Bu çalışma Ulusal Bilim Vakfı hibe CMMI-1300007 tarafından finanse edildi. Joseph J. Pancrazio, Dr. Hamid Charkhkar, Dr. Gretchen Knaack, bu protokollerin tasarımında yardımcı olan önceki laboratuar üyelerini ve beş yıldan uzun süre boyunca kültürlerin bakımını George Mason Üniversitesi&#39;nde kabul etmek istiyoruz. , Dr. Franz Hamilton, Michael Maquera ve Robert Graham.

<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>

Yazarların açıklayacak bir şeyi yok.

Bu protokolde özetlenen adımlar, yeni başlayanlar için kendi nöronal kültürlerini ÇÇ&#39;lar üzerine yerleştirip ağ etkinliklerini kaydetmek için yeterli ayrıntı sağlar. Bu protokol, kültürlerin düzgün bir şekilde yapışmasını sağlamaya, elektrot dizileri üzerinde bir halı hücre tabakası oluşturmaya ve aylardır sağlıklı ve kirletici olmamasına yardımcı olacaktır. Protokolün tüm bölümlerine uymak en iyisi olsa da, süreç boyunca başarılı sonuç için kritik öneme sahip adımlar vardır. Tüm süreç boyunca aseptik tekniğin kullanılması, kültürlerin kontamine olmasını önlemek için zorunludur. Yeni ÇÇA&#39;lar, protokolde tarif edildiği gibi hidrofil hale getirilmelidir veya yoksul hücre yapışması ortaya çıkar. Ayrıştırma sırasında sert pipetleme ve hava kabarcıklarının oluşmasını önlemek, hasar gören hücrelerin sayısını azaltacak ve daha yüksek ve daha sağlıklı bir verim sağlayacaktır. İlk önce DMEM 5/5&#39;ten DMEM +&#39;ye geçmeBeslenme de önemlidir. DMEM 5/5 at serumu içerir; bu, eğer glial hücrelerin devamlı kullanılması halinde kültüre egemenlik kazandıracak ve kültürler zayıf nöronal aktiviteye neden olacaktır, ancak kültürler sağlıklı görünecektir 17 . Kültürleri planlandığı gibi besleme ve bunları uygun kuluçka koşullarında tutmak da çok önemlidir. ÇÇA&#39;larda hücre kültürlerinin plaklanması, optimal sonuçların altında kalmasına neden olabilecek birçok değişkeni içerir. Hedef, hücrelerin mükemmel bir &quot;halı&quot; olmasına rağmen, yukarıda bahsedilen kritik adımları atlama başarısızlığı yetersiz hücre olgunlaşması veya bulaşma ile sonuçlanır. Fakir hücre adezyonu, fakir hücre olgunlaşmasından farklıdır, aynı zamanda bir endişe kaynağıdır. Bunun nedeni, kaplamadan önce zayıf MEA hazırlığı veya eski ortamın kullanılması da dahil olmak üzere birçok faktörden kaynaklanabilir. Eğer nöral hücre kültürü için stabilize edilmiş L-glutamin ve serum içermeyen bir form içeren eski ortam varsa (bkz. Malzeme Tablosu) Kullanılır, hücreler başlangıçta yapışır, ancak yaklaşık iki hafta sonra kaybolurlar. Bakteri kontaminasyonu kalıcı bir sorundaysa, ampisilin veya kalem strep gibi bir antibiyotik ortama ilave edilebilir. Mantar kontaminasyonunu tedavi etmek için mevcut fungisitler de mevcuttur. Bunlar, kültürlerin sonucunu etkileyebilecek daha yaygın değişkenlerden bazılarıdır. Yalnızca zaman ve deneyim sonrasında karşılaşılacak diğer pek çok şey vardır. Cam mikroelektrodlarının kullanımıyla kıyaslandığında, bu teknik ağ dinamikleri ve farmakolojik cevapların incelenmesi için mükemmeldir. Birçok farklı uzaysal ve zamansal uyarılma modelinin kullanılmasını sağlar ve aynı anda birden çok bölgedeki nöronal yanıtların kaydedilmesini sağlar. Önceki gruplar burada açıklananlara benzer protokoller kullanarak ilginç sonuçlar verdi 18 . Kültürler hafta veya ay sürer ve aynı kültürler tekrar kullanılabilir olduğundan, bu teknik aynı zamandaAynı ağ üzerinde zamanla çoklu denemeler için geçerlidir. Bununla birlikte, bu tekniğin sınırları vardır. ÇÇA&#39;lar invaziv değildir. Bu nedenle, yalnızca pipet ile yama tutma veya hücre içi kayıt yerine, hücre dışı aktiviteyi kaydedebilirler. Dahası, bir dizideki her elektrod birkaç hücre ile kaplandığından, tek bir nöronun etkinliğini çözmek mümkün değildir. Tersine, bunlar in vitro kültürler olduğundan, beyindeki ağların yapısal özelliklerini tam olarak üretemezler. Ayrıca, aktivite, hücrelerin pH dengesini korumak için CO2 atmosferi sağlayan bazı mekanizmalar olmaksızın, aynı anda 30 dakikadan az sürede kaydedilebilir. Bu tekniğe hakim olunca, elektrik stimülasyonlu veya elektriksel uyarı olmadan farmakolojik manipülasyonlar araştırılabilir. Nöronal ağlarda öğrenmeyi ve hafıza oluşumunu araştıran yeni protokoller de birlikte tasarlanabilir ve test edilebilirHipokampal veya omurilik ağları için rotokolatlar. Şebekelerin teşvik edilmesi ve eğitimi için protokoller daha önce yayınlanmış ve bunlardan bazıları, Eytan ve diğerleri tarafından önerilen &quot;seçici adaptasyon&quot; gibi in vivo protokollerle daha da geliştirilmiştir . 19 . Birkaç protokol test edildi. Bununla birlikte, sadece burada 2005 yılında Ruaro tarafından önerilen tetanoz prosedürüne yapılan bir değişiklik 14,20&#39;de sunulmuştur.

