Name: organotypic slice cultures for studies of postnatal neurogenesis
Uploaded: 2015-03-04T13:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

HINWEIS: Alle Tierverfahren gemäß den zum Tiergesundheit und Tier Richtlinien der Abteilung für Vergleichende Medizin an der Universität von Toronto. 1. Herstellung von Schnitten des Hippocampus <ol><li> Sterilisieren Sie die folgenden Instrumente mit dem Trocken Autoklaven bei 125 ° C: Skalpellgriff (# 3) (2), Standard-Muster Pinzetten, groß (1), Kleine Dissektor Scheren (abgewinkelt zu Seite) (1), Micro Löffel (Löffel und Flach Spachtel Enden) (1), Mikro-Spatel (abgerundet und aufgerundet verjüngten Enden) (2), Gourmet Pinsel (1), poliert Feuer Pasteur-Pipette (2), Gaze Plätze, 2 x 2 Zoll (5). </li><li> Wenn sterile, legen Sie die Instrumente in einen sterilen Behälter und halten bis zum Gebrauch überdachte. Unmittelbar vor der Dissektion, tauchen Instrumente in einer 70% igen Ethanollösung. </li><li> Bereiten Sie eine 6-Well-Kulturplatte mit Kultureinsatzes vor Beginn der Dissektion Verfahren durch Zugabe von 1 ml Kulturmedium / Vertiefung und Speichern der Platte in den Inkubator bei 35 ° C einnd 5% CO 2. </li></ol> 2. Ordnen Sie Dissection Werkzeuge in sterilen Laminarströmungshaube <ol><li> Sprühen Sie das Steril mit 70% Ethanol und entfernen sterilisiert Dissektionsinstrumente von Alkohol. Ermöglichen, dass die Instrumente zu trocknen, während auf eine sterile Petrischale ruhen, um den Kontakt zwischen dem Alkohol und dem sezierten Hirngewebe zu vermeiden. </li><li> Kaution 5-7 ml sterilisiertem eiskalten Sezieren Lösung in 2 große sterile Petrischalen. Ein Gericht wird zu kühlen und reinigen Sie den Kopf (dirty), die andere zum Kühlen und Spülen der ausgehöhlt Gehirn (clean). Platzieren eines sterilisierten Filterpapier in einer Petrischale Deckel zum Zerlegen das Gehirn. </li><li> Legen Sie eine kleine, sterilisierte Filterpapier in einem der kleinen Petrischale Deckel zum Präparieren Sie das Hippocampus. Kaution 3-5 ml sterilisiertem eiskalten Sezieren Lösung in 2 kleine, sterile Petrischalen. Ein Gericht wird den ausgehöhlt Hippocampus zu halten, wird man Abschnitte während der Abtrennung des Hippocampus Abschnitte haltenunter Dissektionsmikroskop. </li><li> Bereiten Sie das Gewebe Chopper durch Abkleben ein Stück Filterpapier sterilisiert an die Schneidphase und Montage einer sterilen Rasierklinge. Befeuchten Sie das Filterpapier mit sterilisierten Sezieren Lösung. </li><li> Sprühen Sie eine saubere Bio-Beutel mit Alkohol und legen Sie sie in eine laminare Strömung Sterilbank. </li></ol> 3. Hippocampus Dissection <ol><li> Sprühen Sie das P7 Sprague-Dawley Rattenjungen mit 70% Ethanol außerhalb der laminaren Strömung Sterilbank und schnell zu enthaupten, das Tier mit großen sterilen chirurgischen Schere in der Sterilbank. Lassen Sie den Kopf fallen in eiskaltes Sezieren Lösung in einem der Petrischalen. </li><li> In der Petrischale, spülen Sie das Blut und schnell überweisen Sie den Kopf zu sterilisierende Filterpapier, Bauchseite nach unten. </li><li> Verwendung des Skalpell entlang der Rückenfläche in der sagittalen Ebene schneiden die darunterliegende Schädel freizulegen. Schnitt durch die Haut, aber nicht die darunter liegenden Knochen, die weich und bei Ratten t leicht durchlässig istsein Alter. Beiseite dieses &quot;dirty&quot; Skalpell und nicht auf Hirngewebe zu verwenden nicht. </li><li> Mit den kleinen Dissektor Schere (abgewinkelt zu Seite) und Zangen, aufgeschnitten den Schädel entlang Pfeilnaht des Schädels zu Bregma, die anatomische Punkt auf dem Schädel, wo die Kranznaht senkrecht durch die Pfeilnaht durchschnitten. Verwenden einer Pinzette, um Schädel Klappen von der Mittellinie des Schädels auf und davon ziehen. </li><li> Setzen Sie den Mikrolöffel an der Unterseite des Gehirns, unter der Hirnstamm, zu heben, das Gehirn aus dem Schädel. Heben Sie das Gehirn, um die Sehnerven und Riechkolben auf die basale Oberfläche des Gehirns aus. Schneiden Sie diese Strukturen mit einer kleinen Schere vollständig zu lösen, die das Gehirn vor den Schädel. Entfernen Sie und übertragen Sie die intakten Gehirn auf die andere große Petrischale mit eiskaltem Sezieren Lösung. </li><li> Mit dem Mikrolöffel, übertragen das Gehirn auf eine kleine Petrischale Deckel, die steriles Filterpapier. Mit einer sterilen Pasteurpipette gründlich das Gehirn mit ein paar Tropfen disschneidenden Lösung, um Gewebe feucht zu halten. </li><li> Mit Hilfe eines &quot;sauberen&quot; Skalpell schneiden die beiden Halbkugeln auseinander. Übertragen Sie die linke Hemisphäre wieder in großen Petrischale mit Mikrolöffel und legen Hemisphäre pia Seite nach unten in eiskaltem Sezieren Lösung für die spätere Verwendung. </li><li> Sehen Sie sich die medialen Fläche des rechten Hemisphäre und Identifizierung der Randgewölbe, eine herausragende Band aus weißen Substanz entlang der medialen Rand des Hippocampus. Mit einem sterilen Skalpell, einen sagittalen Schnitt durch das Scheidengewölbe, aber darauf achten, denn nur 0,5 cm des Skalpells Spitze wird ausreichen, um den Fornix schneiden. </li><li> Mit 2 Mikro-Spatel, entfernen Sie die ersten Hippocampus vom rechten Hemisphäre, indem Sie den rechten-Spatel auf den Hirnstamm und Anheben des darüber liegenden Kortex mit der linken Spachtel. Heben Sie die Rinde, die Seitenventrikel und mediale Fläche des Hippocampus zu offenbaren. Eine weiße geschwungene Linie, die Fimbria, sollte jetzt sichtbar sein. </li><li> Richten Sie die Krümmung der Spatel mit der Curvatur der Fimbrien und vorsichtig auf die Spachtel unter der Fimbrien. Schieben Sie den Spatel nach links und fahren Sie entlang rostral-kaudale Achse und heben Spachtel in dorsale Richtung Hippocampus zu entfernen. </li><li> Übertragen Sie den Hippocampus zu einem 2. kleinen Petrischale mit eiskaltem Sezieren Lösung. Wiederholen Sie den Vorgang auf der linken Hemisphäre, um den linken Hippocampus zu entfernen. </li><li> Mit einem Mikrospatel, sorgfältig die hippocampi Transfer zum Gewebe Chopper Bühne. Ordnen sie benachbart und parallel zueinander und senkrecht zu der Achse der oszillierenden Klinge. Verwenden Sie einen Pinsel, um das Gewebe zu positionieren und ein paar Tropfen des Präparationslösung auf der Oberseite des Hippocampus. </li><li> Schneiden Sie das Gewebe in 400 &amp; mgr; m Scheiben ohne Pause zu einzelnen Scheiben zu entfernen (in der Regel werden sie nicht an der Klinge haften). Nach den ganzen Hippocampus geschnitten worden ist, verwenden Sie den Pinsel, um vorsichtig zu übertragen die Abschnitte zu einem 2. kleinen Petrischale mit Dissektion Lösung. </li><li> Unter Anzeigeissecting Mikroskop vorsichtig trennen die schwimmenden Scheiben mit amicro-Spachtel und Pinsel. </li><li> Entfernen Sie die vorbereiteten Kulturplatte mit Kultureinsatzes aus dem Inkubator und in eine Sterilbank. </li><li> Mit einer feuerpolierten Pasteur-Pipette, ziehen 