我々 はまた、重要な読書のガブリエルの手を感謝します。

すべての実験は、大阪大学・日本神経科学学会動物愛護委員会によって確立されたガイドラインに従って行われました。
1. 脊髄共培養系視床と大脳皮質の
メモ: 手順については、オリジナルの出版物7,8,13を参照してください。すべてのプロシージャは、無菌条件の下で行わなければなりません。Sprague-dawley (SD) ラットは、神経細胞の培養に使用されます。
<ol><li>
試薬の調製 注:次の試薬のボリュームは、1 匹のラット脳です。<ol>
<li>
変更された N2 サプリメント x 10 の準備13: インスリン (50 μ g/mL)、プロゲステロン (200 nM)、ヒドロコルチゾン (200 nM)、亜セレン酸ナトリウム (300 nM)、トランスフェリン (1 mg/mL)、プトレシン (1 mM)、グルコース (60 mg/mL)</li>
		<li>
準備血清を含む培(10 mL): 85 %dmem/f12 キー、変更 10 x N2 サプリメントは 10%、5% ウシ胎児血清 (FBS)</li>
		<li>
準備無血清培(20 mL): 90 %dmem/f12 キー、10% 変更 10 x N2 サプリメント</li>
		<li>ラット尾コラーゲン溶液0.5 mL を準備します。簡単に言えば、無菌条件下でのラットの尾からの柔らかい繊維を取り、酢酸溶液で一晩インキュベートします。遠心分離の後上澄み (数 mg/mL) を集め、冷蔵庫13します。</li>
	</ol>
</li>
	<li>
培養皿の準備
	<ol>
<li>35 mm のペトリ皿に膜挿入を配置します。</li>
		<li>膜にラット尾コラーゲン溶液の 40-50 μ L を追加し、中心の周りのコラーゲン ソリューションを普及します。</li>
		<li>空気は、クリーン ベンチで完全に膜を乾燥させます。</li>
		<li>膜媒体でビショビショですように、皿に 1.5-2 mL の血清を含む培養液を置きます。</li>
		<li>めっき前にインキュベーター (37 ° C、加湿の 95% の空気と 5% CO2) で培養皿を配置します。</li>
	</ol>
</li>
	<li>
皮質の準備
	<ol>
<li>10 分以上、70% エタノールですべての手術器具を滅菌します。</li>
		<li>生後 (P) 2 からすぐに全脳を解剖 (氷片に数分間浸漬) 冷たい麻酔下ラット。</li>
		<li>場所を 100 mm ペトリ皿冷たいハンクスの 100 mL を含む脳は平衡塩類溶液です。</li>
		<li>微細鉗子で脳から pia 問題を削除し、顕微鏡下のハサミで視覚と体性感覚野から皮質スライス (300-400 μ m 厚) をカット (詳細については、図 1を参照)。 注:双眼顕微鏡の下のグリッドのスケールでは、皮質の厚さはおよそ。 注:また、皮質無菌条件下で vibratome を作ることができます。</li>
		<li>使い捨てプラスチック ピペットと膜挿入にいくつかの皮質スライスを転送します。</li>
		<li>(詳細についてを参照してください8,13) 火磨かれたパスツール ピペットで文化挿入の中心近くスライスの位置を調整し、挿入から余分なメディアを削除します。 注:スライスは、ガスとメディアの両方の十分な供給を受信できるように、スライスの表面の下にわずか媒体のレベルを調整します。これはセル実行可能性にとって重要です。</li>
		<li>視床のブロックを取られ、それの隣に置かれた前に 1 日だけで加湿の 95% 空気と 5% CO2と文化の環境で 37 ° C で血清を含む培地のスライスを維持します。</li>
	</ol>
</li>
	<li>
視床のブロックの準備
	<ol>
<li>10 分以上、70% エタノールですべての手術器具を滅菌します。</li>
		<li>ペントバルビ タール (50 mg/kg) を麻酔薬の入った腹腔内投与による妊娠ラット (15 日) の麻酔します。</li>
		<li>子宮角からそれぞれの胚を解剖し、100 mm ペトリ皿冷たいハンクス平衡塩溶液の 100 mL を含む胚を配置します。</li>
		<li>双眼顕微鏡の下でそれぞれの胚から脳を削除し、核複雑と外側膝状体 (約 0.5 mm × 3)7 (図 1参照) 顕微鏡のはさみを使用してを含む主に視床のブロックをカットします。</li>
	</ol>
</li>
	<li>
文化し、脊髄共培養系を維持
	<ol>
<li>パスツール ピペットを使用して、メンブラン フィルター上に配置されている皮質スライスの心室の表面の横にある視床ブロックを配置します。</li>
		<li>加湿の 95% の空気と 5% CO2の環境で 37 ° C で血清を含む培地の共培養系を維持します。</li>
		<li>2 日間の培養後培養の無血清培地での培地の半分を交換します。その後、2 〜 3 日ごと媒体を交換します。 注:媒体のレベルは、細胞の生存のために重要であるメディア交換後チェック必要があります。</li>
	</ol>
</li>
</ol>2. エレクトロポレーション
すべてのプロシージャは、無菌条件の下で行わなければなりません。
注:エレクトロポレーション 1 日後、ブロックの離脱を防ぐために視床のブロックの準備を実行します。
<ol><li>
プラスミド
	<ol>
<li>プラスミドの混合物を次のようにすること: pCAGGS 下流 2 μ g/μ L。pCAG-SYP EGFP の 3.5 μ g/μ l。 注:エンドトキシン フリーの Maxiprep キットを使用して両方のプラスミッドの浄化し、ハンクス平衡塩溶液中で溶解します。</li>
