The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

Todos os ratos foram mantidos e tratados de acordo com a Lei de Bem-estar animal suíço e em conformidade com os regulamentos do escritório Cantonal Veterinária, Zurique. 1. Preparação da dissecção Material, Meios de Cultura, microfluídicos <ol><li> Autoclave pinças micro-dissecção e tesouras (121 ° C, o tempo de esterilização: 20 min) e armazená-los em um recipiente estéril. </li><li> Esterilizar lamelas de vidro (24 mm x 24 mm), incubando-as em HCl 1 M durante 24 h num agitador orbital a 37 ° C. Lavar três vezes com estéril, H2O destilada e três vezes com etanol a 99%. A seco seguida as lamelas a 37 ° C ou sob câmara de fluxo estéril. Finalmente, autoclave ou expor as lamelas a luz UV (30 min) para completar a esterilização. As lamelas podem então ser armazenados em etanol a 70%. </li><li> Remova cuidadosamente os dispositivos de isolamento AXIS Axon do pacote usando uma pinça estéril e colocá-los em uma placa de Petri estéril. </li&gt; <li> Usando uma biópsia estéril (o: 1 mm) criar um buraco por amostra a ser cultivadas (Figura 1), em correspondência com as câmaras de cultura. NOTA: Não perfurar demasiado perto das estrias, pois eles podem ser danificados pela pressão aplicada. </li><li> Esterilizar os dispositivos de isolamento AXIS Axon por imergindo-los em etanol a 70%. Seca, então AXIS Axon dispositivos de isolamento e lamelas completamente sob uma capela de fluxo estéril. Espere um mínimo de 3 horas antes de prosseguir. NOTA: secagem incompleta resultará em conjunto com defeito de dispositivos microfluídicos. </li><li> Colocar em cada lamela de 35 mm numa caixa de Petri ou num poço numa placa de 6 poços. </li><li> Coloque o dispositivo de isolamento AXIS Axon na lamela e pressione delicadamente mas com firmeza com uma pinça com extremidades dobradas a fim de permitir a adesão completa entre o dispositivo de isolamento ea lamela de vidro. </li><li> Em cada câmara de cultura, pipeta 150 uL de poli-D-lisina (0,1 mg / ml em estéril, destilada H 2O). Colocar os dispositivos de microfluidos sob vácuo durante 5 minutos, a fim de remover todo o ar das câmaras de cultura. </li><li> Se o ar ainda pode ser visto dentro das câmaras, re-pipeta a solução de poli-D lisina para as câmaras. </li><li> Incubar os dispositivos com poli-D-lisina O / N a 37 ° C. </li><li> Lavar três vezes com câmaras estéreis, H2O destilada </li><li> Encha as câmaras com 150 ul de solução de trabalho laminina (Sigma-Aldrich, 5 ug / ml, em PBS ou meio isento de soro), e incubar O / N a 37 ° C. </li><li> Preparar 50 ml de meio de culturas de gânglios do trigémeo 37, composto como se segue: 48 ml de meio Neurobasal, 1 ml de B-27, 100 U de estreptomicina, 2 mM de L-glutamina, fator / ml de penicilina / 5 ng / mL de crescimento do nervo (NGF) , 12:25 citosina arabinósido. </li><li> Preparar 50 ml de meio de culturas de germe de dente 37, a seguinte composição: 40 ml de DMEM-F12, 10 ml de soro fetal bovino (FBS, concentração final: 20%), 100 U / ml de penicilina / estreptomicina, 2 mM de L-glutamina, 150 ug / ml de ácido ascórbico. </li></ol> 2. mouse Embryo Geração e Dissection <ol><li> Determinar a idade embrionária de acordo com plugue vaginal (plug vaginal: dia embrionária de desenvolvimento 0.5, E0.5) e confirmá-la através de critérios morfológicos. Para este protocolo, geralmente usamos embriões E14.5-E17.5 rato. </li><li> Limpe a área de dissecção eo estereoscópio com etanol 70%. </li><li> Sacrifique a mãe grávida via deslocamento cervical. Bloquear o pescoço do rato com o primeiro e segundo dedo dentro de uma grade, e puxar com a decisão da cauda. </li><li> Dissecar a pele ao redor do abdômen inferior, e abrir o abdômen com uma tesoura. Localize o útero: durante estas