Questo studio è stato finanziato in parte dalla Fondazione Ipsen. IMV è un americano Cancer Society Professore di Biologia Molecolare, ed è titolare della cattedra e Joan Irwin Jacobs in Scienze della vita esemplare. Questo lavoro è stato sostenuto in parte da finanziamenti del NIH, Fondazione Leducq, Meriaux Fondazione, Ellison Medical Foundation, Ipsen / Biomeasure, Sanofi Aventis, Prostate Cancer Foundation, e la HN e Frances C. Berger Foundation. PAS è finanziato dal Fondo per McKnight Endowment Neuroscienze e il National Institute on Drug Abuse (R01 DA019022). Gli autori desiderano ringraziare Mark Schmitt. Grafica è stato progettato da Jamie T. Simon. Sponsorizzazione è stato gentilmente fornito da Invitrogen.

1. Organotipica Culture cerebellare: <ol><li> Per generare con successo organotipica culture cerebellare uso topi tra P0-P3 o P8-P12. Per il nostro esperimento abbiamo usato topi nel P8-P12 gamma perché questo stadio di sviluppo è stato più appropriato per il nostro studio. </li><li> Preparare la dissezione e terreno di coltura: <ol><li> Dissezione del cervello e la preparazione fetta sono svolte in condizioni di scarsa artificiale di sodio liquido cerebrospinale (ACSF) contenente cloruro di calcio 1 mm, 10mM di D-glucosio, 4mm cloruro di potassio, cloruro di magnesio 5mm, 26mm bicarbonato di sodio, saccarosio e 246 mm rosso fenolo soluzione (1:1000) , pH 7.3. Per sterilizzare la soluzione, filtrare su filtro a 0,22 micron e conservare a 4 ° C. </li><li> Fette cerebellari sono coltivate nel 75% minimo essenziale mezzo di Eagle (MEM), il 25% di siero siero di cavallo, HEPES 25mm, 1 mM glutammina, 5 mg / ml di glucosio, la penicillina (100 U / ml) e streptomicina (100 U / mL). Filtrare il mezzo attraverso un filtro a 0,22 micron e conservare a 4 ° C per un massimo di due settimane. </li></ol></li><li> Sezionare il cervello in gelide medio ACSF precedentemente bollito con Carbogen (95% di ossigeno e anidride carbonica del 5%) Si noti, che la saturazione con Carbogen cambierà il pH della soluzione in modo che si sposterà da rosso ad arancione. La dissezione deve essere eseguita nel minor tempo possibile facendo attenzione a non danneggiare la struttura del cervello. </li><li> Tagliate il cervello in sezioni sagittali di 400 micron utilizzando un vibratome. <ol><li> Fare un taglio sagittale con una lametta in un emisfero, cercando di tagliare il meno possibile del cervelletto, ciò fornirà una superficie piana per affettare. La corteccia viene utilizzato solo per il supporto per rendere il taglio più facile, ma perché il tempo è una questione importante che è più conveniente tagliare la parte rostrale della corteccia con una lametta. </li><li> Usa cianoacrilato (supercolla) per fissare la posizione del cervello sul piatto vibratome metallo. E &#39;importante per raffreddare la piastra sul ghiaccio prima di aggiungere la colla e di diffondere bene usando la punta del tubo per formare un sottile strato adesivo. Se c&#39;è un eccesso di colla, si staccano dalla piastra quando viene a contatto con il ACSF e il campione sarà perso. </li><li> Una volta che il cervello è attaccato alla piastra vibratome, aggiungere i media ACSF abbastanza per coprire il cervello e mettere il ghiaccio sotto la piastra per mantenerlo freddo. </li><li> Tagliate le fette 400 micron e