Die Autoren danken Giorgio Carlini für die technische Unterstützung bei der Entwicklung der Begrenzungsstruktur und Dott. Ornella LoBrutto für die gründliche Überarbeitung des Manuskripts. Die Forschung, die diese Ergebnisse hat die Finanzierung von 7. Rahmenprogramm der Europäischen Union erhalten (ICT-FET FP7 / 2007-2013, FET Young Explorers Schema) unter Finanzhilfevereinbarung 284.772 BRAIN BOW (www.brainbowproject.eu).

Das experimentelle Protokoll wurde von der Europäischen Animal Care Gesetzgebung (2010/63 / EU), von der italienischen Gesundheitsministerium in Übereinstimmung mit dem DL 116/1992 von den Richtlinien der Universität Genua (Prot genehmigt. N. 13130, Mai 2011). Alle Anstrengungen wurden unternommen, um die Zahl der Tiere, für das Projekt zu reduzieren und ihr Leiden zu minimieren. 1. Herstellung von Materialien und Unterstützungen <ol><li> Konstruieren Sie die Form, um die PDMS (Poly-Dimethyl-Siloxan) Einschränkung mit Hilfe einer CNC (Computer Numerical Control) Fräsmaschine zu bauen. Eine solche Form ist aus Polycarbonat mit dem zentralen Zylinder in Polytetrafluorethylen (PTFE) realisiert. Dieses Material ermöglicht eine einfachere Entnahme der Maske, sobald das PDMS ausgehärtet ist. HINWEIS: Das Design der Form wurde von Computer-Aided-Design (CAD) durchgeführt und dann an die CNC-Fräsmaschine ausgeliefert. </li><li> Bereiten Sie die PDMS-Elastomer. PDMS ist ein organisches Polymer. Er besteht aus zwei Elementen zusammengesetzt istEin Härtungsmittel und ein Polymer. Mischen sie von einem Volumenverhältnis von 1:10, wobei ein Teil des Härtungsmittels und neun Teile Polymer. Setzen Sie die beiden Komponenten in einer Petrischale, schütteln und legen Sie die Mischung in der Vakuumkammer für 10 Minuten, um Luftblasen zu beseitigen. 3 g PDMS ausreichend sind für eine Einschränkung. </li><li> Konstruieren Sie die PDMS-Einschränkung. Legen Sie die PDMS-Material in den Formgeber und legen Sie sie in den Ofen für 30 Minuten bei 120 ° C. Die PDMS-Einschränkung die Form eines idealen Zylinders mit einem äußeren und inneren Durchmesser von 22,0 und 3,0 mm betragen und einer Höhe von 650 um. </li><li> Am Tag vor dem Plattieren Paar der Maske auf den aktiven Bereich der Mikroelektrodenanordnungen (MEAs) mittels einer Pinzette unter einem Stereomikroskop und Sterilisieren der PDMS-Maske in einem Ofen bei 120 ° C. </li><li> Sterilisation der Mikroglaskugeln (Nenndurchmesser von 40 ± 2 um; zertifiziert mittleren Durchmesser von 42,3 ± 1,1 &amp; mgr; m) in 70% Ethanol für 2 h in einem konischen Röhrchen und drehen every 30 Minuten, um alle Mikrokügelchen freizulegen. </li><li> Entfernen Sie die Ethanol-Lösung aus dem Fläschchen und spülen Sie die Mikroperlen zweimal mit sterilem Wasser. </li><li> Konditionieren des zentralen Teils der MEA Bereich begrenzt durch die PDMS Maske (Durchmesser gleich 3,0 mm) mit 24 &amp; mgr; l der gemischten Lösung von Poly-D-Lysin und Laminin bei 0,05 ug / ml. </li><li> Beschichtung der Mikrokügelchen mit Adhäsionsproteine, Laminin und Poly-D-Lysin bei 0,05 ug / ml und lassen Sie sie über Nacht in den Inkubator bei 37 ° C, um eine stabile und langanhaltende neuronale Netz zu erhalten. </li><li> Entfernen der Haftfaktoren sowohl von der Glasoberfläche der MEA und den Mikroglaskugeln. Aspirat etwa 95% der Beschichtungslösung