Gostaríamos de agradecer ao Dr. Jeffrey Rothstein para a prestação de ratos BAC GLT1 EGFP e GLT1 anticorpos; Tufts Center for Neuroscience Research (NIH P30 NS047243; PI, Rob Jackson) para a prestação de importantes instalações valiosos; Nova faculdade de recrutamento de subvenção (NIH P30 5P30NS069254-02 , PI, Phil Haydon) na Tufts Neuroscience Departamento.

1. Assembleia da Câmara Cultura microfluídicos (MCP) <ol><li> MCP (Figura 1) são concebidos para câmaras abertas compartimentados culturas de diferentes tipos de células 4. Geralmente, ela tem dois compartimentos, que são ligados por meio dos canais centrais (3 mm de diâmetro). Assembleia de MCP com vidro de fundo pratos é necessário para preparação de culturas e análise de imagem posterior. </li><li> Em primeiro lugar, revestimento estéreis de vidro com fundo de pratos com poliornitina (Sigma-Aldrich, 1 mg / ml) dissolvido em tetraborato de sódio (Sigma-Aldrich, 10 mM pH 8,5) e foram incubadas durante a noite a 37 ° C. </li><li> No dia seguinte, as revestidas de vidro com fundo de placas foram lavadas três vezes com DDQ 2 O e secou-se sob o capô estéril fumos. Os pratos de vidro com fundo foram então adicionalmente revestidas com laminina (Sigma-Aldrich, 1 mg / ml) e secou-se sob luz UV, durante 1 hora. Pratos revestidos estão prontas para utilização ou podem ser armazenadas à temperatura de -20 ° C até à sua utilização. Estas revestimento de umre necessário para chapeamento neurônios e astrócitos na co-cultura. </li><li> Para a montagem da plataforma de cultura, as plataformas de cultura microfluídicos foram colocados no topo dos com revestimento de vidro com fundo de pratos com canais de ligação centrais no seu lado inferior para formar uma vedação estanque. Neurónio regular ou meios de cultura de astrócitos foram adicionados (a partir das áreas de células de revestimento), em ambos os lados (fig. 1) para a MCP montado e incubadas durante 2-4 horas a 37 ° C, para assegurar que não haja fugas entre a MCP e-glass prato fundo. Nós testamos com o meio antes de as células de revestimento para garantir que não haja vazamento. A montagem de MCP sobre o prato de vidro de fundo deve ser preparado imediatamente antes da utilização. </li></ol> 2. Preparação da cultura neuronal e Indução de axónios neuronais em MCP montado <ol><li> Culturas de células neuronais corticais foram recém-preparado a partir de cérebros embrionários 14-16 dias de idade, de ratinho. O meio de cultura é composto por neurónios meio neurobasal, 2% B27 neurobasal suplemento, glutamina 2 mM pela adição de 1% de 100x GlutaMAX, e 1% de penicilina-streptomysin. Seguindo a dissecção do cérebro do rato, as meninges foram removidas a partir do córtex sob o microscópio de dissecação. O tecido foi então triturado com uma lâmina de barbear para fazer pequenos blocos de tecido de modo a aumentar a área de superfície exposta à tripsina. Os blocos de tecido foram tratadas com tripsina (Sigma-Aldrich, de Tripsina a 0,05%) durante 10 minutos num banho de água a 37 ° C, e depois dissociado por trituração suave com um fogo polida pipeta de Pasteur. As células dissociadas foram filtradas através de um filtro de 70 | iM para recolher a suspensão de células claras neuronal. </li><li> Neurónios (2-3 x 10 6 / ml, 150 ul) foram colocadas em placas sobre as áreas de células de revestimento do lado direito (Figura 1) da MCP montado de modo que os neurónios pode anexar na área de retenção de células (Figura 1). Neurónios recentemente preparadas foram colocadas em placas com meio de plaqueamento neuronal que é suplementado com 5% de soro fetal bovino (Hyclone) para a regulaçãor meio de cultura neuronal. Porque apenas os neurónios que se ligam na área de retenção de células são capazes de crescer em axónios do outro lado da MCP montado por meio de canais centrais, é importante que a densidade da placa elevada de neurónios na área de revestimento de células suficientes para assegurar neurónios estão ligados ao área de retenção de células. Uma imagem representativa de alta densidade de axónios neuronais é mostrado na Figura 2A. </li><li> No dia seguinte, o meio de plaqueamento neuronal foi substituído por meio normal de cultura neuronal. Alterando médio foi realizado por meio de aspiração cuidadosamente a partir da área de revestimento da câmara de célula, mas não a partir da área de retenção de células da MCP montado, de modo a evitar bolhas de ar na área de retenção de células e os canais de ligação centrais. As bolhas de ar vai inibir seriamente o crescimento axonal ea subsequente entrada nos canais centrais no MCP. Glial-cell factor neurotrófico derivado (GDNF, 10-20 ng / ml) foi também adicionado no lado (esquerdo) outrosda câmara no mesmo dia, para facilitar a indução de crescimento axonal 5 a partir do lado neuronal e transversal através de canais centrais da MCP montada (Figura 2A). Quantidade de crescimento de neurites espontânea sem GDNF é também observada a partir do lado neuronal e transversal através de canais centrais da MCP montado. Axónios que entram no canal central são frequentemente observadas 2-3 dias após o plaqueamento. Uma vez que os axónios entrar no canal central, que normalmente entrar no outro lado no prazo de um dia. Axônios são normalmente intacto durante pelo menos uma semana após a entrada no outro lado. </li></ol> 3. Além de astrócitos cultivados para MCP estabelecer um sistema de co-cultura Compartimentada Culturas primárias de astrócitos foram preparadas a partir do cérebro P1-3 do rato filhote. O procedimento de dissociação cérebro é semelhante ao processo neuronal de células de isolamento descritas acima. Os astrócitos foram inicialmente semeadas em placa de 10 cm, que foram pré-Revestidas com poliornitina (Sigma-Aldrich, 1 mg / ml). O meio de cultura de astrócitos (DMEM, 10% de soro fetal bovino, 1% de penicilina-streptomysin) foi mudado todos os dias durante os dois dias seguintes para remover os detritos. Depois disso, o meio foi trocado a cada três dias. <ol><li> Astrócitos tornam-se confluentes 90% e formam uma monocamada após 7 dias. Astrócitos confluentes foram tripsinizadas e 150 ul de re-suspensas astrócitos (1x10 6 / ml) foram re-plaqueadas em placas de revestimento da área da célula do lado esquerdo do MCP montado quando axónios estão prestes a entrar ou ter acabado de entrar na parte lateral esquerda do o montado MCP. Os astrócitos foram plaqueadas normalmente 4-5 dias depois dos neurónios foram plaqueados. O GDNF foi primeiro removido antes da re-chapeamento dos astrócitos para o lado esquerdo da MCP. Re-plated astrócitos foram fixados na área de retenção de células, como mostrado pela imunocoloração GFAP (Figura 1). Apenas o fluxo mínimo de meio entre os dois lados das câmaras (determinado por fluorescência dsim) foi encontrada na MCP, tal como anteriormente demonstrado 4,6. </li><li> Astrócitos tornam-se confluentes 90% e formam uma monocamada após 7 dias. Astrócitos confluentes foram tripsinizadas e 150 ul de re-suspensas astrócitos (1 x 10 6 / ml) foram re-plaqueadas em placas de revestimento da área da célula do lado esquerdo do MCP montado quando axónios estão prestes a entrar ou ter acabado de entrar na esquerda lado da MCP montado. Os astrócitos foram plaqueadas normalmente 4-5 dias depois dos neurónios foram plaqueados. O GDNF foi primeiro removido antes da re-chapeamento dos astrócitos para o lado esquerdo da MCP. Re-plated astrócitos foram fixados na área de retenção de células, como mostrado pela imunocoloração GFAP (Figura 1). Apenas o fluxo mínimo de meio entre os dois lados das câmaras (determinado por fluorescência de corantes) foi encontrada na MCP, tal como anteriormente demonstrado 4,6. </li><li> Após a re-chapeamento dos astrócitos, axónios que entraram no lado esquerdo da MCP montado interagir directamente com o Astrocytes por contato direto axonal ou liberação de fatores solúveis. A imunocoloração de astroglial GLT1 transportador de membrana plasmática de glutamato e neuronal βIII-tubulina foi utilizada para visualizar a interacção entre os axónios e os astrócitos, conforme mostrado na Figura 2D-F. </li></ol>

