Os autores agradecem Giorgio Carlini pelo apoio técnico no desenvolvimento da estrutura de confinamento e dott. Ornella LoBrutto para a revisão completa do manuscrito. A investigação conducente a estes resultados beneficiaram de financiamento do 7º Programa-Quadro da União Europeia (ICT-FET FP7 / 2007-2013, FET jovens exploradores esquema), sob acordo de subvenção 284.772 CÉREBRO BOW (www.brainbowproject.eu).

O protocolo experimental foi aprovado pelo Animal Care Legislação Europeia (2010/63 / UE), pelo Ministério da Saúde italiano, de acordo com o DL 116/1992 e pelas diretrizes da Universidade de Genova (Prot. N. 13.130, de maio 2011). Foram feitos todos os esforços para reduzir o número de animais para o projeto e para minimizar o seu sofrimento. 1. Preparação de Materiais e Suportes <ol><li> Construir o molde para construir as PDMS (Poly-dimetil-siloxano) de restrição por meio de um CNC (Controle Numérico Computadorizado) fresadora. Tal molde realiza-se em policarbonato com o cilindro central de politetrafluoroetileno (PTFE). Este material permite uma extracção fácil da máscara uma vez que o PDMS tem endurecido. NOTA: O projeto do molde foi realizada por Computer-Aided-Design (CAD) e, em seguida, entregue ao fresadora CNC. </li><li> Prepara-se o elastómero PDMS. PDMS é um polímero orgânico. Ele é constituído por dois elementos, Um agente de cura e um polímero. Misture-os por uma relação de volume de 1:10, uma parte do agente de cura e nove partes de polímero. Colocar os dois componentes em uma placa de Petri, e inserir agitar a mistura na câmara de vácuo durante 10 minutos para eliminar as bolhas de ar. 3 g de PDMS são suficientes para uma restrição. </li><li> Construa a restrição PDMS. Inserir o material PDMS no moldador e colocá-la no forno durante 30 minutos a 120 ° C. A restrição de PDMS tem a forma de um cilindro ideal, com um diâmetro interior e exterior de 22,0 e 3,0 mm, respectivamente, e uma altura de 650 microns. </li><li> Um dia antes do chapeamento, casal a máscara para a área ativa das matrizes Micro-Eletrodo (AMA) por meio de uma pinça sob microscópio estereoscópico e esterilizar a máscara PDMS no forno a 120 ° C. </li><li> Esterilizar a microesferas de vidro (diâmetro nominal de 40 ± 2 uM; diâmetro médio certificado de 42,3 ± 1,1 uM) em etanol a 70% durante 2 horas num frasco cónico e girar every 30 min para expor todas as micropérolas. </li><li> Remover a solução de etanol a partir do frasco e lavar as micro-esferas duas vezes com água esterilizada. </li><li> Condiciona-se a parte central da área de MEA delimitada pela máscara de PDMS (diâmetro igual a 3,0 mm) com 24 ul de solução de mistura de poli-D-lisina e laminina em 0,05 ug / ml. </li><li> Revestir as micropérolas com proteínas de adesão, a laminina e poli-D-lisina na 0,05 ug / ml e deixá-los durante a noite na incubadora a 37 ° C, para se obter uma rede neuronal estável e de longa duração. </li><li> Remover os factores de adesão, tanto a partir da superfície de vidro da MEA e para as microesferas de vidro. Aspirar aproximadamente 95% da solução de revestimento com uma pipeta e aplicar uma pequena volume- 24μl- de água estéril sobre a superfície do MEA. Aspirar novamente mais do que 95% da água e deixar secar o MEA sob o capô laminar durante 1 h antes do plaqueamento das células. NOTA: No caso de micro-esferas em vez disso, o revestimento aspirarsolução e fazer uma primeira lavagem com água esterilizada e um segundo com o meio basal e seu complemento, tais como B27, a fim de condicionar a superfície do vidro, onde os neurónios vai ser revestida. Não deixe que as microesferas de seca. Deixar-los em suspensão no meio de cultura no interior do frasco. </li><li> Distribuir, por meio de uma pipeta -200 μl-, a suspensão de micro-esferas tratadas (cerca de 32.000) numa placa com múltiplas cavidades com pastilha de membrana (diâmetro de poro de 0,4 um), onde eles se auto-montam formando uma camada uniforme. </li><li> Encher cada poço da membrana com 0,5 ml de meio e colocá-lo na incubadora (T = 37,0 ° C, CO2 = 5%) até que os neurónios estão prontos a ser revestida (3,2). </li></ol> 2. Dissecção de Embriões e dissociação de Tissue <ol><li> Ratas adultas Casa (200-250 g) a uma temperatura constante (22 ± 1 ° C) e humidade relativa (50%), sob um horário de luz-escuro normal (em 7 de luz-AM-7 PM) nabiotério. Certifique-se de que os alimentos e água estão disponíveis gratuitamente. </li><li> Anestesiar um rato fêmea grávida após 18 dias de desenvolvimento (E18), utilizando 3% de isoflurano. Então, sacrificar o animal por deslocamento cervical. </li><li> Remover hipocampos de cada embrião de rato e colocá-los em solução salina equilibrada de Hank gelada sem Ca2 + e Mg2 +. No dia 18 de desenvolvimento