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.

Toutes les souris ont été maintenues et gérées selon la loi sur la protection des animaux en Suisse et en conformité avec les règlements de l&#39;office vétérinaire cantonal, Zurich. 1. Préparation de dissection Matériau, Culture Media, dispositifs microfluidiques <ol><li> Forceps autoclave micro-dissection et ciseaux (121 ° C, le temps de stérilisation: 20 min) et de les stocker dans un récipient stérile. </li><li> Stériliser lamelles de verre (24 mm x 24 mm) en les incubant dans HCl 1 M pendant 24 heures sur un agitateur orbital à 37 ° C. Laver trois fois avec eux stérile, H 2 O distillée et trois fois avec de l&#39;éthanol à 99%. Sec, puis les lamelles à 37 ° C ou sous une hotte à flux stérile. Enfin, l&#39;autoclave ou exposer les lamelles à la lumière UV (30 min) pour compléter la stérilisation. Lamelles peuvent ensuite être stockés dans de l&#39;éthanol 70%. </li><li> Retirer soigneusement l&#39;isolement périphériques AXIS Axon du package à l&#39;aide de pinces stériles et les placer dans une boîte de Pétri stérile. </li&gt; <li> En utilisant une biopsie poinçon stérile (ø: 1mm) créer un trou par échantillon pour être cultivées (figure 1) en correspondance des chambres de culture. NOTE: Ne perforer trop près des microsillons, car ils pourraient être endommagés par la pression appliquée. </li><li> Stériliser les Dispositifs d&#39;isolement AXIS Axon en les immergeant dans de l&#39;éthanol 70%. Sèches puis AXIS Axon Dispositifs d&#39;isolement et lamelles complètement sous une hotte à flux stérile. Attendre un minimum de 3 heures avant de procéder. NOTE: séchage incomplet entraînera l&#39;assemblage défectueux des dispositifs microfluidiques. </li><li> Placez chaque lamelle dans une boîte de Pétri de 35 mm ou dans un puits dans une plaque à 6 puits. </li><li> Placez le périphérique Isolation AXIS Axon sur la lamelle et appuyez doucement mais fermement avec une pince à bouts recourbés afin de permettre la pleine adhésion entre le dispositif d&#39;isolement et de la lamelle de verre. </li><li> Dans chaque chambre de culture, H 2 pipette 150 ul de poly-D-lysine (0,1 mg / ml dans stérile distillée,O). Placez les dispositifs microfluidiques sous vide pendant 5 minutes, afin d&#39;éliminer tout l&#39;air des chambres de culture. </li><li> Si de l&#39;air peut encore être vu dans les chambres, re-pipette la solution de poly-D-lysine dans les chambres. </li><li> Incuber les appareils avec O poly-D-lysine / N à 37 ° C. </li><li> Laver les chambres à trois temps avec stérile, H 2 O. distillée </li><li> Remplissez chambres avec 150 ul solution de travail laminine (Sigma-Aldrich, 5 ug / ml, dans du PBS ou un milieu sans sérum), et incuber O / N à 37 ° C. </li><li> Préparer 50 ml de milieu pour les cultures de ganglions de Gasser 37, composé comme suit: 48 ml Neurobasal moyenne, 1 ml B-27, 100 U / ml de pénicilline / streptomycine, 2 mM de L-glutamine, 5 ng / ml de facteur de croissance des nerfs (NGF) , 12:25 cytosine arabinoside. </li><li> Préparer 50 ml de milieu pour les cultures dent germinales 37, composé comme suit: 40 ml de DMEM-F12, 10 ml de sérum fœtal bovin (FBS, concentration finale: 20%), 100 U / ml de pénicilline / streptomycine, 2 mM de L-glutamine, 150 ug / ml d&#39;acide ascorbique. </li></ol> 2. Génération d&#39;embryon de souris et Dissection <ol><li> Déterminer l&#39;âge embryonnaire selon bouchon vaginal (bouchon vaginal: jour de développement embryonnaire 0,5, 0,5 ME) et le confirmer par l&#39;intermédiaire des critères morphologiques. Pour ce protocole, nous utilisons généralement des embryons de souris E14.5-E17.5. </li><li> Nettoyer la zone de dissection et le stéréoscope avec de l&#39;éthanol 70%. </li><li> Sacrifiez la mère enceinte par dislocation cervicale. Bloquer le col de la souris avec la première et la deuxième doigt sur une grille, et tirez avec la décision de la queue. </li><li> Disséquer la peau autour de l&#39;abdomen inférieur, et d&#39;ouvrir l&#39;abdomen avec des ciseaux. Situer l&#39;utérus: au cours de ces derniers stades de la