Name: conditional genetic transsynaptic tracing in the embryonic mouse brain
Uploaded: 2014-12-22T14:00:00.000+00:00
Duration: 11 min 3 s
Description: Watch this Scientific Journal Video about Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain at JoVE.com

We thank Michael Candlish for critical comments on the manuscript. This project was supported by the Deutsche Forschungsgemeinschaft grants BO1743/6 and SFB/TRR 152 P11 and Z02 to Ulrich Boehm.

注：倫理声明：動物を対象とする手順は、ハンブルク大学とザールラント大学の動物福祉委員会によって承認された。 1.準備と胚組織の固定<ol><li>胚を解剖し、動物を犠牲にする前に、組織のその後の固定のためのソリューションを準備するために必要なすべての機器を配置します。 注：常に（pHを7.4に調整し、0.1 Mリン酸緩衝生理食塩水（PBS）中4％PFA）新鮮な4％パラホルムアルデヒド（PFA）溶液を調製する。 </li><li>倫理的に承認された手順とタイミングを妊娠したメスのマウスを安楽死させる。 </li><li>プラットフォーム上にマウスを転送します。 70％エタノールで腹部の腹側を湿らせ、腹腔を露出させ、鋭いハサミを使って垂直切開する。 </li><li>胚チェーンを特定し、慎重に鈍鉗子を使用してそれを引き出し、氷冷PBSにそれを配置します。 </li><li>から個々の胚を削除する細かいハサミとピンセットを用いて、胚チェーン。胚の周りに余分な組織を除去し、氷冷PBSでそれらを洗ってください。 </li><li> （PFAに胚を転送する前に）小さな尾の生検を取り、性別の識別とトレーサー対立遺伝子の遺伝子型決定PCR用テール溶解バッファーでそれをインキュベートする。 </li><li>ほとんどの側面に胚か​​ら開始し、シェーカー上で氷の上の氷冷4％PFAを含むチューブに別々に各胚を移す。 </li><li>一定に振盪しながら氷上で1.5時間E13.5歳以下の年齢で胚を浸す。一定に振盪しながら氷上で2.5時間、年齢E14.5とE15.5で胚を浸す。 </li><li>胚の年齢E16.5歳以上の場合は、首を切るとPFA溶液に頭を浸漬する前に、外側の皮膚を除去。 E16.5胚の頭部が一定に振盪しながら氷上で4.5時間浸漬することができます。 </li><li>適切な固定後、氷冷PBSで胚を3回洗浄する。それらは沈むまで4℃でPBS溶液中の30％スクロース中に胚を移し下へ。 </li></ol> 2.凍結<ol><li>クライオ金型、クライオ手袋、フェザー級昆虫学鉗子、マーカーペン、最適切削温度化合物（OCT）：凍結プロセスを開始する前に必要なすべての材料を配置</li><li> （組織、日付と遺伝子型の向きに言及）恒久的なアルコール耐性マーカーでクライオモールドラベルを付けます。砕いたドライアイスのスラッシュとアイスバケットで100％エタノールを準備します。イソペンタンを含む内部のガラスビーカーを置きます。イソペンタンを冷却するために10分間待ちます。 </li><li>一方、ショ糖溶液から組織を除去する実験室のワイプを用いて組織の周りに余分なショ糖を拭き取る。組織をカバーするために十分な10月で予め標識低温金型に組織を移す。 10月中に気泡を避ける。特に組織の周りに。 </li><li>組織の損傷を避けるために、フェザー級昆虫学の鉗子を使用して、OCTで組織を向けます。クライオモルを転送することによって、凍結開始予め冷却したイソペンタン浴中dは。それが適切に凍結しないように10月にイソペンタンをかけないでください。 -80℃でアルミ箔とストア内のサンプルを包みます。 </li></ol> 3.凍結切片化<ol><li>クライオスタットを用いて、5のシリーズの中で14μmの薄い連続切片をカットし、免疫蛍光（IF）分析のためのSuperFrost®プラスガラススライド上に収集する。使用するまで-80℃でスライドを保管してください。 </li></ol>チラミドシグナル増幅（TSA）プロトコルを使用して4トレーサーの可視化<ol><li>染色に必要なバッファーと試薬を準備します。常に新鮮に利用当日にトリスNaClのトゥイーン（TNT）のバッファを準備します。 <ol><li> 1 Lの最終容量までのNaClとのddH 2 O 5の1 Mトリス塩酸pH7.5の、30ミリリットルの100ミリリットルを使用して、TNT緩衝液を準備する1ミリリットルピペットを用いてのTween 20の500を添加する。