The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

全てのマウスは、チューリッヒ、スイスの動物福祉法に、州立獣医局の規制に準拠してに従って維持し、取り扱いました。 解剖素材、文化メディア、マイクロ流体デバイスの作製<ol><li>オートクレーブマイクロ解剖ピンセットやハサミ（121℃、滅菌時間：20分）、滅菌容器に保管してください。 </li><li> 37℃でオービタルシェーカー上で24時間、1 M HCl中でそれらをインキュベートすることにより、ガラスカバースリップ（24ミリメートル×24 mm）を滅菌します。 99％エタノールで滅菌蒸留H 2 O 3回とそれらを3回洗浄します。乾燥後、37℃または滅菌流フードの下でカバースリップ。最後に、滅菌を完了するためにUV光（30分）にカバースリップをオートクレーブまたは露出します。カバースリップは、次いで、70％エタノールに保存することができます。 </li><li>滅菌ピンセットを使用してパッケージから慎重AXIS軸索分離デバイ​​スを取り外し、滅菌ペトリ皿に置きます。 </LI&gt; <li>滅菌生検パンチ（φ：1ミリメートル）を使用すると、培養室に対応して（ 図1）で培養するサンプルあたり一つの穴を作成します。 注：それらが適用された圧力によって損傷する可能性があるとして、微小溝に近すぎるパンチしないでください。 </li><li> 70％エタノールでそれらを浸すことにより、AXIS軸索分離デバイ​​スを滅菌します。完全無菌流フードの下で乾燥した後、AXIS軸索分離デバイ​​スとカバーガラス。先に進む前に、3時間以上の間隔をあけてください。 注：不完全な乾燥は、マイクロ流体デバイスの組立不良になります。 </li><li> 6ウェルプレート内で35ミリメートルペトリ皿にまたはウェルに各カバースリップを置きます。 </li><li>絶縁装置とカバーガラスとの間に完全な接着を可能にするために曲がった端部を有するピンセットで慎重にしっかりとカバーガラスとプレスにAXIS軸索遮断装置を配置します。 </li><li>ポリ-D-リジンの各培養室では、ピペットを150μl（滅菌蒸留H 2で0.1 mg / mlのO）。培養チャンバからすべての空気を除去するために、5分間真空下でのマイクロ流体デバイスを置きます。 </li><li>空気は、依然としてチャンバへの再ピペットポリ-D-リジン溶液、チャンバ内見られる場合。 </li><li> 37℃で、ポリ-D-リジンO / Nを有するデバイスをインキュベートします。 </li><li>滅菌蒸留H 2 Oで室を3時間を洗います</li><li> 150（PBS中のシグマアルドリッチ、5μg/ mlの、または無血清培地）μlのラミニン作業溶液とチャンバーを記入し、37℃でO / Nインキュベート。 </li><li> 48ミリリットルたNeurobasal培地、1mLのB-27、100 U / mlペニシリン/ストレプトマイシン、2mMのL-グルタミン、5 ngの/ mlの神経成長因子（NGF）：三叉次のように構成される神経節培養37のための培地50mlを準備、0.25のPMシトシンアラビノシド。 </li><li> 40ミリリットルDMEM-F12 10mlのウシ胎児血清（FBS、最終濃度：20％）を以下のように構成され、歯胚培養37培地50mlを調製し、100 U / mlペニシリン/ストレプトマイシン、2mMのL-グルタミン、150 / mlのアスコルビン酸。 </li></ol> 2.マウスの胚の発生と解剖<ol><li>膣プラグ（膣プラグ：開発0.5、E0.5の胚日）に応じて、胚の年齢を判断し、形態学的基準を介して確認します。このプロトコルのために、私たちは一般的にE14.5-E17.5マウス胚を使用しています。 </li><li>解剖エリアとエタノール70％でステレオスコープを清掃してください。 </li><li>頚椎脱臼を経由して妊娠中の母親を生け贄に捧げます。グリッド上に第一および第二指でマウスの首をブロックし、決定に尾を引きます。 </li><li>下腹部の周りの皮膚を切開し、はさみを使って腹部を開きます。子宮の位置を確認します：妊娠のような後期の段階で、子宮が豊富に腹腔を埋めます。 </li><li>氷上でPBSで満たされたチューブ内の子宮と場所を解剖。氷の上で、組織はいくつかの時間のために残すことができます。機関のガイドラインに従って、母の死体を捨てます</li> &lt;LI&gt;子宮から胚を解剖し、それらの胚体外組織からそれらを解放。氷上でPBS中で胚を置きます。 </li><li>はさみを使用して胚を刎ねると、マイクロ解剖ハサミ（ 図2A）を使用して、ヘッドの残りの部分から下顎を分離します。三叉神経節を損傷することなく、正確に下顎を外します。後者は下顎に近接して局在しているように、それらの偶発的な損傷が可能です。氷上で、冷PBS中で下顎と頭の残りの部分を保持します。 </li><li> TGを分析するには、頭を取ると、以前に冷PBSで満たされた解剖ガラスシャーレ上に置きます。鉗子を使用して、皮膚や頭蓋骨を削除します。終脳とリフトの下に鉗子を配置することにより、次に終脳と小脳を削除します。終脳と小脳が露出頭蓋骨の底部を残して、一緒に反転します。 </li><li> （ 図2Bに示されている）三叉神経節をローカライズ。鉗子を使用してください三叉神経からTGを分離します。ナイフのように解剖針を使用して、三叉神経突起の残党を排除します。冷PBSで満たされたペトリ皿に解剖TGを配置し、氷上で保管してください。 </li><li> 、胚の歯を解剖下顎を配置するには、以前に冷PBSで満たされた解剖ガラスシャーレ上に、頭蓋骨から分離。ナイフのように解剖針を使用して、舌と顎の周囲の皮膚を取り除きます。顎の正中線に沿って切断することにより、左右の半顎を分離します。 図1Cに示すように、歯胚は、簡単に可視化されます。解剖針を使用して歯胚を分離し、非歯の組織の過剰を取り除きます。冷PBSで満たされたペトリ皿に解剖歯胚を置き、氷上で保管してください。 </li></ol> 3.マイクロ流体共培養<ol><li>解剖後、マイクロ流体デバイスからラミニンを削除します。 respecti200μlのチャンバーを埋めますメディアVEの。 </li><li>ピンセットで、（ 図1D）を打ち抜いて作成した穴の中に静かに解剖し、TGと歯胚を転送します。歯胚が浮いていないことを確認し、彼らがカバーガラスに連絡するまで、彼らは沈むこと。 </li><li>培養を37℃、5％CO 2のインキュベーター内のサンプル。 </li><li>培地を48時間ごとに変更します。完全にチャンバーを空にしないでください、培養室に直接ピペットではありません。チャンバの完全な排出はチャンバ内の気泡の形成をもたらします。チャンバ内への直接ピペッティングは軸索損傷をもたらすであろう。これらの問題を回避するために、井戸の外側に向かってピペットポインティング培地を除去し、同様に、チャンバの反対側に位置井戸の側に新鮮な培地をピペット。 </li><li>培養期間中、同時培養を簡単タイムラプス顕微鏡によって画像化することができます。共培養物を10日間維持することができます。 </li><li>培養後のペリオD、室につき1つのウェルにPBSを150μlをピペットし、チャンバーを3回PBSを流すことにより、チャンバーを洗浄します。 </li><li> PBSを除去し、チャンバー当たり1つのウェルに（PBS中）パラホルムアルデヒド150μlの4％をピペットでサンプルを修正。室温で15分間インキュベートします。 </li><li> 3.6で説明したようにPBSで2回チャンバーを洗浄します。 </li><li>さらに分析を進めます。 </li></ol>