fases tardias da gravidez, o útero abundantemente encher a cavidade abdominal. </li><li> Dissecar o útero e coloque em um tubo cheio de PBS em gelo. Quando em gelo, o tecido pode ser deixado para várias horas. Descarte o cadáver da mãe de acordo com as diretrizes institucionais </li> &lt;li&gt; Dissecar os embriões do útero e libertá-los de seus tecidos extra-embrionário. Coloque os embriões em PBS em gelo. </li><li> Decapitar os embriões utilizando uma tesoura, e separar o maxilar inferior do resto da cabeça com uma tesoura de micro-dissecção (Figura 2A). Remover precisamente o maxilar inferior, sem danificar o gânglio trigeminal; em que esta última está localizada na proximidade da mandíbula inferior, o seu dano acidental é possível. Preservar a mandíbula e o resto da cabeça em PBS frio, em gelo. </li><li> Para dissecar TG, pegue a cabeça e colocá-lo em uma placa de Petri de vidro dissecação, anteriormente preenchido com PBS frio. Usando fórceps, remover a pele e o crânio. Remove então o telencéfalo e ao cerebelo, colocando uma pinça abaixo do telencéfalo e elevador; do telencéfalo e ao cerebelo vai virar em conjunto, deixando a parte inferior da cabeça exposta. </li><li> Localizar o gânglio trigeminal (mostrado na Figura 2B). Use a pinçapara separar o TG a partir dos nervos trigeminais. Elimine os restos das projeções do trigêmeo utilizando as agulhas de dissecação como facas. Inserir o TG dissecados numa caixa de Petri cheio com PBS frio e mantê-los em gelo. </li><li> Para dissecar dentes embrionárias, coloque a mandíbula inferior, previamente separado do crânio, sobre o vidro dissecção placa de Petri, cheio de frio PBS. Usando agulhas de dissecação como facas, retirar a língua ea pele ao redor da mandíbula. Separa-se o hemi-mandíbulas esquerdo e direito através do corte ao longo da linha média da mandíbula. Os germes dentários são facilmente visíveis, como mostrado na Figura 1C. Isolar os germes dentários utilizando agulhas de dissecação e remover o excesso de tecidos não dentais. Coloque os germes dentários dissecados em uma placa de Petri preenchido com PBS frio e mantê-los no gelo. </li></ol> 3. microfluídicos Co-culturas <ol><li> Após a dissecção, remova laminina a partir dos dispositivos microfluídicos. Encha as câmaras com 200 ul da respective mídia. </li><li> Com uma pinça, transferir suavemente os TG e dente germes dissecados nos orifícios criados por perfuração (Figura 1D). Certifique-se de que os germes dentários não flutuam e que eles afundam até que entre em contato com as lamelas. </li><li> Cultura as amostras em estufa a 37 ° C, 5% de CO 2. </li><li> Alterar o meio de cultura a cada 48 horas. Não esvaziar as câmaras completamente, e não pipetar diretamente para as câmaras de cultura. O esvaziamento completo de câmaras que resultam na formação de bolhas de ar no interior das câmaras; pipetagem direto para a câmara iria resultar em lesão axonal. Para evitar estes problemas, o meio de remover a pipeta apontando para o lado externo das cavidades; Do mesmo modo, pipetar o meio fresco no lado das cavidades localizadas de frente para as câmaras. </li><li> Durante o período de cultura, co-culturas podem ser facilmente visualizados por microscopia de lapso de tempo. Co-culturas pode ser mantida por mais de 10 dias. </li><li> Após a cultura period, lavar as câmaras por pipetagem de 150 ul de PBS por poço para uma câmara, e permitindo PBS fluir através das câmaras de três vezes. </li><li> Remover o PBS e fixar as amostras por pipetagem de 150 uL de paraformaldeído a 4% (em PBS) por poço em uma câmara; incubar à TA durante 15 min. </li><li> Lavam-se as câmaras duas vezes com PBS como descrito em 3.6. </li><li> Avançar com a análise. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