すべてのTween 20であることを保証するためにゆっくりとツイーン20を加え、上下に数回ピペットをフラッシュソリューション。 </li><li>ブロッキング試薬5グラムを追加1 Lの最終容量まで1 Mトリス塩酸pH7.5の、30のNaClとのddH 5 mlの2 Oの100ミリリットルを使用してトリスのNaClブロッキング（TNB）のバッファを準備します（付属キット）をゆっくりバッファに少しずつ攪拌しながら。 （これは30〜60分より長い時間がかかるべきではありません）完全にブロッキング試薬を溶解するために連続的に撹拌しながら55℃まで徐々にソリューションを熱し。均質な加熱は55℃の水浴を使用し達成するために。ソリューションは、乳白色に表示されます。使用する前に室温にソリューションを持参してください。長期保存のために-20℃で小分けし、保存。 </li><li>分子生物学/ HPLCグレードDMSO中（キットに付属）ビオチンチラミド試薬を再構成する。これに応じて、キットは、DMSOの適切な量は変化し得るために使用される。 4℃で保存溶液を保管してください。 注：それぞれのCreおよびR26対立遺伝子についてヘテロ接合性動物からの切片は、回路解析のために使用することができる。使用Sネガティブコントロールとして、それぞれ1 のCreまたは1 R26対立遺伝子を運ぶ同腹仔からections、。正確にダブルヘテロ接合動物からのスライドのようなネガティブコントロールスライドを扱います。また、ダブルヘテロ接合動物から一つの制御スライドを含むが、TSA試薬（増幅されていないコントロール）を除外。 </li></ol></li><li>鋭い新鮮なメスは、組織切片の周囲の過剰なOCTを除去し、PAPペンでセクションの周りに境界をマークする使用。 </li><li>室温（RT）で2〜3分間、スライドを乾燥させる。 5分ごとにTNTでRT 3Xでスライドを洗う。内因性ペルオキシダーゼ活性をクエンチし、氷冷メタノール中で0.3％H 2 O 2溶液中に室温で30分間スライドをインキュベートする。 </li><li>室温で5分間ごとに、PBSで洗浄3X。組織透過性のためにPBS中0.5％トリトンX-100で10分間インキュベートする。 5分ごとにTNTでRT 3Xでスライドを洗う。 </li><li> TNBとのブロック30（スライドあたり、200〜300μlのブロッキング溶液）加湿チャンバー中室温で分。すべてのセクションが完全にTNBでカバーされていることを確認してください。スライドはステップ間で完全に乾くせてはいけない。 </li><li>過剰のブロッキングバッファーを切るとトレーサー認識する一次抗血清を適用するRTまたは一晩、4℃で2時間、TNB中に希釈し（1：1,000ヤギ抗WGA BLのため、1 15000ウサギGTT抗GFP）を。完全に組織切片（スライドあたり200〜300μl）をカバーするために十分なボリュームを使用してください。 注：ここで推奨プライマリ抗血清希釈液を調整しなければならないことがある。 </li><li> 5分で攪拌しながらそれぞれのTNTでRT 3Xでスライドを洗う。 </li><li> （：抗WGAのための500ウマ抗ヤギIgG、1：RTで1時間TNBにおける抗GFPのために5000ヤギ抗ウサギIgG 1）最初の抗血清のホストを認識し、ビオチン化二次抗血清でのセクションをインキュベートします。 </li><li> 5分で撹拌しながら、各RTでTNTを洗浄3X。 100ストレプトアビジン（SA）結合西洋ワサビペルオキシダーゼ（SA-HRP、提供のWi：1でインキュベートする加湿チャンバー内で室温で30分間、TNB中キット）番目。 5分ごとにTNTでRT 3Xでスライドを洗う。一方、TSAを解凍。 </li><li> （キットに付属、BLの可視化とGTTのためのTSA +キット用TSAキットを使用）TSA増幅希釈剤中TSA 1:50に希釈する。 RTで正確に10分間、希釈したTSAでの組織切片をインキュベートします。使用直前にTSAを希釈し、理想的に前の洗浄工程の間に、将来の実験のために、残りの希釈されたTSA試薬を使用しないでください。常に新鮮な準備。 </li><li> 5分で攪拌しながらそれぞれのTNTでRT 3Xでスライドを洗う。 RTで30分間TNBで500のAlexa Fluor（登録商標）546ストレプトアビジンコンジュゲート：1でインキュベートする。 5分で攪拌しながらそれぞれのTNTでRT 3Xでスライドを洗う。 </li><li>核染色のために室温でPBS中の5％ビスベンズイミド溶液中で10分間インキュベートする。 5分で攪拌しながらそれぞれのTNTでRT 3Xでスライドを洗う。 Fluromount Gでマウントし、エピ蛍光または共焦点顕微鏡法を行う。 <em&gt;トラブルシューティング：トレーサー転送の有効性は、（使用のCreドライバ線によって決定される）を産生する細胞におけるROSA26遺伝子座の発現レベルに依存し、神経回路上（トレーサー、収束等の産生ニューロン数など ）を分析します。