	</ol>
</li>
	<li>
エレクトロポレーション機器のセットアップ 刺激物やアイソレータなど電気計測器は、図 2に示すように接続してください。<ol>
<li>相性アイソレータに刺激を接続します。</li>
		<li>電極 (銀線 0.2 mm 径) をアイソレータの負の端子に接続します。</li>
		<li>電極 (銀線 1 mm 径) をシリーズに 100 Ω 抵抗とアイソレータの肯定的なターミナルに接続します。</li>
		<li>オシロ スコープ電流増幅器で電圧を測定することによって抵抗の両端で渡されるかどうかを監視するためにアンプを接続します。</li>
	</ol>
</li>
	<li>
ガラス ピペットの準備 注: ガラス ピペットの 2 種類は、プラスミド溶液の排出と電気パルスの応用に使用されます。<ol>
<li>電極引き手とガラス管からピペットを作る。</li>
		<li>(先端径が 20-50 μ m であるべき) 放出ピペットの先端を破る。</li>
		<li>挽くし、サンドペーパー火研磨 (内径は 50-200 μ m であるべき) 続いて電極ピペットの先端を研磨します。 注:それ直接組織に触れると、電極先端部は平らで滑らかでをする必要があります。</li>
	</ol>
</li>
	<li>
電気穿孔法の手順
	<ol>
<li>マニピュレーターにイジェクトや電極のピペットを接続します。</li>
		<li>電極ピペットに銀線 (0.2 mm 径) を挿入します。</li>
		<li>プラスチック製のチューブを 1 mL の注射器に取り出しピペットを取り付けます。</li>
		<li>取る > 注射器吸引吐出ピペットにプラスミド溶液 5 μ L。</li>
		<li>エレクトロポレーション ステージ共を入れて培養培地に 1 mm 径の電極を挿入します。</li>
		<li>視床外植体に放出ピペットを配置します。</li>
		<li>先端が視床のブロックの表面に触れる後に注射器を手動でプッシュします。 注:プラスミッドの 0.5 μ L についてソリューションは 1 つの視床ブロックあたり通常使用されます。</li>
		<li>イジェクション ピペットの撤回後すぐに視床のブロックに電極ピペットを配置します。</li>
		<li>電気パルス (50-400 μ A、200 Hz で 1 ms の 200 の矩形パルスの 3 に 5 列車) を提供します。振幅とオシロ スコープの列車の数を監視します。 注:電流の振幅は、電極ピペット13の内径に依存します。</li>
		<li>電極ピペットを撤回し、インキュベーターに皿を返します。</li>
	</ol>
</li>
	<li>
顕微鏡観察
	<ol>
<li>EGFP の 2 つのフィルター セット装備直立した共焦点顕微鏡の顕微鏡ステージ上培養皿を置く (励起、488 nm; 排出, 515 nm) とした DsRed (励起、514 nm; 排出, 565 nm)。</li>
		<li>10-25 光学セクション 1-7 μ m のステップ サイズ、20 X 長作動距離の対物レンズでの画像を取る。個別に区別のラベル付き軸索した DsRed、SYP EGFP の両方を表現するを選択します。 注:観測地は約 0.7 × 0.7 mm (つまり 1024 x 1024 ピクセルに対応する、0.68 μ m/ピクセル)。</li>
		<li>毎日イメージングのためのすべての 24 h の画像をキャプチャします。室温で 10 分間以内に完了し、インキュベーターに文化を返します。短い間隔の時間タイムラプス イメージングのための環境湿度 95% 空気と 5% CO2 37 ° C で維持する培養室で文化を維持します。</li>
	</ol>
</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+plasticity+of+the+primary+visual+cortex.">Development and plasticity of the primary visual cortex.</a> Neuron. 75 (2), 230-249 (2012).</li><li>Portera-Cailliau, C., Weimer, R. M., De Paola, V., Caroni, P., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+axon+elaboration+in+the+developing+neocortex.">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS Biol. 3 (8), 272 (2005).</li><li>Holtmaat, A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+structural+synaptic+plasticity+in+the+mammalian+brain.">Experience-dependent structural synaptic plasticity in the mammalian brain.</a> Nat. Rev. Neurosci. 10 (9), 647-658 (2009).</li><li>Bhatt, D. H., Zhang, S., Gan, W. 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著者が明らかに何もありません。

