Los autores agradecen a Giorgio Carlini por el apoyo técnico en el desarrollo de la estructura de confinamiento y Dott. Ornella LoBrutto para la revisión a fondo del manuscrito. La investigación que lleva a estos resultados ha recibido financiación del 7º Programa Marco de la Unión Europea (TIC-FET FP7 / 2007-2013, FET joven Exploradores esquema) bajo acuerdo de subvención 284.772 CEREBRO DEL ARCO (www.brainbowproject.eu).

El protocolo experimental fue aprobado por la Legislación Europea de Cuidado de Animales (2010/63 / UE), por el Ministerio de Salud de Italia, de conformidad con el DL 116/1992 y por las directrices de la Universidad de Génova (Prot. N. 13130, de mayo 2011). Se hicieron todos los esfuerzos para reducir el número de animales para el proyecto y para minimizar su sufrimiento. 1. Preparación de Materiales y Apoyos <ol><li> Construir el molde para construir el PDMS (poli-dimetil-siloxano) de restricciones por medio de un CNC (Control Numérico Computarizado) fresadora. Tal molde se realiza en policarbonato con el cilindro central en politetrafluoroetileno (PTFE). Este material permite una extracción más fácil de la máscara una vez que el PDMS ha endurecido. NOTA: El diseño del molde se ha realizado por Computer-Aided-Design (CAD) y luego entregado a la fresadora CNC. </li><li> Preparar el elastómero PDMS. PDMS es un polímero orgánico. Se compone de dos elementos, Un agente de curado y un polímero. Mezclar por una relación en volumen de 1:10, una parte del agente de curado y nueve partes de polímero. Ponga los dos componentes en una placa de Petri, agitar e introduzca la mezcla en la cámara de vacío durante 10 min para eliminar las burbujas de aire. 3 g de PDMS son suficientes para una restricción. </li><li> Construir la restricción PDMS. Inserte el material de PDMS en el moldeador y ponerlo en el horno durante 30 minutos a 120 ° C. La restricción de PDMS tiene la forma de un cilindro ideal, con un diámetro externo e interno de 22,0 y 3,0 mm, respectivamente y una altura de 650 m. </li><li> El día antes de la galvanoplastia, acoplar la máscara a la zona activa de las matrices de micro-electrodos (AMUMA) por medio de una pinza bajo un microscopio estereoscópico y esterilizar la máscara de PDMS en el horno a 120 ° C. </li><li> Esterilizar las microesferas de vidrio (diámetro nominal de 40 ± 2 micras; diámetro medio certificada de 42,3 ± 1,1 micras) en etanol al 70% durante 2 horas en un vial cónico y girar every 30 minutos para exponer todas las microesferas. </li><li> Retirar la solución de etanol del vial y enjuagar las microperlas dos veces con agua esterilizada. </li><li> Acondicionar la parte central de la zona MEA delimitada por la máscara de PDMS (diámetro igual a 3,0 mm) con 24 l de solución mixta de poli-D-lisina y laminina en 0.05 g / ml. </li><li> Escudo las microperlas con proteínas de adhesión, laminina y poli-D-lisina en 0,05 g / ml y se dejan durante la noche en la incubadora a 37 ° C, para obtener una red neuronal estable y de larga duración. </li><li> Retire los factores de adhesión tanto de la superficie de vidrio de la MEA y de las microesferas de vidrio. Aspirar aproximadamente 95% de la solución de revestimiento con una pipeta y se aplica una pequeña en volumen 24μl- de agua estéril en la superficie MEA. Aspirar de nuevo más de 95% del agua y dejar que el MEA seca bajo el capó laminar durante 1 hora antes en placas las células. NOTA: En el caso de las microesferas lugar, aspirar el recubrimientosolución y hacer un primer lavado con agua esterilizada y una segunda con el medio basal y su suplemento tales como B27, con el fin de acondicionar la superficie del vidrio en el que se sembraron las neuronas. No dejes que las microesferas secas. Deja ellos en suspensión en el medio de cultivo en el interior del vial. </li><li> Distribuir, por medio de una pipeta -200 μl-, la suspensión de microperlas tratados (aproximadamente 32.000) en una placa de múltiples pocillos con inserción de membrana (diámetro de poro 0,4 micras) donde se auto-ensamblan formando una capa uniforme. </li><li> Llene cada pocillo de la membrana con 0,5 ml de medio y ponerlo en la incubadora (T = 37,0 ° C, CO 2 = 5%) hasta que las neuronas están listos para ser plateado (3,2). </li></ol> 2. La disección de embriones y la disociación de Tejidos <ol><li> Ratas hembras Casa adultas (200-250 g) a una temperatura constante (22 ± 1 ° C) y humedad relativa (50%) en un horario de luz-oscuridad normal (luz de 7 a.m.