Nous tenons à remercier le Dr Jeffrey Rothstein de fournir BAC GLT1 souris eGFP et GLT1 anticorps; Tufts Center for Neuroscience Research (NIH P30 NS047243; PI, Rob Jackson) pour fournir des installations de base précieuses; New recrutement de professeurs de subvention (NIH P30-02 5P30NS069254 , PI, Phil Haydon) à Tufts Neuroscience Department.

1. Assemblée de la Chambre Culture microfluidique (MCP) <ol><li> MCP (figure 1) sont conçus pour les chambres ouvertes cultures compartimentées de différents types de cellules 4. Il a généralement deux compartiments qui sont reliés par les canaux centraux (3 m de diamètre). Assemblée des MCP avec fond de verre plats est nécessaire pour la préparation de cultures et d&#39;analyse d&#39;images ultérieur. </li><li> Tout d&#39;abord, manteau stériles à fond de verre plats de polyornithine (Sigma-Aldrich, 1 mg / ml) dissous dans le tétraborate de sodium (Sigma-Aldrich, 10 mM, pH 8,5) et on a incubé une nuit à 37 ° C. </li><li> Le lendemain, les enduits à fond de verre plats ont été lavés trois fois avec ddH 2 O et séché sous la hotte stérile fumées. Les plats à fond de verre ont ensuite été en outre revêtues de laminine (Sigma-Aldrich, 1 mg / ml) et séché sous lumière UV pendant 1 heure. Plats couchés sont prêts à l&#39;emploi ou peut être conservé à -20 ° C jusqu&#39;à utilisation. Ce revêtement d&#39;unre nécessaire pour les neurones et les astrocytes de placage dans la co-culture. </li><li> À assembler la plate-forme de la culture, les plates-formes de culture microfluidiques ont été placés au-dessus des enduits à fond de verre plats centraux avec des canaux de liaison sur son côté inférieur pour former un joint étanche. Neurone régulière ou milieux de culture d&#39;astrocytes ont été ajoutées (des zones de placage de cellules) sur les deux côtés (figure 1) dans le MCP assemblé et mis à incuber pendant 2-4 heures à 37 ° C, pour s&#39;assurer qu&#39;il n&#39;ya pas de fuite entre le MCP et le verre Plat à fond. Nous avons testé avec support avant étalement des cellules pour s&#39;assurer qu&#39;il n&#39;y ait pas de fuite. L&#39;assemblage de MCP sur le plat à fond de verre doit être fraîchement préparée avant utilisation. </li></ol> 2. Préparation de la culture neuronale et induction de axones neuronaux en assemblée MCP <ol><li> Cultures de cellules neuronales corticales ont été fraîchement préparés à partir de cerveaux de souris embryonnaires 14-16 jours d&#39;âge. Le milieu de culture neurone est composé du milieu neurobasal, 2% B27 neurobasaSupplément l, 2 mM de glutamine en ajoutant 1% de Glutamax 100x, et 1% de pénicilline-streptomysin. Après dissection du cerveau de la souris, les méninges a été retiré du cortex sous la loupe binoculaire. Le tissu est ensuite haché avec une lame de rasoir pour faire les petits blocs de tissus en vue d&#39;augmenter l&#39;aire de surface exposée à la trypsine. Blocs de tissus ont été traitées à la trypsine (Sigma-Aldrich, la trypsine 0,05%) pendant 10 min dans un bain à 37 ° C de l&#39;eau, puis dissociées par trituration doucement avec une pipette Pasteur polie au feu. Les cellules dissociées sont filtrées à travers un filtre 70 microns pour recueillir clair suspension cellulaire neuronale. </li><li> Neurones (2-3 x 10 6 / ml, 150 pi) ont été étalées sur les zones de placage de cellules sur le côté droit (figure 1) de la MCP assemblés de sorte que les neurones peuvent joindre dans la zone de rétention cellulaire (Figure 1). Neurones fraîchement préparés ont été étalées avec un milieu de placage neurone qui est supplémenté avec 5% de sérum fœtal bovin (Hyclone) dans la réglementationmilieu de culture neurone r. Parce que seuls les neurones qui se fixent dans la zone de rétention des cellules sont capables de croître axones vers l&#39;autre côté de l&#39;assemblée MCP par les voies centrales, il est important de densité élevée plaque de neurones dans la zone de placage cellulaire pour assurer suffisamment de neurones sont attachés à la cellule de zone de rétention. Une image représentative de la densité élevée des axones neuronaux est représenté sur la figure 2A. </li><li> Le jour suivant, le milieu d&#39;étalement neurone a été remplacé