Name: purkinje cell survival in organotypic cerebellar slice cultures
Uploaded: 2019-12-18T12:00:00
Description: Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

Les travaux d'imagerie ont été réalisés au Northwestern University Center for Advanced Microscopy généreusement soutenu par NCI CCSG P30 CA060553 décerné au Robert H Lurie Comprehensive Cancer Center. Nous remercions Sean McDermott pour son assistance technique et son soutien, et Maya Epstein pour les illustrations dessinées à la main présentées à la figure 1.

Toutes les expériences impliquant des animaux ont été réalisées conformément au comité d'études animales de l'Université Northwestern.
1. Préparation avant les cultures de tranches de cervelateur organotypiques
<ol><li>Dans une hotte de culture cellulaire pulvérisée avec 70% d'éthanol à l'avance, préparer 200 ml de milieu de culture dans un filtre à vide de 250 ml fixé à un récepteur de bouteille stérile. Ajouter 100 ml d'aigle moyen basal (BME), 50 ml de solution de sel ...

<ol>
	<li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>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=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> Journal of Neuroscience Methods. 4 (4), 329-342 (1981).</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>Dusart, I., Airaksinen, M. S., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje+cell+survival+and+axonal+regeneration+are+age+dependent:+an+in+vitro+study.">Purkinje cell survival and axonal regeneration are age dependent: an in vitro study.</a> Journal of Neuroscience. 17 (10), 3710-3726 (1997).</li><li>Wiegreffe, C., Feldmann, S., Gaessler, S., Britsch, S. <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+Confocal+Imaging+of+Migrating+Neurons+in+Organotypic+Slice+Culture+of+Embryonic+Mouse+Brain+Using+In+Utero+Electroporation.">Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation.</a> Journal of Visualized Experiments. (125), e55886 (2017).</li><li>Daviaud, N., 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=Modeling+nigrostriatal+degeneration+in+organotypic+cultures,+a+new+ex+vivo+model+of+Parkinson's+disease.">Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease.</a> Neuroscience. 256, 10-22 (2014).</li><li>Ghoumari, A. 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=Mifepristone+(RU486)+protects+Purkinje+cells+from+cell+death+in+organotypic+slice+cultures+of+postnatal+rat+and+mouse+cerebellum.">Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7953-7958 (2003).</li><li>Labombarda, F., 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=Neuroprotection+by+steroids+after+neurotrauma+in+organotypic+spinal+cord+cultures:+a+key+role+for+progesterone+receptors+and+steroidal+modulators+of+GABA(A)+receptors.">Neuroprotection by steroids after neurotrauma in organotypic spinal cord cultures: a key role for progesterone receptors and steroidal modulators of GABA(A) receptors.</a> Neuropharmacology. 71, 46-55 (2013).</li><li>Gao, 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=P2X7+receptor-sensitivity+of+astrocytes+and+neurons+in+the+substantia+gelatinosa+of+organotypic+spinal+cord+slices+of+the+mouse+depends+on+the+length+of+the+culture+period.">P2X7 receptor-sensitivity of astrocytes and neurons in the substantia gelatinosa of organotypic spinal cord slices of the mouse depends on the length of the culture period.</a> Neuroscience. , 195-207 (2017).</li><li>Sundstrom, L., Morrison, B., Bradley, M., Pringle, 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+cultures+as+tools+for+functional+screening+in+the+CNS.">Organotypic cultures as tools for functional screening in the CNS.</a> Drug Discovery Today. 10 (14), 993-1000 (2005).