4-5 Scheiben in die Pipette und Transfer Scheiben an die apikale Oberfläche der Kultureinsatzes Membran. Als nächstes stellen Sie die Position mit einem Pinsel und lassen Raum zwischen einzelnen Abschnitte und der Grenze des Kultureinsatzes. </li><li> Mit einer sterilen Pasteur-Pipette, entfernen Sie das überschüssige Sezieren Lösung von apikalen Oberfläche der Membran. HINWEIS: Beim Entfernen Lösung vermeiden Zeichnung Gewebeschnitte in die Pipette. Alternativ können Sie einen regelmäßigen Pipette (P200 oder P1000) mit sterilisierten Pipettenspitzen langsam entfernen Lösung. </li><li> Platzieren der Kulturplatte mit serumhaltigem Kulturmedium und den Hippocampus wieder in den Inkubator bei 35 ° C und 5% CO 2. HINWEIS: Wenn Experiment Anrufezur Erzeugung von Kulturen aus verschiedenen Tieren, gründlich zu reinigen die laminare Strömung Sterilbank zwischen Dissektionen. Zurück Instrumente Alkohollösung und ersetzen Sie alle Petrischalen, Filterpapiere und sterilen Rasierklingen vor der zweiten Sektion. </li></ol> 4. Fütterung und Pflege von organotypischen Slices <ol><li> Futterkulturen in einem sterilen laminaren Strömung Sterilbank. </li><li> Führen Sie die erste Fütterung der kultivierten Abschnitten 2 Tage nach der Präparation. Saugen Sie alte Kulturmedium unter Verwendung sterilisiert Glaspipette. </li><li> Sterile 5 ml serologische Pipette 1 ml frisches, steriles, Serum enthaltendem Medium zu den Vertiefungen hinzu. </li><li> Ersetzen Sie vorsichtig die Kultureinsatzes und kümmern uns um die Luftblasen, die unter Membranoberfläche gebildet haben können. </li><li> Nach der ersten Fütterung, jeden zweiten Tag geändert Medium. </li></ol> 5. Inkubation von Gewebeschnitten mit Thymidin-Analoga zu Newborn Neuronen Beschriften <ol><li> Um zu untersuchen,die Reifung und Integration von dentatus Körnerzellen im Hippocampus, brüten die organotypische Scheiben mit einer Thymidinanalogon wie Bromodeoxyuridine (BrdU) oder Chlorodeoxyuridine (CldU). Hier verwenden wir CldU aufgrund seiner höheren Löslichkeit in Salzlösung. </li><li> Nach 3DIV wird mit 1 ul 10 mg / ml Stammlösung CldU in Kochsalzlösung auf 1 ml Kulturmedium bis zu einer Endkonzentration von 10 ug / ml. Wenn BrdU verwendet wird, korrigieren Sie die Konzentration für die Unterschiede im Molekulargewicht. Hinzufügen Medium mit CldU Kultur Vertiefungen inkubieren Gewebes mit CldU haltige Medium für 2 Stunden bei 35 ° C. </li><li> Nach 2 Stunden Inkubation bei 35 ° C, entfernen Sie die CldU haltigen Mediums und Ersetzen mit regulären Fütterungsmedium zur normalen Ernährungsplan (siehe oben) wieder aufzunehmen. </li></ol> 6. Gewebefixierung und Lagerung <ol><li> Stellen Sie einen Zeitplan für die Anwendung Behandlungen und Befestigungsgewebeproben vor Beginn der Kulturexperimenten (2B). </li> &lt;li&gt; Bei einem vorbestimmten Tag nach der Dissektion, bereiten Sie die folgenden in einem Laborabzug: a 10-50 ml Becherglas mit 4% Paraformaldehyd (PFA) in phosphatgepufferter Kochsalzlösung (PBS); ein leeres Becherglas verworfen Kulturmedium; kleine Zange; und ein 1.000 ml-Pipette mit Einwegspitzen. </li><li> Entfernen Sie die Kulturplatte (n) aus Inkubationskammer und Transfer zu einem Abzug. Kippen Sie die einzelnen Well-Platte Einsätze mit einer Pinzette. Als nächstes mit einer Pipette Kulturmedium und in ein Becherglas zur Verfügung zu entfernen. Schwenk der Kulturplatte in einem Winkel, um sicherzustellen, dass alle Kulturmedium