This work was partially supported by National Institute of Deafness and Other Communications Disorders Awards R03-DC-012125 to D. A. Llano and F31-DC-013501 to B. J. Slater as well as the Carver Foundation.
The authors would like to thank Jason MacLean and Matthew Banks for technical advice with calcium imaging.

すべての手順は、イリノイ大学の施設内動物管理使用委員会によって承認されました。全ての動物は、実験動物管理の認定のためのアメリカ協会によって承認された動物管理施設に収容しました。すべての試みが使用される動物の数を最小限にするために、研究のすべての段階での苦しみを軽減させました。 1.準備やスライスのためのマウスからの脳の除去<ol><li>灌流およびスライスのインキュベーションのために準備します。 <ol><li>約30分スライスする前に、記録またはイメージングする前に、スライスのインキュベーションのために高スクロース切削液と低カルシウムaCSFのを準備します。 </li><li>すべての必要なツール（大平滑末端はさみ、小さな春のはさみ、解剖鉗子、大きなハサミやギロチン、アイリスはさみ、宝石商の鉗子、27Gのx 1/2インチの針で10mlシリンジ、1センチメートル×2センチフィルタの小片をレイアウト紙、SLのための壊れひっくり返したパスツールピペットアイス灌流およびスライス用の転送）、および灌流トレイを準備します。 </li><li> 32°Cの水浴とソリューションを切断で満たされた培養皿に酸素化aCSFのでチャンバーをインキュベート設定します。 </li></ol></li><li>スライスのための段階を切断準備します。 <ol><li>脳のためのバックストップとして使用する3％寒天約1cm 3の小片をカットし、1.5センチメートルX 1.5ミリメートルのICを下支えするために使用されるバンプ用×3 mmです。 </li><li>シアノアクリレート系接着剤と同様に、彼らは80 Oの角度を形成するように、バックストップの右側上のバンプと、ステージ上にバックストップを接着。 </li></ol></li><li>脳を削除します。 <ol><li>深くP12-P20ケタミンを100mg / kgおよびxylaxineに3mg / kgの（または同等の倫理委員会承認手続き）と（理想的にp14-18）は、マウスを麻酔。 注：つま先のピンチに応答の欠如を経由して、適切な麻酔レベルを確認してください。動物が麻酔簡単に下にあるように、眼軟膏は不要です。 </li><li>鈍使用はさみを終了し、剣状突起から首に胸郭を公開します。 <ol><li>剣状突起を検索し、約1.5 cmで水平カットを行います。 </li><li>肩に前の水平カットの端から2つの垂直カットを行い、約1.5センチメートルそれぞれ。 </li></ol></li><li>心臓を露出するダイアフラムとcostochondral接合を通してカット。 </li><li>小さな春のはさみで、心の側を背ために腹側から、右心房にある小さな2〜5ミリのカットを行います。 </li><li> 27Gのx 1/2インチ針を10ミリリットル注射器を使用して、左心室を注入し、迅速に高スクロース切断溶液で動物を灌流。 </li><li>血液が明確で実行されると、頭部を削除するには、大きなハサミを使用しています。 注：私たちは、体系的灌流の有用性を評価されていません。