Este trabalho foi apoiado pelo BRIANBOW Projeto Europeia (FP7 Jovens Exploradores, Os autores gostariam de agradecer ao Dr. Jacopo Tessadori para comentários úteis sobre o manuscrito, e Silvia Chiappalone por sua ajuda na produção dos gráficos usados ​​no vídeo.

O procedimento foi realizado em conformidade com as normas do NIH para o cuidado e uso de animais de laboratório e foi aprovado pelo cuidado e uso Comitê de Tel-Aviv University Animal (número de autorização - L-14-019). 1. Preparação de Instrumentos e PDMS <ol><li> Preparar a bolacha (Tabela de Materiais, ou encomendar a bolacha a partir de um laboratório de microfabricação), um bisturi, e um par de pinças - a esterilização não é necessária. </li><li> Adicione solução de poli-D-lisina (PDL) de acordo com as seguintes condições: 4 mg / ml em tampão borato 0,1 M, pH 8, e armazenar a -20 ° C. </li><li> Prepare stencils PDMS de acordo com o seguinte procedimento: <ol><li> Prepare de polidimetilsiloxano (PDMS, elastómero de silicone) misturando em conjunto a base e o agente de cura com um rácio de 10: 1, em seguida, agitar as duas substâncias de aproximadamente 5 min. </li><li> Lugar na câmara de vácuo duas vezes durante 15 minutos de cada vez e verificar bolhas sumiram. </li><li> Quandoo PDMS está pronto, preparar rotação revestidor: botões de azoto abertos para permitir o uso de gás, colocar a pastilha de silício (Figura 1A) na fieira, e usar a vácuo para impedi-lo de se mover. </li><li> Verta sobre bolacha de PDMS e activar o botão rotativo durante 1 min a 1000 rpm (aproximadamente 100 pm cria altura). </li><li> Coloque bolacha sobre uma placa quente a 100 ° C durante 30 min. </li><li> Quando o PDMS tem endurecido, delinear as fronteiras do estêncil com PDMS, utilizando pipeta Pasteur. Coloque bolacha sobre uma placa quente a 100 ° C durante 30 min. </li><li> Quando as fronteiras PDMS ter endurecido, usar um bisturi para cortar stencils de acordo com as fronteiras e retire o estêncil de silício do wafer (a PDMS quadrados contendo um único padrão). </li></ol></li></ol> 2. Disco de Petri e tampa de deslizamento Preparação <ol><li> Prepare 23 milímetros lamelas de vidro quadrado e limpá-los de acordo com a seguinte ordem: <ol><li> Lava-se com água destilada, etanol a 70%, e acetona. </li><html><li> Lavar com isopropanol e seca rápido com nitrogênio. </li></ol></li><li> Coloque estampados PDMS stencils na lamela, e pressione suavemente para verificar se eles estão firmemente ligado à superfície do vidro. Coloque na câmara de vácuo durante 15 min. </li><li> Aplicar 1 ml da PDL em stencil. Inserir a lamela na câmara de vácuo duas vezes durante 20 min de cada vez, e deixar PDL O / N para secar. </li><li> Prepare uma placa de Petri para criar uma rede de células de apoio: <ol><li> Cobrir a superfície do prato (3.5 cm) com 1 ml de PDL durante 2 horas em RT. Remover o PDL gota, usando uma pipeta. </li><li> Lavar com água destilada e deixar secar. Colocar uma pequena gota de massa de silicone em cada canto da lamela. </li></ol></li><li> Coloque a lamela no centro da placa de Petri (ou seja, os PDMS deve estar voltado para cima) e pressione delicadamente para verificar anexo. </li><li> Remova cuidadosamente PDMS de lamela usando uma pinça. Para a esterilização, expor a iluminação UV para 7 min. </li></ol>le &quot;&gt; 3. multieletrodos Array (MEA) Preparação <ol><li> Limpe MEA nesta ordem (Tabela de Materiais): <ol><li> Lavar com água debaixo da torneira e sonicate em um detergente concentrado, enzimática 3 vezes. </li><li> Sonicate em água destilada 3 vezes. </li><li> Lavar com água destilada (na capa, 1.000 ul ponta) por 3 vezes e colocar sob UV por 30 min. </li></ol></li><li> Apoiar a preparação da rede: <ol><li> Prepare o molde rede de apoio projetado. <ol><li> Pour PDMS em uma placa de 12 poços (22 mm de diâmetro). </li><li> Quando o PDMS é endurecido retirar o molde e, usando um bisturi, cortar um furo no meio para criar uma forma de anel. </li><li> Coloque um molde de rede de suporte concebido em a centro da MEA (Figura 2A) e cobrir o resto da superfície com PDL durante 2 horas à temperatura