Name: collection of frozen rodent brain regions for downstream analyses
Uploaded: 2020-04-23T13:00:00
Description: Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Dette arbeidet ble støttet av NIH, DA043982 og DA046196.

Dyr som brukes i denne studien ble behandlet på en etisk og human måte som fastsatt av Indiana University's Institutional Animal Care and Use Committee (IACUC) og National Institutes of Health (NIH) retningslinjer.
MERK: Alle verktøy og overflater skal vaskes med et passende løsningsmiddel for å fjerne nukleone18 før du starter noe arbeid.
1. Lagring av hjerner
<ol><li>Fjern raskt hjernen fra eutaniserte voksne CD1 wildtype mus som ve...

<ol>
	<li>. Allen Mouse Brain Atlas Available from: <a target="_blank" href="https://mouse.brain">https://mouse.brain</a> (2008)</li><li>Kuleshova, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+-+New+Potentials+for+Electrophysiology.">Optogenetics - New Potentials for Electrophysiology.</a> Neuroscience and Behavioral Physiology. 49 (2), 169-177 (2019).</li><li>Scanziani, M., H&#228;usser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+in+the+age+of+light.">Electrophysiology in the age of light.</a> Nature. 461, 930 (2009).</li><li>Shimono, M., Beggs, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Clusters,+Hubs,+and+Communities+in+the+Cortical+Microconnectome.">Functional Clusters, Hubs, and Communities in the Cortical Microconnectome.</a> Cerebral cortex. 25 (10), 3743-3757 (2015).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Kebschull, J. 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M., Lau, H. T., So, K. F., Chang, R. C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-dissection+of+Rat+Brain+for+RNA+or+Protein+Extraction+from+Specific+Brain+Region.">Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region.</a> JoVE. (7), e269 (2007).</li><li>Aring, L., S, S., Marcus, K., May, C., Murran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Capture Microdissection. Methods in Molecular Biology. 1723, 2457 (2018).</li><li>Bj&#246;rk, 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=Glutathione-S-transferase+expression+in+the+brain:+possible+role+in+ethanol+preference+and+longevity.">Glutathione-S-transferase expression in the brain: possible role in ethanol preference and longevity.</a> The FASEB Journal. 20 (11), 1826-1835 (2006).</li><li>Jeong, H., 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=Gene+Network+Dysregulation+in+the+Trigeminal+Ganglia+and+Nucleus+Accumbens+of+a+Model+of+Chronic+Migraine-Associated+Hyperalgesia.">Gene Network Dysregulation in the Trigeminal Ganglia and Nucleus Accumbens of a Model of Chronic Migraine-Associated Hyperalgesia.</a> Frontiers in Systems Neuroscience. 12 (63), (2018).</li><li>Spijker, S., Li, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Rodent+Brain+Regions.">Dissection of Rodent Brain Regions.</a> Neuroproteomics, Neuromethods. 57, 13-26 (2011).</li><li>Palkovits, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+removal+of+hypothalamic+or+other+brain+nuclei+of+the+rat.">Isolated removal of hypothalamic or other brain nuclei of the rat.</a> Brain Research. 59, 449-450 (1973).</li><li>Palkovits, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 103, 368-376 (1983).</li><li>Paxinos, G., Franklin, K. B. 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> The Mouse Brain in Stereotaxic Coordinates. 2 edn. , (2001).</li><li>Paxinos, G., 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=.">.</a> The Rat Brain in Stereotaxic Coordinates. 4 edn. , (1998).</li><li>RNaseZap. Ambion Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/">https://assets.thermofisher.com/TFS-Assets/LSG/</a> (2008)</li><li>. RNA Integrity Number (RIN)- Standardization of RNA Quality Control Available from: <a target="_blank" href="https://www.agilent.com/cs/library/">https://www.agilent.com/cs/library/</a> (2016)</li><li>Scheyer, A. 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=Cannabinoid+Exposure+via+Lactation+in+Rats+Disrupts+Perinatal+Programming+of+the+Gamma-Aminobutyric+Acid+Trajectory+and+Select+Early-Life+Behaviors.">Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors.</a> Biological Psychiatry. , (2019).</li><li>Choi, D. W., Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+glutamate+neurotoxicity+in+hypoxic-ischemic+neuronal+death.">The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death.</a> Annual Review of Neuroscience. 13 (1), 171-182 (1990).</li><li>Guhaniyogi, J., Brewer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mRNA+stability+in+mammalian+cells.">Regulation of mRNA stability in mammalian cells.</a> Gene. 265 (1-2), 11-23 (2001).</li><li>Sampaio-Silva, F., Magalh&#227;es, T., Carvalho, F., Dinis-Oliveira, R. J., Silvestre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+RNA+degradation+for+estimation+of+post+mortem+[corrected]+interval.">Profiling of RNA degradation for estimation of post mortem [corrected] interval.</a> PloS One. 8 (2), 56507 (2013).</li><li>Yamagata, 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=Signs+of+biological+activities+of+28,000-year-old+mammoth+nuclei+in+mouse+oocytes+visualized+by+live-cell+imaging.">Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.</a> Scientific Reports. 9 (1), 4050 (2019).</li><li>Allen Brain Atlas: Developing Mouse Brain. Allen Institute Available from: <a target="_blank" href="https://developingmouse.brain-map.org/static/atlas">https://developingmouse.brain-map.org/static/atlas</a> (2008)</li></ol>

