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

As obras presentes foram apoiadas pelo Instituto Nacional de bolsas de saúde (NIH), HD043680, MH106392, DA013137 e NS100624. Fundo parcial foi fornecido por uma concessão de treinamento de NIH T32 na ciência biomédica-comportamental.

O protocolo experimental foi aprovado pelo Comitê de uso (IACUC) na Universidade da Carolina do Sul e cuidado Animal (número de garantia federal: A3049-01).
1. preparação das seções do cérebro congelado fresco
<ol><li>Usar a cepa de ratos de F344/N: três ratos de cada sexo, 13 meses de idade, peso cerca de 320 g de corpo.</li>
	<li>Ajuste a concentração de sevoflurano para 5% (overdose de sevoflurano). Continue o sevoflurano exposição após a respiração para por um minuto adici...

<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>

Neste protocolo, descrevemos uma nova técnica de hibridação in situ por fatias de cérebro fresco congelado avaliar a expressão de receptores Drd1α no núcleo accumbens (NAc) e Tirosina Hidroxilase (TH) na região de substantia nigra (SNR). Nós também fornecemos métodos de análise de dados para diferentes padrões de coloração: "ponto discreto" ou "clusters".
Os passos críticos para um ensaio de sucesso fluorescência multiplex do RNA em situ da hibridação são ...

Disfunção do sistema de dopamina striatal está envolvida na progressão dos sintomas clínicos observados em várias doenças neurocognitivos. Receptores de dopamina D1 estão presentes no córtex pré-frontal (PFC) e regiões striatal do cérebro e fortemente influenciam processos cognitivos1, incluindo o trabalho de memória, processamento temporal e comportamento locomotiva2,3 , 4 , 5 , 6 , 7. estudos anterior elucidado que alterações de receptores de dopamina D1 foram associadas com a progressão da atenção défice-Hiperatividade Transtorno (TDAH)8, os sintomas neurocognitivos em esquizofrenia9, 10 e stress susceptibilidade11. Especificamente, na esquizofrenia, estudos de tomografia por emissão de pósitrons (PET) indicaram que a capacidade de ligação dos receptores de dopamina D1 nos córtices pré-frontal foi altamente relacionada com déficits cognitivos e a presença de sintomas negativos11. O crescimento dendrítica de neurônios excitatórios no córtex pré-frontal, regulado pelo receptor de dopamina D1 alivia o stress susceptibilidade. Além disso, o nocaute do receptor D1 em neurônios corticais pré-frontal medial (mPFC) poderá reforçar a derrota social induzida por estresse evitação social12.
Aqui, apresentamos uma nova técnica de hibridação in situ RNA Visualizar moléculas de RNA única numa cela com amostras de tecido fresco congelado. A presente técnica tem várias vantagens sobre os métodos que existem dentro da literatura atual. Primeiro, o procedimento atual preserva o contexto espacial e morfológico do tecido e foi realizado em amostras de tecido fresco congelado, para que outros procedimentos que exigem fresco, tecidos não-incorporado podem ser combinados com os métodos atuais. Procedimentos semelhantes em tecidos fixados em formalina e parafina ilustraram que resolução única transcrição pode ser conseguida usando um RNA em situ da hibridação técnica13. Detecção de RNA no nível de simples transcrição fornece sensibilidade superior à expressão de número de cópia baixa, bem como a oportunidade de comparar a expressão do gene no nível de células individuais que não podem ser alcançados por outros métodos de detecção do ácido nucleico, tais como técnicas de reação em cadeia (PCR) de polimerase. Além disso, o método atual mantém imagens com uma alta relação sinal-ruído através de sondas de RNA altamente específicas que são hibridizadas de transcritos de RNA único alvo e sequencialmente limite com uma cascata de moléculas de amplificação de sinal na identificação sistema. Finalmente, a tecnologia atual oferece a oportunidade de avaliar vários sistemas biológicos com suas sondas proprietárias de destino específico, ao invés de limitar a nossa investigação a apenas uma classe de marcadores relacionados ao sistema, como a detecção de proteínas por métodos de imuno-histoquímica.
Em nosso estudo, usamos esta novela RNA em situ da hibridação para avaliar a expressão de receptores Drd1α no núcleo accumbens (NAc) e Tirosina Hidroxilase (TH) na substância negra (SNR) de ratos de F344/N tanto masculinos como femininos. A inovador RNA em situ hibridação permitiu-nos investigar mecanismos influenciando DA fixação e DA liberação, simultaneamente, melhorar a nossa compreensão das complexidades do sistema striatal DA. Aqui, descrevemos o procedimento para o cérebro fresco congelado fatias e fornecer métodos de análise de dados para diferentes padrões de coloração: "ponto discreto" ou "clusters".

