Cette étude a été financée en partie par la Fondation IPSEN. IMV est professeur American Cancer Society of Molecular Biology, et titulaire de la Chaire Irwin et Joan Jacobs en sciences de vie exemplaire. Ce travail a été soutenu en partie par des subventions du NIH, Leducq Foundation, Fondation Mériaux, Ellison Medical Foundation, Ipsen / Biomeasure, Sanofi Aventis, Prostate Cancer Foundation, et le HN et Frances C. Berger Foundation. Le PAS est financé par McKnight Fonds de dotation pour les neurosciences et le National Institute on Drug Abuse (R01 DA019022). Les auteurs tiennent à remercier Mark Schmitt. Art graphique a été conçue par Jamie T. Simon. Parrainage a été aimablement fourni par Invitrogen.

1. Cultures organotypiques cérébelleuse: <ol><li> Pour réussir à produire des cultures organotypiques cérébelleuse utiliser des souris entre P0-P3 ou P8-P12. Pour notre expérience, nous avons utilisé des souris de la gamme P8-P12 parce que ce stade de développement était plus approprié pour notre étude. </li><li> Préparer la dissection et le milieu de culture: <ol><li> La dissection du cerveau et de préparation de tranches sont réalisées dans artificielle de sodium liquide céphalo-bas (ACSF) contenant du chlorure de calcium 1 mM, 10 mM D-glucose, 4mm chlorure de potassium, chlorure de magnésium 5 mM, du bicarbonate de sodium 26mm, 246mm de saccharose et le phénol solution rouge (1:1000) , pH 7,3. Pour stériliser la solution, elle filtre à travers un filtre 0,22 um et stocker à 4 ° C. </li><li> Tranches du cervelet sont cultivées dans 75% minimum Essential Medium Eagle (MEM), 25% de sérum de cheval inactivé à la chaleur, HEPES 25 mM, 1 mM de glutamine, 5mg / ml de glucose, la pénicilline (100 U / ml) et streptomycine (100 UI / ml). Filtrer le support à travers un filtre de 0,22 um et stocker à 4 ° C pour un maximum de deux semaines. </li></ol></li><li> Disséquer cerveau dans glacée ACSF moyennes préalablement à barboter avec carbogène (95% d&#39;oxygène et de dioxyde de carbone de 5%) Notez que la saturation avec Carbogen va changer le pH de la solution de sorte qu&#39;il passera du rouge à l&#39;orange. La dissection doit être effectuée dans le minimum de temps possible en faisant attention à ne pas endommager la structure du cerveau. </li><li> Trancher le cerveau dans des coupes sagittales de 400 um en utilisant un vibratome. <ol><li> Faites une coupe sagittale avec une lame de rasoir dans un hémisphère, en essayant de couper aussi peu que possible du cervelet, ce qui donnera une surface plane pour le tranchage. Le cortex est utilisé uniquement pour le soutien pour faire le découpage plus facile, mais puisque le temps est une question importante qu&#39;il est plus commode de couper la moitié rostrale du cortex avec une lame de rasoir. </li><li> Utilisez cyanoacrylate (superglue) pour fixer la position du cerveau sur la plaque de métal vibratome. Il est important de refroidir la plaque sur la glace avant d&#39;ajouter la colle et à le répandre ainsi l&#39;aide de l&#39;extrémité du tube pour former une couche de colle mince. Si il ya un excès de superglue, il va se détacher de la plaque quand il entre en contact avec l&#39;ACSF et l&#39;échantillon sera perdu. </li><li> Une fois que le cerveau est attaché à la plaque vibratome, ajouter suffisamment de supports ACSF pour couvrir le cerveau et mettre de la glace en dessous de la plaque pour le garder froid. </li><li> Couper les tranches 400 um et de les transférer à une pipette Pasteur en plastique (couper la pointe de l&#39;élargir et d&#39;éviter d&#39;endommager les tranches) pour une plaque de 6 puits de culture cellulaire contenant ACSF sur la glace. </li><li> Séparez le cervelet du reste du cerveau avec l&#39;aide de deux seringues à insuline (seringues avec petit diamètre de 28 aiguilles de calibre) sous la loupe binoculaire. Certaines tranches aura Superglue fixés sur les bords: c&#39;est le moment d&#39;essayer de l&#39;enlever car il peut affecter la viabilité de la tranche. Il est important de le faire avec soin, sans endommager la structure du cervelet. </li></ol></li><li> Transférer les tranches de cervelet de plaques de 6 puits contenant 30mm inserts plaque de culture avec 0,4 um pores. Ajouter 1 mL de milieu de culture par puits, qui a été pré-conditionnée par incubation pendant au moins 2 heures à 37 ° C et 5% de CO 2 (dans la incubateur de culture cellulaire). Placer les inserts sur le dessus du support en vous assurant qu&#39;il n&#39;ya pas de bulles d&#39;air piégées en dessous et puis placer les tranches de cervelet (1-3 par plaquette) sur le dessus des inserts en s&#39;assurant qu&#39;ils sont en pleine extension et qu&#39;aucun liquide n&#39;est en les couvrant. Aspirer l&#39;excès des médias, si nécessaire. Incuber à 37 ° C et 5% de CO 2 change le milieu de culture tous les 2 ou 3 jours. Il est préférable de ne remplacer qu&#39;une partie du milieu (par exemple ½ volume) à la fois, puisque milieu utilisé contient des facteurs trophiques tranche de dérivés qui sont importantes pour la survie cellulaire. </li></ol> 2. Traitement de Fas et de coloration des tranches du cervelet: <ol><li> Pour l&#39;expérience du challenge apoptotiques, nous les tranches première culture pendant 5 jours médiatique en pleine