Mikro elektrot dizileri (MEA&#39;lar) ilaç toksisitesini araştırmak, yeni nesil kişiselleştirilmiş tıp için tasarım paradigmalarını ve nöronal kültürlerde ağ dinamikleri incelemek için kullanılabilir 1 . Yalnızca tek bir hücreden aktiviteyi kaydedebilen ya da elektrodu tek bir alanda çevreleyen nöronlardan hücre dışı tepkileri kaydedebilen cam pipetle alan kaydı gibi daha geleneksel yöntemlerin aksine, MEA&#39;lar aynı anda Her elektrotu tek tek yerleştirmenin zorlu görevini gerektirmeden hücre kültüründe birden fazla bölgeden kayıt yapabilirsiniz. Bu, o kültür içinde bir ağ oluşturan hücreler grubu arasındaki dinamik etkileşimlerin çalışılmasına olanak tanır. Ayrıca, 2 , 3 , 4 , 5 şebeke ateşleme modelleri ve şebeke kontrolü için elektriksel uyarıların etkisi 6 &lt;/ Sup&gt; nöronal kültürlerde iyi belgelenmiştir ve sayısız konfigürasyon elektrik stimülasyonu ve kontrolleri, aynı deney düzeneğinde kolayca uygulanabilir ve geniş bir aralıktaki uzaysal-zamansal dinamiklerin araştırılmasına izin verilir. Bu in vitro araştırmalarda ilgi çekici anahtar dinamiklerden biri, kültürlü şebekelerin öğrenmeyi gösteren özellikleri gösteren 7 , 8 , 9 , 10 , 11 , 12 , 13 olmuştur. Peixoto Laboratuarı daha önce, yüksek frekanslı eğitim sinyallerinin etkilerini Ruaro ve ark. 14 , mikroelektrod dizilerine kaplanmış fare nöronlarının ağlarında 15 . Bu deneylerde, şebekeler yanıt izlemede artış gösterdiElektriksel uyarım ile indüklenen g eğitimi. Artan tepki, uyarıcı tanıma yoluyla öğrenme biçimi olarak kabul edildi; ağlar belirli bir uyarı ( örn., Eğitim) protokolünün uygulanmasından sonra uyarandaki bir değişime tutarlı bir şekilde yanıt verdi. Bu protokol, ÇÇ&#39;larda nöronal hücrelerin nasıl kültürlendiğini, kaplanan yemeklerin% 95&#39;inden başarıyla kaydedildiğini, uyarı modellerine cevap vermek için ağları eğitmenin, tek üniteli aktiviteleri sıralamanın, histogramların çizilmesini ve sonuçların nasıl yorumlanacağını gösteren bir protokol oluşturmayı göstermektedir. Böyle deneyler. Nöronal kültürlerin uyarılması ve kaydedilmesi için tescilli bir sistemin kullanılması ( Malzeme Tablosuna bakınız ) yanı sıra, nöronal üniteleri sıralamak için yazılım paketlerinin uygulanması (Malzemelerin Tablosuna bakınız ) gösterilmiştir . Özel tasarlanmış bir grafik kullanıcı arabirimi (Malzeme Tablosuna bakınız) post-s&#39;leri görselleştirmek için kullanılırTimulus zamanı histogramları, inter-burst aralıkları ve patlamanın süresini belirlemek ve ayrıca bir antrenman protokolü öncesi ve sonrasında uyarıya hücresel cevabı karşılaştırmak için kullanılmıştır.