Dette arbeidet beskriver en teknikk for å isolere små, spesifikke områder av hjernen samtidig som de begrenser nedbrytning av nukleinsyre og protein. Skade på hjernevev skjer raskt når en organisme dør. Dette skyldes delvis en rask oppbygging av ekstracellulær glutamat og den resulterende eksitotoksisiteten som oppstår21. Messenger RNA er spesielt utsatt for nedbrytning22,23. Nedbrytning av protein og nukleinsyre reduseres sterkt v...

Dette arbeidet illustrerer disseksjon av frosne hjerneregioner for utvinning av nukleinsyre eller protein av høy kvalitet ved hjelp av en referanse, for eksempel Allen Mouse Brain Atlas1, som en guide. I denne teknikken blir hjernen flash-frosset og lagret ved -80 °C for senere snitting og disseksjon mens de opprettholdes i frossen tilstand. Denne prosessen gjør det mulig for forskeren å høste et stort antall hjerner i en økt og senere dissekere dem for en nøyaktig samling av flere hjerneregioner.
Den nøyaktige samlingen av hjerneregioner av interesse (ROIer) er ofte nødvendig når du svarer på spørsmål knyttet til gen- og proteinuttrykk. Mens farmakologi, elektrofysiologi og optogenetikk kan brukes på villtype eller genmodifiserte gnagere for å bidra til å belyse molekylære endringer underbygge observert atferd2,3,4, målingen av induserte endringer i transkripsjoner og proteomer brukes ofte til å støtte disse funnene. Teknikker som kvantitativ omvendt transkripsjon polymerase kjedereaksjon (RT-qPCR), western blotting, RNAseq5, MAPSeq6 og HPLC7 er robuste og relativt lave i pris, slik at mange laboratorier kan studere indusertmolekylære endringer i små hjerneregioner2,4,5,6.
Det er flere måter å trekke ut og rense nukleinsyre eller protein fra hjerneregioner8,9,10,11,12. Mange laboratorier høster hjerneregioner ved å kjøle og kutte hjerner på is på tidspunktet for innhøsting9,13. Selv om denne tilnærmingen kan resultere i kjernesyre og protein av høy kvalitet, er det noe tidsbegrenset som nedbrytning i mikromiljøet i vevet kan finne sted ved disse temperaturene. Dette kan være spesielt sant når du prøver å dissekere et stort antall dyr eller ROIer i en sittende. Holde prøver frosset bidrar til å opprettholde labile målmolekyler samtidig som forskeren tid til å nøye sammenligne landemerker på begge sider av hver seksjon i arbeidet med å samle relativt rene prøver. Laserfangst er en annen måte å samle vev for RNA eller proteinanalyse fra hjerneområder10. Denne prosedyren er bedre enn mekanisk disseksjon ved at svært små og uregelmessig formede ROIer kan identifiseres og isoleres. Laserfangst er imidlertid begrenset av bruk av dyrt utstyr og reagenser, er tidkrevende og kan også være mer utsatt for prøvenedbrytning.
Mikropunch disseksjon på frosne vev er ikke nytt. Tidlige papirer av Miklos Palkovits og andre beskriver de grunnleggende teknikkene i detalj14,15. Denne presentasjonen følger i stor grad det opprinnelige arbeidet, med noen forbedringer for å lette effektiviteten og redusere kostnadene for utstyret som trengs. For eksempel er hjerneseksjoner laget i en frossen hjerneblokk i stedet for på en kryostat. Dette gir tykkere seksjoner som reduserer antall seksjoner som trengs for å samle inn avkastningsprøver. Denne metoden dissekerer også prøver på en frossen glassplate som sitter på tørris i en isolert boks. Dette gir en sub-frysing scenen på benken som å jobbe. Seksjoner dissekert på denne måten er lett manipuleres, slik at forskeren kan sammenligne begge sider av hver seksjon med en referanse for å begrense forurensning fra regioner utenfor ønsket avkastning.
Fordelene med denne protokollen er at 1) hjernen holdes i en frossen tilstand gjennom hele prosessen, noe som bidrar til å bevare protein og nukleinsyre og gir forskeren tid til å nøye høste ROIer, og 2) reagensene som kreves er billig og finnes i de fleste molekylærbiologilaboratorier. I denne prosessen er hele hjernen delt til 0,5–1,0 mm i en hjernematrise og plassert på en frossen glassplate som kontinuerlig avkjøles med tørris. Landemerker funnet i Allen Brain Atlas1 eller andre hjerneatlas16,brukes 17 til å identifisere områder av interesse, som deretter dissekeres ved hjelp av enten et kaldt slag eller skalpell. Fordi vevet aldri tines, gir regioner høstet på denne måten høy kvalitet RNA og protein for nedstrøms analyser.