mutation, le jour in vitro de 3 (DIV3). Sur DIV5 cultures sont traitées avec 0,5 pg / ml d&#39;anticorps de l&#39;Jo2 Fas agoniste en l&#39;ajoutant directement aux médias dans le but d&#39;avoir un fond propre de la mort cellulaire depuis les changements de médias peuvent induire une certaine mortalité dans les cultures. La moitié des puits ne sont pas traitées comme témoins. </li><li> Après 24 heures retirer le support sous le inserts par aspiration. Pour fixer les tranches, ajouter 1 ml de glace froide PFA 4% en dessous et 1 ml dessus de l&#39;insert. Incuber 10 minutes à température ambiante. </li><li> Après 10 minutes retirer le PFA et laver rapidement avec du PBS froid. </li><li> Ensuite enlevez le PBS et ajouter 1 ml et en dessous de 1 mL au-dessus de l&#39;insert de 20% du méthanol glacé dans le PBS et incuber pendant 5 minutes à température ambiante. </li><li> Laver à nouveau avec du PBS et ajouter 1 mL dessous et 1 ml dessus de l&#39;insert de 0,5% de Triton-X-100 dans du PBS pour la perméabilisation. Incuber pendant un minimum de 12 heures à 4 ° C. </li><li> Après perméabilisation, bloc avec 20% de BSA dans du PBS pour un minimum de4 heures à température ambiante ou une nuit à 4 ° C. A ce stade, les tranches peuvent être conservés dans une solution de blocage d&#39;au moins 3 jours à 4 ° C. </li><li> Afin de colorer les tranches soigneusement découpé le morceau de la membrane d&#39;insérer attaché à la tranche du cervelet et le transférer à des plaques 24 puits contenant du PBS. </li><li> L&#39;immunomarquage est réalisé de manière séquentielle, d&#39;abord avec le réactif TUNEL pendant une heure et ensuite avec l&#39;anticorps Calbidin nuit. <ol><li> Aspirer le PBS et ajouter 250 uL hors réactif TUNEL dans chaque puits de s&#39;assurer qu&#39;il couvre toute la surface de la tranche. Incuber à 37 ° C pendant une heure dans l&#39;obscurité. </li><li> Après une heure retirer le mélange TUNEL et laver avec du PBS à trois reprises pendant 10 minutes. </li><li> Après le dernier lavage, retirez le PBS et ajouter 250 ul d&#39;une dilution de 1 / 1000 de l&#39;anticorps calbindine dans du PBS et incuber une nuit à 4 ° C dans l&#39;obscurité. </li><li> Retirer la solution d&#39;anticorps primaire et laver avec du PBS à trois reprises pendant 10 minutes. </li><li> Ajouter 250 ul d&#39;anticorps secondaire dilué au 1 / 500 en PBS et laisser incuber pendant au moins trois heures à température ambiante dans l&#39;obscurité. Le kit de TUNEL, nous avons utilisé est étiqueté avec TMR rouge, donc notre anticorps secondaire pour calbindine est conjugué à Alexa-488. </li><li> Laver trois fois pendant 10 minutes avec du PBS et monter les tranches sur une lame de microscope en verre en utilisant un média de montage qui contient DAPI. </li><li> Les tranches d&#39;image avec un microscope confocal en utilisant les 488 nm longueur d&#39;onde d&#39;excitation et 561 nm pour calbindine et TUNEL respectivement. </li></ol></li></ol> 3. Les résultats représentatifs: Le principal défi de ce protocole est d&#39;être capable de générer des cultures saines tranche de cervelet, surtout depuis que notre lecture finale sera la mort cellulaire. Une saine culture apparaît comme l&#39;un où la couche de cellules du corps est translucide et vous permet de voir la structure en feuillets du cervelet sous le microscope (figure 1). Après quelques jours dans la culture des tranches de commencer à cellules minces et non-neuronales peuvent être observés migrer loin de la marge de la tranche. Les cellules rondes sombre (la plupart des macrophages probable) qui couvrent les tranches après quelques jours de culture, finira par diminuer en nombre après 10-14 jours in vitro (figure 2) 5. La preuve ultime de la viabilité des tranches est de coloration pour les différents marqueurs cellulaires, telles que calbindine pour les cellules de Purkinje. La figure 3 montre une image au microscope confocal à représentatifs de la couche de cellules de Purkinje avec plusieurs noyaux TUNEL positif. Il est important de considérer que même si certaines tranches reçu un stimulus apoptotique, elles étaient seulement exposés pendant 24 heures. Cela a permis de suffisamment de temps pour observer la fragmentation de l&#39;ADN dans les cellules de mourir avec la coloration TUNEL, mais une couche de cellules de Purkinje défini était encore présent. <img src="/files/ftp_upload/2564/2564fig1.jpg" alt="Figure 1" /> Figure 1. Healthy tranche de cervelet au DIV2. Cette image montre la couche de cellules translucides corps et la structure en feuillets du cervelet dans une tranche saine. <img src="/files/ftp_upload/2564/2564fig2.jpg" alt="Figure 2" /> Figure 2. Healthy tranche de cervelet au DIV 7. Dans cette image on peut encore observer la structure feuilletée du cervelet et de l&#39;apparence des organes de cellule sombre. <img src="/files/ftp_upload/2564/2564fig3.jpg" alt="Figure 3" /> Figure 3. Représentant l&#39;image d&#39;une tranche de Fas-traitée cérébelleux. Cette image confocale montre les cellules de Purkinje colorés avec calbindine (vert) et les cellules apoptotiques positifs pour la coloration TUNEL (rouge). Les cellules de Purkinje ne semblent pas en une seule rangée, mais sont regroupés en formant une structure ressemblant à folium.