Anterior estudos in vitro de inervação do dente foram baseados em co-culturas convencionais de gânglios do trigémeo e tecidos dentais ou células 26,28,29. Estes estudos foram conduzidos para investigar principalmente os efeitos atraentes dessas células ou tecidos em axônios sensitivos 38. Apesar de trazer avanços significativos no campo, várias questões técnicas foram levantadas. Germes dentários começar a degenerar após alguns dias de cultura 37. Com base nessas o...

Inervação desempenha um papel chave no desenvolvimento, homeostase e regeneração de órgãos e tecidos 1,2. Além disso, a inervação está envolvido na regulação da proliferação de células estaminais, diferenciação e mobilização 3-5. Com efeito, estudos recentes realizados em tecidos do complexo orofacial têm mostrado que os nervos parassimpáticos são necessários para a função de células progenitoras epiteliais durante o desenvolvimento e regeneração das glândulas salivares 6,7. Da mesma forma, foi demonstrado que a inervação é necessário para o desenvolvimento e manutenção de paladar 8-11. Assim, é importante analisar as funções ainda negligenciadas de inervação no desenvolvimento de outros órgãos e tecidos orofaciais importantes, tais como os dentes. Apesar da rica inervação dos dentes adultos, e em contraste com todos os outros órgãos e tecidos do corpo, desendentes de ping começar a ser inervados nas primeiras fases pós-natais. Os dentes se desenvolvem como resultado de interações seqüenciais e recíprocas entre o ectoderma oral e cranial mesênquima derivado da crista neural. Estas interacções dar origem ao derivado ameloblastos-epiteliais e mesenquimais derivadas de odontoblastos que são responsáveis ​​pela formação do esmalte e dentina, respectivamente 12. Nervos sensoriais do gânglio trigeminal e nervos simpáticos do gânglio cervical superior inervam os dentes adulto 13-15. Durante a embriogênese, fibras nervosas que emana do projeto gânglios trigeminal para os germes dentários em desenvolvimento e, progressivamente, cercá-los, mas eles não penetram no mesênquima papila dentária 13. As fibras nervosas entrar no mesênquima polpa dental em estágios mais avançados de desenvolvimento que se correlacionam com diferenciação de odontoblastos e matriz dentinária deposição 16. Inervação polpa dentária é complETED logo após a erupção dos dentes na cavidade oral 13. Estudos anteriores revelaram que vários semaforinas e neurotrofinas estão envolvidos na regulação da inervação durante odontogenesis 16-19. Estudos anteriores demonstraram claramente que a inervação é um pré-requisito para a formação dos dentes em peixes 20. Estudos mais recentes têm mostrado que a homeostase das células-tronco mesenquimais dentária em incisivos de rato é regulada por nervos sensoriais através da secreção de sonic hedgehog (Shh) 21. No entanto, o papel da inervação em dente de iniciação, desenvolvimento e regeneração ainda é altamente controversa em mamíferos 22-24. Uma infinidade de estudos in vivo têm fornecido informações importantes sobre os padrões de inervação dos tecidos dentários durante os processos de vários modelos animais 13,25,26 desenvolvimento e reparação. No entanto, a maioria destes Approdores não são ideais para destacar a base molecular das interacções entre as fibras nervosas e os órgãos e tecidos alvo. Co-culturas constituem um método valioso para estudar e manipular as interacções entre as fibras nervosas e os dentes num ambiente controlado e isolado 26-29. Ao mesmo tempo, a co-cultura é sujeita a várias adaptações técnicas. Por exemplo, nervos e tecidos dentais específicos (por exemplo, polpa dentária, folículo dental, epitélio dental) muitas vezes requerem diferentes meios de cultura, a fim de garantir a sobrevivência do tecido por longos períodos de tempo de 30-32. Nas últimas décadas, co-culturas convencionais, utilizando o mesmo meio de cultura foram efectuadas por períodos muito curtos (por exemplo, dois dias) para investigar os efeitos atractivas ou repulsivas de desenvolvimento de tecidos orais e dentais em fibras nervosas sensoriais 27-29.No entanto, a extensão do período de cultura é necessária para investigar os efeitos da inervação na morfogénese e citodiferenciação dente, e para estudar a dinâmica de fibras nervosas de ramificação dentro de órgãos alvo. Portanto, não contíguas co-culturas seria mais apropriada para realizar estudos sobre as interacções de tecidos neuronais-dentário. Sistemas microfluidicos permitir co-culturas de neurónios e diferentes tipos de células no seu meio de cultura apropriado. Nestes dispositivos, os tecidos dentais e neurónios são separados em diferentes compartimentos, permitindo que o crescimento dos axónios dos corpos celulares neuronais através de microcanais para o compartimento que contém o seu tecido alvo 33. Microcanais têm já sido usadas para estudar as interacções entre neurónios e microglia 34,35, bem como de células para as interacções das células no cancro e neovascularização 35. Além disso, estes sistemas têm sido usados ​​para estudar as interacções entre dorsal gânglios da raiz e osteoblastos 36. Nós demonstramos recentemente que gânglios trigeminais (TG) e os dentes são capazes de sobreviver durante longos períodos de tempo quando co-cultivadas em microcanais 37. Além disso, demonstrámos que os dentes de diferentes estádios de desenvolvimento em manter estas condições in vitro, os mesmos efeitos repulsivos ou atractivos da inervação do trigémio que mostram in vivo 37. Este protocolo fornece informações sobre uma maneira simples, poderosa e flexível para co-cultura gânglios / nervos e tecidos-alvo e para estudar os papéis de moléculas específicas sobre tais interações em um ambiente controlado e isolado.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AXIS Axon Isolation Devices</td>
			<td>Millipore</td>
			<td>AX15010-TC</td>
			<td>Microchannels of different lenght are available</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse beta-NGF</td>
			<td>R&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;D Systems) are equivalent</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

