Name: cerebellar regional dissection for molecular analysis
Uploaded: 2020-12-05T14:30:00
Description: Watch this Scientific Journal Video about Cerebellar Regional Dissection for Molecular Analysis at JoVE.com

Vi er taknemmelige for Austin Ferro og Juao-Guilherme Rosa i Cvetanovic lab for deres hjælp til fejlfinding dissektioner og i RNA udvinding og RTqPCR. Denne forskning er finansieret af M. Cvetanovic, R01 NS197387; HHS | Nationale institutter for sundhed (NIH).

1. Opsætning
<ol><li>Saml nødvendigt udstyr, herunder halshugning saks, stump tang, dissektion saks, vaskulær saks, mikrospatula, sagittal mus hjerne matrix, barberblade, 200 μL pipet tips, glas petriskål, glas dias, og is spand. Læg alt udstyr på en absorberende pude.</li>
	<li>Placer petriskål, glasplade og hjernematrix på is.</li>
	<li>Ved hjælp af et barberblad i vinkelret vinkel skal du trimme ca. 5 mm af spidsen af en 200 μL pipetspids. Dette gør størrelsen af åbningen i slutningen ...

<ol>
	<li>Herculano-Houzel, 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+glia/neuron+ratio:+How+it+varies+uniformly+across+brain+structures+and+species+and+what+that+means+for+brain+physiology+and+evolution.">The glia/neuron ratio: How it varies uniformly across brain structures and species and what that means for brain physiology and evolution.</a> Glia. 62 (9), 1377-1391 (2014).</li><li>Schmahmann, J. D., Caplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognition,+enotion,+and+the+cerebellum.">Cognition, enotion, and the cerebellum.</a> Brain. 129 (2), 290-292 (2006).</li><li>Badura, 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=Normal+cognitive+and+social+development+require+posterior+cerebellar+activity.">Normal cognitive and social development require posterior cerebellar activity.</a> eLife. 7, 36401 (2018).</li><li>Diedrichsen, J., King, M., Hernandez-Castillo, C., Sereno, M., Ivry, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+Transform+or+Multiple+Functionality?+Understanding+the+Contribution+of+the+Human+Cerebellum+across+Task+Domains.">Universal Transform or Multiple Functionality? Understanding the Contribution of the Human Cerebellum across Task Domains.</a> Neuron. 102 (5), 918-928 (2019).</li><li>Strick, P. L., Dum, R. P., Fiez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellum+and+Nonmotor+Function.">Cerebellum and Nonmotor Function.</a> Annual Review of Neuroscience. 32 (1), 413-434 (2009).</li><li>King, M., Hernandez-castillo, C. R., Poldrack, R. A., Ivry, R. B., Diedrichsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+boundaries+in+the+human+cerebellum+revealed+by+a+multi-domain+task+battery.">Functional boundaries in the human cerebellum revealed by a multi-domain task battery.</a> Nature Neuroscience. 22, 1371-1378 (2019).</li><li>Buckner, R. L., Krienen, F. M., Castellanos, A., Diaz, J. C., Yeo, B. T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+the+human+cerebellum+estimated+by+intrinsic+functional+connectivity.">The organization of the human cerebellum estimated by intrinsic functional connectivity.</a> Journal of neurophysiology. 106 (5), 2322-2345 (2011).</li><li>Schmahmann, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+movement+to+thought:+Anatomic+substrates+of+the+cerebellar+contribution+to+cognitive+processing.">From movement to thought: Anatomic substrates of the cerebellar contribution to cognitive processing.</a> Human Brain Mapping. 4 (3), 174-198 (1996).</li><li>Kelly, R. M., Strick, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+Loops+with+Motor+Cortex+and+Prefrontal+Cortex+of+a+Nonhuman+Primate.">Cerebellar Loops with Motor Cortex and Prefrontal Cortex of a Nonhuman Primate.</a> The Journal of Neuroscience. 23 (23), 8432-8444 (2003).</li><li>Stoodley, C. J., Macmore, J. P., Makris, N., Sherman, J. C., Schmahmann, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Location+of+lesion+determines+motor+vs.