Cette recherche a été soutenue par des subventions de la recherche Possibilités programme de premier cycle à l&#39;UCI (AR et SP) et des subventions de l&#39;Etat de Californie, l&#39;initiative la maladie d&#39;Alzheimer et les National Institutes of Health accorde pas. HD38466, et le Centre de recherche sur les maladies d&#39;Alzheimer accorde pas. AG16573 (JB)

1. La cryoconservation de tissu cortical Blocks Préparation des matériaux <ol><li> Préparer la solution 1X Hank saline tamponnée (HBSS) en diluant la solution mère à 10X dans de l&#39;eau stérile (1:9). HBSS magasin à 4oC. </li><li> Préparer 10% milieu de congélation du DMSO en diluant DMSO dans HBSS (1:9). Le milieu de congélation doivent être préparées avant la cryoconservation et conservés à 4 ° C jusqu&#39;à refroidissement. </li><li> Une lame de rasoir en acier est utilisé pour le hacher systématique des tissus. Avant l&#39;utilisation, la lame de rasoir est stérilisé par immersion dans l&#39;éthanol à 70% pendant 2 heures. Immédiatement avant le traitement des tissus, rincer la lame de rasoir à l&#39;eau stérile 3 fois. Évitez de laisser le rasoir dans l&#39;eau stérile pour une quantité excessive de temps, car il est sensible à l&#39;oxydation. </li><li> Un conteneur de congélation Nalgene est chargé avec cryotubes (2,5 ml) et ensuite amené dans une armoire biologique stérile. 1 ml de milieu de congélation glacée est ajoutée à chaque flacon. Le conteneur de congélation est alors placé à 4 ° C pendant au moins 2 heures. </li></ol> Nettoyage, découper, et le gel <ol><li> Le tissu cérébral à congeler est nettoyée de la membrane méningée et les vaisseaux sanguins. Utilisez conseils aiguille stérile pour enlever soigneusement les débris du tissu tout en travaillant sur le dessus d&#39;un sac de glace. Tissus nettoyables doivent être rincés doucement avec HBSS froid et transféré à un nouveau plat de Pétri pour hacher. </li><li> Travail sur le dessus d&#39;un sac de glace, utilisez une lame de rasoir stérilisée pour rapidement couper le tissu dans environ 1 mm 3 blocs. Travailler avec de petites portions de tissus nettoyés au temps entraîne un meilleur contrôle sur la procédure de hachage, résultant en des tailles de bloc plus homogène. </li><li> Ajouter lentement les tissus hachés dans 50 ml de HBSS dans un tube conique de 50 ml. Rincer délicatement la boîte de Pétri avec HBSS afin de collecter tous les blocs de tissus restants. Autoriser les blocs de tissus pour descendre au fond du tube, créant un bloc de tissu légèrement tassées à granulés. </li><li> Alors que le tissu est de décantation, mettre le récipient préalablement refroidi congélation et cryotubes dans l&#39;armoire de sécurité biologique. Débouchez toutes les cryotubes de hâter le processus d&#39;ajout de tissu. </li><li> Après tous les blocs de tissus se sont installés, doucement aspirer le HBSS excès, laissant une très fine couche de média-dessus du culot. La centrifugation est déconseillée car elle va provoquer des blocs de tissus d&#39;adhérer les unes aux autres, diminuant de manière significative l&#39;efficacité de congélation. </li><li> Lentement recueillir 200 uL du fond de la dérive des tissus bloc granulés en utilisant la pointe de pipette avec des orifices larges (coupe la pointe avec des ciseaux stériles). Transfert du matériel à un cryovial et de passer à la suivante, des tissus à nouveau la collecte à partir du bas de la pastille. Il est essentiel que la procédure d&#39;attribution des tissus ensemble ne prend pas plus de 3-4 min par conteneur de congélation. Cela garantit que le tissu est exposé à du DMSO pour seulement une courte période de temps avant la congélation. Après le transfert du tissu pour les flacons, placez le récipient