trasferirli con una pipetta Pasteur di plastica (tagliare la punta di ampliare ed evitare danni al fette) per un 6-pozzetti di coltura cellulare contenente ACSF sul ghiaccio. </li><li> Separare il cervelletto dal resto del cervello con l&#39;aiuto di due siringhe da insulina (siringhe di piccolo diametro 28-gauge aghi) sotto il microscopio da dissezione. Alcune fette avrà supercolla attaccato ai bordi: questo è il momento di provare a rimuoverlo in quanto può influenzare la vitalità della fetta. E &#39;importante farlo con cura, senza danneggiare la struttura del cervelletto. </li></ol></li><li> Trasferire le fettine cerebellare a 6-pozzetti contenenti inserti di 30 millimetri piastra di coltura con 0,4 micron pori. Aggiungere 1 ml di terreni di coltura per pozzetto, che è stato condizionato dalla pre-incubazione per almeno 2 ore a 37 ° C e 5% di CO 2 (in colture cellulari incubatore). Posizionare gli inserti sulla parte superiore del supporto assicurandosi che non ci siano bolle d&#39;aria intrappolate sotto e poi posto le fette cerebellare (1-3 per inserto) sulla parte superiore del inserti assicurandosi che siano completamente estesi e che nessun liquido li copre. Aspirare l&#39;eccesso di mezzi di comunicazione, se necessario. Incubare a 37 ° C e 5% di CO 2 modifica del mezzo di coltura ogni 2 o 3 giorni. È meglio sostituire solo una porzione del mezzo (ad esempio il volume ½) in una sola volta, dal momento che contiene mezzo utilizzato fetta derivati ​​fattori trofici che sono importanti per la sopravvivenza delle cellule. </li></ol> 2. Fas trattamento e colorazione di sezioni cerebellare: <ol><li> Per l&#39;esperimento sfida apoptotico, abbiamo prima cultura le fette per 5 giorni la sostituzione del supporto il giorno in vitro 3 (DIV3). Su DIV5 culture sono trattati con 0,5 mg / mL di anticorpi Jo2 Fas agonista aggiungendolo direttamente sul supporto in modo da avere uno sfondo pulito di morte cellulare in quanto i cambiamenti dei media possono indurre alcuni mortalità nelle culture. La metà dei pozzi sono lasciati non trattati come controlli. </li><li> Dopo 24 ore togliere la carta sotto gli inserti di aspirazione. Per risolvere le fette, aggiungere 1 ml di ghiaccio freddo PFA 4% e 1 mL sotto sopra l&#39;inserto. Incubare per 10 minuti a temperatura ambiente. </li><li> Dopo 10 minuti togliere il PFA e lavare brevemente con PBS freddo. </li><li> Avanti rimuovere il PBS e aggiungere 1 sotto mL e 1 mL sopra l&#39;inserto del 20% ghiacciata metanolo in PBS e incubare per 5 minuti a temperatura ambiente. </li><li> Lavare nuovamente con PBS e aggiungere 1 mL e 1 mL sotto sopra l&#39;inserto del 0,5% Triton X-100 in PBS per permeabilizzazione. Incubare per un minimo di 12 ore a 4 ° C. </li><li> Dopo permeabilizzazione, blocco con il 20% di BSA in PBS per un minimo di4 ore a temperatura ambiente o durante la notte a 4 ° C. In questa fase le fette può essere conservato in soluzione di blocco per almeno 3 giorni a 4 ° C. </li><li> Al fine di macchiare le fette tagliate con cura la parte della membrana inserto allegato alla fetta cerebellare e trasferirlo a 24-pozzetti contenenti PBS. </li><li> L&#39;immunocolorazione è fatta in modo sequenziale, prima con reagente TUNEL per un&#39;ora e poi con l&#39;anticorpo Calbidin durante la notte. <ol><li> Aspirare il PBS e aggiungere 250 