We are grateful for the technical support from the Neural Circuit Electrophysiology Core at Academia Sinica. This work was supported by the National Science Council (102-2320-B-001-026-MY3 and 100-2311-B-001-003-MY3) and Neuroscience Program of Academia Sinica.

動物被験体が関与する手順は、施設内動物管理と活用委員会、中央研究院、台北、台湾によって承認されました。 1.多電極アレイ記録のための実験的なソリューションと機器を準備<ol><li>人工脳脊髄液（aCSFの調製95％のO 2を通気124ミリモルのNaCl、4.4ミリモルのKCl、1mMののNaH 2 PO 3、2mMのMgSO 4を、2ミリモルのCaCl 2、25mMのNaHCO 3を、10 mMグルコース、および5％CO 2）。 </li><li> 6×10の平面MEAと8×8 MEA：MEAプローブの2種類を使用してください。前者のようなプローブは、皮質、線条体、および視床を備える領域を覆います。後者のプローブは、唯一の皮質領域をカバーしています。 </li><li> 0.1 Hzから1200増幅で3 kHzの間に設定したバンドパスフィルタで60チャンネルアンプを使用してください。 10 kHzのサンプリングレートでデータを取得します。 </li><li> DCS用MEA室内2のAgClコート銀ワイヤを配置します。 AgClを使用してくださいコー​​ティングされた銀ワイヤは、単離された刺激装置によって生成される電界を生成します。 </li><li>タングステン電極（;長さ、7.62センチメートル; 8°ACテーパーチップ;直径、127μmの抵抗、5MΩ）を配置視床刺激のために、およびMEAのチャンバー内の参照電極を配置します。パルス発生器によって制御され、単離された刺激装置を用いて、タングステン電極の電流を供給。 </li></ol> 2.脳スライスの準備<ol><li> 4-8週齢の雄のC57BL / 6Jマウスを、使用してください。食料と水を自由にアクセスできるハウスエアコンの動物（12時間/ 12時間の明/暗サイクル、午前8:00に点灯21-23°C;湿度50％）。 </li><li>ステップ1.1で調製したaCSFの250mlのアリコートを取り、氷を含んでいるビーカーに入れてください。同時に、95％O 2および5％CO 2で構成されている連続的なガスを供給する。 </li><li>手術<ol><li>ガラス中の4％イソフルランで動物を麻酔約3分間の箱。動物は（つま先のピンチへの応答の欠如によって示される）麻酔の外科的な深さに達すると、砕いた氷で満たされている浅いトレイの上に置き、はさみを使用してヘッドを取り外します。 </li><li>頭蓋骨を露出させ、残りの筋肉を切り落とします。次に、骨鉗子を使用して、脳から頭蓋骨の背側表面をはがします。骨鉗子を用いて、頭蓋骨の側面を切り取ります。 75％エタノール溶液で手術器具の全てを滅菌します。 </li><li>スパチュラを用いて、脳の腹側表面に沿って嗅球と神経接続を切断し、脳を取り出します。断頭後、速やかに氷のように冷たい酸素化にaCSFで満たされたビーカーに脳を転送します。 </li></ol></li><li>内側視床（MT）-ACC脳スライスの作製 注：ACC 13へのMTからの経路が含まれているスライスを準備します。 <ol><li>ハンドカットの2矢状カット各半球における正中線に横2.0ミリメートルと脳のブロックを皮質下の解剖学的構造を表示します。