+cognitive+consequences+in+patients+with+cerebellar+stroke.">Clinical Location of lesion determines motor vs. cognitive consequences in patients with cerebellar stroke.</a> NeuroImage: Clinical. 12, 765-775 (2016).</li><li>Guo, C. C., Tan, R., Hodges, J. R., Hu, X., Sami, S., Hornberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network-selective+vulnerability+of+the+human+cerebellum+to+Alzheimer's+disease+and+frontotemporal+dementia.">Network-selective vulnerability of the human cerebellum to Alzheimer's disease and frontotemporal dementia.</a> Brain. 139 (5), (2016).</li><li>Bocchetta, M., Cardoso, M. J., Cash, D. M., Ourselin, S., Warren, J. D., Rohrer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+regional+cerebellar+atrophy+in+genetic+frontotemporal+dementia.">Patterns of regional cerebellar atrophy in genetic frontotemporal dementia.</a> NeuroImage: Clinical. 11, 287-290 (2016).</li><li>Sillitoe, R. V., Fu, Y., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellum.">Cerebellum.</a> The Mouse Nervous System. , 360-397 (2012).</li><li>Nguyen-Minh, V. T., Tran-Anh, K., Luo, Y., Sugihara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Excitability+and+Parallel+Fiber+Synaptic+Properties+of+Zebrin-Positive+and+-Negative+Purkinje+Cells+in+Lobule+VIII+of+the+Mouse+Cerebellar+Slice.">Electrophysiological Excitability and Parallel Fiber Synaptic Properties of Zebrin-Positive and -Negative Purkinje Cells in Lobule VIII of the Mouse Cerebellar Slice.</a> Frontiers in Cellular Neuroscience. 12, 513 (2019).</li><li>Manto, M., Oulad Ben Taib, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+nuclei:+Key+Roles+for+Strategically+Located+Structures.">Cerebellar nuclei: Key Roles for Strategically Located Structures.</a> The Cerebellum. 9 (1), 17-21 (2010).</li><li>Driessen, T. M., Lee, P. J., Lim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathway+analysis+towards+understanding+tissue+vulnerability+in+spinocerebellar+ataxia+type+1.">Molecular pathway analysis towards understanding tissue vulnerability in spinocerebellar ataxia type 1.</a> eLife. , (2018).</li><li>Grabert, K., 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=Microglial+brain+region+-+dependent+diversity+and+selective+regional+sensitivities+to+aging.">Microglial brain region - dependent diversity and selective regional sensitivities to aging.</a> Nat Neurosci. 19 (3), 504 (2016).</li><li>Ingram, 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=Cerebellar+Transcriptome+Profiles+of+ATXN1+Transgenic+Mice+Reveal+SCA1+Disease+Progression+and+Protection+Pathways.">Cerebellar Transcriptome Profiles of ATXN1 Transgenic Mice Reveal SCA1 Disease Progression and Protection Pathways.</a> Neuron. 89 (6), 1194-1207 (2016).</li><li>Cendelin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> From mice to men : lessons from mutant ataxic mice. , 1-21 (2014).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+RNA+Using+TRIzol+(TRI+Reagent).">Purification of RNA Using TRIzol (TRI Reagent).</a> Cold Spring Harbor Protocols. 2010 (6), (2010).</li><li>Kim, J. H., Lukowicz, A., Qu, W., Johnson, A., Cvetanovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglia+contribute+to+the+pathogenesis+of+spinocerebellar+ataxia+Type+1+(SCA1)+in+a+biphasic,+stage-of-disease+specific+manner.">Astroglia contribute to the pathogenesis of spinocerebellar ataxia Type 1 (SCA1) in a biphasic, stage-of-disease specific manner.</a> Glia. 66 (9), 1972-1987 (2018).</li><li>Chopra, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Capicua+expression+drives+regional+Purkinje+neuron+vulnerability+through+ion+channel+gene+dysregulation+in+spinocerebellar+ataxia+type+1.">Altered Capicua expression drives regional Purkinje neuron vulnerability through ion channel gene dysregulation in spinocerebellar ataxia type 1.</a> Human Molecular Genetics. , (2020).</li></ol>