したがって、ここに推奨1次抗血清の希釈は、低または過剰な信号を防止するために調整されなければならない。背景は、常に適切な対照スライド（上記参照）を使用して分析されるべきである。 </li></ol>胚セクション5.τlacZ染色<ol><li>染色に必要なバッファーと試薬を準備します。常に新鮮に使用前のLacZバッファーCを準備します。 <ol><li> 1ミリリットルの70％ジメチルホルムアミド（DMF）のX-galを40gのを添加することにより5-ブロモ-4-クロロ-3-インドリルβ-D-ガラクトピラノシド（X-galの）ストック溶液（40 mg / mlの）を調製。長期保存のために-20℃でアルミホイルや店舗で覆い。 </li><li>準備し、ニトロブルーテトラゾリウム（NBT）番目1ミリリットルの70％DMFにNBTを50g添加することによりOCK溶液（50 mg / mlで）。長期保存のために-20℃でアルミホイルや店舗で覆い。 </li><li> LacZの25％グルタルアルデヒドを80μl、4％PFAの5ミリリットル、1 MのMgCl 2、最終的に最大500のEGTAを100μl、1ミリリットルM 1のリン酸緩衝液pH7.4とのddH 2 O20μlのを使用して固定液の準備容量10ml。 </li><li> LacZを500 mMのEGTAを用いて、1 mlの1 MのMgCl 2、5ミリリットル、500ミリリットルの最終容量までの1 Mリン酸緩衝液（pH7.4）とのddH 2 Oを50mlのバッファ準備する。 </li><li> 1mlのMののMgCl 2、500 mMのEGTA 5mlの5mlの1％デオキシコール酸ナトリウム、10％のNP-40を100ml、50 1 Mリン酸緩衝液（pH7.4）の溶液とのddH 2 Oを使用してLacZのバッファBを準備500ミリリットルの最終容量まで。 </li><li>使用直前に新鮮な作り、アルミホイルで覆ったLacZ緩衝液Bの1mlにNBTストック溶液10μlおよびX-galストック溶液12.5μLを添加することにより、LacZの緩衝液Cを調製する。 </li></OL&gt; </li><li> （その間PAPペンでスライドを準備する）は、少なくとも10〜15分間室温でスライドを乾燥させます。 </li><li> LacZのセクションでは、化学ヒュームフード内部の加湿チャンバー内で室温で2分間固定液固定する。 PBSで3回洗浄を、2mMのMgCl 2を補った。 </li><li> LacZを持つスライドを室温で10分間、3回ずつ洗浄緩衝液のLacZ緩衝液Aでスライドを洗浄します。 5分間ずつのLacZ緩衝液Bで2回洗浄する。加湿チャンバー内で6時間または一晩30〜37℃でのLacZ緩衝液Cでスライドをインキュベートする。 注：LacZのバッファCの十分なボリュームを追加します。スライドは、インキュベーション中に完全に乾くべきではありません。 </li><li>適切なインキュベーションの後、PBSでスライドを洗浄は、2のMgCl 2を補った。のddH 2 Oで簡単にセクションをすすぐマイヤーのグリシンゼラチンでカバースリップ。微分干渉コントラスト（DIC）イメージング·セットアップを搭載した光学顕微鏡に適しています。 </li></ol> 6. GENDERおよびトレーサージェノタイピング<ol><li> 10ミリリットルの最終容量を補うために、1Mトリス塩酸pH8.5の、500 mMのEGTA、10％SDS、5 M NaClおよびのddH 2 O400μlの200μlの100μlを1ミリリットルを使用してテール溶解バッファーを準備します。 </li><li>尾の生検を含む各エッペンドルフチュー​​ブにテール溶解バッファー500μlのを追加します。を100mg / mlの最終濃度にプロテイナーゼKを加える。 55℃でシェーカー上で一晩インキュベートする。 </li><li> RTで10分間、17000×gで遠心分離サンプル。新しい遠心チューブに上清を移し。上清に500μlのイソプロパノールを加える。アップ及びダウンチューブを反転させて、少なくとも5分間混ぜる。 </li><li> RTで5分間17000×gで遠心機でスピン。上清を注ぐ。 70％エタノール200μlを追加します。少なくとも5分間振る。 </li><li> 5分間17000×gで遠心分離する。ピペットマン（オフかけないでください）​​で上清を取り除きます。 15〜30分間のウォーム室（37℃）でペレットを乾燥させる。 </li><li> 100μlの再懸濁ペレット低TE pHが8.0または水の広口径フィルターチップを使用。適切な溶解に一晩で1時間、55℃に入れ、37℃で。 4℃で保存。 </li><li> PCR反応（尾DNA1μlの、各プライマー2.5μlのDMSO、10 PM、25の各dNTPμM、及び1Mベタインを含む50μlの反応混合物）のためのDNA1μlのを使用してください。ジェノタイピングと性別識別のためのPCRプライマーは、 マテリアルの表に記載されている。 </li></ol>