</li><li>Jankowski, J., Miething, A., Schilling, K., Baader, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+purkinje+cell+death+is+spatiotemporally+organized+in+the+developing+mouse+cerebellum.">Physiological purkinje cell death is spatiotemporally organized in the developing mouse cerebellum.</a> Cerebellum. 8 (3), 277-290 (2009).</li><li>Ghoumari, A. M., Wehrle, R., Bernard, O., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+Bcl-2+and+Caspase-3+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture:+an+in+vitro+model+to+study+apoptosis.">Implication of Bcl-2 and Caspase-3 in age-related Purkinje cell death in murine organotypic culture: an in vitro model to study apoptosis.</a> European Journal of Neuroscience. 12 (8), 2935-2949 (2000).</li><li>Ghoumari, A. M., Wehrle, R., De Zeeuw, C. I., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+kinase+C+prevents+Purkinje+cell+death+but+does+not+affect+axonal+regeneration.">Inhibition of protein kinase C prevents Purkinje cell death but does not affect axonal regeneration.</a> Journal of Neuroscience. 22 (9), 3531-3542 (2002).</li><li>Ghoumari, A. 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=Neuroprotective+effect+of+mifepristone+involves+neuron+depolarization.">Neuroprotective effect of mifepristone involves neuron depolarization.</a> FASEB Journal. 20 (9), 1377-1386 (2006).</li><li>Repici, M., Wehrle, R., Antoniou, X., Borsello, T., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Jun+N-terminal+kinase+(JNK)+and+p38+play+different+roles+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture.">c-Jun N-terminal kinase (JNK) and p38 play different roles in age-related Purkinje cell death in murine organotypic culture.</a> Cerebellum. 10 (2), 281-290 (2011).</li><li>Rakotomamonjy, J., Ghoumari, 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=Brain-Derived+Neurotrophic+Factor+Is+Required+for+the+Neuroprotective+Effect+of+Mifepristone+on+Immature+Purkinje+Cells+in+Cerebellar+Slice+Culture.">Brain-Derived Neurotrophic Factor Is Required for the Neuroprotective Effect of Mifepristone on Immature Purkinje Cells in Cerebellar Slice Culture.</a> International Journal of Molecular Sciences. 20 (2), (2019).</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>Linkert, 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Uesaka, N., 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+coculture+preparation+for+the+study+of+developmental+synapse+elimination+in+mammalian+brain.">Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain.</a> Journal of Neuroscience. 32 (34), 11657-11670 (2012).</li><li>Falsig, 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=Prion+pathogenesis+is+faithfully+reproduced+in+cerebellar+organotypic+slice+cultures.">Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.</a> PLoS Pathogens. 8 (11), 1002985 (2012).</li><li>Bouslama-Oueghlani, L., Wehrle, R., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+loss+of+the+ability+of+Purkinje+cells+to+regenerate+their+axons+occurs+in+the+absence+of+myelin:+an+in+vitro+model+to+prevent+myelination.">The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination.</a> Journal of Neuroscience. 23 (23), 8318-8329 (2003).</li><li>Apuschkin, M., Ougaard, M., Rekling, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+calcium+waves+in+granule+cells+in+cerebellar+slice+cultures.">Spontaneous calcium waves in granule cells in cerebellar slice cultures.</a> Neuroscience Letters. 553, 78-83 (2013).</li><li>Audinat, E., Knopfel, T., 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=Responses+to+excitatory+amino+acids+of+Purkinje+cells'+and+neurones+of+the+deep+nuclei+in+cerebellar+slice+cultures.">Responses to excitatory amino acids of Purkinje cells' and neurones of the deep nuclei in cerebellar slice cultures.</a> Journal of Physiology. 430, 297-313 (1990).</li></ol>

