Diese Arbeit wurde von NINDS finanziert - NIH R01NS060756

1. Vor dem Start der Vorbereitung von Schnitten des Hippocampus. <ol><li> Bereiten Sie das Gewebe Allesschneider, indem ein Stück Teflon Blatt und Montage einer neuen Klinge. </li><li> Wischen Sie die Gewebekultur (TC) Kapuze mit 70% Ethanol und stellen Sie die Präpariermikroskop innen. Sterilisieren der Haube, Mikroskop, Gewebe Hobel und alle Sezierbesteck für 15 Minuten mit UV-Licht. </li><li> Bereiten Sie sechs gut TC-Platten. Add 750 ul Stück Kultur Medien (SCM) pro Vertiefung und Ort Zellkultureinsätzen in jede Vertiefung. Sicherstellen, dass die Membranen sind völlig nass ohne Blasen darunter. Legen Sie die Platten im Brutschrank bei 35 ° C mit 5% CO 2 begast, bis sie benötigt. </li><li> Gießen Sie 50 mL niedrigen Na + ACSF (Sezieren Lösung) in einem 100 mL Becherglas und stellen Sie es auf dem Eis-Salz-Mischung. Blase den niedrigen Na + ACSF mit 5% CO 2 / 95% O 2 bis zum Farbumschlag und ACSF bildet einen Schlamm-Mix von gefrorenen und flüssigen Lösung (10-20 min). </li><li> Get a P5-P7 Jungtier. Bis zu drei Welpen eingesetzt werden kann. </li></ol> 2. Schnitten des Hippocampus Vorbereitung. <ol><li> Schneiden Sie den Kopf des Tieres mit scharfen Schere Dienstprogramm. Schneiden Sie die Haut und setzen den Schädel. Öffnen des Schädels durch Schneiden von Seite zu Seite entlang der interauralen Linie und dann entlang der Sagittalnaht mit einer kleinen Schere. Eine optionale Schnitt von Seite zu Seite in der Front gemacht zu erleichtern das Entfernen der Knochen und der Exposition des Gehirns sein. Scoop aus dem Gehirn schnell mit einem abgerundeten Löffel Mikrospatel und legen Sie sie in die Gülle zu sezieren Lösung für ~ 1 Minute chill. Gießen Sie ca. 10 mL eiskaltem Sezieren Lösung auf eine 60 mm Schale und den Transfer des Gehirns aus dem Becher in die Schale. Das Gehirn soll mit Sezieren Lösung abgedeckt werden. </li><li> Unter dem Binokular: Place des Gehirns und halten Sie ihn an der Mittellinie mit der Pinzette gedrückt, um den unteren Rand des 60 mm Schale. Verwenden Sie den Hippocampus Sezieren Werkzeug, um die Halbkugeln Weglassen des Mittelhirns zu trennen. Der Hippocampus werden dann auf jeder Hemisphäre ausgesetzt. Dann vorsichtig mit einer Schaufel Hippocampus mit dem Hippocampus Sezieren Werkzeug. Verwenden Sie die Präpariernadel völlig zu isolieren Hippocampus und reinigen Sie sie so weit wie möglich. </li><li> Mit einem snipped Spitze eines P1000 Filter Pipettenspitze vorsichtig aspirieren Hippocampus und überträgt es auf die Teflon Folie auf das Gewebe Schneidemaschine. Position des Hippocampus auf der konkaven Seite. </li><li> Richten Sie die hippocampi senkrecht zur Klinge koronalen Abschnitte zu erhalten und Drain überschüssige Flüssigkeit. </li><li> Schneiden Sie die hippocampi alle 400 um. </li><li> Gießen Sie ca. 10 ml kaltem SCM in eine 60 mm Schale und den Transfer in Scheiben geschnitten hippocampi vom Allesschneider mit anderen snipped P1000 Filter Spitze und kalten SCM. Vermeiden Sie Luftblasen. </li><li> Mit Hilfe eines Iris-Spachtel und einer geraden Spatel vorsichtig trennen die Scheiben voneinander. </li><li> Separate gut definiert und unbeschädigt Scheiben von beschädigten Scheiben. </li></ol> 3. Schnitten des Hippocampus Kultur <ol><li> Bringen Sie die Sechs-Well-Platten mit SCM-und Zellkultur-Einsätze aus dem Inkubator. Mit Hilfe eines anderen snipped P1000 Filter Spitze, Transfer einzelnen Scheiben auf die Membran. Legen Sie 4-5 Scheiben pro Membran. Achten Sie darauf, statt der Scheiben entweder an die Wand legen nahe oder dicht beieinander. Wenn nötig, verwenden Iris Spatel zu trennen Scheiben schneiden. Entfernen Sie überschüssiges Medium. Touch-Scheiben