著者たちは、Ofer Feinerman、Fred Wolf、Menahem Segal、Andreas Neef、Eitan Reuvenyに非常に有益な議論に感謝します。著者は、この技術の初期バージョンの開発について、Ilan BreskinとJordi Sorianoに感謝します。作者は理論的概念の助けを借りてTsvi TlustyとJean-Pierre Eckmannに感謝します。この研究は、ミネルバ財団、イスラエル科学技術省、イスラエル科学財団助成金1320/09およびバイナショナル科学財団助成金2008331によって支援された。

<ol>
	<li>Nagarajan, S. S., Durand, D. M., Warman, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+induced+electric+fields+on+finite+neuronal+structures:+a+simulation+study.">Effects of induced electric fields on finite neuronal structures: a simulation study.</a> IEEE Trans Biomed Eng. 40 (11), 1175-1188 (1993).</li><li>Nowak, L. G., Bullier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axons+but+not+cell+bodies,+are+activated+by+electrical+stimulation+in+cortical+gray+matter.+II.+Evidence+from+selective+inactivation+of+cell+bodies+and+axon+initial+segments.">Axons but not cell bodies, are activated by electrical stimulation in cortical gray matter. II. Evidence from selective inactivation of cell bodies and axon initial segments.</a> Exp Brain Res. 118 (4), 489-500 (1998).</li><li>Ranck, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+elements+are+excited+in+electrical+stimulation+of+mammalian+central+nervous+system:+a+review.">Which elements are excited in electrical stimulation of mammalian central nervous system: a review.</a> Brain Res. 98 (3), 417-440 (1975).</li><li>Rattay, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+mechanism+for+the+electrical+stimulation+of+the+nervous+system.">The basic mechanism for the electrical stimulation of the nervous system.</a> Neuroscience. 89 (2), 335-346 (1999).</li><li>Stern, S., Agudelo-Toro, A., Rotem, A., Moses, E., Neef, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronaxie+Measurements+in+Patterned+Neuronal+Cultures+from+Rat+Hippocampus.">Chronaxie Measurements in Patterned Neuronal Cultures from Rat Hippocampus.</a> PLoS One. 10 (7), e0132577 (2015).</li><li>Brunelin, J., 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=Examining+transcranial+direct-current+stimulation+(tDCS)+as+a+treatment+for+hallucinations+in+schizophrenia.">Examining transcranial direct-current stimulation (tDCS) as a treatment for hallucinations in schizophrenia.</a> Am J Psychiatry. 169 (7), 719-724 (2012).</li><li>Cruccu, G., 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=EFNS+guidelines+on+neurostimulation+therapy+for+neuropathic+pain.">EFNS guidelines on neurostimulation therapy for neuropathic pain.</a> Eur J Neurol. 14 (9), 952-970 (2007).</li><li>Kennedy, S. H., 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=Canadian+Network+for+Mood+and+Anxiety+Treatments+(CANMAT)+Clinical+guidelines+for+the+management+of+major+depressive+disorder+in+adults.+IV.+Neurostimulation+therapies.">Canadian Network for Mood and Anxiety Treatments (CANMAT) Clinical guidelines for the management of major depressive disorder in adults. IV. Neurostimulation therapies.</a> J Affect Disord. 117, S44-S53 (2009).</li><li>Minzenberg, M. J., Carter, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+treatments+for+impaired+cognition+in+schizophrenia.">Developing treatments for impaired cognition in schizophrenia.</a> Trends Cogn Sci. 16 (1), 35-42 (2012).</li><li>Vaidya, N. A., Mahableshwarkar, A. R., Shahid, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuation+and+maintenance+ECT+in+treatment-resistant+bipolar+disorder.">Continuation and maintenance ECT in treatment-resistant bipolar disorder.</a> J ECT. 19 (1), 10-16 (2003).</li><li>Bartlett, W. P., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electron+microscopic+study+of+the+development+of+axons+and+dendrites+by+hippocampal+neurons+in+culture.+II.+Synaptic+relationships.">An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. II. Synaptic relationships.</a> J Neurosci. 4 (8), 1954-1965 (1984).</li><li>Bartlett, W. P., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electron+microscopic+study+of+the+development+of+axons+and+dendrites+by+hippocampal+neurons+in+culture.+I.+Cells+which+develop+without+intercellular+contacts.">An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts.</a> J Neurosci. 4 (8), 1944-1953 (1984).</li><li>Beggs, J. M., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+avalanches+in+neocortical+circuits.">Neuronal avalanches in neocortical circuits.</a> J Neurosci. 23 (35), 11167-11177 (2003).</li><li>Breskin, I., Soriano, J., Moses, E., Tlusty, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percolation+in+living+neural+networks.">Percolation in living neural networks.</a> Phys Rev Lett. 97 (18), (2006).</li><li>Feinerman, O., Moses, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+information+along+unidimensional+layered+networks+of+dissociated+hippocampal+neurons+and+implications+for+rate+coding.">Transport of information along unidimensional layered networks of dissociated hippocampal neurons and implications for rate coding.</a> J Neurosci. 26 (17), 4526-4534 (2006).