現在および過去のルーベルの研究室のメンバー。

1。エリアの解剖の準備<ol><li> 95％O 2 / 5％CO 2の混合物（7.2から7.4の間のpH、浸透圧295から310 mOsm / L）で連続的にバブル人工脳脊髄液（ACSF）。 </li><li> ACSFは、70％EtOHでクリーンな作業領域をバブリングしている間。ビブラトームとスライス刃をも清掃してください。蒸留水（のdH 2 O）で刃をすすぎます。 </li><li>適切な解剖ツールで領域を解剖にクリーンな液体吸収パッドを配置。 </li><li>ビブラトームスライシングチャンバーの段階に接着剤の寒天ブロック（のdH 2 O 40 mgのアガロース/ mLの、すなわち4％）* *ヒント：解剖する前に寒天を準備。冷蔵庫で覆われたペトリ皿内の既製の寒天を保管してください。アガロースは、EMDケミカルズまたはInvitrogenから購入した。 </li></ol> 2。培養液で6ウェルプレートの準備<ol><li>無菌フードの下、35℃培養液とインキュベート1mlで6ウェルプレートの動物ごとに良く℃、5％のCO 2を 1埋める。 </li><li>多くの文化膜インサートとして、必要に応じて準備し、後で使用するためのフードの中に置いてください。 </li></ol> 3。チキンの聴性脳幹の分離<ol><li>胚（通常は卵の大側）の空気充填空間の空隙を決定するために明るい光源の下に場所の卵。 </li><li>膜嚢を露出させる卵の大きな側をこじ開ける。メスと膜嚢のスライスを作成し、静かにニワトリの胚の頭部を取り除く。 </li><li>急速にはさみで頭を刎ねる。 </li><li>へと目の間にわずかに後方かみそりの刃の代わりに先端。わずかな圧力をかけて頭蓋骨を介して吻側から尾側正中切開を行う* *ヒント：適用圧力が胚の年齢に依存している（すなわち、若い=小さい、古い=より） </li><li>お肌と頭蓋骨を露出し、正中線のカットを確認するために羽を押しのける。 </li><li>かみそりの刃でスライスして頭蓋骨の吻側部をブロックします。目に後方と急速に全体の頭蓋骨と脳の組織を介して切断位置のブレード* *ヒント：強い圧力が完全に吻側セクションを削除する必要があります。 </li><li>頭の両側に見える首の筋肉へ少し前頭骨の尾部領域、の小さなハサミで正中線から外側切開を加えます。頭蓋骨と小脳と脳を公開するために組織を引き離します。 </li><li>そっと小さなはさみで添付された組織を切断して頭蓋底から脳幹を削除します。すべての組織が切断されると、脳幹は頭蓋骨から離れて自由に移動する必要があります* *ヒント：添付された組織を切断するために頭蓋骨（脳の下）を反転する必要があります。 </li></ol> 4。スライシングのための脳幹の準備<ol><li>一度頭蓋骨から削除、視蓋を通して脳幹を突き止める。 </li><li>完全に第4脳室底を露出させる、優しく小さなハサミで花梗をカットして小脳を削除します。 </li><li>ピンセットで脳幹の表面から膜様組織と血管を削除し、脳幹を分離するために視蓋を通して横カットを行います。 </li><li>寒天ブロックの前でビブラトームステージ（ビブラトームのブレードに向かって）にスーパー接着剤の少量を置きます。 </li><li>ピンセットを使用すると、静かにかつ迅速ビブラトームのブレードに向かって吻側面を下にし、背側にスーパー接着剤上に脊髄と場所の組織で脳幹を持ち上げる* *ヒント：余分な接着剤は、次のステップの前に金ワイプまたはろ紙に吸収されるべきである。 </li><li>優しくビブラトームステージに酸素ACSFを注ぐ* *注意：このACSFを連続的にO 2 / CO 2でバブリングすることができます。しかし、脳幹スライスに不要な動きのソリューションの結果のような小さな体積（および損傷）にACSFのバブリング。迅速に実行した場合、解剖から既に酸素ACSFで十分です。 </li><li>ビブラトームブレードは、20〜22℃の角度になっているはず。スタートビブラトーム（振動が最大振幅に設定する必要があります）とは、組織のトップは、スライスする前に刃と平行になるようにブレードに向かってステージを移動する。 </li><li>すぐに脳幹の組織に向けてブレードを移動する。かなり組織と刃の接触直前にスローダウン。 </li><li>最も遅い可能性前進運動と組織をスライス始める。かつて組織の全体冠状セクションを通じて、軽くブラシや割れたガラスでブレードからスライスを離れて移動する、洗練されたピペットはゴム球を一端に合わせ、発射。 </li><li>再び300から500μmのステップとスライスの下段。解剖学的な署名は脳幹の組織で表示されるまで繰り返します。この時点で、200-1000μmで聴覚構造を含むスライスの脳幹の組織は、組織や実験的なニーズの年齢に応じて、厚さ。 </li><li>慎重にスライスの順番で使用可能なスライスを維持する、すなわち、1回目のスライスは、回路の最も尾部領域（低周波音の処理）を表し、最後のスライスは最も吻側部（高周波processiを表します。NG）* *注：文化用のスライスの配置については、セクション6に進みます。 体外生理学ストレージの場合、次のセクションを参照してください。 </li></ol> 5。 体外生理学のスライスストレージ<ol><li>ニワトリ胚やスライスの厚さの年齢に応じて、各動物は1〜6のスライスを提供する必要があります。 </li><li>ゆっくりとゴム球を一端に取り付けた火災研磨したガラスピペットを用いてチャンバー内の個々のスライスを配置します。チャンバーには番号がされるべきである。いくつかtonotopic特異性（例えば、チャンバー1内の最も尾側のスライスと最後のチャンバー内の最も尾側のスライスを）維持するために、ウェルあたり1つのスライスを置きます。チャンバーは、ACSFを充填し、連続して95％O 2 / 5％CO 2液（pH7.4、浸透圧295から310 mOsm / L）の混合物をバブリングしてください。 </li><li>スライスは約1時間温浴（36℃）に室を置くことによって平衡させる。 </li><li>温かいお風呂からチャンバーを取り外し、約半時間室温で休ませることができます。 </li><li>室温で半時間後、スライスは保持室から電気生理学実験のための顕微鏡にマウントされている〜0.5mLの記録室に転送することができます。 </li><li>スライスは、温かいお風呂から取り外した後〜6時間まで使用することができます。 </li></ol> 6。器官切片培養8の準備<ol><li>すぐにスライス手順は、次の（セクション6を参照）氷上で48ウェルプレートにゴム球を一端に取り付けた火災研磨したガラスピペットを用いて〜500μLのACSFでスライスを転送する。ウェルあたりつのスライス*. *ヒント：スライスが少なくとも300μmおよび最大1000μmの厚さにする必要がある。 </li><li>スライスが収集された後、ボンネットに48ウェルプレートに移す。 </li><li>インキュベーターから培養培地を含む6ウェルプレートを取り外し、ボンネットに転送します。 </li><li>直前に培地とよくに（セクション2.2を参照）膜上に単一の脳、調製した膜挿入の一箇所からのスライスを配置する。に気泡が膜下に存在していないことを確認してください。 </li><li>ガラスピペットを用いて、培養膜上にACSFのドロップでスライスを転送し、マイクロピペットで過剰ACSFを削除します。残りのスライスに対して、この手順を繰り返します（膜あたり最大4スライス）* *ヒント：彼らは、膜の縁に接触しないようにスライスが膜上で中央集約的にお互いに触れると場所をスライスしていないことを確認してください。 </li><li>必要な数の脳切片に対して繰り返します*. *ヒント：培養は後で時点で操作される場合、培養はインキュベーターから削除される時間を削減するとして、それは6ウェルプレートあたり3膜の量を制限するために有利かもしれない。 </li><li>ボンネットの下に週3回培養液を変更する：メンブレンインサートをピンセットでうまくから持ち上げている間に無菌の先端に真空管を使用して、古いメディアを削除します。メンブレンインサートをウェルから持ち上げている間にも新鮮な培養培地1mLを追加。すべてのメディアが変更されると、インキュベーター内にプレートを戻す。 </li></ol> 7。代表的な結果： <img src="/files/ftp_upload/2527/2527fig1.jpg" alt="図1" /> 図1。鶏の聴覚脳幹のバイノーラル回路。チキンの聴覚脳幹のスライス画像、体外で （）冠状断面の模式図と（BF）。 （A）の回路では、核magnocellularis（NM）と核グナ（NA）に聴覚神経（）からの求心性興奮性入力を示します。 NMは、脳幹の両側の核laminaris（NL）への二国間の興奮性入力を投影します。上オリーブ核（SON）プロジェクトからNA、NMとNLへの抑制性入力。のインビトロスライス （BF）は、連続した画像の高周波領域の低にして表す尾から（B）鶏の聴覚脳幹の吻側（F）になる（各200μM）は、、それぞれです。回路が音源定位のために主に使用される音の時間的なプロパティをコーディングする責任があります。のスケールバー（B）= 500μmとの画像（CF）に適用されます。 NLの単一の細胞体層に（D）点の矢印。 <img src="/files/ftp_upload/2527/2527fig2.jpg" alt="図2" /> 図2。培養神経細胞は、正常な生理学的応答の特性を開発する。急性（A＆C）と4（B）と7（D）文化の日（DIC）での培養組織からの現在の手順を過分極し、脱分極に応答して膜電位の変化をスーパーインポーズ。年齢同等の急性組織に比べてスライス培養のアプローチで同じように開発する電圧&quot;サグ&quot;、外向き電流の大幅低減、および単一のAPの焼成を過分極の注意の変更。現在の注射は200ミリ秒、50から100 pAのステップだった。 RMPS、-55、-60 mVの間であった。 <img src="/files/ftp_upload/2527/2527fig3.jpg" alt="図3" /> 図3。文化のSLIの解剖学的構造CES。文化の中で7日後のE11の鶏（DIC）の聴覚脳幹の左半分。相馬と樹状突起で発生する微小管関連タンパク質2（MAP2）、に対する抗体は、緑のラベルが貼られています。核magnocellularis（NM）の神経細胞とその軸索のクラスタは、アレクサデキストラン色素のエレクトロポレーションを経由して赤で表示されています。 NMとNLの細胞株の両方が表示されます。同側のNMの軸索末端は、背側NLの樹状突起（白矢頭）に投影見ることができます。