Den her beskrevne metode gør det muligt at vurdere det underliggende genekspression og molekylære mekanismer inden for fire forskellige cerebellarområder – den dybe cerebellarkerner (DCN), vermis '(CCaV) forreste cerebellarbark , vermis 's bageste cerebellar cortex (CCpV) og halvkuglernes lillehjernen cortex (CCH). Evnen til at vurdere disse regioner separat vil udvide vores viden om heterogeniteten i specifikke cerebellar-regioner og muligvis kaste lys over deres bidrag til forskellige adfærd.
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Den lillehjernen indeholder over halvdelen af neuroner i hjernen og har historisk været omtalt som en motorisk kontrol og balance center i hjernen1. For nylig har undersøgelser vist, at lillehjernen spiller en central rolle i forskellige andre funktioner, herunder kognition, belønningsbehandling og påvirker2,3,4,5.
Lillehjernen har velbeskrevet anatomi: cortex-regionen består af granulat, Purkinje og molekylære lag. Granulatceller danner granulatcellelaget og sender input via parallelle fibre til Purkinje-celledecititterne i det molekylære lag, som også modtager input fra klatrefibre, der stammer fra den ringere oliven. Purkinje celler sende hæmmende fremskrivninger til celler i den dybe cerebellar kerner (DCN), der tjener som den vigtigste produktion fra lillehjernen. Produktionen af dette lillehjernskredsløb moduleres yderligere af aktiviteten af de hæmmende interneuroner i cerebellar cortex, herunder Golgi, stellate og kurvceller4. Denne lillehjernen funktionelle enhed er fordelt over alle lobules af cerebellar cortex. På trods af dette relativt ensartede kredsløb på tværs af lillehjernen indikerer beviser fra menneskelig neuroimaging litteratur og patientundersøgelser funktionel heterogenitet af lillehjernen6,7.
Den lillehjernen cortex kan opdeles i to hovedregioner: den midterlinje-definerede vermis, og den laterale halvkugler. Vermis kan yderligere opdeles i forreste og bageste lobules. Disse forskellige regioner i lillehjernen har været impliceret i at bidrage til forskellige adfærd. Opgave-fremkaldte eller opgave-fri aktivitetsmønstre impliceret, at forreste regioner i vermis bidrage mere til motorisk funktion, mens posterior vermis bidrager mere til kognition6,7. Vermis er også forbundet med påvirkning og følelser, mens cerebellar halvkugler bidrager til udøvende, visuel-rumlige, sprog og andre mnemoniske funktioner8. Desuden gav anatomiske undersøgelser bevis for, at funktionelt adskilte cerebellar regioner er forbundet med forskellige kortikale regioner9. Lesion-symptom kortlægning viste, at patienter med slagtilfælde påvirker forreste lobules (strækker sig ind i lobule VI) havde dårligere resultater på finmotoriske opgaver, mens patienter med skader på bageste lap regioner og halvkugler udstillet kognitive underskud i mangel af cerebellar motor syndrom10. Endelig, regionale cerebellar patologi i sygdom viser, at funktionelt forskellige cerebellar regioner er også forskelligt modtagelige for sygdom11,12.
Mens langt mindre udforsket, foreløbige beviser viser forskellige genekspression underskrifter på tværs af cerebellar kortikale regioner. Purkinje celle udtryk zebrin II viser region specifikke mønstre i vermis sådan, at der er flere Zebrin II positive celler i posterior lobules og færre i forreste lobules13. Dette korrelerer også med regionalt forskellige fysiologiske funktion som Zebrin II negative Purkinje celler viser højere hyppighed af tonic fyring end Purkinje celler, der er Zebrin II positive14.
Ud over cerebellar cortex omfatter lillehjernen de dybe cerebellarkerner (DCN), der tjener som den primære udgang for lillehjernen. Kernerne består af mediale (MN), interposed (IN), og lateral kerner (LN). Funktionel billeddannelse og patientundersøgelser har vist, at DCN også deltager i forskellige adfærdsformer15, men meget få undersøgelser undersøger genekspressionsændring i DCN.
