Nous reconnaissons K. Mikic, qui ont contribué à la phase initiale de ce film et M. Nicolescu pour l&#39;image miR. C. Huber a été soutenue par une bourse de la IZKF du Universtitätsklinikum Tübingen, A. Alwin Prem Anand par le programme fortune de la Universtitätsklinikum Tübingen.

Une. Exigences pour les ovo En électroporation <ol><li> Préparation de la construction de vecteur: miR amorces sens et antisens ont été conçus après les instructions de Ambion, recuits et ligaturées dans Apal / digéré par EcoRI U6.1 vecteur pSilencer. Après une transformation réussie des clones résultants a été cultivée et pSilencer plasmide a été isolé par la méthode de la lyse alcaline à l&#39;aide d&#39;un kit de préparation Qiagen midi. </li><li> Électrodes: Les électrodes peuvent être achetés ou facilement être construits 13. Nous utilisons des électrodes de self-made de fil de platine (Φ = 0,2, 0,25 ou 0,5 mm de diamètre) ou, dans certains cas, un fil-électrode de tungstène encore plus faible (Φ = 0,1 mm) 8 en fonction de la zone que nous voulons pour l&#39;électroporation. Pour éviter de brûler du tissu, nous utilisons en règle générale la plus proche du tissu le plus mince des électrodes. Pour large électroporation de mésencéphale, nous utilisons 0,5 fils de platine mm soudées à un tube laiton / acier inoxydablearbre et fixée à un porte-électrode avec un rayon de 5.3 mm (figure 1). Pour l&#39;électroporation des domaines spécifiques, on utilise 0,25 mm anodes en fil de platine de 0,2 mm et un fil de platine ou de tungstène de 0,1 mm de la cathode à une distance d&#39;environ 1 mm ou moins. </li><li> Solutions: Demandez les solutions suivantes Prêt: <ol><li> Assortie de poulet à l&#39;autoclave, pH 7,5 (9,0 g NaCl, 0,42 g de KCl, 0,24 g de CaCl2, dissoudre dans 1 L d&#39;eau distillée). </li><li> PBS (NaCl 8 g, 0,2 g de KCl, 0,24 g de KH 2 PO 4, 1,44 g de Na 2 HPO 4, dissoudre dans 1 litre de H 2 O distillée). </li><li> Fast Green: solution mère à 10 mg / ml H 2 O distillée </li><li> Encre de séchage lent (moins de laque et donc moins empoisonnement de l&#39;embryon). Remarque: un filtre dichroïque bleu fixé à une lampe à fibre optique tel que décrit dans Canto-Soler et Adler 14 améliore le contraste et peut éliminer la nécessité d&#39;injecter de l&#39;encre. </li></ol></li></ol><img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg"/> Figure 1. Diagramme schématique des électrodes et du porte-électrode. (A, B, C), le fil de platine est soudé à un côté d&#39;un arbre tubulaire en acier inoxydable. Un trou a été percé dans l&#39;autre extrémité de l&#39;arbre tubulaire ainsi que des fiches à broches peuvent s&#39;adapter po (A) et de fils de couleur rouge bleu ont été connectées aux fiches à broches. À l&#39;autre extrémité du fil a été installé sur les fiches bananes qui travaillent avec les prises électriques de la électroporateur. (A, C) Le fil de platine a été plié en un ange de 90 °. Une couche de vernis à ongles isolé l&#39;arbre au-dessus du coude. 2. Préparatifs directement devant Dans ovo électroporation <ol><li> Préparer le matériel suivant: <ol><li> Diluer l&#39;encre avec du poulet sonnerie 01:06, ajouter la solution antibiotique-antimycosique (100x de GIBCo) 1:100. </li><li> Préparer la solution de vecteur: 1-2 pg / pl par ADN spécifique en H 2 O complété avec Green rapide (1:20). </li><li> Stériliser pinces, ciseaux, aiguilles de tungstène et des électrodes avec 70% d&#39;éthanol. </li><li> Tirer micropipettes pour l&#39;injection de l&#39;acide nucléique à partir de capillaires en verre de borosilicate (longueur à diamètre extérieur: 100 mm x 0,9 mm). Nous utilisons simple extracteur PUL-1 (WPI, Berlin) avec les paramètres indiqués dans le tableau 1. </li><li> Régler les paramètres de l&#39;électroporateur comme indiqué dans le tableau 2. </li></ol></li></ol><table border="1" ><tbody><tr><td fo:width="1in"> Paramètres </td><td fo:width="1in"> Valeurs </td></tr><tr><td> Chaleur </td><td> 350 </td></tr><tr><td> Tirer </td><td> 100 </td></tr><tr><td> Vitesse </td&gt; <td> 50 </td></tr><tr><td> Retard </td><td> 200 </td></tr></tbody></table> Tableau 1. Réglages pour PUL-1 <table border="1"><tbody><tr><td fo:width="1.5in"> Paramètres </td><td fo:width="1.5in"> Valeurs </td></tr><tr><td> Fréquence </td><td> 50 Hz </td></tr><tr><td> Retard </td><td> 20-50 msec * </td></tr><tr><td> Largeur </td><td> 20 msec </td></tr><tr><td> Tension </td><td> 8-25 V * </td></tr><tr><td> Impulsion </td><td> 3 - 5 fois </td></tr><tr><td colspan="2"> * Selon le diamètre de l&#39;électrode et de la distance. S&#39;il vous plaîten tenir compte lors du changement de l&#39;un des paramètres. </td></tr></tbody></table> Tableau 2. Réglages pour le stimulateur d&#39;impulsions <ol start="2"><li> Préparer des embryons de poulet pour l&#39;électroporation: <ol><li> Incuber des oeufs de poule fécondés, de leur côté plus de 37 ° C avec 65% d&#39;humidité pour 39 h d&#39;acquérir embryons entre Hamburger &amp; Hamilton (HH) met en scène 15 9-11. L&#39;embryon s&#39;installe à la partie supérieure de l&#39;œuf, de sorte que vous devrait marquer avec un trait de crayon. </li><li> Désinfecter les œufs avec 70% d&#39;éthanol. </li><li> Percer l&#39;œuf à son côté plus large, où la cavité d&#39;air devrait être. </li><li> Retirez 1-2 ml de blanc d&#39;oeuf. Cela détacher l&#39;embryon de la coquille. </li><li> Cello-bande la coquille pour éviter les éclats d&#39;obus tombent sur l&#39;embryon. Utilisez une paire de ciseaux pour couper une petite fenêtre dans la coquille autour de la ligne de crayon. </li><li> Injecter un peu d&#39;encreen dessous de la zone opaca de visualiser l&#39;embryon. </li><li> Faire une coupe dans la membrane vitelline avec une aiguille de tungstène aiguisées pour obtenir une meilleure accessibilité de l&#39;embryon pour l&#39;injection de l&#39;acide nucléique et l&#39;électroporation. </li></ol></li></ol> 3. Électroporation et incubation <ol><li> Ajouter quelques gouttes de solution de Ringer chaud sur les embryons. Cela permettra de diminuer les embryons, d&#39;éviter toute surchauffe, le collage des électrodes au tissu et à fournir un support pour le courant entre les deux électrodes. </li><li> Injecter la solution de vecteur dans la lumière du tube neural au niveau du mésencéphale. Si le vecteur d&#39;expression d&#39;ADN ne contient pas de gène rapporteur (par exemple EGFP), ajouter un vecteur de gène rapporteur légèrement inférieure concentré (1 ug / ul miR vecteur exprimant à 0,9 ug / vecteur du gène rapporteur ul) à la solution d&#39;ADN. Cela permettra d&#39;assurer que vous reconnaissez cellules transfectées plus tard 8. Nous utilisons le vecteur soi-disant pCAX, un cadeau aimable vientm C. Krull 13. </li><li> Pour une large transfection du mésencéphale, placer les électrodes gauche et à droite de l&#39;embryon à la hauteur du mésencéphale. La distance entre les électrodes doit être d&#39;environ 3-5 mm. Les autres paramètres sont 15V, 20 impulsions ms et 50 ms de retard pour HH 10. Le changement de position des deux électrodes légèrement le long de la (DV) axe dorso-ventral du mésencéphale assure une plus large transfection de cellules de la transfection de l&#39;axe de DV. </li><li> Pour la transfection ventrale, ajouter un peu de sonnerie de relever la tête de la membrane du jaune et placer l&#39;anode sous mésencéphale ventral. Si la tête ne dépasse pas pousser l&#39;électrode sous la membrane qui sépare le jaune et l&#39;embryon d&#39;éviter de brûler du tube neural. La cathode doit être placé au-dessus du mésencéphale dorso-latérale opposée à l&#39;anode. Ne touchez pas le tube neural. Nous transfecter la moitié ventrale du mésencéphale gauche pour éviter des difficultés avec le développement du cœur. La distance entre les électrodesest d&#39;environ 1-0,5 mm, le reste des paramètres sont 10V, 20 msec de large et 50 ms de retard. </li><li> Appliquer une goutte de solution saline sur des embryons pour la refroidir et enlever les bulles d&#39;air. </li><li> Sceller les oeufs avec la bande et incuber pendant au moins 4 heures, le temps minimum requis pour l&#39;expression de vecteur. </li></ol> 4. Fixation des embryons de poulet <ol><li> Retirez les embryons de leurs œufs. </li><li> Embryons nettoyer sous un stéréomicroscope et d&#39;évaluer l&#39;ampleur de la transfection avec un microscope stéréo fluorescent. </li><li> Fixer les embryons pendant la nuit dans 4% PFA à 4 ° C. Procéder à la coupe et / ou coloration (hybridation in situ, immunohistochimie) pour analyser les changements dans le domaine transfectées. </li></ol>