La culture de tranches de céveltove est un outil puissant pour étudier la mort cellulaire programmée de Purkinje pendant le développement postnatal. Cette technique peut être utilisée pour dépister rapidement les molécules candidates pour leur potentiel neuroprotecteur. Le principal avantage est que la configuration est simple et très rentable, et ne nécessite qu'un investissement modeste dans l'équipement (un vibratome peut coûter jusqu'à 3 fois plus cher qu'un hélico de tissu). En outre, 10 à 15 tranches...

La modélisation in vitro est un outil essentiel de la recherche biomédicale. Il permet aux chercheurs d'étudier et de contrôler étroitement des mécanismes spécifiques dans les types de cellules restreintes, ou dans des systèmes/organes isolés. La culture de tranches organotypiques est une technique in vitro largement utilisée, en particulier dans le domaine des neurosciences1. La méthode a d'abord été mise en place par le président Dehwiler, qui a cultivé des tranches de cerveau en utilisant la technique du tube à rouleaux2, puis modifiée par Yamamoto et coll., qui ont introduit l'utilisation d'une membrane microporeuse pour effectuer des cultures de tranches corticales3. Comparéaux aux cultures cellulaires primaires, les cultures de tranches organotypiques présentent l'avantage de préserver la cytoarchitecture du tissu, ainsi que les connexions cellulaires-cellules indigènes dans le plan de la section tissulaire.
Les tranches organotypiques ont été cultivées à partir de nombreuses parties du système nerveux central, comme l'hippocampe4, cortex5, striatum6, cervelet4,7, et la moelle épinière8,9, entre autres. Ils se sont avérés être un outil puissant dans les études de découverte de médicaments10. Les effets des molécules neuroactives peuvent être évalués de plusieurs façons : survie et neurodégénérescence à l'aide d'analyses d'immunostaining et de biochimie, formation de circuits neuronaux ou perturbation à l'aide de l'électrophysiologie et de l'imagerie vivante.
Le but de ce travail est de décrire une méthode simple pour effectuer la culture de tranche de cervelateur organotypic, qui est connu pour être un modèle pertinent pour imiter le développement cérécolal in vitro. En particulier, nous nous sommes concentrés sur l'étude de la mort développementale de cellules de Purkinje. In vivo, les cellules de Purkinje subissent l'apoptose au cours de la première semaine postnatale, avec un pic au jour postnatal 3 (P3)11. Le même modèle est observé dans la culture de tranche de céveineux, avec des neurones purkinje mourir par apoptose quand les cerveleux sont pris des animaux entre P1 et P8, avec un pic à P34,12. L'utilisation des cultures de tranches de cervelage organotypiques a permis d'identifier plusieurs molécules neuroprotectrices7,13, ainsi que de comprendre une partie des mécanismes impliqués dans cette mort cellulaire programmée14,15,16. Ici, nous décrivons un protocole basé sur l'étude de Stoppini et autres17 dans l'hippocampe, et adapté au cervelet par Dusart et autres4 Il inclut la dissection rapide et le hachage du cerebella postnatal ; trancher la culture sur un insert de culture cellulaire contenant une membrane microporeuse, avec ou sans traitement neuroprotecteur; et la coloration d'immunofluorescence pour évaluer la survie neuronale.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody</td>
			<td>ThermoFisher scientific</td>
			<td>A21203</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal Medium Eagle (BME)</td>
			<td>ThermoFisher scientific</td>
			<td>21010046</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet Class II, Type A2</td>
			<td>NuAire</td>
			<td>NU-540-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Calbindin D-28K antibody (CB-955)</td>
			<td>Abcam</td>
			<td>ab82812</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NuAire</td>
			<td>NU-5700</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom 6-well Cell Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blades</td>
			<td>Fisher Scientific</td>
			<td>50949411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-127</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100X</td>
			<td>ThermoFisher scientific</td>
			<td>25030149</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose solution</td>
			<td>ThermoFisher scientific</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks' Balanced Salt Solution</td>
			<td>ThermoFisher scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated, New Zealand origin</td>
			<td>ThermoFisher scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Fisher Scientific</td>
			<td>NC9914528</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobes</td>
			<td>Fisher Scientific</td>
			<td>08-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts</td>
			<td>Millipore Sigma</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL</td>
			<td>ThermoFisher scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal laser microscope system</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Imaging Software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>41678-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8403</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker Belly Dancer</td>
			<td>IBI Scientific</td>
			<td>BDRLS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish, 60 mm w/ grip ring</td>
			<td>Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 well</td>
			<td>Falcon/Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes, sterile</td>
			<td>ThermoFisher scientific</td>
			<td>13-711-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

purkinje cell survival in organotypic cerebellar slice cultures

La culture de tranches organotypiques est un modèle in vitro puissant qui mimicks in vivo conditions plus étroitement que les cultures cellulaires primaires dissociées. Au début du développement postnatal, les cellules cérévelaires de Purkinje sont connues pour passer par une période vulnérable, au cours de laquelle elles subissent la mort cellulaire programmée. Ici, nous fournissons un protocole détaillé pour effectuer la culture de tranche de cervelateur organotypic de souris pendant ce temps critique. Les tranches sont également étiquetées pour évaluer la survie des cellules de Purkinje et l'efficacité des traitements neuroprotecteurs. Cette méthode peut être extrêmement utile pour dépister de nouvelles molécules neuroactives.