しかし、我々の経験では、この若いマウスで経心臓灌流は、ほぼ100％の成功で行うことができ、赤BLを排除するという利点を持っています蛍光を発するとイメージングを妨害することがOOD細胞。 </li><li>頭蓋骨を露出するために正中線を皮膚を削減。 </li><li>曲がったアイリスのはさみを使用して、その後、慎重に下に脳に損傷を与えないように注意して正中縫合糸に沿って後方の前方からカット目の間のカットから出発し、目の間の頭蓋骨を切りました。 注意：硬膜は通常、頭蓋骨と外れます。必要に応じて、慎重に脳を削除する前に硬膜を削除します。 </li><li>宝石の鉗子を使用して、プライ頭蓋骨を開き、慎重に脳を削除し、次いでこの溶液を切断中に脳を置きます。皮質の損傷を防ぐために注意してください。 </li></ol></li></ol> 2.スライスのための脳の準備<ol><li>ビブラトームステージに取り付けるための脳を準備。 <ol><li> 90 Oの2つのラインが付いているスライドと左上から右下へ17 Oで対角線を使用して、2つの線が交わる使用して、アップ脳の背側を配置し、交差かみそりの刃は、平坦な表面を作成吻端脳2-4ミリ削除します。 </li><li>新しく作成された平らな面に脳尾側を上に置き、水平および垂直線と脳の正中線と脳の背側表面の位置を合わせます。 </li><li> （水平面と冠状から60 oから 17 o）の 、17 Oラインにカミソリの刃を合わせ30 Oの角度でカミソリを傾け、ダブル斜めカットで右皮質の約3mmを削除します。 </li></ol></li><li>ビブラトームステージ上で脳をマウント。 <ol><li>長い寸法が正中線に垂直になるようにろ紙脳の腹側に約1cm×2センチの小片を置きます。 </li><li>慎重にバックストップ（バンプの左側）の前の領域にシアノアクリレート接着剤の少量を適用します。 </li><li>脳と後脳の尾の部分があるようにPR接着剤上に二重斜めにカット脳の顔を配置バンプ時にoppedと脳の右側はバックストップに反しています。 </li><li>繊細全面をスライスチャンバーに接触していることを確認して、脳を押し下げます。 </li></ol></li></ol> 3. Colliculo-視床皮質スライスの取得<ol><li>すぐに、ビブラトームで切断段階と場所を取るソリューションを切断して、ステージを埋めます。 </li><li>脳の上部（腹側）とブレードの位置を合わせます。 </li><li> 、脳の上から1〜1.5ミリメートルを削除300-500ミクロンのスライスを削除し、除去した後の深さを評価します。 注：IC、MGB、および外側膝状核（LGN）がすべて表示され、および歯状回は、「C」形状は、2つの600ミクロンのスライスに1を取る形成する場合。 図3Aを参照してください。 </li><li>チャンバーを保持する加熱中（32 O C）のスライスを配置します。 </li></ol>スライスの4イメージング<ol><li>フラビンタンパク質の自己蛍光イメージングのための準備。 <ol> &lt;LI&gt;、標準の電気生理学的記録チャンバーにスライスの灌流のために95％O 2/5％CO 2でバブルaCSFのをaCSFのを準備します。 </li><li>刺激のためのガラス電極を引き出します。複数の異なる刺激電極と構成（ガラス、タングステン、炭素繊維、単極、双極）すべてはフラビンタンパク質の自家蛍光イメージングと一緒に非常にうまく機能していることに注意してください。 </li><li>コンピュータ、カメラ、マイクロマニピュレーター、光源、刺激ソフトウェア、画像キャプチャソフトウェアをオンにします。 </li><li>チャンバ内のスライスを上昇させるために、カスタム・ブリッジ（補足資料で利用可能なデザイン）を置き、酸素化aCSFので記録チャンバーを灌流。 注意：灌流速度が設定したすべての個人と異なります。 