<ol>
	<li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+ependymal+cells+in+tissue+cultures.">Human fetal ependymal cells in tissue cultures.</a> Anat Rec. 99, 523-529 (1947).</li><li>Hogue, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+studies+of+human+fetal+brain+cells+in+tissue+cultures.">Review of studies of human fetal brain cells in tissue cultures.</a> Etudes Neonatales. 1, 1-13 (1952).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocol+for+organotypic+hippocampal+slice+cultures.">Staining protocol for organotypic hippocampal slice cultures.</a> Nat Protoc. 1, 2452-2456 (2006).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Beat, H., G&#228;hwiler, S. M. T., McKinney, R. A., Debanne, D., Robertson, R. T., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic Slice Cultures of Neural Tissue in Culturing Nerve Cells. , 461-498 (1998).</li></ol>

Tous les travaux menées sur des animaux a été effectué conformément aux politiques établies par le IACUC. Le Salk Institute est un centre accrédité AAALAC.

Cette méthode décrit l&#39;une des nombreuses applications possibles des cultures organotypiques neuronale et il nous a permis d&#39;étudier l&#39;effet de stimuli apoptotiques de type sauvage et mutant tranches cérébelleux. La partie la plus critique de cette expérience est d&#39;être capable de générer constamment de saines cultures tranche de cervelet. Les facteurs clés sont la dissection et la technique de tranchage et la capacité d&#39;exécuter le protocole dans le moins de temps possible, mais en même temps, en faisant attention à ne pas endommager les tranches. Un autre facteur crucial est la préparation et la composition des milieux de culture, ces cultures sont très sensibles, alors nous avons dû tester plusieurs recettes et les marques de médias. Même les différents lots de sérum ou de tout autre composant du média pourrait affecter la viabilité des tranches. Nous avons obtenu les meilleurs résultats avec le sérum de cheval à partir du lot # 480116 Invitrogen. Une fois les tranches de cervelet sont générés, on peut étudier le développement du cervelet, de l&#39;électrophysiologie, la survie cellulaire, seul ou en réponse à des stimuli apoptotiques, excitotoxicité ou la privation d&#39;oxygène. Des études comparatives à l&#39;aide de type sauvage et mutant tranches cérébelleuses ont été très perspicaces dans de nombreux aspects de la fonction cérébelleuse et le développement.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11090</td>
<td>&nbsp;</td>
<tr>
<td>Horse Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/ Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Superglue</td>
<td>&nbsp;</td>
<td>Krazy Glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well / 24 well cell culture plates</td>
<td>&nbsp;</td>
<td>Corning Incorporated</td>
<td>3506/ 3527</td>
<td>&nbsp;</td>
<tr>
<td>30mm culture plate inserts (0.4-&mu;m pore)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
<tr>
<td>Purified NA/LE Hamster anti-mouse CD95 (Jo2)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>554254</td>
<td>&nbsp;</td>
<tr>
<td>In situ cell death detection kit, TMR red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>12 156 792 910</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-Calbindin D-28k antibody</td>
<td>&nbsp;</td>
<td>Swant</td>
<td>CB-38a</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 488-goat anti-rabbit IgG secondary antibody</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular probes)</td>
<td>A-11008</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield Mounting Medium with DAPI</td>
<td>&nbsp;</td>
<td>Vector labs</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Zeiss Stemi SV 11</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica TCS SP2 AOBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic cerebellar cultures: apoptotic challenges and detection

