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 arbejde blev støttet af NIH, DA043982 og DA046196.

Dyr, der anvendes i denne undersøgelse blev behandlet på en etisk og human måde som fastsat af Indiana University's Institutional Animal Care and Use Committee (IACUC) og National Institutes of Health (NIH) retningslinjer.
BEMÆRK: Alle værktøjer og overflader skal vaskes med et passende opløsningsmiddel til at fjerne nuklatermer18, før arbejdet påbegyndes.
1. Opbevaring af hjerner
<ol><li>Fjern hurtigt hjernen fra euthaniserede vok...

<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. 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=High-throughput+mapping+of+single+neuron+projections+by+sequencing+of+barcoded+RNA.">High-throughput mapping of single neuron projections by sequencing of barcoded RNA.</a> bioRxiv. , 054312 (2016).</li><li>Mitulovi&#263;, G., Mechtler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+techniques+for+proteomics+analysis&#8212;a+short+overview+of+latest+developments.">HPLC techniques for proteomics analysis&#8212;a short overview of latest developments.</a> Briefings in Functional Genomics. 5 (4), 249-260 (2006).</li><li>Bettscheider, M., Murgatroyd, C., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+DNA+and+RNA+isolation+from+brain+punches+for+epigenetics.">Simultaneous DNA and RNA isolation from brain punches for epigenetics.</a> BMC Research Notes. 4, 314 (2011).</li><li>Chiu, K., Lau, W. 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 arbejde beskriver en teknik til at isolere små, specifikke områder af hjernen og samtidig begrænse nedbrydningen af nukleinsyre og protein. Skader på hjernevæv sker hurtigt, når en organisme dør. Dette skyldes til dels en hurtig ophobning af ekstracellulær glutamat og den deraf følgende excitotoxicity, der opstår21. Messenger RNA er særligt sårbart over for nedbrydning22,23. Nedbrydning af protein og nukleinsyre er stærk...

Dette arbejde illustrerer dissektion af frosne hjerne regioner til udvinding af høj kvalitet nukleinsyre eller protein ved hjælp af en reference, såsom Allen Mouse Brain Atlas1, som en guide. I denne teknik, hjerner er flash-frosset og opbevares ved -80 °C til senere skæring og dissektion, mens de opretholdes i en frossen tilstand. Denne proces gør det muligt for forskeren at høste et stort antal hjerner i en session og senere dissekere dem for en nøjagtig samling af flere hjerneregioner.
Den nøjagtige indsamling af hjerneregioner af interesse (ROI'er) er ofte påkrævet, når der besvares spørgsmål relateret til gen- og proteinekspression. Mens farmakologi, elektrofysiologi og optogenetik kan bruges på vilde type eller genetisk modificerede gnavere til at hjælpe med at belyse molekylære ændringer understøtter observeret adfærd2,,3,,4, måling af inducerede ændringer i transkriptioner og proteomes bruges ofte til at støtte disse resultater. Teknikker såsom kvantitativ omvendt transskription polymerase kædereaktion (RT-qPCR), vestlige blotting, RNAseq5, MAPSeq6 og HPLC7 er robuste og relativt lave i omkostninger, så mange laboratorier til at studere inducerede molekylære ændringer inden for små hjerne regioner2,4,5,6.
Der er flere måder at udtrække og rense nukleinsyre eller protein fra hjerneregioner8,9,10,11,12. Mange laboratorier høst er hjerneregioner ved at køle hjerner på is på tidspunktet for høsten9,,13. Mens denne tilgang kan resultere i høj kvalitet nukleinsyre og protein, det er noget tidsbegrænset som nedbrydning inden for mikromiljøet af vævet kan finde sted ved disse temperaturer. Dette kan især være tilfældet, når man forsøger at dissekere et stort antal dyr eller roi'er på ét møde. Holde prøver frosne hjælper med at opretholde labile mål molekyler og samtidig give forskeren tid til omhyggeligt at sammenligne landemærker på begge sider af hver sektion i bestræbelserne på at indsamle relativt rene prøver. Laser capture er en anden måde at indsamle væv til RNA eller protein analyse fra hjernen områder10. Denne procedure er bedre end mekanisk dissektion i, at meget små og uregelmæssigt formede ROI'er kan identificeres og isoleres. Laseropsamling er dog begrænset ved brug af dyrt udstyr og reagenser, er tidskrævende og kan også være mere modtagelig for prøvenedbrydning.
Micropunch dissektion på frosset væv er ikke nyt. Tidlige papirer af Miklos Palkovits og andre beskriver de grundlæggende teknikker i detaljer14,15. Denne præsentation følger i vid udstrækning det oprindelige arbejde, med nogle forbedringer for at lette effektiviteten og mindske bekostning af det nødvendige udstyr. For eksempel er hjerne sektioner lavet i en frossen hjerneblok snarere end på en kryoat. Dette giver tykkere sektioner, hvilket reducerer antallet af sektioner, der er nødvendige for at indsamle ROI-prøver. Denne metode dissekerer også prøver på en frossen glasplade, der sidder på tøris i en isoleret kasse. Dette giver en sub-frysning fase på bænken, som at arbejde. Sektioner dissekeret på denne måde er let manipuleres, så forskeren til at sammenligne begge sider af hvert afsnit med en henvisning for at begrænse forurening fra regioner uden for den ønskede ROI.
Fordele ved denne protokol er, at 1) hjernen holdes i en frossen tilstand i hele processen, som hjælper med at bevare protein og nukleinsyre og giver forskeren tid til omhyggeligt at høste ROI'er, og 2) de nødvendige reagenser er billige og findes i de fleste molekylærbiologiske laboratorier. I denne proces er hele hjerner opdelt til 0,5-1,0 mm i en hjernematrix og placeres på en frossen glasplade, der løbende køles med tøris. Vartegn fundet i Allen Brain Atlas1 eller andre hjerne atlasser16,17 bruges til at identificere regioner af interesse, som derefter dissekeres ved hjælp af enten en kold punch eller skalpel. Fordi vævet aldrig er optøet, regioner høstet på denne måde giver høj kvalitet RNA og protein til downstream 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

