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 los ratones fueron mantenidos y manejados de acuerdo a la Ley de Protección de los Animales de Suiza y en cumplimiento de la normativa de la oficina cantonal Veterinaria, Zurich. 1. Preparación de Material de disección, Medios de Cultivo, dispositivos de microfluidos <ol><li> Fórceps Autoclave micro-disección y tijeras (121 ° C, tiempo de esterilización: 20 min) y almacenarlos en un recipiente estéril. </li><li> Esterilizar cubreobjetos de vidrio (24 mm x 24 mm) mediante su incubación en HCl 1 M durante 24 h en un agitador orbital a 37 ° C. Lavar tres veces con ellos, H 2 O destilada estéril y tres veces con etanol 99%. Seco entonces los cubreobjetos a 37 ° C o bajo campana de flujo estéril. Por último, en autoclave o exponer los cubreobjetos a la luz UV (30 min) para completar la esterilización. Los cubreobjetos se pueden almacenar entonces en etanol al 70%. </li><li> Retire con cuidado los dispositivos de aislamiento AXIS Axon del paquete usando pinzas estériles y colocarlos en una placa de Petri estéril. </li&gt; <li> Usando un punzón de biopsia estéril (ø: 1 mm) crear un agujero por muestra a ser cultivados (Figura 1) en correspondencia de las cámaras de cultivo. NOTA: No perfore demasiado cerca de los microsurcos, ya que podrían ser dañados por la presión aplicada. </li><li> Esterilizar los dispositivos de aislamiento AXIS Axon sumergiéndola en etanol al 70%. Seque luego Dispositivos de aislamiento AXIS Axon y cubreobjetos completamente bajo una campana de flujo estéril. Espere un mínimo de 3 horas antes de proceder. NOTA: secado incompleto resultará en el montaje defectuoso de los dispositivos de microfluidos. </li><li> Coloque cada cubreobjetos en una placa de Petri de 35 mm o en un pozo dentro de una placa de 6 pocillos. </li><li> Coloque el aislamiento de dispositivos AXIS Axon en el cubreobjetos y presione suavemente pero con firmeza con un fórceps con extremos doblados con el fin de permitir la plena adherencia entre el dispositivo de aislamiento y el cubreobjetos de vidrio. </li><li> En cada cámara de cultivo, pipeta 150 l de poli-D-lisina (0,1 mg / ml en estéril, destilada H 2O). Coloque los dispositivos de microfluidos bajo vacío durante 5 min, a fin de eliminar todo el aire de las cámaras de cultivo. </li><li> Si el aire todavía se puede ver dentro de la cámaras, re-pipeta la solución de poli-D-lisina en las cámaras. </li><li> Incubar los dispositivos con O poli-D-lisina / N a 37 ° C. </li><li> Lávese las cámaras tres veces con estéril, H2O destilada </li><li> Rellene cámaras con 150 l solución laminina trabajo (Sigma-Aldrich, 5 mg / ml, en PBS o medio libre de suero), e incubar O / N a 37 ° C. </li><li> Preparar 50 ml de medio para cultivos de ganglios del trigémino 37, compuesta como sigue: 48 ml Neurobasal medio, 1 ml B-27, 100 U de estreptomicina, 2 mM L-glutamina, 5 ng / ml de factor de penicilina / / ml de crecimiento nervioso (NGF) , arabinósido de citosina 24:25. </li><li> Preparar 50 ml de medio para cultivos germinales diente 37, compuesta como sigue: 40 ml de DMEM-F12, 10 ml de suero fetal bovino (FBS, concentración final: 20%), 100 U / ml de penicilina / estreptomicina, 2 mM L-glutamina, 150 mg / ml de ácido ascórbico. </li></ol> 2. Generación de embriones de ratón y disección <ol><li> Determinar la edad embrionaria acuerdo con tapón vaginal (tapón vaginal: día embrionario de desarrollo 0.5, E0.5) y confirmarla mediante criterios morfológicos. Para este protocolo, por lo general usamos embriones de ratón E14.5-E17.5. </li><li> Limpie el área de disección y el estereoscopio con etanol al 70%. </li><li> Sacrificar la madre embarazada a través de la dislocación cervical. Bloquear el cuello del ratón con el primer y segundo dedo sobre una rejilla y tire con la decisión de la cola. </li><li> Diseccionar la piel alrededor del abdomen inferior, y abrir el abdomen con unas tijeras. Busque el útero: durante tales etapas finales del embarazo, el útero abundantemente llenar la cavidad abdominal. </li><li> Diseccionar el útero y colocar en un tubo lleno de PBS en hielo. Cuando en el hielo, el tejido se puede dejar para varias horas. Deseche el cadáver