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+to+GABAergic+inhibition+dependent+on+increases+in+Kv1-type+K++currents.">A developmental switch to GABAergic inhibition dependent on increases in Kv1-type K+ currents.</a> J Neurosci. 27, 2112-2123 (2007).</li><li>Kuba, H., Koyano, K., Ohmori, H. <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+membrane+conductance+improves+coincidence+detection+in+the+nucleus+laminaris+of+the+chicken.">Development of membrane conductance improves coincidence detection in the nucleus laminaris of the chicken.</a> J Physiol. 540, 529-542 (2002).</li><li>Gao, H., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+intrinsic+and+synaptic+properties+of+chicken+nucleus+laminaris+neurons.">Early development of intrinsic and synaptic properties of chicken nucleus laminaris neurons.</a> Neuroscience. 153, 131-143 (2008).</li><li>Sanchez, J. 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A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Reyes, A. D., Rubel, E. W., Spain, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+properties+underlying+the+firing+of+neurons+in+the+avian+cochlear+nucleus.">Membrane properties underlying the firing of neurons in the avian cochlear nucleus.</a> J Neurosci. 14, 5352-5364 (1994).</li><li>Monsivais, P., Rubel, E. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+elimination+of+endbulb+synapses+in+the+chick+cochlear+nucleus.">Development and elimination of endbulb synapses in the chick cochlear nucleus.</a> J Neurosci. 27, 808-8017 (2007).</li><li>Kuba, H., Yamada, R., Fukui, I., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonotopic+specialization+of+auditory+coincidence+detection+in+nucleus+laminaris+of+the+chick.">Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick.</a> J Neurosci. 25, 1924-1934 (2005).</li><li>Trussell, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+for+preservation+of+timing+in+central+auditory+pathways.">Cellular mechanisms for preservation of timing in central auditory pathways.</a> Curr Opin Neurobiol. 7, 487-492 (1997).</li><li>Trussell, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+mechanisms+for+coding+timing+in+auditory+neurons.">Synaptic mechanisms for coding timing in auditory neurons.</a> Annu Rev Physiol. 61, 477-496 (1999).</li><li>Sorensen, S. A., Rubel, E. 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利害の衝突は宣言されません。

