私たちは、映画への彼女のサポートのための解剖手法と藤田保健大学亜紀宮川の彼女の命令のために京都大学で博士陽子鍋島に感謝。日本の文部科学省から、とで - この作品は、医薬基盤、優先エリア - 統合脳研究（Shien）科学研究費補助金の国立研究所の健康科学の基礎的研究の推進のためのプログラムによってサポートされていました日本科学技術振興機構のCRESTからの補助金。

海馬歯状回の解剖<ol><li>深く麻酔マウスでは、慎重に頭蓋骨から脳を摘出し、氷冷リン酸緩衝生理食塩水（PBS）に配置します。 </li><li>氷冷したPBSを含むペトリ皿に、メスを用いて大脳縦裂に沿って脳を切断し、ラムダ（中脳、後脳、小脳）の後方の領域を切り取ります。 </li><li>大脳半球の内側面を上に配置し、鉗子を使用して、慎重に解剖顕微鏡下で間脳（視床と視床下部）を取り外します。これは、歯状回の可視化を可能にする、海馬の内側を公開します。歯状回では、それらの間のギャップによってアモンの角から区別される。海馬や周辺地域への損傷は、それがより困難に海馬歯状回を分離することになります。 </li><li>歯状回（歯状回とアンモン角の境界、図1）の両側に鋭い針先（例えば、27ゲージの針を）挿入し、隔離する海馬のsepto - 時間軸に沿って表面的に針をスライドさせ歯状回。 </li><li>針やピンセットを使用して分離海馬歯状回をピックアップし、サンプル管に入れてください。得られた歯状回の組織サンプルは、任意のアッセイのために直ちに使用するか、後で使用するための深い冷凍庫に保存することができます。 </li><li>同じ方法を使用して他の大脳半球から海馬歯状回を分離する。 </li></ol>定量的リアルタイムPCR 歯状回は、上記の方法を用いて単離され、残りの海馬は、野生型マウスからアモンのホーンのサンプルとして解剖した。 β-アクチン、TDO2、DSP、Mrg1bとTyro3のリアルタイムPCRは、以前は次の1に記載のように海馬歯状回とアンモン角の試料を用いて行われた。プライマー5&#39; - CTGGCGAGATCACGATGACGと5&#39; - AAGCTACGCTGTTGTCTAACCはTyro3ためMrg1b、およびGCCTCCAAATTGCCCGTCAと5&#39; - CCAGCACTGGTACATGAGATCAのために使用されていました。 マイクロアレイ解析 （ -マウスα- CaMKII + /）前述1に記載マイクロアレイ実験は、雄の野生型マウスとカルシウム/カルモジュリン依存性プロテインキナーゼIIのα-アイソフォームのためのヘテロ接合体マウスを用いて行った。簡単に言えば、全体海馬または野生型と変異マウスの海馬歯状回から単離されたRNAは、マウスゲノム430 2.0アレイ（Affymetrix社、サンタクララ、カリフォルニア州）とハイブリダイズさせた、とそれぞれのGeneChipはアフィメトリクスGeneChipスキャナー3000（GCS3000）によってスキャンされた。 GeneChip解析は、マイクロアレイ解析スイートバージョン5.0で実施した。