dans un gel-80oC congélateur pendant au moins 4 heures. Alternativement, le récipient de congélation peut être laissé une nuit à -80 oC. Répétez ce processus pour tous les conteneurs de congélation restantes. </li><li> Transférer les cryotubes dans le conteneur de congélation (s) à un cryobox et placer dans un réservoir d&#39;azote liquide pour stockage à long terme. </li></ol> 2. La décongélation et la culture de Frozen Blocks tissu cortical Préparation des matériaux <ol><li> Un jour avant le dégel des échantillons de tissus, de poly-L-lysine revêtement boîtes de Petri / lamelles sont préparés. En général, pour les neurones corticaux nous préparons des plats revêtus avec 500 ug / ml ou 1 mg / ml. Ajouter suffisamment d&#39;acide poly-L-lysine solution (faites dans un tampon de borate) pour couvrir entièrement le fond du plat. Si lamelles de verre sont nécessaires, s&#39;assurer qu&#39;ils sont complètement submergés sous la solution de poly-L-lysine. Incuber pendant au moins 12 hr. Avant l&#39;utilisation, rincer la vaisselle avec une généreuse quantité d&#39;eau stérile 3 fois, 5 minutes chacun. </li><li> HBSS chaud est utilisé pour nettoyer les tissus décongelés ainsi que de dissocier le tissu dans des suspensions cellulaires. Le volume de HBSS nécessaire variera en fonction de la quantité de tissu à être décongelés, mais typiquement une cryovial va diluer dans 50 ml de HBSS et seront dissociées dans 10 ml de HBSS. </li><li> Eagle modifié par Dulbecco moyenne avec 10% des suppléments de fer du sérum de veau bovin (EBS) et 1% d&#39;antibiotique / antimycotique (AA) mélange (DMEM (++)) devrait être placé dans un bain à 37 ° C l&#39;eau. </li><li> Milieu d&#39;étalement (NB (+++)) doivent être préparées avant l&#39;utilisation. Ce milieu est préparé en ajoutant 1% d&#39;antibiotique / antimycotique ainsi B27 et les compléments de N2. </li></ol> Remarque: Les noms des médias en annexe avec des croix (+) indique l&#39;inclusion d&#39;additifs à la composition de base des médias. Dans ce texte, DMEM (+ +) indique DMEM plus 10% EBS majoré de 1% AA, tandis NB indique (+++) NB plnous 1% AA et B27 ainsi que N2. La décongélation et la culture <ol><li> Retirez les cryotubes du réservoir d&#39;azote liquide et de dégel rapide à 37 ° C dans un bain d&#39;eau glacée jusqu&#39;à ce que seul une petite pastille est observée à l&#39;intérieur du contenu du flacon. </li><li> Délicatement le transfert le contenu du flacon de 50 ml de HBSS au chaud dans un tube conique. Utiliser un embout large orifice pour doucement déloger les blocs de tissus restent coincés dans le cryovial. Inverser le tube 3-4 fois et laissez le tissu à recueillir lentement vers le bas. Les tissus doivent régler rapidement, mais si les blocs sont trop petits cela peut ne pas se produire et la lumière de centrifugation (~ 200 x g) est recommandée. </li><li> Aspirer HBSS excès. </li><li> Ajouter 10 ml de HBSS chaude pour le tissu et de culot 300 pi de trypsine à 0,25% et de 50 uL d&#39;ADNase. Incuber dans un bain d&#39;eau à 37 ° C pendant 5 min, puis placer dans un agitateur orbital à 80 rpm jeu et 37 ° C pendant 5 minutes supplémentaires. </li><li> Après cette période, amener les blocs de tissus dans une enceinte de sécurité biologique et l&#39;utilisation d&#39;une pipette 10 ml de bien agiter le blocs de tissus jusqu&#39;à une suspension de cellules trouble est formé. Désactiver la trypsine en ajoutant 10 ml de DMEM chaude (+ +) à la suspension cellulaire. </li><li> Centrifuger les cellules dissociées à 1200 x g pendant 5 min. Aspirer et jeter le surnageant, resuspendre le culot cellulaire dans 10 ml d&#39;eau tiède DMEM (+ +) et de quantifier le nombre de cellules en utilisant un hématimètre. </li><li> Cellules de