Name: identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology
Uploaded: 2018-03-27T15:00:00
Description: Watch this Scientific Journal Video about Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology at JoVE.com

De nuværende værker blev støttet af nationale kontorer i Health (NIH) tilskud HD043680, MH106392, DA013137 og NS100624. Delvis Fonden blev leveret af en NIH T32 uddannelse grant i biomedicinsk-adfærdsmæssige videnskab.

Den eksperimentelle protokollen blev godkendt af Animal Care og brug udvalg (IACUC) på University of South Carolina (føderale assurance nummer: A3049-01).
1. forberedelse af frisk frosset hjernen sektioner
<ol><li>Bruge F344/N rat stamme: tre rotter af hvert køn, 13 måneder i alder, kropsvægt ca 320 g.</li>
	<li>Justere Sevofluran koncentration på 5% (overdosis af Sevofluran). Fortsætte Sevofluran eksponering efter vejrtrækning stopper for en ekstra minut.</li>
	<li>Hug hovedet af ro...

<ol>
	<li>Goldman-Rakic, P. S., Castner, S. A., Svensson, T. H., Siever, L. J., Williams, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+dopamine+D1+receptor+in+schizophrenia:+insights+for+cognitive+dysfunction.">Targeting the dopamine D1 receptor in schizophrenia: insights for cognitive dysfunction.</a> Psychopharmacology (Berl). 174 (1), 3-16 (2004).</li><li>Beaulieu, J. M., Gainetdinov, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology,+signaling,+and+pharmacology+of+dopamine+receptors.">The physiology, signaling, and pharmacology of dopamine receptors.</a> Pharmacol Rev. 63, 182-217 (2011).</li><li>Zahrt, J., Taylor, J. R., Mathew, R. G., Arnsten, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supranormal+stimulation+of+D1+dopamine+receptors+in+the+rodent+prefrontal+cortex+impairs+spatial+working+memory+performance.">Supranormal stimulation of D1 dopamine receptors in the rodent prefrontal cortex impairs spatial working memory performance.</a> J Neurosci. 17, 8528-8535 (1997).</li><li>Floresco, S. B., Magyar, O., Ghods-Sharifi, S., Vexelman, C., Tse, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+dopamine+receptor+subtypes+in+the+medial+prefrontal+cortex+of+the+rat+regulate+set-shifting.">Multiple dopamine receptor subtypes in the medial prefrontal cortex of the rat regulate set-shifting.</a> Neuropsychopharmacology. 31, 297-309 (2006).</li><li>Arnsten, A. F., Girgis, R. R., Gray, D. L., Mailman, 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=Novel+dopamine+therapeutics+for+cognitive+deficits+in+schizophrenia.">Novel dopamine therapeutics for cognitive deficits in schizophrenia.</a> Biol Psychiatry. 81, 67-77 (2017).</li><li>Ellenbroek, B. A., Budde, S., Cools, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prepulse+inhibition+and+latent+inhibition:+the+role+of+dopamine+in+the+medial+prefrontal+cortex.">Prepulse inhibition and latent inhibition: the role of dopamine in the medial prefrontal cortex.</a> Neuroscience. 75 (2), 535-542 (1996).</li><li>Parker, K. L., Alberico, S. L., Miller, A. D., Narayanan, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+D1+dopamine+signaling+is+necessary+for+temporal+expectation+during+reaction+time+performance.">Prefrontal D1 dopamine signaling is necessary for temporal expectation during reaction time performance.</a> Neuroscience. 255, 246-254 (2013).</li><li>Manduca, A., Servadio, M., Damsteegt, R., Campolongo, P., Vanderschuren, L. J., Trezza, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+Neurotransmission+in+the+Nucleus+Accumbens+Modulates+Social+Play+Behavior+in+Rats.">Dopaminergic Neurotransmission in the Nucleus Accumbens Modulates Social Play Behavior in Rats.</a> Neuropsychopharmacology. 41 (9), 2215-2223 (2016).</li><li>Okubo, Y., 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=Decreased+prefrontal+dopamine+D1+receptors+in+schizophrenia+revealed+by+PET.">Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET.</a> Nature. 385 (6617), 634-636 (1997).</li><li>Abi-Dargham, 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=Prefrontal+dopamine+D1+receptors+and+working+memory+in+schizophrenia.">Prefrontal dopamine D1 receptors and working memory in schizophrenia.</a> J Neurosci. 22 (9), 3708-3719 (2002).</li><li>Shinohara, 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=Dopamine+D1+receptor+subtype+mediates+acute+stress-induced+dendritic+growth+in+excitatory+neurons+of+the+medial+prefrontal+cortex+and+contributes+to+suppression+of+stress+susceptibility+in+mice.">Dopamine D1 receptor subtype mediates acute stress-induced dendritic growth in excitatory neurons of the medial prefrontal cortex and contributes to suppression of stress susceptibility in mice.</a> Mol Psychiatry. 19, (2017).</li><li>Okubo, Y., 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=Decreased+prefrontal+dopamine+D1+receptors+in+schizophrenia+revealed+by+PET.">Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET.</a> Nature. 385, 634-636 (1997).</li><li>Wang, 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diagn. 14 (1), 22-29 (2012).</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. , (2014).</li></ol>

I denne protokol beskriver vi en roman teknik i situ hybridisering til frisk frosset hjernen skiver til at evaluere Drd1α receptor udtryk i nucleus accumbens (NAc) og tyrosin hydroxylase (TH) udtryk i regionen substantia nigra (SNR). Vi leverer også metoder til analyse af data for forskellige farvning mønstre: "diskret dot" eller "klynger".
De kritiske trin til en vellykket multiplex fluorescens RNA i situ hybridisering assay er inkluderet. Når hugger rotten og fjerne hje...

Dysfunktion af striatal dopamin system er involveret i udviklingen af kliniske symptomer observeret i flere neurokognitive sygdomme. Dopamin D1 receptorer findes i det præfrontale cortex (PFC) og striatal regioner af hjernen og påvirke stærkt kognitive processer1, herunder arbejder hukommelse, tidsmæssige forarbejdning og lokomotiv adfærd2,3 , 4 , 5 , 6 , 7. tidligere undersøgelser belyst, at ændringer af dopamin D1 receptorer var forbundet med progression af opmærksomhed underskud hyperaktivitet lidelse (ADHD)8, neurokognitive symptomer på skizofreni9, 10 og stress modtagelighed11. Specifikt, i skizofreni vist positron emissions tomografi (PET) undersøgelser, at bindende evne til dopamin D1 receptorer i de præfrontale cortex var stærkt relateret til kognitiv underskud og tilstedeværelsen af negative symptomer11. Dendritiske væksten af excitatoriske neuroner i præfrontal cortex reguleret af dopamin D1 receptor lindrer stress modtagelighed. Derudover kunne knockdown af D1 receptor i mediale præfrontale kortikale (mPFC) neuroner forbedre sociale nederlag stress-induceret sociale unddragelse12.
Her introducerer vi en ny teknik af RNA i situ hybridisering til at visualisere enkelt RNA molekyler i en celle med frisk frosset vævsprøver. Den nuværende teknik har flere fordele i forhold til metoder, der findes i den aktuelle litteratur. Først, den nuværende procedure bevarer den rumlige og morfologiske sammenhæng af væv og blev udført på frisk frosset vævsprøver, således at andre procedurer, der kræver friske, ikke-indlejrede væv kan kombineres med de nuværende metoder. Lignende procedurer i formalin-fast og paraffin-embedded væv har illustreret, at enkelt transskription opløsning kan opnås ved hjælp af en RNA i situ hybridisering teknik13. Påvisning af RNA på enkelt transskription plan giver overlegen følsomhed til lav kopi nummer udtryk samt mulighed for at sammenligne genekspression i forbindelse med individuelle celler, der ikke kan opnås ved andre metoder til påvisning af nukleinsyre såsom polymerase kædereaktion (PCR) teknikker. Desuden, fastholder den nuværende metode billeder med en høj signal-støj-forholdet gennem meget specifikke RNA-sonder, der er hybridiseret at enkelt mål RNA udskrifter, og sekventielt bundet med en kaskade af signal forstærkning molekyler i afsløring system. Endelig, den nuværende teknologi giver mulighed for at evaluere flere biologiske systemer med sine mål-specifikke proprietære sonder, snarere end at begrænse vores undersøgelse til kun én klasse af system-relaterede mærker såsom protein påvisning af Immunhistokemi metoder.
I vores undersøgelse brugte vi denne roman RNA i situ hybridisering til at evaluere Drd1α receptor udtryk i nucleus accumbens (NAc) og tyrosin hydroxylase (TH) udtryk i substantia nigra (SNR) af både mandlige og kvindelige F344/N rotter. Den innovative RNA i situ hybridisering aktiveret os til at undersøge mekanismerne påvirker både DA optagelsen og DA release samtidigt, forbedre vores forståelse af striatal DA systemets kompleksitet. Her, vi beskrive proceduren for frisk frosset hjernen skiver og give metoder til analyse af data for forskellige farvning mønstre: "diskret dot" eller "klynger".