Comme le montre la figure 4, ce protocole produit des cultures de tranches de cervelateurs organotypiques dans lesquelles la survie des cellules de Purkinje peut être évaluée après les étapes d'immunofluorescence et d'acquisition d'image. Les cellules purkinje ont été étiquetées avec une combinaison d'anticorps anti-Calbindin D-28K (dilution 1/200) et d'anticorps anti-souris Alexa594 (dilution 1/300). La couture d'image a été faite automatiquement par le logiciel d'acquisition de ...

Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Les cultures de tranches organotypiques sont un outil puissant pour étudier les processus neurodéveloppementaux ou dégénératifs/régénérateurs. Ici, nous décrivons un protocole qui modélise la mort neurodéveloppementale des cellules de Purkinje dans les cultures de tranche de cévedrillon de souris. Cette méthode peut bénéficier à la recherche dans la découverte de médicaments neuroprotecteurs.

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

La culture des tranches organotypiques est un outil puissant pour étudier les processus neurodéveloppementaux et dégénératifs ou régénérateurs. Cette technique peut être utilisée pour dépister rapidement les molécules candidats pour leur potentiel neuroprotecteur. Cette méthode imite étroitement les conditions in vivo, par rapport aux cultures cellulaires primaires dissociées, car l’architecture des ensembles tissulaires et les connexions cellulaires indigènes sont préservées dans les plans des sections.Ici nous démontrons l’étude de la mort cellulaire de Purkinje dans le cervelet en développement. Mais la culture organotypique de tranche est également appropriée pour modéliser des maladies neurodegenerative dans presque chaque région centrale de système nerveux. Sean McDermott, technicien de mon laboratoire, démontrera la procédure avec Jennifer Rakotomamonjy.Avant de récolter les tranches de cerebellar, dans une armoire de biosécurité stérilisée, remplissez chaque puits d’une plaque de culture de six puits d’un millilitre de milieu de culture filtré sous vide, et ajoutez l’agent pharmacologique d’intérêt aux puits de traitement, et un volume égal de véhicule aux puits de contrôle. À l’aide de forceps stériles, placez un filtre à membrane PTFE de taille pore de 0,4 micromètre inséré dans chaque puits, en prenant soin d’éviter les bulles à l’interface de chaque membrane et de chaque milieu, et équilibrez le milieu dans un incubateur de 37 degrés Celsius et 5 % de dioxyde de carbone pendant deux heures. Pour récolter le cervelet, utilisez des pinces droites pour saisir la tête de chiot par le nez, et utilisez des ciseaux droits pour ouvrir le cuir chevelu de l’extrémité postérieure latéralement à la ligne médiane.Coupez le crâne de la même manière, en pointant les pointes de ciseaux vers l’extérieur pour éviter d’endommager le cervelet, et utilisez une spatule pour transférer le cerveau dans un plat de 60 millimètres contenant du HBSS froid et cinq milligrammes par millilitre de glucose. Utilisez les forceps droits pour disséquer soigneusement le cervelet et utilisez des forceps fins incurvés stériles pour placer le tissu sur un disque en plastique sur la table de coupe d’un hachoir à tissus. Faites pivoter la table de coupe pour orienter le tissu pour permettre l’acquisition de sections parasagittal, et tirez le bouton de dégagement de table vers la droite pour déplacer la table de coupe jusqu’à ce que la lame soit placée au bord du tissu.Ajustez ensuite l’épaisseur de la tranche à 350 micromètres, et la vitesse de la lame à moyen, et démarrez l’hélico. Lorsque tout le cervelet a été tranché, éteignez l’hélico. À l’aide de forceps stériles, maintenez le disque sur un nouveau plat de 60 millimètres.Utilisez une pipette de transfert pour rincer hbss plus le glucose sur le disque de sorte que les tranches tombent dans le plat. Ensuite, en touchant les échantillons le moins possible, utilisez un microprobe pour séparer les tranches. Utilisez la pipette de transfert pour sélectionner des sections du cervelet près du verme sur des inserts individuels de culture cellulaire dans la plaque de six puits, et utilisez le microprobe pour positionner les tranches au centre de chaque insert.Lorsque toutes les tranches ont été placées, aspirez soigneusement tout excès de HBSS plus le glucose, et retournez la plaque à l’incubateur de culture cellulaire. Pour les taches d’immunofluorescence, retirez le surnatant de chaque puits et lavez les inserts avec du PBS. Fixer les tranches avec un millilitre de paraformaldéhyde froid de 4% dans le puits de chaque insert, et 500 microlitres sur chaque insert pendant une heure.