entfernt. </li><li> Beenden Entfernen Medium für eine Kulturplatte zu einer Zeit. Als Medium wurde entfernt, 1 ml von 4% PFA zu jeder Kultur gut und Siegel gut mit Parafilm zu plattieren. Wiederholen für so viele Kulturplatten als notwendig und Übertragungsplatten in den Kühlschrank bei 4 ° C für 24 Stunden. </li><li> Nach 24 Stunden, bereiten Sie die folgenden in einem Abzug: 10-50 ml Becherglas mit 0,1% Natriumazid in PBS; eine leereBecherglas verworfen PFA (folgen Sie Sicherheitsvorkehrungen, wenn das Verwerfen giftige Stoffe); kleine Zange; und 1000 ml-Pipette mit Einwegspitzen. </li><li> Gehen Sie wie unter 6.4 für das Hinzufügen von PFA umrissen, aber 1 ml PBS mit Natriumazid statt. Einmal vollständig übertragen, Dichtungs Mikrotiterplatten für die zukünftige Verwendung durch Umwickeln Kanten in Parafilm und Lagerung im Kühlschrank bei 4 ° C. </li></ol> 7. Schnitte Gewebe für die Immunhistochemie <ol><li> Führen Schneiden mit einem Vibratom Gewebe. Die folgende Reihe von Schritten zur Maximierung der Ausbeute an verwertbarem Gewebeschnitte von organotypischen Kulturen. HINWEIS: Unmittelbar im Anschluss an hippocampale Dissektion und beschichteten Scheiben, hat das Gewebe eine Dicke von etwa 400 um. Jedoch nach 2-3 Wochen in der Inkubationskammer werden Gewebeschnitte beginnen zu glätten, was in einem letzten Abschnitt Dicke von 150-300 &amp; mgr; m. </li><li> Bereiten Sie Folgendes vor dem Kapitel gezüchtete Gewebe: ein # 11 Skalpell blade und Griff, eine Glaspetrischale mit eiskaltem PBS, eine Mikrodissektion Zangen, und eine saubere Schnitt Vibratom Bühne, um Gewebe zu befestigen. </li><li> Verwenden ein Skalpell vorsichtig entlang des Umfangs des kreisförmigen Einsatz Membran geschnitten, so dass sie von den Kunststoffeinsatz Gehäuse gelöst werden. Lassen Sie genügend Raum zwischen der kultivierten Scheibe und dem Skalpell. </li><li> Übertragen Sie die freistehende Einsatz Membran auf eine Petrischale mit PBS. Nach dem Spülen in PBS verwenden Zange, um die Membran zu der Vibratom Montagetisch zu übertragen. </li><li> Als Nächstes verwenden Sie das Skalpell, um überschüssige Membran umgibt kultivierten Scheiben zu beseitigen und schneiden Sie überschüssiges Material um saubere Kanten zu schaffen. Dieser Schritt wird die Membran sicherzustellen ist flach und kann leicht an der Schnittfläche entsprechen. </li><li> Zeigen 1-2 Tropfen Kleber auf der Vibratom Schneid Bühne und verteilt in gleichmäßige Schicht mit einer 22 G-Nadel. Verbreiten Klebstoff in einer rechteckigen Form mit der langen Seite parallel zu der Schneidklinge der Vibratom. Führen Sie diesen Schrittschnell, um den Klebstoff vor dem Austrocknen zu verhindern. </li><li> Verwenden einer Pinzette, um das zugeschnittene Membran, die Hippocampus-Schnitten an der Schneidphase zu übertragen und vorsichtig positionieren Sie die Membran auf Kleber und sicherzustellen, dass sich keine Luftblasen. </li><li> Wie der Sekundenkleber trocknet, übertragen Sie die Vibratom Bühne mit dem Klebemembran zurück in die PBS, die Petrischale. Bereiten Sie die Vibratom Klinge und eine 48 Well-Platte mit Natriumazid, um Gewebeschnitte zu speichern. </li><li> Verwenden Sie die Vibratom bis 30 um Abschnitte der organotypischen Gewebe und in ein 48-Well-Platte, die Natriumazid enthalten, abgespeichert und dann immunhistochemischen Färbung zu erzeugen. </li><li> Siehe Protokolle zur immunhistochemischen Färbung 26,29 vorbestehenden. </li></ol>