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No conflict of interest declared.

遺伝的に定義されたニューロン集団の神経回路をトレースする導入遺伝子として経シナプストレーサーを発現するトレーサーまたはneurotopicウイルスの定位注射と比較していくつかの利点を有する。まず、トレーサーは、内因性タンパク質として産生されるので、任意の免疫応答を誘発しないと選択的な神経経路は、再現性の高い別の動物で分析することができる。これは、非侵襲的な方法で...

解剖パストレースは、脳と行動1との間の関係を解読するための最も一般的に使用さツールの一つです。神経回路のトレース技術の進歩は、マウス2における遺伝的に特定されニューロン集団から神経回路をトレースする能力を持つ神経科学を授けている。これらの技術的進歩にもかかわらず、それは特に、胚成熟中の神経回路の形成を解明するために依然として厳しい。現在までに開発さトレースの方法の多くは経シナプストレーサーまたは遺伝的に改変された神経向性ウイルスの定位注射（ 図1）2,3に基づいているためである。これらの技術は、発達中の脳、注射部位の再現性、注射部位の電位の炎症と最もimporに、接続性の空間的および時間的分解能を達成するような技術的に困難なトレーサー注射などのいくつかの固有の制限ものの神経向性ウイルスによるtantlyの細胞毒性は、それらの使用4を制限する。 別の方法は、遺伝子改変マウスでの導入遺伝子として経シナプストレーサーを表明することにある。我々は最近、この手法を変更し、すべての遺伝的に特定されニューロン集団5の神経回路をマッピングするために、バイナリ遺伝経シナプストレーシングシステムを開発した。 Cre介在した後に私たちの実験的な戦略は、双方向のトレーサー大麦レクチン（BL）6またはROSA 26座からGFP（GTT）7に融合した逆行性トレーサー破傷風毒素フラグメントCのいずれかを発現する二つの新しいノックインマウス系統に基づいており、再結合。ここでは、選択的にキスペプチンを作る神経細胞のBLとGTT、生殖軸8,9の成熟の調節に関与している神経ペプチドを表現するために、これらのマウス系統を使用していました。我々は、この技術は、キスの発達および成熟を可視化するために適していることを証明する雌マウスの脳5の胚発生時にpeptin神経回路。 育種戦略 R26-BL-IRES-τlacZ（BIZ）とR26-GFP-TTC（GTT）トレーサーラインがノックインされた組換えROSA26対立遺伝子を運ぶ菌株5。 R26-BIZとR26-GTT対立遺伝子は、2つのloxP部位5が隣接している強力な転写停止信号の存在に転写的に沈黙している。 BIZとGTT導入遺伝子の発現は、転写停止信号のCre媒介除去により活性化される。 R26-BIZとR26-GTT対立遺伝子は、単にのCreドライバラインと交配することによって独立して使用することができる。分析動物についてそれぞれCreおよびR26対立遺伝子についてヘテロ接合を使用することができる。 1 のCreまたは1 R26対立遺伝子を有する同腹仔は、それぞれ、対照として使用されるべきである。あるいは、トンを生成することも可能であるノックインriple のCre、R26-BIZとR26-GTT対立遺伝子を有する動物が、これは一つの追加のクロスが必要になります。

