This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

注：すべての動物の手順は、トロント大学の比較医学部の動物の健康と福祉のガイドラインに適合。 海馬スライスの調製<ol><li> 125℃で乾燥したオートクレーブを用いて、以下の機器を滅菌：メスハンドル（＃3）（2）、標準パターン鉗子、大（1）、小切開はさみ（左右に角度を付け）（1）、マイクロスプーン（スプーンとフラットスパチュラ端）（1）、丸みを帯びたマイクロスパチュラ（およびテーパ端部を四捨五入）（2）、ファイン絵筆（1）、火災研磨パスツールピペット（2）は、ガーゼ正方形、2×2インチ（5）。 </li><li>場合には、滅菌、無菌の容器に器具を入れ、使用するまで覆われ続ける。すぐに解剖する前に、70％エタノール溶液中で楽器を浸す。 </li><li> 35°C aでインキュベーター中でプレートをウエル/培養培地1mlを添加し、記憶することによって切開手順を開始する前に、培養インサートを6ウェル培養プレートを準備しND 5％CO 2。 </li></ol> 2.無菌層流フード中で解剖ツールを配置<ol><li> 70％エタノールで層流フードをスプレーし、アルコールから滅菌解剖器具を取り外します。アルコールと解剖脳組織との接触を避けるために、無菌のペトリ皿に載っている間楽器が乾燥することができます。 </li><li> 2つの大きな滅菌ペトリ皿で​​滅菌氷冷解剖溶液の堆積物5-7ミリリットル。一皿は、寒さと頭（汚い）、冷却とえぐら脳（クリーン）をすすぐための他の1をきれいにします。脳を解剖用シャーレのふたの1で滅菌ろ紙を置きます。 </li><li>海馬を解剖するための小さなペトリ皿のふたの一つに小さな、滅菌ろ紙を置きます。 2小さな、滅菌ペトリ皿中の滅菌氷冷解剖溶液の堆積物3-5ミリリットル。一皿は1つが、海馬のセクションの分離中のセクションを保持する、えぐら海馬を開催します解剖顕微鏡下。 </li><li>切削ステージに滅菌ろ紙の一部をテーピングし、滅菌カミソリの刃を装着することにより組織チョッパーを準備します。滅菌解剖溶液とろ紙を湿ら。 </li><li>アルコールできれいなバイオバッグをスプレーし、層流クリーンベンチに入れてください。 </li></ol> 3.海馬解剖<ol><li>層流クリーンベンチ外の70％エタノールとP7のSprague Dawleyラット子犬をスプレーし、す​​ぐに層流ベンチの内側に大型無菌の外科用ハサミを用いて動物を首を切る。頭がペトリ皿の1中の氷冷解剖ソリューションにドロップしてみましょう。 </li><li>ペトリ皿に、血を洗い流し、すぐに滅菌ろ紙、腹側を下に頭を転送する。 </li><li>メスを用いて、下にある頭蓋を露出させるように矢状面内の背側表面に沿って切断する。 Tのラットで柔らかく、容易に貫通可能で、基礎となる骨、皮膚を通してカットではなく、彼の年齢。さておき、この &quot;汚い&quot;メスを設定し、脳組織には使用しないでください。 </li><li>小さな切開（側に傾斜し）はさみや鉗子を使用して、ブレグマに頭蓋骨の矢状縫合に沿って頭蓋骨を開き、冠状縫合を矢状縫合によって垂直に交差する頭蓋骨上の解剖学的な点をカット。頭蓋骨の正中線から持ち上げて頭蓋骨フラップを引っ張って鉗子を使用してください。 </li><li>そっと頭蓋骨から脳を持ち上げるために、脳幹の下に、脳の下側にマイクロスプーンを置きます。脳の基底面に視神経と嗅球を露出するために脳を持ち上げます。完全に頭蓋骨から脳を分離するために、小さなハサミでこれらの構造をカット。取り外し、氷冷解剖溶液を含む他の大きなペトリ皿に無傷の脳を転送する。 </li><li>マイクロスプーンを使用して、無菌のろ紙を含む小さなペトリ皿の蓋に脳を転送する。無菌のパスツールピペットを用いて、DIS数滴の脳をすすぐ湿った組織を維持するためのソリューションをsecting。 </li><li> 「クリーン」なメスの刃を使用して、離れて2半球を切った。マイクロスプーンでバック大型シャーレに左半球を転送し、その後の使用のための氷冷解剖溶液に半球PIA側を下に置きます。 </li><li>右半球の内側の顔を見るとエッジ円蓋、海馬の内側縁に沿って白質の顕著なバンドを特定する。