grossesse, l&#39;utérus remplir abondamment la cavité abdominale. </li><li> Disséquer l&#39;utérus et le placer dans un tube rempli de PBS sur la glace. Lorsque sur la glace, le tissu peut être laissé pour plusieurs heures. Jeter le cadavre de la mère selon les directives institutionnelles </li> &lt;li&gt; disséquer les embryons de l&#39;utérus et de les libérer de leurs tissus extra-embryonnaires. Placez les embryons dans PBS sur la glace. </li><li> Décapiter les embryons avec des ciseaux, et séparer la mâchoire inférieure du reste de la tête avec des ciseaux micro-dissection (figure 2A). Retirer précisément la mâchoire inférieure, sans endommager les ganglions de Gasser; comme celles-ci sont localisées à proximité immédiate de la mâchoire inférieure, leur endommagement accidentel est possible. Préserver la mâchoire inférieure et le reste de la tête dans du PBS froid, sur la glace. </li><li> Pour disséquer TG, prendre la tête et placez-le sur une boîte de Pétri en verre de dissection, préalablement rempli avec du PBS froid. Utilisation de la pince, enlever la peau et le crâne. Retirer ensuite le télencéphale et le cervelet en plaçant une pince ci-dessous le télencéphale et un ascenseur; le télencéphale et au cervelet se retourner, en laissant la partie inférieure du crâne exposé. </li><li> Localiser le ganglion trijumeau (représenté sur la figure 2B). Utilisez la pincepour séparer la TG des nerfs trijumeau. Éliminer les restes de les projections du trijumeau utilisant les aiguilles de dissection comme couteaux. Placez le TG disséqué dans une boîte de Pétri remplie de PBS froid et de les garder sur la glace. </li><li> Pour disséquer dents embryonnaires, placer la mâchoire inférieure, préalablement séparée du crâne, sur le verre de dissection boîte de Pétri, rempli avec du PBS froid. En utilisant des aiguilles de dissection comme couteaux, retirez la langue et la peau entourant la mâchoire. Séparer l&#39;hémi-mâchoires gauche et droite en coupant le long de la ligne médiane de la mâchoire. Les germes dentaires sont facilement accessibles, comme le montre la figure 1C. Isoler les germes dentaires en utilisant des aiguilles de dissection et éliminer l&#39;excès de tissus non-dentaires. Placez les germes dentaires disséqués dans une boîte de Pétri remplie de PBS froid et de les garder sur la glace. </li></ol> 3. microfluidiques Co-cultures <ol><li> Après dissection, retirez la laminine des dispositifs microfluidiques. Remplir les chambres avec 200 ul de la respective médias. </li><li> Avec des pinces, transférer doucement les germes TG et dents disséqués dans les trous créés par poinçonnage (figure 1D). Assurez-vous que les germes dentaires ne flottent pas et qu&#39;ils coulent jusqu&#39;à ce qu&#39;ils touchent les lamelles. </li><li> Culture des échantillons dans l&#39;incubateur à 37 ° C, 5% CO 2. </li><li> Changer le milieu de culture toutes les 48 heures. Ne pas vider les chambres complètement, et ne pas pipeter directement dans les chambres de culture. Vidange complète de chambres se traduirait par la formation de bulles d&#39;air à l&#39;intérieur des chambres; pipetage directe dans la chambre de causer des dommages axonale. Pour éviter ces problèmes, éliminer le milieu pointant la pipette vers le côté externe des puits; de même, la pipette sur du milieu frais du côté des puits situés à l&#39;opposé des chambres. </li><li> Pendant la période de culture, co-cultures peuvent être facilement visualisés par microscopie time-lapse. Co-cultures peuvent être maintenues pendant plus de 10 jours. </li><li> Après la pério de cultured, laver les chambres en pipetant 150 pi de PBS dans un puits par chambre, et en laissant PBS circuler à travers les chambres à trois reprises. </li><li> Retirez le PBS et fixer les échantillons par pipetage 150 pi de paraformaldéhyde 4% (dans du PBS) dans un puits par chambre; incuber à température ambiante pendant 15 min. </li><li> Laver les chambres deux fois avec PBS comme décrit dans 3.6. </li><li> Procéder à une analyse plus approfondie. </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.