</li><li>Soriano, J., Rodriguez Martinez, ., Tlusty, M., T, E., Moses, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+input+connections+in+neural+cultures.">Development of input connections in neural cultures.</a> Proc Natl Acad Sci U S A. 105 (37), 13758-13763 (2008).</li><li>Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics.">Optogenetics.</a> Nat Methods. 8 (1), 26-29 (2011).</li><li>Fenno, L., Yizhar, O., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+application+of+optogenetics.">The development and application of optogenetics.</a> Annu Rev Neurosci. 34, 389-412 (2011).</li><li>Williams, S. C., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics.">Optogenetics.</a> Proc Natl Acad Sci U S A. 110 (41), 16287 (2013).</li><li>Rotem, A., Moses, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+stimulation+of+curved+nerves.">Magnetic stimulation of curved nerves.</a> IEEE Trans Biomed Eng. 53 (3), 414-420 (2006).</li><li>Rotem, A., Moses, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+stimulation+of+one-dimensional+neuronal+cultures.">Magnetic stimulation of one-dimensional neuronal cultures.</a> Biophys J. 94 (12), 5065-5078 (2008).</li><li>Feinerman, O., Moses, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+picoliter+'fountain-pen'+using+co-axial+dual+pipettes.">A picoliter 'fountain-pen' using co-axial dual pipettes.</a> J Neurosci Methods. 127 (1), 75-84 (2003).</li><li>Feinerman, O., Segal, M., Moses, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+propagation+along+unidimensional+neuronal+networks.">Signal propagation along unidimensional neuronal networks.</a> J Neurophysiol. 94 (5), 3406-3416 (2005).</li><li>Papa, M., Bundman, M. C., Greenberger, V., Segal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+dendritic+spine+development+in+primary+cultures+of+hippocampal+neurons.">Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons.</a> J Neurosci. 15 (1 Pt 1), 1-11 (1995).</li><li>Bikson, 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=Effects+of+uniform+extracellular+DC+electric+fields+on+excitability+in+rat+hippocampal+slices+in+vitro.">Effects of uniform extracellular DC electric fields on excitability in rat hippocampal slices in vitro.</a> J Physiol. 557 (1), 175-190 (2004).</li><li>Rahman, 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=Cellular+effects+of+acute+direct+current+stimulation:+somatic+and+synaptic+terminal+effects.">Cellular effects of acute direct current stimulation: somatic and synaptic terminal effects.</a> J Physiol. 591 (10), 2563-2578 (2013).</li><li>Stern, S., Segal, M., Moses, E. <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+Potassium+and+Cation+Channels+in+Hippocampal+Abnormalities+of+Embryonic+Ts65Dn+and+Tc1+Trisomic+Mice.">Involvement of Potassium and Cation Channels in Hippocampal Abnormalities of Embryonic Ts65Dn and Tc1 Trisomic Mice.</a> EBioMedicine. 2 (9), 1048-1062 (2015).</li><li>Rotem, 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=Solving+the+orientation+specific+constraints+in+transcranial+magnetic+stimulation+by+rotating+fields.">Solving the orientation specific constraints in transcranial magnetic stimulation by rotating fields.</a> PLoS One. 9 (2), e86794 (2014).</li><li>Feinerman, O., Rotem, A., Moses, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+neuronal+logic+devices+from+patterned+hippocampal+cultures.">Reliable neuronal logic devices from patterned hippocampal cultures.</a> Nat Phys. 4 (12), 967-973 (2008).</li><li>Kleinfeld, D., Kahler, K. H., Hockberger, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+outgrowth+of+dissociated+neurons+on+patterned+substrates.">Controlled outgrowth of dissociated neurons on patterned substrates.</a> J Neurosci. 8 (11), 4098-4120 (1988).</li><li>Bugnicourt, G., Brocard, J., Nicolas, A., Villard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+surface+topography+reshapes+neuronal+growth+in+culture.">Nanoscale surface topography reshapes neuronal growth in culture.</a> Langmuir. 30 (15), 4441-4449 (2014).</li><li>Roth, S., 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=Neuronal+architectures+with+axo-dendritic+polarity+above+silicon+nanowires.">Neuronal architectures with axo-dendritic polarity above silicon nanowires.</a> Small. 8 (5), 671-675 (2012).</li><li>Peyrin, J. 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=Axon+diodes+for+the+reconstruction+of+oriented+neuronal+networks+in+microfluidic+chambers.">Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers.</a> Lab Chip. 11 (21), 3663-3673 (2011).</li><li>Renault, 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=Combining+microfluidics,+optogenetics+and+calcium+imaging+to+study+neuronal+communication+in+vitro.">Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro.</a> PLoS One. 10 (4), e0120680 (2015).</li><li>Roth, B. J., Basser, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+the+stimulation+of+a+nerve+fiber+by+electromagnetic+induction.">A model of the stimulation of a nerve fiber by electromagnetic induction.</a> IEEE Trans Biomed Eng. 37 (6), 588-597 (1990).</li></ol>

