この作品は、国家自然科学基金、中国の (31201086、31470759、31671219) と上海自然科学財団 (12ZR1436900) からの補助金によって支えられました。

c57bl/6 マウス特定病原体フリーの条件で飼育しています。すべての動物実験、動物実験、第二軍医大学の倫理委員会で承認されたガイドラインに準拠しています。
 1。 郭清の生後開発下垂 <ol><li>実行麻酔: 低体温症を誘導するために砕いた氷を新生児マウス (P0 - P5) を配置。マウスの年齢の 5 日間以上、麻酔を誘導するために 5% ウレタン (体重の 30 μ L/g) を腹腔内注入します。テール/足指つまみレスポンスを評価します。マウスが有害な刺激への応答後にのみ続行します</li>。
	<li>そっと前足をテーピングで (上向きの顔、背中に横たわって) 仰臥位でマウスをセキュリティで保護し、あることが作業面にグルタミ固定化学発煙のフードの中に (すなわち、発泡スチロール).</li>
	<li>Transcardial 潅流を行う。
	<ol><li>マウス胸部を中心 (やその他の胸部臓器) を公開する手術ハサミで切っています</li>。
		<li>は鈍い鉗子との心臓の鼓動をセキュリティで保護、26 G (0.45 mm) 針を左心室に挿入します。針が満ちているヘパリン リン酸緩衝生理食塩水 10 mL のシリンジに接続されている (PBS; 亜硝酸ナトリウム 5 mg/mL と 10 U/mL のヘパリンを添加した、pH 7.4 では、使用する前に 37 ° C に暖かい)。すぐに高級ハサミで右房をカットし、右心房を出る流体がクリアされるまで、温かみのある PBS で体を灌流を開始 。
		注: おおよそ 5-10 mL の PBS を新生児マウスに必要な大人のマウスの 10-20 mL PBS</li>。
		<li>の場所で針し 4% パラホルムアルデヒド (PFA; pH 7.4) で満たされた別の注射器に変更します。10 mL と 20 mL の定着剤を灌流新生児と P21 マウスそれぞれ 。
		注意: PFA は有毒です。それは、皮膚と呼吸器の炎症、目の損傷を引き起こすかもしれません。手袋を着用し、化学のフードの下で動作します</li>。
	</ol></li> <li>PFA 固定マウスの首をはねるし、頭蓋骨骨ハサミで切り開いて。
	<ol><li>は、小さな鉗子で頭蓋底から後脳を慎重に持ち上げます。トルコ鞍の最初の兆候で停止解除しますが、保持する菱脳、下垂体茎と高級ハサミで脳の基部に接続する神経線維に切る。下垂体は脳と離れて解除されていないことを確認重要なこの手順です</li>。
		<li>は、脳を持ち上げておいてし、下垂体腺を完全に公開する脳全体を削除します。横の三叉神経下垂体など全体セッラ地域をカットとハサミ (約 3 × 5 mm 2) で蝶形骨下</li>。
	</ol></li> <li>35 mm ディッシュや冷たい 4 %2 mL を含む 6 ウェル プレートの 1 つの井戸に切り裂かれたティッシュを入れて PFA (pH 7.4)。P0 - 脳下垂体 P7、P14 脳下垂体、P21 - P28 脳下垂体および大人の脳下垂体の 3 h の 1.5 h の 1 h の 40 分のための 4 ° C で組織を修正します</li>。
	<li>下垂体のさらに解離 <ol><li>洗浄 10 ml の PBS、固定ティッシュ 5 変更、各 15 分。その周囲の組織とともに新生児の下垂と蝶形骨の下には、脱水症状に直接処理できます。5 日間よりも古いマウスから下垂体腺を分離する必要がありますただし、実体顕微鏡下で更なる</li>。
		<li>は、1 mL の PBS を含む 35 mm ディッシュに固定組織を置きます。P5 の-P14 脳下垂体の微細鉗子、はさみ、骨と神経の結合膜を削除し、慎重に鞍を残して、全体として一緒に横方向の三叉神経下垂を分離します。結合膜による分離腺と神経が接続されている、&#34; H &#34;-外観 ( 図 1 b) を希望します</li>。
		<li>の P21 および大人脳下垂体神経および下垂体周囲の結合膜を削除し、周囲の組織から腺を無料します。レンズ クリーニング ティッシュ ペーパーと下垂体組織をラップし、埋め込みカセットに入れて 。
		注: 折り返しの小さな下垂体でレンズのティッシュ ペーパーを洗浄標本の潜在的な損失を防ぐため、パラフィン包埋手順で組織の整合性を維持するのに役立ちます。それは低温埋め込み手順で小瓶で処理される場合、下垂体をラップする必要がない可能性があります</li>。
	</ol></li></ol> 2。埋め込みと押しますパラフィン <ol><li>脱水 15 分 50%、65%、75%、95% のエタノール溶液でカセットを埋め込むことで標本。P21 と古い腺は、純粋なエタノール、P0 - の 3 分の 3 つの変更によって腺 P4、P5 の-P14 腺と各 5 分の 4 分を脱水後。キシレンと明確 3、P0 から各 3 分腺 P4、P5 の-P14 腺と各 5 分の 4 分の変更 P21 および古い腺。P0 - P4 腺の溶融パラフィン ワックス、3 変更、7 分に潜入し、8 分 2 回続いて 6 分一度 P5 の古い腺 。