現在のプロトコルはまた TC 投影11以外の軸索の成長の発達的側面を研究する強力なツールです。例えば、皮質スライス培養とエレクトロポレーション技術の組み合わせは、皮質ニューロンと長期観測9,18の個々 の軸索形態を視覚化することができます。
現在のプロトコルを使用しては、蛍光タンパク質および?...

哺乳類の脳 (TC) 視床皮質投射は、軸索ガイダンスとターゲットのメカニズムを検討する適切なシステムです。開発時に TC の感覚軸索は皮質骨板やフォーム枝や大脳皮質1,2の一次感覚野のシナプスで優先的に層 IV で育ちます。基本的な接続の確立後も環境の変化3,4によって軸索のアーバーおよびシナプス終末が改造されています。ただし、TC 軸索形態を動的に変更する方法がよくわかっていません。主な理由の 1 つは、単一細胞レベルでの構造変化を観察する適切な方法がないです。2 光子顕微鏡などの顕微鏡の最近の進歩は、生活皮質ニューロンの生体内での直接観測を許可している、全体的な TC の軌跡5,をキャプチャするためまだ技術的な限界があります。6。 したがって、体外TC 軸索のライブ イメージング法は、軸索分岐およびシナプス形成の構造解析のための強力なツールを提供します。
初めて私たちのグループは、透過膜7静的スライス培養法を設立しました。このメソッドを使用すると、ラット脳スライスされた感覚視床ブロックと遊走し、ラミナ特異 TC 接続されたこの脊髄共培養系7,8で締めくくっています。蛍光タンパク質を持つスパース ラベリングさらに TC の軸索の成長や枝の形成9,10,11を観察するができました。最近では、我々 は分岐の同時可視化手法を開発しているし、切片における個々 の TC の軸索のシナプス形成 cocultures12。TC 軸索およびシナプス前のサイトを同時に視覚化するには、下流と EGFP タグ シナプトフィジン (SYP EGFP) は切片培養電気穿孔法による視床のニューロンの数が少ないに cotransfected でした。現在のメソッドは TC 軸索の形態素解析を容易に、軸索分岐およびシナプス形成の因果関係を示すため使用できます長期観察できます。