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			<td height="23" style="height:23px;width:329px;">HybEZ Oven system</td>
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			<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>
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			<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>
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			<td height="23" style="height:23px;">RNAscope Fluorescent Multiplex Reagent Kit</td>
			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
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			<td height="23" style="height:23px;">ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratory</td>
			<td>H-4000</td>
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			<td height="23" style="height:23px;">SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%&#160;</td>
			<td></td>
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			<td height="23" style="height:23px;">4% paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
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			<td height="23" style="height:23px;">Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
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			<td height="23" style="height:23px;">Tissue-Tek vertical 24 slide rack</td>
			<td>Fisher Scientific</td>
			<td>NC9837976</td>
			<td></td>
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			<td height="23" style="height:23px;">Tissue-Tek staining dish</td>
			<td>Fisher Scientific</td>
			<td>NC0731403</td>
			<td></td>
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			<td height="23" style="height:23px;">Precision General Purpose Baths</td>
			<td>ThermoFisher Scientific</td>
			<td>TSGP28</td>
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			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
			<td>parts of kits</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology

No sistema nervoso central, os receptores do subtipo de D1-alfa (Drd1α) é o mais abundante receptor de dopamina (DA), que desempenha um papel vital na regulação de desenvolvimento e crescimento neuronal. No entanto, os mecanismos subjacentes a anormalidades do receptor de Drd1α mediando respostas comportamentais e modulando a função de memória de trabalho são ainda pouco claras. Usando um romance RNA em situ da hibridação do ensaio, o atual estudo identificou dopamina Drd1α receptor Tirosina Hidroxilase (TH) RNA expressão e do circuito DA relacionados na área de núcleo accumbens (NAc) e região de substantia nigra (SNR), respectivamente. Drd1α expressão no NAc mostra um "ponto discreto" padrão de coloração. Observaram-se diferenças de sexo claro na expressão de Drd1α. Em contraste, o TH mostra um padrão de coloração "cluster". Em relação a expressão TH, ratos fêmeas exibida uma maior expressão de sinal por célula em relação a animais machos. Os métodos aqui apresentados fornecem uma novela em situ técnica de hibridização para investigar alterações na disfunção do sistema de dopamina durante a progressão de doenças do sistema nervoso central.

O atual estudo observou um "pontos discretos" padrão para a expressão de RNA nos receptores de dopamina D1-alfa (Drd1α) de coloração do CCN em ratos F344/N (figura 1A). Sinais de fluorescência individuais foram identificados facilmente e podem ser vistos como único "pontos", cada um deles representa um único transcrito de RNA dentro da célula. Para imagens de NAc que exibem os "pontos discretos" padrão de coloração, avaliamos a gama dinâmica de e...

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

Identificação de Receptor de dopamina D1-alfa dentro roedores núcleo Accumbens por uma inovador RNA In Situ tecnologia da deteção

Identificação de receptor de dopamina D1-alfa no núcleo accumbens é fundamental para esclarecer a disfunção do receptor D1 durante uma doença do sistema nervoso central. Realizamos um novela RNA em situ da hibridação do ensaio para visualizar moléculas de RNA única em uma área específica do cérebro.

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 ...