Som vores forståelse af neurobiologi har udviklet sig, molekylære analyser udføres ofte på små hjerneområder såsom den mediale præfrontale cortex (mPFC) eller kerne accumbens. Udfordringen i dette arbejde er at dissekere det korrekte område og samtidig bevare det mikromiljø, der skal undersøges. I dette papir beskriver vi en enkel, billig metode ved hjælp af ressourcer, der er let tilgængelige i de fleste laboratorier. Denne metode bevarer nukleinsyre og proteiner ved at holde vævet frosset gennem hele processen. Hjerner skæres i 0,5-1,0 mm sektioner ved hjælp af en hjernematrix og arrangeres på en frossen glasplade. Landemærker inden for hvert afsnit sammenlignes med en reference, såsom Allen Mouse Brain Atlas, og regioner dissekeres ved hjælp af en kold skalpel eller biopsi punch. Væv opbevares derefter ved -80 °C indtil brug. Gennem denne proces rotte og mus mPFC, kerne accumbens, dorsale og ventrale hippocampus og andre regioner er blevet analyseret med succes ved hjælp af qRT-PCR og vestlige analyser. Denne metode er begrænset til hjerneregioner, der kan identificeres ved klare landemærker.

For at validere denne metode, den mediale præfrontale cortex blev indsamlet fra voksne CD1 wildtype mandlige mus og RNA og protein blev udvundet og karakteriseret. RNA blev analyseret ved kapillær elektroforese. Forringet RNA viser et tab i intensiteten af 28S og 18S ribosomal bands og viser også nedbrydningsprodukter som en smear mellem 25 og 200 nukleotider (Figur 5A, prøve 1). Rna af høj kvalitet viser forskellige ribosombånd med lidt eller intet signal i det lavere molekylvægtomr?...

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 procedure beskriver indsamling af diskrete frosne hjerneregioner for at opnå protein og RNA af høj kvalitet ved hjælp af billige og almindeligt tilgængelige værktøjer.

