部揺動条件では、MEAの上のスライスを育成における当社の成功のための最も重要な鍵だった。 （現在ではない市販）Multichannelsystemsの社内デザインのオリジナルに基づいて、内部のロッキングデバイスには二つの大きな側面の車輪に接続されているトレイで構成されています。ステッピングモータとコンピュータのコントロールは、（速度の揺動角度を揺らし、、と断続的に一時停止します）正確なロッキングの軌跡を可能にする。最終的に、スライス培養は遅い交替の雰囲気と文化メディアに公開する必要があります。伝統的なアプローチは、徐々にそこに長い軸に沿って回転する細いチューブにスライス培養を配置することです。ここで、ゆっくりと回転は回転自体に起因する機械的ストレスを生成しませんし、回転速度が約5の最適な&quot;摂食/呼吸&quot;サイクルを得るために十分に高いです - 10分の時間を。 MEA室、〜2 ccのその合計量、そして組織自体が培地空調に必要な小規模な培養培地の容積のよりコンパクトなインテリアは、重要な課題となっています。 ±〜70 °の角度（サイクルタイム：〜200秒）の間にMEAを揺らすことによって、している液相と大気との間の文化の遷移に応じ揺動速度を減速し、大気への長時間曝露のために極端な角度で揺動を停止文化の生存に必須であった。 </li></ol> 2。神経雪崩を研究するために皮質の文化の発達年齢ラットの大脳皮質からの急性スライスは一般的にPND 0で取られる - 1とMEA上で多くの週間培養する。初期の研究は明らかに単一の皮質の切片培養は、in vitroで数週間後、容易にin vivoでの細胞のクラス18,18-21 のに比較することができる識別可能な細胞の種類と階層構造を維持することを明らかにした。 in vitroの系でこの中の層状組織が ​​便利に視床を研究するために使用されています開発22〜24時の大脳皮質の神経支配、だけでなく、線条体25,26のような皮質下構造を駆動するための。実際には、脳の領域内および組織間の神経接続の形成の特異性は、例えば皮質-大脳基底核回路27から30までのもの、多数の詳細な投影システムを奪還in vitro系における複合体の構築が可能になります。 4の後に- in vitroでの6週間、単一の皮質スライス31および皮質スライスが線条体26または視床32と共培養自発的には、上下を示す典型的にはウレタン麻酔ラット33に生体内で使用されるステート 。これらのアップ状態の微細な時間的な組織では、錐体ニューロンと速いスパイクGABA作動性介在ニューロン31の電気生理学的に成熟したネットワークのネストされたθ-とγ-振動を示すの特徴を負いません。重要なのは、ドーパミンD2受容体刺激の非存在下で、パルブアルブミン陽性皮質介在ニューロンの成熟は、大脳皮質のスライス培養34に約2週間遅れで表示しています。皮質スライスをする投射ドーパミン作動性ニューロンを含む腹側被蓋野（VTA）、共培養しているときにこれらの知見に沿って、ネストされたθ-、β-およびγ-振動の発達時間のコースが生体内でそれに一致さ皮質6。 これらの研究は極めて成熟した高速なGABA作動性抑制に依存すると皮質2,4の浅層に配置されている神経雪崩を、勉強するときに、細心の注意が皮質組織の適切な成熟を ​​確保するために取られる必要があることを示している。神経雪崩が2の時の経過とともに、単一の皮質培養で発生しながら- 5週間4、in vivoでの開発のためにマッチした開発時間の経過を必要とするときに、皮質スライスは、共培養皮質スライスによって、例えば適切なドーパミン受容体の刺激を、必要VTA 6と。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>統合された平面多電極アレイ</td><td>マルチチャンネルシステム（ <a href="http://www.multichannelsystems.com">www.multichannelsystems.com</a> ） ALAサイエンティフィックインスツルメンツ （www.alascience.com） </td><td> <a href="http://www.multichannelsystems.com/products-mea/product-details/products/158/20030ir-ito-wo.html">200/30iR-ITO-w/o</a> </td><td>チタンの硝酸塩（TiN）の電極（直径30mm）は、低インピーダンス（1 kHzで〜1.5kΩの）と優れたワイドバンドレコーディング（ - リングなしW / O）で、その結果大規模な表面を持っている</td></tr><tr><td>チャンバーガラス</td><td> www.aceglass.com </td><td> 7620〜32 </td><td>スレッドのガラスシリンダー</td></tr><tr><td>チャンバーキャップ</td><td> www.aceglass.com </td><td> 7622-114 </td><td>テフロンインサート付きプラスチック製キャップ</td></tr><tr><td> Sylgard 184 </td><td> www.wpiinc.com </td><td> SYLG184 </td><td> 2つの部分から成るシリコーンエラストマー</td></tr><tr><td>ポリ- D -リジン</td><td>シグマアルドリッチ</td><td> P6407 - 5mgの</td><td> γ線照射、凍結乾燥粉末、テストされ培養された細胞。使用前に5ミリリットルの脱イオン水で再構成。 </td></tr><tr><td> Geyの平衡塩類溶液</td><td>シグマアルドリッチ</td><td> G9779 - 500mLに</td><td>滅菌濾過し、培養試験</td></tr><tr><td>鶏血漿</td><td>シグマアルドリッチ</td><td> P3266 - 5mLの</td><td>使用する前に、5 mLの脱イオン水で凍結乾燥、再構成する。 </td></tr><tr><td>トロンビン</td><td>シグマアルドリッチ</td><td> T6634 - 1,000個</td><td>ウシ血漿、凍結乾燥粉末の形から。 </td></tr><tr><td>馬血清</td><td>シグマアルドリッチ</td><td> H1138 - 100mLの</td><td>ドナーの群れ、熱不活性化、細胞培養テスト済み</td></tr><tr><td>基礎培地イーグル</td><td>インビトロジェン</td><td> 21010-046 </td><td> 1 ×、500ミリリットル - （+）アールの塩、（ - ）L -グルタミン）、 </td></tr><tr><td>ハンクス緩衝生理食塩溶液</td><td>インビトロジェン</td><td> 24020-117 </td><td> 500ミリリットル - （+）マグネシウム、（+）、カルシウム、W /フェノールレッド） </td></tr><tr><td>チャンバースライド</td><td>無菌ラボテックチャンバースライドW /カバー（Nunc社）、 </td><td> 177429 </td><td></td></tr><tr><td>ウリジン</td><td>シグマアルドリッチ</td><td> U3003 </td><td></td></tr><tr><td> ARA - Cシトシン-β- D -アラビノフラノシド</td><td>シグマアルドリッチ</td><td> C6645 </td><td></td></tr><tr><td> 5 - フルオロ-2&#39; - デオキシウリジン</td><td>シグマアルドリッチ</td><td> F0503 </td><td></td></tr></tbody></table>