-7 p.m.) en elanimalario. Asegurar que los alimentos y el agua son de libre acceso. </li><li> Anestesiar una rata hembra embarazada después de 18 días de desarrollo (E18) con 3% de isoflurano. Luego, sacrificar al animal por dislocación cervical. </li><li> Retire hipocampos de cada embrión de rata y colocarlos en una solución salina equilibrada helada de Hank sin Ca 2+ y Mg 2+. En el día 18 de hipocampo de desarrollo y los tejidos de la corteza son muy suaves y no tienen que ser cortado en trozos pequeños. Más detalles se pueden encontrar 18,19. </li><li> Disociar el tejido en 0,125% de solución de tripsina / de Hank que contiene 0,05% de ADNasa durante 18-20 min a 37 ° C. </li><li> Eliminar la solución sobrenadante con una pipeta Pasteur y detener la digestión enzimática mediante la adición de medio con suero bovino fetal al 10% (FBS) durante 5 min. </li><li> Eliminar el medio con FBS y lavar una vez con el medio con su suplemento, 1% de L-glutamina, gentamicina 10 mg / ml. Quite de nuevo el medio con un pip Pasteurette y vuelva a llenar de nuevo con una pequeña cantidad (500 l) de medio de crecimiento con su suplemento, 1% de L-glutamina, gentamicina 10 mg / ml. </li><li> Disociar el sedimento de tejido mecánicamente con una pipeta Pasteur estrecha hasta una suspensión lechosa de las células es evidente. No es necesario centrifugar la suspensión celular. </li><li> Diluir el pequeño volumen de suspensión de células con el medio de crecimiento para obtener un volumen final de 2,0 ml. Contar la concentración celular obtenida con una cámara de hemocitómetro. Diluir esta concentración a 1: 5 con el fin de obtener la concentración celular deseada de 600-700 células / mL. </li></ol> 3. celular Chapado <ol><li> Células de placas a una densidad de aproximadamente 2.000 células / mm 2 en el área activa del MEA, definidos por la restricción de PDMS para crear una red neuronal 2D. NOTA: Cada hipocampo contiene aproximadamente 5 x 10 5 células, (1 x 10 6 para un solo embrión). Diseccionar 6 embriones para conseguir un total de 6 x 106 células en 2 ml, y una concentración estimada de 3.000 células / mL. Diluir esta concentración a 1: 5 con el fin de obtener la concentración deseada de células y la placa aproximadamente de 600-700 células / l. Si el área de MEA delimitada por la restricción de PDMS es de aproximadamente 7,065 mm 2, y el número total de células cultivadas en placas células 600 / l x 24 l = 14.400 células, la densidad final de 2.038 células / mm 2. </li><li> Coloque los dispositivos de los AMUMA en la incubadora con humidificado atmósfera de CO 2 (5%) a 37 ° C. </li><li> Distribuir 160 l de la suspensión con una concentración celular de 600-700 células / l (aproximadamente 100.000 células) sobre la superficie de la monocapa microperlas posicionado dentro de las placas de múltiples pocillos para completar el paso preliminar para la construcción de cultivo tridimensional. Coloque las placas de múltiples pocillos en la incubadora con humidificado atmósfera de CO 2 (5%) a 37 ° C. </li></ol> Construcción 4. Red Neuronal 3D <ol><li> 6-8 horas después de la galvanoplastia, transferir la suspensión (microperlas con neuronas) de las placas de múltiples pocillos con mucho cuidado dentro del área delimitada por la restricción PDMS mediante el uso de una pipeta fijada para un volumen de alrededor de 30-40 l. Después de cada transferencia, espere alrededor de medio minuto, para permitir que las microesferas de auto-ensamblan en una estructura compacta hexagonal. </li><li> Una vez que todas las capas se depositan y espontáneamente se reunieron, vuelva a llenar con una gran caída de unos 300 l de medio con la parte superior de la zona delimitada por la restricción PDMS. </li><li> Ponga la estructura 3D acoplado a la MEA en la incubadora (T = 37.0 o C, CO 2 = 5%) durante 48 horas antes de añadir un volumen final (aproximadamente 1 ml) del crecimiento medio de cultivo con su suplemento. NOTA: Tenga en cuenta el número total de cuentas en las placas de múltiples pocillos con inserto de membrana (30.000) y el número de cuentas en una sola capa sobre el dispositivo MEA (6000). La estructura 3D resultante se compone de 5 capas de microbeads y células. Teniendo en cuenta que la red neuronal 3D no es geométricamente perfecto, la estructura 3D resultante podría estar compuesto por capas 5-8. </li><li> El día después de la siembra, añadir con cuidado el volumen final del medio (aproximadamente 900 l) en el interior del anillo de MEA. Mantener las culturas 3D en una atmósfera de CO2 humidificado (5%) a 37 ° C durante 4-5 semanas. Reemplazar la mitad del medio una vez por semana. </li></ol> 5. Microscopía Confocal Adquisición <ol><li> Fijar las muestras biológicas a la platina del microscopio en un soporte. Ajuste el láser (argón, 496-555 nm), la velocidad de exploración (400 Hz) en el que adquirir la imagen, la ganancia y el offset (-0,3%) de PMT para cada imagen para capturar el filtro de emisión de ancho de banda correcto y con el fin para evitar la saturación y para aumentar la relación señal a ruido. La sección de resultados informa de los valores utilizados para adquirir las imágenes de la Figura 4. </li><li> Para la adquisición de la z -stack secuencia, abetost seleccionar, el valor de la posición z de la parte superior y la capa inferior de la muestra, a continuación, seleccione el tamaño de paso para hacer la adquisición. </li></ol>