Fremskridt inden for molekylære teknikker har gjort det muligt at vurdere regionale genekspression i hjernen og har afdækket heterogenitet på tværs af og inden for forskellige hjerneområder i både fysiologiske og sygdomstilstande16. Sådanne undersøgelser implicerer, at lillehjernen er forskellig fra andre hjerneregioner. For eksempel er forholdet mellem neuroner og gliaceller omvendt i lillehjernen sammenlignet med andre hjerneområder1. Selv under normale fysiologiske forhold er ekspressionen af proinflammatoriske gener upreguleret i lillehjernen sammenlignet med de andre hjerneområder17. Molekylære teknikker har også været meget nyttige til at identificere de veje, der bidrager til patogenese af cerebellarsygdomme. For eksempel identificerede RNA-sekventering af hele cerebellarekstrakterne gener, der var ændret i en Purkinje-cellespecifik transgen musemodel af spinocerebellar ataxia type 1 (SCA1) sammenlignet med deres vilde typekontroller. Sådanne beviser har afsløret vigtige molekylære veje, der ligger til grund for patogenese i cerebellar Purkinje-celler og har bidraget til at identificere potentielle terapeutiske mål18. Nylige undersøgelser tyder dog på, at der er forskelle i sårbarheden over for sygdomme i de lillehjernen regioner11,12,19. Dette kan indikere, at der sker vigtige ændringer i forskellige cerebellarområder, som kan maskeres eller ikke registreres med hele cerebellarekstrakter. Der er således behov for at udvikle teknikker, der gør det muligt for forskere at undersøge molekylære profiler i forskellige cerebellarregioner.
Den teknik, der foreslås her, beskriver en reproducerbar metode til at dissekere fire forskellige regioner i musebellum for at isolere RNA fra disse regioner og udforske regionale forskelle i genekspression. Den skematiske af musen lillehjernen i Figur 1A fremhæver vermis i blåt, og halvkugler i gult. Nærmere bestemt denne teknik gør det muligt at isolere fire regioner: dyb cerebellarkerner (DCN) (rød-prikket kasser i figur 1A), den lillehjernen cortex af forreste vermis (CCaV) (mørk blå i figur 1A), den lillehjernen cortex af den bageste vermis (CCpV) (lyseblå i figur 1A), og cerebellar cortex af halvkugler (CCH) (gul i figur 1A). Ved at vurdere genekspressionen af disse regioner separat vil det være muligt at undersøge molekylære mekanismer, der ligger til grund for diskrete funktioner i disse forskellige regioner, samt potentielle forskelle i deres sårbarhed i sygdom.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1.5 Microcentrifuge tubes</td>
			<td >ThermoScietific</td>
			<td >3456</td>
			<td ></td>
		</tr>
		<tr >
			<td >100% Isopropyl Alcohol</td>
			<td >VWR Life sciences</td>
			<td >1106C361</td>
			<td></td>
		</tr>
		<tr >
			<td >200 ul Pipet tips</td>
			<td >GeneMate</td>
			<td >P-1237-200</td>
			<td></td>
		</tr>
		<tr >
			<td >Adult Mouse Brain Matrix Sagittal</td>
			<td >Kent Scientific Corporation</td>
			<td >RBMA-200S</td>
			<td></td>
		</tr>
		<tr >
			<td >Blunt forceps</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Chloroform</td>
			<td >Macron</td>
			<td >220905</td>
			<td></td>
		</tr>
		<tr >
			<td >Decapitation Scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting Scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl Alcohol</td>
			<td >Pharmco</td>
			<td >111000200</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Slide (for electrophoresis)</td>
			<td >BIORAD</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Homogenizer</td>
			<td >Kimble</td>
			<td >6HAZ6</td>
			<td></td>
		</tr>
		<tr >
			<td >Ice Bucket</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Insulin Syringe (.5ml)</td>
			<td >BD</td>
			<td >329461</td>
			<td></td>
		</tr>
		<tr >
			<td >iScript Adv cDNA kit for RT-qPCR</td>
			<td >BIORAD</td>
			<td >1725037</td>
			<td></td>
		</tr>
		<tr >
			<td >Micro Spatula</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Nose forceps</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Pyrex</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer for Aldolase C</td>
			<td >IDT</td>
			<td >Mm.PT.58&#62;43415246</td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer for Kcng4</td>
			<td >IDT</td>
			<td >Mm.PT.56a.9448518</td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer for Parvalbumin</td>
			<td >IDT</td>
			<td >Mm.PT.58.7596729</td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer Rps18&#160;</td>
			<td >IDT</td>
			<td >Mm.PT.58.12109666</td>
			<td></td>
		</tr>
		<tr >
			<td >Single Edge Rzor Blades</td>
			<td >Personna GEM</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sterile, sigle-use pestles</td>
			<td >FisherScientific</td>
			<td >12141364</td>
			<td></td>
		</tr>
		<tr >
			<td >TRIzol Reagent</td>
			<td >Ambion by Life technologies</td>
			<td >15596018</td>
			<td></td>
		</tr>
		<tr >
			<td >Vascular Scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