<table>
	<tbody>
		<tr>
			<td>0.5 mm Mouse coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 505C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>0.5 mm Rat coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 705C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>1.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Dot Scientific</td>
			<td>229443</td>
			<td>For storing frozen ROIs</td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-03</td>
			<td></td>
		</tr>
		<tr>
			<td>4-12% NuPage gel</td>
			<td>Invitrogen</td>
			<td>NPO323BOX</td>
			<td>protein gradient gel</td>
		</tr>
		<tr>
			<td>Bioanalyzer System</td>
			<td>Agilent</td>
			<td>2100</td>
			<td>RNA analysis system</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Millipore Sigma</td>
			<td> 
			D8938</td>
			<td>Glass tissue homogenizer</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Lite</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>Model 180</td>
			<td>Cool lamp</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>LabRepCo</td>
			<td>11074010</td>
			<td></td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>ThermoFisher</td>
			<td>78440</td>
			<td>protease inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Low profile blades</td>
			<td>Sakura Finetek USA Inc.</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-actin antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>JLA20</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td>2000C</td>
			<td>Used in initial RNA purity analysis</td>
		</tr>
		<tr>
			<td>No. 15 surgical blade</td>
			<td>Surgical Design Inc</td>
			<td>17467673</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking buffer</td>
			<td>LiCor Biosciences</td>
			<td>927-40000</td>
			<td>Western blocking reagent</td>
		</tr>
		<tr>
			<td>Omni Tissue Master 125</td>
			<td>VWR</td>
			<td>10046-866</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>rabbit anti-KCC2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>94725S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>RNA Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>73034</td>
			<td>Used to extract RNA from small tissue samples</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Excelta Corp.</td>
			<td>16050103</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard razor blades</td>
			<td>American Line</td>
			<td>66-0362</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596026</td>
			<td>Used to extract RNA from tissue</td>
		</tr>
	</tbody>
</table>

collection of frozen rodent brain regions for downstream analyses

Etter hvert som vår forståelse av nevrobiologi har utviklet seg, utføres molekylære analyser ofte på små hjerneområder som medial prefrontal cortex (mPFC) eller nucleus accumbens. Utfordringen i dette arbeidet er å dissekere riktig område samtidig som mikromiljøet undersøkes. I denne artikkelen beskriver vi en enkel, rimelig metode ved hjelp av ressurser som er lett tilgjengelige i de fleste laboratorier. Denne metoden bevarer nukleinsyre og proteiner ved å holde vevet frosset gjennom hele prosessen. Hjernen er skåret i 0,5–1,0 mm seksjoner ved hjelp av en hjernematrise og ordnes på en frossen glassplate. Landemerker i hver del sammenlignes med en referanse, for eksempel Allen Mouse Brain Atlas, og regioner dissekeres ved hjelp av en kald skalpell eller biopsipunch. Vev lagres deretter ved -80 °C til bruk. Gjennom denne prosessen rotte og mus mPFC, nucleus accumbens, dorsal og ventral hippocampus og andre regioner har blitt vellykket analysert ved hjelp av qRT-PCR og vestlige analyser. Denne metoden er begrenset til hjerneregioner som kan identifiseres av klare landemerker.