	注: ティッシュの処理のパラメーターを最適経験的調整できます。高品質のセクションを得るためには、十分以上の脱水は避けてください。同じはキシレンを使用して組織をクリアするためです</li>。
	コンソール システムを埋め込み組織に 埋め込み時の向き <ol><li><li>カセットから取り外し、溶融パラフィン ワックスで半分いっぱい基本金型に入れます。埋め込みの下垂体の適切な向きはコロナ セクションを満たすため不可欠です</li>。
		<li>の P21 および成人下垂体腺は、その短い軸基本型 (断面) の底面に垂直に下垂を配置します。蝶形骨と三叉神経解剖学的ランドマークとして P0 - P4 と P5 の-P14 脳下垂体をそれぞれ使用します。三叉神経または基本型の底面に垂直な蝶形骨の横の板片を方向づける</li>。
		ワックスが冷却板に半固体になるまで、<li>では目的の位置に温めた微細鉗子で優しくティッシュを保持します。溶融パラフィン ワックス金型をトップします。ワックスが完全に固化するまでを冷却するためパラフィン ブロックを許可します 。
		注: 18.5 日下垂体 (E18.5) は新生児下垂体と同じ方法で扱うことができます。脳下垂体 (ラトケ &#39; s ポーチ) 未満の E16.5 を頭全体が埋め込まれ、基本型の底面に垂直な (後頭部の口) から矢状軸に指向する必要があります</li>。
	</ol></li> <li>10 〜 20 分のための-20 ° C でパラフィン ブロックを寒さし、切ってミクロトームに薄膜 (4 μ m の厚みが望ましい) のスライス。満足のいくコロナ セクションを取得するには、断面中にパラフィン ブロックの位置を微調整します。次の手順でスライスの抜け防止のためポリリジン コート ガラス スライド上に組織スライスをマウントします 。
	注: また、標本 15% で脱水されることができます 30% (W/V) ショ糖液 (PBS) で同じ向き法を用いた低温埋め込み化合物に埋め込まれ、すぐにドライアイスで凍結、底に沈むまで 4 ° c。クライオスタットを用いた 8-10 μ m のスライスに凍結するティッシュのブロックをカットします。ただし、低温部には、形態はパラフィン セクションに劣る頻繁</li>。
</ol> 3。H &#38; E 染色と蛍光ラベリング <ol><li>暖かい熱スライド 55 ° c ブロック、キシレン、2 つの変更、4 分は、各 dewax。95% エタノール二度と 75% エタノール 2 回、各 3 分サンプルを脱水します。シェーカー上で 5 分間蒸留水でスライドを洗うです</li>。
	<li>の H &#38; 染色、ミョウバン ヘマトキシリン溶液 6 分間のセクションを染色、スケルトンスナイパーの洗浄45 s と再度 2 分間流水ですすぎの 0.2% アンモニア水を 2 分間 ing 水道水を区別します。1 分と 2 分のエオシン染色順次脱水、消去、およびマウントの 95% エタノールでセクションを脱水します</li>。
	蛍光標識 <ol><li><li>は、メタノールを含む 3% H 2 O 2 と 0.01% ナン 3 内因性ペルオキシダーゼを不活化する室温で 20 分間のセクションを扱います。沸騰 2 分加温の圧力鍋で検索バッファー (1 mM EDTA と 1 mM トリス-HCl、pH 7.4) のセクション 37 で 30 分のブロック バッファー (PBS で 5% ヤギ血清) のセクションによって抗原を取得 ° C</li>
		<li>4 で一晩ブロック バッファーで希釈したウサギ抗成長ホルモン (GH) 抗体 (1:2, 000) またはウサギ抗グリア線維性酸性蛋白 (GFAP) 抗体 (1:2, 000) セクションをインキュベート ° C</li>
		<li>37 で 35 分間インキュベート二次抗体 (ヤギ抗うさぎ IgG HRP、ブロック バッファーに 1: 300) とセクション ° C</li>
		<li>、室温で 15 分間ビオチニル パスチラミドシグナル (1:50 0.3% H 2 O 2 を含むトリス緩衝生理食塩水) を使用して信号を増幅してストレプトアビジン Alexa594 または 488 (37 ° C、30 分バッファーのブロックに 1: 300) を視覚化します</li>。
		<li>Counterstain 室温で 5 分間の核を DAPI (PBS で 50 mg/L) と 。
		注: パスチラミドシグナル信号増幅 (TSA) システムを使用して信号を増幅する必要ない場合があります。信号を可視化する蛍光標識二次抗体を使用ことができますまたは、します</li>。
	</ol></li> <li>蛍光顕微鏡を用いて取得画像搭載 DAPI、テキサス赤と FITC フィルター セット × 4/0.13 × 40/0.75 目標と 5.0 メガピクセルのカメラ。それぞれ、イメージング DAPI、アレクサ 488-Alexa 594 ステンドのセクションに、360、488、および 594 nm のレーザー光の波長を使用しています。レーザー パワーを最適化 (一般に 0.8 を使用) と各チャンネルの所望のレベルに露光時間。画像集録ソフトウェアの制御の下でイメージをキャプチャし、その後画像処理ソフトウェアを用いた蛍光画像をオーバーレイします</li>。
</ol>