<table><tbody><tr><td>Bisbenzimide (Hoechst 33258 dye)</td><td>Sigma</td><td>14530-100MG</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>32205-1L</td><td></td></tr><tr><td>Cryo mold (Peel-a-way)</td><td>Polyscience Inc.</td><td>18646A-1</td><td>22 x 22 x 20mm</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418-100ML</td><td></td></tr><tr><td>Dimethyl Formamide (DMF)</td><td>VWR Chemicals</td><td>23470,293</td><td></td></tr><tr><td>EGTA</td><td>ROTH</td><td>3054.3</td><td></td></tr><tr><td>Fluoromount G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G5882-50ML</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma</td><td>34988-7</td><td></td></tr><tr><td>Isopentane (Methyl 2-butane)</td><td>Sigma</td><td>M32631-2.5L</td><td></td></tr><tr><td>Kaiser&#039;s glycine gelatin</td><td>Merck</td><td>1092420100</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>494437-1L</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M2670-100G</td><td></td></tr><tr><td>NaCl</td><td>ROTH</td><td>HN00.2</td><td></td></tr><tr><td>NBT</td><td>Sigma</td><td>298-83-9</td><td></td></tr><tr><td>Nonidet P40 substitute</td><td>Fluka</td><td>743.85</td><td></td></tr><tr><td>OCT</td><td>Leica</td><td>14020108926</td><td></td></tr><tr><td>PAP pen</td><td>Dako</td><td>S2002</td><td></td></tr><tr><td>Parafarmaldehyde</td><td>Sigma</td><td>P6148-1KG</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>D6750-25G</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903-1KG</td><td></td></tr><tr><td>Superfrost slides</td><td>Thermo Scientific</td><td>FT4981GLPLUS</td><td></td></tr><tr><td>TSA kit</td><td>PerkinElmer</td><td>NEL700</td><td></td></tr><tr><td>TSA plus kit</td><td>PerkinElmer</td><td>NEL749A001KT</td><td></td></tr><tr><td>Tris</td><td>ROTH</td><td>AE15.2</td><td></td></tr><tr><td>Triton-X 100</td><td>ROTH</td><td>3051.2</td><td></td></tr><tr><td>Tween 20</td><td>ROTH</td><td>9127.1</td><td></td></tr><tr><td>X-gal</td><td>ROTH</td><td>2315.1</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>na</td><td></td></tr><tr><td>Light microscope equipped with DIC imaging</td><td>Zeiss</td><td>Axioskop2 equipped with Axio Vision software</td><td></td></tr><tr><td>Fluroscence microscope</td><td>Zeiss</td><td>Axioskop2 equipped with Axio Vision software</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td>PS6</td><td></td></tr><tr><td>Goat anti-WGA (recognizes BL)</td><td>Vector Laboatories</td><td>AS-2024</td><td></td></tr><tr><td>Biotinylayted horse anti-goat IgG</td><td>Vector Laboatories</td><td>BA-9500</td><td></td></tr><tr><td>Biotinylated goat anti-rabbit IgG</td><td>Vector Laboatories</td><td>BA-1000</td><td></td></tr><tr><td>Rabbit anti-GFP (recognizes GTT)</td><td>Invitrogen</td><td>A11122</td><td></td></tr><tr><td>Rabbit anti-GnRH</td><td>Affinity Bio Reagent</td><td>PA1-121</td><td></td></tr><tr><td>Dylight488-donkey anti-rabbit IgG</td><td>Thermo Scientific</td><td>SA5-10038</td><td></td></tr><tr><td>SA-Alexa Fluor 546</td><td>Life Technologies</td><td>S-11225</td><td></td></tr><tr><td colspan="4">Primers</td></tr><tr><td>BL Fwd (for BIZ genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>ATGAAGATGATGAGCACCAGGGC</td></tr><tr><td>BL Rev (for BIZ genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>AGCCCTCGCCGCAGAACTC</td></tr><tr><td>Cre Fwd (for Cre genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>GTCGATGCAACGAGTGATGAGGTTCG</td></tr><tr><td>Cre Rev (for Cre genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>CCAGGCTAAGTGCCTTCTCTACACCTGC</td></tr><tr><td>TTC Fwd (for GTT genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>AGCAAGGGCGAGGAGCTGTT</td></tr><tr><td>TTC Rev (for GTT genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>GTCTTGTAGTTGCCGTCGTCCTTGAA</td></tr><tr><td>XY Fwd (for gender genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>TGAAGCTTTTGGCTTTGA</td></tr><tr><td>XY Rev (for gender genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>CCGCTGCCAAATTCTTTG</td></tr><tr><td>ROSA26 Fwd</td><td>Eurofins MWG Operon</td><td></td><td>CGAAGTCGCTCTGAGTTGTTATC</td></tr><tr><td>ROSA26 Rev</td><td>Eurofins MWG Operon</td><td></td><td>GCAGATGGAGCGGGAGAAAT</td></tr><tr><td>SA Rev</td><td>Eurofins MWG Operon</td><td></td><td>CGAAGTCGCTCTGAGTTGTTATC</td></tr></tbody></table>