数十年の間、鶏の脳幹の急性スライス標本は、聴覚処理9、10の研究に使用されています。このアプローチは、両方の発達および成熟の状態4、11、12からバイノーラル処理上のin vitro生理データの膨大な量を提供しています。多くは、この高度に専門化された回路と、各核が音13、14の時間的な処理で果たす役割についてはほとんど知られていない。実際には、こ?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Chicken ACSF</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M-11624</td>
<td>130 mM</td>
</tr>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M-10576</td>
<td>26 mM</td>
</tr>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-9333</td>
<td>3 mM</td>
</tr>
<tr>
<td>NaH2PO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-8282</td>
<td>1.25 mM</td>
</tr>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7528</td>
<td>10 mM</td>
</tr>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M33-500</td>
<td>1 mM</td>
</tr>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>423525000</td>
<td>2 mM</td>
</tr>
<tr>
<td>Chicken culture medium (store at 4&deg;C)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>advanced minimum essential medium (with NEAA, sodium pyruvate at 110 mg/l, without L-glutamate)</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen</td>
<td>12492-013</td>
<td>48.00%</td>
</tr>
<tr>
<td>Earl's balanced salt solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E-2888</td>
<td>24.00%</td>
</tr>
<tr>
<td>L-glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7513</td>
<td>1.00%</td>
</tr>
<tr>
<td>Glucose solution (200 g/l, sterile filtered)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7528</td>
<td>2.75%</td>
</tr>
<tr>
<td>horse serum (heat inactivated, sterile filtered)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-1138</td>
<td>24.00%</td>
</tr>
<tr>
<td>Penicillin-streptomycin*</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-0781</td>
<td>1.00 ml 
* = Add to 100 ml culture medium if needed to prevent contamination</td>
</tr>
<tr>
<td>Specific equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millicell-CM cell culture inserts</td>
<td>PICMORG50</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well plate</td>
<td>6 Well Cell Culture Cluster</td>
<td>Corning Incorporated</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Forma Scientific CO2 Water Jacketed Incubator</td>
<td>Forma Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation and culture of chicken auditory brainstem slices

鶏の聴性脳幹は広く控えめな開発1-4の期間だけでなく、5月7日 、中枢神経系における時間コーディングのためのメカニズムで聴覚処理の解剖学と生理学を研究するために使用されている十分に確立されたモデルシステムです。 ここでは、急性実験手順または長期的な実験操作のための文化器官のスライスに使用することができる鶏の聴性脳幹スライスを準備する方法を提示する。 鶏の聴性脳幹は核、グナmagnocellularis、laminarisと優れたオリーブで構成されています。これらの原子核は、バイノーラル音響処理と単冠状スライス標本全体の回路を維持する責任があります。最終的に、器官切片培養では、シナプス活動などのいくつかの発達のパラメータを操作する機会、事前およびシナプス後成分の発現、興奮性および異なる遺伝子発現を制御する側面の表現を提供することができますこのアプローチは、神経回路の開発、改良と熟成に関する一般的な知識を広げるために使用することができます。

Watch this Scientific Journal Video about Preparation and Culture of Chicken Auditory Brainstem Slices at JoVE.com

Preparation and Culture of Chicken Auditory Brainstem Slices

鶏の聴性脳幹は、バイノーラルサウンド処理に責任を核で構成されています。培養のアプローチは、分子、細胞およびネットワークレベルで神経細胞の構造と聴覚機能の発達を研究するユニークな準備を提供しながら、単一冠状スライス標本は、回路全体を維持します。

チキンの聴性脳幹スライスの準備と文化

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

preparation-and-culture-of-chicken-auditory-brainstem-slices

Research

JoVE Journal

Neuroscience

11.0K ビュー.  University of Washington. 鶏の聴性脳幹は、バイノーラルサウンド処理に責任を核で構成されています。培養のアプローチは、分子、細胞およびネットワークレベルで神経細胞の構造と聴覚機能の発達を研究するユニークな準備を提供しながら、単一冠状スライス標本は、回路全体を維持します。

ビデオ: Preparation and Culture of Chicken Auditory Brainstem Slices

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

β-アミロイドオリゴマーの調製 1から42まで</sub海馬スライスでのシナプス可塑性の減損の>と誘導

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

神経科学

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

カルシウムイメージングの急性脳室下帯スライスの調製

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

ニワトリ胚脊髄スライス培養プロトコル

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

長期増強の非常に安定しており、再現性録音のための改良された準備と海馬マウススライスの保全

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

聴覚神経の光遺伝学的刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

前根刺激のための斜め脊髄スライスの調製

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

聴性脳幹反応と生後ラットの外有毛細胞細胞パッチ ・ クランプ記録

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

人工内耳手術と c57bl/6 マウスに電気的誘発脳幹の応答録音

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

リズミカルにアクティブな準備体外新生児齧歯動物脳幹-脊髄損傷で薄くスライス

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...