À la fin de la fixation, laver les inserts quatre fois pendant 10 minutes avec un millilitre de PBS frais sous chaque insert et 500 microlitres de PBS frais sur le dessus de chaque insert par lavage sur un shaker orbital. Pré-remplir chaque puits d’une plaque de 24 puits avec 500 microlitres de PBS-TB, et utiliser un pinceau pour transférer les tranches de cerebellar de chaque insert de culture cellulaire dans les puits individuels d’une plaque de 24 puits. Perméabilizez et bloquez les tranches à température ambiante pendant une heure.À la fin de l’incubation, étiquetez les cellules avec 200 microlitres de l’anticorps primaire d’intérêt dilués dans PBS-TB par puits pendant la nuit à quatre degrés Celsius sur le shaker orbital. Le lendemain matin, lavez les tranches quatre fois pendant 10 minutes et 500 microlitres de PBS frais par lavage sur le shaker, avant d’étiqueter les échantillons avec les anticorps secondaires conjugués au fluorophore appropriés pendant deux heures à température ambiante sur le shaker, à l’abri de la lumière. À la fin de l’incubation, contrestain les sections avec 500 microlitres d’une tache nucléaire appropriée par puits pendant 10 minutes à température ambiante, à l’abri de la lumière.Et utilisez une pipette de transfert pour monter les tranches sur des toboggans en verre. Ensuite, laissez sécher complètement les sections avant de les réhydrater avec du PBS et de monter les tissus avec des couvertures recouvertes d’environ 80 microlitres de milieu de montage. Une fois que le support de montage s’est durci, les sections cerebellar sont prêtes à être imaged.Au sixième jour postnatal, la survie cellulaire de Purkinje est faible dans les échantillons de contrôle, compatible avec leur fenêtre de vulnérabilité connue. La survie augmente à mesure que l’animal donneur vieillit et sort de cette période critique. Le traitement des tranches de cerebellar avec une forte concentration de chlorure de potassium a comme conséquence l’induction réussie de leur dépolarisation et survie.Pour obtenir des résultats cohérents et reproductibles, il est essentiel de sélectionner des sections de cerebellar saines et d’effectuer la configuration de la culture aussi efficacement que possible. Les applications post-culture vont au-delà de l’immunofluorescence. Comme les tranches organotypiques peuvent être utilisées pour les études d’expression des protéines du génome, et pour la surveillance de l’activité du circuit neuronal à l’aide de l’électrophysiologie et de l’imagerie en direct du calcium.

purkinje-cell-survival-in-organotypic-cerebellar-slice-cultures

Research

JoVE Journal

Neuroscience

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

Culture organotypique d&#39;exprimant la GFP embryons de souris pour les images en temps réel d&#39;excroissance des nerfs périphériques

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Recherche

Neurosciences

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.

Cultures organotypiques d&#39;hippocampe Slice

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

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. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Cultures organotypiques cérébelleuse: Défis apoptotiques et détection

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

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

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

Enregistrement à partir de cellules entières une préparation de tranches organotypiques du néocortex

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

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

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

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

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Cultures organotypique de mésencéphale ventral embryonnaire: Un système pour étudier le développement neuronal dopaminergique In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

L&#39;analyse de la morphologie de Purkinje cellules dendritiques dans les cultures organotypique

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

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

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

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

Étiquetage immunologique post-enrobage des protéines synaptiques dans l&#39;hippocampe cultures Slice

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

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Cultures organotypique de tranche d&#39;étudier Oligodendrocyte Dynamics et myélinisation

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

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

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

L&#39;analyse de neurovasculaire Rénovation dans Entorhino-hippocampe cultures organotypique de tranche

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Déplétion sélective des cellules microgliales du cervelet granules de cultures cellulaires utilisant la L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...