Inervação desempenha um papel chave no desenvolvimento, homeostase e regeneração de órgãos e tecidos. No entanto, os mecanismos subjacentes a esses fenômenos não são bem compreendidas ainda. Em particular, o papel de inervação no desenvolvimento e regeneração de dente é negligenciado. Vários estudos in vivo têm fornecido informações importantes sobre os padrões de inervação dos tecidos dentários durante os processos de vários modelos animais de desenvolvimento e reparação. No entanto, a maioria destas abordagens não são ideais para destacar a base molecular das interacções entre as fibras nervosas e os órgãos e tecidos alvo. Co-culturas constituem um método valioso para estudar e manipular as interacções entre as fibras nervosas e os dentes num ambiente controlado e isolado. Nas últimas décadas, co-culturas convencionais, utilizando o mesmo meio de cultura foram efectuadas por períodos muito curtos (por exemplo, dois dias)investigar os efeitos atractivas ou repulsivas de desenvolvimento de tecidos orais e dentais em fibras nervosas sensoriais. No entanto, a extensão do período de cultura é necessária para investigar os efeitos da inervação sobre a morfogénese do dente e citodiferenciação. Sistemas microfluidicos permitir co-culturas de neurónios e diferentes tipos de células no seu meio de cultura apropriado. Nós demonstramos recentemente que gânglios trigeminais (TG) e os dentes são capazes de sobreviver durante um longo período de tempo quando co-cultivadas em microcanais, e que eles se mantêm em estas condições o mesmo padrão de inervação que mostram in vivo. Nesta base, nós descrevemos como isolar e co-cultura em desenvolvimento e dos gânglios trigêmeo germes dentários em um protocolo de co-cultura microfluídicos system.This descreve uma forma simples e flexível para co-cultura gânglios / nervos e tecidos-alvo e para estudar os papéis moléculas de específicas sobre estas interacções em um controlled e ambiente isolado.

Estes resultados mostram que os gânglios do trigémeo isoladas podem crescer num compartimento do dispositivo de microfluidos e, além disso, que o desenvolvimento das bactérias isoladas de dentes é mantida por um longo período de tempo no outro compartimento do dispositivo de microfluidos. Diferentes meios de cultura são usados ​​nos dois compartimentos, e as micro-ranhuras entre os dois compartimentos de permitir a extensão do axónio do gânglio trigeminal no sentido de os germes de dentes em desenvolviment...

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

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

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 ...

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Neuroscience

8.2K visualizações.  University of Zurich. 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.

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

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

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

A Unidade Funcional Motor na placa de cultura: Co-cultivo de explantes da Medula Espinhal e as células musculares

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

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

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

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

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

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 ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funcional e morfológica de Avaliação de Diafragma Innervation por frênico Neurônios Motores

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

High-resolution Volume Imaging of Neurons by the Use of Fluorescence eXclusion Method and Dedicated Microfluidic Devices

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).

Volume is an important parameter regarding physiological and pathological characteristics of cells. We describe a fluorescent exclusion method allowing full-field measurement of in vitro neuronal volume with sub-micrometric axial resolution required for the analysis of neurites and dynamic structures implied in neuronal growth.

De alta resolução Volume Imaging de neurônios pela exclusão da utilização da fluorescência método e dedicado Microfluidic dispositivos

Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Aquisição e análise de dados na audiometria de resposta evocada de tronco encefálico em camundongos

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Migração, Quimio-Atração e Ensaios de Cocultura para Células Endoteliais derivadas de células-tronco humanas e neurônios gabaergicos

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

Geração de Oligodendrocytes e Oligodendrocyte-Conditioned Medium para Experimentos de Cocultura

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Transporte Axonal de Organelas em Culturas de Neurônios Motores usando sistema de câmaras microfluidas

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

Visualizando a Interiorização Imunoreativa Do Peptídeo Relacionado ao Gene calcitonina do Rato Cranial Dura Mater com Imunofluorescência e Rastreamento Neural

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the ...