do hipocampo e do córtex tecidos são muito suaves e não precisa de ser cortado em pequenos pedaços. Mais detalhes podem ser encontrados 18,19. </li><li> Dissociar o tecido em solução de 0,125% de tripsina / de Hank contendo 0,05% de DNAse, durante 18-20 min a 37 ° C. </li><li> Remover a solução de sobrenadante com uma pipeta Pasteur e parar a digestão enzimática pela adição de meio com 10% de soro fetal de bovino (FBS) durante 5 min. </li><li> Remover o meio com FBS e lavar uma vez com o meio com suplemento a, 1% de L-glutamina, gentamicina 10 ug / ml. Retirar novamente o meio com um pip Pasteurette e encher de novo com uma pequena quantidade (500 uL) de meio de crescimento com o seu complemento, 1% de L-glutamina, gentamicina 10 ug / ml. </li><li> Dissociar o sedimento tecido mecanicamente com uma pipeta Pasteur estreita até uma suspensão leitosa de células é aparente. Não é necessário centrifugar a suspensão de células. </li><li> Dilui-se a pequeno volume de suspensão de células com o meio de crescimento para se obter um volume final de 2,0 ml. Contagem celular a concentração obtida com uma câmara de hemocitómetro. Dilui-se esta concentração de 1: 5 a fim de se obter a concentração desejada de células de 600-700 células / mL. </li></ol> 3. celular Galvanização <ol><li> As células em placas a uma densidade de cerca de 2.000 células / mm 2 sobre a área activa do MEA, definidos pela restrição PDMS para criar uma rede neuronal 2D. Nota: Cada hipocampo contém cerca de 5 x 10 5 células, (1 x 10 6 para um único embrião). Dissecar 6 embriões para obter um montante total de 6 x 106 células em 2 ml e uma concentração estimada de 3.000 células / mL. Dilui-se esta concentração de 1: 5 a fim de se obter a concentração de células desejada e placa cerca de 600-700 células / mL. Se a área de MEA delimitado pela restrição PDMS é de cerca de 7.065 mm 2, e o número total de células plaqueadas 600 células / mL x 24 mL = 14.400 células, a densidade final de 2.038 células / mm 2. </li><li> Colocar os dispositivos de MEA na incubadora humidificada com CO 2 ambiente (5%) a 37 ° C. </li><li> Distribuir 160 ul da suspensão com uma concentração de células de 600-700 células / ul (cerca de 100.000 células) sobre a superfície da monocamada micropérolas posicionado no interior das placas com múltiplas cavidades para completar o passo preliminar para a construção de cultura tri-dimensional. Colocar as placas com múltiplas cavidades na incubadora humidificada com CO 2 ambiente (5%) a 37 ° C. </li></ol> 4. Construção 3D Rede Neuronal <ol><li> 6-8 horas após o plaqueamento, transferir a suspensão (micropérolas com neurónios) a partir das placas com múltiplas cavidades com muito cuidado dentro da área delimitada pela restrição PDMS usando uma pipeta para definir um volume de cerca de 30-40 ul. Depois de cada transferência, espere por cerca de meio minuto, para permitir que as microesferas para se juntam em uma estrutura compacta hexagonal. </li><li> Uma vez que todas as camadas são depositadas e espontaneamente montado, encher com uma grande gota de cerca de 300 ul de meio do topo da área delimitada pela restrição de PDMS. </li><li> Coloque a estrutura 3D acoplado a MEA na incubadora (T = 37,0 ° C, CO2 = 5%) durante 48 horas antes da adição de um volume final (cerca de 1 ml) de meio de cultura de crescimento com o seu complemento. NOTA: Considere o número total de grânulos sobre as placas com múltiplas cavidades com pastilha de membrana (30,000) e o número de pérolas numa única camada sobre o dispositivo MEA (6000). A estrutura 3D resultante é composto por 5 camadas de microbeads e células. Levando-se em conta que a rede neuronal 3D não é geometricamente perfeita, a estrutura em 3D resultante pode ser composta de 5-8 camadas. </li><li> No dia após o plaqueamento, adicionar cuidadosamente o volume final do meio (cerca de 900 uL) no interior do anel de MEA. Manter as culturas 3D em uma atmosfera humidificada de CO 2 (5%) a 37 ° C durante 4-5 semanas. Substituir metade do meio de uma vez por semana. </li></ol> 5. Microscopia Confocal Aquisição <ol><li> Fixar as amostras biológicas para a fase de microscópio de um suporte. Defina a laser (argônio, 496-555 nm), a velocidade de verificação (400 Hz) em que adquirir a imagem, o ganho e offset (-0,3%), da PMT para cada imagem para capturar o filtro de emissão de banda correta e em ordem para evitar a saturação e para aumentar a relação sinal-ruído. A seção de resultados informa os valores utilizados para adquirir as imagens da Figura 4. </li><li> Para a aquisição da sequência Z -stack, abetost selecionar, o valor da posição z da parte superior e a camada inferior da amostra, e em seguida, selecione o tamanho do passo para fazer a aquisição. </li></ol>