la plaque de poly-L-lysine plats revêtus. Typiquement, un plat de 60 mm sera plaqué avec 1x10 6 cellules. </li><li> Laisser les cellules d&#39;attacher au plat / lamelles d&#39;environ 1 h dans l&#39;incubateur de culture tissulaire. Il est recommandé de suivre les progrès de l&#39;attachement dans une plaque toutes les 10 min jusqu&#39;à ce l&#39;attachement effectif est observé. Puis, doucement remplacer le milieu avec du DMEM frais (++). </li><li> Après 24 h, remplacer le DMEM (+ +) avec les chaudes NB (+++). Changements de milieu partielle (50%) sont effectuées tous les 5 jours avec frais NB (+++). Cultures saines montrent habituellement des signes de différenciation et de croissance des processus après 24 heures. Dans ces conditions, et effectuer des changements partiels moyenne tous les 4-5 jours, les cultures peuvent être maintenues pendant des périodes prolongées jusqu&#39;à 4-6 semaines. </li><li> Pour la génération des cellules précurseurs neuronales (CPN), un protocole similaire est utilisé à l&#39;exception que, après centrifugation, DMEM sans EBS est utilisé. Les cellules sont étalées dans le T-25 flacons de DMEM/F12 (1:3) complété avec B27 et l&#39;EGF (20 ng / ml) pour permettre la formation d&#39;neuropheres. Pour différencier les PNJ, neurosphères sont étalées sur la laminine (10 ug / ml) recouvert de lamelles de DMEM/F12 (1:3) milieu supplémenté avec N2. </li></ol> 3. Les résultats représentatifs Cultures saines montrera une croissance importante et de différenciation après 5 jours post-décongélation et seront généralement stabiliser dans sa croissance en 10 jours (figure 1). Coloration Immuncocytochemical de ces cultures révèle de nombreux astrocytes (figure 2A) et les neurones (figure 2B) pour les humains et les rats des cultures primaires de neurones. Ce protocole est également approprié pour la génération de PNJ que flottant neurosphères (figure 3A), qui dans des conditions de différenciation, le résultat dans les cultures mixtes de haute qualité (figure 3B, C). <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="Figure 1" /> Figure 1. Des cultures cellulaires de tissu cortical humain congelé. Frozen humaine blocs de tissus corticaux sont décongelées et plaquées sur la poly-lysine lamelles couché et cultivées pendant 10 jours. Dès le jour 5 expansion cellulaire est apparente et par jour 10 confluence est atteinte. Barre d&#39;échelle: 100 um. <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="Figure 2" /> Figure 2. Coloration immunocytochimique de cultures cellulaires. (A) Les cultures ont été colorés avec le marqueur protéine gliale acide fibrillaire gliale (GFAP, flèches épaisses). (B) Les neurones ont été détectés avec le marqueur neuronal bêta-tubuline III (TIII, flèches fines). (C) Les deux rats et des cultures humaines montrent gliales et neurones abondante après 10 jours de culture. Anticorps primaire: la souris anti-GFAP (1:1000) et de lapin anti-TIII (1:1000). Anticorps secondaire: anti-souris Alexa 594 (1:500) et anti-lapin Alexa 488 (1:500). Coloration des noyaux: Hoestch bleue (1:1000). Barre d&#39;échelle: 50 um. <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="Figure 3" /> Figure 3. Génération de neurosphères. (A) des tissus congelés a été traitée comme décrit ci-dessus et a permis de se propager comme neurosphères. (B) neurosphères ont été étalées sur des lamelles de verre dans des conditions de différenciation et cultivées pendant 10 jours, ce qui entraîne la migration des cellules loin de la Neurosphère. (C) Les deux neurones et les astrocytes sont présents dans la frange d&#39;une expansion Neurosphère différencier. Contre-coloration nucléaire, les anticorps primaires et secondaires utilisés comme dans la figure 2.

