Name: modeling neuronal death and degeneration in mouse primary cerebellar granule neurons
Uploaded: 2017-11-06T16:30:00
Description: Watch this Scientific Journal Video about Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons at JoVE.com

Dette arbejde er støttet af naturvidenskab og Engineering Research Council of Canada og den canadiske institutter for sundhedsforskning tilskud til AJ-A.

denne protokol er baseret på ændringer af procedurer, der er blevet beskrevet tidligere 18 , 24 , 25 , 26 , 27. denne protokol godkendes af Animal Care Udvalget på McGill University.
 1. eksperimentel forberedelse Bemærk: de følgende stamopløsninger kan forberedes og opretholdt indtil brug....

<ol>
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Her giver vi en simpel metode til dyrkning af primære mus cerebellare granulet neuroner (CGNs), tab og gevinst på funktion studier og modellering forskellige ordninger for celledød. Adskillige faktorer påvirker reproducerbarhed af resultaterne ved hjælp af denne procedure, som kræver nøje overvågning. Disse omfatter renheden af den kultur, herunder eliminering af glial celler i kulturen, sammenløbet af kultur, og opretholde sunde celler. At indføre variation i disse faktorer kan bias resultaterne og udfordre re...

Cerebellare granulet neuroner (CGNs) er godt præget i kultur og har tjent som en effektiv model til at studere neuronal død og udvikling 1,2,3,4,5, 6. den tidlige udtryk for N-methyl-D-aspartat (NMDA) receptorer i CGN kulturer i vitro gør dem en attraktiv model til at studere NMDA-induceret signalering. Aktivering af disse receptorer med NMDA sammenholdt med membran depolarisering bruges til at modellere fysiologiske neuronal aktivitet stimulation, og har gjort det muligt for forskning i mekanismer af synaptisk plasticitet 7,8. Tværtimod kan over-stimulation af disse receptorer af NMDA ligand bruges til at modellere excitotoxicity, en vigtig mekanisme af neuronal tabet i akutte skader på hjernen og neurodegenerative sygdomme 9. En mekanisme til induktion af excitotoxicity er gennem ATP sult med reduceret ilt, som ses med akut neuronal skade. Dette fører til membran depolarisering og forhøjede niveauer af glutamat frigive på synapser. Den efterfølgende overstimulering af NMDA-receptor af forhøjede glutamat resulterer i overdreven Ca2 + tilstrømning via disse receptorer, som igen aktiverer flere veje herunder Ca2 +-aktiveret proteaser, phospholipases, og endonucleases, der resulterer i ukontrolleret nedbrydningen af kritiske cellulære komponenter og celledød. Derudover, fører høje intracellulære Ca2 + til generation af ilt frie radikaler og mitokondriel skader 10,11.
Mens fleste af neuronal tab efter NMDA-induceret neuronal excitotoxicity skyldes calcium tilstrømning og er Bax/Bak uafhængige, kan andre mekanismer for celledød udelukkes fra denne model. Udseendet af begge nekrotisk og apoptotisk celledød på grund af excitotoxicity er delvist skabelsen af reaktive ilt arter (ROS) og DNA-skader forårsaget af høje intracellulære Ca2 + niveauer 12. DNA-skader medfører neuronal død gennem apoptotiske mekanismer, at være korreleret med kendetegnene ved apoptotisk celledød, såsom fremkomsten af kromatin masserne og apoptotiske organer. Induktion af apoptose er medieret gennem frigivelse af cytokrom c fra mitokondrier, og har vist sig at være afhængig af Bax/Bak oligomerisering 13. Bax/Bak oligomerisering fremmer pore dannelse i den ydre membran, som resulterer i cytokrom c frigivelse og aktivering af pro-apoptotiske lovgivere som set med mild iskæmisk skade 14.
Generation af ROS er et vigtigt spørgsmål i hjernen på grund af de lave endogene niveauer af antioxidanter, kombineret med store ilt kravet om neuronal fungerende 15. Når de udsættes for en iskæmisk begivenhed, er nitrogenoxid syntase upregulated, producerer nitrogenoxid og øge reaktive ilt arter 14. Den øgede koncentration af ilt radikaler kan forårsage DNA-skader og indirekte forårsage energi sult. Høje niveauer af DNA dobbelt-strenget pauser er afhjulpet ved aktivering af poly ADP-ribose polymerase-1 (PARP-1), en eukaryot kromatin-bundet protein ansvarlig for katalysere overførsel af ADP-ribose enheder fra NAD+, en integreret del af processen DNA reparation 16. Men med alt for store skader på grund af oxidativt stress, PARP-1 aktivering kan forårsage energi sult på grund af den øgede afløb på NAD+, et nødvendigt substrat til produktion af ATP ved oxidativ fosforylering. I sidste ende, oxidativ stress vil udløse apoptose i Bax/Bak afhængig måde fører til mitokondrie cytokrom c release, og har vist sig at fremkalde mitokondrie ombygning i CGNs 17.
Endelig, ændringer i koncentrationen af kaliumchlorid (KCl) i CGN kulturer kan bruges til at modellere lavt kalium/depolarisering medieret apoptose 18,19,20. Når udsat for lave niveauer af K+, gennemgå CGNs forskellige fysiologiske ændringer, hvilket resulterer i reduktion af både mitokondrie respiration og glykolyse, tilskrives nedsat cellulær efterspørgsel 21, samt reduktion i niveauet af nuklear factor-κB (NFκB) som regulerer aktiviteter herunder betændelse og synaptisk transmission 22. Denne model er af særlig interesse for studiet af celledød under neuronal udvikling. Lav K+ miljøet mere nøje ligner fysiologiske tilstande, og forårsager kendetegnende for celledød ses under neuronal udvikling 23.
I Resumé giver CGNs en mangeårige model til at undersøge de underliggende molekylære mekanismer af neuronal død og degeneration. Følgende protokol giver mulighed for isolering og dyrkning af CGNs, udtryk eller undertrykkelse af en bestemt genetisk pathway ved hjælp af vira og induktion af neuronal død via forskellige mekanismer der repræsenterer neuronal skade og degeneration.