滅菌メスを使用すると、円蓋の矢状カットを作るが、メス先端のわずか0.5センチ円蓋をカットするのに十分であるので、注意してください。 </li><li> 2マイクロスパチュラを使用して、脳幹に右手へらを配置し、左側のヘラで上を覆う皮質を持ち上げて右半球からの第1の海馬を削除します。静かに海馬の側脳室および内側の表面が露出する皮質を持ち上げます。白い曲線、采は、表示されるようになるはずです。 </li><li> CURVAとへらの曲率を合わせます采のトゥーレと優しく采下にへらを押してください。左のヘラをスライドさせ、吻側 - 尾側軸に沿って乗って、次に海馬を削除するには、背側方向にへらを持ち上げる。 </li><li>氷のように冷たい解剖溶液と第 2小ペトリ皿に海馬を転送します。左海馬を削除するには左半球で同じ手順を繰り返します。 </li><li>マイクロスパチュラを使用して、慎重に組織チョッパーステージに海馬を転送する。チョッパーブレードの軸に対して互いに垂直それらを隣接して平行に配置する。組織を置き、海馬の上に解剖ソリューションの数滴を追加するために絵筆を使用してください。 </li><li>個々のスライスを削除するには一時停止することなく400μmの切片で組織を切断する（通常、彼らは、ブレードに付着しない）。全海馬が切断された後、穏やかに解離溶液と第 2小ペトリ皿にセクションを転送するために絵筆を使用。 </li><li>広告の下で顕微鏡をissecting、慎重にamicro-へらと絵筆を使用して浮動スライスを分離する。 </li><li>層流フード内インキュベーターと場所からカルチャーインサートが事前に準備された培養プレートを外します。 </li><li>ファイアーポリッシュパスツールピペットを用いて、カルチャーインサート膜の頂端表面にピペットと転送スライスに4-5スライスを描く。次に、絵筆を持つ位置を調整し、個々のセクションと文化インサートの境界線間にスペースを入れてください。 </li><li>無菌のパスツールピペットを用いて、膜の頂端表面から余分な解剖溶液を除去する。 注：ピペットに組織切片を描画ソリューションの回避を除去しながら。代わりに、ゆっくりと溶液を除去するために滅菌ピペットチップとの定期的なピペット（P200またはP1000）を使用します。 </li><li>血清含有培地と35℃のバックインキュベーターに海馬スライスし、5％CO 2で培養プレートを置きます。 注：実験呼び出す場合複数の動物からの文化を生成するために、徹底的に解剖の間に層流クリーンベンチをきれいに。アルコール溶液に楽器を戻し、すべてのペトリディッシュ、ろ紙及び第二解剖前に無菌のかみそりの刃を交換してください。 </li></ol> 4.供給および器官型スライスを維持<ol><li>無菌層流クリーンベンチ内の文化を養う。 </li><li>培養されたセクションの最初の給餌2日後に解剖を行います。滅菌したガラスピペットを用いて吸引し、古い培地。 </li><li>ウェルに新鮮な、無菌の、血清含有培地の1ミリリットルを追加するために、滅菌5ミリリットルの血清学的ピペットを使用してください。 </li><li>静かにカルチャーインサートを交換し、膜表面の下に形成された可能性のある気泡を除去するように注意してください。 </li><li>最初の給餌の後、一日おきに培地を変更します。 </li></ol>新生ニ​​ューロンをラベルにチミジン類似体5.インキュベート組織スライス<ol><li>研究するために海馬の歯状回顆粒細胞の成熟と統合は、そのようなブロモデオキシウリジン（BrdU）のまたはChlorodeoxyuridine（CldU）などのチミジン類似体と器官のスライスを、インキュベートする。ここでは、あるため、生理食塩水での高い溶解度のCldUを使用しています。 </li><li> 3DIVした後、10μg/ mlの最終濃度のために培地を1mlの生理食塩水中の10mg / mlのCldUストック溶液1μlを加える。 BrdUを使用する場合は、分子量の差についての濃度を補正する。培養ウェルにCldUとメディアを追加し、35℃で2時間CldU含有培地で組織をインキュベートする。 </li><li> 35℃でのインキュベーションの2時間後、CldU含有培地を除去し、（上記に概説）通常の給餌スケジュールを再開するために定期的に送り媒体と交換してください。 </li></ol> 6.組織固定およびストレージ<ol><li>治療を適用し、培養実験（図2B）を起動する前に、組織試料を固定するためのタイムラインを確立する。 </li> &lt;所定の日後の切開で李&gt;は、実験室のドラフト内で次の準備：10〜50ミリリットルのビーカーに、リン酸緩衝生理食塩水（PBS）中4％パラホルムアルデヒド（PFA）を含有する。廃棄された培養液のための空のビーカー。小さな鉗子。使い捨てチップとし、千ミリリットルピペット。 </li><li>インキュベーションチャンバーから培養プレート（S）を取り外し、ヒュームフードに転送。ピンセットで個々のウェルプレートインサートを傾けます。次に、廃棄ビーカーに、培養培地と転送を削除するには、ピペットを使用しています。全ての培地を除去していることを確認するために角度を培養プレートを傾ける。 </li><li>一度に1つの培養プレート用媒体を除去完了します。培地が除去されたときに、各培養ウェルに4％PFAを1mlを追加し、パラフィルムでプレートウェルシールする。 24時間4℃の冷蔵庫に必要な、転送プレートと同じ数の培養プレートのために繰り返します。 </li><li> 24時間後、ヒュームフードに次のように準備します。10-50 PBS中の0.1％アジ化ナトリウムを含むミリリットルビーカー。空の廃棄されたPFA（有害物質を廃棄する際に安全上の注意に従ってください）​​用のビーカー。小さな鉗子。使い捨てチップとし、千ミリリットルピペット。 </li><li> PFAを追加するための6.4で説明する手順に従いますが、その代わりにアジ化ナトリウムを含むPBSの1ミリリットルを追加します。一度完全にパラフィルムのエッジをラップし、4℃の冷蔵庫に保存することによって、将来の使用のためのシールウェルプレートに移した。 </li></ol> 7.免疫組織化学のための組織をセクショニング<ol><li>ビブラトームを使用して切片組織を実行します。以下の一連の工程は、器官培養物から使用可能な組織切片の収率を最大限に高める。 注：直ちに海馬解剖とスライスのメッキ以下では、組織は、約400μmの厚さを有している。しかし、インキュベーションチャンバー内の2〜3週間後に、組織切片は、150〜300ミクロンの最後のセクションの厚さで、その結果、平らにし始めます。 </li><li> ＃11メスB：セクション培養組織に以下の項目を準備クレード、ハンドル、氷冷PBS、マイクロ解剖鉗子、および組織をマウントする段階をカットクリーンビブラトームを含むガラスシャーレ。 </li><li>それはプラスチック製インサートハウジングから取り外すことができるように、外科用メスで慎重に円形インサート膜の周囲に沿って切断する。培養されたスライスと、メスの間に十分なスペースを入れてください。 </li><li> PBSを含むペトリ皿に切り離され、インサートメンブレンを転送します。 PBSですすいだ後、ビブラトーム取り付けステージに膜を転送するために鉗子を使用しています。 </li><li>次に、培養されたスライスを囲む過剰膜を除去し、クリーンなエッジを作成するために余分な材料を切り取るためにメスを使用しています。このステップでは、膜は平坦であり、容易に切断面に付着することが確実になる。 </li><li>ビブラトーム切削ステージ上に接着剤の1-2滴を置き、さらには22 G針を使用して層内に広がった。ビブラトームの切断刃に長辺と平行な矩形状に接着剤を広げた。このステップを実行しますすぐに、乾燥からの接着を防止する。 </li><li>切削ステージに海馬スライスを含むトリミング膜を転送し、軽く糊上の膜を配置し、気泡がないことを確認するために鉗子を使用してください。 </li><li>瞬間接着剤が乾燥したように、バックPBS含むペトリ皿に接着膜でビブラトーム舞台を移す。ビブラトームブレードと組織切片を格納するためのアジ化ナトリウムを含有する48ウェルプレートを準備する。 </li><li>貯蔵およびその後の免疫組織化学的染色のためにアジ化ナトリウムを含む48ウェルプレートに器官スライス組織と転送の30μmのセクションを生成するためにビブラトームを使用してください。 </li><li>免疫組織化学染色26,29のために、既存のプロトコルを参照してください。 </li></ol>