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The authors declare that they have no competing financial interests.

Neste trabalho, um romance experimental em plataforma vitro composta de 3D ​​engenharia culturas neuronais acoplados a AAM para eletrofisiologia rede foi apresentada. A utilização de micropérolas como andaime para permitir que o crescimento neurítico ao longo da -axis Z foi adaptado para ser integrado com o MEA planar. Desta forma, os resultados obtidos micro-sistema em um modelo in vitro 3D válido e confiável para estudar a dinâmica eletrofisiológicos emergentes 16.<...

Em bidimensionais redes neurais (2D) vitro acoplados a Micro-Arrays eletrodo (AMA) arethe modelo experimental padrão-ouro adotada para estudar a interação entre a dinâmica neuronal e a conectividade subjacente. Durante o desenvolvimento, os neurônios recriar redes complexas que mostrar bem actividade definedspatio-temporalpatternsof 1,2 (ou seja, explosões, rajadas de rede, atividade spiking aleatório). AAM registrar a atividade eletrofisiológica de muitos sites (de dezenas a milhares de microeletrodos), permitindo uma investigação detalhada da dinâmica expressos no nível da rede. Além disso, a utilização de culturas dissociadas faz possibile para conceber-redes modificadas. É mais fácil de entender, desta forma as relações funcionais entre a atividade eletrofisiológica registradas e os parâmetros da organização em rede como densidade de células 3, grau de modularidade 4,5, presença de pop neuronal heterogêneoulations 6, etc. No entanto, todos os estudos in vitro em células cultivadas dissociados são baseados em redes neuronais 2D. Esta abordagem conduz a simplificações em relação à (, intrinsecamente 3-dimensional 3D) Sistema in vivo: (I) num modelo em 2D, somata e cones de crescimento se encontram achatados e a excrescência axónios-dendrites não pode propagar em todas as direcções 7. (ii) 2D in vitro redes exposição estereotipada dinâmica eletrofisiológicos dominadas por atividade envolvendo a maior parte dos neurônios da rede 8 estourando. Recentemente, soluções diferentes têm sido desenvolvidos para permitir que a construção in vitro de 3D ​​redes neuronais dissociadas. A idéia comum consiste na criação de um andaime onde os neurônios pode crescer de uma forma 3D. Um tal andaime pode ser realizada com géis de polímeros e matrizes sólidas porosas 9-13. Ao explorar as propriedades mecânicas dos polímeros, é possível incorporar no interior das células Tse estruturas através da definição de um bloco uniforme de culturas 3D de neurospheres 11. A principal característica dessa abordagem é a propriedade mecânica rígida das neuroesferas 9,12. No entanto, estes materiais têm porosidade limitada, e eles não garantem a migração de células no interior da matriz. Para superar esta desvantagem, uma possível solução consiste em cortar a matriz em módulos «unidade». Infelizmente, o tamanho e forma das partículas de diversidade pode dificultar a embalagem em estruturas em camadas regulares. Em 7 de Cullen e colaboradores projetou uma construção neuronal 3D feito de neurônios e / ou astrócitos dentro de um extracelular andaime bioativo à base de matriz. Um tecido neural, tais engenharia permitida em investigações in vitro para estudar e manipular as respostas neurobiológicas dentro de micro-ambientes 3D. Este modelo consistiu em neurónios e glia distribuídas por toda a matriz extracelular (ECM) e / ou andaimes de hidrogel (500-600 mm de espessura). Neste condition, uma viabilidade celular óptima (maior do que 90%) foi encontrado por plaqueamento de células, a uma densidade de cerca de 3,750 definitiva - 5000 células / mm3. Deve-se notar que um tal valor de densidade é muito menor do que a da condição in vivo, onde a densidade das células do córtex cerebral do rato é de cerca de 90.000 células / mm 3 14. Para superar esta limitação Pautot e colaboradores 15 realizado um sistema 