de la madre según las directrices institucionales </li> &lt;li&gt; diseccionar los embriones del útero y liberarlos de sus tejidos extraembrionarias. Coloca los embriones en PBS en hielo. </li><li> Decapitar a los embriones con unas tijeras, y separar la mandíbula inferior del resto de la cabeza con unas tijeras micro-disección (Figura 2). Eliminar precisamente la mandíbula inferior, sin dañar los ganglios del trigémino; como este último se localizan en las proximidades de la mandíbula inferior, su daño accidental es posible. Preservar la mandíbula inferior y el resto de la cabeza en PBS frío, en hielo. </li><li> Para diseccionar TG, toma la cabeza y colocarla en una placa de Petri de vidrio de la disección, previamente llenado con PBS frío. Usando las pinzas, quitar la piel y el cráneo. Retire entonces el telencéfalo y el cerebelo colocando fórceps debajo del telencéfalo y ascensor; el telencéfalo y el cerebelo le dará la vuelta juntos, dejando la parte inferior del cráneo expuesta. </li><li> Localizar los ganglios del trigémino (que se muestra en la Figura 2B). Utilice las pinzaspara separar el TG de los nervios trigémino. Elimine los restos de las proyecciones del trigémino usando las agujas de disección como cuchillos. Coloque el TG diseccionado en una placa de Petri llena de PBS frío y mantenerlos en hielo. </li><li> Para diseccionar dientes embrionarias, colocar la mandíbula inferior, separado previamente del cráneo, en la placa de Petri de vidrio disección, lleno de PBS frío. El uso de agujas de disección como cuchillos, retire la lengua y la piel que rodea la mandíbula. Separar el los hemi-mandíbulas izquierdo y derecho cortando a lo largo de la línea media de la mandíbula. Los gérmenes de los dientes son fácilmente visibles, como se muestra en la Figura 1C. Aislar los gérmenes de dientes utilizando agujas de disección y retirar el exceso de tejidos no dentales. Coloca los gérmenes de dientes disecados en una placa de Petri llena de PBS frío y mantenerlos en hielo. </li></ol> 3. microfluidos Co-culturas <ol><li> Después de la disección, laminina eliminar de los dispositivos de microfluidos. Llenar las cámaras con 200 l de la respecticinco medios de comunicación. </li><li> Con unas pinzas, transfiera suavemente los TG y diente gérmenes disecados en los agujeros creados por la perforación (Figura 1D). Asegúrese de que los gérmenes de dientes no flotan y que se hunden hasta que entran en contacto con los cubreobjetos. </li><li> Cultura las muestras en incubadora a 37 ° C, 5% de CO 2. </li><li> Cambie el medio de cultivo cada 48 horas. No vaciar las cámaras por completo, y no pipetear directamente en las cámaras de cultivo. El vaciado completo de las cámaras daría lugar a la formación de burbujas de aire dentro de las cámaras; pipeteado directa en la cámara podría resultar en daño axonal. Para evitar estos problemas, se elimina el medio que señala la pipeta hacia el lado externo de los pozos; Del mismo modo, pipetear el medio fresco en el lado de los pozos situados frente a las cámaras. </li><li> Durante el período de cultivo, co-cultivos pueden ser fácilmente imágenes de microscopía de lapso de tiempo. Co-culturas se pueden mantener por más de 10 días. </li><li> Después de que el perio culturad, lavar las cámaras con la pipeta 150 l de PBS en un pocillo por cámara, y dejando PBS fluya a través de las cámaras de tres veces. </li><li> Retire el PBS y fijar las muestras con la pipeta 150 l de paraformaldehído al 4% (en PBS) en un pozo por cada cámara; incubar a TA durante 15 min. </li><li> Lavar las cámaras dos veces con PBS como se describe en 3.6. </li><li> Continúe con su posterior análisis. </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 en estudios in vitro de la inervación de los dientes estaban basados ​​en co-cultivos convencionales de los ganglios del trigémino y los tejidos o células 26,28,29 dentales. Estos estudios se realizaron para investigar principalmente los efectos atractivos de estas células o tejidos en los axones sensoriales 38. Aunque trayendo avances significativos en el campo, se plantearon varias cuestiones técnicas. Gérmenes de dientes empiezan a degenerar después de pocos...