5-15ミリリットル/分、ここで使用されます。 </li><li>間の経路上でハープの文字列を配置しないように注意しながら、スライスの上に特別に構築されたハープを配置しながら橋をリフトオフからスライスを防止するために、チャンバ内の液体レベルを下げ、チャンバ内のスライスを配置EST（ 図1A）。スライスの上下にaCSFの流れを維持するために、スライス上の約1〜2 mmの液体レベルを上げます。 注：再配置が必要な場合は、切断端パスツールピペットを使用するか、またはaCSFのレベルが上昇し、細心の注意を払って鉗子を使用することができます。 </li><li> （ 図1A参照 ）、aCSFので満たされたガラス電極に塩化銀電極を挿入マイクロマニピュレーターにガラス電極を挿入し、アイソレータを刺激し、慎重に視床につながるIC /白質にガラス電極を配置するために接続します。 </li></ol></li><li>自家蛍光画像取得をフラビンタンパク質。 <ol><li>電気的に刺激しながら照射しつつ（各刺激は0.05 Hzで40 Hzの2ミリ秒パルス組織の1秒の5回からなる）105秒間4ヘルツ（無限遠補正2Xマクロ対物レンズ（NA 0.13）を使用して）、カメラで画像を収集青色光470〜490 nmおよび515 nmの上に捕獲で組織。画像であることを確認してくださいどちらにO暗く、また吹き出さ参照図3は、参考のために列を残しました。 注：収集時間、収集頻度、および刺激周波数は、実験に合わせて変更することができ、副資材に含まれるカスタムプログラムは、次のような変形が可能です。 </li><li>輸出のMatlabの画像、0.05 Hzで画像のスペクトルパワーを分析するための補足資料に使用可能なカスタム書かれたプログラムを使用して。結果として得られる画像は、接続された経路が表示されます。 注：カスタムプログラムは、高速フーリエ変換画像を生成するために、時系列の画素値の変換を使用します。 </li></ol></li><li>カルシウムイメージングのための準備。 <ol><li> C. O 32 aCSFのスライスの上部と下部の両方に到達させるために3センチ培養皿と隆起培養膜を用いた小型インキュベーションチャンバーを準備し、そして加熱パッド上の場所は、ほぼ温度を維持します</li><li> 95％Oで暖かいaCSFの、ゆっくりと泡と小さなインキュベーションチャンバーを埋めます2/5％CO 2。 </li><li> 2プルロニックF-127酸μlのDMSOを48μlの溶解のFura-2AMの50μgのミックス。 </li><li>隆起した膜と慎重にマイクロピペットを用いて上に小さなインキュベーションチャンバーに入れスライスは染色直接MGB上記混合物（または関心のある他の構造）を追加します。 </li><li>撮像前に退色を防止するために、スライスをカバーし、スライスを45分間インキュベートすることを可能にする - 1時間。 </li><li>余分な材料を洗浄するために10〜15分間加熱保持室にスライスを削除します。 </li><li>刺激するための準備や画像colliculo-視床皮質スライスのために4.1.6にステップ4.1.1に従ってください。 </li></ol></li><li>カルシウム画像取得。 <ol><li> 365nmの光で組織を照射し、510 nmの上に蛍光を捕捉しながら、電気的に0.2 Hzで（各刺激が1 2ミリ秒パルスからなる）5回組織を刺激しながら25秒間（20X水浸対物レンズを使用して）10Hzで画像を収集します。 </lI&gt; <li> MATLABおよび補足資料で使用可能なカスタム書かれたプログラムを使用するエクスポート画像が0.2 Hzで画像のスペクトルパワーを分析します。結果として得られる画像は、アクティブセルが表示されます。 </li></ol></li></ol>

