我々は、解剖や文化技術と有用なアドバイスのためにA. Hadjantonakis（スローンケタリング研究所）の研究室とL. Niswander（コロラ​​ド - デンバーのU）の研究室に感謝します。この作品は、L. NiswanderとJ.ナドー（システム生物学研究所）と共同でMERにJDGとRO1NS05897にNRSA NS059562によってサポートされています。

1。培地の調製 - すべての試薬を表1に記載されています<ol><li> 37雄ラットの血清を融解℃を</li><li> 55℃30分間熱不活性化するラットの血清° C </li><li>室温で5分のための10Kでの遠心ラットの血清</li><li>上清を除去し、フェノールレッドを含まないDMEM W /高グルコースで50:50を混ぜる</li><li> 0.45 UMシリンジフィルターを使用してメディアを滅菌</li><li>メディア/胚の少なくとも1ミリリットルを用意する必要があります</li></ol> 2。妊娠ダムから子宮の単離<ol><li>動物実験および使用委員会（IACUC）によって承認された手順を使用して、妊娠中のダムは最初の麻酔ですし、頚椎脱臼により屠殺</li><li> 70パーセントエタノールで腹部を消毒する</li><li>大規模な鉗子で、ちょうど乳首のラインの上に、正中線に沿って皮膚の小さなパスをつまんで、大きなハサミを使って鉗子の下に腹部を開きます。任意の内部構造を傷つけないように注意してくださいの</li><li>腹部全体が開いているように、マウスの各側に向かって最初の開口部から横方向にカットするあなたのはさみを使用して、 </li><li>腸と余分な脂肪パッドはしばしば子宮を隠蔽したりすることができますし、これらは、子宮と卵巣の上部は両側に観察できるように脇に移動する必要があります</li><li>卵巣下子宮の上部をつまんで、他の卵巣に子宮の一方の端からカット、脂肪から子宮を分離するためにあなたのはさみを使用して</li><li>生理食塩水とペトリ皿の中であなたの鉗子と場所と子宮を持ち上げ</li></ol> 3。子宮から胚の除去<ol><li>余分な血液を除去し、外から余分な脂肪をトリミングする生理食塩水で子宮をすすぐ</li><li>室温で清潔な皿に移す。可視化を改善するために生理食塩水Tyrodes </li><li>実体顕微鏡を使用して、慎重に離れて子宮の皮に小さなハサミまたは必要に応じて細かい場合（＃5）鉗子のいずれかを使用してそれぞれの胚を区切ります。 </li> &lt;LI&gt;胚ではないピアスに注意して、子宮壁と脱落膜と背面皮子宮壁との間の空間に微細な鉗子を挿入</li></ol> 4。胚からの脱落膜の除去<ol><li>オリエント暗い部分（胎盤）はあなたから離れて手前にして脱落膜</li><li>あまりにも深く切らないように注意しながら、軽い部分から脱落膜の暗い部分を分ける線に沿って鉗子で切開を加えます</li><li>栄養膜の円錐（EPC）を引き裂くように注意しながら、卵黄嚢の上から胎盤の分離を完了。この時点で、卵黄嚢にReicherts膜を可視化することができるはずです</li><li>ではないピアス卵黄嚢に注意して全体の脱落膜の（軽い部分）を削除し続けます</li><li>そっとつまみ、EPCから、それを離れて剥離によってReichertsを削除します。 EPCが損傷したり、卵黄嚢から分離されている場合は、胚はE8.5 - E9.5から正常にオンに失敗します。 </li><li&gt; Reichertsでは、胚は卵黄嚢を介して簡単に見えなければならないとsomitiesをカウントすることにより、上演することができる除去。 </li></ol> 5。文化システムのセットアップ<ol><li>胚の卵黄嚢に損傷を与えることなくピペッティングすることができるように、開口部の直径を大きくするハサミでプラスチック製トランスファーピペットの先端をカット</li><li>胚あたりのメディアの1 mlのクリーンローラーボトルを埋める。 3月6日胚の最大が使用されてのローラーボトルの種類に応じて、それぞれのボトルにロードすることができます。 </li><li>できるだけ少ないTyrodesとして繰り越し、メディア充填ローラーボトルに、カットのピペットを用いて胚のようにメディアを希薄化しないように転送する</li><li>ガラスのローラーボトルの上部にゴム製のプラグ（栓）を添付する</li><li>タイトなシールがプラグとドラムの間に形成されるようにローラー培養ドラムにローラーボトルを挿入</li><li>一度、すべてのボトルが添付されており、ドラム内の任意の空のスロットは、wを封印されているi番目のゴム製ストッパー、ローラーを活性化</li><li> CO 2とO 2タンクを開き、出口弁1秒に泡を放出されるように、約2 psiにそれらを調整する。 E8.5 - E10胚の場合、20％O 2および5％CO 2を使用する必要があります。 </li><li>インキュベーターを37に設定されていることを確認℃、覆ったり、胚を光から遮蔽されるようにインキュベーターを閉じる。 </li><li>胚はsomitiesのほか、回転の進行、および神経管の閉鎖の程度によって、その発展を評価するために定期的に点検してください</li><li>文化の中で2〜3時間後に、薬理学的阻害剤または他の治療は、培地に添加することができます</li><li>研究デザインは、車両や研究試薬の不活性類似体などのコントロールを含める必要があります。 </li></ol> 6。