著者らは、閉じ込め構造とdottの開発に技術的なサポートのためにジョルジオCarliniに感謝します。原稿の徹底した見直しのためのオルネラLoBrutto。これらの結果につながる研究は、欧州連合（EU）の7 番目のフレームワークプログラムから資金提供を受けた（ICT-FET FP7 / 2007年から2013年、FETヤング探検方式）付与契約284772脳弓下（www.brainbowproject.eu）。

実験プロトコルは、DL 1992分の116とし、ジェノバ大学（Protののガイドラインにより応じた保健のイタリア語省によって、ヨーロッパの動物保護法制（63分の2010 / EU）によって承認された。N. 13130​​、月を2011）。すべての努力は、プロジェクトのための動物の数を減少させ、その苦痛を最小限にするために行われました。 材料と支持体の作製<ol><li> CNC（コンピュータ数値制御）フライス盤を用いて、PDMS（ポリジメチルシロキサン）の制約を構築するための金型を作成します。このような金型は、ポリテトラフルオロエチレンで中心筒（PTFE）とポリカーボネートで実現されます。この材料は、PDMSが硬化した後にマスクを容易に抽出することができます。 注：金型の設計は、コンピュータ支援設計（CAD）によって行われ、その後、CNCフライス盤に配信されています。 </li><li> PDMSエラストマーを準備します。 PDMSは、有機ポリマーです。これは、二つの要素から構成されています、硬化剤及びポリマー。 1：10の体積比で混合して、硬化剤の一部とポリマー9部。ペトリ皿に二つの成分を入れ、気泡を除去するために10分間真空チャンバ内で混合を振るとインサート。 PDMSの3グラムは、つの制約のために十分です。 </li><li> PDMS制約を構築します。成形機にPDMS材料を挿入し、120℃で30分間オーブンに入れて。 PDMS制約はそれぞれ22.0と3.0ミリメートルの外部および内部直径650ミクロンの高さの理想的な円筒の形状を有しています。 </li><li>メッキ前日、カップル実体顕微鏡下でピンセットを用いた微小電極アレイ（MEAの）の活性領域にマスクと120℃のオーブンで、PDMSマスクを滅菌します。 </li><li>円錐バイアル中で2時間、70％エタノールおよびEVを回転させ、ガラスビーズ（42.3±1.1ミクロンの認定平均直径40±2μmの呼び径）滅菌すべてのマイクロビーズを公開するERY 30分。 </li><li>バイアルからのエタノール溶液を除去し、滅菌水でマイクロビーズを2回すすいでください。 </li><li> 0.05μgの/ mlのポリ-D-リジンおよびラミニンの混合溶液の24μlの（直径3.0ミリメートルに等しい）PDMSマスクによって画定MEA領域の中央部を条件付けます。 </li><li>コー​​トは0.05μg/で接着タンパク質、ラミニンおよびポリ-D-リジンとのマイクロビーズはmlであり、安定的かつ長期的な神経ネットワークを得るために、37℃のインキュベーター内で一晩置いておきます。 </li><li> MEAのガラス表面からガラスマイクロビーズの両方から接着因子を削除します。吸引するピペットでコーティング溶液の約95％とは、MEAの表面に滅菌水の小さなVOLUME-24μl-を適用します。吸引し、再び水の95％以上と細胞をプレーティングする前に1時間層状フードの下で、MEAの乾燥をしてみましょう。 注：マイクロビーズの場合は代わりに、コーティングを吸引ニューロンはメッキされたガラス表面を調整するために、滅菌水や、B27などの基礎培地とサプリメント第1と第一の洗浄を、ソリューションとなります。マイクロビーズを乾燥させないでください。バイアル内部の培養液中の懸濁液のままにしておきます。 </li><li>彼らは均一な層を形成する自己集合する膜インサート（細孔直径0.4ミクロン）とのマルチウェルプレートにピペット-200μl-、処理されたマイクロビーズの懸濁液（約32,000）を用いて、配布します。 </li><li>培地0.5mlを有する膜の各ウェルを記入し、インキュベーターに入れて（T = 37.0°Cの 、CO 2 = 5％）ニューロンは（3.2）メッキすることの準備が整うまで。 </li></ol> 2.胚の解剖と組織の解離<ol><li>中（点灯午前7時 - 午後7時）、通常の明暗スケジュールの下で一定温度（22±1℃）および相対湿度（50％）でハウス大人雌ラット（200〜250グラム）動物施設。食料と水は自由に利用可能であることを確認してください。 </li><li> 3％のイソフルランを使用して、開発の18日（E18）の後に妊娠したメスのラットを麻酔。その後、頸椎脱臼により動物を生け贄に捧げます。 </li><li>各ラットの胚から海馬を削除およびCa 2+およびMgのない氷冷ハンクス液2+に入れます。日目に開発海馬および皮質組織の18は非常に柔らかく、小片に切断する必要はありません。