<ol>
	<li>Yamasaki, N., Maekawa, M., Kobayashi, K., Kajii, Y., Maeda, J., Soma, M., Takao, K., Tanda, K., Ohira, K., Toyama, K., Kanzaki, K., Fukunaga, K., Sudo, Y., Ichinose, H., Ikeda, M., Iwata, N., Ozaki, N., Suzuki, H., Higuchi, M., Suhara, T., Yuasa, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-CaMKII+deficiency+causes+immature+dentate+gyrus,+a+novel+candidate+endophenotype+of+psychiatric+disorders.">Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.</a> Mol. Brain. 1, 6-6 (2008).</li><li>Insausti, A. M., Megias, M., Crespo, D., Cruz-Orive, L. M., Dierssen, M., Vallina, I. F., Insausti, R., Florez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+neuronal+number+in+Ts65Dn+mice:+a+murine+model+of+Down+syndrome.">Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of Down syndrome.</a> Neurosci. Lett. 253, 175-175 (1998).</li><li>Redwine, J. M., Kosofsky, B., Jacobs, R. E., Games, D., Reilly, J. F., Morrison, J. H., Young, W. G., Bloom, 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=Dentate+gyrus+volume+is+reduced+before+onset+of+plaque+formation+in+PDAPP+mice:+a+magnetic+resonance+microscopy+and+stereologic+analysis.">Dentate gyrus volume is reduced before onset of plaque formation in PDAPP mice: a magnetic resonance microscopy and stereologic analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 1381-1381 (2003).</li><li>Lein, E. S., Zhao, X., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+molecular+atlas+of+the+hippocampus+using+DNA+microarrays+and+high-throughput+in+situ+hybridization.">Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization.</a> J. Neurosci. 24, 3879-3879 (2004).</li><li>Matsuo, N., Yamasaki, N., Ohira, K., Takao, K., Toyama, K., Eguchi, M., Yamaguchi, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+changes+underlying+the+working+memory+deficit+in+alpha-CaMKII+heterozygous+knockout+mice.">Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice.</a> Front. Behav. Neurosci. 3, 20-20 (2009).</li><li>Franklin, K. B. J., Paxinos, G. <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 Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