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The authors declare that they have no competing financial interests.

En esta obra, una novela experimental en la plataforma vitro formado por 3D diseñado cultivos neuronales, junto a los acuerdos ambientales multilaterales para electrofisiología red se ha presentado. El uso de microperlas como andamio para permitir la consecuencia a lo largo del eje x neurítico z se ha adaptado para ser integrado con el MEA planar. De esta manera, los resultados obtenidos micro-sistema en un modelo in vitro de 3D ​​válido y fiable para estudiar la din?...

In vitro de dos dimensiones (2D) redes neuronales acoplados a micro-electrodos Arrays (AMUMA) arethe modelo experimental de oro-estándar adoptado para estudiar la interacción entre la dinámica neuronal y la conectividad subyacente. Durante el desarrollo, las neuronas recrean redes complejas que muestran bien definedspatio temporalpatternsof actividad 1,2 (es decir, las explosiones, las explosiones de red, actividad clavar al azar). AAM registran la actividad electrofisiológica de muchos sitios (de decenas a miles de microelectrodos), lo que permite una investigación detallada de la dinámica expresadas a nivel de red. Además, el uso de cultivos disociados hace possibile para diseñar-redes de ingeniería. Es más fácil de entender de esta manera las relaciones funcionales entre la actividad electrofisiológica registrada y los parámetros de la organización de la red, como la densidad de células 3, grado de modularidad 4,5, presencia de pop neuronal heterogéneaulations 6, etc. Sin embargo, todos los estudios in vitro sobre células cultivadas disociadas se basan en redes neuronales 2D. Este enfoque conduce a simplificaciones con respecto al sistema in vivo (intrínsecamente 3-dimensional, 3D): (I) en un modelo conos, somata de crecimiento y 2D son aplanadas y la consecuencia axones-dendritas no puede extenderse en todas las direcciones 7. (ii) 2D in vitro redes exhibición estereotipada dinámica electrofisiológicos dominadas por la actividad que implica la mayor parte de las neuronas de la red 8 reventar. Recientemente, diferentes soluciones han sido desarrolladas para permitir la construcción de in vitro 3D disociada redes neuronales. La idea común consiste en crear un andamio donde las neuronas pueden crecer en una forma 3D. Un andamio de este tipo puede realizarse con geles de polímeros y matrices porosas sólidas 9-13. Mediante la explotación de las propiedades mecánicas de los polímeros, es posible incrustar dentro de las células Tse estructuras mediante la definición de un bloque uniforme de las culturas 3D de neuroesferas 11. La característica principal de este enfoque es la propiedad mecánica rígida de las neuroesferas 9,12. Sin embargo, estos materiales han porosidad limitada, y no garantizar la migración de células dentro de la matriz. Para superar este inconveniente, una posible solución consiste en cortar la matriz en módulos &quot;unidad&quot;. Por desgracia, el tamaño y la forma de las partículas de diversidad podría obstaculizar el embalaje en estructuras en capas regulares. En 7, Cullen y sus colaboradores diseñaron un constructo neuronal 3D compuesto por neuronas y / o astrocitos dentro de un andamiaje basado en la matriz extracelular bioactiva. Tal tejido neural ingeniería permitido en investigaciones in vitro para estudiar y manipular las respuestas neurobiológicas dentro de micro-ambientes 3D. Este modelo consistía en neuronas y glia distribuidos por toda la matriz extracelular (ECM) y / o andamios de hidrogel (de 500-600 m de espesor). En este condition, una viabilidad celular óptima (mayor que 90%) se encontró en placas células a una densidad final de aproximadamente 3.750 - 5.000 células / mm 3. Debe tenerse en cuenta que un valor tal densidad es mucho menor que el que está en la condición in vivo, donde la densidad de células de la corteza cerebral de ratón es de aproximadamente 90.000 células / mm 3 14. Para superar esta limitación Pautot y compañeros de