Questo studio è stato finanziato dalla Divisione del programma di ricerca intramurale (DIRP) del National Institute of Mental Health, National Institutes of Health.

<ol>
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M., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+avalanches+are+diverse+and+precise+activity+patterns+that+are+stable+for+many+hours+in+cortical+slice+cultures.">Neuronal avalanches are diverse and precise activity patterns that are stable for many hours in cortical slice cultures.</a> J Neurosci. 24, 5216-5229 (2004).</li><li>Stewart, C. V., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverted-U+profile+of+dopamine-NMDA-mediated+spontaneous+avalanche+recurrence+in+superficial+layers+of+rat+prefrontal+cortex.">Inverted-U profile of dopamine-NMDA-mediated spontaneous avalanche recurrence in superficial layers of rat prefrontal cortex.</a> J. Neurosci. 26, 8148-8159 (2006).</li><li>Stewart, C. 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Nessun conflitto di interessi dichiarati.

1. Questioni tecniche: <ol><li> Tecnica sterile. La preparazione del MEA e preparazione culturale sono tutti eseguiti all&#39;interno di una cappa a flusso laminare in condizioni sterili. Gli antibiotici, che influenzano l&#39;attività neuronale, non vengono utilizzati in qualsiasi momento durante il processo di preparazione e cultura. </li><li> Plasma / trombina coagulazione e l&#39;aderenza del tessuto sul MEA. Tissue sopravvivenza sulla MEA richiede un attento equilibrio tra il tempo richiesto per il plasma / trombina coagulazione e il tempo di esposizione dei tessuti per l&#39;atmosfera. Un tempo di coagulazione breve rischi prematuro distacco di sezioni dalla MEA, mentre l&#39;esposizione prolungata in atmosfera provoca una degenerazione dei tessuti. Perché la forza della soluzione di trombina determina la velocità del processo di coagulazione, è un parametro molto importante per attaccare con successo, le culture sane in superficie MEA. Si ottengono risultati migliori con 1000 unità (1KU; 1 unità NIH = 0,324 ± 0,073 mg). Importante, incompleta miscelazione del plasma / soluzione di trombina risultati nella coagulazione spazialmente eterogeneo promozione plasma rotture durante la coltura. &#39;Buchi&#39; queste plasma gravemente nuocere alla salute della cultura e staccare la cultura dal microelettrodi, compromettendo così la qualità della registrazione elettrofisiologica. Lavorare con piastre di raffreddamento durante la MEA / tessuto assemblaggio rallenta la coagulazione e consente la miscelazione omogenea del plasma / soluzione di trombina e il posizionamento corretto delle sezioni di tessuto. Allo stesso modo, con l&#39;aggiunta di medie gocce solo alla camera di cultura di sommergere la cultura dopo soli 5 minuti di coagulazione, riduce notevolmente il rischio di distacchi del tessuto a causa di tensioni superficiali. Una cultura di successo si appiattisce sulla MEA e leggermente ampliare mostrando una crescita sana per settimane, senza alcun segno importante di incompleta tessuto-MEA contatto o degenerazione dei tessuti (es. fig. 1D). </li><li> Dissezione dei tessuti. Coltelli Micro hanno notevolmente migliorato il nostro processo di dissezione. Noi usiamo due lame di rasoio bordo (Strumenti Belle Scienza - fragili lame di bisturi - 100050-00), da cui abbiamo diviso ~ 2 mm di larghezza &#39;lame&#39; con una pinza, e tenerli con un supporto bisturi. Campioni di tessuto sezionato dalla fetta coronale con una superficie liscia, movimento verticale fermo della lama quindi