Name: organotypic slice cultures to study oligodendrocyte dynamics and myelination
Uploaded: 2014-08-25T15:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.

OPMERKING: Alle dierlijke procedures worden volgens de richtlijnen en hebben door de Institutional Animal Care en gebruik Comite (IACUC) aan de Universiteit van Connecticut is goedgekeurd. LET OP: Voor deze experimenten constitutieve NG2cre 18 (JAX # 008533) en induceerbare NG2creER 16 (JAX # 008538) transgene muizen gekruist tot Z / EG 19 (JAX # 003920) of gtRosa26: YFP reporter 20 (JAX # 006148) lijnen respectievelijk werden gebruikt om beeld NG2 cellen en hun nageslacht. Om het beeld ma...

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	<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), (1991).</li><li>Gogolla, N., Galimberti, I., DePaola, V., Caroni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+organotypic+hippocampal+slice+cultures+for+long-term+live+imaging.">Preparation of organotypic hippocampal slice cultures for long-term live imaging.</a> Nature Protocols. 1 (3), 1165-1171 (2006).</li><li>G&#228;hwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A., Thompson, 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=Organotypic+slice+cultures:+a+technique+has+come+of+age.">Organotypic slice cultures: a technique has come of age.</a> Trends in Neurosciences. 20 (10), 471-477 (1997).</li><li>Yamamoto, N., Kurotani, T., Toyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+connections+between+the+lateral+geniculate+nucleus+and+visual+cortex+in+vitro.">Neural connections between the lateral geniculate nucleus and visual cortex in vitro.</a> Science. 245 (4914), 192-194 (1989).</li><li>Bahr, B. A., Kessler, 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=Stable+maintenance+of+glutamate+receptors+and+other+synaptic+components+in+long-term+hippocampal+slices.">Stable maintenance of glutamate receptors and other synaptic components in long-term hippocampal slices.</a> Hippocampus. 5 (5), 425-439 (1995).</li><li>Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+derived+from+radial+glial+cells+establish+radial+units+in+neocortex.">Neurons derived from radial glial cells establish radial units in neocortex.</a> Nature. 409 (6821), 714-720 (2001).</li><li>Noctor, S. C., Mart&#237;nez-Cerde&#241;o, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nature Neuroscience. 7 (2), (2004).</li><li>Kapfhammer, J. P., Gugger, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+purkinje+cell+dendritic+morphology+in+organotypic+slice+cultures.">The analysis of purkinje cell dendritic morphology in organotypic slice cultures.</a> Journal of Visualized Experiments JoVE. (61), (2012).</li><li>Elias, L., Kriegstein, 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+slice+culture+of+E18+rat+brains.">Organotypic slice culture of E18 rat brains.</a> Journal of Visualized Experiments JoVE. (6), e235 (2007).</li><li>Nishiyama, A., Komitova, M., Suzuki, R., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polydendrocytes+(NG2+cells):+multifunctional+cells+with+lineage+plasticity.">Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity.</a> Nature Reviews Neuroscience. 10 (1), 9-22 (2009).</li><li>Haber, M., Vautrin, S., Fry, E. J., Murai, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-specific+oligodendrocyte+dynamics+in+organotypic+culture.">Subtype-specific oligodendrocyte dynamics in organotypic culture.</a> Glia. 57 (9), 1000-1013 (2009).</li><li>Bin, J. M., Leong, S. Y., Bull, S. -. J., Antel, J. P., Kennedy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+precursor+cell+transplantation+into+organotypic+cerebellar+shiverer+slices:+a+model+to+study+myelination+and+myelin+maintenance.">Oligodendrocyte precursor cell transplantation into organotypic cerebellar shiverer slices: a model to study myelination and myelin maintenance.</a> PloS one. 7 (7), (2012).</li><li>Birgbauer, E., Rao, T. S., Webb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysolecithin+induces+demyelination+in+vitro+in+a+cerebellar+slice+culture+system.">Lysolecithin induces demyelination in vitro in a cerebellar slice culture system.</a> Journal of Neuroscience Research. 78 (2), 157-166 (2004).</li><li>Harrer, M. D., von Budingen, H. C., 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=Live+imaging+of+remyelination+after+antibody-mediated+demyelination+in+an+ex+model+for+immune+mediated+CNS+damage.">Live imaging of remyelination after antibody-mediated demyelination in an ex model for immune mediated CNS damage.</a> Experimental Neurology. 216 (2), 431-438 (2009).</li><li>Fancy, S. P. J., Harrington, E. P., 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=Axin2+as+regulatory+and+therapeutic+target+in+newborn+brain+injury+and+remyelination.">Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination.</a> Nature Neuroscience. 14 (8), 1009-1016 (2011).</li><li>Zhu, X., Hill, R. A., Dietrich, D., Komitova, M., Suzuki, R., Nishiyama, A. <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+fate+and+lineage+restriction+of+single+NG2+cells.">Age-dependent fate and lineage restriction of single NG2 cells.</a> Development. 138 (4), 745-753 (2011).</li><li>Hill, R. A., Patel, K. D., Medved, J., Reiss, A. M., Nishiyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2+Cells+in+White+Matter+But+Not+Gray+Matter+Proliferate+in+Response+to+PDGF.">NG2 Cells in White Matter But Not Gray Matter Proliferate in Response to PDGF.</a> Journal of Neuroscience. 33 (36), 14558-14566 (2013).