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			<td style="width:215px;">Sigma Aldrich</td>
			<td style="width:219px;">#M-3262</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phenol Red Solution&#160;</td>
			<td style="width:215px;">Sigma Aldrich</td>
			<td style="width:219px;">#P-0290</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Trypsin&#160;</td>
			<td style="width:215px;">Sigma Aldrich</td>
			<td style="width:219px;">#T-4549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 3000</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000-008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p3000 enhancer reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000-008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl&#160;</td>
			<td style="width:215px;">VWR</td>
			<td style="width:219px;">#CABDH9258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl&#160;</td>
			<td style="width:215px;">VWR</td>
			<td style="width:219px;">#CABDH9286</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4H2O&#160;</td>
			<td style="width:215px;">VWR</td>
			<td style="width:219px;">#CABDH9298</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly D-lysine&#160;</td>
			<td style="width:215px;">VWR</td>
			<td style="width:219px;">#89134-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">DMEM</td>
			<td style="width:215px;">Wisent</td>
			<td style="width:219px;">#319-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">FBS</td>
			<td style="width:215px;">Wisent</td>
			<td style="width:219px;">#080-450</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling neuronal death and degeneration in mouse primary cerebellar granule neurons

Cerebellare granulet neuroner (CGNs) er et almindeligt anvendte neuronal model, danner en rigelige homogen befolkning i lillehjernen. I lyset af deres postnatal udvikling, overflod, og adgangen er CGNs en ideel model til at studere neuronal processer, herunder neuronal udvikling, neuronal migration og fysiologiske neuronal aktivitet stimulation. Derudover giver CGN kulturer en fremragende model for at studere forskellige former for celledød, herunder excitotoxicity og apoptose. Inden for en uge i kultur express CGNs N-methyl-D-aspartat (NMDA) receptorer, en bestemt ionotropic glutamat receptor med mange kritiske funktioner i neuronal sundhed og sygdom. Tilsætning af lave koncentrationer af NMDA sammenholdt med membran depolarisering til gnaver primære CGN kulturer har været brugt til at modellere fysiologiske neuronal aktivitet stimulation, mens tilsætning af høje koncentrationer af NMDA kan være ansat til at modellere excitotoxic neuronal skade. Her beskrives en metode til isolering og dyrkning af CGNs fra 6 dage gamle hvalpe samt genetisk manipulation af CGNs af adenovirus og lentiviruses. Vi også nuværende optimerede protokoller om, hvordan man stimulerer NMDA-induceret excitotoxicity, lav-kalium-induceret apoptose, oxidativ stress og DNA-skader efter transduktion af disse neuroner.

Med omhyggelig dissektion, intakt hjernen, bør fjernes med minimal skade som det ses i figur 1A-B. Indsats bør træffes for at minimere hjerneskader under fjernelse, især skader på lillehjernen. Beskadige lillehjernen gør for vanskeligere identifikation og fuldstændig fjernelse af meninges, og øger risikoen for kontaminering af neuronal kultur. Når meninges er blevet fjernet, kan lillehjernen dissekeret fra de resterende væv, som det...

Watch this Scientific Journal Video about Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons at JoVE.com

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Denne protokol beskriver en simpel metode til isolering og dyrkning af primære mus cerebral granulet neuroner (CGNs) fra 6-7 dage gamle hvalpe, effektiv transduktion af CGNs for tab og gevinst på funktion undersøgelser, og modellering NMDA-induceret neuronal excitotoxicity, lav-kalium-induceret celledød, DNA-skader og oxidativt stress ved hjælp af den samme kultur model.

Modeling Neuronal død og Degeneration i musen primære cerebellare granulet neuroner

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Research

JoVE Journal

Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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Genetisk manipulation af Cerebellær granuleneuroner<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; At studere Neuronal morfologi og Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging af Postnatal cerebellare Granule Cell Migration Brug Konfokal makroskopi

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

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

Selektiv Udtømning af Mikroglia fra cerebellare Granule cellekulturer anvendelse af L-leucin-methylester

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

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Vurdering af neuronal levedygtighed ved anvendelse af fluoresceindiacetat-propidiumiodid dobbeltfarvning i cerebellar granulærneuronkultur

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

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Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

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It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...