riducendo notevolmente lo stress meccanico a causa della trazione del tessuto complessivo miglioramento della salute della cultura. </li><li> Tessuto di raffreddamento. Il corretto raffreddamento delle fette e sezioni di tessuto durante la preparazione è essenziale per il successo della cultura. Noi usiamo piatti freddi su misura costruito su elementi Peltier attaccato sotto ad un disco di metallo. Il calore prodotto dall&#39;elemento Peltier viene rimosso attraverso la perfusione acqua fredda. Questo riduce notevolmente i tempi di preparazione e standardizza il raffreddamento durante ogni fase della preparazione (Labs Dold &amp; Engineering 131 Plantation Dr. Seguin, TX 78155;. (830) 560-1471)). </li><li> Condizione incubatrice. Una misura incubatore con precise condizioni a dondolo interno è stato fondamentale per il nostro successo nel coltivare le fette su MEA. Sulla base di un originale in-house di progettazione Multichannelsystems (attualmente non disponibile in commercio), il dispositivo a dondolo interno è costituito da vassoi che sono collegati a due ruote laterali di grandi dimensioni. Motori passo-passo e controlli computerizzati consentono una precisa traiettoria a dondolo (angolo a dondolo, dondolo velocità e intermittenti pause). In ultima analisi, culture fetta devono essere esposti a medio ambiente e la cultura in alternanza lento. L&#39;approccio tradizionale è quello di posizionare le culture fetta in tubi stretti che ruotano lentamente lungo ci asse più lungo. Qui, lenta rotazione non produce sollecitazioni meccaniche dovute alla stessa rotazione e la velocità di rotazione è abbastanza alta da ottenere un ottimale &#39;alimentazione / respirare&#39; ciclo di circa 5 - durata 10 min. L&#39;interno, più compatto della camera di MEA, il suo volume totale di circa 2 cc, e il piccolo volume di terreno di coltura richiesta per il condizionamento di media dal tessuto stesso, pone una sfida significativa. Facendo scorrere l&#39;MEA tra ± ~ 70 ° angolo (tempo di ciclo: ~ 200 s), rallentando la velocità a dondolo come le transizioni cultura tra la fase liquida e l&#39;atmosfera, e ad arrestare la dondolo agli angoli estremi per la prolungata esposizione all&#39;atmosfera è stato essenziale per la sopravvivenza della cultura. </li></ol> 2. Età evolutiva di culture corteccia per studiare valanghe neuronale Fette acuta da corteccia di ratto sono comunemente prese a PND 0-1 e colto per molte settimane sulla MEA. I primi studi hanno chiaramente dimostrato che singole culture fetta corteccia, dopo diverse settimane in vitro, mantenere una struttura a strati con tipi di cellule identificabili che possono essere facilmente rispetto alle classi di cellule in vivo 18,18-21. L&#39;organizzazione a strati in questo sistema in vitro è stato convenientemente utilizzata per lo studio del talamoinnervazione della corteccia durante lo sviluppo 22-24, così come per la guida delle strutture sottocorticali, come lo striato 25,26. In effetti, la specificità nella formazione di connessioni neuronali all&#39;interno e tra regioni del cervello permette per la costruzione di complessi sistemi in vitro che riprendono numerosi sistemi di proiezione dettagliate, ad esempio quello della corteccia, dei gangli basali circuiti 27-30. Dopo 4 - 6 settimane in vitro, solo fette corteccia 31 e fettine di corteccia co-coltura con striato 26 o 32 talamo manifestazione spontanea e GIÙ stati tipicamente presenti in vivo sul ratto anestetizzato uretano 33. L&#39;organizzazione bel temporale di questi Stati-up porta il marchio