mit einer Pipette und eine kleine Volumen- 24μl- sterilem Wasser auf der Oberfläche MEA. Saugen Sie wieder mehr als 95% des Wassers und lassen Sie die MEA trocken unter der laminaren Haube 1 Stunde vor dem Ausplattieren der Zellen. HINWEIS: Im Fall von Mikrokügelchen statt absaugen BeschichtungLösung und eine erste Wäsche mit sterilisiertem Wasser und ein zweiter mit dem Grundmedium und seine Ergänzung wie B27, um die Glasoberfläche, wo Neuronen plattierenden konditionieren. Lassen Sie sich nicht die Mikroperlen trocken. Lassen sie in Suspension in dem Kulturmedium im Inneren der Ampulle. </li><li> Verteilen, mittels einer Pipette -200 μl- die Suspension der behandelten Mikrokügelchen (ca. 32.000) auf einer Multiwellplatte mit Membraneinsatz (Porendurchmesser 0,4 um), wo sie selbstorganisieren Bildung einer gleichförmigen Schicht. </li><li> Füllen Sie jede auch der Membran mit 0,5 ml Medium, und steckte es in den Inkubator (T = 37,0 o C, CO 2 = 5%), bis die Neuronen sind bereit, plattiert werden (3.2). </li></ol> 2. Dissection von Embryonen und Dissoziation von Tissue <ol><li> Haus erwachsene weibliche Ratten (200-250 g) bei einer konstanten Temperatur (22 ± 1 ° C) und die relative Luftfeuchtigkeit (50%) im Rahmen eines regelmäßigen Hell-Dunkel-Zeitplan (licht auf 7.00 Uhr bis 19.00 Uhr) in derTierhaltung. Stellen Sie sicher, dass Lebensmittel und Wasser sind frei verfügbar. </li><li> Anesthetize eine schwangere weibliche Ratten nach 18 Tagen Entwicklung (E18) unter Verwendung von 3% Isofluran. Dann, zu opfern das Tier durch Genickbruch. </li><li> Entfernen hippocampi voneinander Rattenembryo und legen Sie sie in eiskaltes Hanks ausgewogener Salzlösung ohne Ca 2+ und Mg 2+. An Tag 18 der Entwicklung Hippocampus und Cortex Geweben sind sehr weich und müssen nicht in kleine Stücke geschnitten werden. Weitere Details können gefunden 18,19 werden. </li><li> Dissoziation des Gewebes in 0,125% Trypsin / Hanks-Lösung, die 0,05% DNAse für 18-20 min bei 37 ° C. </li><li> Entfernen der überstehenden Lösung mit einer Pasteurpipette und Stoppen der enzymatischen Verdau durch Zugabe von Medium, das mit 10% fötalem Rinderserum (FBS) für 5 min. </li><li> Entfernen Sie das Medium mit FBS und einmal mit dem Medium mit seiner zu ergänzen, zu 1% L-Glutamin, Gentamycin 10 ug / ml zu waschen. Entfernen Sie wieder das Medium mit einer Pasteur-pipette und füllen es wieder mit einer kleinen Menge (500 ul) des Wachstumsmediums mit seiner Ergänzung, 1% L-Glutamin, Gentamycin 10 ug / ml. </li><li> Dissoziieren die Gewebepellet mechanisch mit einem schmalen Pasteurpipette bis eine milchige Suspension von Zellen ist offensichtlich. Es ist nicht notwendig, die Zellsuspension zentrifugiert. </li><li> Verdünnen des geringen Volumens der Zellsuspension mit dem Wachstumsmedium, um ein Endvolumen von 2,0 ml zu erhalten. Zählen Sie die erhaltenen Zellkonzentration mit einem Hämocytometer Kammer. Verdünnte diese Konzentration in einer 1: 5, um die gewünschte Zellkonzentration von 600-700 Zellen / &amp; mgr; l zu erhalten. </li></ol> 3. Zell Plating <ol><li> Platten Zellen in einer Dichte von etwa 2000 Zellen / mm 2 auf die aktive Fläche der MEA von der PDMS Einschränkung definiert, um eine 2D neuronalen Netzwerks. Hinweis: Jeder Hippocampus etwa 5 x 10 5 Zellen enthält, (1 x 10 6 für einen einzigen