その後、2角度付きカットを行います。線条体における可視光ファイバ管への最初のクロスカット、平行してください。 </li><li> thalamocingulate経路に腹側と平行で前交連と視索の間の中間点に小脳と視覚野との間の接続から第二のクロスカットを行います。 </li><li>シアノアクリレート系接着剤で、角板（〜120°）に脳のブロックを取り付け、単に経路の転換点以上のカットを行います。プレートを広げ、それを平らにし、ビブラトームのチャンバステージの上にのり。 </li><li>内側視床-ACC脳切片（厚さの500μm）を作成した後、記録室に氷のように冷たい酸素aCSF.Transferスライスでそれらを浸し、そして1のために酸素aCSFので連続灌流（12ミリリットル/分）下で32℃で維持時間。 </li></ol></li></ol>多電極アレイ記録用灌流チャンバーの調製<ol><li>準備灌流チャンバーの<ol><li>マルチチャネルシステム上MEAプローブを配置し、蠕動ポンプにプローブを接続するために2つの別々のポリエチレン管を使用しています。 MEA室と室の外にaCSFを導くために、他のチューブにaCSFのを案内するために、1つのチューブを使用してください。最後に、継続的に暖かい（29-30℃）で酸素化にaCSF（8 ml /分）で準​​備を灌流。 </li></ol></li><li> MEAに脳スライスを転送します。湿った綿棒を使用して、MEAの脳切片を押したままにします。慎重にACCが電極上に指向されていることを確認するために脳スライスを移動します。 </li><li>スライスアンカーキットを使用して、脳切片を押してダウンを保持します。このステップは、スライスと電極との間の良好な電気的接続を確実にします。 </li></ol> DCSによって電場の4世代注：電界配向の定義はACCにおける軸索樹状突起間の軸の方向に基づいていました。樹状突起と細胞体コンパートメントの向きがありましたゴルジ染色12を使用して確認しました。 <ol><li> ACCにAgCl電極（アノードとして定義される）近位に配置し、ACCの遠位に（陰極として定義される）他の電極を配置します。 MEAによる2つの電界の向き（ACC軸索樹状突起間繊維に平行および垂直）によって生成される電界強度を記録し、刺激を用いた電界の電流を供給します。 </li><li> AgCl電極（約1.5〜2センチメートル）の距離を修正し、ミリアンペア0.5〜2 DCSを作るために刺激装置の現在の強さを調整します。 </li></ol> 5.電気的に誘発された皮質シナプス応答注：プログラム可能な電気刺激発生器は、長方形の二相性の電流パルスを生成するには、MTに電気刺激によってACCにおけるシナプス応答を誘導します。 <ol><li>上記の繰り返しセクション3。 </li><li> MTでタングステン電極を配置し、サールに刺激からのパルスを届けます双極タングステン電極を介して、スライスのアミック地域。 </li><li> ACC応答を誘発するしきい値を決定するために、さまざまな現在の強度を使用してください。ここでは、ほとんどのスライスでACCで80％最大応答を誘発した200マイクロ秒の±150μAの強度および持続時間を、使用しています。 </li><li>最適な応答プロファイルを得るために、MT-ACCスライスに（脳梁にMTから）thalamocingulate経路に沿ってタングステン電極を移動します。 </li><li> ACC応答の10-20スイープを作成し、MTの刺激により誘発されるACCのすべてを自動的に平均化するためのソフトウェアを使用しています。 MT-ACC経路によってMT刺激から誘導されたACCにおけるシナプス応答ISS結果。 </li></ol> 6.