3D in vitro em que a densidade celular e a conectividade de rede são controladas para se assemelhar a condições in vivo, permitindo simultaneamente imagens em tempo real da rede. Na prática, este método baseia-se no conceito de que os neurónios dissociados em cultura são capazes de crescer em microesferas de sílica. Estas contas fornecem uma superfície de crescimento grande o suficiente para os corpos celulares neuronais para aderir e para as suas arborizações para crescer, amadurecer, se estendem, e definem contatos sinápticos para outros neurônios. Este método explora as propriedades espontâneas de montagem de contas monodispersas para form 3D em camadas matrizes hexagonais contendo subconjuntos distintos de neurônios em diferentes camadas com conectividade restrita entre os neurônios em diferentes esferas. A densidade celular obtida com este método foi de cerca de 75.000 células / mm3. Recentemente, nós adaptamos o método de Pautot aos MEA 16: os resultados obtidos mostram que a atividade eletrofisiológica 3D apresenta um repertório mais vasto de actividades do que aquela expressa por redes 2D. Culturas maduras 3D exibem uma dinâmica reforçada em que ambos estouro de rede e aleatória coexistem pico de atividade. Da mesma forma, Tang-Schomer e colaboradores 17 realizado um andaime poroso à base de proteína de seda que mantém uma cultura primária cortical in vitro para alguns meses, e registada a actividade electrofisiológica por meio de um eléctrodo de tungsténio. Neste trabalho, os procedimentos experimentais para construir redes neuronais 3D acoplados aos MEA será descrito.

<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.

. Neste procedimento experimental, um papel relevante é reproduzida pelas microesferas que definem o andaime mecânica para o crescimento da rede neuronal 3D Figuras visor 1A e B, o arranjo das microesferas (diâmetro nominal de 40 ± 2 uM; diâmetro médio autenticada 42,3 ± 1,1 uM) no plano. A particularidade deste tipo de estruturas é que micropérolas espontaneamente auto-montar definindo uma geometria hexagonal compacto (Figura 1B e C). O andaime assim gerado ga...

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.

Interface 3D Engineered culturas neuronais para Micro-Arrays eletrodo: An Innovative<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 visualizações.  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.

Vídeo: 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.

Multieletrodos Recordings Array of Avalanches Neuronal em culturas organotípicas

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

Neurociência

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.

Organotípicas culturas de mesencéfalo ventral Slice Embrionárias: Um Sistema para o Estudo de Desenvolvimento Neuronal dopaminérgicos 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, uma abordagem inovadora Automatizada de Análise 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.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração 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.

Um Procedimento para Implantação de Arrays organizado de microfios para gravações a única unidade em Awake, comportando-se Animais

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.

Manipulação genética de Cerebelar Granule Neurônios<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Estudar Neuronal Morfologia e Migração

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 Propagação em uma Unfolded Hippocampal Preparação com um penetrante Micro-eletrodo 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.

Aumento Time-dependente na resposta da rede à estimulação de culturas de pilha neuronais em matrizes do micro-elétrodo

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.

Preparação de fatia de cunha in vitro para imitar conectividade do circuito neuronal vivo

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