ambiente. </li></ol></li><li> Remover PDL utilizando uma pipeta e lava-se com água destilada. </li><li> Retirar o molde e deixar secar (Figura 2A). &lt;/ Li&gt; </ol></li><li> Alinhar estêncil de MEA da seguinte forma: <ol><li> Coloque stencil sobre micro manipulador designado. Usar um microscópio invertido para alinhar com precisão a estrutura modelada eléctrodos, e diminuir o estêncil até que seja colocado na superfície MEA (Figura 2B). Nota: Entre em contato com um MEA bem limpo proporciona aderência de concorrência entre as PDMS e do próprio MEA. </li><li> Levantar o micromanipulador e, se necessário, utilizar uma pinça para aplicar uma pequena quantidade de pressão acima do PDMS para evitar que desprendam do MEA. </li><li> Pressione suavemente stencil à superfície MEA, e usar microscópio para verificar se ele está bem encaixada, e bem alinhados. (Figura 2C-D). </li></ol></li><li> Coloque MEA em câmara de vácuo durante 15 min; colocar uma gota 1 ml de PDL em stencil. </li><li> Insira na câmara de vácuo 2 vezes por 20 min cada. Deixar secar PDL O / N na incubadora. </li><li> Antes de chapeamento, retire as PDMS stencils da AAM e lavagem usando estéril water. Coloque sob iluminação UV para 7 min. </li></ol> 4. Dissecção e Cultura <ol><li> Prepare culturas como descrito na Herzog et al 2011 21.. </li></ol> 5. Galvanização <ol><li> Calcular o número de células necessário para efeitos de revestimento, usando um hemocitómetro (densidade óptima de acordo com o tamanho do circuito como discutido nos resultados). <ol><li> Certifique-se que a câmara de contagem (hemocitômetro) é limpa e coloque uma lamínula sobre ele (o uso de álcool para limpar). </li><li> Diluir 10 uL da suspensão de células em 190 ul de meio de plaqueamento (diluição de 1:20). </li><li> Carregar 10 ml de células diluídas para a borda da câmara de contagem e lentamente pipetar as células para fora permitindo que a câmara encher-se. </li><li> Usando um microscópio invertido, visualizar a grade hemocitï¿½etro. Determinar o número de células na câmara através da contagem direta (células saudáveis ​​devem ser redondos). Contar as células dentro da grande witho quadradout aqueles que cruzam as bordas. </li><li> Calcula-se a concentração de células: Número total de células / 1000 ul = Total de células contadas × fator de diluição × 10 4 (área da hemocitômetro). Nota: Por exemplo, se o factor de diluição foi de 20 e o total de células contadas foi de 100: 100 × 20 × 10 4 = 0,1 x 10 7 células / ul ou 1 1,000 x 10 6/100 ul. A fim de placa de 0,75 x 10 6 células, tomar 75 ul para fora das células. </li></ol></li><li> Tirar o número de células necessário para o chapeamento por MEA único ou numa caixa de Petri, ressuspender as células para evitar a agregação e placa-los no centro, em cima da área do patternate (se necessário, é possível diluir as células no meio) . Para o MEA, colocar uma gota 100 uL no meio. Para tampa de deslizamento, coloque uma gota de 1.000 l no meio. </li><li> Incubar as células plaqueadas a 37 ° C durante 40 min. Adicionar meio de plaqueamento para as células plaqueadas, até 1ml por MEA e 2 ml por tampa de deslizamento. </li><li> Manter a 37 ° C e, a cada 4 dias, dilui-se com meio de crescimento fresco enriquecido com 0,5 Pen-Strep, 2% de B-27 e 0,75% glutamax. </li><li> De 6/7 DIV, depois de ver ligações entre as ilhas, dilua com meio de 10 ul / ml de FUDR (25 mg desoxiuridina + 62,5 mg de uridina em 12,5 ml de MEM (Meio Mínimo Essencial de Eagle)) ou qualquer outro agente anti-mitótico para evitar glial supercrescimento. </li></ol>

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Um protocolo para crescer redes neuronais modulares 2D in vitro composto por circuitos funcionalmente interligadas é descrito. O procedimento baseia-se moldando uma camada adesiva celular. A padronização é conseguido com PDMS stencils reproduzindo a característica negativa da arquitectura da rede desejada. Stencils PDMS definir as áreas onde a camada adesiva celular é depositada. Uma vez que as células são banhados, eles se reúnem espontaneamente para as ilhas revestidos e auto-organizar-se em circuit...