電気的に誘発される発作のような活動発作様活性は、4-アミノピリジンの適用により誘導された（4-AP; 250μM）およびビククリン（5μM）：注意してください。前回コントロール研究は、最大かつ安定した応答が現れたことを示しました薬物適用14後の2〜3時間。 <ol><li>上記の繰り返しセクション5。 </li><li>灌流液に薬物を追加します。 4-AP（250μM）を使用し、ビククリン（5μM）。均一に薬を混ぜて、2-3時間灌流を続けます。 </li><li>発作様活性を容易にするために、また、pH勾配のビルドアップを防ぐことができ、比較的速い灌流速度（8ミリリットル/分）、で灌流ポンプを維持します。 </li><li> MTでタングステン電極を配置し、ACCの応答プロファイルを得るために、電気刺激（150μA、200マイクロ秒の持続時間）を提供します。 </li><li> 10-20スイープを作成し、応答を平均します。 </li><li>薬物を洗い流すために、新鮮なaCSFの灌流液を交換してください。繰り返しステップ6.5。 </li></ol>誘発皮質応答に対するDCS 7.テスト効果<ol><li>繰り返しセクション3と4は、Mの内側に配置された2つの並列のAgClコート銀ワイヤ間の電流を流すことにより生成される均一な電界を確認してくださいEA室。問題がない場合、DCSは0.5と2ミリアンペアの間にとどまります。 </li><li> DCSの電源をオフにして、（150μA、200マイクロ秒の持続時間を±）視床を刺激するためにタングステン電極を配置します。 ACCの最大シナプス応答を得るために、10月20日スイープを作成し、応答を平均します。 </li><li>同時に、DCS（2 MV / mmのDCSの強さ）と視床刺激（350μA、200マイクロ秒の持続時間）をオンにします。 DCS中の視床刺激誘発ACC応答の振幅の変化を評価します。 </li><li> DCSの電源をオフにし、灌流液に4-AP（250μM）およびビククリン（5μM）を追加します。その後、2〜3時間を待ちます。薬は脳切片に影響を与える場合には、スライスは、皮質発作応答を生成します。 </li><li> ACC応答の10-20スイープを行い、その後、電気誘発皮質発作応答の振幅と持続時間を測定します。 </li><li>ステップ7.5の後、同時にDCS（2 MV / mmのDCSの強さ）と視床刺激（150μA、200 duratiをオンにしますマイクロ秒に）。 DCSアプリケーションの間に誘発された皮質の発作応答の振幅と持続時間の変化を評価します。 </li><li>薬物を洗い流すために、新鮮なaCSFの灌流液を交換し、リピートは7.2と7.3を繰り返します。 </li><li>異なる実験条件にデータを記録するすべてのデータを収集し、グループ。異なる実験条件の下で皮質発作応答の振幅と持続時間を評価します。 </li></ol> 8.データ解析<ol><li> （ 例えば 、MCラックソフトウェア）が自動的に記録された応答を平均化するためのソフトウェアを使用して、スプレッドシートに生データをエクスポートします。生データの振幅と持続時間を分析し、色の数値を生成します。 </li><li>振動発作イベントを検出するために、ベースライン値と標準偏差（SD）を測定するためのソフトウェアを使用しています。閾値として、ノイズレベルの3 SDを設定します。このしきい値を超える振動イベント中のピークの振幅が自動的に検出されていますエド。 </li><li> スチューデントのt検定を用いて統計分析を行います。 </li><li>スライスの数を示すnのエクスプレスの測定値と平均値±SEとしてテキストで分散分析（ANOVA）の結果の一方向分析は、12を検討しました 。 </li></ol>