par du milieu de culture neurone régulière. Milieu changeant a été soigneusement réalisé par aspiration du milieu de la zone de placage cellule de la chambre, mais pas de la zone de retenue de cellules de la MCP assemblés, afin d&#39;éviter les bulles d&#39;air dans la zone de retenue de cellules et les canaux centraux de liaison. Bulles d&#39;air fortement inhiber la croissance axonale et l&#39;entrée subséquente dans les canaux centraux de la MCP. Cellules gliales facteur neurotrophique dérivé de cellules (GDNF, 10-20 ng / ml) a été ajoutée à l&#39;autre (gauche)de la chambre le même jour pour faciliter l&#39;induction de la croissance axonale 5 de la partie neuronale et traverser les canaux centraux de l&#39;assemblée MCP (figure 2A). Mal de la croissance des neurites spontanée sans GDNF est également observé de côté neuronale et transversale à travers les canaux centraux de la MCP assemblé. Les axones qui entrent dans le canal central sont souvent observées 2-3 jours après placage. Une fois que les axones entrer dans le canal central, ils entrent habituellement dans l&#39;autre en une journée. Les axones sont normalement intactes pendant au moins une semaine après la saisie de l&#39;autre côté. </li></ol> 3. Outre une culture d&#39;astrocytes à MCP pour établir une co-culture compartimentée système Cultures primaires d&#39;astrocytes ont été préparés à partir du cerveau de la souris P1-3 chiot. La procédure de dissociation cerveau est similaire à la procédure d&#39;isolement de cellules neuronales décrit ci-dessus. Les astrocytes ont d&#39;abord été étalées dans des boîte de 10 cm qui ont été préRevêtu de polyornithine (Sigma-Aldrich, 1 mg / ml). Le milieu de culture des astrocytes (DMEM, 10% de sérum de veau fœtal, 1% de pénicilline-streptomysin) a été changé tous les jours pendant les deux prochains jours pour enlever les débris. Après cela, le milieu a été changé tous les trois jours. <ol><li> Astrocytes deviennent confluentes et 90% d&#39;une monocouche après 7 jours. Astrocytes confluentes ont été traitées à la trypsine et 150 ul de re-suspension des astrocytes (1x10 6 / ml) ont été ré-étalées dans la zone de placage cellule du côté gauche de l&#39;assemblée GPE lorsque les axones sont sur ​​le point d&#39;entrer ou venez d&#39;entrer dans le côté gauche de l&#39;assemblée MCP. Les astrocytes sont généralement plaquées 4-5 jours après que les neurones étaient plaqués. GDNF a été éliminée avant la première ré-plaquage des astrocytes dans le côté gauche de la MCP. Re-plaqué astrocytes ont été fixés dans le domaine de la conservation de cellules, comme le montre l&#39;immunomarquage GFAP (Figure 1). Seulement un débit minimal de fluide entre les deux côtés des chambres (déterminée par fluorescence doui) a été trouvé dans le MCP, comme indiqué précédemment 4,6. </li><li> Astrocytes deviennent confluentes et 90% d&#39;une monocouche après 7 jours. Astrocytes confluentes ont été traitées à la trypsine et 150 ul de re-suspension des astrocytes (1 x 10 6 / ml) ont été ré-étalées dans la zone de placage cellule du côté gauche de l&#39;assemblée GPE lorsque les axones sont sur ​​le point d&#39;entrer ou venez d&#39;entrer dans la gauche côté de l&#39;assemblée MCP. Les astrocytes sont généralement plaquées 4-5 jours après que les neurones étaient plaqués. GDNF a été éliminée avant la première ré-plaquage des astrocytes dans le côté gauche de la MCP. Re-plaqué astrocytes ont été fixés dans le domaine de la conservation de cellules, comme le montre l&#39;immunomarquage GFAP (Figure 1). Seulement un débit minimal de fluide entre les deux côtés des chambres (déterminé par des colorants de fluorescence) a été trouvé dans le MCP, comme précédemment indiqué 4,6. </li><li> Après le re-dépôt des astrocytes, les axones qui sont entrées dans la partie gauche de la MCP assemblé interagir directement avec le astrocytes par axonale soit un contact direct ou libération de facteurs solubles. Immunomarquage de la membrane plasmique GLT1 astrogliale glutamate transporteur et neuronale βIII-tubuline a été réalisée afin de visualiser l&#39;interaction entre les axones et les astrocytes, comme le montre la figure 2D-F. </li></ol>