cerebellar regional dissection for molecular analysis

Cerebellum spiller en vigtig rolle i flere nøglefunktioner, herunder kontrol af bevægelse, balance, kognition, belønning og indflydelse. Imaging undersøgelser viser, at forskellige cerebellar regioner bidrager til disse forskellige funktioner. Molekylære undersøgelser, der undersøger regionale cerebellarforskelle, halter, da de for det meste udføres på hele cerebellarekstrakter og derved maskerer enhver sondring på tværs af specifikke cerebellar-regioner. Her beskriver vi en teknik til reproducerbart og hurtigt dissekere fire forskellige cerebellar regioner: den dybe cerebellar kerner (DCN), forreste og bageste vermal cerebellar cortex, og cerebellar cortex af halvkugler. Dissekering af disse forskellige regioner giver mulighed for udforskning af molekylære mekanismer, der kan ligger til grund for deres unikke bidrag til balance, bevægelse, indflydelse og kognition. Denne teknik kan også bruges til at undersøge forskelle i patologisk modtagelighed af disse specifikke regioner på tværs af forskellige musesygdomsmodeller.

Til disse eksperimenter blev der anvendt fire elleve uger gamle vilde kvindelige C57/Black6-mus. En mus blev brugt til at foretage en fuld cerebellar dissektion, der kaldes 'bulk cerebellum' og gav mulighed for sammenligning af RNA-niveauer i dissekerede regioner til en fuld dissektion. De tre andre mus blev brugt til at udføre den cerebellar dissektion, der er beskrevet i denne protokol. Ved hjælp af tre mus gør det muligt at sikre, at de tendenser, der opdages i niveauerne af RNA, ka...

Watch this Scientific Journal Video about Cerebellar Regional Dissection for Molecular Analysis at JoVE.com

Cerebellar Regional Dissection for Molecular Analysis

Forskellige cerebellar regioner har været impliceret til at spille en rolle i forskellige adfærdsmæssige output, men de underliggende molekylære mekanismer forbliver ukendt. Dette arbejde beskriver en metode til reproducerbart og hurtigt dissekere cerebellar cortex af halvkugler, forreste og bageste regioner i vermis, og den dybe cerebellar kerner med henblik på at sonde for molekylære forskelle ved at isolere RNA og test for forskelle i genekspression.

Cerebellar regional dissektion til molekylær analyse

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies ...