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Cette vidéo montre une méthode efficace pour transfecter le plasmide dans les cellules neuro-épithéliales des zones spécifiques du cerveau moyen de poussin. Impulsions électriques rectangulaires de basse tension peuvent introduire de l&#39;ADN dans des cellules du tube neural de poussin in ovo 6,16. Toutefois, la précision de ciblage de l&#39;ADN est souvent entravée par le champ électrique de large, qui s&#39;élève à travers les relativement grandes électrodes (Φ = 0,5 mm). Nous avons ...

La reconnaissance de petits ARN non-codants que les joueurs supplémentaires pour l&#39;expression du gène a lancé une nouvelle complexité à la réglementation génomique programmation / de gène. Différentes espèces d&#39;ARN non-codants ont une importance fonctionnelle dans les cellules neuronales, y compris les petits ARN non-codant 1-4. Les microARN miR (ou miARN) par exemple montrent des profils d&#39;expression distincts et l&#39;évolution dans le cerveau en développement 5. Ciblée dans électroporation in ovo d&#39;embryons de poulet offre une occasion unique pour le contrôle temporelle et spatiale de l&#39;expression génique et de la répression au cours du développement. Cette vidéo montre les différentes étapes de réalisation d&#39;expression ectopique de mirs dans des zones spécifiques du cerveau moyen de poussin en utilisant l&#39;électroporation in ovo 6-10. Pour assurer un effet de longue durée de ces petits ARN non codants dans les cellules, la séquence d&#39;ADN de mirs ont été clones dans des vecteurs de mono-ou bi-cistroniques. Pour en électroporation in ovo, miR contenant vecteur est injecté dans le tube neural par exposition du mésencéphale embryonnaire après avoir effectué une petite fenêtre dans la coquille de l&#39;oeuf. Pour transfecter des zones spécifiques du petit plus mésencéphale (anode) et moins (cathode) électrodes de platine sont placés à des positions spécifiques. Pour la transfection du mésencéphale ventral, l&#39;anode est placée sous le mésencéphale ventral gauche et la cathode au-dessus de la moitié droite du mésencéphale avant l&#39;application d&#39;un courant. L&#39;ouverture dans la coquille d&#39;oeuf est fermé avec la bande et les embryons sont incubés pendant aussi longtemps que nécessaire pour toute l&#39;analyse. Cette méthode a été décrite par Muramatsu et al. 6 et amélioré par Momose et al. 8 pour la zone transfection spécifique. <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> Vue d&#39;ensemble schématique. <ol><li> L&#39;embryon dans l&#39;œuf est exposée en coupant une petite fenêtre en ee coquille. </li><li> Le vecteur (s) en solution est injecté dans le mésencéphale en utilisant un micro-capillaire. </li><li> Deux électrodes placées parallèlement - ou en dessous et au-dessus de l&#39;embryon, - générer un champ électrique pulsé. </li><li> Le champ électrique crée temporairement des pores dans la membrane cellulaire, ce qui facilite l&#39;entrée dans la cellule par l&#39;ADN chargé négativement (ou d&#39;ARN) attirés par l&#39;anode 11,12. </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