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ネズミの脳下垂体を開発するための小さくて壊れやすい機能と解剖学的特徴6,8により適切なコロナ セクションを得ることが技術的に困難だった。いくつかの研究グループは従って萌芽期・新生児下垂体11,12の形態を分析する半ば矢状のセクションを選んだ。下垂体の半ば矢状断面が前方、中間を示すこと...

下垂体または下垂体が恒常性1,2のために不可欠なホルモンを分泌する重要な内分泌器官です。解剖学的, 下垂体腺、後葉と下垂から成る「2 台」構造です。これらの部品がある異なる胚起源と機能非常に異なる。後葉は神経外胚葉由来、オキシトシンと抗利尿ホルモンを分泌します。下垂ラトケ嚢に由来、成長ホルモン、プロラクチン、甲状腺刺激ホルモン、卵胞刺激ホルモン、黄体形成ホルモン、副腎皮質刺激ホルモンを含むホルモンのリリースを担当し、メラニン細胞刺激ホルモンの3,の4,5。
下垂体腺は、マウス頭蓋骨の蝶形骨 (鞍) の背側表面にかかっている、壊れやすい茎によって脳の床にアタッチされます。三叉神経と前方視交叉6、7それは横方向に囲まれています。腺は細長い楕円形で、その軸に垂直に頭を。背側表面の後葉と下垂することができます簡単に区切られ、背側内側領域と腹の横と、後者の拡張を占める前者で。生後発達期下垂体のサイズが誕生8,9後最初の月に急速に増加します。それにもかかわらず、マウス下垂体は、まだ 1.9 mg の平均重量と 〜 3 mm の長軸径サイズで非常に小さい大人10, 生後 21 (P21) で 2 〜 2.5 mm と P0 でわずか 1 〜 1.5 mm の長軸径。
マウス下垂体の単一のスライスで下垂前葉と後葉の両方を表示する冠状断面お勧めします。ただし、いくつかの技術的なスキルは、非常に小さなサイズと異なる解剖学マウスを開発から下垂体腺のコロナ セクションを満たすを取得する必要があります。このビデオの記事でマウス下垂体腺を分析し、さまざまな開発段階で下垂体のコロナ セクションを準備する方法を示します。