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	<li>Buchs, P. A., Stoppini, L., 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=Structural+modifications+associated+with+synaptic+development+in+area+CA1+of+rat+hippocampal+organotypic+cultures.">Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures.</a> Brain research. Developmental Brain Research. 71 (1), 81-91 (1993).</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> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Opitz-Araya, X., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures.">Organotypic hippocampal slice cultures.</a> Journal of Visualized Experiments. (48), (2011).</li><li>Muller, D., Buchs, P. A., Stoppini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+synaptic+development+in+hippocampal+organotypic+cultures.">Time course of synaptic development in hippocampal organotypic cultures.</a> Developmental Brain Research. 71 (1), 93-100 (1993).</li><li>Rio, J. A., Heimrich, B., Soriano, E., Schwegler, H., Frotscher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+glial+fibrillary+acidic+protein-immunoreactive+glial+cells+in+organotypic+slice+cultures+of+rat+hippocampus.">Proliferation and differentiation of glial fibrillary acidic protein-immunoreactive glial cells in organotypic slice cultures of rat hippocampus.</a> Neuroscience. 43 (2-3), 335-347 (1991).</li><li>Ziemka-Nalecz, M., Stanaszek, L., Zalewska, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-glucose+deprivation+promotes+gliogenesis+and+microglia+activation+in+organotypic+hippocampal+slice+culture:+involvement+of+metalloproteinases.">Oxygen-glucose deprivation promotes gliogenesis and microglia activation in organotypic hippocampal slice culture: involvement of metalloproteinases.</a> Acta Neurobiologiae Experimentalis. 73 (1), 130-142 (2013).</li><li>Subramanian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+Lhx2+is+necessary+and+sufficient+to+suppress+astrogliogenesis+and+promote+neurogenesis+in+the+developing+hippocampus.">Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (27), E265-E274 (2011).</li><li>Strassburger, M., Braun, H., Reymann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+treatment+with+the+p38+mitogen-activated+protein+kinase+inhibitor+SB239063+is+neuroprotective,+decreases+the+number+of+activated+microglia+and+facilitates+neurogenesis+in+oxygen-glucose-deprived+hippocampal+slice+cultures.">Anti-inflammatory treatment with the p38 mitogen-activated protein kinase inhibitor SB239063 is neuroprotective, decreases the number of activated microglia and facilitates neurogenesis in oxygen-glucose-deprived hippocampal slice cultures.</a> European Journal Of Pharmacology. 592 (1-3), 55-61 (2008).</li><li>Sadgrove, M. P., Chad, J. E., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kainic+acid+induces+rapid+cell+death+followed+by+transiently+reduced+cell+proliferation+in+the+immature+granule+cell+layer+of+rat+organotypic+hippocampal+slice+cultures.">Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures.</a> Brain Research. 1035 (2), 111-119 (2005).</li><li>Wise-Faberowski, L., Robinson, P. N., Rich, S., Warner, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+glucose+deprivation+in+an+organotypic+hippocampal+slice+model+of+the+developing+rat+brain:+the+effects+on+N-methyl-D-aspartate+subunit+composition.">Oxygen and glucose deprivation in an organotypic hippocampal slice model of the developing rat brain: the effects on N-methyl-D-aspartate subunit composition.</a> Anesthesia and Analgesia. 109 (1), 205-210 (2009).</li><li>Cho, S., Wood, A., Brain Bowlby, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=slices+as+models+for+neurodegenerative+disease+and+screening+platforms+to+identify+novel+therapeutics.">slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics.</a> Current Neuropharmacology. 5 (1), 19-33 (2007).</li><li>Noraberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures+for+studies+of+brain+damage,+neuroprotection+and+neurorepair.+Current+Drug+Targets.">Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Current Drug Targets.</a> CNS And Neurological Disorders. 4 (4), 435-452 (2005).</li><li>Berdichevsky, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K-Akt+signaling+activates+mTOR-mediated+epileptogenesis+in+organotypic+hippocampal+culture+model+of+post-traumatic+epilepsy.">PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 33 (21), 9056-9067 (2013).</li><li>Koyama, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+excitation+after+febrile+seizures+induces+ectopic+granule+cells+and+adult+epilepsy.">GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.</a> Nature Medicine. 18 (8), 1271 (2012).</li><li>Staley, K. J., White, A., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+spikes:+harbingers+or+causes+of+epilepsy.">Interictal spikes: harbingers or causes of epilepsy.</a> Neuroscience Letters. 497 (3), 247-250 (2011).</li><li>Lee, H., Lee, D., Park, C. H., Ho, W. K., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+mediates+the+network+activity-dependent+facilitation+of+axonal+outgrowth+from+the+newborn+granule+cells+in+the+early+postnatal+rat+hippocampus.">GABA mediates the network activity-dependent facilitation of axonal outgrowth from the newborn granule cells in the early postnatal rat hippocampus.</a> The European Journal Of Neuroscience. 36 (6), 2743-2752 (2012).</li><li>Raineteau, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+labeling+of+newborn+granule+cells+to+visualize+their+integration+into+established+circuits+in+hippocampal+slice+cultures.">Conditional labeling of newborn granule cells to visualize their integration into established circuits in hippocampal slice cultures.</a> Molecular and Cellular Neurosciences. 32 (4), 344-355 (2006).</li><li>Raineteau, O., Rietschin, L., Gradwohl, G., Guillemot, F., Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+hippocampal+slice+cultures.">Neurogenesis in hippocampal slice cultures.</a> Molecular And Cellular Neurosciences. 26 (2), 241-250 (2004).</li><li>Kamada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+spontaneous+neurogenesis+in+the+postnatal+slice+culture+of+rat+hippocampus.">Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.</a> The European Journal Of Neuroscience. 20 (10), 2499-2508 (2004).</li><li>Kim, H., Kim, E., Park, M., Lee, E., Namkoong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+culture+from+the+adult+mouse+brain:+a+versatile+tool+for+translational+neuropsychopharmacology.">Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Legradi, A., Varszegi, S., Szigeti, C., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+hippocampal+slices+as+in+vitro+models+for+neurodegeneration:+Studies+on+cell+viability+and+apoptotic+processes.">Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes.</a> Brain Research Bulletin. 84 (1), 39-44 (2011).</li><li>Sadgrove, M. P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Nach CldU (oder BrdU) Verabreichung kann die Zeitleiste der Anwendung von pharmakologischen Mitteln ausgewählt werden, um neugeborene DGCs während bestimmter Entwicklungs Fenster Ziel. Zum Beispiel kann eine hypothetische Mittel während der zweiten Woche nach der CldU Injektion, die vorgeschlagen wird, mit dem Alter der unreifen Neuronen, die zu einem Entwicklungsstadium sind, wo GABA depolarisierende übereinstimmen angewendet werden. Zukünftige Studien unter Verwendung dieses Protokolls kann die pharmakologische M...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