Embryo). Sezieren 6 Embryonen auf einen Gesamtbetrag von 6 x 10 bekommen6 Zellen in 2 ml, und einer geschätzten Konzentration von 3.000 Zellen / &amp; mgr; l. Verdünnen Sie diese Konzentration bei 1: 5, um die gewünschte Zellkonzentration zu erhalten und die Platte ca. 600-700 Zellen / ul. Wenn die MEA Bereich durch die PDMS-Einschränkung begrenzt ist etwa 7.065 mm 2, und die Gesamtzahl der plattierten Zellen 600 Zellen / ul x 24 &amp; mgr; l = 14.400 Zellen, die endgültige Dichte 2.038 Zellen / mm 2. </li><li> Platzieren der MEA Vorrichtungen in den Brutschrank mit befeuchteter Atmosphäre CO 2 (5%) bei 37 ° C. </li><li> Verteilen 160 ul der Suspension mit einer Zellkonzentration von 600-700 Zellen / ul (etwa 100.000 Zellen) auf die Oberfläche der Monoschicht Mikrokugeln im Inneren der Multiwell-Platten angeordnet, um die Vorstufe für den Aufbau von dreidimensionalen Kultur abzuschließen. Setzen die Multiwell-Platten im Brutschrank mit befeuchteter Atmosphäre CO 2 (5%) bei 37 ° C. </li></ol> 4. 3D-Neuronal Network Construction <ol><li> 6-8 Stunden nach der Plattierung, übertragen Sie die Suspension (Mikroperlen mit Neuronen) von den Multiwell-Platten sehr sorgfältig in den durch die PDMS-Einschränkung begrenzt durch die mit einer Pipette für ein Volumen von etwa 30-40 &amp; mgr; l eingestellt. Nach jeder Übertragung, warten Sie etwa eine halbe Minute, um die Mikroperlen zur Selbstorganisation in einem sechseckigen kompakten Aufbau zu ermöglichen. </li><li> Nachdem alle Schichten abgeschieden werden und sich spontan zusammengebaut, füllen Sie mit einem großen Tropfen von etwa 300 ul Medium die Spitze des von der PDMS-Einschränkung begrenzt. </li><li> Setzen die 3D-Struktur an die MEA gekoppelt im Inkubator (T = 37,0 ° C, CO 2 = 5%) für 48 Stunden, bevor eine endgültige Volumen (etwa 1 ml) des Wachstumsmediums Kultur mit Ergänzung. HINWEIS: Beachten Sie die Gesamtzahl der Perlen auf der Multiwell-Platten mit Membraneinsatz (30.000) und die Anzahl der Perlen auf einer einzigen Schicht auf die MEA-Gerät (6000). Die resultierende 3D-Struktur besteht aus 5 Schichten zusammengesetzt microbeads und Zellen. Unter Berücksichtigung, dass die 3D neuronalen Netzwerk nicht geometrisch vollkommen, könnte das resultierende 3D-Struktur von 5-8 Schichten bestehen. </li><li> Am Tag nach der Beschichtung, sorgfältig fügen Sie das endgültige Volumen des Mediums (etwa 900 ul) in der MEA-Ring. Aufrechterhaltung der 3D-Kulturen in einem befeuchteten CO 2 -Atmosphäre (5%) bei 37 ° C für 4-5 Wochen. Ersetzen Sie die Hälfte des Mediums einmal pro Woche. </li></ol> 5. konfokale Mikroskopie Acquisition <ol><li> Fixierung der biologischen Proben auf der Bühne des Mikroskops in einer Halterung. Stellen Sie den Laser (Argon, 496 bis 555 nm), der Abtastgeschwindigkeit (400 Hz), bei dem, um das Bild, das Gain und Offset (-0,3%) der PMT für jedes Bild zu erwerben, um die richtige Bandbreite Emissionsfilter und um zu erfassen um eine Sättigung zu vermeiden und um das Signal-Rausch-Verhältnis zu erhöhen. Ergebnisteil meldet die verwendet werden, um die Bilder von 4 zu erwerben Werte. </li><li> Für den Erwerb der z -stack Sequenz, Tannest wählen, den Wert der z-Position der oberen und der unteren Schicht der Probe, und wählen Sie dann die Schrittweite auf die Übernahme vorzunehmen. </li></ol>

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The authors declare that they have no competing financial interests.

In dieser Arbeit wird eine neuartige experimentelle in vitro-Plattform aus 3D gemacht entwickelt neuronalen Kulturen auf MEAs für Netzwerk-Elektrophysiologie wurde vorgestellt gekoppelt. Die Verwendung von Mikrokügelchen als Gerüst, um das Auswachsen neuritischen entlang der z-Achse zu ermöglichen wurde maßgeschneidert, um mit der planaren MEA integriert werden. Auf diese Weise können die erhaltenen Mikrosystem ergibt eine gültige und zuverlässige in vitro- 3D-Modells, um die austreten...

In vitro zweidimensionale (2D) neuronale Netze zu Micro-Elektroden-Arrays (MEAs) gekoppelt arethe Goldstandard angenommen, um das Zusammenspiel von neuronaler Dynamik und der zugrunde liegenden Konnektivität studieren experimentellen Modell. Bei der Entwicklung, Nervenzellen neu zu komplexen Netzwerken, die auch angezeigt werden definedspatio-temporalpatternsof Tätigkeit 1,2 (dh platzt, Netzwerk-Bursts, zufällige spiking Aktivität). MEAs erfassen die elektrophysiologische Aktivität von vielen Websites (von zehn bis Tausende von Mikroelektroden), so dass eine detaillierte Untersuchung der Dynamik zum Ausdruck auf Netzwerkebene. Darüber hinaus ist die Verwendung von dissoziierten Kulturen macht possibile, um technisch-Netzwerke zu entwerfen. Es ist einfacher, auf diese Weise zu verstehen, die funktionalen Beziehungen zwischen dem aufgezeichneten elektrophysiologische Aktivität und die Parameter der Netzwerkorganisation, wie Zelldichte 3 Grad an Modularität 4,5, Gegenwart heterogener neuronalen populations 6 usw. Aber alle in vitro-Studien auf distanzierte kultivierten Zellen werden auf 2D neuronale Netze. Dieser Ansatz führt zu Vereinfachungen in Bezug auf die in vivo (eigen 3-dimensional, 3D-System): (i) in einem 2D-Modell, Somata und Wachstumskegeln sind abgeflacht und die Axone-Dendriten Auswuchs kann nicht in allen Richtungen 7 verbreiten. (ii) 2D-in-vitro-Netzwerke weisen stereoelektrophysiologischen Dynamik durch Platzen Tätigkeit, die die meisten Neuronen des Netzes 8 dominiert. In jüngster Zeit verschiedene Lösungen entwickelt worden, um die Konstruktion von in-vitro-3D dissoziierten neuronalen Netzen zu ermöglichen. Die gemeinsame Idee besteht in der Schaffung eines Gerüsts, wo Nervenzellen können in einer 3D-Mode wachsen. Ein solches Gerüst kann mit Polymer-Gelen und festen porösen Matrizen 9-13 realisiert werden. Durch Ausnutzung der mechanischen Eigenschaften der Polymere ist es möglich, Zellen in thes einbettene Strukturen durch einen einheitlichen Block von 3D-Kulturen von Neurosphären 11 definieren. Das Hauptmerkmal dieser Methode ist die starre mechanische Eigenschaft der Neurosphären 9,12. Jedoch haben diese Materialien beschränkt Porosität, und sie garantieren nicht die Zellmigration in der Matrix. Um diesen Nachteil zu überwinden, ist eine mögliche Lösung besteht im Trennen der Matrix in &quot;Einheit&quot; Modulen. Leider konnte die Größe und Form der Partikel Vielfalt Packung in regulären Schichtstrukturen zu behindern. In 7, Cullen und Mitarbeiter entwickelt einen 3D-neuronalen Konstrukt up von Neuronen und / oder Astrozyten innerhalb eines bioaktiven extrazellulären Matrix-basierten Gerüst gemacht. Eine solche konstruiert Nervengewebe in vitro Untersuchungen erlaubt, zu studieren und zu manipulieren neurobiologischen Reaktionen innerhalb von 3D-Mikro-Umgebungen. Dieses Modell besteht aus Neuronen und Gliazellen im gesamten extrazellulären Matrix (ECM) und / oder Hydrogel Gerüste (500-600 &amp; mgr; m dick) verteilt. In dieser Co5000 Zellen / mm 3 - ndition eine optimale Zelllebensfähigkeit (mehr als 90%) wurde durch Ausplattieren Zellen bei einer endgültigen Dichte von ungefähr 3,750 gefunden. Es ist zu beachten, dass eine solche Dichtewert ist viel niedriger als die in der in-vivo-Zustand, in dem die Zelldichte der Maus Hirnrinde als etwa 90000 Zellen / mm 3 14 werden. Um diese Einschränkung zu überwinden und Mitarbeiter Pautot 15 realisiert ein 3D in vitro System, in dem die Zelldichte und die Netzwerkverbindung gesteuert werden, um in vivo-Bedingungen ähnlich und ermöglichen eine Echtzeitabbildung des Netzwerks. In der Praxis wird diese Methode auf dem Konzept, das dissoziiert kultivierten Neuronen können auf Siliciumdioxid-Mikroperlen wachsen basiert. Diese Kügelchen eine Wachstumsoberfläche groß genug für neuronalen Zellkörpern zu haften und ihre Verzweigungen zu wachsen, reifen, zu erweitern und zu definieren synaptische Kontakte zu anderen Neuronen. Diese Methode nutzt die spontane Versammlung Eigenschaften von monodispersen Perlen form 3D-Schichten-hexagonalen Arrays mit verschiedenen Untergruppen von Neuronen auf verschiedenen Ebenen mit eingeschränkter Konnektivität zwischen Nervenzellen auf verschiedenen Perlen. Die erzielte Zelldichte bei diesem Verfahren betrug etwa 75.000 Zellen / mm 3. Vor kurzem haben wir Pautot Methode angepasst MEAs 16: Die erhaltenen Ergebnisse zeigen, dass die 3D-elektrophysiologische Aktivität stellt einen größeren Repertoire von Aktivitäten als die von 2D-Netze ausgedrückt. 3D reife Kulturen weisen eine verbesserte Dynamik in der sowohl Netzwerk-Burst und zufällige Spike-Aktivität koexistieren. Ähnlich Tang-Schomer und Mitarbeiter 17 realisiert eine Seidenproteinbasis poröse Gerüst, das eine primäre kortikale Kultur in vitro für einige Monate beibehält, und nahm die elektrophysiologische Aktivität mittels einer Wolfram-Elektrode. In dieser Arbeit wurden die experimentellen Verfahren, um 3D-neuronalen Netzen, um MEAs gekoppelt bauen beschrieben.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25050-014</td>
			<td align="left">0.125% diluted 1:2 in HBSS w\o CA++, MG++</td>
		</tr>
		<tr>
			<td>DNAase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td>0.05% diluted&#160; in Hanks solution</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco Invitrogen</td>
			<td>21103049</td>
			<td>culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco Invitrogen</td>
			<td>17504044</td>
			<td>2% medium supplent</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F-2442</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Glutamax-I&#160;</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G.1272</td>
			<td>5mg/liter</td>
		</tr>
		<tr>
			<td>Poly-Dimethyl-Siloxane (PDMS)</td>
			<td>Corning Sigma</td>
			<td>481939</td>
			<td>curing agent and the polymer</td>
		</tr>
		<tr>
			<td>Micro-Electrode Arrays</td>
			<td>Multi Channel Systems (MCS)</td>
			<td>60MEA200/30-Ti-pr</td>
			<td>MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 &#181;m, respectively; plastic ring without thread</td>
		</tr>
		<tr>
			<td>Microbead</td>
			<td>Distrilab-Duke Scientific</td>
			<td>9040</td>
			<td>&#160;1gr Glass Part.Size Stds 40 &#181;m</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Costar Sigma</td>
			<td>CLS 3413</td>
			<td>multiwell plates with membrane insert (6.5 mm diameter porous 0.4 &#181;m)</td>
		</tr>
		<tr>
			<td>HBSS w\o Ca++ ,Mg++&#160;</td>
			<td>Gibco Invitrogen</td>
			<td>14175-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Buffer Solution&#160;</td>
			<td>Sigma&#160;</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon</td>
			<td>Sigma</td>
			<td>430935-5G</td>
			<td>polytetrafluoroethylene</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Sprague Dawley</td>
			<td>Wistar Rat</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscopy Upright</td>
			<td>Leica</td>
			<td>TCS SP5 AOBS</td>
			<td></td>
		</tr>
		<tr>
			<td>20.0x0.50 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>40.0x0.80 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>25.0x0.95 WATER objective</td>
			<td>Leica</td>
			<td>HCX IRAPO L</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

. In diesem experimentellen Verfahren wird eine relevante Rolle der Mikrokügelchen, die die mechanische Gerüst für das Wachstum des 3D neuronales Netzwerk definieren gespielt 1A und B-Anzeige, die die Anordnung der Mikrokügelchen (Nenndurchmesser von 40 ± 2 um; zertifiziert mittlere Durchmesser 42,3 ± 1,1 &amp; mgr; m) in der Ebene. Die Besonderheit solcher Strukturen besteht darin, daß Mikrokügelchen spontan selbst zusammensetzen, die einen kompakten hexagonalen Geometrie (1B und C).</s...

Watch this Scientific Journal Video about Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model at JoVE.com

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.

Schnittstellen 3D Engineered neuronalen Kulturen auf Mikroelektrodenarrays: ein innovatives<em&gt; In-vitro-</em&gt; Experimental Modell

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

interfacing-3d-engineered-neuronal-cultures-to-micro-electrode-arrays

Research

JoVE Journal

Neuroscience

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

9.9K Aufrufe.  University of Genova. 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.

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

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.

Multi-Elektroden-Array Recordings neuronaler Lawinen in organotypischen Kulturen

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

Forschung

Neurowissenschaften

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotypischen Kulturen von embryonalen ventralen Mittelhirn: Ein System zur Dopaminerge Neuronale Development Study In-vitro-</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker, eine innovative automatisierte Ansatz zur oculomotorius Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

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.

Schnelle Micro-Iontophorese von Glutamat und GABA: Ein nützliches Tool, um Synaptic Integration Untersuchen

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

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

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

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

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

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

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

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

Ein Verfahren für die Implantation von Mikrodrähte Organisierte Arrays für Einzel-Einheit Recordings in Awake, Behaving Tiere

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

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Genetische Manipulation von Kleinhirnkörnerzellen<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt;, Um neuronale Morphologie und Migration Studieren

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

Neuronale Aktivitätsausbreitung in einem ungefalteten Hippocampus Vorbereitung mit einem durchdringenden Mikroelektroden-Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity 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.

Zeitabhängige Erhöhung der Netzwerkreaktion auf die Stimulation von neuronalen Zellkulturen auf Mikroelektroden-Arrays

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

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In Vitro Wedge Slice Vorbereitung für Diemimitieren In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...