Indsamling af frosne gnaver brain regioner for downstream analyser

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

Denne procedure beskriver indsamlingen af diskrete frosne hjerneregioner for at opnå protein og RNA af høj kvalitet ved hjælp af billige og almindeligt tilgængelige værktøjer. Fordi hjernen regioner holdes frosset fra høst gennem dissektion, målmolekyler bevares, og forskeren har tid til omhyggeligt dissekere og gemme regioner af interesse. Det kan i første omgang være en udfordring at identificere interesseregioner.Ved hjælp af fælles hjerne landemærker, som de opstår og trække sig tilbage gennem sektioner i forhold til de ønskede regioner vil gøre identifikation klar. Efter fjernelse af hjerner fra aflivede voksne CD-1 vilde type mus, flash fryse vævet i 60 sekunder i enten flydende nitrogen eller isopentane. Pre-chill med tøris og gemme det på minus 80 grader Celsius.24 timer før dissekere vævet, placere en ren hjerne matrix på en stak optøede fryser pakker. Og sandwich siderne til matrix mellem to frysepakker og sørg for at forlade cirka en halv centimeter mellem bunden af razor slots og toppen af pakkerne. Opsæt en frossen glasplade til dissektionen ved at fylde en isoleret kasse med is op til ca. fem centimeter fra toppen.Derefter placere en 2,5 centimeter lag tøris på toppen. Og dække det med sort plastfolie. Anvend en glasplade oven på plasten.Og tøris omkring pladens grænser. Fjern den frosne hjerne matrix fra fryseren og indsætte hjernen, cortex side op, i matrix. Lad vævet komme i ækvivalens med temperaturen i kassen i 10 minutter.Hold låget åbent i dette tidsrum. Brug kolde kraftvipper til at justere hjernens position i matrixen, så sagittal sinus og tværgående sinus linje op med de vinkelrette riller af blokken. Når hjernen er på plads, skal du placere et kølet barberblad i nærheden af dens centrum og trykke det cirka en millimeter ind i vævet.Dernæst placere en kølet klinge i hver ende af hjernen og tryk hele vejen ned i matrixen. Begynd derefter at tilføje kølede knive i rostralenden, placere dem i rillerne en ad gangen og forsigtigt trykke dem en millimeter ind i vævet. Fortsæt med at tilføje knive med en millimeter mellemrum, arbejder mod caudal ende.Når alle knive er på plads, skal du trykke ned på toppen med fingre, håndflade eller en stump genstand og sten dem langsomt fra side til side for at flytte dem gennem vævet. Når knivene har nået bunden af rillerne, skal du gribe fat i hver side af gruppen af vinger og befri dem fra matrixen ved at rokke frem og tilbage. Efter at have befriet gruppen af knive placere dem rostral side op på glaspladen, og sætte tøris ved siden af eller på toppen af stakken for yderligere at fryse prøverne for lettere adskillelse.Placer derefter stakken med skarpe kanter ned og adskaf knivene ved at flytte stakken mellem tommel og fingre. Line up sektionen på glaspladerne fra rostral til hale og adskille vævet fra knivene ved at bøje dem mellem fingrene, eller ved at adskille dem med en anden kølet klinge. Til tider kan væv holde sig til begge sider af en klinge og skal være forsigtige med at opretholde rostral til caudal orientering.Før du starter dissektionen, skal du åbne Allen Mouse Brain Atlas eller en anden reference og finde de landemærker, der er nødvendige for at identificere interesseområder. Brug kølet kraft og klinger til at vende afsnittet. Og sørg for, at området af interesse er konsekvent i hele afsnittet.Skær ind i afsnittet med en ren skalpel eller en punch, skubbe metal forsigtigt, men fast i vævet. Rokkende den frem og tilbage for at lave snittet. Efter høst regionen af interesse, placere den i mærket pre-kølet 1,5 millimeter rør og gemme dem på minus 80 grader Celsius.For at behandle vævet tilsættes kold RIPA-buffer til proteinekstraktion eller et guanidinium, der indeholder opløsningsmiddel til RNA-ekstraktion, og det homogeniseres straks med en glasnedsættelse eller mekanisk homogenisator. For at validere denne metode, den mediale præfrontale cortex blev indsamlet fra voksne CD-1 vilde type mandlige mus, og RNA og protein blev karakteriseret. Når det analyseres med kapillær elektroforese, har forringet RNA et tab i intensiteten af 28S og 18S ribosomale bånd samt en udtværing mellem 25 og 200 nukleotider.Mens høj kvalitet RNA viser forskellige ribosomal bånd lidt at intet signal i den lavere molekylvægt region. RNA-rnaet ved hjælp af den frosne dissektionsmetode blev sammenlignet med RNA fremstillet af friskhøstet væv. Begge metoder producerer RNA med høje integritetstal og stærk ribosomal banding.For at bekræfte, at denne metode kan bruges til at bevare mikromiljø af dissekeret væv til senere analyse, RNA blev udvundet fra dissekeret mediale præfrontale cortex, der var blevet opbevaret over flere uger. Alle prøver producerede RNA af høj kvalitet med forskellige ribosommale bånd og RNA-integritetsnumre over otte. For at bekræfte proteinintegriteten af de frosne dissekerede prøver blev proteinet indsamlet, overført til nitrocellulose og sonde med antistof mod det høje molekylvægtmål KCC2 og den lavere molekylvægtprotein actin.I alle tilfælde var båndene skarpe og forskellige uden mærkbare nedbrydningsprodukter. Det er vigtigt at holde væv frosset gennem hele processen, da dette bevarer mikromiljø af sektionerne og vedligeholde sektion morfologi til sammenligning med hjernen atlas billeder. Regioner af interesse indsamlet på denne måde kan opbevares i flere måneder på negative 80 grader Celsius og kan udnyttes i mange downstream applikationer, herunder RT-qPCR, RNA-Seq, Western blot analyse, og HPLC.Denne metode er blevet brugt til at udforske molekylære ændringer i de limbiske systemer af perinatale gnavere udsat for THC og andre cannabinoider til bedre at forstå, hvordan disse stoffer kan påvirke hjernens udvikling.

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.

Ikke-Laser Capture Mikroskopi Tilgang til mikrodissektion af diskrete Mouse Brain Regioner for Total RNA Isolation og downstream næste generation sekventering 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 af Retina Sektioner

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.

Voksende neurale stamceller fra konventionelle og ikke-konventionelle regioner Adult gnaverhjerne

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 af Rodent centralnervesystemet

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 Mouse Brain Regioner Efter Auditiv diskrimination 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 nåle til Microinjections i gnavere 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-styrede Laser fange Microdissection af hjerneregioner ramt af 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 af Synapse tæthed i hippocampus gnaver hjernen skiver

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