<table><tbody><tr><td>DMEM/F12</td><td>GIBCO</td><td>11320-033</td><td></td></tr><tr><td>Hanks&amp;rsquo; balanced salt solution (HBSS)</td><td>Nissui</td><td>5905</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Scientific</td><td>SH30396-03</td><td>Hyclone</td></tr><tr><td>Insulin</td><td>Sigma</td><td>I6634</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma&amp;nbsp;</td><td>H0888</td><td></td></tr><tr><td>Sodium selenite</td><td>Wako Pure&lt;br/&gt; Chemical Industries</td><td>192-10843</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma</td><td>T1147</td><td></td></tr><tr><td>Putrescine&amp;nbsp;</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Glucose</td><td>Wako&lt;br/&gt; Pure Chemical Industries</td><td>16806-25</td><td></td></tr><tr><td>35 mm petri dishes</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>Millicell-CM insert</td><td>Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>100 mm petri dishes</td><td>BIO-BIK</td><td>I-90-20</td><td>petri dish sterrile</td></tr><tr><td>HiPure Plasmid Maxiprep Kit</td><td>Invitrogen</td><td>K210006</td><td></td></tr><tr><td>Disposable sterile plastic pipettes</td><td></td><td>202-IS</td><td>transfer pipets sterile</td></tr><tr><td>Glass capillary: OD 1.2 mm</td><td>Narishige</td><td>&amp;nbsp;G-1.2</td><td>inner diameter, 1.2 mm</td></tr><tr><td>Silver wire: 0.2 and 1 mm&amp;nbsp;</td><td>Nilaco</td><td>AG-401265 (diameter, 0.2 mm), AG-401485 (diameter, 1.0 mm)</td><td></td></tr><tr><td>1 mL syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Stimulator&amp;nbsp;</td><td>A.M.P.I</td><td>Master 8</td><td></td></tr><tr><td>Biphasic isolator&amp;nbsp;</td><td>BAK ELECTRONICS</td><td>BSI-2</td><td></td></tr><tr><td>Amplifier&amp;nbsp;</td><td>A-M Systems</td><td>Model 1800</td><td></td></tr><tr><td>Oscilloscope</td><td>Hitachi</td><td>VC-6723</td><td></td></tr><tr><td>Manipulator</td><td>Narishige</td><td>SM-15</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MO-10</td><td></td></tr><tr><td>Stereomicroscope&amp;nbsp;</td><td>Olympus</td><td>SZ40</td><td></td></tr><tr><td>Universal stand&amp;nbsp;</td><td>Olympus</td><td>SZ-STU2</td><td></td></tr><tr><td>Light illumination system&amp;nbsp;</td><td>Olympus</td><td>LG-PS2, LG-DI, HLL301</td><td></td></tr><tr><td>Electrode puller&amp;nbsp;</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Digital eclipse C1 laser</td><td></td></tr><tr><td>x20 objective</td><td>Nikon</td><td>ELWD 20x/0.45</td><td></td></tr><tr><td>Culture chamber</td><td>Tokai Hit</td><td>UK A16-U</td><td></td></tr><tr><td>Sprague-Dawley (SD) rat</td><td>Japan SLC and Nihon-Dobutsu</td><td></td><td></td></tr><tr><td>Microsurgery scissors</td><td>Natsume&amp;nbsp;</td><td>MB-54-1</td><td></td></tr></tbody></table>

visualization of thalamocortical axon branching and synapse formation in organotypic cocultures

軸索分岐とシナプスの形成は、精密な神経回路を確立するための重要なプロセスです。開発中には、感覚視床皮質 (TC) 軸索は大脳皮質の特定のレイヤーで枝とシナプスを形成します。にもかかわらず、軸索分岐とシナプス形成間明白な相関、因果関係はよくわかっていません。この問題に対処するため、我々 は最近脊髄共培養系における軸索 TC 個々 の分岐とシナプス形成の同時イメージング法を開発しました。
このプロトコルでは、切片培養とエレクトロポレーションの組み合わせから成っている方法について説明します。層構成などの特徴的な構造を維持する軸索のプロセスの遺伝子操作と観測の視床と大脳皮質脊髄共培養系を促進します。エンコードした DsRed と EGFP タグ シナプトフィジン (SYP EGFP) 2 つの明瞭なプラスミドはエレクトロポレーション法による視床のニューロンの数が少ないに cotransfected だったこのメソッドでは、個々 の軸索組織の TC ニューロンとシナプス前サイトを同時に可視化することができました。メソッドには、軸索分岐およびシナプス形成の因果関係を明らかにした長期的な観測も有効になります。

ここで TC 軸索分岐とシナプス形成との関係を明らかにすることを目的に記載されているの実験。同時に脊髄共培養系における少数の視床細胞やシナプス前のサイトは、単一の場所軸索の軌跡を視覚化するには、SYP EGFP とした DsRed エレクトロポレーションを使用して 2 つのプラスミドを導入させた。文化の第 2 週の間に軸索 TC 個別に区別はっきりした DsRed (<strong class=...