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

1Dパターニングは、様々な用途に使用することができる重要なツールです。例えば、我々は、ラット海馬ニューロン5のクロナキシーおよび基電流を測定するために、より最近ニューロン培養物29からの論理ゲートを作成するための1次元パターニングを使用している、とと比較してダウン症候群海馬ニューロンにおける発火活動の信号伝搬速度の減速します...

ニューロンおよび外部電場との相互作用は、基本的な意味合いだけでなく、実用的なものを持っています。それは外部から印加される電界が組織を励起することができるボルタの時代から知られているが、ニューロンにおける結果として得られる活動電位の生成を担うメカニズムは、ごく最近、4 3、2、1解明され始めています。これは、電場2、5に応答ニューロンにおける膜電位の脱分極、膜特性のとイオンチャネルの役割、さらには地域の原因となるメカニズムに関する質問への回答を見つけることが含まれます。治療的神経刺激6、7、8、9、 <supクラス=「外部参照」は&gt; 10個の方法論は、被災地を標的とすると治療の結果を理解するために重要であることができ、この情報に特に依存しています。このような理解はまた、治療プロトコルと、脳内の異なる領域を刺激するための新しいアプローチを開発するのに役立ちます。 in vivoでの脳内の相互作用を測定することは、この理解に重要な要素が追加されますが、頭蓋骨内の測定の不正確さと低制御性によって妨げられています。対照的に、培養物中の測定を容易に高精度、ノイズ性能に優れた信号と再現性と制御性の高い大量に行うことができます。集合的ネットワーク動作のニューロン特性の多種多様を14、13、12、11明らかにすることができる培養物を使用して、 </sup&gt; 15、16。同様に、このよく制御されたシステムは、他の刺激方法がoptogeneticallyアクティブニューロン17、18の光刺激の間のチャネル開口部、19は活動電位を生成するための責任がある方法、例えば、動作するメカニズムを解明するには非常に効率的であることが期待されます。 ここでの焦点は、効率的に外部電界を経由してニューロンを励起することができるツールの開発と理解を記述するにあります。本稿では、バス電極によって異なる構成と直接印加される電界の向きを使用して、刺激を二次元の調製と一次元パターン化された海馬培養物を記述し、そして最終的に二次元の刺激とによって一次元培養をパターニング時間的に変化する電界を誘起する磁界を、5、20、21。

