Cette étude a été financée par la Division du programme de recherche intra-muros (PRID) de l&#39;Institut national de santé mentale, National Institutes of Health.

<ol>
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Aucun conflit d&#39;intérêt déclaré.

1. Problèmes techniques: <ol><li> Technique stérile. La préparation de l&#39;AME et la préparation de la culture sont toutes effectuées dans une hotte à flux laminaire dans des conditions stériles. Les antibiotiques, qui affectent l&#39;activité neuronale, ne sont pas utilisées à tout moment pendant le processus de préparation et de la culture. </li><li> Plasma / thrombine coagulation et l&#39;adhérence des tissus sur la survie des AEM. Tissu sur la MEA exige un équilibre délicat entre le temps requis pour le plasma / thrombine coagulation et le temps d&#39;exposition des tissus à l&#39;atmosphère. Un temps de coagulation courte risques décollement prématuré des sections de la MEA, tandis que l&#39;exposition prolongée à l&#39;atmosphère provoque la dégénérescence des tissus. Parce que la force de la solution de thrombine détermine la vitesse du processus de la coagulation, il est un paramètre très important pour réussir à fixer, des cultures saines à la surface de la MEA. Nous obtenons de meilleurs résultats à l&#39;aide de 1000 unités (1KU; 1 unité = 0,324 ± NIH 0,073 mg). Surtout, mélange incomplet des résultats solution de plasma / thrombine dans la coagulation du plasma spatialement hétérogènes promotion-ruptures en cours de culture. «Trous» Ces plasmatique gravement affecter la santé de la culture et de détacher la culture de microélectrodes, compromettant ainsi la qualité de l&#39;enregistrement électrophysiologiques. Travailler avec des plaques de refroidissement lors de la MEA / tissu montage ralentit la coagulation et permet un mélange homogène de la solution de plasma / thrombine et le bon positionnement des sections de tissus. De même, en ajoutant le milieu dans des gouttelettes unique pour la chambre de culture de submerger la culture, après seulement 5 min de la coagulation, réduit considérablement le risque de détachements de tissu due à des tensions de surface. Une culture réussie va aplatir sur la MEA et légèrement expansion montrant une croissance saine pendant des semaines, sans aucun des signes importants de tissus incomplète-MEA contact ou la dégénérescence des tissus (par exemple, Fig. 1D). </li><li> Dissection des tissus. Couteaux Micro ont grandement amélioré notre processus de dissection. Nous utilisons des lames de rasoir à double pointe (Outils Fine Science - cassables les lames de bistouri - 100050-00), à partir de laquelle nous avons divisé ~ 2 mm de large &quot;lames&quot; en utilisant une pince, et les maintenir avec un support de scalpel. Des échantillons de tissus disséqués de la tranche coronale en utilisant une surface lisse, mouvement ferme verticale de la lame ce qui réduit considérablement les contraintes mécaniques dues à la traction du tissu améliorer la santé globale de la culture. </li><li> Refroidissement des tissus. Refroidissement correct des tranches et des coupes de tissus lors de la préparation est essentielle pour la réussite de la culture. Nous utilisons des faits sur des assiettes froides construites d&#39;éléments Peltier attaché en dessous pour un disque de métal. La chaleur produite par l&#39;élément Peltier est enlevé par une perfusion d&#39;eau froide. Ceci réduit considérablement le temps de préparation et standardise le refroidissement pendant chaque étape de la préparation (Labs Dold &amp; Engineering 131 Plantation Dr Seguin, TX 78155;. (830) 560-1471)). </li><li> Incubateur état. Un incubateur sur mesure avec précision les conditions de bascule interne a été essentielle à notre succès à cultiver les tranches sur les AEM. Basé sur un original en interne la conception par Multichannelsystems (actuellement non disponible dans le commerce), l&#39;appareil bascule interne est composé de plateaux qui sont attachés aux deux roues grand côté. Les moteurs pas à pas et des contrôles informatisés permettant une trajectoire précise à bascule (rocking angle, bascule la vitesse, et pauses intermittentes). Finalement, les cultures tranche besoin d&#39;être exposés à moyenne atmosphère et la culture en alternance lente. L&#39;approche traditionnelle est de placer les cultures tranche dans des tubes étroits qui tournent lentement le long il axe le plus long. Ici, la rotation lente ne produit pas de contraintes mécaniques dues à la rotation elle-même, et la vitesse de rotation est suffisamment élevée pour obtenir un optimal »d&#39;alimentation / respiration» du cycle d&#39;environ 5 à 10 min durée. L&#39;intérieur plus compact de la chambre de la MEA, son volume total d&#39;environ 2 cc, et le volume de culture petit moyen nécessaire pour le conditionnement de support par le tissu lui-même, représente un défi important. En basculant la MEA entre ± ~ 70 ° d&#39;angle (temps de cycle: ~ 200 s), ce qui ralentit la vitesse de balancement que les transitions de la culture entre la phase liquide et l&#39;atmosphère, et l&#39;arrêt de la bascule au niveau des angles extrêmes d&#39;une exposition prolongée à l&#39;atmosphère a été essentielle pour