動物実験は、ケアと実験動物の使用のための健康ガイドの国立研究所にしたがって行われ、藤田保健大学で動物のケアと使用に関する委員会によって承認された。

歯状回は海馬体2,3の容積の約25％〜30％を占めている。それは、ユニークな細胞組成を有し、様々な脳機能において重要な役割を果たしている。したがって、歯状回を分離する技術は、この地域で特に発生するイベントを分析するのに便利です。 ここで、我々は効率的に成体マウスの海馬から海馬歯状回を分析する手順を明らかにし、技術の精度を確認した。最初に、組織学的研究は、歯状回は、純粋な歯状回のサンプルが準備できることを示す、他の地域で汚染（図1）に分離していることが判明した。 第二に、我々は、解剖組織は、歯状回特異的遺伝子、TDO2やDSP、そしてアモンの角に富んだ遺伝子、Mrg1bとTyro3 4（図2）のリアルタイムPCRを行うことにより、歯状回であることを確認した。 TDO2のmRNA発現（P = 0.000023であり、nの= 4、4、それぞれ）とDSP（P = 0.0000030; nのそれぞれ= 4、4、）歯状回のサンプルでは、明らかに高いレベルで検出されたのに対し、Mrg1b（P = 0.000080であり、nの= 4、4、それぞれ）とTyro3（P = 0.00017、nのそれぞれ= 4と4は、）アモンの角の試料のものよりも、低いレベルであった。 （; Nの= 4と4、 それぞれ 、p = 0.11）β-アクチンの発現レベルは、これらのサンプルでは差は認められなかった。従って、我々は、海馬歯状回が正確にこのような単純なリアルタイムPCR実験を行うことにより、外切開されたかどうかをチェックすることもできます。 この解剖法の有用性を評価するために、第三に、我々は、海馬歯状回のように全体の海馬のmRNA発現レベルを比較した。マウス（それぞれnの= 18、4、）マイクロアレイ解析のために処理され、すべての遺伝子が得点のために、フォールド - 野生型（Nの= 9、4、それぞれ）とα- CaMKII + /は、から得られる全体の海馬と歯状回変更は、野生型の値によって変異体値で割ることにより算出した。結果は、特にそのようなDSPとTDO2などの歯状回、特定の分子のmRNA発現の変化が、、全体の海馬サンプル（表1）と比較して歯状回の試料で感度が5倍に増加すると、最大検出されたことが示された。このようなワーキングメモリの赤字と誇張されたinfradianリズム1,5のような人間の精神疾患に関連したマウスの展示行動-我々は以前にα- CaMKII + /ことを示した。さらに、変異マウスの海馬歯状回の神経細胞の形態学的および電気生理学的特徴は、これらの変異マウスの神経細胞が成熟1に開発するために失敗したことを示す、通常のげっ歯類の未成熟歯状回の神経細胞のそれらと非常に似ています。未熟な歯状回とダウンレギュレートDSPの発現とα- CaMKIIのTDO2 mRNAは、+ / - マウスでは、DSPとTDO2は歯状回（大平らに成熟顆粒細胞のマーカーとして使用することができるという知見と一致している、未発表。データ）。 一緒になって、この便利で正確な解剖の技術は確実に歯状回に焦点を当てた研究にも使用できます。この方法を用いて得られた歯状回の組織では、プロテオミクス、細胞生物学の分析を含むだけでなく、分析の他のタイプに適用可能である。 <img src="/files/ftp_upload/1543/1543fig1.jpg" alt="図1" /> 図1組織学的研究により、孤立歯状回の検証。歯状回を分離後の脳の冠状断面をニッスル染色（左パネル）のために処理、およびマウスの脳atlas6から適応の模式図は、左側のパネル（右パネル）に示すようにセクションのほぼ同じレベルを表していた。矢印は針先端の挿入の方向を示している。スケールバーは1mm。 <img src="/files/ftp_upload/1543/1543fig2.jpg" alt="図2" /> 図2リアルタイムPCRにより単離した海馬歯状回の検証。 four野生型マウスから得られた海馬歯状回とアンモンの角はβ-アクチン、TDO2、DSP、Mrg1bとTyro3のリアルタイムPCRのために処理した。結果は平均± SEMとして表示されます。統計分析のために、学生のt検定が用いられた、とp値が続いている：β-アクチン、P = 0.11; TDO2、P = 0.000023（** 1）、DSP、P = 0.0000030（** 2）; Mrg1b、P = 0.000080（** 3）、及びTyro3、P = 0.00017（** 4）。 表1。 。全体の海馬と歯状回のマイクロアレイ解析遺伝子が差α- CaMKIIの歯状回と全体の海馬で発現する+ / -マウスは野生型マウスで検出されたことから倍の変化を計算することにより決定された。マウス-データは、野生型とα- CaMKII + /の間にスチューデントの t検定を用いて統計的有意性について分析した。その発現が&lt;0.05 の p を示した遺伝子の中でα- CaMKII + /の歯状回 - 野生型マウスのそれと比較してマウスは、トップ50の遺伝子が記載されています。歯状回のためのサンプル数が全体の海馬のものよりはるかに少ないことに注意してください。 AffyID、アフィメトリクスプローブ識別子、CKII、α- CaMKII + / - マウス、WT、野生型マウス。 <table border="1"><tbody><tr><td></td><td></td><td></td><td></td><td colspan="2">歯状回（P &lt;0.05） WT：N = 4、CKII + / - ：n = 4の</td><td></td><td colspan="2">全体海馬 WT：N = 9、CKII + / - ：n = 18と</td></tr><tr><td>遺伝子のタイトル</td><td>ジーンバンク</td><td> AffyID </td><td></td><td>倍の変化</td><td> p値</td><td></td><td>倍の変化</td><td> p値</td></tr><tr><td>デスモプラキン</td><td> AV297961 </td><td> 1435494_s_at </td><td></td><td> 0.011018913 </td><td> 7.02694E - 06 </td><td></td><td> 0.037021003 </td><td> 1.86126E - 13 </td></tr><tr><td>デスモプラキン</td><td> AV297961 </td><td> 1435493_at </td><td></td><td> 0.014369734 </td><td> 7.86747E - 06 </td><td></td><td> 0.04232106 </td><td> 1.00579E - 12 </td></tr><tr><td>トリプトファン2,3 - ジオキシゲナーゼ</td><td> AI098840 </td><td> 1419093_at </td><td></td><td> 0.020986484 </td><td> 5.23546E - 09 </td><td></td><td> 0.101037776 </td><td> 4.14823E - 13 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452106_at </td><td></td><td> 0.075479901 </td><td> 1.05191E - 08 </td><td></td><td> 0.234001154 </td><td> 1.66301E - 15 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452107_s_at </td><td></td><td> 0.079457767 </td><td> 1.40433E - 07 </td><td></td><td> 0.177974715 </td><td> 3.9758E - 12 </td></tr><tr><td>甲状腺刺激ホルモンは、ホルモン受容体をリリース</td><td> M59811 </td><td> 1449571_at </td><td></td><td> 0.103105815 </td><td> 0.003093796 </td><td></td><td> 0.801412732 </td><td> 0.283994361 </td></tr><tr><td>リアノジン受容体1、骨格筋</td><td> X83932 </td><td> 1427306_at </td><td></td><td> 0.104825517 </td><td> 3.38513E - 07 </td><td></td><td> 0.650685017 </td><td> 0.000308462 </td></tr><tr><td>不可知のヘリックスループヘリックス1 </td><td> NM_010916 </td><td> 1419533_at </td><td></td><td> 9.431896 </td><td> 6.7979E - 06 </td><td></td><td> 4.078815314 </td><td> 5.27E - 11 </td></tr><tr><td> copine家族メンバーIX </td><td> BB274531 </td><td> 1454653_at </td><td></td><td> 9.159157 </td><td> 7.99492E - 06 </td><td></td><td> 1.797304153 </td><td> 0.000296375 </td></tr><tr><td> doublecortinのようなキナーゼ3 </td><td> BB326709 </td><td> 1436532_at </td><td></td><td> 0.109336662 </td><td> 1.95278E - 07 </td><td></td><td> 0.56697229 </td><td> 2.62633E - 08 </td></tr><tr><td>カルパイン3 </td><td> AF127766 </td><td> 1426043_a_at </td><td></td><td> 0.111269769 </td><td> 8.07053E - 06 </td><td></td><td> 0.370956608 </td><td> 2.04421E - 14 </td></tr><tr><td>成人男性の線条体cDNAを、理研フルレングス濃縮ライブラリ、クローン：C030023B07製品：分類できない、完全なインサート配列</td><td> BB357628 </td><td> 1460043_at </td><td></td><td> 0.118712341 </td><td> 6.16926E - 07 </td><td></td><td> 0.682339204 </td><td> 2.33001E - 06 </td></tr><tr><td>コラーゲンとカルシウム結合EGFドメイン1 </td><td> AV264768 </td><td> 1437385_at </td><td></td><td> 0.124043978 </td><td> 3.65669E - 05 </td><td></td><td> 0.488394112 </td><td> 4.05538E - 06 </td></tr><tr><td>アミロイドベータ（A4）前駆体タンパク質結合、家族、メンバー2結合タンパク質</td><td> AK013520 </td><td> 1431946_a_at </td><td></td><td> 7.7986307 </td><td> 1.2098E - 06 </td><td></td><td> 2.099164713 </td><td> 1.67047E - 06 </td></tr><tr><td>カルビンジン- 28K </td><td> BB177770 </td><td> 1456934_at </td><td></td><td> 0.130255444 </td><td> 3.32186E - 06 </td><td></td><td> 0.572605751 </td><td> 1.99157E - 10 </td></tr><tr><td>転写された遺伝子座</td><td> AV328597 </td><td> 1443322_at </td><td></td><td> 0.133290835 </td><td> 5.43583E - 06 </td><td></td><td> 0.562767164 </td><td> 7.56544E - 06 </td></tr><tr><td>ニューロペプチドY受容体Y2 </td><td> NM_008731 </td><td> 1417489_at </td><td></td><td> 0.135319609 </td><td> 0.000113407 </td><td></td><td> 0.781498474 </td><td> 0.00394504 </td></tr><tr><td> RAS応答性エレメント結合タンパク質1 </td><td> BE197381 </td><td> 1428657_at </td><td></td><td> 0.138235114 </td><td> 7.93691E - 07 </td><td></td><td> 0.651220705 </td><td> 2.94209E - 05 </td></tr><tr><td>アルファ2神経栄養因子ファミリーの受容体に由来グリア細胞株</td><td> BB284482 </td><td> 1433716_x_at </td><td></td><td> 0.1390​​62563 </td><td> 2.35371E - 06 </td><td></td><td> 0.669544709 </td><td> 0.000214146 </td></tr><tr><td> preproenkephalin 1 </td><td> M13227 </td><td> 1427038_at </td><td></td><td> 6.9850435 </td><td> 2.39074E - 08 </td><td></td><td> 1.766018828 </td><td> 0.000250501 </td></tr><tr><td>理研cDNAを1810010H24遺伝子</td><td> BI729991 </td><td> 1428809_at </td><td></td><td> 6.8658915 </td><td> 1.88516E - 05 </td><td></td><td> 2.77573142 </td><td> 6.81865E - 09 </td></tr><tr><td>リアノジン受容体1、骨格筋</td><td> BG793713 </td><td> 1457347_at </td><td></td><td> 0.151364292 </td><td> 3.35612E - 05 </td><td></td><td> 0.503144617 </td><td> 4.32907E - 05 </td></tr><tr><td>プロト21 </td><td> NM_130878 </td><td> 1418304_at </td><td></td><td> 0.152671849 </td><td> 8.57783E - 06 </td><td></td><td> 0.670714726 </td><td> 1.56309E - 05 </td></tr><tr><td>コルニッションのホモ​​ログ3（ショウジョウバエ） </td><td> NM_028408 </td><td> 1419517_at </td><td></td><td> 0.153724144 </td><td> 8.90755E - 06 </td><td></td><td> 0.95780695 </td><td> 0.661055608 </td></tr><tr><td>腹切、BCL2相互作用するタンパク質（のみBH3ドメインを含む） </td><td> BQ175572 </td><td> 1439854_at </td><td></td><td> 0.154284407 </td><td> 2.0118E - 05 </td><td></td><td> 0.56516812 </td><td> 4.86925E - 09 </td></tr><tr><td>炭水化物（N -アセチルガラクトサミン4-0）硫酸転移酵素9 </td><td> AK017407 </td><td> 1431897_at </td><td></td><td> 0.155238951 </td><td> 5.37423E - 06 </td><td></td><td> 1.14910007 </td><td> 0.215637733 </td></tr><tr><td>カルパイン3 </td><td> AI323605 </td><td> 1433681_x_at </td><td></td><td> 0.160871988 </td><td> 1.07655E - 05 </td><td></td><td> 0.477164757 </td><td> 1.33753E - 11 </td></tr><tr><td>ジンクフィンガー、5を含むCCHCドメイン</td><td> BQ126004 </td><td> 1437355_at </td><td></td><td> 0.161812078 </td><td> 3.08262E - 06 </td><td></td><td> 0.421252632 </td><td> 0.01152969 </td></tr><tr><td>ロリクリン</td><td> NM_008508 </td><td> 1448745_s_at </td><td></td><td> 0.165129967 </td><td> 1.86362E - 05 </td><td></td><td> 0.639733409 </td><td> 0.000729772 </td></tr><tr><td> spondin 1、（F - spondin）細胞外マトリックスタンパク質</td><td> BC020531 </td><td> 1451342_at </td><td></td><td> 0.168035879 </td><td> 6.67867E - 07 </td><td></td><td> 0.821042412 </td><td> 0.023650765 </td></tr><tr><td>理研cDNAをA930035E12遺伝子</td><td> AV348640 </td><td> 1429906_at </td><td></td><td> 5.9086795 </td><td> 1.747E - 07 </td><td></td><td> 1.470383201 </td><td> 0.104085454 </td></tr></tbody><tbody><tr><td> BB247294理研フルレングス濃縮、7日新生児小脳ハツカネズミcDNAクローンA730018G18 3&#39;、mRNA配列。 </td><td> BB247294 </td><td> 1447907_x_at </td><td></td><td> 5.9047494 </td><td> 1.04931E - 05 </td><td></td><td> 1.968147585 </td><td> 0.00010636 </td></tr><tr><td> 3を含むFERMドメイン</td><td> BB099015 </td><td> 1437075_at </td><td></td><td> 5.860216 </td><td> 0.000345581 </td><td></td><td> 2.780297178 </td><td> 1.83072E - 06 </td></tr><tr><td&gt;神経細胞ペントラキシン2 / / /仮想タンパク質LOC100044234 </td><td> NM_016789 </td><td> 1420720_at </td><td></td><td> 5.7568517 </td><td> 1.34227E - 06 </td><td></td><td> 