Evidências experimentais e teóricas suporta a possibilidade de que o cérebro opera através da activação coordenada de conjuntos de células de 1-5, que pode ser considerado como unidades funcionais dinâmicos que transitoriamente interagem uns com os outros, para modelar e subjacentes estados cerebrais diferentes. Modularidade funcional também é dependente e associado com a organização estrutural de modular os circuitos cerebrais 6,7. Como a função e estrutura dos circuitos cerebrais mutuamente moldar o outro ainda é uma das principais questões em aberto na neurociência. Para fornecer uma compreensão mais profunda da questão, é importante para identificar estruturas experimentais ótimas, onde é possível resolver, pelo menos em parte, esses problemas. Desde a manipulação da dinâmica espaço-temporal de redes neuronais controlada em experimentos in vivo é um desafio, o desenvolvimento de modelos in vitro redes neuronais é de interesse significativo devido à sua acc fácilessibility, monitoramento, manipulação e modelagem de 8,9. Nos últimos anos, no domínio das tecnologias in vitro apoiados por métodos substrato padronização avançados têm permissão para induzir redes neuronais para desenvolver uma gama de estruturas modulares pré-definidos e 3 para estudar as propriedades funcionais das redes com topologias impostas 10. Em particular, os métodos foram recentemente usados ​​para organizar redes através da imposição de restrições físicas 4,11. Com efeito, para estudar a relação entre a estrutura e função em redes neuronais e para fornecer uma representação simplificada mas plausível de interagir conjuntos neuronais, em sistemas in vitro devem proporcionar sub-populações neuronais interligados. Culturas neuronais homogêneos 2D amplamente estudados não impor quaisquer restrições espaciais sobre a fiação emergente auto-organizada dos circuitos. Portanto, uma possível abordagem para modelar conjuntos de células artificialmente interconectadas é posicionar diferentes populações neuronais em brigaially áreas distintas. A distância entre estas áreas não impede que as inter conexões assembléias. Esta abordagem, assegurando ao mesmo tempo um controlo considerável sobre a complexidade da rede, tem sido mostrado para fornecer um repertório mais rica de modelos de sincronização 6,7,12. A fim de facilitar a cultura reprodutível de assembléias neuronais modulares, um protocolo para montar a auto-organização das redes em agrupamentos neuronais ligados por axônios e dendritos é apresentado e descrito. A estrutura polimérica para o confinamento físico de culturas neuronais foi criado a partir de polydimtheylsiloxane (PDMS). PDMS é um elastómero amplamente utilizado para aplicações biomédicas devido à sua biocompatibilidade, a transparência e a permeabilidade a gases 13. O PDMS é preparado e excluídos do SU8 microusinado 2075 14,15 estruturas por um PDMS líquido sobre um &quot;mestre&quot;, como descrito anteriormente em Jackman et al-spin coating. 16 TEle alcançou modelado redes neuronais são compostos de módulos interligados de tamanho diferente e eles têm sido obtidos com êxito em ambas as lamelas e Microeletrodos Arrays (AMA) 17-20. A densidade das ligações entre os módulos podem alterar as características da rede de sincronização, a partir de uma rede totalmente sincronizados, típico das culturas uniformes, para estados transientes de sincronização entre os módulos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PDMS, Sylgard 184</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Vacuum Chamber</td>
			<td>Thermo</td>
			<td>5305-0609</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine PDL</td>
			<td>Sigma</td>
			<td>P7886</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell - Technologies Corporation</td>
			<td>WS-650-23</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well culture plate</td>
			<td>Sigma</td>
			<td>CLS3336</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8217;-deoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA1060-Inv-BC</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC02</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Biological Industries Beit Haemek</td>
			<td>03-033-1c</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaMAX</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Minimum Essential Medium-Eagle</td>
			<td>Biological Industries Beit Haemek</td>
			<td>01-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Electrode Arrays 4Q</td>
			<td>Multi Channel Systems</td>
			<td>60-4QMEA1000iR-Ti-pr</td>
			<td>cleaning manual: http://www.multichannelsystems.com</td>
		</tr>
		<tr>
			<td>silicon wafer</td>
			<td>microchem</td>
			<td>SU8-2075</td>
			<td>Preparation protocol: www.microchem.com</td>
		</tr>
	</tbody>
</table>