<table><tbody><tr><td>5-chloro-2&#039;-deoxyuridine (CldU)</td><td>MP Biomedicals</td><td>105478</td><td>Hazardous, Carcinogenic</td></tr><tr><td>Cell culture inserts, 30 mm diameter, 0.4 &amp;micro;m pore size</td><td>Thermo scientific&amp;nbsp;</td><td>140660</td><td>Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td></tr><tr><td>Conical Centrifuge tubes (sterile)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Dissector scissors (angled to side)</td><td>Fine Science Tools&amp;nbsp;</td><td>14082-09</td><td></td></tr><tr><td>Minimum essential medium (MEM)</td><td>Gibco</td><td>11095; liquid</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Eclipse Ni-U fluorescent microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Glue for tissue</td><td>Krazy Glue</td><td>KG585</td><td>Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS) (500 ml)</td><td>Gibco</td><td>14025-092</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Horse Serum Heat Inactivated (500 ml)</td><td>Gibco</td><td>16050-122</td><td>Make 50 ml aliquots and store at -20 &amp;deg;C</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clarke</td><td>TW 31KYPBX</td><td></td></tr><tr><td>Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td><td></td><td></td><td></td></tr><tr><td>Petri Dish (100 mm x 15 mm) and (60 mm x 15 mm)</td><td>Fisher Brand</td><td>FB0875712 and FB0875713A</td><td></td></tr><tr><td>Scalpel blades #11</td><td>Fine Science Tools</td><td>10011-00</td><td></td></tr><tr><td>Scalpel handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Serological Pipettes</td><td>Sorfa Medical Plastic Co.</td><td>P8050</td><td></td></tr><tr><td>Standard Pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Sterile vacuum filter</td><td>Thermo-Scientific</td><td>565-0020</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14054-13</td><td></td></tr><tr><td>Syringe driven filter unit</td><td>Millipore-Millex</td><td>SLGP033RS</td><td></td></tr><tr><td>Tissue chopper with moveable stage</td><td>Stoelting&amp;nbsp;</td><td>51425</td><td></td></tr><tr><td>Fine tip paintbrush</td><td></td><td></td><td></td></tr></tbody></table>