Traditional molecular studies of the cerebellum have been done on whole cerebellar extracts, which may mask any distinctions across specific cerebellar regions. This protocol makes it possible to assess distinct regions of the cerebellum separately and allows for the exploration of molecular mechanisms that may underlie their unique contributions to a variety behaviors and disease progression. The main advantage of this technique is that it allows for the reproducible and quick dissection of four cerebellar regions, the deep cerebellar nuclei, the anterior and posterior cerebellar cortex of the Vermis and the cerebellar cortex of the hemispheres.After decapitating the euthanized mouse, make an incision with a razor blade along the medial sagittal line of the head, starting at the nose and continuing all the way back. Separate the skin and use the razor blade to cut away the muscle on each side, cutting down past the ear canals. Using dissecting scissors, trim many spinal cord regions up to where the brain stem meets the cerebellum, taking care to not damage the cerebellum.Insert one of the vascular scissor blades into the space between the brainstem and vertebral column and cut toward the ear canal, lifting upward while cutting to limit damage to the tissue. Continue to cut along the edge of the skull up toward the olfactory bulbs. Using the blunt forceps, gently peel off the back of this skull to uncover the posterior region of the brain and cerebellum.Position the blunt forceps along the cut edge of the skull and peel the skull up and over the brain. Trim the rest of the skull with the vascular scissors and blunt forceps, clearing most of it from the top of the brain. Slightly lift the brain with the micro spatula to remove the olfactory bulbs from the remaining skull and disconnect optic tract fibers.The brain should come free easily at this point. Place the brain into the Petri dish, sitting on ice and remove any remaining skull or other debris. Gently place the brain into the brain matrix with the dorsal side up.Make sure that it is set level in the matrix and that the midline is in the center. Place one razor blade along the sagittal midline, making sure that the blade pushes all the way to the bottom of the matrix. Place another razor blade one millimeter to the side of the first blade.Place two more blades resulting in three blades placed on one side of the brain, all one millimeter apart. Repeat this on the other side. carefully grab the front and back end to the razorblades, and lift them straight up out of the matrix.Discard the tissue on the outside of the razor blades. Slowly, separate one razor blade at a time from the others being careful not to damage the tissue sections. Slide the tissue section off the razor blade and onto the glass slide with the micro spatula.Six sagittal brain sections should be collected. The foremost lateral sections will have DCN visible. To isolate the DCN, hold a trim to 200 microliter pipette tip perpendicularly over the DCN and push down through the tissue firmly rocking in all directions.Lift the pipette up and visually confirm the presence of the tissue in the tip. Place one finger at the top of the tip and push down, causing the tissue to bulge out. Insert the tip into a correctly labeled microfiche tube and ensure that the tissue punch is placed in the bottom of the tube.Repeat this for the remaining three sections, placing the DCN punches in the same tube. Flash freeze the tube in liquid nitrogen. Push away the rest of the brain tissue around the cerebellum in the sections where the DCN was extracted.Use blunt forceps to gently pick up these hemisphere cerebellar cortex sections and place them into respective microfiche tubes, then flash freeze them. For the last two vermal sections, push away surrounding brain tissue to leave only the cerebellum. Using a razor blade, make a cut separating the anterior labials from the posterior labials.Ensure that the cut is just after the formation of labial six and does not include labial 10. Using blunt forceps, carefully placed the anterior cerebellar cortex sections and posterior cerebellar cortex sections into their respective microfiche tubes and flash freeze them by leaving the tubes in liquid nitrogen for five minutes. Place the microfiche tubes on ice to keep the tissue from thawing too quickly and apply 150 microliters of cold RNA isolation solution in the microfiche tube, then homogenize with a sterilized pestle.Once the tissue is homogenized pipette the solution up and down to ensure there is no remaining intact tissue. Further, break up any small tissue pieces by pulling it up into an insulin syringe a few times. Add another 350 microliters of the reagent, pipette up and down to mix thoroughly and let it sit at room temperature for five minutes.Add 150 microliters of chloroform to the tube and shake it vigorously, then let it rest for two to three minutes. Centrifuge at 12, 000 times G at 15 degrees Celsius for 10 minutes. Carefully remove the tubes from the centrifuge and set the temperature of the centrifuge to four degrees Celsius.Transfer only the clear aqueous phase into a new tube, taking care not to disrupt the opaque interphase. Save or discard the remaining solution in the tubes. Add 100%isopropyl alcohol at a one to two ratio and mix thoroughly by pipetting up and down.Let the sample rest at room temperature for 10 minutes to precipitate the RNA out of the solution. Centrifuge at 12, 000 times G and four degrees Celsius for 10 minutes, placing all tubes in the same orientation to make it easier to visualize the pellet. After centrifugation, carefully remove the tubes and remove the supernatant with the pipette, making sure to not disrupt the pellet.The pellet is gel like and difficult to see, so estimate where it is based on the orientation of the tubes in the centrifuge. After removing all supernatant, add 500 microliters of 75%ethanol, vortex briefly and centrifuge at 7, 500 times G and four degrees Celsius for five minutes. Remove the supernatant carefully, without disrupting the pellet.Leave the caps open to dry the sample for five to 10 minutes. Once dry resuspend the pellet in DNA's free water at 20 microliters to the samples for DCN and 30 microliters for all others. Store the samples at minus 80 degrees Celsius until further analysis.This protocol was used to dissect four distinct regions of the mouse cerebellum and to explore regional differences in gene expression. The expression levels of Aldolase C, parvalbumin and KCNG4 were assessed using real-time quantitative PCR. Aldolase C is more highly expressed in the posterior cerebellar vermis.But lower in the DCN and the anterior region of the vermis when compared to the bulk cerebellar dissection. Parvalbumin is similarly present in the DCN anterior vermis, posterior vermis and hemisphere cerebellar cortices, when compared to bulk cerebellar extracts. KCNG4 is significantly enriched in the DCN and the interior vermis, but not in the posterior vermis or hemispheres when compared to the bulk extraction.To directly compare the expression of aldolase C across the cerebellar cortex, the expression levels were compared to the anterior vermis. The expression level of aldolase C was significantly higher in the posterior Vermis and trending higher in the cerebellar hemispheres. The RNA extracted from these regions can also be used in RNA sequencing, experiments.RNA seq comparing these for cerebellar regions would provide more detailed information on molecular mechanisms, underlying discrete functions of these regions. As well as potential differences in their vulnerability in disease.

Research

JoVE Journal

Neuroscience

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

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Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

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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 ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

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Laser Nanosurgery of Cerebellar Axons In Vivo

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A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

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En simpel en-trin Dissektion Protokol for Whole-mount Forberedelse af Voksen<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...