さらなる詳細は18,19を見ることができます。 </li><li> 37℃で18〜20分間のDNAse 0.05％を含むトリプシン/ハンク溶液の0.125％で組織を解離します。 </li><li>パスツールピペットを用いて上澄み液を除去し、5分間、10％ウシ胎児血清（FBS）を含む培地を加えることによって、酵素消化を停止します。 </li><li> FBSで培地を除去し、そのサプリメント、1％L-グルタミン、ゲンタマイシンを10μg/ mlの培地で1回洗浄します。パスツールピップで再びメディアを削除しますETTEおよび10μg/ mlのゲンタマイシン、そのサプリメント、1％L-グルタミンを有する成長培地の少量（500μL）で再度補充。 </li><li>細胞の乳白色の懸濁液が明らかになるまで、狭いパスツールピペットで機械的に組織ペレットを解離します。これは、細胞懸濁液を遠心分離する必要はありません。 </li><li> 2.0 mlの最終容積を得るために、増殖培地で細胞懸濁液の少量を希釈します。血球計数器室で得られた細胞濃度を数えます。 /μL600-700細胞の所望の細胞濃度を得るためには5：1で、この濃度を希釈します。 </li></ol> 3.セルメッキ<ol><li> PDMSの制約によって定義され、MEAの活性領域上に約2,000細胞/ mm 2の密度でプレートの細胞は、2次元神経回路網を作成します。 注：各海馬は約5×10 5細胞を含む、（単一胚のための1×10 6）。 6×10の合計量を取得するために6胚を解剖6 2ml中の細胞、および/μlの3000細胞の推定濃度。 /μlの所望の細胞濃度およびプレート約600-700の細胞を得るために、5：1で、この濃度を希釈します。 PDMSの制約によって区切られたMEAの面積は約7.065ミリメートル2、播種した細胞600個/μL×24μL= 14,400細胞/ mm 2で2038細胞の最終濃度の合計数である場合。 </li><li> 37℃の加湿CO 2雰囲気下（5％）でインキュベーターにMEAデバイスを配置します。 </li><li>三次元培養の構築のための予備的なステップを完了するために、マルチウェルプレートの内側に位置するマイクロビーズ単層の表面に600〜700個/μlの（約100,000細胞）の細胞濃度で懸濁液の160μlのを配布します。 37℃の加湿CO 2雰囲気下（5％）でインキュベーターでマルチウェルプレートを置きます。 </li></ol> 4. 3D神経ネットワークの構築<ol><li> 6-8時間、めっき後、約30〜40マイクロリットルの容量に設定されたピペットを用いて、PDMSの制約によって区切られた領域の内側に非常に慎重に、マルチウェルプレートからサスペンション（ニューロンとマイクロビーズ）を転送します。各転送後、自己集合六角形のコンパクトな構造にするマイクロビーズを可能にするために、約半分の分を待ちます。 </li><li>すべての層が堆積され、自然に組み立てられると、媒体の約300μlのPDMS制約で区切られた領域の上部の大滴で補充します。 </li><li>インキュベーター内（T = 37.0をO℃、CO 2 = 5％）をMEAに結合された三次元構造を入れ48時間のサプリメントで成長培地培養物の最終体積（約1ml）を添加する前に。 注：メンブレンインサート（3万）とMEAデバイス（6000）上に単層のビーズの数をマルチウェルプレート上のビーズの総数を考えてみましょう。得られた三次元構造は、MIの5 層から構成されていますcrobeads細胞。 3D神経ネットワークが幾何学的に完全ではないことを考慮すると、結果として得られる3次元構造は、5-8の層で構成することができます。 </li><li>メッキ後の日は、慎重に、MEAのリングの内側に培地（約900μL）の最終容量を追加します。 4-5週間、37℃の加湿CO 2雰囲気下（5％）での3次元培養を維持します。週に一度培地の半分を交換してください。 </li></ol> 5.共焦点顕微鏡の取得<ol><li>ホルダーに顕微鏡のステージに、生物学的サンプルを修正。正しい帯域幅発光フィルターをキャプチャするイメージごとに、順番にPMTの（-0.3％）画像を取得することで、レーザー（アルゴン、496から555 nm）で、スキャン速度（400 Hz）を設定し、ゲインとオフセット飽和を回避し、信号対雑音比を増加させます。結果セクションは 、図4の画像を取得するために使用される値を報告します。 </li><li> Z -stackシーケンスの取得については、モミSTは、上部とサンプルの底層のz位置の値を選択し、取得を行うためのステップサイズを選択します。 </li></ol>

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The authors declare that they have no competing financial interests.