<ol>
	<li>Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+signaling+in+the+development+and+plasticity+of+neural+circuits.">Neuron-astrocyte signaling in the development and plasticity of neural circuits.</a> Neuro-Signals. 16, 278-288 (2008).</li><li>Paixao, S., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+communication+and+synaptic+plasticity.">Neuron-astrocyte communication and synaptic plasticity.</a> Current opinion in neurobiology. 20, 466-473 (2010).</li><li>Fields, R. D., Stevens-Graham, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+neuron-glia+communication.">New insights into neuron-glia communication.</a> Science. 298, 556-562 (2002).</li><li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature. 1, 2128-2136 (2006).</li><li>Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuromuscular+synapse+development+by+glial+cell+line-derived+neurotrophic+factor+and+neurturin.">Regulation of neuromuscular synapse development by glial cell line-derived neurotrophic factor and neurturin.</a> The Journal of biological chemistry. 277, 10614-10625 (2002).</li><li>Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+astroglial+excitatory+neurotransmitter+transporter+GLT1.">Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1.</a> Neuron. 61, 880-894 (2009).</li><li>Regan, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+promoter+activity+reveal+a+differential+expression+and+physiology+of+glutamate+transporters+by+glia+in+the+developing+and+mature+CNS.">Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS.</a> The Journal of neuroscience. 27, 6607-6619 (2007).</li><li>Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+glutamate+transporter+subtype+expression+in+astrocytes.">Neuronal regulation of glutamate transporter subtype expression in astrocytes.</a> The Journal of neuroscience. 17, 932-940 (1997).</li><li>Schlag, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+glial+Na+-dependent+glutamate+transporters+by+cyclic+AMP+analogs+and+neurons.">Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.</a> Molecular pharmacology. 53, 355-369 (1998).</li><li>Ponomarev, E. D., Novikova, M., Maresz, K., Shriver, L. P., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+culture+system+that+supports+adult+microglial+cell+proliferation+and+maintenance+in+the+resting+state.">Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state.</a> Journal of immunological. 300, 32-46 (2005).</li><li>Espinosa-Jeffrey, A., Wakeman, D. R., Kim, S. U., Snyder, E. Y., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+system+for+rodent+and+human+oligodendrocyte+specification,+lineage+progression,+and+maturation.">Culture system for rodent and human oligodendrocyte specification, lineage progression, and maturation.</a> Current protocols in stem cell biology. Chapter 2, (2009).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li><li>Debanne, D., Rama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+shape+axonal+signaling.">Astrocytes shape axonal signaling.</a> Science signaling. 4, pe11 (2011).</li></ol>