</li><li>Zhu, X., Bergles, D. E., Nishiyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2+cells+generate+both+oligodendrocytes+and+gray+matter+astrocytes.">NG2 cells generate both oligodendrocytes and gray matter astrocytes.</a> Development. 135 (1), 145-157 (2008).</li><li>Novak, A., Guo, C., Yang, W., Nagy, A., Lobe, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Z/EG,+a+double+reporter+mouse+line+that+expresses+enhanced+green+fluorescent+protein+upon+Cre-mediated+excision.">Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.</a> Genesis. 28 (3-4), 147-155 (2000).</li><li>Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+lacZ+expression+with+the+ROSA26+Cre+reporter+strain.">Generalized lacZ expression with the ROSA26 Cre reporter strain.</a> Nature Genetics. 21 (1), 70-71 (1999).</li><li>Hirrlinger, P. G., Scheller, 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=Expression+of+reef+coral+fluorescent+proteins+in+the+central+nervous+system+of+transgenic+mice.">Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice.</a> Molecular and Cellular Neuroscience. 30 (3), 291-303 (2005).</li><li>Nave, K. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+and+support+of+axonal+integrity+by+glia.">Myelination and support of axonal integrity by glia.</a> Nature. 468 (7321), 244-252 (2010).</li><li>Rivers, L. E., Young, K. 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=PDGFRA/NG2+glia+generate+myelinating+oligodendrocytes+and+piriform+projection+neurons+in+adult+mice.">PDGFRA/NG2 glia generate myelinating oligodendrocytes and piriform projection neurons in adult mice.</a> Nature Neuroscience. 11 (12), 1392-1401 (2008).</li><li>Dimou, L., Simon, C., Kirchhoff, F., Takebayashi, H., Gotz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progeny+of+Olig2-expressing+progenitors+in+the+gray+and+white+matter+of+the+adult+mouse+cerebral+cortex.">Progeny of Olig2-expressing progenitors in the gray and white matter of the adult mouse cerebral cortex.</a> The Journal of Neuroscience. 28 (41), 10434-10442 (2008).</li><li>Kang, S. H., Fukaya, M., Yang, J. K., Rothstein, J. D., Bergles, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2++CNS+glial+progenitors+remain+committed+to+the+oligodendrocyte+lineage+in+postnatal+life+and+following+neurodegeneration.">NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration.</a> Neuron. 68 (4), 668-681 (2010).</li><li>Ioannidou, K., Anderson, K. I., Strachan, D., Edgar, J. M., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+the+dynamics+of+CNS+glial-axonal+interactions+in+vitro+and+ex+vivo.">Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo.</a> PloS one. 7 (1), e30775 (2012).</li><li>Ghoumari, A. M., Baulieu, E. E., Schumacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+increases+oligodendroglial+cell+proliferation+in+rat+cerebellar+slice+cultures.">Progesterone increases oligodendroglial cell proliferation in rat cerebellar slice cultures.</a> Neuroscience. 135 (1), 47-58 (2005).</li><li>Azim, K., Butt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GSK3&#946;+negatively+regulates+oligodendrocyte+differentiation+and+myelination+in+vivo.">GSK3&#946; negatively regulates oligodendrocyte differentiation and myelination in vivo.</a> Glia. 59 (4), 540-553 (2011).</li><li>Hussain, R., El-Etr, 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=Progesterone+and+nestorone+facilitate+axon+remyelination:+a+role+for+progesterone+receptors.">Progesterone and nestorone facilitate axon remyelination: a role for progesterone receptors.</a> Endocrinology. 152 (10), 3820-3831 (2011).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nature Reviews. Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Lubetzki, C., Demerens, C., 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=Even+in+culture,+oligodendrocytes+myelinate+solely+axons.">Even in culture, oligodendrocytes myelinate solely axons.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (14), 6820-6824 (1993).</li><li>Demerens, C., Stankoff, B., 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=Induction+of+myelination+in+the+central+nervous+system+by+electrical+activity.">Induction of myelination in the central nervous system by electrical activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (18), 9887-9892 (1996).</li><li>Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+sensory+axons+are+mitogenic+for+Schwann+cells.">Evidence that sensory axons are mitogenic for Schwann cells.</a> Nature. 256 (5519), 662-664 (1975).</li><li>Stevens, B., Tanner, S., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+myelination+by+specific+patterns+of+neural+impulses.">Control of myelination by specific patterns of neural impulses.</a> The Journal of Neuroscience. 18 (22), 9303-9311 (1998).</li><li>Stevens, B., Porta, S., Haak, L. L., Gallo, V., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine:+a+neuron-glial+transmitter+promoting+myelination+in+the+CNS+in+response+to+action+potentials.">Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials.</a> Neuron. 36 (5), 855-868 (2002).</li><li>Wake, H., Lee, P. R., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+local+protein+synthesis+and+initial+events+in+myelination+by+action+potentials.">Control of local protein synthesis and initial events in myelination by action potentials.</a> Science. 333 (6049), 1647-1651 (2011).</li><li>Rosenberg, S. S., Kelland, E. E., Tokar, E., De la Torre, A. R., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geometric+and+spatial+constraints+of+the+microenvironment+induce+oligodendrocyte+differentiation.">The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14662-14667 (2008).</li></ol>