<ol>
	<li>Llano, D. A., Slater, B. J., Lesicko, A. M., Stebbings, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+auditory+colliculothalamocortical+brain+slice+preparation+in+mouse.">An auditory colliculothalamocortical brain slice preparation in mouse.</a> J. Neurosci. 111, 197-207 (2014).</li><li>Agmon, A., Connors, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamocortical+responses+of+mouse+somatosensory+(barrel)+cortex+in+vitro.">Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro.</a> Neuro. 41, 365-379 (1991).</li><li>Cruikshank, S. J., Rose, H. J., Metherate, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+Thalamocortical+Synaptic+Transmission+In+Vitro.">Auditory Thalamocortical Synaptic Transmission In Vitro.</a> J Neurophysiol. 87, 361-384 (2002).</li><li>Lee, C. C., Sherman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+and+physiology+of+ascending+streams+in+the+auditory+tectothalamic+pathway.">Topography and physiology of ascending streams in the auditory tectothalamic pathway.</a> PNAS. 107, 372-377 (2010).</li><li>MacLean, J. N., Fenstermaker, V., Watson, B. O., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+visual+thalamocortical+slice.">A visual thalamocortical slice.</a> Nat meth. 3, 129-134 (2006).</li><li>Crabtree, J. W., Isaac, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+intrathalamic+pathways+allowing+modality-related+and+cross-modality+switching+in+the+dorsal+thalamus.">New intrathalamic pathways allowing modality-related and cross-modality switching in the dorsal thalamus.</a> J. Neurosci. 22, 8754-8761 (2002).</li><li>McAlonan, K., Cavanaugh, J., Wurtz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Modulation+of+Thalamic+Reticular+Neurons.">Attentional Modulation of Thalamic Reticular Neurons.</a> J. Neurosci. 26, 4444-4450 (2006).</li><li>Weese, G. D., Phillips, J. M., Brown, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Orienting+Is+Impaired+by+Unilateral+Lesions+of+the+Thalamic+Reticular+Nucleus+in+the+Rat.">Attentional Orienting Is Impaired by Unilateral Lesions of the Thalamic Reticular Nucleus in the Rat.</a> J. Neurosci. 19, 10135-10139 (1999).</li><li>Zikopoulos, B., Barbas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+Projections+to+the+Thalamic+Reticular+Nucleus+form+a+Unique+Circuit+for+Attentional+Mechanisms.">Prefrontal Projections to the Thalamic Reticular Nucleus form a Unique Circuit for Attentional Mechanisms.</a> J. Neurosci. 26, 7348-7361 (2006).</li><li>Kalatsky, V. A., Stryker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Paradigm+for+Optical+Imaging:+Temporally+Encoded+Maps+of+Intrinsic+Signal.">New Paradigm for Optical Imaging: Temporally Encoded Maps of Intrinsic Signal.</a> Neuron. 38, 529-545 (2003).</li><li>Llano, D. A., Turner, J., Caspary, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+cortical+inhibition+in+an+aging+mouse+model+of+chronic+tinnitus.">Diminished cortical inhibition in an aging mouse model of chronic tinnitus.</a> J. Neurosci. 32, 16141-16148 (2012).</li><li>Ehret, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+studies+on+auditory+development+in+mammals+in+relation+to+higher+nervous+system+functioning.">Behavioural studies on auditory development in mammals in relation to higher nervous system functioning.</a> Acta otolaryngol. 99, 31-40 (1985).</li><li>Mikaelian, D., Ruben, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+hearing+in+the+normal+CBA-J+mouse:+correlation+of+physiological+observations+with+behavioral+responses+and+with+cochlear+anatomy.">Development of hearing in the normal CBA-J mouse: correlation of physiological observations with behavioral responses and with cochlear anatomy.</a> Acta. 59, 451-461 (1965).</li></ol>

