この研究は、国立科学財団グラントCMMI-1300007によって資金提供されました。ジョセフ・メイソン大学でジョセフ・J・パンクラジオ博士、ハミド・チャルカカル博士、グレッチェン・クナック博士（Dr. Gretchen Knaack）博士の5年間にわたり、これらのプロトコールの設計と5年以上の文化の維持に協力してきた先の研究員に感謝したい。 、フランツ・ハミルトン博士、マイケル・マケラ、ロバート・グラハム。

<ol>
	<li>Charkhkar, H., Frewin, C., 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=Use+of+cortical+neuronal+networks+for+in+vitro+material+biocompatibility+testing.">Use of cortical neuronal networks for in vitro material biocompatibility testing.</a> Biosens Bioelectron. 53, 316-323 (2014).</li><li>Bologna, L. L., Nieus, T., Tedesco, M., Chiappalone, M., Benfenati, F., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-frequency+stimulation+enhances+burst+activity+in+cortical+cultures+during+development.">Low-frequency stimulation enhances burst activity in cortical cultures during development.</a> Neuroscience. 165 (3), 692-704 (2010).</li><li>Fr&#246;hlich, F., McCormick, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+electric+fields+may+guide+neocortical+network+activity.">Endogenous electric fields may guide neocortical network activity.</a> Neuron. 67 (1), 129-143 (2010).</li><li>Anastassiou, C. A., Perin, R., Markram, H., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephaptic+coupling+of+cortical+neurons.">Ephaptic coupling of cortical neurons.</a> Nature Neurosci. 14 (2), 217-223 (2011).</li><li>Ozen, S., Sirota, 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=Transcranial+electric+stimulation+entrains+cortical+neuronal+populations+in+rats.">Transcranial electric stimulation entrains cortical neuronal populations in rats.</a> J. Neurosci. 30 (34), 11476-11485 (2010).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, 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=Controlling+bursting+in+cortical+cultures+with+closed-loop+multi-electrode+stimulation.">Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.</a> J. Neurosci. 25 (3), 680-688 (2005).</li><li>Shahaf, G., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+networks+of+cortical+neurons.">Learning in networks of cortical neurons.</a> J. Neurosci. 21 (22), 8782-8788 (2001).</li><li>Marom, S., Shahaf, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+learning+and+memory+in+large+random+networks+of+cortical+neurons:+lessons+beyond+anatomy.">Development, learning and memory in large random networks of cortical neurons: lessons beyond anatomy.</a> Q Rev Biophys. 35 (1), 63-87 (2002).</li><li>Marom, S., Eytan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+ex-vivo+developing+networks+of+cortical+neurons.">Learning in ex-vivo developing networks of cortical neurons.</a> Prog Brain Res. 147, 189-199 (2005).</li><li>Li, Y., Zhou, W., Li, X., Zeng, S., Luo, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+learning+in+cultured+neuronal+networks+with+antagonists+of+glutamate+receptors.">Dynamics of learning in cultured neuronal networks with antagonists of glutamate receptors.</a> Biophys. J. 93 (12), 4151-4158 (2007).</li><li>Li, Y., Zhou, W., Li, X., Zeng, S., Liu, M., Luo, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+synchronized+bursts+in+cultured+hippocampal+neuronal+networks+with+learning+training+on+microelectrode+arrays.">Characterization of synchronized bursts in cultured hippocampal neuronal networks with learning training on microelectrode arrays.</a> Biosens Bioelectron. 22 (12), 2976-2982 (2007).</li><li>Chiappalone, M., Massobrio, P., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+plasticity+in+cortical+assemblies.">Network plasticity in cortical assemblies.</a> Eur. J. Neurosci. 28 (1), 221-237 (2008).</li><li>le Feber, J., Stegenga, J., Rutten, W. L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+slow+electrical+stimuli+to+achieve+learning+in+cultured+networks+of+rat+cortical+neurons.">The effect of slow electrical stimuli to achieve learning in cultured networks of rat cortical neurons.</a> PloS one. 5 (1), 8871 (2010).</li><li>Ruaro, M. E., Bonifazi, P., Torre, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+neurocomputer:+image+processing+and+pattern+recognition+with+neuronal+cultures.">Toward the neurocomputer: image processing and pattern recognition with neuronal cultures.</a> IEEE Trans Biomed Eng. 52 (3), 371-383 (2005).</li><li>Hamilton, F., Graham, R., 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=Time-Dependent+Increase+in+Network+Response+to+Stimulation.">Time-Dependent Increase in Network Response to Stimulation.</a> PloS one. 10 (11), 0142399 (2015).</li><li>. Guidelines for Euthanasia of Rodents Using Carbon Dioxide Available from: <a target="_blank" href="https://oacu.oir.nih.gov/sites/default/files/uploads/arac-guidelines/rodent_euthanasia_adult.pdf">https://oacu.oir.nih.gov/sites/default/files/uploads/arac-guidelines/rodent_euthanasia_adult.pdf</a> (2016)</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells: Business Source. , (1998).</li><li>Gullo, F., Maffezzoli, A., Dossi, E., Wanke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-latency+cross-and+autocorrelation+identify+clusters+of+interacting+cortical+neurons+recorded+from+multi-electrode+array.">Short-latency cross-and autocorrelation identify clusters of interacting cortical neurons recorded from multi-electrode array.</a> J. Neurosci. Methods. 181, 186-198 (2009).</li><li>Eytan, D., Brenner, N., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Adaptation+in+Networks+of+Cortical+Neurons.">Selective Adaptation in Networks of Cortical Neurons.</a> J. Neurosci. 23 (28), (2003).</li><li>Bonifazi, P., Ruaro, M. E., Torre, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+properties+of+information+processing+in+neuronal+networks.">Statistical properties of information processing in neuronal networks.</a> Eur. J. Neurosci. 22 (11), 2953-2964 (2005).</li></ol>