<ol>
	<li>Robbins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+brain+tissue.">Cryopreservation of human brain tissue.</a> Exp Neurol. 107, 208-213 (1990).</li><li>Ware, C. B., Nelson, A. M., Blau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-rate+freezing+of+human+ES+cells.">Controlled-rate freezing of human ES cells.</a> Biotechniques. 38, 879-880 (2005).</li><li>Paynter, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practical+issues+for+cryopreservation+of+nerve+cells.">Principles and practical issues for cryopreservation of nerve cells.</a> Brain Res Bull. 75, 1-14 (2008).</li><li>Thirumala, S., Gimble, J. M., Devireddy, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+stromal+vascular+fraction+of+adipose+tissue+in+a+serum-free+freezing+medium.">Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.</a> J Tissue Eng Regen Med. 4, 224-232 (2010).</li></ol>

Cryoconservation offre une opportunité précieuse à la banque d&#39;échantillons de tissus du cerveau pour une utilisation future. Nous décrivons ici un protocole simple mais efficace pour générer deux neurones enrichi les cultures et les cellules précurseurs des neurones à partir des blocs congelés tissu cérébral. Cette procédure évite les coûts économiques des techniques de cryoconservation traditionnelles qui utilisent plus cher vitesse contrôlée congélateurs. Le protocole permet la génération de cultures de neurones viables après décongélation en fournissant un moyen rapide et efficace pour geler les blocs de tissus. Le processus de congélation entier peut prendre aussi peu que 20 minutes. En plus de cultures neuronales primaires, en utilisant cette méthode de blocs de tissus peuvent également être décongelés à générer des PNJ grandi comme libre neurosphères flottant. À cet égard, le manque de sérum dans notre milieu de congélation assure que les cellules sont conservés dans un état indifférencié. Dans des conditions de différenciation, neurosphères produite à partir de l&#39;expansion des tissus congelés et montrent des taux de différenciation très comparable à neurosphères générés à partir de tissus frais.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995-073</td>
<td>High gluclose 1X</td>
</tr>
<tr>
<td>Bovine calf serum supplemented</td>
<td>&nbsp;</td>
<td>HYCLONE</td>
<td>SH30072.03</td>
<td>For culture medium </td>
</tr>
<tr>
<td>Neurobasal-A Medium (1X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>1088-022</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>N2 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>Trypsin (10X)</td>
<td>&nbsp;</td>
<td>Cellgro </td>
<td>25-054CI </td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Deoxyribonuclease 1 from bovine pancreas (DNase)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4527-30KU</td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) (10X)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14065</td>
<td>Cleaning tissue, washes, and freezing medium</td>
</tr>
<tr>
<td>Poly-L-Lysine- 500mg</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2636</td>
<td>Substrate for adhesion of neuronal cells</td>
</tr>
<tr>
<td>antibiotic-antimycotic (100X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>To prevent contamination</td>
</tr>
<tr>
<td>Large orifice pipette Tips (1-200 ul)</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>02-681-141</td>
<td>To prevent shear stress to cells</td>
</tr>
<tr>
<td>Graduated pipette tips (101-1000 ul)</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1111-2721</td>
<td>&nbsp;</td>
<tr>
<td>21 G1 precision guide needles</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>305165</td>
<td>To clean tissue</td>
</tr>
<tr>
<td>10 ml pipette </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1071-0810</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>926-9-04</td>
<td>&nbsp;</td>
<tr>
<td>Single edge razor blade</td>
<td>&nbsp;</td>
<td>Smith Brand</td>
<td>67-0238</td>
<td>To chop tissue</td>
</tr>
<tr>
<td>60 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>For general culture</td>
</tr>
<tr>
<td>100 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0010</td>
<td>For cleaning tissue</td>
</tr>
<tr>
<td>Cryogenic box </td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5026-1010</td>
<td>&nbsp;</td>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5100-0001</td>
<td>"Mr. Frosty"</td>
</tr>
<tr>
<td>2.0 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5012-0020</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>D128-500</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Ethanol 200 proof</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>For sterilizing razor blade</td>
</tr>		</tbody>
		</table>
		</div>

