Name: organotypic hippocampal slice cultures
Uploaded: 2011-02-03T17:30:00
Description: Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures at JoVE.com

Questo lavoro è stato finanziato dal NINDS - NIH R01NS060756

1. Prima di avviare la preparazione di fettine di ippocampo. <ol><li> Preparare l&#39;affettatrice tessuto mettendo un pezzo di foglio di teflon e montare una nuova lama. </li><li> Pulire la coltura del tessuto (TC) cappuccio con il 70% di etanolo e impostare l&#39;interno microscopio da dissezione. Sterilizzare il cofano, microscopio, affettatrice tessuti e tutti gli strumenti di dissezione per 15 minuti con la luce UV. </li><li> Preparare sei piastre TC. Aggiungere 750 microlitri terreni di coltura slice (SCM) per b...

<ol>
	<li>Martin, S. J., Grimwood, P. D., Morris, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+and+memory:+an+evaluation+of+the+hypothesis.">Synaptic plasticity and memory: an evaluation of the hypothesis.</a> Annu Rev Neurosci. 23, 649-711 (2000).</li><li>Bliss, T. V., Collingridge, G. L., Morris, R. G. I. n. t. r. o. d. u. c. t. i. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+potentiation+and+structure+of+the+issue.">Long-term potentiation and structure of the issue.</a> Philos Trans R Soc Lond B Biol Sci. 358, 607-611 (2003).</li><li>Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The synaptic organization of the brain. , (2004).</li><li>Malinow, R., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+enhancement+shown+by+whole-cell+recordings+of+long-term+potentiation+in+hippocampal+slices.">Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices.</a> Nature. 346, 177-180 (1990).</li><li>Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+green+fluorescent+protein+(GFP)+into+hippocampal+neurons+through+viral+infection.">Introduction of green fluorescent protein (GFP) into hippocampal neurons through viral infection.</a> Cold Spring Harb Protoc. , (2010).</li><li>Shi, S., Hayashi, Y., Esteban, J. A., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-specific+rules+governing+AMPA+receptor+trafficking+to+synapses+in+hippocampal+pyramidal+neurons.">Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons.</a> Cell. 105, 331-343 (2001).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li><li>Esteban, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKA+phosphorylation+of+AMPA+receptor+subunits+controls+synaptic+trafficking+underlying+plasticity.">PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity.</a> Nat Neurosci. 6, 136-143 (2003).</li><li>Mainen, Z. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+in+living+brain+slices.">Two-photon imaging in living brain slices.</a> Methods. 18, 231-239 (1999).</li><li>Barria, A., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-specific+NMDA+receptor+trafficking+to+synapses.">Subunit-specific NMDA receptor trafficking to synapses.</a> Neuron. 35, 345-353 (2002).</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>Simoni, A. D. e., Griesinger, C. B., Edwards, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+rat+CA1+neurones+in+acute+versus+organotypic+slices:+role+of+experience+in+synaptic+morphology+and+activity.">Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity.</a> J Physiol. 550, 135-147 (2003).</li></ol>