全胚培養の後に開発を評価する<ol><li>ガスの電源をオフにし、ローラーを停止し、インキュベーターからボトルを取り外し</li><li>タイロードに戻す胚を転送する実体顕微鏡下で評価のためのペトリ皿の中のsまたはPBS </li><li>卵黄嚢は、風船のようなハートビートが見えるはずと心拍数が&gt; 120分あたりの拍数、および流通が明らかでなければならないはず表示されます</li><li>必要に応じて、卵黄嚢除去の前に写真の胚</li><li> EPCの近くに穴を切削し、胚の頭部と臀部を介して卵黄嚢と羊膜を反転させる胚から卵黄嚢を削除します。 </li><li>臍帯は、胚から卵黄嚢を分離するためにカットすることができます、と卵黄嚢は、必要に応じて胚を遺伝子型に使用することができます。 </li><li>体節の数と、オープン頭蓋ひだのような神経管欠損、顔面及び/またはオープン尾側神経孔の不完全閉鎖のための胚を調べます。胚は（複数の形態形成変化のタイラーの分類に応じてステージングできます<a href="http://genex.hgu.mrc.ac.uk/Databases/Anatomy/MAstaging.html">遊んで住む/ home.htmlのをhttp://www.emouseatlas.org/~~V</a> 、アゲイン）の写真記録を胚の開発に便利です。 </li></ol> 7。ノート<ol><li>どんなニックネームや卵黄嚢に涙が成功した転換を防ぎ、正常な発達を阻害するので、高品質の解剖は、不可欠です。 EPCは、ライヒェルトの膜（RM）の除去中に損傷することができる、とEPCが部分的にでも卵黄嚢から分離されている場合、胚は正常に発育しません。しかし、RMの不完全な除去胚は、および/またはRMセルを一緒に固執する原因となるには正常な発達を妨げるような、どちらも、文化に生い茂ると卵黄嚢の拡張を収縮させることができる。 </li><li>鈍いや曲がり鉗子の手順が著しく困難になるため、クリーンでシャープな鉗子は、良好な解剖を取得するための秘密です。楽器の蛋白質の堆積物は、組織の損傷につながる。 </li><li>クリーン、無菌ガラスびんはまた、側壁に付着し、損傷から胚を防ぐために不可欠です。実験を開始する前に、ボトルには、SCRにする必要があります、石鹸と水でubbedのdH 2 Oで洗浄し、水の沸騰するまで、各ボトルに少量の水でマイクロ波処理。瓶は、その後70％エタノールでリンスし、空気乾燥を許可されます。 </li><li>早く胚を培養培地に転送することができる、37に戻っ° Cより良い生存を暖めているため、可能な限り迅速に動作する。 </li><li>それぞれの胚が胚の成長の矛盾の発生を防ぐために、同じ時間、室温になるようにローラーボトルに、任意の胚を転送する前に全体のゴミを解剖完了</li><li>文化の細菌汚染を防ぐために無菌環境を維持する。私たちの培養実験は、抗生物質なしで行われますが、多くのラボでは、細菌の増殖を防ぐために、彼らのメディアへの抗生物質に追加されています。それは実験開始時だったので培地は実験終了時に明確に表示されます。メディアが白濁しているか、目に見える沈殿物がある場合は、CONがある可能性がありますあなたの文化に感染しているtamination。 </li><li>効果のないガス交換には、胚の発達を遅らせるので、連続的な配信を保証する信頼性の高いレギュレータを持っていることを確認することができます。 </li><li>胚は、ex vivoでの培養で約50％遅く、開発、従ってそれはあなたが実験の完了のための開発の適切な段階に達していることを保証するために体節を数えるために不可欠です。 </li></ol> 8。代表的な結果： 胚の出現前後のローラーの培養は、図1に図示されています。解剖の時に、胚は尾が頭のひだの背後にあるあらゆる手段を設定した（図1A、D）で指定する必要があります。文化の36時間後、胚は、彼らは尾は頭部の前面（図1Bを、C、E、F）になっているC -カーブ、胎児の位置、になるように回し完了しておく必要があります。 RhoAのキナーゼ阻害剤（Y - 27632）、中に収斂伸長の既知の阻害剤と薬理学的操作神経胚形成（Ybot -ゴンザレス、2007）、それらの吻側 - 尾側軸に沿った胚の短縮の結果（図1E、F）および頭蓋神経襞閉鎖を阻害する。私たちのデータは、Y - 27632の増量は徐々に頭蓋倍クロージャを損なう（図2A）と下流型RhoAは頭蓋神経胚形成と収斂伸長におけるシグナル伝達の役割と一致して体の軸（図2B）を、短縮することを示している。 <img alt="図1" src="/files/ftp_upload/3132/3132fig1.jpg" /> 図1。薬理学的阻害剤Y - 27632と文化の胚と操作の外観。前の全胚培養へのE8.5で（、D）ディテールに秘められた胚（B、E）ローラーの文化の36hrsに続いて、まだそのまま卵黄嚢とE10での胚。 （C、F）卵黄嚢が成功した転機と神経管閉鎖を説明するために削除されています。 Rhoキナーゼ阻害剤Y - 27632（E、F）で処理した胚は、適切な収斂伸長を受け、説明するために失敗しました。短縮体軸。 <img alt="図2" src="/files/ftp_upload/3132/3132fig2.jpg" /> 図2。頭部神経管閉鎖と軸の伸長に対するRhoAのキナーゼ阻害剤の効果。 （A）その頭蓋ひだ（％NTC =パーセンテージ神経管閉鎖）を正常に閉じることができた胚の割合は、培養培地に添加Y - 27632の投与量と比較されます。 （B）耳胞と前肢の間の距離が大幅にY - 27632の増量（P &lt;0.05）で減少した。