著者は何も開示することはない。

このプロトコールで概説されている手順は、初心者がMEA上で自身のニューロン培養物をプレートし、ネットワーク活動を記録するのに十分な詳細を提供する。このプロトコールは、培養物が適切に付着し、電極アレイ上に細胞のカーペット層を形成し、数ヶ月間健康で汚染物質を含まないことを確実にするのに役立つ。 プロトコルのすべての部分を順守することが最善の方法ですが、成果を上げるために重要なプロセスがあります。培養が汚染されるのを防ぐためには、プロセス全体を通して無菌技術を使用することが不可欠です。プロトコールに記載されているように、新しいMEAを親水性にしなければならない。そうでなければ、細胞接着不良が生じる。厳しいピペッティングおよび解離中の気泡の形成を回避することにより、損傷した細胞がプレーティングされる回数を減少させ、より高く、より健康的な収量をもたらす。最初の後にDMEM 5/5からDMEM +に切り替える摂食も重要です。 DMEM 5/5にはウマ血清が含まれており、連続的に使用するとグリア細胞が培養上支配的になり、培養が正常に見えるものの、ニューロン活性が低下します17 。スケジュールどおりに培養物に給餌し、適切なインキュベーション条件に保つこともまた重要である。 MEA上の細胞培養物をプレーティングすることは、最適ではない結果をもたらす可能性がある多くの変数を伴う。目標は細胞の完全な「カーペット」であるが、上記の重要なステップに対処できなければ、細胞の成熟が悪くなったり、汚染されたりする。乏しい細胞の成熟とは異なる貧弱な細胞接着もまた懸念される。これは、メッキ前の貧弱なMEA調製または古い培地の使用を含むいくつかの要因によって引き起こされる可能性がある。神経細胞培養のための安定化形態のL-グルタミンおよび無血清補充物を含有する古い培地（ 材料 表）が使用されると、細胞は最初に接着するが、約2週間後に浮遊する。細菌汚染が持続的な問題である場合、アンピシリンまたはペン - ストレプトンなどの抗生物質を培地に添加することができる。真菌汚染を処理するために利用できる殺菌剤もある。これらは、文化の成果に影響する可能性があるより一般的な変数のいくつかです。時間と経験の後にのみ遭遇する多くのものがあります。 ガラス微小電極の使用と比較して、この技術はネットワークのダイナミクスおよび薬理学的応答を研究するのに優れている。これは、多くの異なる時空間刺激パターンの使用を可能にし、同時に複数の領域からの神経応答の記録を可能にする。以前のグループは、ここで説明したものと同様のプロトコルを使用して興味深い結果を示しています18 。培養は数週間または数ヶ月続き、同じ培養物を再使用することができるので、この技術もまた同じネットワーク上で時間の経過とともに複数の実験が繰り返されます。 しかし、この手法には限界があります。 MEAは非侵襲性である。したがって、パッチクランプまたはピペットによる細胞内記録とは対照的に、細胞外活性のみを記録することができる。さらに、アレイ中の各電極はいくつかの細胞によって覆われているので、単一のニューロンの活性を分解することは不可能である。逆に、これらはインビトロ培養であるため、脳内のネットワークの構造的特性を完全に再現することはできません。また、細胞がそれらのpHバランスを維持するためのCO 2雰囲気を提供する機構がなければ、活性は一度に30分未満しか記録できない。 この技術が習得されると、電気刺激を伴うまたは伴わない薬理学的操作が探究され得る。ニューロンネットワークにおける学習および記憶形成を突き止めるための新しいプロトコルもまた、p海馬または脊髄ネットワークのためのプロトコル。ネットワークの刺激および訓練のためのプロトコルは以前に公開されており、これらのうちのいくつかは、Eytan らによって提案された「選択的適応」などのインビボプロトコルにさらに発展した 。 19 。いくつかのプロトコルがテストされました。しかし、2005年にRuaroによって提案された破傷風手技の修正の結果のみがここに提示される14,20。