la survie de la culture. </li></ol> 2. Le stade de développement des cultures à l&#39;étude du cortex avalanches neuronale Tranches de cortex de rats aiguë sont souvent prises au PND 0-1 et cultivées pendant plusieurs semaines sur la MEA. Les premières études ont clairement démontré que seule tranche de cultures cortex, après plusieurs semaines in vitro, de maintenir une structure en couches avec différents types cellulaires identifiables qui peuvent être facilement comparées aux classes de cellules in vivo 18,18-21. L&#39;organisation en couches dans ce système in vitro a été utilisée pour étudier commodément thalamiqueinnervation du cortex pendant le développement 22-24, ainsi que pour la conduite des structures sous-corticales comme le striatum 25,26. En fait, la spécificité de la formation des connexions neuronales au sein et entre les régions du cerveau permet la construction de complexes systèmes in vitro que la récupération de nombreux systèmes de projection détaillée, par exemple, que des ganglions de la base du cortex-circuits 27-30. Après 4 - 6 semaines in vitro, seule tranches et des tranches de cortex 31 cortex co-cultivées avec striatum 26 ou 32 montrent thalamus spontanée haut et le bas on trouve habituellement dans les États vivo chez le rat anesthésié uréthane 33. L&#39;organisation temporelle fine de ces états-place porte le poinçon de imbriquées θ et γ-oscillations indicative d&#39;un réseau électrophysiologique des neurones pyramidaux matures et fast-dopage interneurones GABAergiques 31. Surtout, en l&#39;absence de dopamine récepteur D2 de stimulation, la maturation de la parvalbumine-positif interneurones corticaux est retardée d&#39;environ deux semaines dans les cultures tranche de cortex 34. En ligne avec ces résultats, le cours du temps de développement de imbriquées θ-, β-et γ-oscillations est adaptée à celle in vivo lorsque tranches cortex sont co-cultivées avec l&#39;aire tegmentale ventrale (VTA), qui contient les neurones dopaminergiques se projetant vers l&#39; corticale 6. Ces études indiquent que lorsque l&#39;on étudie les avalanches neuronale, qui dépendent essentiellement matures inhibition GABAergique rapide et sont situés dans les couches superficielles du cortex 2,4, le plus grand soin doit être pris pour assurer la maturation adéquate du tissu cortical. Alors que les avalanches surviennent dans les cultures neuronales du cortex seule au cours du temps de 2 - 5 semaines 4, en exigeant un cours à temps de développement qui est adaptée à la développement in vivo, des tranches de cortex ont besoin de stimulation d&#39;un récepteur approprié de la dopamine, par exemple, par tranches de cortex co-culture avec le VTA 6.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Intégré array multiélectrodes planaire </td><td> Systèmes multicanaux ( <a href="www.multichannelsystems.com">www.multichannelsystems.com</a> ) ALA Scientific Instruments (Www.alascience.com) </td><td> <a href="http://www.multichannelsystems.com/products-mea/product-details/products/158/20030ir-ito-wo.html">200/30iR-ITO-w/o</a> </td><td> Titane nitrates (TiN) électrodes (30 mm de diamètre) ont une surface importante résultant en une faible impédance (~ 1,5 kQ à 1 kHz) et une excellente bande large enregistrements (w / o - sans anneau) </td></tr><tr><td> Chambre en verre </td><td> www.aceglass.com </td><td> 7620-32 </td><td> Cylindre de verre fileté </td></tr><tr><td> Chambre bouchon </td><td> www.aceglass.com </td><td> 7622-114 </td><td> Bouchon en plastique avec garniture en Teflon </td></tr><tr><td> Sylgard 184 </td><td> www.wpiinc.com </td><td> SYLG184 </td><td> Élastomère de silicone en deux parties </td></tr><tr><td> Poly-D-lysine </td><td> Sigma-Aldrich </td><td> P6407-5mg </td><td> γ-irradiés, poudre lyophilisée, cellule cultivée testés. Reconstitué avec 5 ml d&#39;eau désionisée avant usage. </td></tr><tr><td> Gey solution saline équilibrée </td><td> Sigma-Aldrich </td><td> G9779-500ml </td><td> filtré stérile et cultivé testé </td></tr><tr><td> de poulet de plasma </td><td> Sigma-Aldrich </td><td> P3266-5mL </td><td> Lyophilisée, reconstituer avec 5 ml d&#39;eau déminéralisée avant utilisation. </td></tr><tr><td> thrombine </td><td> Sigma-Aldrich </td><td> T6634-1KU </td><td> à partir de plasma bovin, sous forme de poudre lyophilisée. </td></tr><tr><td> sérum de cheval </td><td> Sigma-Aldrich </td><td> H1138-100mL </td><td> troupeau donneur, inactivé par la chaleur, la culture de cellules testées </td></tr><tr><td> Medium base de Eagle </td><td> Invitrogen </td><td> 21010-046 </td><td> 1x, 500 ml - (+) des sels de Earle, (-) L-glutamine), </td></tr><tr><td> Hank solution saline tamponnée </td><td> Invitrogen </td><td> 24020-117 </td><td> 500 ml - (+) Magnésium, (+) de calcium, w / rouge de phénol) </td></tr><tr><td> Diapositives Chambre </td><td> Lab-Tek Chamber-Glissez w / couvercle (Nunc), stérile </td><td> 177429 </td><td></td></tr><tr><td> Uridine </td><td> Sigma-Aldrich </td><td> U3003 </td><td></td></tr><tr><td> ARA-C-β-cytosine D-arabinofuranoside </td><td> Sigma-Aldrich </td><td> C6645 </td><td></td></tr><tr><td> 5-fluoro-2&#39;-désoxyuridine </td><td> Sigma-Aldrich </td><td> F0503 </td><td></td></tr></tbody></table>