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O neurônio MCP baseado e astrócitos sistema de co-cultura de neurônio permite dissecção detalhada para as vias de sinalização astroglia permitindo que somente os axônios passam os canais centrais e interagindo com as células gliais. Este sistema de co-cultura pode ser convenientemente configurado com o neurônio convencional e procedimentos de cultura de astrócitos. Nós também descrita uma aplicação prática deste sistema de co-cultura utilizando um repórter baseado eGFP para demonstrar a activação depe...

<table border="1"><tbody><tr><td> De soro fetal bovino </td><td> Hyclone </td><td> SH30070.03 </td><td> para revestimento do neurônio para neurônio médio cutlure </td></tr><tr><td> De soro fetal bovino </td><td> Sigma-Aldrich </td><td> F4135 </td><td> por meio de cultura de astrócitos </td></tr><tr><td> Fator nervo gliais derivadas </td><td> R &amp; D de sistemas </td><td> 212-GD </td><td> Aplicar 10-20 ng / ml para neurónio lado da câmara </td></tr><tr><td> Dulbecco Eagle modificado meio de glucose elevada </td><td> Sigma-Aldrich </td><td> 11995 </td><td></td></tr><tr><td> 70 milímetros filtro celular </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Prato fundo de vidro esterilizado </td><td> MatTek Corporação </td><td></td><td></td></tr><tr><td> Microfluídicos plataformas cultura </td><td> Xona microfluídica LLC </td><td> SND150 </td><td></td></tr><tr><td> 6 cavidades da placa de cultura </td&gt; <td> Cellstar </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> Neuron meio de cultura <ul><li> Meio Neurobasal </li><li> 2% B27 suplemento Neurobasal </li><li> 2 mM de glutamato pela adição de 1% de 100x GlutaMAX </li><li> 1% penicilina streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Neurônio meio de cultura para células de revestimento <ul><li> Meio Neurobasal </li><li> 2% B27 suplemento Neurobasal </li><li> 2 mM de glutamato pela adição de 1% de 100x GlutaMAX </li><li> 1% penicilina streptomysin </li><li> 5% de soro fetal bovino SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> Meio de cultura de astrócitos <ul><li> Dulbecco modificado eaglucose do meio gle alta </li><li> 10% de soro bovino fetal F4135 </li><li> 1% penicilina streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Materiais utilizados na cultura de microfluidos axon plataforma baseada neuronal e da glia do sistema de co-cultura. </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