Questo metodo si basa sul primo metodo descritto da Stoppini et al. 11 e offre un modo rapido a fette cultura dell&#39;ippocampo. L&#39;aspetto più importante di questo protocollo è quello di mantenere fette sterile, quindi è fondamentale utilizzare appropriate tecniche sterili e per disinfettare correttamente e sterilizzare tutto il materiale a contatto con il tessuto. Diverse fonti sieri possono influenzare la qualità delle fette. Si consiglia di provare diversi lot...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell culture inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM03050</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Slicer</td>
<td>&nbsp;</td>
<td>Stoelting</td>
<td>51425/51415</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZX7-ILLD2-100</td>
<td>&nbsp;</td>
<tr>
<td> Hippocampus dissecting tool</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>10099-15</td>
<td>&nbsp;</td>
<tr>
<td>Large utility scissors. Perfection</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>37500-00/37000-00 Right/ Left handed</td>
<td>&nbsp;</td>
<tr>
<td>Iris Spatula</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>10093-13</td>
<td>&nbsp;</td>
<tr>
<td>Straight spatula</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>10094-13</td>
<td>&nbsp;</td>
<tr>
<td>Rounded spoon micro spatula</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57949-039</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting single cutting edge needle</td>
<td>&nbsp;</td>
<td>Electron Microscopy Science</td>
<td>72946</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting tweezers</td>
<td>&nbsp;</td>
<td>Dummont</td>
<td>#2</td>
<td>&nbsp;</td>
<tr>
<td>Small dissecting scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>14060-10</td>
<td>&nbsp;</td>
<tr>
<td>MEM Eagle medium</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>50-019 PB</td>
<td>&nbsp;</td>
<tr>
<td>Horse serum heat inactivated</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26050-88</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3881</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4 (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2773</td>
<td>&nbsp;</td>
<tr>
<td>Insulin (1 mg/ml), dissolved in HCl 0.01 N</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Ascorbic Acid, solution (25%)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4544</td>
<td>&nbsp;</td>
<tr>
<td>D-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6014</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7523</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5016</td>
<td>&nbsp;</td>
<tr>
<td>Phenol Red Solution 0.5% in DPBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P0290</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 (1 M)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9272</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic hippocampal slice cultures

L&#39;ippocampo, un componente del sistema limbico, gioca un ruolo importante nella memoria a lungo termine e la navigazione spaziale 1. Neuroni dell&#39;ippocampo può modificare la forza delle loro connessioni, dopo brevi periodi di forte attivazione. Questo fenomeno, noto come potenziamento a lungo termine (LTP), può durare per ore o giorni ed è diventato il meccanismo miglior candidato per l&#39;apprendimento e la memoria 2. Inoltre, l&#39;anatomia ben definito e connettività dell&#39;ippocampo 3 ha fatto un sistema modello per lo studio classico trasmissione sinaptica e 4 plasticità sinaptica. Come la nostra comprensione della fisiologia delle sinapsi dell&#39;ippocampo crebbe e giocatori molecolare è stato identificato, la necessità di manipolare proteine ​​sinaptiche è diventato un imperativo. Organotipica culture dell&#39;ippocampo offrono la possibilità di manipolazione genetica facile e preciso l&#39;intervento farmacologico, ma mantenere l&#39;organizzazione sinaptica che è fondamentale per comprendere la funzione sinapsi in un contesto più naturalistico di routine cultura metodi dissociato neuroni. Qui vi presentiamo un metodo per la preparazione e la cultura fettine di ippocampo, che può essere facilmente adattato ad altre regioni del cervello. Questo metodo permette di raggiungere facilmente le fette di manipolazione genetica utilizzando approcci diversi, come infezioni virali o 5,6 biolistics 7. Inoltre, fette può essere facilmente recuperato per le analisi biochimiche 8, o trasferiti ad microscopi per l&#39;imaging 9 o esperimenti elettrofisiologici 10.

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures at JoVE.com

Organotypic Hippocampal Slice Cultures

Descriviamo un metodo per preparare organotipica fettine di ippocampo che può essere facilmente adattato ad altre regioni del cervello. Sezioni di cervello sono previste sulle membrane porose e terreni di coltura è consentito costituiscono l&#39;interfaccia. Questo metodo consente di mantenere l&#39;architettura lordo dell&#39;ippocampo per un massimo di 2 settimane in cultura.

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 ...

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Il organotipica ippocampale Cultura Modello Slice per l&#39;esame di danno neuronale

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Ricerca

Neuroscienze

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Registrazione intera cella da un Preparazione Tagliare organotipica della neocorteccia

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

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

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 ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Post-embedding Etichettatura immuno di proteine ​​sinaptiche in Culture Slice ippocampali

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

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 ...

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

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 ...

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

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

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 ...