For å validere denne metoden ble den mediale prefrontale cortex samlet inn fra voksne CD1 wildtype hannmus og RNA og protein ble ekstrahert og karakterisert. RNA ble analysert av kapillær elektroforese. Degradert RNA viser et tap i intensiteten av 28S og 18S ribosomal band og viser også nedbrytningsprodukter som et smør mellom 25 og 200 nukleotider (Figur 5A, eksempel 1). Rna av høy kvalitet viser distinkte ribosomale bånd med lite eller ingen signal i den lavere molekylvektregionen (<...

Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Collection of Frozen Rodent Brain Regions for Downstream Analyses

Denne prosedyren beskriver samlingen av diskrete frosne hjerneregioner for å oppnå protein av høy kvalitet og RNA ved hjelp av billige og allment tilgjengelige verktøy.

Samling av frosne gnagerhjerneregioner for nedstrøms analyser

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Denne prosedyren beskriver samlingen av diskrete frosne hjerneregioner for å oppnå protein og RNA av høy kvalitet ved hjelp av billige og allment tilgjengelige verktøy. Fordi hjerneregioner holdes frosset fra høst gjennom disseksjon, bevares målmolekyler og forskeren har tid til å nøye dissekere og lagre områder av interesse. Å identifisere interesseområder kan i utgangspunktet være utfordrende.Ved hjelp av vanlige hjernelandemerker som de dukker opp og går tilbake gjennom seksjonene i forhold til de ønskede regionene vil gjøre identifikasjon klart. Etter å ha fjernet hjernen fra eutaniserte voksne CD-1 villtypemus, fryse vevet i 60 sekunder i enten flytende nitrogen eller isopentane. Pre-chill med tørris og lagre den på minus 80 grader Celsius.24 timer før dissekere vevet, plasser en ren hjernematrise på en stabel med tinte frysepakker. Og smør sidene til matrisen mellom to frysepakker som sørger for å forlate omtrent en halv centimeter mellom bunnen av barberhøvelsporene og toppen av pakkene. Sett opp en frossen glassplate for disseksjonen ved å fylle en isolert boks med is opp til omtrent fem centimeter fra toppen.Legg deretter et 2,5 centimeter lag tørris på toppen. Og dekk den med svart plastfolie. Plasser en glassplate på toppen av plasten.Og tørris rundt grensen til platen. Fjern den frosne hjernematrisen fra fryseren og sett hjernen, cortex side opp, inn i matrisen. La vevet likevekte til temperaturen i boksen i 10 minutter.Holde lokket åpent i løpet av denne tiden. Bruk kalde tang til å justere hjernens posisjon i matrisen slik at sagittal sinus og tverrgående sinus linje opp med vinkelrett spor av blokken. Når hjernen er i posisjon, plasser et kjølt barberblad nær midten og trykk det omtrent en millimeter inn i vevet.Deretter plasserer du ett kjølt blad i hver ende av hjernen og trykker helt ned i matrisen. Begynn deretter å legge til kjølte blader i rostralenden, plassere dem i sporene en om gangen og forsiktig trykke dem en millimeter inn i vevet. Fortsett å legge til blader med ett millimeter intervaller, arbeider mot den kaudale enden.Når alle bladene er på plass, trykk ned på toppen med fingre, håndflate, eller en stump gjenstand og rock dem sakte fra side til side for å flytte dem gjennom vevet. Når bladene har nådd bunnen av sporene, ta tak i hver side av gruppen av blader og frigjøre dem fra matrisen ved å gynge frem og tilbake. Etter å ha frit gruppen av blader plassere dem rostral side opp på glassplaten, og sette tørris ved siden av eller på toppen av stabelen for å ytterligere fryse prøvene for enklere separasjon.Plasser deretter stabelen med skarpe kanter ned og skill bladene ved å flytte stabelen mellom tommelen og fingrene. Linje opp avsnittet på glassplatene fra rostral til caudal og skille vevet fra bladene ved å bøye dem mellom fingrene, eller ved å skille dem med et annet kjølt blad. Til tider kan vev holde seg til begge sider av et blad og det må tas hensyn til å opprettholde rostral