<ol>
	<li>Schiller, Y., Najjar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+response+to+antiepileptic+drugs:+effect+of+past+treatment+history.">Quantifying the response to antiepileptic drugs: effect of past treatment history.</a> Neurology. 70 (1), 54-65 (2008).</li><li>Fregni, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+controlled+clinical+trial+of+cathodal+DC+polarization+in+patients+with+refractory+epilepsy.">A controlled clinical trial of cathodal DC polarization in patients with refractory epilepsy.</a> Epilepsia. 47 (2), 335-342 (2006).</li><li>Auvichayapat, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+direct+current+stimulation+for+treatment+of+refractory+childhood+focal+epilepsy.">Transcranial direct current stimulation for treatment of refractory childhood focal epilepsy.</a> Brain Stimul. 6 (4), 696-700 (2013).</li><li>Chung, M. G., Lo, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+brain+stimulation:+the+potential+for+use+in+the+rehabilitation+of+pediatric+acquired+brain+injury.">Noninvasive brain stimulation: the potential for use in the rehabilitation of pediatric acquired brain injury.</a> Arch Phys Med Rehabil. 96 (4 Suppl), S129-S137 (2015).</li><li>Del Felice, A., Magalini, A., Masiero, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow-oscillatory+Transcranial+Direct+Current+Stimulation+Modulates+Memory+in+Temporal+Lobe+Epilepsy+by+Altering+Sleep+Spindle+Generators:+A+Possible+Rehabilitation+Tool.">Slow-oscillatory Transcranial Direct Current Stimulation Modulates Memory in Temporal Lobe Epilepsy by Altering Sleep Spindle Generators: A Possible Rehabilitation Tool.</a> Brain Stimul. 8 (3), 567-573 (2015).</li><li>Garnett, E. O., Malyutina, S., Datta, A., den Ouden, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Use+of+the+Terms+Anodal+and+Cathodal+in+High-Definition+Transcranial+Direct+Current+Stimulation:+A+Technical+Note.">On the Use of the Terms Anodal and Cathodal in High-Definition Transcranial Direct Current Stimulation: A Technical Note.</a> Neuromodulation. , (2015).</li><li>Biraben, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fear+as+the+main+feature+of+epileptic+seizures.">Fear as the main feature of epileptic seizures.</a> J. Neurol. Neurosurg. Psychiatry. 70 (2), 186-191 (2001).</li><li>Zaatreh, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontal+lobe+tumoral+epilepsy:+clinical,+neurophysiologic+features+and+predictors+of+surgical+outcome.">Frontal lobe tumoral epilepsy: clinical, neurophysiologic features and predictors of surgical outcome.</a> Epilepsia. 43 (7), 727-733 (2002).</li><li>Karim, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+truth+about+lying:+inhibition+of+the+anterior+prefrontal+cortex+improves+deceptive+behavior.">The truth about lying: inhibition of the anterior prefrontal cortex improves deceptive behavior.</a> Cereb. Cortex. 20 (1), 205-213 (2010).</li><li>Keeser, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+transcranial+direct+current+stimulation+changes+connectivity+of+resting-state+networks+during+fMRI.">Prefrontal transcranial direct current stimulation changes connectivity of resting-state networks during fMRI.</a> J. Neurosci. 31 (43), 15284-15293 (2011).</li><li>Nelson, J. T., McKinley, R. A., Golob, E. J., Warm, J. S., Parasuraman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+vigilance+in+operators+with+prefrontal+cortex+transcranial+direct+current+stimulation+(tDCS).">Enhancing vigilance in operators with prefrontal cortex transcranial direct current stimulation (tDCS).</a> Neuroimage. 85 (Pt 3), 909-917 (2014).</li><li>Chang, W. P., Lu, H. C., Shyu, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+direct-current+stimulation+against+cingulate+seizure-like+activity+induced+by+4-aminopyridine+and+bicuculline+in+an+in+vitro+mouse+model.">Treatment with direct-current stimulation against cingulate seizure-like activity induced by 4-aminopyridine and bicuculline in an in vitro mouse model.</a> Exp. Neurol. 265, 180-192 (2015).</li><li>Lee, C. M., Chang, W. C., Chang, K. B., Shyu, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+organization+and+input-specific+short-term+plasticity+in+anterior+cingulate+cortical+neurons+with+intact+thalamic+inputs.">Synaptic organization and input-specific short-term plasticity in anterior cingulate cortical neurons with intact thalamic inputs.</a> Eur. J. Neurosci. 25 (9), 2847-2861 (2007).</li><li>Chang, W. P., Shyu, B. C., Pandalai, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+thalamocingulate+pathway+in+the+regulation+of+cortical+seizure+activity.">Involvement of the thalamocingulate pathway in the regulation of cortical seizure activity.</a> Recent Research Developments in Neuroscience. 4, 1-27 (2013).</li><li>Brummer, S. B., Turner, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+considerations+for+safe+electrical+stimulation+of+the+nervous+system+with+platinum+electrodes.">Electrochemical considerations for safe electrical stimulation of the nervous system with platinum electrodes.</a> IEEE Trans. Biomed. Eng. 24 (1), 59-63 (1977).</li><li>Durand, D. M., Bikson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+and+control+of+epileptiform+activity+by+electrical+stimulation:+a+review.">Suppression and control of epileptiform activity by electrical stimulation: a review.</a> Proc. IEEE. 89 (7), 1065-1082 (2001).</li><li>Fritsch, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+current+stimulation+promotes+BDNF-dependent+synaptic+plasticity:+potential+implications+for+motor+learning.">Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning.</a> Neuron. 66 (2), 198-204 (2010).</li></ol>