design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits

O cérebro opera por meio da ativação coordenada e comunicação dinâmica das assembléias neuronais. A questão em aberto é importante como um vasto repertório de motivos dinâmicos, que são a base mais diversas funções do cérebro, pode emergir de uma organização fixa topológico e modular dos circuitos cerebrais. Em comparação com estudos in vivo de circuitos neuronais que apresentam dificuldades experimentais intrínsecas, preparações in vitro oferece uma possibilidade muito maior para manipular e sondar as propriedades estruturais, dinâmicos e químicos de sistemas neuronais experimentais. Este trabalho descreve uma metodologia experimental in vitro que permite a cada vez maior de redes modulares compostos por, assembléias neuronais funcionalmente interligadas espacialmente distintas. O protocolo permite o controlo da arquitectura bidimensional (2D) da rede neuronal a diferentes níveis de complexidade topológica. A padronização rede desejada pode serconseguido tanto em lamelas regulares e substrato incorporado matrizes micro eletrodos. Micromachined estruturas estão em alto relevo sobre uma bolacha de silício e utilizada para criar matrizes poliméricas biocompatíveis, que incorporam as características negativas do arquitectura de rede desejada. As matrizes são colocados sobre os substratos de cultura durante o processo de revestimento da superfície com uma camada molecular para promover a adesão celular. Após a remoção das matrizes, os neurónios são plaqueadas e eles espontaneamente redireccionado para as áreas revestidas. Diminuindo a distância entre o compartimento, é possível obter os circuitos neuronais isolados ou interligados. Para promover a sobrevivência celular, as células são co-cultivadas com uma rede neuronal de suporte, que está localizado na periferia da placa de cultura. Registros eletrofisiológicos e ópticas da atividade de redes modulares obtidos, respectivamente, utilizando substrato incorporado micro matrizes de eletrodos e de imagem de cálcio são apresentados. Enquanto cada módulo mostra espontsincronizações globais aneous, a ocorrência de sincronização entre módulos é regulada pela densidade de ligação entre os circuitos.

A SU8-2075 molde sobre uma pastilha de silício, com espessura característica de aproximadamente 100 mm foi utilizada para moldar as PDMS. O padrão foi composta por quadrados de várias dimensões, com um comprimento lateral e distância que varia entre 200 e 700 um (Figura 1B). O tamanho do quadrado foi escolhida para se adaptar ao campo de visão de um 10X (por ilhas com um comprimento lateral &lt;800 um) e de uma objectiva de 20x (por ilhas com um comprimento lateral &lt;400 um). Três parâmetros,...

Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Este manuscrito descreve um protocolo para crescer em redes modulares vitro que consistem em espacialmente confinados, circuitos neuronais funcionalmente inter-relacionados. Uma máscara polimérico é utilizado para uma camada de padrão de proteína para promover a adesão celular sobre o substrato de cultura. Neurônios Banhado crescer em áreas revestidas estabelecendo conexões espontâneas e exibem atividade eletrofisiológica.

Desenho, Tratamento de Superfícies, Cellular Galvanização e Cultura de modulares Neuronal de redes formadas por Circuitos Funcionalmente Inter-conectados

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

design-surface-treatment-cellular-plating-culturing-modular-neuronal

Research

JoVE Journal

Neuroscience

8.4K visualizações.  Tel-Aviv University.  Este manuscrito descreve um protocolo para crescer em redes modulares vitro que consistem em espacialmente confinados, circuitos neuronais funcionalmente inter-relacionados. Uma máscara polimérico é utilizado para uma camada de padrão de proteína para promover a adesão celular sobre o substrato de cultura. Neurônios Banhado crescer em áreas revestidas estabelecendo conexões espontâneas e exibem atividade eletrofisiológica. 

Vídeo: Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Mapeamento Inibitória Circuitos Neuronal por Laser Scanning fotoestimulação

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Neurociência

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 ...

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 ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

De circuito fechado experiências neuro-robótico para testar as propriedades computacionais da Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Rewiring Neuronal Circuits: um novo método para Fast Neurite Extension e Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Determinando a expressão da superfície celular e a taxa endocítica de proteínas em culturas primárias de astrocitos usando a biotinilação

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Estimulação elétrica transcraniana crônica e gravação Intracortical em ratos

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastamento de Theta Hippocampal oscilações em comportar-se ratos

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Imagem Simultânea da Dinâmica Microglial e Atividade Neuronal em Camundongos Acordados

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...