organotypic slice cultures for studies of postnatal neurogenesis

Hier beschreiben wir eine Technik zur Untersuchung Hippocampus postnatale Neurogenese im Gehirn von Nagetieren mit dem organotypischen Kulturtechnik. Diese Methode behält die charakteristische topografische Morphologie des Hippocampus und ermöglicht die direkte Anwendung von pharmakologischen Mitteln für die Entwicklungs Gyrus dentatus des Hippocampus. Zusätzlich können Gewebekulturen für bis zu 4 Wochen aufrechterhalten werden, und somit erlauben, die Reifung von neugeborenen Körnerzellen zu untersuchen. Schnittkulturen ermöglichen eine effiziente pharmakologische Manipulation von hippocampalen Schnitten unter Ausschluss komplexen Variablen wie Unsicherheiten in den tiefen anatomischen Lage des Hippocampus sowie der Bluthirnschranke stehen. Aus diesen Gründen haben wir versucht, organotypischen Kulturen für postnatale Neurogenese Forschung gezielt zu optimieren.

Feststellen, ob organotypischen Kulturen wäre geeignet für adulte Neurogenese Forschung erforderlich, dass sie zwei Hauptkriterien erfüllen: 1), dass Scheiben halten charakteristischen morphologischen Merkmale der Hippocampus nach 10-21 Tagen in vitro (DIV), und 2) dass neugeborene DGCs quantifiziert werden mit Standard-immunhistochemischen Techniken, die üblicherweise in der adulten Neurogenese Forschung beschäftigt. In Bezug auf das erste Kriterium, 1A und 1B markieren S...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Hier beschreiben wir eine Technik zur Untersuchung Hippocampus postnatale Neurogenese mit dem organotypischen Kulturtechnik. Dieses Verfahren ermöglicht die in vitro Manipulation von Neurogenese und ermöglicht die direkte Anwendung von pharmakologischen Wirkstoffen an die kultivierten Hippocampus.

Organotypischen Kulturen für Studien der postnatale Neurogenese

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

Research

JoVE Journal

Developmental Biology

12.2K Aufrufe.  University of Toronto.  Hier beschreiben wir eine Technik zur Untersuchung Hippocampus postnatale Neurogenese mit dem organotypischen Kulturtechnik. Dieses Verfahren ermöglicht die in vitro Manipulation von Neurogenese und ermöglicht die direkte Anwendung von pharmakologischen Wirkstoffen an die kultivierten Hippocampus. 

Serie: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Organotypischen Kultur der E18 Gehirn von Ratten

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Forschung

Biologie

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.

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 ...

Neurowissenschaften

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Ein organotypischen Assay für High-Resolution Time-Lapse Imaging neuronaler Migration in der postnatalen Gehirn

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

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 ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

Methoden zur Untersuchung der neuronalen Morphogenese:<em&gt; Ex-vivo-</em&gt; RNAi Elektroporation in embryonale Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

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. ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Kurzfristig frei schwebende Scheibenkulturen aus dem erwachsenen menschlichen Gehirn

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Ex Vivo Okulomotor Slice Culture von embryonalen GFP-ausdrückenden Mäusen für Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Purkinje Zell Überleben in organotypischen Zerebellar Slice Kulturen

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

Organotypischen Kulturen für Studien der postnatale Neurogenese

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