Les ARN non codantes sont des acteurs supplémentaires dans la régulation de l&#39;expression génique. Ciblée dans électroporation in ovo de domaines spécifiques fournit un outil unique pour le contrôle spatial et temporel de l&#39;expression de microARN extra-utérine. Cependant, les structures du cerveau comme le mésencéphale ventral ventrales sont plutôt difficiles à atteindre pour tous les manipulations. Ici, nous démontrons un moyen efficace pour l&#39;électroporation des miARN dans le mésencéphale ventral en utilisant des électrodes de platine minces. Cette méthode offre un moyen fiable pour transfecter des zones spécifiques du cerveau moyen et un outil utile pour les études in vivo.

L&#39;étendue de la région mésencéphalique transfectée avec miR est visible au travers de l&#39;expression de GFP à partir d&#39;un vecteur rapporteur injectée en même temps que le vecteur exprimant miR (figure 2). En utilisant des électrodes en platine d&#39;un diamètre de 0,5 mm et placée parallèlement à l&#39;axe AP du conduit mésencéphale normalement à la transfection d&#39;une large zone le long de la et DV-AP-axe, dont rhombencéphale, le mésencéphale, et parfois diencéphale <st...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

L&#39;expression ectopique est une technique permettant d&#39;éclaircir le rôle des microARN dans le développement du cerveau. Cependant, le ciblage des zones spécifiques à l&#39;aide de l&#39;électroporation in ovo est un défi. Ici, nous montrons de manière efficace à des régions du mésencéphale ventral et dorsal sélectivement l&#39;électroporation.

Dans Expression in ovo de microARN dans ventrale Poussin mésencéphale

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

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Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

10.4K vues.  University of Tübingen.  L'expression ectopique est une technique permettant d'éclaircir le rôle des microARN dans le développement du cerveau. Cependant, le ciblage des zones spécifiques à l'aide de l'électroporation in ovo est un défi. Ici, nous montrons de manière efficace à des régions du mésencéphale ventral et dorsal sélectivement l'électroporation. 

Vidéo: In ovo Expression of MicroRNA in Ventral Chick Midbrain

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 ...

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In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

En électroporation in ovo dans Mésencéphale Poussin pour étudier la fonction des gènes dans le développement neurone dopaminergique

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Profilage global de régulation de la dopamine dans la substance noire et aire tegmentale ventrale

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Deux enregistrements électrophysiologiques des synapses-réponses évoquées astrogliales et neuronales aiguës dans des coupes d&#39;hippocampe

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

ARN-Seq analyse de l&#39;expression génique différentielle dans électroporation embryon de poulet de la moelle épinière

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Modulations environnementaux du mésencéphale Nombre de dopamine neurones chez la souris adulte

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

Identification fiable de la vie Neurones Dopaminergiques dans les cultures Mésencéphale utilisant l&#39;ARN Séquençage et TH-promoteur-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Un modèle In Ovo pour tester les composés insulino-mimétiques

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Traçage anatomique et fonctionnel in vivo combiné des terminaux de glutamate de la zone tegmentale ventrale dans l’hippocampe

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Quantification spontanée Ca2+ Fluxes et leurs effets en aval dans les neurones midbrains de souris primaires

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...