multi-electrode array recordings of neuronal avalanches in organotypic cultures

Le cortex est spontanément actif, même en l&#39;absence de toute entrée ou sortie particulière du moteur. Lors du développement, cette activité est importante pour la migration et la différenciation des types de cellules du cortex et de la formation des connexions neuronales 1. Dans l&#39;animal adulte, l&#39;activité continue reflète le passé et l&#39;état actuel d&#39;un animal dans lequel les stimuli sensoriels sont parfaitement intégrés pour calculer les actions futures. Ainsi, une compréhension claire de l&#39;organisation de cours de l&#39;activité spontanée savoir est une condition préalable pour comprendre la fonction du cortex. De nombreuses techniques d&#39;enregistrement a révélé que l&#39;activité continue dans le cortex est constitué de nombreux neurones dont les activités individuelles transitoirement somme à des événements plus importants qui peuvent être détectés dans le potentiel de champ local (LFP) avec des microélectrodes extracellulaires, ou de l&#39;électroencéphalogramme (EEG), le magnétoencéphalogramme (MEG ), et le signal BOLD de l&#39;imagerie par résonance magnétique fonctionnelle (IRMf). La LFP est actuellement la méthode de choix lorsque l&#39;on étudie l&#39;activité de la population neuronale avec une résolution temporelle et spatiale à l&#39;échelle mésoscopique (plusieurs milliers de neurones). Lors de la microélectrode extracellulaire, localement synchronisées activités de résultat neurones spatialement neighbored de déflexions rapides dans la LFP jusqu&#39;à plusieurs centaines de microvolts. Lorsque vous utilisez un tableau de microélectrodes, les organisations de tels détournements peuvent être facilement surveillés dans l&#39;espace et du temps. Avalanches neuronale décrire l&#39;organisation invariante d&#39;échelle spatio-temporelle de l&#39;activité neuronale dans le cerveau continue 2,3. Elles sont spécifiques à des couches superficielles du cortex comme établi in vitro, 4,5, in vivo chez le rat anesthésié 6, et chez le singe éveillé 7. Surtout, les études théoriques et empiriques suggèrent que les avalanches 2,8-10 neuronale montrent une dynamique bien équilibré état ​​critique du cortex qui optimise le transfert de l&#39;information et de traitement de l&#39;information. Afin d&#39;étudier les mécanismes du développement neuronal avalanches, l&#39;entretien et la réglementation, dans des préparations in vitro sont très bénéfiques, car ils permettent des enregistrements d&#39;activité stable avalanches dans des conditions contrôlées avec précision. L&#39;actuel protocole décrit comment étudier les avalanches neuronales in vitro, en profitant du développement de la couche superficielle du cortex dans les cultures organotypiques, les cultures tranche-dire, cultivé sur planaires, les tableaux de microélectrodes intégré (AME; voir aussi 11-14).

Watch this Scientific Journal Video about Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures at JoVE.com

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Une manière robuste à l&#39;étude des avalanches neuronale, c&#39;est à dire invariante d&#39;échelle éclate l&#39;activité spatio-temporelle, indicative de la dynamique de l&#39;état critique dans le cortex. Avalanches émergent spontanément dans le développement de couches superficielles du cortex culture qui permet de mesures à long terme de l&#39;activité avec des tableaux intégrés planaires multi-électrodes (MEA) dans des conditions contrôlées avec précision.

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 ...

multi-electrode-array-recordings-neuronal-avalanches-organotypic

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Neuroscience

26.3K vues.  National Institute of Mental Health. Une manière robuste à l'étude des avalanches neuronale, c'est à dire invariante d'échelle éclate l'activité spatio-temporelle, indicative de la dynamique de l'état critique dans le cortex. Avalanches émergent spontanément dans le développement de couches superficielles du cortex culture qui permet de mesures à long terme de l'activité avec des tableaux intégrés planaires multi-électrodes (MEA) dans des conditions contrôlées avec p...

Vidéo: Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

Enregistrements tableau multiélectrodes de l&#39;épithélium Vomeronasal

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

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Neurosciences

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 ...

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 ...

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 ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

Conception et de fabrication de Ultralight Poids, réglables sondes multi-électrodes pour électrophysiologiques enregistrements chez la souris

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Appariés enregistrements de cellules entières dans organotypiques coupes d&#39;hippocampe

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Stimulation à courant continu et multi-réseau d&#39;électrodes d&#39;enregistrement de la capture comme activité chez la souris Cerveau Slice Préparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...