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	<li>Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+signaling+in+the+development+and+plasticity+of+neural+circuits.">Neuron-astrocyte signaling in the development and plasticity of neural circuits.</a> Neuro-Signals. 16, 278-288 (2008).</li><li>Paixao, S., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+communication+and+synaptic+plasticity.">Neuron-astrocyte communication and synaptic plasticity.</a> Current opinion in neurobiology. 20, 466-473 (2010).</li><li>Fields, R. 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Aucun conflit d&#39;intérêt déclaré.

Le MCP neurone base et les astrocytes du système de co-culture de neurones permet dissection détaillée de astroglies voies de signalisation en n&#39;autorisant que les axones passent les canaux centraux et interagissant avec les cellules astrocytaires. Ce système de co-culture peut être facilement mis en place avec des neurones classiques et des procédures de culture d&#39;astrocytes. Nous avons également décrit une application pratique de ce système de co-culture en employant un journaliste eGFP base pour dém...

<table border="1"><tbody><tr><td> Sérum de veau fœtal </td><td> Hyclone </td><td> SH30070.03 </td><td> pour le placage neurone neurone à moyen cutlure </td></tr><tr><td> Sérum de veau fœtal </td><td> Sigma-Aldrich </td><td> F4135 </td><td> pour un milieu de culture des astrocytes </td></tr><tr><td> Gliale facteur de nerf dérivée </td><td> R &amp; D systèmes </td><td> 212-GD </td><td> Appliquer 10-20 ng / ml à côté de la chambre de neurone </td></tr><tr><td> Dulbecco modifié Eagle milieu glucose à haute </td><td> Sigma-Aldrich </td><td> 11995 </td><td></td></tr><tr><td> 70 mm tamis cellulaire </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Stérile à fond de verre plat </td><td> MatTek Corporation </td><td></td><td></td></tr><tr><td> Plates-formes de culture microfluidiques </td><td> Xona Microfluidique LLC </td><td> SND150 </td><td></td></tr><tr><td> 6 puits de la plaque de culture </td&gt; <td> Cellstar </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> Milieu de culture neurone <ul><li> Milieu Neurobasal </li><li> 2% supplément B27 Neurobasal </li><li> 2 mM de glutamate en ajoutant 1% 100x Glutamax </li><li> 1% de pénicilline-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Milieu de culture cellulaire neurone pour placage <ul><li> Milieu Neurobasal </li><li> 2% supplément B27 Neurobasal </li><li> 2 mM de glutamate en ajoutant 1% 100x Glutamax </li><li> 1% de pénicilline-streptomysin </li><li> 5% de sérum fœtal bovin SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> Milieu de culture des astrocytes <ul><li> Dulbecco modifié eagle de glucose moyen-élevé </li><li> 10% de sérum bovin fœtal F4135 </li><li> 1% de pénicilline-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Matériaux tableau 1. Utilisés dans la culture à base de plate-forme microfluidique axone des neurones et cellules gliales du système de co-culture. </td></tr></tbody></table>