microlitri off reagente TUNEL in ciascun pozzetto facendo attenzione che copre l&#39;intera superficie della fetta. Incubare a 37 ° C per un&#39;ora al buio. </li><li> Dopo un&#39;ora rimuovere il mix TUNEL e lavare con PBS per tre volte per 10 minuti. </li><li> Dopo l&#39;ultimo lavaggio, rimuovere il PBS e aggiungere 250 microlitri di una diluizione 1 / 1000 della dell&#39;anticorpo calbindin in PBS e incubare una notte a 4 ° C al buio. </li><li> Rimuovere la soluzione primaria di anticorpi e lavare con PBS per tre volte per 10 minuti. </li><li> Aggiungere 250 microlitri di anticorpo secondario diluito 1 / 500 in PBS e incubare per almeno tre ore a temperatura ambiente al buio. Il kit TUNEL che abbiamo usato è etichettato con TMR rosso, quindi il nostro anticorpo secondario per calbindin è coniugato con Alexa-488. </li><li> Lavare tre volte per 10 minuti con PBS e montare le fette su un vetrino di vetro con un altro supporto di montaggio che contiene DAPI. </li><li> Immagine le fette con un microscopio confocale utilizzando il 488 nm e 561 nm lunghezza d&#39;onda di eccitazione per calbindin e TUNEL, rispettivamente. </li></ol></li></ol> 3. Rappresentante dei risultati: La sfida principale di questo protocollo è quello di essere in grado di generare sano culture fetta cerebellare, soprattutto perché la nostra lettura finale sarà la morte delle cellule. Una sana cultura appare come quella in cui lo strato corpo cellulare è trasparente e permette di vedere la struttura lamellare del cervelletto al microscopio (Figura 1). Dopo pochi giorni di cultura le fette inizio alle cellule sottili e non-neuronali possono essere osservati migrare lontano dai margini della fetta. Le cellule tondo scuro (macrofagi più probabile) che coprono le fette dopo pochi giorni di cultura, alla fine diminuzione del numero dopo 10-14 giorni in vitro (Figura 2) 5. La prova finale per la vitalità delle fette è a macchia di differenti marcatori cellulari, come calbindin per le cellule di Purkinje. La figura 3 mostra un&#39;immagine rappresentativa microscopio confocale dello strato di cellule di Purkinje con diversi nuclei TUNEL positivi. E &#39;importante considerare che, anche se alcune fette ricevuto uno stimolo apoptotico, sono stati esposti solo per 24 ore. Questo ha permesso il tempo sufficiente per osservare la frammentazione del DNA nelle cellule morenti con la colorazione TUNEL, ma un determinato strato di cellule di Purkinje era ancora presente. <img src="/files/ftp_upload/2564/2564fig1.jpg" alt="Figura 1" /> Figura 1. Sana fetta cerebellare a DIV2. Questa immagine mostra il corpo traslucido strato di cellule e la struttura lamellare cerebellare in una fetta sano. <img src="/files/ftp_upload/2564/2564fig2.jpg" alt="Figura 2" /> Figura 2. Sana fetta cerebellare a DIV 7. In questa immagine possiamo ancora osservare la struttura lamellare del cervelletto e la comparsa di corpi cellulari buio. <img src="/files/ftp_upload/2564/2564fig3.jpg" alt="Figura 3" /> Figura 3. Immagine rappresentativa di una fetta Fas trattati cerebellare. Confocale Questa immagine mostra le cellule di Purkinje colorate con calbindin (verde) e le cellule apoptotiche positivo per la colorazione TUNEL (rosso). Cellule di Purkinje non appaiono in una singola riga, ma sono raggruppati formando una struttura simile folium.