The authors declare that they have no competing interests.

本研究では、ACC発作様活性にDCSの持続時間と方向の効果を試験しました。マウス脳切片で安定したデータを取得するには、どのようにMT-ACC経路の整合性を保つために、それは、2角度を付け腹カットと皮質の背側カットが行われている、特にステップキーである損傷を避けるために。また、脳スライスを準備する時間も新鮮で強い脳を維持するための最短時間であるべき脳切片の活性に影響を...

Epilepsy is a common neurological disorder. Thirty percent of patients with epilepsy suffer from drug-resistant seizures1. Transcranial direct-current stimulation (tDCS) provides a noninvasive approach to control or alter network activities across large brain areas, such as seizures. Clinical studies have shown that tDCS effectively treats intractable seizures2 and can produce both short- and long-term suppressive effects on seizures3-5. However, the therapeutic mechanism of tDCS actions is still unclear. The brain slice model presented is an in vitro method to investigate how the therapeutic mechanism of tDCS actions alters the symptoms of seizure-like brain activities. Accordingly, to achieve its optimal effects, specific stimulation parameters including orientation, field strength, and stimulation duration need to be tested in an experimental model. Previous studies have shown that the orientation of the electric field is important to obtain therapeutic effects6. Thus, testing and arranging the orientation of electrodes relative to the position of the tested brain slice are feasible.
Frontal lobe epilepsy and anterior cingulate cortex (ACC) seizures are often drug-resistant7,8. Some studies have reported the application of tDCS in the cingulate cortex9-11. tDCS is shown to affect vigilance, decision making and emotion through alteration of ACC activities, and can modulate neuronal excitability and seizure activity in this brain region12. Therefore, suppressive effects of tDCS on ACC seizures might be helpful for clinical treatment and the evaluation of alternative treatments.
The present protocol describes the preparation of an electrode in the recording chamber for DCS of a brain slice and its effect on seizure-like activity recording with a multielectrode array (MEA).

<table border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1017px;">Anesthetic:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Halocarbon Products Corporation&#160;</td>
			<td>NDC 12164-002-25</td>
			<td style="width:187px;">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">aCSF (total:1L):</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-Glucose</td>
			<td>MERCK</td>
			<td>1.08337.1000</td>
			<td style="width:187px;">10 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydrogen carbonate</td>
			<td>MERCK</td>
			<td>1.06329.0500</td>
			<td style="width:187px;">25 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>MERCK</td>
			<td>1.06404.1000</td>
			<td style="width:187px;">124 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(+)-Sodium L-ascorbate, &#62;=98%</td>
			<td>SIGMA</td>
			<td>A4034-100G</td>
			<td style="width:187px;">0.15 g / 2 c.c</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium sulfate, anhydrous,ReagentPlus</td>
			<td>SIGMA</td>
			<td>M7506-500G</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>MERCK</td>
			<td>1.02382.1000</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dihydrogen phosphate monohydrate</td>
			<td>MERCK</td>
			<td>1.06346.1000</td>
			<td style="width:187px;">1 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>May &#38; Baker LTD Dagenham England</td>
			<td>MS 7616</td>
			<td style="width:187px;">4.4 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Drugs:</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Bicuculline</td>
			<td>TOCRIS</td>
			<td>0130</td>
			<td style="width:187px;">5 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Aminopyridine</td>
			<td>TOCRIS</td>
			<td>0940</td>
			<td style="width:187px;">250 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Brain slice Preparation:</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Vibratome</td>
			<td>Vibratome</td>
			<td>Series 1000</td>
			<td style="width:187px;">Block slicing into 500 &#181;m thick slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">MEA system:</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:355px;">Multielectrode array (MEA) probes: 6 x 10 planar MEA</td>
			<td>Multi Channel Systems</td>
			<td style="width:184px;">60MEA500/30iR-Ti-pr MEAS 6x10</td>
			<td style="width:187px;">electrode diameter, 30 &#181;m; electrode spacing, 500 &#181;m; impedance, 50 k&#937; at 200 Hz</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:355px;">Multielectrode array (MEA) probes: 8 x 8 MEA&#160;</td>
			<td>Ayanda Biosystems</td>
			<td style="width:184px;">60MEA200/10iR-Ti-pr MEAS 8x8</td>
			<td style="width:187px;">pyramidal-shaped electrode; diameter, 40 &#181;m; tip height, 50 &#181;m; electrode spacing, 200 &#181;m; impedance, 1000 k&#937; at 200 Hz</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:355px;">A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1200X amplification</td>
			<td>Multi-Channel Systems</td>
			<td>MEA-1060-BC</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MC Rack software at a 10 KHz sampling rate</td>
			<td>Multi-Channel Systems</td>
			<td></td>
			<td style="width:187px;">Software for data collect and recordings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">control of a pulse generator</td>
			<td>Multi-Channel Systems</td>
			<td>STG 1002</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">slice anchor kits and hold-downs</td>
			<td>Warner Instruments</td>
			<td style="width:184px;">SHD-26H/10; WI64-0250</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump-minipuls3</td>
			<td>Gilsom</td>
			<td style="width:184px;">MINIPULS3</td>
			<td>perfusion rate : 8 ml/min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Stimulation system:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isolated stimulator</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
			<td style="width:187px;">intensity of &#177;350 &#956;A , duration of 200 &#956;s</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten electrode</td>
			<td>A-M Systems</td>
			<td>575300</td>
			<td style="width:187px;">placed in thalamus</td>
		</tr>
	</tbody>
</table>

direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation

陰極経頭蓋直流刺激（TDCの）は、薬剤耐性発作の抑制効果を誘導します。効果的なアクションを実行するために、刺激パラメータ（ 例えば 、配向、磁界強度、および刺激期間）は、マウスの脳スライス標本で検査する必要があります。試験マウスの脳切片の位置に電極の向きを配置も可能です。本発明の方法は、前帯状皮質の発作のような活動にDCSの効果を評価するthalamocingulate経路を保持します。マルチチャンネル・アレイ・レコーディングの結果は陰極DCSが大幅に4-アミノピリジン及びビククリン誘発性発作様活性の刺激誘発応答の振幅および持続時間を減少することが示されました。この研究はまた15分で陰極DCSアプリケーションがthalamocingulate経路の長期不況を引き起こしたことがわかりました。本研究は、thalamocingulat上のDCSの影響を調査します電子シナプス可塑性および急性発作のような活動。現在の手順は、in vitroマウスモデルにおける配向、磁界強度、および刺激期間を含む最適な刺激パラメータをテストすることができます。また、この方法は、両方の携帯電話やネットワークレベルでの皮質発作のような活動にDCSの効果を評価することができます。

ThalamocingulateスライスとMEA記録システムのセットアップの準備マウスからのMT-ACCスライスはthalamocingulate経路の電気生理学的特性の探査を可能にする特殊なスライス標本である。 図1Aは、MT-ACCスライスが用意された方法を示しています。マウスの脳を速やかに除去し、（ 図1A、B）クール酸素aCSFの中...

Watch this Scientific Journal Video about Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation at JoVE.com

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

マウス脳スライスの準備中に発作のような活動の直流刺激とマルチ電極アレイ録音

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

direct-current-stimulation-multi-electrode-array-recording-seizure

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.4K ビュー.  Academia Sinica. Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

ビデオ: Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

スライスの準備、器官組織培養と視交叉上核における時計遺伝子の活動のルシフェラーゼ記録

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

神経科学

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

経頭蓋直流電流刺激で電極の位置とモンタージュ

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官培養における神経雪崩のマルチ電極アレイの録音

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

コンピュータ支援多電極パッチクランプシステム

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

における発作様活性を分析し、移動のための低コストの方法<em&gt;ショウジョウバエ</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

マウスの電気生理学的記録のための設計と超軽量重量の作製、調整可能なマルチ電極プローブ

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

マウスにおける経頭蓋直流電流刺激 (tDCS)

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, causal evidence of brain area activation leading to a behavior is often left missing. Transcranial direct current stimulation (tDCS), which can temporarily alter brain cortical excitability and activity, is a noninvasive neurophysiological tool used to study causal relationships in the human brain. High-definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation (NIBS) technique that produces a more focal current compared to conventional tDCS. Traditionally, the stimulation location has been roughly determined through the 10-20 EEG system, because determining precise stimulation points can be difficult. This protocol uses a 3D digitizer with HD-tDCS to increase accuracy in determination of stimulation points. The method is demonstrated using a 3D digitizer for more accurate localization of stimulation points in the right temporo-parietal junction (rTPJ).

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

高精細経頭蓋直流刺激を用いた3Dデジタイザーを用いた刺激位置決定

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

生体内でのトランス脊髄直流刺激時の型同定されたラット脊髄モネニューロンの細胞内記録

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.

マウス脳スライスの準備中に発作のような活動の直流刺激とマルチ電極アレイ録音

要約

さらに動画を探す

この動画の章