<ol>
	<li>Cockroft, D. L., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+culture+of+post+implantation+embryos.">Dissection and culture of post implantation embryos.</a> Mammalian Postimplantation Embryos A Practical Approach. , (1990).</li><li>Gray, J. D., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+folate+supplementation+from+Crooked+tail+and+other+NTD-prone+mutant+mice.">Mechanistic insights into folate supplementation from Crooked tail and other NTD-prone mutant mice.</a> Birth Defects Res. A. Clin. Mol. Teratol. 85, 314-321 (2009).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1992).</li><li>Ybot-Gonzalez, P., Savery, D., Gerrelli, D., Signore, M., Mitchell, C. E., Faux, C. H., Greene, N. D., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension,+planar-cell-polarity+signalling+and+initiation+of+mouse+neural+tube+closure.">Convergent extension, planar-cell-polarity signalling and initiation of mouse neural tube closure.</a> Development. 134, 789-799 (2007).</li><li>Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interactions,+folate+metabolism+and+the+embryonic+nervous+system.">Gene-environment interactions, folate metabolism and the embryonic nervous system.</a> Wiley Interdiscip Rev Syst Biol Med. 2, 471-480 (2010).</li></ol>

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その開発中に胚に固有なものから母体、子宮内の要因を分離する能力は、胚発生の全段階を研究するための重要なツールです。ここでは、それによって薬剤の母体の代謝の変数を削除し、頭蓋神経胚形成の元の子宮内でのRhoAのキナーゼの小分子阻害剤の効果を分析した。この薬理学的操作では、頭蓋神経胚形成と収斂伸長に大きな影響を与える。この化合物に対する感受性が異なる遺伝的変異マウスの間で比較することができます。ここで紹介する方法は、試薬のさまざまな方法を使って胚の細胞機能を直接操作できるように、開発中の他の分子経路の研究にも適用できます。