<table><tbody><tr><td>&lt;strong&gt;Tools/Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Surgical scissors-straight</td><td>JinZhong</td><td>J21010</td><td>can be purchased from other vendors</td></tr><tr><td>Fine scissors-strainght</td><td>JinZhong</td><td>WA1010</td><td>can be purchased from other vendors</td></tr><tr><td>Blunt forceps</td><td>JinZhong</td><td>JD1020</td><td>can be purchased from other vendors</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>RS-5015</td><td>for isolation of the pituitary</td></tr><tr><td>26G (0.45mm) needle</td><td>HongDa</td><td></td><td>for transcardial perfusion</td></tr><tr><td>Syringe (1 mL)</td><td>BD</td><td>300841</td><td>can be purchased from other vendors</td></tr><tr><td>Syringe (10 mL)</td><td>BD</td><td></td><td>can be purchased from other vendors</td></tr><tr><td>35mm dish</td><td>Corning</td><td>430165</td><td>can be purchased from other vendors</td></tr><tr><td>Lens cleaning paper</td><td>ShuangQuan</td><td></td><td>can be purchased from other vendors</td></tr><tr><td>Anatomical microscope</td><td>OLYMPUS</td><td>SZX-ILLB2-200</td><td>can be purchased from other vendors</td></tr><tr><td>Embedding cassette</td><td>Thermo Fisher</td><td>22-272423</td><td>can be purchased from other vendors</td></tr><tr><td>Tissue embedding console system</td><td>KEDEE</td><td>KD-BM11</td><td>can be purchased from other vendors</td></tr><tr><td>Microtome</td><td>Thermo Fisher</td><td>HM315R</td><td>can be purchased from other vendors</td></tr><tr><td>Superfrost-Plus slides</td><td>Thermo Fisher</td><td>22-037-246</td><td>can be purchased from other vendors</td></tr><tr><td>Cover glass</td><td>Thermo Fisher</td><td>12-543</td><td>can be purchased from other vendors</td></tr><tr><td>Fluorescence microscope</td><td>OLYMPUS</td><td>BH2-RFCA</td><td>can be purchased from other vendors</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Urethane</td><td>BBI</td><td>EB0448</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>for PBS</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td>for PBS</td></tr><tr><td>Na2HPO4.12H2O</td><td>Sigma</td><td>71650</td><td>for PBS</td></tr><tr><td>K2HPO4</td><td>Sigma</td><td>P2222</td><td>for PBS</td></tr><tr><td>NaNO2</td><td>Sigma</td><td>237213</td><td></td></tr><tr><td>Heparin Sodium Injection</td><td>SPH</td><td>H31022051</td><td>for perfusion saline</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Ethanol</td><td>SCR</td><td>10009218</td><td></td></tr><tr><td>Xylene</td><td>SCR</td><td>10023418</td><td></td></tr><tr><td>Paraffin</td><td>Thermo Fisher</td><td>8330</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma</td><td>H9627</td><td>for H&amp;amp;E staining</td></tr><tr><td>Eosin Y</td><td>Sigma</td><td>E4009</td><td>for H&amp;amp;E staining</td></tr><tr><td>rabbit anti growth hormone (GH)</td><td>National Hormone</td><td></td><td>for immunostaining</td></tr><tr><td>antibody</td><td>Pituitary Program</td><td></td><td></td></tr><tr><td>Rabbit anti-mouse GFAP antibody</td><td>Sigma</td><td>G9269</td><td>for immunostaining</td></tr><tr><td>Goat anti-rabbit IgG, HRP</td><td>Jackson</td><td>111-035-003</td><td>for immunostaining</td></tr><tr><td>TSA system</td><td>NEN Life Science Products</td><td>NEL700</td><td>for immunostaining</td></tr><tr><td>Streptavidin, Alexa Fluor 594</td><td>Thermo Fisher</td><td>S32356</td><td>for immunostaining</td></tr><tr><td>Anti-FITC Alexa Fluor 488</td><td>Thermo Fisher</td><td>A11090</td><td>for immunostaining</td></tr></tbody></table>