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W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature protocols. 1 (4), 2128-2136 (2006).</li><li>Hosmane, S., Tegenge, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll/interleukin-1+receptor+domain-containing+adapter+inducing+interferon-&#946;+mediates+microglial+phagocytosis+of+degenerating+axons.">Toll/interleukin-1 receptor domain-containing adapter inducing interferon-&#946; mediates microglial phagocytosis of degenerating axons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 32 (22), 7745-7757 (2012).</li><li>Delamarche, E., Tonna, N., Lovchik, R. D., Bianco, F., Matteoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+on+microfluidics:+multimodal+analysis+for+studying+celll-cell+interaction.">Pharmacology on microfluidics: multimodal analysis for studying celll-cell interaction.</a> Current opinion in pharmacology. 13 (5), 821-828 (2013).</li><li>Neto, E., Alves, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+neurons+and+osteoblasts:+close+partners+in+a+microfluidic+environment.">Sensory neurons and osteoblasts: close partners in a microfluidic environment.</a> Integrative Biology. , (2014).</li><li>Pagella, P., Neto, E., Jim&#233;nez-Rojo, L., Lamghari, M., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

歯の神経支配のインビトロ研究における前は三叉神経節及び歯の組織または細胞26,28,29の従 ​​来の共培養に基づいていました。これらの研究は主に感覚軸索38にこれらの細胞または組織の魅力的な効果を調査するために行きました。フィールドに大きな進歩をもたらしたが、いくつかの技術的な問題が提起されました。歯胚を培養37の数日後に退化し始...

神経支配は、臓器や組織1,2の発達、ホメオスタシスおよび再生に重要な役割を果たしています。 5 -さらに、神経支配は、幹細胞の増殖、分化及び動員3の調節に関与します。実際には、口腔顔面複合体の組織で実現最近の研究は、上皮前駆細胞は唾液腺6,7の開発と再生中の機能のために副交感神経が必要であることを示しています。 11 -同様に、神経支配が味蕾8の開発及び維持のために必要であることが実証されています。したがって、このような歯のような他の重要な口腔顔面器官および組織の発達における神経支配のまだ無視役割を分析することが重要です。 大人の歯の豊富な神経支配にもかかわらず、ボディ、develo他のすべての臓器や組織とは対照的に、pingの歯は、最も初期の出生後の段階で神経支配され始めます。歯は口腔外胚葉および頭蓋神経堤由来の間葉間シーケンシャルと逆数の相互作用の結果として発生します。これらの相互作用は、それぞれ12、上皮由来エナメル芽細胞とエナメル質や象牙質の形成に関与している間葉由来の象牙芽細胞を生じさせます。 15 -上頸神経節から三叉神経節および交感神経からの感覚神経は大人の歯13を支配します。胚発生時には、神経線維は、現像歯胚に向けて三叉神経節プロジェクトから発せられると次第にそれらを囲むが、彼 ​​らは歯乳頭間葉13に浸透しません。神経線維は、象牙芽細胞分化と象牙質マトリックスの沈着16と相関より高度な発達段階に歯髄間葉を入力してください。歯髄神経支配はCOMPLですすぐに口腔13内の歯の噴火後にeted。 19 -これまでの研究では、様々なセマフォリンおよびニューロトロフィンは、歯牙16の間に神経支配の調節に関与することを明らかにしました。以前の研究は明らかに神経支配が魚20で歯の形成のための前提条件であることを実証しました。より最近の研究は、ソニックヘッジホッグ（SHH）21の分泌を介して感覚神経によって調節されるマウス切歯歯科間充織幹細胞の恒常性を示しました。 24 -それにもかかわらず、歯の開始、開発、再生中の神経支配の役割は、哺乳類22にまだ非常に論争です。 インビボ研究の過多は、様々な動物モデル13,25,26の開発および修復プロセス中に歯の組織の神経支配のパターンに関する重要な情報を提供しています。しかし、これらのほとんどは、アプロ痛みは、神経線維と標的器官と組織との間の相互作用の分子的基礎を強調するために最適ではありません。 29 -共培養は、制御され、隔離された環境26に神経線維と歯の間の相互作用を調査し、操作するための貴重な方法を構成しています。同時に、共培養は、種々の技術的な調整を受けます。 32 -例えば、神経および特定の歯の組織（ 例えば、歯髄、歯小嚢、歯科上皮）は、多くの場合、時間は30の長期間のための組織の生存を保証するために、異なる培地を必要とします。 29 -過去数十年間で、同じ培地を用いた従来の共培養は、感覚神経線維27の口腔および歯の組織を開発するの魅力や反発効果を調査するための非常に短い期間（ 例えば 、2日）のために行われています。しかし、培養期間の延長は、歯の形態形成および細胞分化における神経支配の影響を調査するために、および標的器官内の分岐神経線維の動態を研究するために必要とされます。したがって、非連続的な共培養は、神経、歯の組織の相互作用の研究を実行することがより適切であろう。 マイクロ流体システムは、適切な培養培地中のニューロンと異なる細胞型の共培養を可能にします。それらの標的組織33を含む区画に向かってマイクロチャネルを介して神経細胞体からの軸索の成長を可能にしながら、これらのデバイスでは、歯の組織およびニューロンは、異なる区画に分離されます。マイクロ流体デバイスは、すでにがんや血管新生35で細胞の相互作用へのニューロンとミクログリア34,35との間の相互作用、ならびに細胞を研究するために使用されてきました。さらに、これらのシステムは、ドースの間の相互作用を研究するために使用されていますアル根神経節および骨芽細胞36。 我々は最近、三叉神経節（TG）と歯が時間共培養した場合、マイクロ流体デバイス37での長期間生存することができることを実証しています。また、我々は、異なる発達段階から歯がこれらのin vitroの条件彼らは生体内 37 に示す三叉神経支配で同じ反発や魅力的な効果に維持することを実証しました。このプロトコルは、単純な強力かつ柔軟な方法する共培養神経節/神経と標的組織と制御され、隔離された環境でのこのような相互作用の特定の分子の役割を研究するための情報を提供します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AXIS Axon Isolation Devices</td>
			<td>Millipore</td>
			<td>AX15010-TC</td>
			<td>Microchannels of different lenght are available</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse beta-NGF</td>
			<td>R&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;D Systems) are equivalent</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