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

Neurone propre à l&#39;interaction glie est essentielle à la fonction physiologique du système nerveux central (SNC). Cette communication bidirectionnelle est subtilement médiée par les voies de signalisation spécifiques entre les neurones et les cellules gliales 1,2. L&#39;identification et la caractérisation de ces voies de signalisation est essentielle à la compréhension de la façon dont l&#39;interaction neurone glie formes physiologie du système nerveux central. Auparavant, des cultures de neurones et de cellules gliales mixtes ont été largement utilisés pour tester et caractériser les voies de signalisation entre les neurones et les cellules gliales. Ce que nous avons appris de ces préparations et autres outils in vivo, cependant, a suggéré que la signalisation mutuelles entre neurones et cellules gliales sont souvent produits dans des compartiments spécifiques à l&#39;intérieur des neurones (c.-à axones, dendrites, ou soma) 3. Il est donc important de développer un nouveau système de culture qui permet la séparation des compartiments neuronaux et étudie spécifiquement l&#39;interaction entre les cellules gliales et neuroaxones internes / dendrites. En outre, le système de culture conventionnel mixte n&#39;est pas capable de différencier les facteurs solubles et directs signaux de contact membranaire entre les neurones et les cellules gliales. En outre, la grande quantité de neurones et des cellules gliales dans le classique système de co-culture n&#39;a pas la résolution nécessaire pour observer l&#39;interaction entre un axone unique et une cellule gliale. Dans cette étude, nous décrivons une nouvelle axone et les cellules gliales du système de co-culture avec l&#39;utilisation d&#39;une plate-forme microfluidique culture (MCP). Dans ce système de co-culture, les neurones et les cellules gliales sont cultivées dans deux chambres séparées qui sont connectées par le biais de plusieurs canaux centraux. Dans cette plate-forme microfluidique la culture, seuls les processus neuronaux (en particulier les axones) peut entrer dans le côté gliale à travers les canaux centraux. En combinaison avec marquage des protéines fluorescentes puissant, ce système permet un examen direct des voies de signalisation entre les interactions axonales / dendritiques et des cellules gliales, une telles axone médiée par la régulation transcriptionnelle dans les cellules gliales, les cellules gliales trafic des récepteurs induite dans les terminaux de neurones, les cellules gliales et médiée par la croissance axonale. Le petit diamètre de la chambre également interdit de manière significative l&#39;écoulement du fluide enrichi en neurone dans la chambre gliale, facilitant palpage de la protéine membranaire directe interaction entre les axones / dendrites et les surfaces gliales.

L&#39;analyse d&#39;imagerie time-lapse de l&#39;activation induite par axone promoteur GLT1 dans les astrocytes Le neurone compartimenté et co-culture d&#39;astrocytes système ne permet que les processus neuronaux, notamment des axones, d&#39;interagir sélectivement avec les astrocytes. Suite à la mise en place réussie de l&#39;axone et des astrocytes (cellules gliales ou d&#39;autres) co-culture dans l&#39;assemblée des MCP, différents types d&#39;interactions axone-cellules gliales pe...

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

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

Cette étude décrit les procédures de mise en place d&#39;un axone des neurones et roman (astro) glie plate-forme de co-culture. Dans ce système de co-culture, la manipulation de l&#39;interaction directe entre un axone unique (et unique cellule gliale) devient possible, ce qui permet une analyse mécaniste du neurone mutuelle à la signalisation gliales.

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 ...

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

Research

JoVE Journal

Neuroscience

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

14.9K vues.  Tufts University. Cette étude décrit les procédures de mise en place d'un axone des neurones et roman (astro) glie plate-forme de co-culture. Dans ce système de co-culture, la manipulation de l'interaction directe entre un axone unique (et unique cellule gliale) devient possible, ce qui permet une analyse mécaniste du neurone mutuelle à la signalisation gliales.

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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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

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

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 ...

Recherche

Neurosciences

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

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

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

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 ...

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

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

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

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

Le Neuron-astrocyte indirecte coculture Assay: Un<em&gt; In vitro</em&gt; Mise en place pour l&#39;investigation détaillée des interactions Neuron-glie

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

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

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

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

Isolement et culture des neurones primaires et glia de la vessie urinaire adulte de rat

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

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

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

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

Lésion neuronale ciblée pour la déconnexion non invasive des circuits cérébraux

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

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

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

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

Dissection et isolement de la glie murine de plusieurs régions du système nerveux central

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

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

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

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

Un modèle de culture cellulaire pour étudier le rôle des interactions neurone-glia dans l’ischémie

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

In vivo Calcium Imaging in Mouse Inferior Olive

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

In vivo Calcium Imaging in Mouse Inferior Olive

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

In vivo Imagerie du calcium chez la souris Olive inférieure

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

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

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

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

Visualiser les changements sur le circuit neurone-glie avec la technique d’imagerie calcique

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

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

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

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

Transplantation de microglies dérivées de cellules souches pluripotentes induites par l’homme dans le cerveau de souris immunocompétentes par voie transnasale non invasive

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