The overall goal of this novel RNA in-situ hybridization assay is to visualize single RNA molecules in a specific brain area which are critical for clarifying dopaminergic dysfunction during central nervous system disease. This method can help answer key questions in the neuroscience field such as dopaminergic abnormalities mediating behavioral responses and modulating working memory function. The main advantage of this method is that it provides a novel in-situ hybridization technique to investigate dopamine system dysfunction during the progression of central nervous system diseases.Though this method can provide insight into overall expression levels of a target mRNA in a given brain region, it can also be applied to determine how mRNA transcripts are distributed throughout the cells of a given region. To begin this procedure, within five minutes after harvesting an adult rat brain, submerge it in liquid nitrogen for 15 seconds. Next, equilibrate the brain at minus 20 degrees Celsius in a cryostat for one hour.After an hour, cut 30 micron sections containing nucleus accumbens from 2.76 millimeters to 2.28 millimeters anterior to bregma and mount them onto the slides. Then dry the sections at minus 20 degrees Celsius for 10 minutes. Subsequently, immerse the slides in pre-chilled 4%paraformaldehyde for one hour at four degrees Celsius.Next, place the slides in an increasing ethanol gradient at room temperature then transfer the slides to fresh 100%ethanol for another five minutes. Afterward, place the slides on the absorbent paper to allow air dry. Draw a barrier around each section with a barrier pen.Let the barrier dry completely for one minute. To pre-treat the brain sections, turn on the oven and set the temperature to 40 degrees Celsius. Add three drops of pre-treatment reagent on each brain section.Then incubate the sections for 30 minutes at room temperature. Next, submerge the slides in 1X PBS for one minute at room temperature and transfer the slides to fresh 1X PBS for another minute. Slides should not stay in 1X PBS for longer than 15 minutes.To run the multiplex assay, warm the target probe for 10 minutes at 40 degrees Celsius in a water bath and then cool to room temperature. Place the RNA reagent at room temperature. Remove excess liquid from the slides with absorbent paper and place them back on the slide rack.Then add three drops of the Drd1alpha as probe on each sample slice and incubate the samples for two hours at 40 degrees Celsius. Afterward, wash the slides in 1X wash buffer for two minutes at room temperature twice. Then remove excess liquid from the slides with absorbent paper and place them back on the slide rack.Do not let brain sections dry out between incubation steps. Add three drops of AMP 1FL on each sample slice and incubate the samples for 30 minutes at 40 degrees Celsius. Afterward, wash the slides twice in 1X wash buffer for two minutes each time at room temperature.Then remove excess liquid from the slides with absorbent paper and place them back on the slide rack. Subsequently, add three drops of AMP 2FL on each sample slice. Incubate the samples for 15 minutes at 40 degrees Celsius.Next, submerge the slides in 1X wash buffer for two minutes at room temperature twice. Remove excess liquid from the slides with absorbent paper and place them back on the slide rack. Add three drops of AMP 3FL on each sample slice and incubate the samples for 30 minutes at 40 degrees Celsius.Next, wash the slides in 1X wash buffer for two minutes at room temperature twice. Remove excess liquid from the slides with absorbent paper and place them back on the slide rack. Then add three drops of AMP 4FL ALT A on each sample slice and incubate the samples for 15 minutes at 40 degrees Celsius.Wash the slide in 1X wash buffer for two minutes at room temperature twice. Remove excess liquid from the slides and immediately place two drops of mounting reagent onto each section. Next, place a 22 millimeter by 22 millimeter coverslip over each brain section.Then store the slides in the dark at two to eight degrees Celsius until dry. For imaging, turn on the confocal microscope and switch to a 60X objective. Obtain Z stack images and acquire images of Drd1alpha-labeled cells in the nucleus accumbens region.For images from the nucleus accumbens that displayed the discrete dots staining pattern, the dynamic range of expression was assessed using a semi-quantitative analysis. Each cell was scored from zero to nine based on the number of dots per cell. This hybridized signal score method can thus assess the extent and variability for Drd1alpha expression across individual cells.Here are the representative confocal images of Drd1alpha expression in male and female rats which have the discrete dots staining pattern. To account for the nested data structure, average count values were calculated for each category and used for statistical analysis. A Mann-Whitney U test was conducted to assess differences in median cell count in male and female rats.The frequency of cells in each category is analyzed using relative frequency and cumulative frequency. Female animals displayed a significant shift in the distribution with the greater frequency of cells in lower ordered categories relative to male animals. Overall, the results suggest clear sex differences in Drd1alpha expression in the nucleus accumbens.Once mastered, this technique can be done in seven hours if it is performed properly. While attempting this procedure, it's important to remember to work quickly as temperature changes or drying out of the tissue can damage the mRNA and hinder our ability to obtain a reliable signal. After watching this video, you should have a good understanding of how to perform the in-situ hybridization technique in cortical tissues, fluorescently image signal mRNA transcripts within chosen brain regions, and characterize the distribution of mRNA transcripts for statistical analysis.

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

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A identificação fiável de Vida neurônios dopaminérgicos em culturas mesencéfalo Usando RNA Sequenciamento e TH-driven-promotor eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Localização de Genes de Receptor de Odorantes em Antenas de Locustídeo por RNA<em&gt; In Situ</em&gt; Hibridação

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Avaliação da homeostase Dopaminergic em camundongos pelo uso de análise de cromatografia líquida de alta performance e captação da dopamina Synaptosomal

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...