multi-electrode array recordings of neuronal avalanches in organotypic cultures

皮質はさらに、特定の入力またはモーターの出力がない状態で、自発的にアクティブになります。開発中は、この活動は、皮質の細胞型と神経の接続1の形成の遊走と分化に重要である。成熟した動物では、継続的な活動は、過去と感覚刺激がシームレスに将来の行動を計算するために統合された動物の現状を反映している。このように、継続的な、すなわち自発活動の組織を明確に理解することは、皮質の機能を理解することが前提条件です。 数々の記録技術は、皮質における継続的な活動が、その個々の活動一過性に細胞外微小電極で局所電場電位（LFP）で検出することができる大規模なイベントに合計、または脳波（EEG）、脳磁気図（MEGの多くのニューロンから構成されていることを明らかに）、そして機能的磁気共鳴画像（fMRI）からBOLD信号。メゾスコピックスケール（ニューロン数千）での高時間および空間分解能で神経集団の活動を研究するときにLFPは、現在の選択の方法です。細胞外微小電極では、局所的にマイクロボルトから数百までのLFPの急激なたわみの空間的neighboredニューロンの結果の活動を同期化。微小電極のアレイを使用する場合は、このような偏向の団体は、便利な空間と時間で監視することができます。 神経雪崩は脳2,3の継続的なニューロン活動のスケール不変時空間構成について説明します。 in vitroで 4,5 で 、麻酔ラット6のin vivoで 、と目を覚ましサル7に設立されたとして、それらは皮質の浅層に固有のものです。重要なことは、理論的実証的研究2,8-10両方の神経雪崩の情報伝達と情報の処理を最適化する皮質の絶妙バランスの臨界状態のダイナミクスを示すことを示唆している。 彼らは正確に制御された条件下で雪崩の活動の安定的な録音を可能と神経雪崩の開発、保守、および規制のメカニズムを研究するためには、in vitroでの製剤で 、非常に有益です。現在のプロトコルは、平面、統合された微小電極アレイ（;も11-14を参照してMEA）上に成長した器官皮質の培養、すなわちスライス培養で浅層の開発を活用することによって、in vitroで神経雪崩を勉強する方法について説明します。

Watch this Scientific Journal Video about Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures at JoVE.com

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

皮質における臨界​​状態のダイナミクスを示す神経雪崩、すなわちスケール不変時空間アクティビティのバーストを、勉強するための確実な方法。雪崩は、正確に制御された条件下で平面統合マルチ電極アレイ（MEA）との活動の長期的な測定を可能にする培養皮質の浅層の開発に自発的に出現する。

器官培養における神経雪崩のマルチ電極アレイの録音

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

multi-electrode-array-recordings-neuronal-avalanches-organotypic

Research

JoVE Journal

Neuroscience

26.3K ビュー.  National Institute of Mental Health. 皮質における臨界​​状態のダイナミクスを示す神経雪崩、すなわちスケール不変時空間アクティビティのバーストを、勉強するための確実な方法。雪崩は、正確に制御された条件下で平面統合マルチ電極アレイ（MEA）との活動の長期的な測定を可能にする培養皮質の浅層の開発に自発的に出現する。

ビデオ: Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

鋤鼻上皮の多電極アレイ録音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

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 ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

マウスの電気生理学的記録のための設計と超軽量重量の作製、調整可能なマルチ電極プローブ

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

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 ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

マルチ電極アレイ上に成長させた器官脊髄共培養における機能的再生の調査

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

マウス脳スライスの準備中に発作のような活動の直流刺激とマルチ電極アレイ録音

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

オルガ鼻記小脳スライス培養におけるプルキンジェ細胞生存

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...