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

Tutto il lavoro svolto animale è stato fatto in accordo con le politiche stabilite dal IACUC. L&#39;Istituto Salk è una struttura AAALAC accreditati.

Questo metodo descrive una delle molte applicazioni possibili di culture neuronali organotipica e ci ha permesso di studiare l&#39;effetto degli stimoli apoptotici nel wild-type e mutanti fette cerebellare. La parte più critica di questo esperimento è quello di essere in grado di generare costantemente sano culture fetta cerebellare. I fattori chiave sono la dissezione e la tecnica di taglio e la capacità di eseguire il protocollo nel minor tempo possibile, ma allo stesso tempo facendo attenzione a non danneggiare le fette. Un altro fattore cruciale è la preparazione e la composizione dei terreni di coltura, queste culture sono molto sensibili, quindi abbiamo dovuto provare diverse ricette e marche di supporti. Anche diversi lotti di siero o di altri componenti dei mass media possono influenzare la vitalità delle fette. Abbiamo ottenuto i migliori risultati con il siero di cavallo da Lot Invitrogen # 480116. Una volta che le fette cerebellare si generano si può studiare lo sviluppo del cervelletto, elettrofisiologia, la sopravvivenza delle cellule da soli o in risposta a stimoli apoptotici, eccitotossicità o mancanza di ossigeno. Studi comparativi con wild-type e mutanti fette cerebellare sono stati molto perspicace in molti aspetti della funzione cerebellare e sviluppo.

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

Culture organotipica del tessuto neuronale sono stati introdotti da Hogue nel 1947 1,2 e hanno costituito un importante passo avanti nel campo delle neuroscienze. Da allora, la tecnica è stata sviluppata ulteriormente e attualmente ci sono molti modi diversi per preparare culture organotipica. Il metodo presentato qui è stato adattato da quello descritto da Stoppini et al. Per la preparazione delle fette e da Gogolla et al. Per la procedura di colorazione 3,4. Una caratteristica unica di questa tecnica è che permette di studiare diverse parti del cervello come l&#39;ippocampo e cervelletto nella loro struttura originale, garantendo un grande vantaggio rispetto culture dissociato in cui sono sconvolto tutta l&#39;organizzazione cellulare e reti neuronali. Nel caso del cervelletto è ancora più vantaggiosa perché consente lo studio delle cellule di Purkinje, estremamente difficili da ottenere come dissociata coltura principale. Questo metodo può essere utilizzato per studiare alcune caratteristiche dello sviluppo del cervelletto in vitro, come pure per studi elettrofisiologici e farmacologici, sia wild-type e topi mutanti. Il metodo qui descritto è stato progettato per studiare l&#39;effetto degli stimoli apoptotici, come ligando Fas nel cervelletto di sviluppo, utilizzando colorazione TUNEL per misurare la morte cellulare. Se la colorazione TUNEL è combinato con marcatori tipo di cellule specifiche, come calbindin per le cellule di Purkinje, è possibile valutare la morte cellulare in maniera specifica popolazione di cellule. La colorazione calbindin ha inoltre lo scopo di valutare la qualità delle culture del cervelletto.

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

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Questo metodo descrive la generazione di organotipica culture cerebellare e l&#39;effetto di alcuni stimoli apoptotici sulla vitalità dei diversi tipi di cellule del cervelletto.

Organotipica Culture cerebellare: sfide apoptotiche e rilevamento

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

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

Research

JoVE Journal

Neuroscience

18.1K Views.  The Salk Institute for Biological Studies. Questo metodo descrive la generazione di organotipica culture cerebellare e l'effetto di alcuni stimoli apoptotici sulla vitalità dei diversi tipi di cellule del cervelletto.

Video: Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic Hippocampal Slice Cultures

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

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

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 ...

Ricerca

Neuroscienze

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

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

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

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 ...

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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

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

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

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

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

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

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

L&#39;analisi di neurovascolare ritocca in Entorhino-ippocampali Culture Organotipica Slice

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

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

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

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

L&#39;esaurimento selettiva di microglia da cerebellare granuli colture cellulari Utilizzando L-leucina estere metilico

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

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

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

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

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 ...

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

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

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

Colture Hippocampal Fetta organotipiche come modello di studio Neuroprotection e l'invasività delle cellule del tumore

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

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

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

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

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

Ex Utero Elettroporazione e colture Organotypic fetta di cervelli embrionali del Mouse per vivere-Imaging di migrazione interneuroni GABAergici

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