Précédente dans des études in vitro de la dent innervation étaient basées sur des co-cultures conventionnelles de ganglions de Gasser et les tissus dentaires ou des cellules 26,28,29. Ces études ont été menées pour étudier principalement les effets attractifs de ces cellules ou de tissus sur axones sensoriels 38. Bien amener des avancées significatives dans le domaine, plusieurs questions techniques ont été soulevées. germes de dents commencent à dégénérer après...

Innervation joue un rôle clé dans le développement, l&#39;homéostasie et la régénération des organes et des tissus 1,2. En outre, l&#39;innervation est impliquée dans la régulation de la prolifération des cellules souches, la mobilisation et la différenciation 3-5. En effet, des études récentes réalisées dans les tissus du complexe orofaciale ont montré que les nerfs parasympathiques sont nécessaires pour la fonction épithéliale de cellules souches au cours du développement et de la régénération glandes salivaires 6,7. De même, il a été démontré que l&#39;innervation est nécessaire pour le développement et l&#39;entretien des papilles 8-11. Ainsi, il est important d&#39;analyser le rôle de l&#39;innervation encore négligées dans le développement d&#39;autres organes et tissus oro-faciales importants tels que des dents. En dépit de la richesse de l&#39;innervation dents d&#39;adulte, et contrairement à tous les autres organes et tissus du corps, developing dents commencent à être innervée au stade de la post-natales premiers. Les dents se développent en raison des interactions séquentielles et réciproques entre l&#39;ectoderme oral et crânienne neuronal mésenchyme de la crête dérivés. Ces interactions donnent lieu à adamantoblastes épithéliales dérivées et les odontoblastes mésenchymateuses dérivées qui sont responsables de la formation de l&#39;émail et de la dentine, 12 respectivement. Les nerfs sensoriels du ganglion de Gasser et les nerfs sympathiques du ganglion cervical supérieur innervent les dents d&#39;adultes de 13 à 15. Au cours de l&#39;embryogenèse, les fibres nerveuses émanant du projet de ganglions de Gasser vers les germes dentaires développement et progressivement entourer eux, mais ils ne pénètrent pas dans la papille mésenchyme dentaire 13. Les fibres nerveuses pénètrent dans la pulpe dentaire mésenchyme à des stades de développement plus avancés qui sont en corrélation avec la différenciation des odontoblastes et la matrice de la dentine dépôt 16. Pulpe dentaire innervation est complETED peu de temps après l&#39;éruption des dents dans la cavité buccale 13. Des études antérieures ont révélé que divers sémaphorines et neurotrophines sont impliqués dans la régulation de l&#39;innervation cours odontogenèse 16-19. Des études antérieures ont clairement démontré que l&#39;innervation est une condition préalable à la formation des dents chez les poissons 20. Des études plus récentes ont montré que l&#39;homéostasie des cellules souches mésenchymateuses dentaire dans incisives de souris est réglementée par les nerfs sensoriels via la sécrétion de sonic hedgehog (Shh) 21. Néanmoins, le rôle de l&#39;innervation de la dent initiation, le développement et la régénération est encore très controversée chez les mammifères 22 - 24. Une pléthore d&#39;études in vivo ont fourni des informations importantes sur les motifs de l&#39;innervation des tissus dentaires au cours des processus de développement et de réparation de divers modèles animaux 13,25,26. Cependant, la plupart de ces Approdes maux ne sont pas optimales pour mettre en évidence la base moléculaire des interactions entre les fibres nerveuses et les organes et tissus cibles. Co-cultures constituent une méthode utile pour étudier et manipuler les interactions entre les fibres nerveuses et des dents dans un environnement contrôlé et isolé de 26 à 29. Dans le même temps, la co-culture est soumise à diverses adaptations techniques. Par exemple, les nerfs et les tissus dentaires spécifiques (par exemple, la pulpe dentaire, follicule dentaire, l&#39;épithélium dentaire) nécessitent souvent différents milieux de culture afin de garantir la survie des tissus pendant de longues périodes de temps 30-32. Dans les dernières décennies, les co-cultures conventionnelles utilisant le même milieu de culture ont été effectués pour des périodes très courtes (par exemple, deux jours) pour étudier les effets attractives ou répulsives de développer des tissus buccaux et dentaires sur les fibres nerveuses sensorielles 27-29.Cependant, l&#39;extension de la période de culture est nécessaire pour étudier les effets de l&#39;innervation sur la morphogenèse de la dent et cytodifférenciation, et d&#39;étudier la dynamique de fibres nerveuses de branchement dans les organes cibles. Par conséquent, les co-cultures non-contigus serait plus approprié de réaliser des études sur des tissus interactions neuronales-dentaire. systèmes microfluidiques permettent des co-cultures de neurones et de différents types de cellules dans leur milieu de culture approprié. Dans ces dispositifs, les tissus dentaires et les neurones sont séparés dans des compartiments différents, tout en permettant la croissance des axones dans les corps cellulaires neuronaux à travers des microcanaux vers le compartiment contenant leur tissu cible 33. Les dispositifs microfluidiques ont déjà été utilisées pour étudier les interactions entre les neurones et les microglies 34,35, ainsi que des cellules à des interactions cellulaires dans le cancer et la néovascularisation 35. En outre, ces systèmes ont été utilisés pour étudier les interactions entre dorsal ganglions de la racine et les ostéoblastes 36. Nous avons démontré récemment que les ganglions de Gasser (TG) et les dents sont capables de survivre pendant de longues périodes de temps en cas de co-cultivées dans des dispositifs microfluidiques 37. De plus, nous avons démontré que les dents de différents stades de développement de maintenir dans ces conditions in vitro les mêmes effets répulsifs ou attractifs sur innervation trijumeau qu&#39;ils montrent in vivo 37. Ce protocole fournit des informations sur un moyen simple, puissant et flexible de co-culture ganglions / nerfs et les tissus cibles et d&#39;étudier les rôles des molécules spécifiques sur ces interactions dans un environnement contrôlé et isolé.