Neurónio adequado para interacção glia é crítica para a função fisiológica do sistema nervoso central (SNC). Esta comunicação bidirecional é sofisticada específicas mediadas por vias de sinalização entre neurônios e células gliais 1,2. Identificação e caracterização de tais vias de sinalização é essencial para a compreensão de como a fisiologia neurônio glia interacção CNS formas. Anteriormente, neurônios e células gliais culturas mistas têm sido amplamente utilizados para testes e caracterização de vias de sinalização entre neurônios e células gliais. O que aprendemos com essas preparações e outras ferramentas in vivo, no entanto, sugeriu que a sinalização mútuas entre neurônio e glia que ocorreram geralmente em compartimentos específicos dentro dos neurônios (axônio, dendrito, ou soma) 3. Isto torna importante o desenvolvimento de um sistema de cultura novo, que permite a separação dos compartimentos neuronais e, especificamente, examina a interacção entre as células gliais e neuroaxônios NAL / dendritos. Além disso, o sistema de cultura convencional mista não é capaz de diferenciar os factores solúveis e os sinais de membrana por contacto directo entre neurónios e células gliais. Além disso, a grande quantidade de neurónios e células gliais no sistema de co-cultura convencional não tem a resolução necessária para observar a interacção entre um único axónio e uma célula glial. Neste estudo, descrevemos uma nova axónio e glia do sistema de co-cultura com a utilização de uma plataforma de cultura de microfluidos (MCP). Neste sistema de co-cultura, os neurónios e células gliais são cultivadas em duas câmaras separadas, que estão ligados através de múltiplos canais centrais. Nesta plataforma de cultura de microfluidos, apenas processos neuronais (especialmente axónios) pode entrar no lado glial através dos canais centrais. Em combinação com a marcação de proteínas poderoso fluorescente, este sistema permite a análise directa das vias de sinalização entre as interações axonais / dendrítica e glial, tals regulação axônio mediada transcrição em glia tráfico receptor, mediada por células gliais em terminais neuronais, e glia mediada crescimento do axônio. O diâmetro estreito da câmara também significativamente proíbe o fluxo do meio para neurónios enriquecidos para a câmara de glial, facilitando sondagem da interacção proteína-membrana directa entre axónios / dendritos e superfícies gliais.

Lapso de tempo de análise de imagem de axônio induzida por ativação do promotor GLT1 em astrócitos O neurónio compartimentado e sistema de co-cultura de astrócitos permite que apenas os processos neuronais, particularmente axónios, os seletivamente interagir com os astrócitos. Após o estabelecimento com sucesso de axónio e astrócito (ou outras células da glia) co-cultura na MCP montados, diferentes tipos de interacções axónio-glia pode ser estudado, tais como; axonal induzida po...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Este estudo descreve os procedimentos de criação de um axônio neuronal e romance (astro) glia plataforma de co-cultura. Neste sistema de co-cultura, a manipulação da interacção directa entre um único axónio (e única célula glial) torna-se possível, permitindo a análise mecanicista do neurónio mútuo de sinalização gliais.

Análise de imagens de Neuron Glia para Interação em Plataforma Cultura microfluídicos (MCP) baseado Axon Neuronal e Sistema Glia co-cultura

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

imaging-analysis-neuron-to-glia-interaction-microfluidic-culture

Research

JoVE Journal

Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

14.9K visualizações.  Tufts University. Este estudo descreve os procedimentos de criação de um axônio neuronal e romance (astro) glia plataforma de co-cultura. Neste sistema de co-cultura, a manipulação da interacção directa entre um único axónio (e única célula glial) torna-se possível, permitindo a análise mecanicista do neurónio mútuo de sinalização gliais.

Vídeo: Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Neurociência

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Análise de Desenvolvimento de dente Germ Innervation Utilizar dispositivos microfluídicos Co-cultura

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

A indireta Neuron-astrócitos Coculture Ensaio: An<em&gt; In Vitro</em&gt; Set-up para a investigação detalhada da Relação Neuron-glia

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Isolamento e Cultura dos Neurônios Primários e Glia da Bexiga Urinária de Ratos Adultos

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Lesão Neuronal Direcionada para a Desconexão Não Invasiva do Circuito Cerebral

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissecção e isolamento da glia murina de múltiplas regiões do sistema nervoso central

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Um modelo de cultura celular para estudar o papel das interações neurônio-glia na isquemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imagem de cálcio em rato de azeitona inferior

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Visualizando mudanças no Circuito Neuron-Glia com a Técnica de Imagem de Cálcio

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Transplante de Microglia Pluripotente Pluripotente Induzido Humano em Microglia De camundongos imunocompetentes através da Rota Transnasal Não Invasiva

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...