Myelination in het centrale zenuwstelsel is essentieel voor efficiënte neuronale communicatie en axonaal overleving 22. NG2 cellen continu genereren myeliniserende oligodendrocyten in de volwassenheid met behoud van een bevolking in de meeste gebieden van de hersenen 16,23 - 25. Sommige genetische en moleculaire mechanismen tot regeling van de differentiatie van deze cellen zijn beschreven, maar er is nog veel te ontdekken. Organotypische slice culturen zijn een handig hulpmiddel om deze mechanism...

Organoytpic slice cultures van het centrale zenuwstelsel zijn uitermate bruikbaar voor het bestuderen neuron en gliale celbiologie in een semiintact systeem 1 te zijn - 4. Deze culturen zijn relatief eenvoudig vast te stellen en te behouden vele voordelen van primaire gedissocieerde celculturen, zoals manipulatie van de extracellulaire omgeving en gemakkelijke toegang voor herhaalde langdurige live cell imaging en elektrofysiologische opnames 5-9. Bovendien slice cultures onderhouden 3-dimensionele tissue cytoarchitecture, regionale neurale verbindingen en meest belangrijke celtypen aanwezig zijn in het systeem. Deze eigenschappen maken deze culturen een uniek en handig systeem om enkele cel gedrag en fysiologie met cellulaire en milieu-interacties te onderzoeken. NG2 cellen een populatie van gliale cellen in het centrale zenuwstelsel die blijven prolifereren en genereren myelinating oligodendrocyten tijdens de embryonale en postnatale ontwikkeling 10. Zij zijn uitgebreid bestudeerd in gedissocieerde primaire celculturen en recente ontwikkeling van transgene muizen lijnen met NG2 celspecifieke expressie van fluorescerende eiwitten heeft vergemakkelijkt in vivo lot mapping en elektrofysiologische opnames in acute slice preparaten. Zelfs met deze studies is er weinig bekend over de temporele dynamiek van NG2 celproliferatie en oligodendrocyt differentiatie. Hoewel gedissocieerde celkweek op grote schaal gebruikt voor de relatieve faciliteit farmacologische en genetische manipulaties, is het niet geschikt voor het ondervragen functionele verschillen van deze cellen in verschillende hersengebieden bijzonder wanneer het wenselijk is om de context van de cellulaire micro-omgeving behouden. Plak culturen een eenvoudig alternatief dat vatbaar is voor farmacologische manipulaties en zijn gebruikt voor oligodendrocyten myelinisatie 11,12 onderzoeken cellular reactie na lysolecithine (LPC) of antilichamen geïnduceerde demyelinisatie 13,14 en inductie van remyelinisatie via farmacologische behandeling 15. Werkwijze onderzoeken en live imaging en vast weefsel (of postfixatie) analyse van NG2 celproliferatie en oligodendrocyt differentiatie in organotypische slice culturen uit zowel de voorhersenen en cerebellum beschreven. Dit is een krachtige methode die gebruikt kan worden om de cel lot van NG2 enkelvoudige cellen te bestuderen na splitsing 16 en regio- en leeftijdsafhankelijke verschillen groeifactor geïnduceerde celproliferatie NG2 17 ontdekken. Deze relatief eenvoudige techniek breed toegankelijk verder te onderzoeken cel intrinsieke en / of ecologische mechanismen reguleren van de fysiologie van deze gliacellen en hun respons op neuronale activiteit of myelinebeschadiging.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Slice Culture Medium</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>11090</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Invitrogen</td>
			<td>14175</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H-4034</td>
			<td>25mM</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>1mg/L</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>0.4mM</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Hyclone</td>
			<td>SH30074.03</td>
			<td>25%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>124mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>3.004mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>1.25mM</td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>4.004mM</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>26mM</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A-0278</td>
			<td>2.0mM</td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A4036</td>
			<td>0.075mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents and Supplies</td>
		</tr>
		<tr>
			<td>4-hydroxy tamoxifen (4OHT)</td>
			<td>Sigma-Aldrich</td>
			<td>H7904</td>
			<td>100nM</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>EM Sciences</td>
			<td>19200</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L-6027</td>
			<td>0.1M</td>
		</tr>
		<tr>
			<td>Sodium Metaperiodate</td>
			<td>Sigma-Aldrich</td>
			<td>S-1878</td>
			<td>0.01M</td>
		</tr>
		<tr>
			<td>Rabbit anti NG2 antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>AB5320</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Mouse anti CC1 antibody</td>
			<td>Calbiochem (Millipore)</td>
			<td>OP80</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Mouse anti Ki67 antibody</td>
			<td>Vision Biosystems</td>
			<td>NCL-Ki67-MM1</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Mouse anti NeuN antibody</td>
			<td>Chemicon (Millipore)</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Species specific secondary antibodies</td>
			<td>Jackson Immunoresearch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td></td>
			<td></td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture membrane inserts 0.45&#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filters</td>
			<td>Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2 gas mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile six well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting (hippocampal) or weighing spatulas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manual or automatic tissue chopper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm and 60mm sterile Petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Phase Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microcope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures to study oligodendrocyte dynamics and myelination