<table><tbody><tr><td>APV</td><td>Sigma-Aldrich</td><td>A8054</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>B27 supp</td><td>Gibco</td><td>17504-044</td><td>Plating medium. Mentioned in Section 1.1.1</td></tr><tr><td>bicuculline</td><td>Sigma-Aldrich</td><td>14343</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>Borax (sodium tetraborate decahydrate)</td><td>Sigma-Aldrich</td><td>S9640</td><td>Borate buffer. Mentioned in Section 1.1.2</td></tr><tr><td>Boric acid</td><td>Frutarom LTD</td><td>5550710</td><td>Borate buffer. Mentioned in Section 1.1.2</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; , 1 M</td><td>Fluka&amp;nbsp;</td><td>21098</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>CNQX</td><td>Sigma-Aldrich</td><td>C239</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>COMSOL</td><td>COMSOL Inc</td><td>Multiphysics 3.5</td><td>Numerical simulation. Mentioned in Section 3.5.2</td></tr><tr><td>D-(+)-Glucose, 1 M</td><td>Sigma-Aldrich</td><td>65146</td><td>Plating medium, Extracellular recording solution. Mentioned in Sections 1.1.1 and 1.5.2</td></tr><tr><td>D-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>Cell Cultures. Mentioned in Sections 1.2.4 and 1.2.6</td></tr><tr><td>FCS (FBS)</td><td>Gibco</td><td>12657-029</td><td>Plating medium. Mentioned in Section 1.1.1</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td>Bio Coating. Mentioned in Section 1.2.6</td></tr><tr><td>Fluo4AM</td><td>Life technologies</td><td>F14201</td><td>Imaging of spontaneous or evoked activity. Mentioned in Sections 1.5.1, 1.5.3, and 1.5.5</td></tr><tr><td>FUDR</td><td>Sigma-Aldrich</td><td>F0503</td><td>Changing medium. Mentioned in Section 1.4.1</td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1272</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td></tr><tr><td>GlutaMAX 100x</td><td>Gibco</td><td>35050-038</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td></tr><tr><td>Hepes, 1 M</td><td>Sigma-Aldrich</td><td>H0887</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>HI HS</td><td>BI</td><td>04-124-1A</td><td>Plating medium, Changing medium, Final medium. Mentioned in Sections 1.1.1, 1.4.1, and 1.4.2</td></tr><tr><td>KCl,&amp;nbsp; 3 M</td><td>Merck</td><td>1049361000</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Laminin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L2020</td><td>Bio Coating. Mentioned in Section 1.2.6</td></tr><tr><td>MEM x 1</td><td>Gibco</td><td>21090-022</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1&amp;nbsp;&amp;nbsp;&amp;nbsp; 1.4.2</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; , 1 M</td><td>Sigma-Aldrich</td><td>M1028</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>NaCl, 4 M</td><td>Bio-Lab</td><td>19030591</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Octadecanethiol</td><td>Sigma-Aldrich</td><td>01858</td><td>Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3</td></tr><tr><td>Pluracare F108 NF Prill</td><td>BASF Corparation&amp;nbsp;</td><td>50475278</td><td>Bio-Rejection Coating, Bio Coating. Mentioned in Sections 1.2.4 and 1.2.6</td></tr><tr><td>Poly-L-lysine 0.01% solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;P47075</td><td>Promote cell division. Mentioned in Section 1.1.4</td></tr><tr><td>Sucrose, 1 M</td><td>Sigma-Aldrich</td><td>S1888</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Thiol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>1858</td><td>Bio-Rejection Coating. Mentioned in Section 1.2.3</td></tr><tr><td>URIDINE</td><td>Sigma-Aldrich</td><td>U3750</td><td>Changing medium. Mentioned in Section 1.4.1</td></tr><tr><td>Sputtering machine</td><td>AJA International, Inc</td><td>ATC Orion-5Series&amp;nbsp;</td><td>coating glass with thin layers of metal. Mentioned in Section 1.2.2</td></tr><tr><td>Pen plotter&amp;nbsp;</td><td>Hewlett Packard&amp;nbsp;</td><td>HP 7475A</td><td>Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5</td></tr><tr><td>Electrodes wires&amp;nbsp;</td><td>A-M Systems, Carlsborg WA</td><td>767000</td><td>Electric stimulation of neuronal cultures. Mentioned in Sections 2.1, 2.2, 2.3, and 2.4.5</td></tr><tr><td>Signal generator</td><td>BKPrecision</td><td>4079</td><td>Shaping of the electric signal. Mentioned in Section 2.3</td></tr><tr><td>Amplifier</td><td>Homemade</td><td></td><td>Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3</td></tr><tr><td>Power supply</td><td>Matrix&amp;nbsp;</td><td>MPS-3005 LK-3&amp;nbsp;</td><td>Power supply to the sputtering machine. Mentioned in Section 1.2.2.3</td></tr><tr><td>Transcranial magnetic stimulation</td><td>Magstim, Spring Gardens, UK</td><td>Rapid 2</td><td>Magnetic stimulation of neuronal culture. Mentioned in Sections 3.1, 3.3, and 3.4</td></tr><tr><td>Epoxy</td><td>Cognis</td><td>Versamid 140</td><td>Casting of homemade coils. Mentioned in Section 3.4</td></tr><tr><td>Epoxy</td><td>Shell</td><td>EPON 815&amp;nbsp;</td><td>Casting of homemade coils. Mentioned in Section 3.4</td></tr><tr><td>Platinum wires 0.005&#039;&#039; thick; A-M Systems,&amp;nbsp;</td><td>&amp;nbsp;Carlsborg WA</td><td>&amp;nbsp;767000</td><td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1</td></tr><tr><td>Circular magnetic coil</td><td>Homemade</td><td></td><td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.3</td></tr><tr><td>WaveXpress SW</td><td>B&amp;amp;K Precision&amp;nbsp;</td><td></td><td>Waveform editing software. Mentioned in Section 2.1.32</td></tr><tr><td>Xion Ultra 897</td><td>Andor</td><td></td><td>Sensitive EMCCD camera. Mentioned in Section 2.4.4</td></tr></tbody></table>