マイクロ電極アレイ（MEA）は、薬物の毒性、次世代パーソナライズド医薬品の設計パラダイム、神経細胞培養におけるネットワークダイナミクスの研究に使用できます1 。単一の細胞からの活動のみを記録できるパッチクランプ法や、単一のサイトで電極を囲むニューロンからの細胞外応答を記録できるガラスピペットによるフィールド記録などの従来の方法とは対照的に、MEAは同時に各電極を個々に配置するという煩雑な作業を必要とすることなく、細胞培養中の複数の部位から記録することができる。これは、その培養物内のネットワークを形成する細胞群間の動的相互作用の研究を可能にする。さらに、ネットワーク発射パターン2,3,4,5およびネットワーク制御6に対する電気刺激の効果は、/ sup&gt;は十分に実証されており、電気的刺激および制御の多数の構成を同じ実験装置内で容易に適用することができ、幅広い時空間ダイナミクスを探究することができる。 これらのインビトロ研究における重要な動力学の1つは、培養されたネットワークが、学習の指標である7,8,9,10,11,12,13を示す程度であった。 Peixoto Labは以前、Ruaro らに記載されているように、高周波トレーニング信号の影響を調べました。 14 、マイクロ電極アレイ15上にプレートされたマウスニューロンのネットワーク上。これらの実験では、ネットワークは応答フォローインの増加を示した電気刺激によって誘導されるg訓練。増加した反応は、刺激認識を介した学習の一形態と考えられ、これにより、ネットワークは、特定の刺激（ すなわちトレーニング）プロトコルの適用後に刺激の変化に対して一貫した方法で応答した。 このプロトコルは、MEA上で神経細胞を培養し、皿の95％以上からの記録を成功させ、刺激のパターンに応答してネットワークを訓練し、単一ユニットの活動を分類し、ヒストグラムをプロットし、そのような実験。ニューロン培養の刺激および記録のための専用システム（ 表を参照 ）の使用、ならびにニューロンユニットを分類するためのソフトウェアパッケージ（ 表の表を参照 ）の適用を実証する 。カスタムデザインのグラフィカルユーザーインターフェイス（ 表の表を参照 ）を使用してポストsを視覚化します時間間隔ヒストグラム、インターバースト間隔、およびバースト持続時間の測定、ならびにトレーニングプロトコルの前後の刺激に対する細胞応答を比較することができる。