di garanzia di annidati θ e γ-oscillazioni indicativo di una rete elettrofisiologico maturo di neuroni piramidali e interneuroni GABAergici fast-spiking 31. È importante sottolineare che, in assenza di recettori della dopamina D2-stimolazione, la maturazione degli interneuroni corticali parvalbumina-positivi è ritardata di circa 2 settimane nelle culture fetta corteccia 34. In linea con questi risultati, la durata dello sviluppo di annidati θ-, β-e γ-oscillazioni corrisponde a quella in vivo, quando fette corteccia sono co-coltura con l&#39;area tegmentale ventrale (VTA), che contiene i neuroni dopaminergici che proiettano la corteccia 6. Questi studi indicano che quando si studia valanghe neuronale, che dipendono essenzialmente dal maturo inibizione veloce GABAergici e si trovano negli strati superficiali della corteccia 2,4, grande cura deve essere presa per assicurare corretta maturazione del tessuto corticale. Mentre valanghe neuronale sorgono nelle culture corteccia singolo nel corso di tempo di 2 - 5 settimane 4, quando si richiede un corso evolutivo tempo che è abbinato al vivo nello sviluppo, fette corteccia bisogno di un appropriato stimolazione del recettore della dopamina, ad esempio di co-coltura fette corteccia con la VTA 6.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Array integrato multielectrode planare </td><td> I sistemi multicanale ( <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> Nitrato di titanio (TiN) elettrodi (30 mm di diametro) hanno grande superficie con conseguente bassa impedenza (~ 1,5 kΩ a 1 kHz) ed eccellenti registrazioni a larga banda (w / o - senza anello) </td></tr><tr><td> Camera di vetro </td><td> www.aceglass.com </td><td> 7620-32 </td><td> Cilindro di vetro filettato </td></tr><tr><td> Camera di tappo </td><td> www.aceglass.com </td><td> 7622-114 </td><td> Cappuccio di plastica con inserto in teflon </td></tr><tr><td> Sylgard 184 </td><td> www.wpiinc.com </td><td> SYLG184 </td><td> Due parti in silicone elastomero </td></tr><tr><td> Poli-D-lisina </td><td> Sigma-Aldrich </td><td> P6407-5mg </td><td> γ-irradiati, polvere liofilizzata, cellule in coltura testato. Ricostituito con 5 ml di acqua deionizzata prima dell&#39;uso. </td></tr><tr><td> Sale Gey La soluzione bilanciata </td><td> Sigma-Aldrich </td><td> G9779-500mL </td><td> sterile filtrata e colta testato </td></tr><tr><td> pollo al plasma </td><td> Sigma-Aldrich </td><td> P3266-5ml </td><td> Liofilizzato, ricostituire con 5 ml di acqua deionizzata prima dell&#39;uso. </td></tr><tr><td> trombina </td><td> Sigma-Aldrich </td><td> T6634-1KU </td><td> dal plasma bovino, sotto forma di polvere liofilizzata. </td></tr><tr><td> siero di cavallo </td><td> Sigma-Aldrich </td><td> H1138-100 ml </td><td> mandria donatore, inattivato con il calore, la cultura cellulari testate </td></tr><tr><td> Basale medio Aquila </td><td> Invitrogen </td><td> 21010-046 </td><td> 1x, 500 ml - (+) Earle Sali, (-) L-glutamina), </td></tr><tr><td> Soluzione salina tamponata di Hank </td><td> Invitrogen </td><td> 24020-117 </td><td> 500 ml - (+) Magnesio, (+) di calcio, w / rosso fenolo) </td></tr><tr><td> Camera di diapositive </td><td> Lab-Tek-Camera scorrere w / cover (Nunc), sterile </td><td> 177429 </td><td></td></tr><tr><td> Uridina </td><td> Sigma-Aldrich </td><td> U3003 </td><td></td></tr><tr><td> ARA-C citosina-β-D-arabinofuranoside </td><td> Sigma-Aldrich </td><td> C6645 </td><td></td></tr><tr><td> 5-fluoro-2&#39;-deossiuridina </td><td> Sigma-Aldrich </td><td> F0503 </td><td></td></tr></tbody></table>