神経支配は、臓器や組織の発達、ホメオスタシスおよび再生に重要な役割を果たしています。しかし、これらの現象の根底にあるメカニズムはまだよく理解されていません。具体的には、歯の発生と再生における神経支配の役割は無視されます。 in vivo試験でのいくつかは、様々な動物モデルの開発および修復プロセス中に歯の組織の神経支配のパターンに関する重要な情報を提供しています。しかし、これらのアプローチのほとんどは、神経線維と標的器官と組織との間の相互作用の分子的基礎を強調するために最適ではありません。 共培養物を制御し、隔離された環境で神経線維と歯の間の相互作用を調査し、操作するための貴重な方法を構成しています。過去数十年間で、同じ培地を用いた従来の共培養物は、非常に短い期間に行われている（ 例えば 、2日）感覚神経線維上の口腔や歯の組織の開発の魅力や反発効果を調査します。しかし、培養期間の延長は、歯の形態形成と細胞分化の神経支配の影響を調査するために必要です。 マイクロ流体システムは、適切な培養培地中のニューロンと異なる細胞型の共培養を可能にします。我々は最近、三叉神経節（TG）と歯が長期間生存することができることを実証している時に共培養マイクロ流体デバイスで、それらは、これらの条件で、それらが生体内で示す同一の神経支配のパターンを維持します。 これに基づき、我々は単離する方法を説明し、共培養マイクロ流体共培養system.Thisプロトコルで三叉神経節と歯の細菌の開発は神経節/神経および標的組織培養共同すると役割を研究するために、シンプルで柔軟な方法について説明しますCONTRでこのような相互作用の特定の分子のolledと隔離された環境。

これらの結果は、単離された三叉神経節は、単離された歯の細菌の開発は、マイクロ流体デバイスの他の区画内の長期間持続すること、加えて、マイクロ流体デバイスの一の区画で成長し得ることを示します。異なる培地は、二つの区画で使用されると、2つの区画間マイクログルーブは、現像歯胚に向かって三叉神経節からの軸索の伸長を可能にする。 図3マウスの共培養にお?...

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

マイクロ流体共培養装置の使用歯胚神経支配の開発の分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

analysis-developing-tooth-germ-innervation-using-microfluidic-co

Research

JoVE Journal

Neuroscience

8.2K ビュー.  University of Zurich. Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

ビデオ: Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

神経科学

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

初代ラット皮質ニューロンとグリアのBilaminarの共培養

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

培養皿内の機能モーターユニット：脊髄外植片と筋細胞の共培養

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

マイクロ流体文化·プラットフォーム（MCP）ベースの神経軸索とグリア共培養系におけるグリア相互作用へのニューロンのイメージング解析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

横隔運動ニューロンによってダイヤフラム神経支配の機能的および形態学的評価

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

高解像度ボリューム イメージングのニューロンを蛍光使用排除法によってと専用マイクロ流体デバイス

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

マウスにおける脳幹誘発応答オーディオメトリーにおけるデータ獲得と分析

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.