so wenig wie möglich, sobald sie auf der Membran sind. </li><li> Bewegen Platte zurück zum Inkubator und Kultur bei 35 ° C und 5% CO 2. </li><li> Ändern SCM alle 48 Stunden innerhalb des TC Haube durch Absaugen des SCM mit einer Pasteurpipette. Add 750 ml frisches vorgewärmtes SCM pro Vertiefung. Achten Sie darauf, keine Blasen unter der Membran gebildet werden. </li></ol> 4. Lösungen <ol><li> Low Na + ACSF - Dissecting Lösung für Slice Kulturen Um deionisiertem und sterilem H 2 O hinzu: <table border="1"><tbody><tr><td></td><td> Für 500 ml </td><td> Für 1000 ml </td><td> Endkonzentration </td></tr><tr><td> CaCl 2 (1 M) </td><td> 0,5 ml </td><td> 1 mL </td><td> 1 mM </td></tr><tr><td> D-Glucose </td><td> 0,901 g </td><td> 1,802 g </td><td> 10 mM </td></tr><tr><td> KCl </td><td> 0,149 g </td><td> 0,298 g </td><td> 4 mM </td></tr><tr><td> MgCl 2 (1 M) </td><td> 2,5 ml </td><td> 5 mL </td><td> 5 mM </td></tr><tr><td> NaHCO 3 </td><td> 1,092 g </td><td> 2,184 g </td><td> 26 mM </td></tr><tr><td> Sucrose </td><td> 40 g </td><td> 80 g </td><td> 234 mm </td></tr><tr><td> Phenolrotlösung 0,5% in DPBS </td><td> 0,5 ml </td><td> 1 mL </td><td> 0,1% v / v </td></tr></tbody></table> Mix ~ 30 min Sterilize Durchgang durch 0,22 &amp;mgr; m-Filter Machen Sie 50 mL aliquotiert und bei 4 ° C nicht länger als 2 Monate. </li><li> Slice-Kulturmedium (SCM) <table border="1"><tbody><tr><td></td><td> Für 500 ml </td><td> Für 1000 ml </td><td> Endkonzentration </td></tr><tr><td> MEM Eagle Medium </td><td> 4,2 g </td><td> 8,4 g </td><td> 8,4 g / l </td></tr><tr><td> Pferdeserum hitzeinaktiviertem </td><td> 100 mL </td><td> 200 mL </td><td> 20% </td></tr><tr><td> L-Glutamin (200 mM) </td><td> 2,5 ml </td><td> 5 mL </td><td> 1 mM </td></tr><tr><td> CaCl 2 (1 M) </td><td> 0,5 ml </td><td> 1 mL </td><td> 1 mM </td></tr><tr><td> MgSO 4 (1 M) </td><td> 1 mL </td><td> 2 mL </td><td> 2 mM </td></tr><tr><td> Insulin (1 mg / mL), gelöst in 0,01 N HCl </td><td> 0,5 ml </td><td> 1 mL </td><td> 1 mg / l </td></tr><tr><td> Ascorbinsäure, Lösung (25% w / v) </td><td> 0,024 ml </td><td> 0,048 ml </td><td> 0,00125% </td></tr><tr><td> D-Glucose </td><td> 1.16g </td><td> 2.32g </td><td> 13 mM </td></tr><tr><td> NaHCO 3 </td><td> 0.22g </td><td> 0,44 g </td><td> 5,2 mm </td></tr><tr><td> Hepes </td><td> 3.58g </td><td> 7.16g </td><td> 30 mM </td></tr></tbody></table> Mix bis gänzlich aufgelöst und auf Raumtemperatur bringen. Der pH-Wert auf 7,27 bis 7,28 mit 1 N NaOH Messen Osmolarität. Passen Sie auf 320 mmol / kg mit deionisiertem und sterilisiert H 2 O. Erwarten Sie etwa 25-40 ml hinzuzufügen. Überprüfen Sie Osmolarität wieder. *** PH-Wert kann etwas ändern beim Einstellen der Osmolarität, das in Ordnung ist, ist es umso wichtiger, dass die Osmolarität im richtigen Bereich (317-323) ist. Sterilisieren durch Passage durch 0,22 um-Filter. Machen Sie 20 mL Aliquots und speichern bis zu 2-3 Wochen bei 4 ° C. Gluatamine Lager: Vorbereitung in einer Konzentration von 200 mM und bei -20 ° C in Aliquots von 2,5 mL. Ascorbinsäure Lager: Vorbereitung in einer Konzentration von 25% (w / v) und bei -20 ° C in Aliquots von 100 ul. </li></ol> 5. Repräsentative Ergebnisse: Scheiben aussehen sollte unter einem Binokular weiß ohne schwarze Flecken und gut definiert und unbeschädigt CA1, CA3 und Gyrus dentatus Regionen. Bakterielle Kontamination ist leicht wie das Verschieben schwarze Flecken in der mittleren oder Trübung des SCM zu sehen. Wenn unter dem Mikroskop gelegt, sollte die Oberfläche der Scheibe schauen nach 4 Tagen in Kultur sauber mit klaren und nachvollziehbaren Zellkörper. Wenn keine klare Zellkörper zu sehen sind und viel Schutt bedeckt die Oberfläche nach 4 Tagen, dann ist keine gesunde Scheibe.