conditional genetic transsynaptic tracing in the embryonic mouse brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

このセクションでは、R26-BIZ（B L-I RES-τlacのZ）とR26-GTT（G FP-TT C）の対立と協力を得ることができる代表的な結果を示している。ここでは、生殖軸を調整する神経回路の成熟を ​​分析するためにR26-BIZとR26-GTT対立遺伝子を使用しています。脊椎動物で複製することは、中央ゴナドトロピン放出ホルモン（GnRH）を分泌する視床​​下部ニューロンの?...

Watch this Scientific Journal Video about Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain at JoVE.com

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

マウス胎児の脳のトレース条件付き遺伝経シナプス

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

conditional-genetic-transsynaptic-tracing-in-the-embryonic-mouse-brain

Research

JoVE Journal

Neuroscience

18.6K ビュー.  University of Saarland School of Medicine. Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution. 

ビデオ: Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

ウイルスベースのCre組換えによる出生後のマウスの脳の発達における遺伝子機能のモザイク解析

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

神経科学

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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

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

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

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

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

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

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

遺伝的に定義された神経回路のトレースウイルス

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

マウスの遺伝子操作は、視床下部を介して開発<em&gt;子宮内</em&gt;エレクトロポレーション

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

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

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

発生中のマウス胚性皮質における有糸分裂のライブイメージング

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

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

二光子<em&gt; in vivoで</em&gt;間引き頭蓋骨の準備を使用して、マウス皮質における樹状突起棘のイメージング

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

脳の外植片にレーザー誘導神経トレース

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング<em&gt;ウテロの</em&gt;エレクトロポレーション

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

マウス胎児の脳のトレース条件付き遺伝経シナプス

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ウイルスベースのCre組換えによる出生後のマウスの脳の発達における遺伝子機能のモザイク解析

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作

遺伝的に定義された神経回路のトレースウイルス

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

マウスの遺伝子操作は、視床下部を介して開発

発生中のマウス胚性皮質における有糸分裂のライブイメージング

二光子

脳の外植片にレーザー誘導神経トレース

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング