Gli autori ringraziano Giorgio Carlini per il supporto tecnico per lo sviluppo della struttura di confinamento e dott. Ornella LoBrutto per l&#39;accurata revisione del manoscritto. La ricerca che ha portato a questi risultati ha ricevuto finanziamenti dal 7 ° programma quadro dell&#39;Unione europea (ICT-FET FP7 / 2007-2013, FET giovani esploratori schema), contratto di sovvenzione 284.772 CERVELLO ARCO (www.brainbowproject.eu).

Il protocollo sperimentale è stato approvato dalla cura degli animali Legislazione Europea (2010/63 / UE), dal Ministero della Salute italiano, secondo la DL 116/1992 e dalle linee guida dell&#39;Università di Genova (Prot. N. 13130, maggio 2011). Sono stati fatti tutti gli sforzi per ridurre il numero di animali per il progetto e per ridurre al minimo la loro sofferenza. 1. Preparazione dei Materiali e supporti <ol><li> Costruire lo stampo per costruire le PDMS (Poly-dimetil-silossano) vincolo mediante un CNC (Computer Numerical Control) fresatrice. Tale stampo è realizzato in policarbonato con cilindro centrale in politetrafluoroetilene (PTFE). Questo materiale consente una più facile estrazione della maschera quando il PDMS è indurito. NOTA: Il design dello stampo è stata eseguita da Computer-Aided-Design (CAD) e poi consegnato alla fresatrice CNC. </li><li> Preparare l&#39;elastomero PDMS. PDMS è un polimero organico. Esso è composto da due elementi, Un agente di indurimento e un polimero. Mescolare con un rapporto volumetrico 1:10, una parte di agente indurente e nove parti di polimero. Mettere i due componenti in una capsula di Petri, agitare e inserire la miscela nella camera a vuoto per 10 minuti per eliminare le bolle d&#39;aria. 3 g di PDMS sono sufficienti per un vincolo. </li><li> Costruire il vincolo PDMS. Inserire il materiale PDMS nel modellatore e metterla in forno per 30 minuti a 120 ° C. Il vincolo PDMS ha la forma di un cilindro ideale, con un diametro esterno e interno di 22,0 mm e 3,0, rispettivamente, e un&#39;altezza di 650 micron. </li><li> Il giorno prima della placcatura, coppia la maschera per l&#39;area attiva delle matrici Micro-elettrodo (MEA) per mezzo di una pinzetta allo stereomicroscopio e sterilizzare la maschera PDMS in forno a 120 ° C. </li><li> Sterilizzare le microsfere di vetro (diametro nominale di 40 ± 2 micron; diametro medio certificato di 42,3 ± 1,1 micron) in etanolo al 70% per 2 ore in un flaconcino conica e ruotare every 30 minuti per esporre tutte le microsfere. </li><li> Rimuovere la soluzione di etanolo dal flaconcino e lavare le microsfere due volte con acqua sterilizzata. </li><li> Condizionare la parte centrale della zona MEA delimitata dalla maschera PDMS (diametro pari a 3,0 mm) con 24 ml di soluzione mista di poli-D-lisina e laminina a 0,05 ug / ml. </li><li> Rivestire le microsfere con proteine ​​di adesione, laminina e Poly-D-lisina a 0,05 mg / ml e lasciare per una notte in incubatore a 37 ° C, per ottenere una rete neuronale stabile e duratura. </li><li> Rimuovere i fattori di adesione sia dalla superficie del vetro del MEA e dalle microsfere di vetro. Aspirare circa il 95% della soluzione di rivestimento con una pipetta e applicare una piccola volume- 24μl- di acqua sterile sulla superficie MEA. Aspirare ancora più del 95% dell&#39;acqua e lasciare che il MEA secca sotto il cofano laminare per 1 ora prima della placcatura cellule. NOTA: Nel caso di microsfere invece, aspirare il rivestimentosoluzione e fare un primo lavaggio con acqua sterilizzata e un secondo con il mezzo basale e il suo supplemento come B27, per condizionare la superficie di vetro in cui verranno piastrate neuroni. Non lasciate che le microsfere asciugare. Lasciarli in sospensione nel mezzo di coltura all&#39;interno del flacone. </li><li> Distribuire, mediante una pipetta -200 μl-, la sospensione di microsfere trattate (circa 32.000) su una piastra multipozzetto con inserto di membrana (diametro dei pori 0,4 micron) in cui si auto-assemblano formare uno strato uniforme. </li><li> Riempire ciascun pozzetto della membrana con 0,5 ml di terreno e metterlo in incubatrice (T = 37,0 ° C, CO 2 = 5%) fino a quando i neuroni sono pronte per essere placcato (3.2). </li></ol> 2. Dissezione degli embrioni e la dissociazione di tessuto <ol><li> Casa ratti femmina adulti (200-250 g) ad una temperatura costante (22 ± 1 ° C) e umidità relativa (50%) sotto una pianificazione luce-buio normale (light-on 7 AM-7 PM) nelstabulario. Assicurarsi che il cibo e l&#39;acqua sono liberamente disponibili. </li><li> Anestetizzare un topo femmina incinta dopo 18 giorni di sviluppo (E18) utilizzando 3% isoflurano. Poi, sacrifica l&#39;animale per dislocazione cervicale. </li><li> Rimuovere ippocampi da ogni embrione di ratto e metterli in una soluzione salina bilanciata ghiacciata di Hank senza Ca 2 + e Mg 2+. Al giorno 18 di ippocampo di sviluppo e dei tessuti della corteccia sono molto morbidi e non hanno bisogno di essere tagliato in piccoli pezzi. Ulteriori dettagli possono essere trovati 18,19. </li><li> Dissociare il tessuto 0,125% di soluzione di tripsina / Hank contenente lo 0,05% di DNAsi per 18-20 minuti a 37 ° C. </li><li> Rimuovere la soluzione surnatante con una pipetta Pasteur e fermare la digestione enzimatica aggiungendo mezzo con siero fetale bovino 10% (FBS) per 5 min. </li><li> Rimuovere il mezzo con FBS e lavare una volta con il mezzo con il suo complemento, 1% L-glutamina, gentamicina 10 ug / ml. Rimuovere di nuovo il supporto con un pip Pasteurette e riempire nuovamente con una piccola quantità (500 ml) di mezzo di crescita con il suo complemento, 1% L-glutamina, gentamicina 10 ug / ml. </li><li> Dissociare il precipitato di tessuto meccanicamente con una pipetta Pasteur stretta fino una sospensione lattiginosa di cellule è evidente. Non è necessario centrifugare la sospensione cellulare. </li><li> Diluire il piccolo volume di sospensione cellulare con il terreno di coltura per ottenere un volume finale di 2,0 ml. Contare il concentrazione cellulare ottenuto con una camera emocitometro. Diluire la concentrazione a 1: 5 per ottenere la concentrazione desiderata di cellule 600-700 cellule / microlitro. </li></ol> 3. cellule placcatura <ol><li> Cellule piastra ad una densità di circa 2.000 cellule / mm 2 sulla zona attiva del MEA, definiti dal vincolo PDMS per creare una rete neuronale 2D. NOTA: Ogni ippocampo contiene circa 5 x 10 5 cellule, (1 x 10 6 per un singolo embrione). Sezionare 6 embrioni per ottenere un importo totale di 6 x 106 cellule in 2 ml, e una concentrazione stimata di 3.000 cellule / microlitro. Diluire la concentrazione a 1: 5 per ottenere la concentrazione desiderata e piastra di cellule circa 600-700 cellule / ml. Se l&#39;area MEA delimitata dal vincolo PDMS è di circa 7.065 mm 2, e il numero totale di cellule piastrate 600 cellule / ml x 24 ml = 14.400 cellule, la densità finale di 2.038 cellule / mm 2. </li><li> Posizionare i dispositivi MEA nel incubatore umidificato con CO2 nell&#39;atmosfera (5%) a 37 ° C. </li><li> Distribuire 160 microlitri della sospensione con una concentrazione cellulare di 600-700 cellule / microlitro (circa 100.000 cellule) sulla superficie del monostrato microsfere posizionato all&#39;interno delle piastre multipozzetto per completare la fase preliminare per la costruzione della cultura tridimensionale. Mettere le piastre a pozzetti multipli nell&#39;incubatore con umidificata CO 2 nell&#39;atmosfera (5%) a 37 ° C. </li></ol> Edilizia 4. 3D Rete neuronale <ol><li> 6-8 ore dopo la placcatura, trasferire la sospensione (microsfere con neuroni) dalle piastre multipozzetto attentamente all&#39;interno dell&#39;area delimitata dal vincolo PDMS utilizzando una pipetta impostato per un volume di circa 30-40 ml. Dopo ogni trasferimento, attendere circa mezzo minuto, per consentire le microsfere di auto-assemblarsi in una struttura compatta esagonale. </li><li> Una volta che tutti gli strati sono depositati e spontaneamente assemblati, riempire con una grande goccia di circa 300 ml di mezzo della parte superiore dell&#39;area delimitata dal vincolo PDMS. </li><li> Mettere la struttura 3D accoppiato al MEA in incubatore (T = 37,0 ° C, CO 2 = 5%) per 48 ore prima di aggiungere un volume finale (circa 1 ml) di crescita terreno di coltura con il suo supplemento. NOTA: Si consideri il numero totale di perline su piastre multipozzetto con inserto membrana (30.000) e il numero di perle su un singolo strato sul dispositivo MEA (6000). La struttura 3D risultante è composta da 5 strati di microbeads e cellule. Tenendo conto che la rete neuronale 3D non è geometricamente perfetta, la struttura 3D risultante potrebbe essere composta di 5-8 strati. </li><li> Il giorno dopo la placcatura, aggiungere con attenzione il volume finale del mezzo (circa 900 ml) all&#39;interno dell&#39;anello MEA. Mantenere le culture 3D in un umidificata CO 2 nell&#39;atmosfera (5%) a 37 ° C per 4-5 settimane. Sostituire la metà del mezzo una volta alla settimana. </li></ol> 5. Microscopia confocale Acquisizione <ol><li> Fissare i campioni biologici alla fase del microscopio in un supporto. Posizionare il laser (Argon, 496-555 nm), la velocità di scansione (400 Hz) di acquisire l&#39;immagine, il guadagno e offset (-0,3%) di PMT per ogni immagine di catturare il filtro per le emissioni di banda corretta e in ordine per evitare la saturazione e per aumentare il rapporto segnale-rumore. La Sezione I risultati sono riportati i valori utilizzati per acquisire le immagini di figura 4. </li><li> Per l&#39;acquisizione del z impili sequenza, abetest selezionare, il valore della posizione z della parte superiore e lo strato inferiore del campione, e quindi selezionare il passo di rendere l&#39;acquisizione. </li></ol>

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The authors declare that they have no competing financial interests.

In questo lavoro, un romanzo sperimentale in vitro piattaforma costituita da 3D progettato colture neuronali accoppiati a MEA per elettrofisiologia rete è stata presentata. L&#39;uso di microsfere come impalcatura per consentire la conseguenza neuritica lungo la z -axis è stata adattata per essere integrato con il MEA planare. In questo modo, i risultati ottenuti micro-sistema in vitro modello 3D valida ed affidabile per studiare le dinamiche emergenti elettrofisiologici 16.</s...

In vitro bidimensionali (2D) reti neurali accoppiati a micro-elettrodi Array (MEA) arethe modello sperimentale gold-standard adottato per studiare l&#39;interazione tra la dinamica neuronale e la connettività di base. Durante lo sviluppo, i neuroni ricreano reti complesse che mostrano ben definedspatio-temporalpatternsof attività 1,2 (cioè, scoppi, esplosioni di rete, attività chiodare casuale). MEA registrare l&#39;attività elettrofisiologica da molti siti (da decine a migliaia di microelettrodi), che consente un esame approfondito delle dinamiche espresse a livello di rete. Inoltre, l&#39;uso di colture dissociate rende possibile progettare-reti ingegnerizzati. È facile capire in questo modo le relazioni funzionali tra dell&#39;attività elettrofisiologica registrata ei parametri dell&#39;organizzazione network come densità cellulare 3, grado di modularità 4,5, presenza di eterogenea pop neuronaleulations 6, ecc, tuttavia, tutti gli studi in vitro su cellule in coltura dissociate sono basati su reti neuronali 2D. Questo approccio porta a semplificazioni rispetto al sistema in vivo (intrinsecamente 3-dimensionale, 3D): (i) in un modello 2D, somata e coni di crescita sono appiattite e la conseguenza assoni-dendriti non può diffondere in tutte le direzioni 7. (ii) 2D in vitro reti mostra stereotipata dinamiche elettrofisiologiche dominati da scoppio attività che comporti la maggior parte dei neuroni della rete 8. Recentemente, diverse soluzioni sono state sviluppate per consentire la costruzione di in vitro 3D dissociato reti neuronali. L&#39;idea comune consiste nella creazione di un ponteggio in cui i neuroni possono crescere in modo 3D. Tale impalcatura può essere realizzato con gel polimerici e solidi matrici porose 9-13. Sfruttando le proprietà meccaniche dei polimeri, è possibile incorporare all&#39;interno delle cellule these strutture definendo un blocco omogeneo di culture 3D di neurosfere 11. La caratteristica principale di questo approccio è la proprietà meccanica rigida delle neurosfere 9,12. Tuttavia, questi materiali hanno limitato la porosità, e non garantisce la migrazione delle cellule all&#39;interno della matrice. Per ovviare a questo inconveniente, una possibile soluzione consiste nel tagliare la matrice in moduli &#39;unità&#39;. Purtroppo, la dimensione e la forma delle particelle diversità potrebbe ostacolare l&#39;imballaggio in strutture stratificate regolare. In 7, Cullen e colleghi hanno progettato un costrutto neuronale 3D fatta di neuroni e / o astrociti all&#39;interno di un ponteggio a matrice extracellulare bioattivi. Tale tessuto neurale progettata consentito in Studi in vitro per studiare e manipolare le risposte neurobiologiche all&#39;interno di micro-ambienti 3D. Questo modello consisteva di neuroni e glia distribuiti in tutta la matrice extracellulare (ECM) e / o ponteggi idrogel (spessore 500-600 micron). In questo condition, una vitalità cellulare ottimale (superiore al 90%) è stato trovato placcando le cellule ad una densità finale di circa 3.750 - 5.000 cellule / mm3. Si deve notare che tale valore di densità è di gran lunga inferiore a quello nella condizione in vivo, in cui la densità delle cellule della corteccia cerebrale mouse è circa 90.000 cellule / mm 3 14. Per superare questa limitazione Pautot e collaboratori 15 realizzato un sistema 3D in vitro dove la densità cellulare e la connettività di rete sono controllati per assomigliare in condizioni in vivo consentendo imaging in tempo reale della rete. In pratica, questo metodo si basa sul concetto che dissociata neuroni in coltura sono in grado di crescere su microsfere di silice. Queste perle forniscono una superficie di crescita abbastanza grande per i corpi cellulari neuronali di aderire e per i loro arborizzazioni per crescere, maturare, estendere, e definire contatti sinaptici con altri neuroni. Questo metodo sfrutta le proprietà di assemblaggio spontanei di perline monodisperse a form 3D strati array esagonale contenenti sottoinsiemi distinti di neuroni su livelli diversi con connettività vincolata tra i neuroni in diverse sfere. La densità cellulare ottenuta con questo metodo è stato di circa 75.000 cellule / mm3. Recentemente, abbiamo adattato il metodo di Pautot di MEA 16: i risultati ottenuti mostrano che l&#39;attività elettrofisiologica 3D presenta un repertorio più ampia di attività rispetto a quello espresso dalle reti 2D. Culture mature 3D mostrano una dinamica rafforzata cui sia scoppio rete e casuale coesistono attività picco. Analogamente, Tang-Schomer e collaboratori 17 realizzato uno scaffold poroso a base di proteina di seta che mantiene una cultura corticale primaria in vitro per alcuni mesi, e registrato l&#39;attività elettrofisiologica mediante un elettrodo di tungsteno. In questo lavoro, le procedure sperimentali per costruire reti neuronali 3D accoppiati a MEA sarà descritta.

<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 questa procedura sperimentale, un ruolo importante è giocato dalle microsfere che definiscono l&#39;impalcatura meccanica per la crescita della rete neuronale 3D Figure 1A e B mostra la disposizione delle microsfere (diametro nominale di 40 ± 2 micron; diametro medio certificata di 42.3 ± 1.1 micron) nel piano. La particolarità di tali strutture è che microsfere spontaneamente auto-assemblano definendo una geometria esagonale compatto (Figura 1B e C). L&#39;impalcatura così g...

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.

Interfacciamento 3D Engineered neuronali Culture a Micro-Electrode Array: Un innovativo<em&gt; In Vitro</em&gt; Modello sperimentale

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 Views.  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.

Video: 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-array di elettrodi Recordings Valanghe neuronali in Culture organotipica

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

Ricerca

Neuroscienze

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.

Culture Slice organotipica del mesencefalo ventrale embrionali: un sistema per studiare dopaminergici sviluppo neuronale 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, un approccio innovativo automatizzato di analisi oculomotoria

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.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

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.

Una procedura per l&#39;impianto Array organizzata di microfili per Recordings singola unità di Awake, Comportarsi Animali

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.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

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.

Neural Activity Propagazione in ippocampale preparazione piegato con un penetrante Micro-elettrodo 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.

Aumento del tempo della risposta alla rete alla stimolazione delle colture cellulari neuronali sulle matrici di microelettrodi

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.

Preparazione della fetta di cuneo in vitro per imitare la connettività del circuito neuronale in vivo

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