The authors have nothing to disclose.

This protocol describes improvements upon a previously described colliculo-thalamocortical brain slice in p12-20 mouse to study information flow in the auditory system1. This method has a number of advantages over other, similar, brain slice preparations by retaining connections between more brain areas in a single slice, which gives investigators new tools to understand the interaction and interplay between auditory nuclei in the forebrain. There have been a few key modifications in this protocol, compared to...

感覚周と下丘間の情報の実質的な処理があるが、それは聴覚皮質に到達する前に聴覚系では、かなりの追加処理があります。私たちは、その処理が行われ、したがって、その変換は、脳が入ってくる感覚情報を解釈することを可能にする方法についてはほとんどされている方法についてはほとんど知りません。嗅覚を除いて、感覚のそれぞれは、周辺の信号と非常によく似た組織は、最初に信号が皮質に上昇として低下忠実に中継されています。皮質は、その後さらに、着信情報を変調するために下側の構造体への投影を送信します。この複雑なシステムは 、インビボならびにインビトロでの製剤の数の様々な方法で研究されています。前者では、すべての接続は、感覚入力を制御し、測定しながら、接続の任意のセットを調べるために研究者を可能にする、完全です任意の与えられた領域で出力されます。このアプローチでは、強烈に複雑な出力を生じさせる他の感覚入力、覚醒、と注意を含む他の入力、多種多様なの制御なしにほとんどありません。in vitroでは、脳切片は、単一のセットのいずれかをキャプチャするためにカットされています突出部、または研究者が様々な求心性神経や脳の領域を刺激し、評価することができ、接続された2つの脳領域の。これらは、多くの場合、皮質への視床や視床に入力し、その出力のいずれかが2-5を保存している視床皮質またはtectothalamicスライスのいずれかです。これらの製剤は、薬理学的、電気的、およびoptogenetic操作の多種多様なことができます。しかし、2つだけの脳領域で、それらは主に情報の伝達を評価し、それが視床通過などの情報の変換を評価する能力を欠いています。また、注目変調6-9に役割を果たしている可能性細網視床投射は、広報でありますこのスライスでESENT。ここでは、どのように視床ゲートとフィルタ情報のユニークな視点を与えるために視床へのさまざまな入力の研究者の制御を可能にする私達の前の準備1、時の改善を示しています。私たち夫婦単一細胞レベルでのさまざまな入力の影響を測定するために、スライスの接続や大規模化分析、ニューロン集団の分析のための視床におけるカルシウムイメージング、および単一細胞記録を評価するためのフラビンタンパク質の自己蛍光イメージングとこの新規スライス標本。 これらの広範囲の接続を維持するのを助けるために、我々はまた、強化された灌流のためのスライスを上昇させるために所定の位置に脳切片とブリッジを保持するための通常のスライスアンカー（別名「ハープ」）の変更の数を開発しました。ハープはスライスを囲むとハープの文字列のカスタマイズ可能なアタッチメントポイントを可能にするように修正された馬蹄形状に設計されています。 3つの文字列があります添付i）1種がii）1つは、ACの尾側縁に、ICの尾端から延びており、III）一つの領域にスライスの内側縁から斜め拡張し、スライスの内側エッジに沿って水平に位置するようにAC（ 図1Aを参照）吻側。ハープ文字列の（シアノアクリレート接着剤で）接着のためのフレームの小さなくぼみ（図1B参照 ）、スライスの整合性を維持するためにスライスに圧力の低下量を可能にします。 3次元印刷を使用することにより、我々はデザインが独自の仕様、ならびに組織の上下の人工脳脊髄液（aCSFの）の理想的な流れを可能にするブリッジにハープカスタムすることができます。これはまた、パッチクランプ電気生理学のための組織を貫通する光のために広い面積を維持します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>High sucrose cutting solution</td>
			<td></td>
			<td></td>
			<td>in mM: 206 sucrose, 10.0 MgCl2, 11.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Low calcium aCSF</td>
			<td></td>
			<td></td>
			<td>in mM: 126 NaCl, 3.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 1.0 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>aCSF</td>
			<td></td>
			<td></td>
			<td>in mM: 126 NaCl, 2.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2.0 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>A360</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td>Lot: 1572C502</td>
		</tr>
		<tr>
			<td>Fura-2AM</td>
			<td>Life Technologies</td>
			<td>F1201</td>
			<td>Lot: 144912</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Life Technologies</td>
			<td>P3000MP</td>
			<td>Lot: 1499369</td>
		</tr>
		<tr>
			<td>Large culture dish</td>
			<td>Fisherbrand</td>
			<td>08-757-13</td>
			<td>100x15mm culture dish</td>
		</tr>
		<tr>
			<td>Small culture dish</td>
			<td>Falcon</td>
			<td align="right">353001</td>
			<td>35x10mm culture dish</td>
		</tr>
		<tr>
			<td>Raised culture membrane</td>
			<td>Millicell</td>
			<td>PICMORG50</td>
			<td>Used to maintain oxygenated fluid perfusion on both sides of slice.</td>
		</tr>
		<tr>
			<td>Flavoprotein imaging fluorescence cube</td>
			<td>Olympus</td>
			<td>UMNIB</td>
			<td>470&#8211;490 nm excitation, 505 nm dichroic, 515 nm emission long pass.&#160; We have found that virtually any green fluorescence protein filter cube will work here.</td>
		</tr>
		<tr>
			<td>Calcium imaging fluorescence cube</td>
			<td>Omega Optical</td>
			<td>BX-18</td>
			<td>XF1005 365nm exitation, XF2001 400nm dichroic, XF3080 510nm emission</td>
		</tr>
		<tr>
			<td>Agar for blocking brain</td>
			<td></td>
			<td></td>
			<td>3% by weight in water</td>
		</tr>
		<tr>
			<td>Viper si Stereo Lithography Apparatus</td>
			<td>3D Systems</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

