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

目前的工作得到了国家卫生研究院 (NIH) 赠款 HD043680、MH106392、DA013137 和 NS100624 的支持。部分基金由 NIH T32 的生物医学行为科学培训补助金提供。

实验性议定书经美国南卡罗来纳州大学动物保育和使用委员会 (IACUC) 批准 (联邦保证号: A3049-01)。
1. 新鲜冰冻脑切片的制备
<ol><li>使用 F344/N 鼠株: 每性别三只大鼠, 13 月龄, 体重约320克。</li>
	<li>将七氟醚浓度调整为 5% (过量七氟醚)。在呼吸停止后继续接触七氟醚。</li>
	<li>斩首鼠, 并删除大脑。</li>
	<li>在5分钟的组织收获中, 将大鼠的大脑浸入液氮中, 十五年代。</li>
	<li>平衡...

<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>

在本协议中, 我们描述了一种新的用于新鲜冷冻脑切片的原位杂交技术, 以评估伏核 (NAc) 和酪氨酸羟化酶 (TH) 在黑质 (信噪比) 区的表达 Drd1α受体表达。我们还提供了不同染色模式的数据分析方法: "离散点" 或 "簇"。
包括了成功的多重荧光 RNA原位杂交试验的关键步骤。一旦斩首鼠, 取出大脑, 在5分钟内将大脑冷冻在液氮中. 保持大脑部分在-20 °c 直到4% 多聚甲醛固?...

巴多巴胺系统的功能失调涉及多种认知疾病的临床症状进展。多巴胺 D1 受体存在于脑前额皮质 (PFC) 和巴区域, 严重影响认知过程1, 包括工作记忆、时间处理和机车行为2,3,4,5,6,7. 以往的研究表明, 多巴胺 D1 受体的变化与注意缺陷多动障碍 (ADHD)8的进展有关, 精神分裂症9的认知症状10和应力易感性11。具体地说, 在精神分裂症中, 正电子发射断层扫描 (PET) 研究表明, 前额皮质多巴胺 D1 受体的结合能力与认知缺陷和阴性症状的存在有很大关系11。多巴胺 D1 受体调节的前额皮质兴奋神经元的树突生长缓解了应激敏感性。此外, D1 受体在内侧前额皮质 (mPFC) 神经元中的击倒可以增强社会失败应激引起的社会回避12。
在这里, 我们介绍了一种新的 rna原位杂交技术, 以可视化的单一 RNA 分子在细胞与新鲜冰冻组织样本。目前的技术比现有文献中存在的方法有多种优势。首先, 目前的程序保留了组织的空间和形态背景, 并进行了新鲜冷冻组织样本, 以便其他程序需要新鲜的, 非嵌入组织可以结合目前的方法。福尔马林固定和石蜡嵌入组织的类似程序表明, 可以使用 RNA原位杂交技术13实现单转录分辨率。RNA 在单一转录水平上的检测提供了对低拷贝数表达的卓越敏感性, 同时也有机会将基因表达与其他核酸检测方法无法实现的单个细胞水平进行比较,如聚合酶链反应 (PCR) 技术。此外, 目前的方法通过高度特异的 rna 探针, 通过与单目标 rna 转录的杂交, 并在检测中按顺序绑定到信号放大分子的级联来维持高信噪比的图像。系统.最后, 目前的技术提供了一个机会, 以评估多个生物系统与其特定目标专有探针, 而不是限制我们的调查只有一类系统相关的标记, 如蛋白质检测免疫组化方法。
在我们的研究中, 我们使用这种新的 RNA原位杂交来评估 Drd1α受体表达在伏核 (NAc) 和酪氨酸羟化酶 (TH) 表达在黑质 (信噪比) 中的男性和女性 F344/N 大鼠。创新的 RNA原位杂交使我们能够同时调查影响 da 吸收和大释放的机制, 提高我们对巴 da 系统复杂性的理解。这里, 我们描述了新鲜冰冻脑切片的程序, 并提供了不同染色模式的数据分析方法: "离散点" 或 "簇"。

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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>
		</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>
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			<td>Fisher Scientific</td>
			<td>12-550-154%&#160;</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>
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			<td height="23" style="height:23px;">Tissue-Tek vertical 24 slide rack</td>
			<td>Fisher Scientific</td>
			<td>NC9837976</td>
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			<td height="23" style="height:23px;">Tissue-Tek staining dish</td>
			<td>Fisher Scientific</td>
			<td>NC0731403</td>
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			<td>Advanced Cell Diagnostics&#160;</td>
			<td>320850</td>
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			<td height="23" style="height:23px;width:329px;">Amp 2-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
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			<td height="23" style="height:23px;width:329px;">Amp 3-FL</td>
			<td>Advanced Cell Diagnostics&#160;</td>
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			<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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			<td height="23" style="height:23px;width:329px;">SAS/STAT Software</td>
			<td style="width:205px;">SAS Institute, Inc.,</td>
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			<td>version 9.4</td>
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</table>

identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology

在中枢神经系统中, D1-alpha 亚型受体 (Drd1α) 是最丰富的多巴胺 (DA) 受体, 在调节神经元生长发育中起着至关重要的作用。然而, Drd1α受体异常调解行为反应和调节工作记忆功能的机制尚不清楚。本研究采用一种新的 RNA原位杂交方法, 从伏隔核 (NAc) 区和黑质地区的大相关电路中确定多巴胺 Drd1α受体和酪氨酸羟化酶 (TH) RNA 的表达,分别.Drd1α在 NAc 中的表达式显示了一个 "离散点" 染色模式。Drd1α表达的性别差异明显。相比之下, TH 显示了 "聚集" 的染色图案。对于 TH 表达, 雌性大鼠比雄性动物显示每个细胞更高的信号表达。本文的方法提供了一种新的原位杂交技术, 用于调查中枢神经系统疾病进展过程中多巴胺系统功能障碍的变化。

本研究观察了 F344/N 大鼠中 NAc 多巴胺 D1-alpha 受体 (Drd1α) 中 RNA 表达的 "离散点" 染色模式 (图 1A)。单个的荧光信号很容易被识别出来, 可以被看作是单个的 "点", 每一个都代表细胞内的一个 RNA 转录。对于显示 "离散点" 染色模式的 NAc 的图像, 我们使用半定量分析 (图 1A、图 2A& 2B) 来评估动态表?...

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

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

在伏隔核中多巴胺 D1-alpha 受体的鉴定对于澄清中枢神经系统疾病中 D1 受体功能障碍至关重要。我们进行了一个新的 rna 原位杂交检测, 以可视化单个 RNA 分子在特定的大脑区域。

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.

Research

JoVE Journal

Neuroscience

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

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Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

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

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

接口3D工程神经细胞微电极阵列：一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

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

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

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

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

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