til caudal orientering.Før du starter disseksjonen, åpner du Allen Mouse Brain Atlas eller en annen referanse og finner landemerkene som er nødvendige for å identifisere områder av interesse. Bruk kjølte tang eller blader til å vende seksjonen. Og sørg for at interesseregionen er konsistent i hele seksjonen.Skjær inn i seksjonen med en ren skalpell eller et slag, og skyv metallet forsiktig, men fast inn i vevet. Rocking det frem og tilbake for å gjøre kuttet. Etter høsting av interesseområdet, legg den i merkede forhåndskjølte 1,5 millimeter rør og lagre dem på minus 80 grader Celsius.For å behandle vevet, tilsett kald RIPA-buffer for proteinekstraksjon eller et guanidinium som inneholder løsningsmiddel for RNA-ekstraksjon og homogeniser det umiddelbart med en glassned eller mekanisk homogenisator. For å validere denne metoden ble den mediale prefrontale cortex samlet inn fra voksne CD-1 villtype hannmus, og RNA og protein ble karakterisert. Når analysert med kapillær elektroforese, degradert RNA viser et tap i intensiteten av 28S og 18S ribosomale bånd, samt et smør mellom 25 og 200 nukleotider.Mens RNA av høy kvalitet viser distinkte ribosomale bånd lite eller ingen signal i det nedre molekylvektsområdet. RNA oppnå ved hjelp av den frosne disseksjonsmetoden ble sammenlignet med RNA tilberedt av nyhøstet vev. Begge metodene produserer RNA med høye integritetstall og sterk ribosomal banding.For å bekrefte at denne metoden kan brukes til å bevare mikromiljøet av dissekert vev for senere analyse, ble RNA hentet fra dissekert mediale prefrontal cortex som hadde blitt lagret over flere uker. Alle prøvene produserte RNA av høy kvalitet med distinkt ribosomal banding og RNA-integritetstall over åtte. For å bekrefte proteinintegriteten til de frosne dissekerte prøvene ble proteinet samlet inn, overført til nitrocellulose og sonde med antistoff mot det høye molekylvektsmålet KCC2, og det lavere molekylvektsproteinet actin.I alle tilfeller var band skarpe og distinkte uten merkbare nedbrytningsprodukter. Det er viktig å holde vevet frosset gjennom hele prosessen, da dette bevarer mikromiljøet av seksjonene og opprettholder seksjonsmorfologi for sammenligning med hjerneatlasbilder. Regioner av interesse samlet på denne måten kan lagres i flere måneder ved negativ 80 grader Celsius og kan benyttes i mange nedstrøms applikasjoner, inkludert RT-qPCR, RNA-Seq, Western blot analyse, og HPLC.Denne metoden har blitt brukt til å utforske molekylære endringer i de limbiske systemene til perinatale gnagere utsatt for THC og andre cannabinoider for å bedre forstå hvordan disse stoffene kan påvirke hjernens utvikling.

collection-of-frozen-rodent-brain-regions-for-downstream-analyses

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Non-Laser Capture Mikroskopi Approach for mikrodisseksjon av Discrete Mouse Brain Regions for Total RNA Isolation og nedstrøms Next-Generation Sekvensering og Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morfometriske Analyser av netthinnens Seksjoner

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Økende nevrale stamceller fra konvensjonelle og ikke-konvensjonelle Regions of Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetic Stimulering av gnagere sentralnervesystemet

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

High Resolution Kvantitativ Synaptic Proteome Profilering av mus hjernen regioner Etter auditiv diskriminering Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Personlig pinner for Microinjections i gnager hjernen

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Stereotactic Atlas-guidede Laser fange Microdissection av hjernen regionene påvirkes av traumatisk skade

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Evaluering av Synapse tetthet i hippocampus gnager hjernen skiver

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...