<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>

Diese Methode basiert auf der Methode zuerst von Stoppini et al beschrieben ist. 11 und bietet eine schnelle Art und Weise zur Kultur Schnitten des Hippocampus. Der wichtigste Aspekt dieses Protokolls ist die Erhaltung Scheiben steril, daher ist es wichtig, geeignete sterile Techniken zu verwenden und richtig zu desinfizieren und sterilisieren das gesamte Material in Kontakt mit dem Gewebe. Verschiedene Seren Quellen können Einfluss auf die Qualität der Scheibe...

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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

Der Hippocampus, einer Komponente des limbischen Systems, spielt eine wichtige Rolle in das Langzeitgedächtnis und die räumliche Navigation 1. Hippocampus-Neuronen können die Stärke ihrer Verbindungen nach kurzen starke Aktivierung ändern. Dieses Phänomen, das als Langzeit-Potenzierung (LTP) bekannt ist, kann für Stunden oder Tage dauern und ist mittlerweile der beste Kandidat Mechanismus für Lernen und Gedächtnis 2. Darüber hinaus hat der gut definierten Anatomie und Konnektivität des Hippocampus 3 hergestellt ist ein klassisches Modellsystem zur synaptischen Übertragung und synaptische Plastizität 4-Studie. Als unser Verständnis der Physiologie des Hippocampus Synapsen wuchs und molekulare Spieler identifiziert wurde, wurde die Notwendigkeit, synaptischer Proteine ​​zu manipulieren unerlässlich. Organotypischen hippokampalen Kulturen bieten die Möglichkeit für eine einfache Genmanipulation und präzise pharmakologische Intervention, sondern pflegen synaptischen Organisation, die entscheidend für das Verständnis Synapse Funktion in einem naturalistischen Kontext als Routine-Kultur dissoziierten Neuronen Methoden ist. Hier präsentieren wir eine Methode zur Vorbereitung und Kultur Schnitten des Hippocampus, die leicht auf andere Hirnregionen angepasst werden können. Diese Methode ermöglicht den einfachen Zugriff auf die Scheiben zur genetischen Manipulation mit unterschiedlichen Ansätzen, wie virale Infektionen oder 5,6 Biolistik 7. Darüber hinaus können Scheiben leicht für biochemische Assays 8 gewonnen werden, oder an Mikroskopen für die Bildgebung 9 oder elektrophysiologischen Experimenten 10.

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

Organotypic Hippocampal Slice Cultures

Wir beschreiben eine Methode zur organotypischen Schnitten des Hippocampus, die leicht auf andere Hirnregionen angepasst werden vorbereiten können. Hirnschnitten sind auf porösen Membranen und Kulturmedien ist erlaubt, um eine Schnittstelle bilden gelegt. Diese Methode schont die grobe Architektur des Hippocampus für bis zu 2 Wochen in Kultur.

Organotypischen hippokampalen Slice-Kulturen

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

36.9K Aufrufe.  University of Washington School of Medicine. Wir beschreiben eine Methode zur organotypischen Schnitten des Hippocampus, die leicht auf andere Hirnregionen angepasst werden vorbereiten können. Hirnschnitten sind auf porösen Membranen und Kulturmedien ist erlaubt, um eine Schnittstelle bilden gelegt. Diese Methode schont die grobe Architektur des Hippocampus für bis zu 2 Wochen in Kultur.

Serie: Organotypic Hippocampal Slice Cultures

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Die Organotypische hippokampalen Slice-Kultur-Modell für die Prüfung neuronaler Schädigung

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

Forschung

Neurowissenschaften

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

Whole Cell Aufnahme von einem organotypischen Vorbereitung der Neocortex

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

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.

Organotypischen Kulturen von embryonalen ventralen Mittelhirn: Ein System zur Dopaminerge Neuronale Development Study 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.

Die Analyse von Purkinje Zelle Dendritische Morphologie in organotypischen Kulturen

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 colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Post-Einbettung Immunogoldmarkierung der synaptischen Proteine ​​in Hippocampusschnitt Kulturen

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 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.

Organotypischen Kulturen zu Oligodendrozyten Dynamik und Myelinisierung Studieren

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 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.

Gepaart ganze Zelle Recordings in Organotypische Hippocampus

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 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.

Die Analyse der neurovaskuläre Remodeling in Entorhino-Hippocampus Organotypischen Kulturen

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. 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.

Organotypischen Hippocampal Slice Kulturen als Modell zur Studie Neuroprotektion und Invasivität der Tumorzellen

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 Elektroporation und organotypischen Slice Kulturen der embryonalen Mäusegehirnen für Live-Aufnahmen von Migration Gabaergen Interneuronen

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