external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures

その膜電位が一定の閾値を超えた場合、ニューロンは活動電位を発射します。脳の典型的な活動では、これは、シナプスへの化学入力の結果として生じます。しかし、ニューロンも課せられ、電界によって励起することができます。特に、最近の臨床応用には、外部から電場を作成することによって、神経細胞を活性化させます。これは、ニューロンが外部磁場に応答して、何が活動電位が発生する方法を検討することが重要です。幸いなことに、外部電場の正確かつ制御されたアプリケーションは、切除解離、および培養物中で増殖させ、胚性神経細胞のために可能です。これは、再現性の高いシステムではこれらの質問の調査を可能にします。 本論文では、ニューロン培養物に対する外部電界の制御された適用のために使用される技術のいくつかが検討されています。ネットワークは、いずれか1次元、 すなわちリネアにパターニングすることができますR形態または基板の全体面上に成長させ、したがって、二次元。また、励起は、流体中に浸漬電極を介して電界の直接適用（バス電極）または磁気パルスのリモート作成を使用して電場を誘導することによって作成することができます。

<html>提示プロトコルは、神経細胞培養の容易なパターニングが可能になります。それは私たちが刺激のために開発され、いくつかの方法と組み合わせると、それは、健康と病気のニューロン27の特性を比較するための関数としての文化を刺激するための最適な方法を見つけるために、そのような時値および基電流5のように、いくつ?...

Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

神経細胞培養は、単一ニューロンまたはニューロンの人口への影響を通じて新興脳刺激技術を研究するための良いモデルです。ここに提示バス電極によって直接生成または時間変化する磁場によって誘起される電界によりパターニング神経細胞培養物の刺激のための異なる方法があります。

電気を使用してニューロンと1次元と2次元培養における磁場の外部励起

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

external-excitation-neurons-using-electric-magnetic-fields-one-two

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13.2K ビュー.  The Salk Institute for Biological Studies. 神経細胞培養は、単一ニューロンまたはニューロンの人口への影響を通じて新興脳刺激技術を研究するための良いモデルです。ここに提示バス電極によって直接生成または時間変化する磁場によって誘起される電界によりパターニング神経細胞培養物の刺激のための異なる方法があります。 

ビデオ: External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

生物工学

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

外部から印加された直流電界における神経前駆細胞の移行動態の解析のための走電性アッセイ

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

神経科学

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

マルチ電極アレイ上のらせん神経節ニューロン外植片培養と電気生理学

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

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

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

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 ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

シリコンナノワイヤと細胞間電気結合の研究のための光刺激

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

マイクロ流体デバイスにおける電界誘発神経前駆細胞分化

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

生体組織の刺激のための電界・磁場装置

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

External Excitation of One-Dimensional Patterned Neuronal Cultures

Source:Stern, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for stimulating line-patterned neuronal cultures with a uniform, unidirectional electric field.

一次元パターン化した神経細胞培養の外部興奮

1. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.

	Prepare a solution of 50 &#181;g calcium sensitive fluorescent ...

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Neurophysiology

Stimulation and Modulation Techniques

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Source: Stern, S., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a method for stimulating neuronal cultures grown in a patterned circular ring on a coverslip, using a time-varying magnetic field.

磁場によるパターン化した神経細胞培養の刺激

1. Preparation of coverslips for 1D cultures.

	Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia ...

Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

Source: Tedesco, M., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53080">Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model.</a> J. Vis. Exp. (2015)
This video demonstrates the development of a 3D neuronal culture coupled to a planar microelectrode array with hippocampal neurons. The isolated neurons form a 2D network on the microelectrode array&#39;s active area and adhere to glass microbeads in a multi-well plate with a membrane insert. Upon transferring these neuron-coated microbeads to the electrode, they organize into an interconnected 3D neural network.