この作品では、3Dで構成された試験管プラ ​​ットフォームにおける新規の実験では、ネットワークの電気生理学のためのMEAに結合された神経細胞培養は、提示された設計しました。 Z -軸に沿って神経突起伸長を可能にするための足場としてのマイクロビーズの使用は、平面MEAと統合するように調整されています。このように、有効かつ信頼性の高いin vitroでの3D?...

微小電極アレイ（MEAの）に結合された試験管 2次元（2D）のニューラルネットワークではニューロンのダイナミクスと基礎接続の間の相互作用を研究するために採用した金標準実験モデルをarethe。開発中に、ニューロンは、よくdefinedspatio-temporalpatternsof活性を示す複雑なネットワーク1,2（ すなわち 、バースト、ネットワークバースト、ランダムスパイク活動を）再作成します。 MEAがネットワークレベルで発現ダイナミクスの詳細な調査を可能にする、多くのサイトから（数十から数千の微小電極に）電気生理学的活動を記録。また、解離文化の使用は、設計、ネットワークを設計することが可能ですになります。これは、異種の神経ポップの存在、このように記録された電気生理学的活性および細胞密度3、モジュール4,5度のようなネットワーク組織のパラメータとの間の機能の関係を理解することは容易ですulations 6などがしかし、解離培養細胞上のすべてのin vitroでの研究は、2D神経回路網に基づいています。このアプローチは 、in vivo（本質的に3次元、3D）システムに関してoversimplificationsにつながる：2Dモデル、細胞体と成長円錐（I）における平坦化され、軸索、樹状突起の伸長は、全方向7に拡散することはできません。 （ii）の2D インビトロネットワークの展示は、ネットワーク8のニューロンの大部分を伴う活動を破裂によって支配電気生理学的ダイナミクスをステレオタイプ。 最近、 インビトロの3次元構造を可能にするために開発されてきた別の解決法は、神経ネットワークを解離しました。一般的な考え方は、ニューロンが、3Dの方式で成長することができます足場を作ることにあります。そのような足場は、高分子ゲル、固体多孔質マトリックス9-13で実現することができます。ポリマーの機械的性質を利用することにより、THES細胞内を埋め込むことが可能ですニューロスフェア11の3次元培養の均一なブロックを定義することにより、電子構造。このアプローチの主な特徴は、神経球9,12の剛性機械的性質です。しかしながら、これらの材料は、多孔性が制限されており、それらは、マトリックス内部の細胞移動を保証しません。この欠点を克服するために、可能な解決策は、「ユニット」モジュールにマトリクスをスライスすることからなります。残念ながら、粒子のサイズおよび形状の多様性は、通常、層状構造への充填を妨げる可能性があります。 7では 、カレンと共同研究者は、生物活性、細胞外マトリックスベースの足場内のニューロンおよび/ ​​またはアストロサイトで構成された3D神経構造物を設計しました。このような操作された神経組織は、3Dマイクロ環境内の神経生物学的応答を研究し、操作するためにインビトロの研究では許可 。このモデルでは、細胞外マトリックス（ECM）および/またはヒドロゲル足場（500-600ミクロン厚）全体に分散ニューロンおよびグリア細胞から成っていました。この共同で5,000細胞/ mm 3 - ndition、最適な細胞生存率（90％より大きい）が約3750の最終密度で細胞をプレーティングすることによって発見されました。そのような濃度値がマウスの脳皮質の細胞密度が90,000細胞/ mm 3 14でのin vivo条件でのものよりもはるかに低いことに留意しなければなりません。この制限を克服し、Pautot 15は、細胞密度とのネットワーク接続は、ネットワークのリアルタイムイメージングを可能にしながら、インビボ条件に似ているように制御される三次元インビトロシステムを実現するために、同僚。実際に、この方法は、培養神経細胞は、 シリカマイクロビーズ上で増殖することができる解離概念に基づいています。これらのビーズは付着する神経細胞体のためと、成熟した、成長し拡張し、他のニューロンへのシナプスの連絡先を定義するために、彼らのarborizationsための十分な大きさの成長表面を提供します。この方法は、FOする単分散ビーズの自発的な組立特性を利用RM 3Dは、異なるビーズ上のニューロンの間で拘束された接続性を有する異なる層のニューロンの明確なサブセットを含む六角形の配列をレイヤード。この方法で達成された細胞密度は約75,000細胞/ mm 3でした 。 最近、我々は、MEAを16にPautotの方法を適応している。得られた結果は、3D電気生理学的活性は、2Dネットワークによって発現される1以上の活動の広いレパートリーを提示することを示しています。 3D成熟した文化がネットワークバーストと、ランダムなスパイク活動の共存の両方強化ダイナミックを示します。同様に、唐-Schomerと同僚17は、数ヶ月のために、in vitroでの一次皮質培養を維持シルクプロテイン系の多孔質足場を実現し、タングステン電極を用いて電気生理学的活性を記録しました。 この作品では、多国間環境協定に結合された3次元神経回路網を構築するための実験手順を説明します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25050-014</td>
			<td align="left">0.125% diluted 1:2 in HBSS w\o CA++, MG++</td>
		</tr>
		<tr>
			<td>DNAase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td>0.05% diluted&#160; in Hanks solution</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco Invitrogen</td>
			<td>21103049</td>
			<td>culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco Invitrogen</td>
			<td>17504044</td>
			<td>2% medium supplent</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F-2442</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Glutamax-I&#160;</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G.1272</td>
			<td>5mg/liter</td>
		</tr>
		<tr>
			<td>Poly-Dimethyl-Siloxane (PDMS)</td>
			<td>Corning Sigma</td>
			<td>481939</td>
			<td>curing agent and the polymer</td>
		</tr>
		<tr>
			<td>Micro-Electrode Arrays</td>
			<td>Multi Channel Systems (MCS)</td>
			<td>60MEA200/30-Ti-pr</td>
			<td>MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 &#181;m, respectively; plastic ring without thread</td>
		</tr>
		<tr>
			<td>Microbead</td>
			<td>Distrilab-Duke Scientific</td>
			<td>9040</td>
			<td>&#160;1gr Glass Part.Size Stds 40 &#181;m</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Costar Sigma</td>
			<td>CLS 3413</td>
			<td>multiwell plates with membrane insert (6.5 mm diameter porous 0.4 &#181;m)</td>
		</tr>
		<tr>
			<td>HBSS w\o Ca++ ,Mg++&#160;</td>
			<td>Gibco Invitrogen</td>
			<td>14175-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Buffer Solution&#160;</td>
			<td>Sigma&#160;</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon</td>
			<td>Sigma</td>
			<td>430935-5G</td>
			<td>polytetrafluoroethylene</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Sprague Dawley</td>
			<td>Wistar Rat</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscopy Upright</td>
			<td>Leica</td>
			<td>TCS SP5 AOBS</td>
			<td></td>
		</tr>
		<tr>
			<td>20.0x0.50 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>40.0x0.80 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>25.0x0.95 WATER objective</td>
			<td>Leica</td>
			<td>HCX IRAPO L</td>
			<td></td>
		</tr>
	</tbody>
</table>