2.652516957 </td><td> 0.000206279 </td></tr><tr><td>転写配列</td><td> BG076361 </td><td> 1460101_at </td><td></td><td> 5.657735 </td><td> 2.5015E - 06 </td><td></td><td> 1.296248831 </td><td> 0.22870031 </td></tr><tr><td> spondin 1、（F - spondin）細胞外マトリックスタンパク質</td><td> BC020531 </td><td> 1424415_s_at </td><td></td><td> 0.17783576 </td><td> 1.01658E - 06 </td><td></td><td> 0.836181248 </td><td> 0.001380141 </td></tr><tr><td>カルビンジン- 28K </td><td> BB246032 </td><td> 1448738_at </td><td></td><td> 0.180317904 </td><td> 1.35961E - 05 </td><td></td><td> 0.647334052 </td><td> 4.12268E - 09 </td></tr><tr><td> MARCKS様1 </td><td> AV110584 </td><td> 1437226_x_at </td><td></td><td> 0.186235935 </td><td> 1.47067E - 06 </td><td></td><td> 0.499291387 </td><td> 2.34984E - 08 </td></tr><tr><td> matrilin 2 </td><td> BB338441 </td><td> 1455978_a_at </td><td></td><td> 0.187783528 </td><td> 6.19122E - 05 </td><td></td><td> 0.8967688 </td><td> 0.282337853 </td></tr><tr><td> matrilin 2 </td><td> BC005429 </td><td> 1419442_at </td><td></td><td> 0.188195795 </td><td> 0.000105295 </td><td></td><td> 0.915528892 </td><td> 0.35282097 </td></tr><tr><td> spondin 1、（F - spondin）細胞外マトリックスタンパク質</td><td> BQ175871 </td><td> 1442613_at </td><td></td><td> 0.189956563 </td><td> 9.41195E - 06 </td><td></td><td> 0.861033222 </td><td> 0.1394266 </td></tr><tr><td>アレスチン3、網膜</td><td> NM_133205 </td><td> 1450329_a_at </td><td></td><td> 5.2130346 </td><td> 2.90599E - 05 </td><td></td><td> 3.944218329 </td><td> 1.07437E - 07 </td></tr><tr><td>理研cDNAをA330050F15遺伝子</td><td> AV325555 </td><td> 1457558_at </td><td></td><td> 0.19186781 </td><td> 0.000119342 </td><td></td><td> 0.660282035 </td><td> 2.47342E - 05 </td></tr><tr><td>コンタクチン3 </td><td> BB559510 </td><td> 1438628_x_at </td><td></td><td> 0.194404608 </td><td> 4.08641E - 07 </td><td></td><td> 0.918742591 </td><td> 0.022545297 </td></tr><tr><td>カルビンジン- 28K </td><td> BB246032 </td><td> 1417504_at </td><td></td><td> 0.196381321 </td><td> 2.24182E - 05 </td><td></td><td> 0.619305124 </td><td> 3.6222E - 06 </td></tr><tr><td>ガストリン放出ペプチド</td><td> BC024515 </td><td> 1424525_at </td><td></td><td> 4.9436426 </td><td> 3.00588E - 05 </td><td></td><td> 2.752845903 </td><td> 5.72954E - 07 </td></tr><tr><td>受容体3を含むソルチリン関連VPS10ドメイン</td><td> AK018111 </td><td> 1425111_at </td><td></td><td> 4.885766 </td><td> 1.03645E - 05 </td><td></td><td> 1.29051599 </td><td> 0.029733649 </td></tr><tr><td>ドーパミン受容体D1A </td><td> BE957273 </td><td> 1455629_at </td><td></td><td> 4.869493 </td><td> 3.77525E - 05 </td><td></td><td> 1.815881979 </td><td> 0.000516498 </td></tr><tr><td>前駆タンパク質転換酵素サブチリシン/科新タイプ5 </td><td> BB241731 </td><td> 1437339_s_at </td><td></td><td> 0.210528027 </td><td> 7.83039E - 05 </td><td></td><td> 0.574126078 </td><td> 9.15496E - 05 </td></tr></tbody><tbody><tr><td>インターロイキン1受容体は、私のように入力します</td><td> NM_008362 </td><td> 1448950_at </td><td></td><td> 0.210572243 </td><td> 9.64524E - 06 </td><td></td><td> 0.241135352 </td><td> 2.79816E - 08 </td></tr></tbody></table>