<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

Innervation joue un rôle clé dans le développement, l&#39;homéostasie et la régénération des organes et des tissus. Cependant, les mécanismes sous-jacents de ces phénomènes ne sont pas encore bien compris. En particulier, le rôle dans le développement de l&#39;innervation de la dent et la régénération est négligée. Plusieurs études in vivo ont fourni des informations importantes sur les motifs de l&#39;innervation des tissus dentaires au cours des processus de développement et de réparation de divers modèles animaux. Cependant, la plupart de ces approches ne sont pas optimales pour mettre en évidence la base moléculaire des interactions entre les fibres nerveuses et les organes et tissus cibles. Co-cultures constituent une méthode utile pour étudier et manipuler les interactions entre les fibres nerveuses et des dents dans un environnement contrôlé et isolé. Au cours des dernières décennies, les co-cultures classiques en utilisant le même milieu de culture ont été effectués pendant des périodes très courtes (par exemple, deux jours)pour étudier les effets attractives ou répulsives de développer des tissus buccaux et dentaires sur les fibres nerveuses sensorielles. Cependant, l&#39;extension de la période de culture est nécessaire pour étudier les effets de l&#39;innervation sur la morphogenèse de la dent et cytodifférenciation. systèmes microfluidiques permettent des co-cultures de neurones et de différents types de cellules dans leur milieu de culture approprié. Nous avons montré récemment que les ganglions trigéminés (TG) et les dents sont en mesure de survivre pendant une longue période de temps lorsqu&#39;ils sont co-cultivées dans des dispositifs microfluidiques, et qu&#39;ils maintiennent dans ces conditions, le même schéma d&#39;innervation ils montrent que in vivo. Sur cette base, nous décrivons comment isoler et de co-culture et le développement de ganglions dents germes trijumeau dans le protocole de co-culture microfluidique décrit un moyen simple et flexible pour la co-culture ganglions / nerfs et les tissus cibles et d&#39;étudier les rôles de molécules spécifiques sur ces interactions dans un controlled et environnement isolé.

Ces résultats montrent que les ganglions trigéminés isolés peuvent se développer dans un compartiment du dispositif microfluidique et, en outre, que le développement des germes dentaires isolées est maintenue pendant une longue période de temps dans l&#39;autre compartiment du dispositif microfluidique. Des milieux de culture différents sont utilisés dans les deux compartiments, et les micro-rainures entre les deux compartiments permettre l&#39;extension de l&#39;axone du ganglion trijumeau vers les germes den...

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.

Analyse de développement germe dentaire innervation Utilisation de périphériques Co-culture microfluidique

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

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Neuroscience

8.2K vues.  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.

Vidéo: 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.

Didermique co-culture de neurones de rat corticale primaire et des cellules gliales

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 ...

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Neurosciences

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.

Une unité fonctionnelle du moteur dans la boîte de culture: La co-culture d&#39;explants de la moelle épinière et les cellules musculaires

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.

Analyse en Imagerie du Neuron à l&#39;interaction Glia dans la plate-forme microfluidique Culture (MCP) à base neuronale et Axon Glia co-culture système

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.

Évaluation fonctionnelle et morphologique de membrane innervation par phrénique motoneurones

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.

Haute résolution Volume Imaging de neurones par l’eXclusion de l’utilisation de la Fluorescence méthode et dédié des dispositifs microfluidiques

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.

Acquisition de données et analyse dans 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 ...

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.

Migrations, Chemo-Attraction et Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons (en anglais)

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.

Génération d’Oligodendrocytes et de médium conditionné par oligodendrocyte s’est conditionné pour les expériences de co-culture

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.

Transport axonal des Organlles dans les cultures de neurones moteurs à l’aide du système de chambres microfluidiques

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.

Visualisation de l’innervation immunoréactive du peptide lié au gène de la calcitonine du dura mater crânien de rat avec immunofluorescence et traçage neuronal

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 ...