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

Dans cette étude, nous présentons un protocole standardisé pour la cryoconservation et la décongélation de succès corticale blocs de tissus du cerveau pour générer hautement enrichi cultures neuronales. Pour ce protocole le milieu de congélation utilisé est de 10% de diméthylsulfoxyde (DMSO) dilué dans une solution saline tamponnée de Hank (HBSS). Les blocs de tissu cortical sont transférés à cryotubes contenant le milieu de congélation et lentement congelés à -1 ° C / min dans un récipient vitesse contrôlée de congélation. Post-traitement de dégel et de la dissociation des blocs de tissus congelés systématiquement produit des cultures enrichies en neurones, qui a affiché une croissance neuritique rapide au cours des 5 premiers jours de culture et de l&#39;expansion importante du réseau neuronal dans les 10 jours. Coloration immunocytochimique avec le marqueur protéique astrocytaire acide fibrillaire gliale (GFAP) et le marqueur de neurones bêta-tubuline de classe III, a révélé un nombre élevé de neurones et les astrocytes dans les cultures. Génération de neurones cultures de cellules précurseurs de tissus après la dissociation conduit à bloquer en pleine expansion neurosphères, qui a produit un grand nombre de neurones et les astrocytes dans des conditions de différenciation. Ce protocole de cryoconservation simple permet de rapides, de conservation efficace et peu coûteux de corticale blocs de tissus du cerveau, qui accorde une flexibilité accrue pour la production ultérieure de neurones, astrocytes, neurones et les cultures de cellules précurseurs.

Watch this Scientific Journal Video about Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures at JoVE.com

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Ici, nous décrivons une méthode pour la cryoconservation efficace et dégel du cortex cérébral des blocs de tissus pour générer hautement enrichi cultures neuronales. Ce protocole simple fournit la flexibilité pour la génération plus tard, des neurones, des astrocytes et des cultures de cellules neuronales précurseur.

La cryoconservation de tissu cortical Blocs pour la génération des cultures de neurones hautement enrichi

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

cryopreservation-cortical-tissue-blocks-for-generation-highly

Research

JoVE Journal

Neuroscience

13.5K vues.  University of California, Irvine. Ici, nous décrivons une méthode pour la cryoconservation efficace et dégel du cortex cérébral des blocs de tissus pour générer hautement enrichi cultures neuronales. Ce protocole simple fournit la flexibilité pour la génération plus tard, des neurones, des astrocytes et des cultures de cellules neuronales précurseur.

Vidéo: Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Génération de cellules souches neurales à partir de tissus humains mis au rebut corticale fœtale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Recherche

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 ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Dérivation des cultures enrichies en oligodendrocytes et Oligodendrocyte / Neuron myélinisantes co-cultures de post-natal tissus murins

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

Un système entièrement automatisé et hautement polyvalent pour Test Multi-Fonctions cognitives et l&#39;enregistrement des activités neuronales chez les rongeurs

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Préparation de neurones co-cultures avec l&#39;unité portable de précision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

L&#39;incubation prolongée de aiguë Neuronal tissus pour électrophysiologie et Calcium-imagerie

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Augmentation de la réponse du réseau à la stimulation des cultures cellulaires neuronales sur les tableaux de microélectrodes

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Génération sans OGM de cellules neuronales dérivées du sang

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

Cultures enrichies en cônes de la rétine d’embryons de poulet pour étudier les interactions cellulaires entre tiges et cônes

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Génération robuste et hautement reproductible d’organoïdes cérébraux corticaux pour la modélisation de la sénescence neuronale cérébrale in vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...