<table border="0" cellpadding="0" cellspacing="0" width="1135">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Ascorbic Acid</td>
			<td style="width:204px;">Sigma Aldrich</td>
			<td style="width:155px;">A4403</td>
			<td style="width:356px;">Make 4mg/mL aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">B-27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>Serum-free supplement for neural cell culture. Make 1 mL aliquots and freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Beakers - glass - 100 mL</td>
			<td>VWR</td>
			<td>13912-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Beakers - glass - 500 mL</td>
			<td>VWR</td>
			<td>10754-956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Blunt-tipped thumb forceps</td>
			<td>World Precision Instruments</td>
			<td>500365-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Cryogenic vial - sterile, 2 mL</td>
			<td>Fisher Scientific</td>
			<td>10-500-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Cryogenic vial - sterile, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>10-500-27</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">DMEM with Glutamax</td>
			<td>Gibco Life Technology</td>
			<td>10569-010</td>
			<td>Cell culture media that contains a stabilized form of L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">DNase</td>
			<td>Worthington Biochemical</td>
			<td>LK003172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Fetal bovine serum (FBS)</td>
			<td>ATCC</td>
			<td>30-2030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Fine-tipped thumb forceps</td>
			<td>World Precision Instruments</td>
			<td style="width:155px;">501324-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Glass Pipets</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">GlutaMAX -- I (100X)</td>
			<td>Gibco Life Technology</td>
			<td>35050-061</td>
			<td>A stabilized form of L-glutamine used as a suplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Hemocytometer chip</td>
			<td>Fisher Scientific</td>
			<td>22-600-101</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">Hibernate EB complete</td>
			<td>BrainBits</td>
			<td>D00118</td>
			<td>Ambient CO2 cell storage media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Horse Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12195H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS filter amplifier</td>
			<td>MultiChannel System</td>
			<td>FA60SBC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS headstage/pre-amplifier</td>
			<td>MultiChannel System</td>
			<td>MEA1060-INV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">MCS microelectrode array</td>
			<td>MultiChannel System</td>
			<td>60MEA200/10iR-ITO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS power supply</td>
			<td>MultiChannel System</td>
			<td>PS20W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS signal divider</td>
			<td>MultiChannel System</td>
			<td>SDMEA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS stimulus generator</td>
			<td>MultiChannel System</td>
			<td>STG4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS temperature controller</td>
			<td>MultiChannel System</td>
			<td>TC02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Media storage bottle - glass 500 mL</td>
			<td>VWR</td>
			<td>10754-818</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Microcentrifuge tubes - 0.4 mL</td>
			<td>Thermo Fisher</td>
			<td>3485</td>
			<td>Autoclave</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:420px;">Microcentrifuge tubes - 2 mL</td>
			<td>Thermo Fisher</td>
			<td>3434ECONO</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 10 uL&#160;</td>
			<td>VWR</td>
			<td>37001-166</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 1000 uL&#160;</td>
			<td>VWR</td>
			<td>13503-464</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 200 uL&#160;</td>
			<td>VWR</td>
			<td>37001-596</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 10 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641170N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 1000 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641210N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 200 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641230N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Papain - 0.22 Filtered</td>
			<td>Worthington Biochemical</td>
			<td>LK003178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Penicillin-Streptomycin (Pen Strep)</td>
			<td>Thermo Fisher</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -100mm disposable</td>
			<td>Fisher Scientific</td>
			<td>08-757-100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -100mm glass</td>
			<td>VWR</td>
			<td>10754-788</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -35 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Phosphate buffer saline (PBS) (1X)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Plasma Cleaning System</td>
			<td>Plasma Etch, Inc.</td>
			<td>PE-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Poly-D-lysine hydrobromide (PDL)</td>
			<td>Sigma Aldrich</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Polyethylene conical (centrifuge) tube -15 mL</td>
			<td>Fisher Scientific</td>
			<td>05-527-90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Polypropylene conical (centrifuge) tube -50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Procedure masks</td>
			<td>Imco</td>
			<td>1530-imc</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P4562-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scalpel blades</td>
			<td>World Precision Instruments</td>
			<td>&#160;500237-G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">Scissors - iris</td>
			<td>World Precision Instruments</td>
			<td>14111-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scissors - large</td>
			<td>World Precision Instruments</td>
			<td>14214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scissors - small surgical</td>
			<td>World Precision Instruments</td>
			<td>&#160;501733-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 1 mL&#160;</td>
			<td>VWR</td>
			<td>89130-892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 2 mL&#160;</td>
			<td>VWR</td>
			<td>89130-894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 25 mL&#160;</td>
			<td>VWR</td>
			<td>89130-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 50 mL&#160;</td>
			<td>VWR</td>
			<td>89130-902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: Matlab GUI</td>
			<td>Peixoto Lab</td>
			<td>npeixoto@gmu.edu</td>
			<td>Used to analyze .mat files.&#160; Available for free upon request.&#160; Contact npeixoto@gmu.edu to request a copy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: MCS MC_Rack</td>
			<td>MultiChannel System</td>
			<td>N/A</td>
			<td>Used for data acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: NeuroExplorer</td>
			<td>Plexon&#160;</td>
			<td>http://www.plexon.com/products/neuroexplorer</td>
			<td>Used to convert .nex files to .mat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: Offline Sorter</td>
			<td>Plexon&#160;</td>
			<td>http://www.plexon.com/products/offline-sorter</td>
			<td>Used to sort neural spikes and convert data files to .plx and then to .nex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: MCS MC_Rack</td>
			<td>MultiChannel System</td>
			<td></td>
			<td>https://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/manuals/MC_Rack_Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: NeuroExplorer</td>
			<td>Plexon&#160;</td>
			<td></td>
			<td>https://www.neuroexplorer.com/downloads/Nex3Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: Offline Sorter</td>
			<td>Plexon&#160;</td>
			<td></td>
			<td>www.plexon.com/system/files/downloads/Offline%20Sorter%20v2.8%20Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Spatula - small double-ended</td>
			<td>World Precision Instruments</td>
			<td>&#160;503440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Stericup 0.22 &#181;m pore filter - 250 mL</td>
			<td>Millipore</td>
			<td>SCGVU02RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Transfer pipette - large bore&#160;</td>
			<td>Thermo Fisher</td>
			<td>335-1S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Transfer pipette - small bore&#160;</td>
			<td>Thermo Fisher</td>
			<td>242-1S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Trypan blue</td>
			<td>Sigma Aldrich</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Whatman filter paper</td>
			<td>Whatman</td>
			<td>1442 150</td>
			<td>Cut into 8 pie wedges and autoclave in a glass Petri dish</td>
		</tr>
	</tbody>
</table>