<table border="1"><tbody><tr><th>の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>解剖用実体顕微鏡</td><td>ライカ</td><td> M165 </td><td></td></tr><tr><td>精密インキュベータ</td><td> BTCエンジニアリング（英国） </td><td> BTC 001 </td><td></td></tr><tr><td>回転ボトルの文化のユニット</td><td> BTC </td><td> BTC 003 </td><td></td></tr><tr><td>ユニットを回転させるためのガラスびん</td><td> BTC </td><td> BTC 005 </td><td></td></tr><tr><td>シリコーンゴム栓</td><td> BTC </td><td> BTC 007 </td><td></td></tr><tr><td>シリコーンゴムのコルク</td><td> BTC </td><td> BTC 008 </td><td></td></tr><tr><td>ガスバブラーの入口</td><td> BTC </td><td> BTC 012 </td><td></td></tr><tr><td>ガスバブラーの入口トラップ</td><td> BTC </td><td> BTC 013 </td><td></td></tr><tr><td>ガスバブラーアウトレット</td><td> BTC </td><td> BTC 014 </td><td></td></tr><tr><td>ガスボンベ</td><td> TechAir </td><td></td><td> 20％O 2、CO 2 5％ </td></tr><tr><td>ガスレギュレータ</td><td> TechAir </td><td></td><td></td></tr><tr><td>雄のラット血清</td><td>ハーラン研究所</td><td> 4520 </td><td></td></tr><tr><td> DMEM W / Oフェノールレッド</td><td>インビトロジェン</td><td> 31053 </td><td></td></tr><tr><td> 10mLのシリンジ</td><td> BD </td><td> 309604 </td><td></td></tr><tr><td>シリンジフィルタ</td><td> Nalgene </td><td> 190-2545 </td><td></td></tr><tr><td>大解剖ハサミ（ブラント先端） </td><td> VWR </td><td> 82027-594 </td><td></td></tr><tr><td>エクストラファインボンはさみ</td><td>ファイン科学ツール（FST） </td><td> 14084〜08 </td><td></td></tr><tr><tD&gt; Dumot＃5鉗子</td><td> FST </td><td> 11252〜50 </td><td></td></tr><tr><td>タイロードの生理食塩水</td><td></td><td></td><td>コックロフト1990年に記載のように</td></tr><tr><td> 10センチメートル使い捨てシャーレ</td><td> BD </td><td> 351029 </td><td></td></tr></tbody></table>

neural tube closure in mouse whole embryo culture

遺伝的マウスモデルでは、哺乳類の神経管閉鎖の研究において重要なツールです（グレー＆ロス、2009;ロス、2010）。しかし、 子宮内でマウスの胚の研究は、直接薬理学的に興味のある試薬で母体の代謝の影響から分離して胚を操作するために私達の無力によって制限されます。食事を介して、または注射により、小分子、組換えタンパク質、またはsiRNA、母親へのこれらの物質の配信を使用するかどうかは、それらが胚に達するのを妨げる可能性のある身体の防御の様々なこれらの不安定な化合物を加えないでしょう。全体の胚の培養物における調査は、開発上の本質的な胎児の影響から母体を分離するのに使用できます。 ここでは、郭清後の通常の神経管閉鎖（クロケット、1990）を可能にするローラーインキュベーター装置で高濃縮媒体を用いて培養マウス胚のための手法を提案する。かつて文化の中で、胚ができます胚が子宮内に残っていた場合以外の場合は不可能なインビトロ技術で 、従来使用して操作する。胚の兄弟は、E7 - 7.5（神経板の形成、神経胚形成の開始直前に）から頭蓋倍と尾側神経孔の終了時E9.5 - 10（に発生、神経胚形成の異なる側面を研究するために種々の時点で収集することができます閉鎖、カウフマン、1992）。このプロトコルでは、我々は、頭蓋神経胚形成の研究に最適なタイムポイントで胚を解剖ための私達の方法を示しています。胚は、神経管閉鎖の開始後、前の胚の転換と頭部神経倍閉鎖へ、E8.5（約10〜12 somities）で解剖し、E10（26-28 somities）まで培養によって維持されるときに頭蓋神経胚形成が完了されるはずです。

Watch this Scientific Journal Video about Neural Tube Closure in Mouse Whole Embryo Culture at JoVE.com

Neural Tube Closure in Mouse Whole Embryo Culture

母体の代謝をバイパスする神経胚形成の間にマウス胚の直接の薬理学的操作を可能にする方法が記載されている。技術は時間のポイントと薬剤を変えることによって、神経胚形成の異なる側面を研究するために適応させることができます。

マウス全胚培養における神経管閉鎖

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

neural-tube-closure-in-mouse-whole-embryo-culture

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Neuroscience

21.4K ビュー.  Weill Cornell Medical College. 母体の代謝をバイパスする神経胚形成の間にマウス胚の直接の薬理学的操作を可能にする方法が記載されている。技術は時間のポイントと薬剤を変えることによって、神経胚形成の異なる側面を研究するために適応させることができます。

ビデオ: Neural Tube Closure in Mouse Whole Embryo Culture

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

行動内分泌学と実験経済学を組み合わせること：テストステロンと社会的意思決定

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

3D神経幹細胞培養におけるローカライズたんぱく質：ハイブリッド可視化手法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

内在性NADH蛍光の二光子イメージングによるマウス大脳皮質におけるMicroregional低酸素の検出

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

ニワトリ胚脊髄スライス培養プロトコル

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

マウスの胚感覚軸索投影のホールマウントイメージング

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...