<ol>
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J., White, A., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+spikes:+harbingers+or+causes+of+epilepsy.">Interictal spikes: harbingers or causes of epilepsy.</a> Neuroscience Letters. 497 (3), 247-250 (2011).</li><li>Lee, H., Lee, D., Park, C. H., Ho, W. K., Lee, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+hippocampal+slice+cultures.">Neurogenesis in hippocampal slice cultures.</a> Molecular And Cellular Neurosciences. 26 (2), 241-250 (2004).</li><li>Kamada, M., 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=Intrinsic+and+spontaneous+neurogenesis+in+the+postnatal+slice+culture+of+rat+hippocampus.">Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.</a> The European Journal Of Neuroscience. 20 (10), 2499-2508 (2004).</li><li>Kim, H., Kim, E., Park, M., Lee, E., Namkoong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+culture+from+the+adult+mouse+brain:+a+versatile+tool+for+translational+neuropsychopharmacology.">Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Legradi, A., Varszegi, S., Szigeti, C., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+hippocampal+slices+as+in+vitro+models+for+neurodegeneration:+Studies+on+cell+viability+and+apoptotic+processes.">Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes.</a> Brain Research Bulletin. 84 (1), 39-44 (2011).</li><li>Sadgrove, M. P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. G., 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=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. 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=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., 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=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. 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The authors declare that they have no competing financial interests.