dissection of hippocampal dentate gyrus from adult mouse

海馬は、メモリの処理と学習におけるその重要な機能的役割、その顕著な神経細胞の可塑性、およびてんかんへの関与、神経変性疾患、精神疾患の脳内で最も広く研究されている地域の一つです。海馬は、明確な領域で構成され、主に顆粒ニューロン、および主に錐体細胞を含み、アモンの角、、と二つの領域を含み、歯状回は、解剖学的および機能的な回路の両方で接続されています。多くの異なるmRNAとタンパク質が選択的に海馬歯状回に発現しており、歯状回では、成体のニューロン新生のサイトですされ、つまり、新しいニューロンは継続的に成人歯状回で生成されます。歯状回への特定のmRNAとタンパク質発現を調べるために、レーザーキャプチャーマイクロダイセクションが用いられることが多い。このメソッドは、そのような特別な装置や複雑な処理手順の必要性として、しかし、いくつかの制限があります。このビデオに記録されたプロトコルでは、我々は実体顕微鏡下で成体マウスから海馬歯状回を削除するための解剖手法を示しています。この手法を用いて調製した海馬歯状回のサンプルでは、​​トランスクリプトームプロテオーム、細胞生物学の分析を含む、任意のアッセイに適しています。我々は、解剖組織は、トリプトファン2,3 - ジオキシゲナーゼ（TDO2）とデスモプラキン（DSP）、およびアモンの角に富んだ遺伝子、メイス関連遺伝子型1b（Mrg1b、歯状回特異的遺伝子のリアルタイムPCRを行うことにより、歯状回であることを確認）とTYRO3タンパク質チロシンキナーゼ3（Tyro3）。 Mrg1bとTyro3はアモンの角の試料のものよりも、低いレベルであったのに対し、歯状回のサンプルでTDO2とDSPのmRNA発現は、明らかに高いレベルで検出された。この方法の利点を実証するために、我々は全体の海馬と歯状回のサンプルを使用して、DNAマイクロアレイ解析を行った。 α- CaMKII + /の全体の海馬では、歯状回で選択的に発現しているTDO2とDSPのmRNA発現、 - マウスは、それぞれ0.037および野生型マウスのそれに比べて0.10倍の変化を、示した。孤立歯状回では、しかしながら、これらの式は、より高い感度で検出することができる歯状回におけるその遺伝子発現の変化を示す、0.011と野生型マウスのそれに比べて0.021倍の変化を示した。一緒になって、この便利で正確な解剖の技術は確実に歯状回に焦点を当てた研究にも使用できます。

Watch this Scientific Journal Video about Dissection of Hippocampal Dentate Gyrus from Adult Mouse at JoVE.com

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

実体顕微鏡下で成体マウスから海馬歯状回の除去のための解剖手法は、このビデオに記録されたプロトコルで実証された。

成体マウスから海馬歯状回の解剖

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

dissection-of-hippocampal-dentate-gyrus-from-adult-mouse

Research

JoVE Journal

Biology

82.2K ビュー.  Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST). 実体顕微鏡下で成体マウスから海馬歯状回の除去のための解剖手法は、このビデオに記録されたプロトコルで実証された。

ビデオ: Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPCのマウス胚の解剖

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

生物学

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

アダルトゼブラフィッシュからの臓器の解剖

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

大人からOenocytesの解剖キイロショウジョウバエ</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

アダルトゼブラフィッシュ腎臓の解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

分離と性質、大人マウス心臓から心臓の幹細胞の分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

成人マウス口蓋からの一次口腔角化細胞の分離と培養

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.