Watch this Scientific Journal Video about Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures at JoVE.com

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

このプロトコルでは、視床皮質軸索分岐およびシナプス形成の脊髄共培養系における視床と大脳皮質の同時イメージング法について説明します。個々 の視床皮質軸索とその神経終末は、下流と GFP タグ シナプトフィジン単細胞エレクトロポレーション法による可視化します。

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

visualization-thalamocortical-axon-branching-synapse-formation

Research

JoVE Journal

Neuroscience

6.4K ビュー.  Kanazawa University. このプロトコルでは、視床皮質軸索分岐およびシナプス形成の脊髄共培養系における視床と大脳皮質の同時イメージング法について説明します。個々 の視床皮質軸索とその神経終末は、下流と GFP タグ シナプトフィジン単細胞エレクトロポレーション法による可視化します。

ビデオ: Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

医学

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

マルチコンパートメントのCNSニューロングリア共培養マイクロ流体プラットフォーム

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

生物学

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

ひと多能性幹細胞からのニューロンとアストロ サイトの三次元共培養系によるシナプス局所回路におけるモデリング

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

発生生物学

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三次元組織配置に発達メカニズムを要約し、神経系の再生を容易にするアストロ サイト ネットワーク

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工学

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

商工会議所のコンポーネントとマルチスケール光イメージングの3次元印刷を組み込むColliculo、視床皮質マウス脳スライスの変更、

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

神経科学

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

平坦化された哺乳類の皮質皮質モジュールの可視化

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

リナル皮質-海馬の組織性スライス培養におけるてんかん発生のモデル

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

その場で 急性 エクスビボ 胚性脳スライス培養における軸索成長および成長円錐ダイナミクスの可視化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

<meta charset="utf8"></head><body>マウス感覚皮質の発達における神経活動マッピング

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

<meta charset="utf8"></head><body>著者スポットライト:2光子顕微鏡と単一ニューロンイメージングによる神経回路形成の解読

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

新生児マウス感覚皮質における自発的活動の単一ニューロン分解能でのin vivo可視化

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

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器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

マルチコンパートメントのCNSニューロングリア共培養マイクロ流体プラットフォーム

ひと多能性幹細胞からのニューロンとアストロサイトの三次元共培養系によるシナプス局所回路におけるモデリング

三次元組織配置に発達メカニズムを要約し、神経系の再生を容易にするアストロサイトネットワーク

2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

商工会議所のコンポーネントとマルチスケール光イメージングの3次元印刷を組み込むColliculo、視床皮質マウス脳スライスの変更、

平坦化された哺乳類の皮質皮質モジュールの可視化

リナル皮質-海馬の組織性スライス培養におけるてんかん発生のモデル

その場で急性エクスビボ胚性脳スライス培養における軸索成長および成長円錐ダイナミクスの可視化

新生児マウス感覚皮質における自発的活動の単一ニューロン分解能でのin vivo可視化

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

要約

さらに動画を探す

この動画の章

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

マルチコンパートメントのCNSニューロングリア共培養マイクロ流体プラットフォーム

ひと多能性幹細胞からのニューロンとアストロ サイトの三次元共培養系によるシナプス局所回路におけるモデリング

三次元組織配置に発達メカニズムを要約し、神経系の再生を容易にするアストロ サイト ネットワーク

2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

商工会議所のコンポーネントとマルチスケール光イメージングの3次元印刷を組み込むColliculo、視床皮質マウス脳スライスの変更、

平坦化された哺乳類の皮質皮質モジュールの可視化

リナル皮質-海馬の組織性スライス培養におけるてんかん発生のモデル

その場で 急性 エクスビボ 胚性脳スライス培養における軸索成長および成長円錐ダイナミクスの可視化

新生児マウス感覚皮質における自発的活動の単一ニューロン分解能でのin vivo可視化

ひと多能性幹細胞からのニューロンとアストロサイトの三次元共培養系によるシナプス局所回路におけるモデリング

三次元組織配置に発達メカニズムを要約し、神経系の再生を容易にするアストロサイトネットワーク

その場で急性エクスビボ胚性脳スライス培養における軸索成長および成長円錐ダイナミクスの可視化