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	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">HybEZ Oven system</td>
			<td style="width:205px;">Advanced Cell Diagnostics&#160;</td>
			<td style="width:135px;">310010</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAscope Probe - Rn-Drd1a</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>317031</td>
			<td>Color channel 1, Green</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAscope Probe - Rn-Th-C2</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>314651-C2</td>
			<td>Color channel 2, Orange</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAscope Fluorescent Multiplex Reagent Kit</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratory</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">4% paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Tissue-Tek vertical 24 slide rack</td>
			<td>Fisher Scientific</td>
			<td>NC9837976</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Tissue-Tek staining dish</td>
			<td>Fisher Scientific</td>
			<td>NC0731403</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Precision General Purpose Baths</td>
			<td>ThermoFisher Scientific</td>
			<td>TSGP28</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Pretreatment 4</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">ProLong Gold anti-fade reagent</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:135px;">P36930</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 1-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 2-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 3-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">Amp 4-FL-Alt A</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">EZ-C1 software package</td>
			<td style="width:205px;">Nikon Instruments</td>
			<td style="width:135px;"></td>
			<td>version 3.81b</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:329px;">SAS/STAT Software</td>
			<td style="width:205px;">SAS Institute, Inc.,</td>
			<td style="width:135px;"></td>
			<td>version 9.4</td>
		</tr>
	</tbody>
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identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology

I det centrale nervesystem er D1-alpha subtype receptor (Drd1α) den mest rigelige dopamin (DA) receptor, som spiller en vigtig rolle i reguleringen af neuronal vækst og udvikling. Mekanismerne bag Drd1α receptor abnormiteter mægle adfærdsmæssige reaktioner og modulerende arbejder hukommelsesfunktion er imidlertid stadig uklart. Ved hjælp af en roman RNA i situ hybridisering assay, identificeret den nuværende undersøgelse dopamin Drd1α receptor og tyrosin hydroxylase (TH) RNA udtryk fra DA-relaterede kredsløb i nucleus accumbens (NAc) område og substantia nigra region (SNR), henholdsvis. Drd1α udtryk i NAc viser en "diskret dot" farvning mønster. Klart sex forskelle i Drd1α udtryk blev observeret. Derimod viser TH en "klynge" farvning mønster. Med hensyn til TH udtryk vises hunrotter en højere signal udtryk pr. celle i forhold til handyr. De metoder, der præsenteres her give en roman i situ hybridisering teknik for at undersøge ændringer i dopamin system dysfunktion under progression af centrale nervesystem sygdomme.

Den nuværende undersøgelse observerede en "diskret prikker" farvning mønster for RNA udtryk i dopamin D1-alpha receptorer (Drd1α) af NAc i F344/N rotter (figur 1A). Enkelte fluorescens signaler blev let identificeres og kan ses som enkelt "prikker", som hver repræsenterer en enkelt RNA udskrift i cellen. Billeder fra NAc, der viser de "diskrete prikker" farvning mønster, vurderet vi det dynamiske område af udtryk ved hjælp af en semi-kvantitative anal...

Watch this Scientific Journal Video about Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology at JoVE.com

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identifikation af dopamin D1-Alpha Receptor inden for gnavere kernen Accumbens af en innovativ RNA In Situ opdagelse teknologi

Identifikation af dopamin D1-alpha receptor i kernen accumbens er kritisk for afklaring D1 receptor dysfunktion under en central nervesystemet sygdom. Vi udførte en roman RNA i situ hybridisering assay for at visualisere enkelt RNA molekyler i en bestemt hjerne område.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

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Neuroscience

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

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