CldU（またはBrdU）の投与後に、薬理学的薬剤の適用のタイムラインは、特定の発生の窓の間に新生児DGCsを標的とするように選択することができる。例えば、仮想的な薬剤は、GABAが脱分極される発達段階にある未成熟ニューロンの年齢と一致することが提案されている第二週後CldU注射中に適用することができる。このプロトコルを使用して今後の研究では、薬剤と「テーラー「目的の特定の?...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

<table><tbody><tr><td>5-chloro-2&#039;-deoxyuridine (CldU)</td><td>MP Biomedicals</td><td>105478</td><td>Hazardous, Carcinogenic</td></tr><tr><td>Cell culture inserts, 30 mm diameter, 0.4 &amp;micro;m pore size</td><td>Thermo scientific&amp;nbsp;</td><td>140660</td><td>Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td></tr><tr><td>Conical Centrifuge tubes (sterile)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Dissector scissors (angled to side)</td><td>Fine Science Tools&amp;nbsp;</td><td>14082-09</td><td></td></tr><tr><td>Minimum essential medium (MEM)</td><td>Gibco</td><td>11095; liquid</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Eclipse Ni-U fluorescent microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Glue for tissue</td><td>Krazy Glue</td><td>KG585</td><td>Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS) (500 ml)</td><td>Gibco</td><td>14025-092</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Horse Serum Heat Inactivated (500 ml)</td><td>Gibco</td><td>16050-122</td><td>Make 50 ml aliquots and store at -20 &amp;deg;C</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clarke</td><td>TW 31KYPBX</td><td></td></tr><tr><td>Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td><td></td><td></td><td></td></tr><tr><td>Petri Dish (100 mm x 15 mm) and (60 mm x 15 mm)</td><td>Fisher Brand</td><td>FB0875712 and FB0875713A</td><td></td></tr><tr><td>Scalpel blades #11</td><td>Fine Science Tools</td><td>10011-00</td><td></td></tr><tr><td>Scalpel handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Serological Pipettes</td><td>Sorfa Medical Plastic Co.</td><td>P8050</td><td></td></tr><tr><td>Standard Pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Sterile vacuum filter</td><td>Thermo-Scientific</td><td>565-0020</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14054-13</td><td></td></tr><tr><td>Syringe driven filter unit</td><td>Millipore-Millex</td><td>SLGP033RS</td><td></td></tr><tr><td>Tissue chopper with moveable stage</td><td>Stoelting&amp;nbsp;</td><td>51425</td><td></td></tr><tr><td>Fine tip paintbrush</td><td></td><td></td><td></td></tr></tbody></table>

organotypic slice cultures for studies of postnatal neurogenesis

ここでは、器官型スライス培養技術を用いて齧歯類の脳内の海馬出生後の神経新生を研究するための技術が記載されている。開発海馬歯状回への薬理学的薬剤の直接適用を可能にしながらこの方法では、海馬の特徴的地形形態を維持します。さらに、スライス培養は、最大4週間維持することができ、したがって、一つは新生児顆粒ニューロンの成熟過程を研究することを可能にする。そのような海馬の深い解剖学的位置だけでなく、血液脳関門に関連する不確実性などの複雑な変数を除外してスライス培養は、海馬スライスの効率的な薬理学的操作を可能にします。これらの理由から、我々は、出生後の神経新生の研究のために特異的器官切片培養を最適化しようとした。

1）スライスは、インビトロで 10-21日間（DIV）の後に海馬スライスの特徴的な形態学的特徴を維持すること、および2）新生児DGCsを定量することができる：器官培養成体神経発生の研究に適しているかどうかを決定する彼らは二つの主な基準を満たすことが必要一般的に成人の神経発生研究に用いられる標準的な免疫組織化学的技術を使用して。第一の基準については、 図1A</stron...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

ここでは、器官型スライス培養技術を用いて海馬の出生後の神経新生を研究するための技術が記載されている。この方法では、成人の神経新生のin vitro操作を可能にし、培養海馬への薬理学的薬剤の直接適用することができます。

生後神経発生の研究のための器官型スライス培養

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

Research

JoVE Journal

Developmental Biology

12.2K ビュー.  University of Toronto. ここでは、器官型スライス培養技術を用いて海馬の出生後の神経新生を研究するための技術が記載されている。この方法では、成人の神経新生のin vitro操作を可能にし、培養海馬への薬理学的薬剤の直接適用することができます。 

ビデオ: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18ラット脳の器官型スライス培養

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

生物学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海馬切片培養

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

神経科学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

出生後の脳における神経細胞の移動の高分解能タイムラプスイメージングのための器官型スライスアッセイ

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚腹側中脳の器官型スライス培養：ドーパミン作動性ニューロンの発達を研究するための体制 in vitroで</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神経形態形成の研究のための方法：<em&gt;エキソビボ</em胎児マウス大脳皮質&gt; RNAiのエレクトロポレーション

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

オリゴデンドロサイトダイナミクスと髄鞘形成を研究するための器官型スライス培養

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

成人人間の脳からの短期自由浮遊スライス培養

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

オキュロ運動神経増殖のタイムラプスイメージングのための胚性GFP発現マウスからのEx Vivo Oculomotorスライス培養

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

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

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

オルガ鼻記小脳スライス培養におけるプルキンジェ細胞生存

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

生後神経発生の研究のための器官型スライス培養

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