Inervación juega un papel clave en el desarrollo, la homeostasis y la regeneración de órganos y tejidos 1,2. Además, la inervación está implicado en la regulación de la proliferación de células madre, la movilización y diferenciación 3 - 5 de. De hecho, estudios recientes realizados en los tejidos del complejo orofacial han demostrado que los nervios parasimpáticos son necesarios para la función de las células progenitoras epiteliales durante el desarrollo y la regeneración de la glándulas salivales 6,7. Del mismo modo, se ha demostrado que la inervación es necesaria para el desarrollo y mantenimiento de las papilas gustativas 8-11. Por lo tanto, es importante analizar los papeles todavía descuidados de la inervación en el desarrollo de otros órganos y tejidos orofaciales importantes tales como dientes. A pesar de la rica inervación de los dientes adultos, y en contraste con todos los demás órganos y tejidos del cuerpo, Develode ping dientes comienzan a ser inervado en las etapas postnatales tempranas. Los dientes se desarrollan como resultado de las interacciones secuenciales y recíproca entre el ectodermo oral y craneal mesénquima derivadas de la cresta neural. Estas interacciones dan lugar a ameloblastos epiteliales derivadas de mesénquima y odontoblastos derivados que son responsables de la formación del esmalte y la dentina, respectivamente 12. Los nervios sensoriales de los ganglios del trigémino y los nervios simpáticos de los ganglios cervicales superiores inervan los dientes adultos 13-15. Durante la embriogénesis, las fibras nerviosas que emana del proyecto ganglios del trigémino hacia los gérmenes dentales en desarrollo y progresivamente los rodean, pero que no penetran en el mesénquima papila dental 13. Las fibras nerviosas entran en el mesénquima de pasta dental en las etapas de desarrollo más avanzados que se correlacionan con la diferenciación de odontoblastos y la matriz de dentina deposición 16. Inervación pulpa dental es complETED poco después de la erupción del diente en la cavidad oral 13. Estudios anteriores han revelado que varios semaforinas y las neurotrofinas están implicadas en la regulación de la inervación durante odontogenesis 16-19. Estudios anteriores han demostrado claramente que la inervación es un requisito previo para la formación de los dientes en los peces 20. Estudios más recientes han demostrado que la homeostasis de las células madre mesenquimales dental en incisivos ratón está regulada por los nervios sensoriales a través de la secreción de sonic hedgehog (Shh) 21. Sin embargo, el papel de la inervación en diente de iniciación, el desarrollo y la regeneración es todavía muy controvertido en mamíferos 22-24. Una gran cantidad de estudios in vivo han proporcionado información importante acerca de los patrones de inervación de los tejidos dentales durante los procesos de desarrollo y reparación de diversos modelos animales 13,25,26. Sin embargo, la mayoría de estos APPROdolores no son óptimas para resaltar las bases moleculares de las interacciones entre las fibras nerviosas y órganos diana y tejidos. Co-culturas constituyen un valioso método para investigar y manipular las interacciones entre las fibras nerviosas y los dientes en un ambiente controlado y aislado 26-29. Al mismo tiempo, co-cultivo está sujeto a diversos ajustes técnicos. Por ejemplo, los nervios y los tejidos dentales específicos (por ejemplo, la pulpa dental, folículo dental, epitelio dental) a menudo requieren diferentes medios de cultivo con el fin de garantizar la supervivencia del tejido durante largos períodos de tiempo 30-32. En las últimas décadas, se han realizado co-cultivos convencionales utilizando el mismo medio de cultivo durante períodos muy cortos (por ejemplo, dos días) para investigar los efectos de atracción o repulsión de desarrollar tejidos bucales y dentales en las fibras nerviosas sensoriales 27-29.Sin embargo, se requiere la extensión del período de cultivo para investigar los efectos de la inervación de la morfogénesis y dientes citodiferenciación, y para estudiar la dinámica de las fibras nerviosas se ramifican en los órganos diana. Por lo tanto, co-cultivos no contiguos serían más adecuados para llevar a cabo estudios sobre neuronales dental tejidos interacciones. Sistemas de microfluidos permiten co-cultivos de neuronas y diferentes tipos de células en su medio de cultivo apropiado. En estos dispositivos, los tejidos dentales y las neuronas se separan en diferentes compartimentos, mientras que permite el crecimiento de los axones de los cuerpos de las células neuronales a través de microcanales hacia el compartimento que contiene su tejido diana 33. Los dispositivos microfluídicos han sido ya utilizado para estudiar las interacciones entre las neuronas y la microglia 34,35, así como interacciones célula a célula en el cáncer y la neovascularización 35. Además, estos sistemas se han utilizado para estudiar las interacciones entre dorsal ganglios de la raíz y osteoblastos 36. Hemos demostrado recientemente que los ganglios del trigémino (TG) y los dientes son capaces de sobrevivir por largos períodos de tiempo cuando se co-cultivaron en dispositivos de microfluidos 37. Por otra parte, hemos demostrado que los dientes de las diferentes etapas de desarrollo mantienen en estas condiciones in vitro los mismos efectos repulsivas o atractivas en la inervación del trigémino que muestran in vivo 37. Este protocolo proporciona información acerca de una forma sencilla, potente y flexible para co-cultura ganglios / nervios y los tejidos diana y para estudiar los papeles de moléculas específicas en tales interacciones en un ambiente controlado y aislado.