dissection and coronal slice preparation of developing mouse pituitary gland

下垂体または下垂体は重要な内分泌臓器の恒常性に不可欠なホルモンを分泌します。別の胚起源と機能と 2 つの腺で構成されています — 後葉と下垂。小型で細長い楕円形と繊細な開発マウス下垂体です。マウス下垂体の単一のスライスで下垂前葉と後葉の両方を表示する冠状断面お勧めします。
このプロトコルの目的は、マウスを開発してからの手入れが行き届いた組織アーキテクチャで適切な下垂体のコロナ セクションを達成することです。このプロトコルでは解剖およびマウスを開発から正常下垂体腺を処理する方法詳細に述べる。まず、マウス解剖前にホルムアルデヒドの transcardial 灌流を固定です。3 つの異なる解剖技術はマウスの年齢に応じてそのまま下垂体腺を取得に適用されます。胎児マウス高齢者胚日 (E) 17.5 18.5 と新生児を 4 日間に、蝶形骨、腺、および三叉神経を含む全体のセッラ地域は解剖します。子犬の高齢生後 (P) 5-14、下垂体腺、三叉神経接続は全体として解剖しました。以上 3 週齢のマウスに、下垂体腺は周囲の組織から無料解剖慎重に。コロナ セクションを満たすを取得するランドマークとして周囲の組織を使用して、適切な方向に下垂体腺を埋め込む方法も表示されます。これらのメソッドは、マウスの開発に下垂体腺の組織学的・発達的特徴を分析に役立ちます。

このプロトコルはマウスを開発から下垂体腺を分析する手法を示す.新生児マウス下垂体, 三叉神経と下を含む全体のセッラ地域の蝶形骨は頭蓋底からうち解剖されました。小型で繊細な下垂体腺はプロセス (図 1 a) の中にそのまま残った。5 日間以上のマウスの横の三叉神経に接続されている下垂体腺は当時孤立しました。マウスがよく保持され...

Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

郭清とコロナのスライス標本マウス下垂体腺の開発

下垂体腺の解剖し、マウスを開発から下垂体のコロナ セクションを準備するためのプロトコルを提案します。

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

dissection-coronal-slice-preparation-developing-mouse-pituitary

Research

JoVE Journal

Developmental Biology

17.3K ビュー.  Second Military Medical University. 下垂体腺の解剖し、マウスを開発から下垂体のコロナ セクションを準備するためのプロトコルを提案します。

ビデオ: Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

発生生物学

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 ...

神経科学

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

不連続パーコール-ショ糖密度勾配を用いてマウスの皮質からSynaptoneurosomesの調製

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

げっ歯類の内側嗅内皮質の背腹軸機構の調査のための矢状スライスの作成

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

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 ...

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

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

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

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

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

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

腫瘍微小環境と標的薬物療法研究のための脳の腫瘍/器官スライス共培養系

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

医学

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

出生後(P1-3)マウス神経起源ニッチからの神経球の分離と拡大

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

機能的および解剖学的に異なる領域へのマウス脳の微小解剖

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.