<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

Mikro elektrod dizileri (MEA&#39;lar) ilaç toksisitesini araştırmak, yeni nesil kişiselleştirilmiş tıp için tasarım paradigmalarını ve nöronal kültürlerde ağ dinamikleri incelemek için kullanılabilir. Tek bir hücreden yalnızca aktivite kaydedebilen yama sıkıştırma gibi geleneksel yöntemlerin aksine, ÇÇ&#39;lar, her bir elektrodun ayrı ayrı yerleştirilmesi zorlu bir görev gerektirmeden, bir ağdaki birden çok alandan aynı anda kayıt yapabilir. Dahası, çok sayıda kontrol ve uyarılma konfigürasyonu, aynı deneysel kurulumda kolayca uygulanabilir ve geniş bir dinamik aralığı keşfedilebilir. Bu in vitro araştırmalarda ilgi çekici anahtar dinamiklerden biri, kültürlü şebekelerin öğrenme göstergesinin özelliklerini ne ölçüde sergilediğidir. MEA&#39;lar üzerinde kültüre edilen fare nöronal hücreleri, elektrik uyarımı ile indüklenen eğitimi takiben yanıtta bir artış sergilemektedir. Bu protokol, ÇKA&#39;lardaki nöronal hücrelerin nasıl kültive edileceğini gösterir; Başarıyla tekrarKaplamalı tabakların% 95&#39;inden fazla kablo; Teşvik modellerine cevap vermek için ağları eğitmek için bir protokol oluşturun; Ve bu tür deneylerden elde edilen sonuçları sıralayabilir, arsa ve yorumlayabilir. Nöronal kültürlerin uyarılması ve kaydedilmesi için tescilli bir sistem kullanılması gösterilmiştir. Yazılım paketleri ayrıca nöronal üniteleri sıralamak için kullanılır. Özel uyarlanmış bir grafik kullanıcı arabirimi, uyarı sonrası zaman histogramlarını, kesintisiz aralıklarla ve patlamanın süresini görselleştirmenin yanı sıra, bir eğitim protokolü öncesi ve sonrasında uyarıya hücresel yanıtı karşılaştırmak için kullanılır. Son olarak, bu araştırma çabasının temsili sonuçları ve gelecekteki yönleri tartışılmaktadır.

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

Çok elektrotlu diziler üzerinde kültürlenen fare nöronal hücreleri, elektrik stimülasyonunu takiben yanıtta bir artış sergilemektedir. Bu protokol, nöronların nasıl kültürlendiğini, aktivitenin nasıl kaydedildiğini ve uyarılma modellerine cevap vermek için ağları eğitmek için nasıl protokol oluşturacağını gösterir.

Mikro Elektrod Dizilerinde Nöronal Hücre Kültürlerinin Uyarılmasına Ağ Yanıtına Zamana Bağlı Artış

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 Görüntüleme.  George Mason University.  Çok elektrotlu diziler üzerinde kültürlenen fare nöronal hücreleri, elektrik stimülasyonunu takiben yanıtta bir artış sergilemektedir. Bu protokol, nöronların nasıl kültürlendiğini, aktivitenin nasıl kaydedildiğini ve uyarılma modellerine cevap vermek için ağları eğitmek için nasıl protokol oluşturacağını gösterir. 

Video: 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.

Varsayılan Modu Ağ inceleyin Transkraniyal Manyetik Uyarım ve fMRI birleştiren

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 ...

Nörobilim

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 Aygıt: FDA yürütülmesi Depresyon Tedavisinde Protokolü Onaylandı

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.

Organotipik Kültürleri nöronal Çığ Çok elektrot Dizi Kayıtlar

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.

Glutamat ve GABA hızlı Micro-iyontoforez: Synaptic Entegrasyon Araştırma yararlı bir araç

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.

İskemik Hücre Ölümü Pathways Eğitim için seks Katmanlı Nöronal Kültürleri

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.

Hayvanlar davranmak, Awake Tek-birim Recordings Microwires Organize Dizileri implantasyon için Usulü

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.

Tek Hücre Hassas Nevronik Co-kültürler hazırlanması

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.

Zebra balığı Larva Görsel Tepki Elektroetinogram Analizi

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.

Mikro Elektrot Diziler 3D Engineered Nöronal Kültürler Arayüz: Yenilikçi<em&gt; İn Vitro</em&gt; Deneysel Model

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.

Kombine Metodolojileri kullanan Akson Dallanman ve Nöronal Morfogenez sırasında Plazma Membran Teslimat Rolü tanımlayın

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