interfacing 3d engineered neuronal cultures to micro-electrode arrays: an innovative in vitro experimental model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

この実験手順では、関連する役割が3Dニューロンネットワークの成長のための機械的な足場を定義するマイクロビーズで再生され、図1A及びBは、マイクロビーズの配置を表示する（40±2μmの公称直径;の認定平均直径平面で42.3±1.1ミクロン）。このような構造の特殊性は、マイクロビーズが自発的にコンパクト六角形の幾何学（ 図1BおよびC）を定義する自己集合すると...

Watch this Scientific Journal Video about Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model at JoVE.com

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

微小電極アレイへの3Dエンジニア神経文化のインタフェース：革新的な<em&gt;インビトロ</em&gt;実験モデル

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

interfacing-3d-engineered-neuronal-cultures-to-micro-electrode-arrays

Research

JoVE Journal

Neuroscience

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

9.9K ビュー.  University of Genova. In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

ビデオ: Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

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 ...

神経科学

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 ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker、眼球運動解析への革新的な自動化されたアプローチ

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

動物を振る舞う、覚醒中のシングルユニット記録のためにマイクロワイヤの組織化された配列を移植するための手順

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脳顆粒ニューロンの遺伝子操作<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;ニューロンの形態と移行を研究するために、

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

地中マイクロ電極アレイと非折り畳み海馬準備神経活動の伝播

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

生体内ニューロン回路接続を模倣するためのインビトロウェッジスライスの準備

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...