P15マウスで得られたcolliculo -視床皮質マウス脳スライスの例を図2に示されている。理想的なスライスは、ICが刺激されたときに、すべてのアクティブ化されている4つの主要な中脳と前脳の聴覚構造IC、MGB、TRN、およびACを、含まれています（ 図2A）。フーリエ解析を使用して、スペクトルパワーを周期的刺激周波数10に同伴されるアクティビティ?...

Watch this Scientific Journal Video about Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging at JoVE.com

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

マウスの仔の脳をカットするために、複数の角度を使用して、我々は主要な聴覚中脳と前脳構造のほとんどの間の接続をキャプチャ前述の急性脳スライスを改善。

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 ...

modification-colliculo-thalamocortical-mouse-brain-slice

Research

JoVE Journal

Neuroscience

8.2K ビュー.  University of Illinois Urbana-Champaign. マウスの仔の脳をカットするために、複数の角度を使用して、我々は主要な聴覚中脳と前脳構造のほとんどの間の接続をキャプチャ前述の急性脳スライスを改善。 

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

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

無傷の3 - D脳の回路のウイルス標的のためのスケーラブルな流体インジェクタの配列

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

神経科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

マウス鋤鼻急性組織スライス標本のイメージングフェロモンセンシング

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

縦断のために脊髄商工を移植するための手順<em&gt;インビボ</emマウス脊髄の&gt;イメージング

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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

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

マウスの胚感覚軸索投影のホールマウントイメージング

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

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

感染ニューロンの3-Dイメージングと分析<em&gt;インビボ</em&gt;と<em&gt;トキソプラズマ</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, typically 3 mm x 3 mm. This protocol introduces a method for creating a substantially larger 7 mm x 6 mm cranial window exposing most of a cerebral hemisphere over the mouse temporal and parietal cortices (e.g., bregma 2.5 - 4.5 mm, lateral 0 - 6 mm). To perform this surgery, the head must be tilted approximately 30&#176; and much of the temporal muscle must be retracted. Due to the large amount of bone removal, this procedure is intended only for acute experiments with the animal anesthetized throughout the surgery and experiment.
The main advantage of this innovative large lateral cranial window is to provide simultaneous access to both medial and lateral areas of the cortex. This large unilateral cranial window can be used to study the neural dynamics between cells, as well as between different cortical areas by combining multi-electrode electrophysiological recordings, imaging of neuronal activity (e.g., intrinsic or extrinsic imaging), and optogenetic stimulation. Additionally, this large craniotomy also exposes a large area of cortical blood vessels, allowing for direct manipulation of the lateral cortical vasculature.

This protocol presents a method for creating a large unilateral craniotomy over the temporal and parietal regions of the mouse cerebral cortex. This is especially useful for real time imaging over an expansive area of a cortical hemisphere.

脳活動のメソスケール広視野光学イメージングのために大きな横開頭術手順

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

マウスの脳の側脳室における上衣繊毛のライブイメージング

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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 ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

少量循環、血流 - と水没型チャンバー システムで管理急性海馬スライスにおけるシナプス可塑性を記録

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...