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

This work was funded by NINDS - NIH R01NS060756

1. Before Starting the Preparation of Hippocampal Slices.
<ol>
<li>Prepare the tissue slicer by placing a piece of Teflon sheet and mounting a new blade.</li>
<li>Wipe the tissue culture (TC) hood with 70% ethanol and set the dissecting microscope inside. Sterilize the hood, microscope, tissue slicer and all dissecting instruments for 15 minutes with UV light.</li>
<li>Prepare six well TC plates. Add 750 &mu;L slice culture media (SCM) per well and place cell culture inserts in each well. Make sure the membran...

<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>

This method is based on the method first described by Stoppini et al. 11 and offers a rapid manner to culture hippocampal slices. The most important aspect of this protocol is to maintain slices sterile; therefore it is critical to use appropriate sterile techniques and to properly disinfect and sterilize all the material in contact with the tissue.
Different serums sources can influence the quality of the slices. We recommend testing several batches first. If contaminat...

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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

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.

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

Organotypic Hippocampal Slice Cultures

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.

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

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Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

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Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

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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

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 ...

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

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

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

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

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

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

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