trabajo 15 se dieron cuenta de un sistema in vitro en 3D donde la densidad celular y la conectividad de red están controlados para asemejarse a las condiciones in vivo al tiempo que permite imágenes en tiempo real de la red. Prácticamente, este método se basa en el concepto de que disocian neuronas cultivadas son capaces de crecer en microperlas de sílice. Estas cuentas proporcionan una superficie de crecimiento lo suficientemente grande para que los cuerpos celulares neuronales para adherirse y para sus arborizaciones para crecer, madurar, ampliar y definir contactos sinápticos con otras neuronas. Este método aprovecha las propiedades de montaje espontáneas de cuentas monodispersados ​​a form 3D en capas matrices hexagonales que contienen distintos subconjuntos de neuronas en diferentes capas con conectividad restringida entre las neuronas en diferentes cuentas. La densidad celular lograda con este método fue de alrededor de 75.000 células / mm 3. Recientemente, hemos adaptado el método de Pautot los AMUMA 16: los resultados obtenidos muestran que la actividad electrofisiológica 3D presenta un repertorio más amplio de actividades que la expresada por las redes 2D. Culturas maduras 3D muestran una dinámica mejorada en la que tanto la explosión de la red y actividad pico aleatoria coexisten. Del mismo modo, Tang-Schomer y compañeros de trabajo 17 se dieron cuenta de un armazón poroso a base de proteínas de seda-que mantiene una cultura cortical primaria in vitro para algunos meses, y registraron la actividad electrofisiológica por medio de un electrodo de tungsteno. En este trabajo, los procedimientos experimentales para construir redes neuronales en 3D, junto a los acuerdos ambientales multilaterales se describirá.

<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.

. En este procedimiento experimental, un papel relevante es interpretado por las microperlas que definen el andamiaje mecánico para el crecimiento de la red neuronal 3D Figuras pantalla 1A y B la disposición de las microperlas (diámetro nominal de 40 ± 2 m; diámetro medio certificada de 42,3 ± 1,1 micras) en el avión. La peculiaridad de tales estructuras es que microperlas espontáneamente se auto-ensamblan definir una geometría hexagonal compacta (Figura 1B y C).</stron...

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.

Interfaz culturas 3D Engineered neuronales a micro-electrodos matrices: un innovador<em&gt; In Vitro</em&gt; Modelo Experimental

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 vistas.  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.

Electrodos múltiples grabaciones matriz de avalanchas neuronal en cultivos organotípicos

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

Investigación

Neurociencias

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.

Culturas organotípicos rebanada de mesencéfalo ventral embrionario: Un sistema para estudiar el desarrollo neuronal dopaminérgica 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 enfoque innovador para el análisis automatizado oculomotor

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.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

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.

Un procedimiento para la implantación de matrices Organizados de Microhilos para grabaciones de una sola unidad en Awake, Comportarse Animales

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.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

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.

Actividad neural propagación en una preparación del hipocampo Desplegado con un penetrante Micro-electrodo de matriz

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.

Incremento dependiente del tiempo en la respuesta de la red a la estimulación de cultivos de células neuronales en matrices de microelectrodos

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 Preparación para imitar la conectividad del circuito neuronal In Vivo

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