multi-electrode array recordings of neuronal avalanches in organotypic cultures

La corteccia è attiva spontaneamente, anche in assenza dei fattori di produzione o di uscita del motore. Durante lo sviluppo, questa attività è importante per la migrazione e la differenziazione dei tipi di cellule della corteccia e la formazione di connessioni neuronali 1. Nel animale adulto, l&#39;attività in corso riflette il passato e il presente stato di un animale in cui gli stimoli sensoriali sono perfettamente integrati per calcolare le azioni future. Così, una chiara comprensione dell&#39;organizzazione del corso dell&#39;attività ovvero spontanea è un prerequisito per capire la funzione della corteccia. Tecniche di registrazione ha rivelato che numerose attività in corso nella corteccia si compone di molti neuroni le cui singole attività transitoriamente somma ai grandi eventi che possono essere rilevati nel potenziale di campo locale (LFP) con microelettrodi extracellulari, o nel elettroencefalogramma (EEG), il magnetoencephalogram (MEG ), e il segnale BOLD dalla risonanza magnetica funzionale (fMRI). La LFP è attualmente il metodo di scelta quando si studia l&#39;attività neuronale popolazione con alta risoluzione temporale e spaziale alla scala mesoscopica (diverse migliaia di neuroni). Al microelettrodo extracellulare, localmente attività sincronizzata del spazialmente neighbored risultato neuroni deviazioni rapido l&#39;LFP fino a diverse centinaia di microvolt. Quando si utilizza una serie di microelettrodi, le organizzazioni di tali deviazioni possono essere comodamente controllati nello spazio e nel tempo. Valanghe neuronale descrivere la scala-invariante organizzazione spazio-temporale in corso di attività neuronale del cervello 2,3. Essi sono specifici per gli strati superficiali della corteccia, come stabilito in 4,5 vitro, in vivo nel ratto anestetizzato 6, e nella scimmia sveglio 7. È importante sottolineare che gli studi teorici ed empirici suggeriscono che le valanghe 2,8-10 neuronale indicano una dinamica squisitamente equilibrato stato critico della corteccia che ottimizza il trasferimento di informazioni e di elaborazione delle informazioni. Al fine di studiare i meccanismi di sviluppo valanga neuronale, la manutenzione e la regolamentazione, in preparati in vitro sono di grande beneficio, in quanto consentono di registrazioni stabile di attività valanghiva a condizioni ben controllate. L&#39;attuale protocollo descrive come studiare valanghe neuronali in vitro, sfruttando lo sviluppo strato superficiale in organotipica corteccia culture, culture fetta cioè, coltivate su planare, array di microelettrodi integrati (MEA, vedi anche 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

Un metodo efficace per studiare valanghe neuronale, cioè scala-invarianti spazio-temporale scoppia attività, indicativa delle dinamiche condizioni critiche nella corteccia. Valanghe emergere spontaneamente nello sviluppo di strati superficiali della corteccia cerebrale in coltura che permette di misure a lungo termine delle attività con gli array planare integrata multi-elettrodi (MEA), a condizioni ben controllate.

Multi-array di elettrodi Recordings Valanghe neuronali in Culture organotipica

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

Research

JoVE Journal

Neuroscience

26.3K Views.  National Institute of Mental Health. Un metodo efficace per studiare valanghe neuronale, cioè scala-invarianti spazio-temporale scoppia attività, indicativa delle dinamiche condizioni critiche nella corteccia. Valanghe emergere spontaneamente nello sviluppo di strati superficiali della corteccia cerebrale in coltura che permette di misure a lungo termine delle attività con gli array planare integrata multi-elettrodi (MEA), a condizioni ben controllate.

Video: 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.

Array registrazioni Multielectrode dell&#39;epitelio Vomeronasal

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

Ricerca

Neuroscienze

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.

Organotipica Culture Slice ippocampale

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.

Culture Slice organotipica del mesencefalo ventrale embrionali: un sistema per studiare dopaminergici sviluppo neuronale 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;analisi di morfologia dendritica delle cellule di Purkinje in Culture Slice organotipiche

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.

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

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.

Progettazione e fabbricazione di Ultralight Peso, regolabili sonde multi-elettrodo per elettrofisiologiche Registrazioni nei topi

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.

Associati a cellule intere registrazioni in organotipiche fettine di ippocampo

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.

Indagare rigenerazione funzionale in organotipiche midollo spinale Co-culture Grown in array multi-elettrodo

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.

Stimolazione diretta correnti e multi-elettrodo Array registrazione di sequestro simile attività nei topi cervello fetta di preparazione

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 ...