NG2 expressie brengende cellen (polydendrocytes, oligodendrocyt precursor cellen) zijn de vierde grote gliale celpopulatie in het centrale zenuwstelsel. Tijdens de embryonale en postnatale ontwikkeling ze actief verspreiden en genereren myeliniserende oligodendrocyten. Deze cellen zijn vaak onderzocht bij primaire los culturen, neuron cocultures, en in vaste weefsel. Met vrijgekomen transgene muizenlijnen snede kweeksystemen kunnen worden gebruikt om de proliferatie en differentiatie van oligodendrocyt afkomstcellen onderzoeken zowel grijze en witte stof gebieden van de voorhersenen en cerebellum. Plak culturen worden bereid uit vroege postnatale muizen en in kweek gehouden gedurende 1 maand. Deze segmenten kunnen meerdere malen worden afgebeeld in de kweekperiode cellulaire gedrag en interacties te onderzoeken. Deze methode maakt visualisatie van NG2 celdeling en de stappen die leiden tot oligodendrocyte differentiatie, terwijl waardoor een gedetailleerde analyse van de regio-afhankelijkent NG2 cel en oligodendrocyte functionele heterogeniteit. Dit is een techniek die kan worden gebruikt om de intrinsieke en extrinsieke signalen beïnvloeden deze cellen in de tijd in een cellulaire omgeving die lijkt op die in vivo onderzoeken.

Voorbeelden van representatieve gegevens worden hieronder gegeven die zijn verkregen met behulp van slice culturen van zowel de voorhersenen en het cerebellum van P8 NG2cre: ZEG, NG2creER: YFP en PLPDsRed transgene muis lijnen. NG2 cellen kunnen worden afgebeeld op dagen voorhersenen en cerebellum segmenten (Figuur 1B-D, Video 1) en het fenotype van deze cellen kan worden bepaald na fixatie en immunokleuring met NG2 en CC1 antilichamen (figuur 1E). Naast leven imaging kan celproliferati...

Watch this Scientific Journal Video about Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination at JoVE.com

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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.

Organotypische Slice culturen om te studeren Oligodendrocyte Dynamics en Myelination

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

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

De organotypische hippocampus Slice Cultuur Model is voor de behandeling Neuronale Letsel

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

Organotypic Hippocampal Slice Cultures

Organotypische hippocampus Slice culturen

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypische Cerebellaire culturen: apoptotische uitdagingen en Detectie

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

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Hele Cell Opnemen van een organotypische Slice Voorbereiding van de neocortex

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

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Multi-electrode array Opnamen van neuronale Lawines in organotypische culturen

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Afleiding van Verrijkt oligodendrocyt Culturen en oligodendrocyt / Neuron Myelinating co-culturen van Post-natale Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

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

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

Organotypische Slice culturen van embryonale ventrale middenhersenen: Een systeem om 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

De Analyse van de Purkinje cel dendritische morfologie in organotypische slice culturen

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

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

De Analyse van Neurovascular Verbouwing in Entorhino-hippocampus Organotypische Slice Culturen

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

Organotypic Hippocampal Slice culturen als een Model voor de studie neuroprotectie en invasiviteit van tumorcellen

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