Cultures organotypiques de tissu neuronal ont d&#39;abord été introduite par Hogue en 1947 1,2 et ont constitué une percée majeure dans le domaine des neurosciences. Depuis lors, la technique a été développée et actuellement il ya de nombreuses façons de préparer des cultures organotypiques. La méthode présentée ici a été adaptée de celle décrite par Stoppini et al. Pour la préparation des tranches et de Gogolla et al. Pour la procédure de 3,4 coloration. Une caractéristique unique de cette technique est qu&#39;elle vous permet d&#39;étudier les différentes parties du cerveau comme l&#39;hippocampe ou du cervelet dans leur structure d&#39;origine, offrant un gros avantage sur les cultures dissociées dans laquelle toute l&#39;organisation cellulaire et les réseaux neuronaux sont perturbés. Dans le cas du cervelet, il est encore plus avantageuse car elle permet l&#39;étude des cellules de Purkinje, extrêmement difficile d&#39;obtenir que la culture principale dissociées. Cette méthode peut être utilisée pour étudier certaines caractéristiques développementales du cervelet in vitro, ainsi que pour des expériences électrophysiologiques et pharmacologiques dans les deux de type sauvage et des souris mutantes. La méthode décrite ici a été conçu pour étudier l&#39;effet de stimuli apoptotiques tels que Fas ligand dans le cervelet en développement, utilisant une coloration TUNEL pour mesurer la mort cellulaire par apoptose. Si coloration TUNEL est combiné avec des marqueurs type de cellule spécifique, comme pour les cellules de Purkinje calbindine, il est possible d&#39;évaluer la mort cellulaire d&#39;une manière population cellulaire spécifique. La coloration calbindine sert aussi le but d&#39;évaluer la qualité des cultures du cervelet.

Watch this Scientific Journal Video about Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection at JoVE.com

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Cette méthode décrit la génération de cultures organotypiques cérébelleux et l&#39;effet de certains stimuli apoptotiques sur la viabilité des différents types de cellules du cervelet.

Cultures organotypiques cérébelleuse: Défis apoptotiques et détection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

organotypic-cerebellar-cultures-apoptotic-challenges-and-detection

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Neuroscience

18.1K vues.  The Salk Institute for Biological Studies. Cette méthode décrit la génération de cultures organotypiques cérébelleux et l'effet de certains stimuli apoptotiques sur la viabilité des différents types de cellules du cervelet.

Vidéo: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Cultures organotypiques d&#39;hippocampe Slice

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

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Neurosciences

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Enregistrements tableau multi-électrodes des Avalanches neuronale dans des cultures organotypiques

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Cultures organotypique de mésencéphale ventral embryonnaire: Un système pour étudier le développement neuronal dopaminergique In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

L&#39;analyse de la morphologie de Purkinje cellules dendritiques dans les cultures organotypique

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Cultures organotypique de tranche d&#39;étudier Oligodendrocyte Dynamics et myélinisation

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

L&#39;analyse de neurovasculaire Rénovation dans Entorhino-hippocampe cultures organotypique de tranche

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Déplétion sélective des cellules microgliales du cervelet granules de cultures cellulaires utilisant la L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Enquêter Régénération fonctionnelle en organotypiques médullaires Co-cultures Cultivé sur des tableaux multi-électrodes

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Hippocampe organotypiques comme modèle d’étude Neuroprotection et son caractère invasif des cellules tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex Utero Électroporation et culture organotypique tranche de cerveaux de souris embryonnaires pour vivre-imagerie de la migration des interneurones GABAergiques

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...