<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

Inervación juega un papel clave en el desarrollo, la homeostasis y la regeneración de órganos y tejidos. Sin embargo, los mecanismos subyacentes a estos fenómenos no son bien entendidos todavía. En particular, se descuida el papel de la inervación en desarrollo de los dientes y la regeneración. Varios estudios in vivo han proporcionado información importante acerca de los patrones de inervación de los tejidos dentales durante los procesos de desarrollo y reparación de diversos modelos animales. Sin embargo, la mayoría de estos enfoques no son óptimas para resaltar las bases moleculares de las interacciones entre las fibras nerviosas y órganos diana y tejidos. Co-culturas constituyen un valioso método para investigar y manipular las interacciones entre las fibras nerviosas y los dientes en un ambiente controlado y aislado. En las últimas décadas, se han realizado co-cultivos convencionales utilizando el mismo medio de cultivo durante períodos muy cortos (por ejemplo, dos días)para investigar los efectos de atracción o repulsión de desarrollar tejidos bucales y dentales en las fibras nerviosas sensoriales. Sin embargo, se requiere la extensión del período de cultivo para investigar los efectos de la inervación de la morfogénesis y dientes citodiferenciación. Sistemas de microfluidos permiten co-cultivos de neuronas y diferentes tipos de células en su medio de cultivo apropiado. Hemos demostrado recientemente que los ganglios del trigémino (TG) y los dientes son capaces de sobrevivir durante un largo período de tiempo cuando se co-cultivaron en dispositivos de microfluidos, y que se mantienen en estas condiciones el mismo patrón de inervación que muestran in vivo. Sobre esta base, se describe cómo aislar y co-desarrollo de la cultura y de los ganglios del trigémino diente gérmenes en el protocolo de un co-cultivo de microfluidos sistema.Este describe una forma simple y flexible para co-cultura ganglios / nervios y los tejidos diana y para estudiar los papeles de moléculas específicas sobre tales interacciones en un controlled y entorno aislado.

Estos resultados muestran que los ganglios del trigémino aislados pueden crecer en un compartimiento del dispositivo de microfluidos y, además, que el desarrollo de los gérmenes de los dientes aislados se mantiene durante un largo período de tiempo en el otro compartimento del dispositivo de microfluidos. Diferentes medios de cultivo se utilizan en los dos compartimentos, y los microsurcos entre los dos compartimentos permitir la extensión de axones desde el ganglio del trigémino hacia los gérmenes dentales en de...

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.

Análisis de Desarrollo del diente Germ Inervación Uso de dispositivos de microfluidos Co-cultura

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

analysis-developing-tooth-germ-innervation-using-microfluidic-co

Research

JoVE Journal

Neuroscience

8.2K vistas.  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.

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Investigación

Neurociencias

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-cultivo de neuronas de rata primaria cortical y la glía

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 ...

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.

Una unidad motora funcional en la placa de cultivo: Co-cultivo de explantes de la médula espinal y las células musculares

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

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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.

Análisis de imágenes de Neuron a la interacción glía en la Plataforma Cultura Microfluidic (MCP)-basada en Axón Neuronal y Sistema Glia Co-cultura

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

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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.

Evaluación funcional y morfológica de Diafragma Inervación por frénico neuronas motoras

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.

Alta resolución volumen imágenes de neuronas por la exclusión de uso de fluorescencia método y dispositivos microfluídicos dedicada

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.

Adquisición y análisis de datos en Brainstem Evocada Audiometría de Respuesta en Ratones

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.

Análisis de Desarrollo del diente Germ Inervación Uso de dispositivos de microfluidos Co-cultura

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