time-dependent increase in the network response to the stimulation of neuronal cell cultures on micro-electrode arrays

マイクロ電極アレイ（MEA）は、薬物毒性、次世代パーソナライズド医薬品の設計パラダイム、神経細胞培養におけるネットワークダイナミクスの研究に使用できます。単一のセルからの活動だけを記録できるパッチクランプのような従来の方法とは対照的に、MEAは、各電極を個別に配置するという厄介な作業を必要とせずに、ネットワークの複数のサイトから同時に記録することができる。さらに、多数の制御および刺激構成を同じ実験装置内で容易に適用することができ、幅広いダイナミクスを探索することが可能になる。これらのインビトロ研究における重要な動力学の1つは、培養されたネットワークが学習を示す特性を示す程度であった。 MEA上で培養されたマウス神経細胞は、電気刺激によって誘発された訓練後の応答の増加を示す。このプロトコルは、MEA上で神経細胞を培養する方法を示す。成功したメッキされた皿の95％以上からのコード;刺激のパターンに応答するようにネットワークを訓練するためのプロトコルを確立する。そのような実験の結果をソートし、プロットし、解釈する。ニューロン培養を刺激および記録するための独自のシステムの使用が実証される。ソフトウェアパッケージは、ニューロンユニットの分類にも使用されます。刺激後時間ヒストグラム、バースト間隔、バースト持続時間を視覚化し、トレーニングプロトコールの前後で刺激に対する細胞応答を比較するために、カスタム設計されたグラフィカルユーザインタフェースが使用される。最後に、この研究努力の代表的な結果と今後の方向性について議論する。

Watch this Scientific Journal Video about Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays at JoVE.com

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

多電極アレイ上で培養されたマウス神経細胞は、電気刺激後の応答の増加を示す。このプロトコルは、ニューロンを培養する方法、活動を記録する方法、および刺激のパターンに応答するようにネットワークを訓練するプロトコルを確立する方法を示す。

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

time-dependent-increase-network-response-to-stimulation-neuronal-cell

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.8K ビュー.  George Mason University. 多電極アレイ上で培養されたマウス神経細胞は、電気刺激後の応答の増加を示す。このプロトコルは、ニューロンを培養する方法、活動を記録する方法、および刺激のパターンに応答するようにネットワークを訓練するプロトコルを確立する方法を示す。 

ビデオ: Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

デフォルトモードのネットワークを検査する経頭蓋磁気刺激とfMRIを組み合わせ

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

神経科学

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMSデバイス：うつ病の治療薬としてFDA承認されたプロトコルを実施

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

動物を振る舞う、覚醒中のシングルユニット記録のためにマイクロワイヤの組織化された配列を移植するための手順

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

単セルの精度で神経共培養の調製

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

ゼブラフィッシュ幼虫における視覚反応の電位図解析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

微小電極アレイへの3Dエンジニア神経文化のインタフェース：革新的な<em&gt;インビトロ</em&gt;実験モデル

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...