Name: identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology
Uploaded: 2018-03-27T15:00:00.000+00:00
Duration: 7 min 25 s
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>平衡大脑到-20 °c 在恒温器1小时。</li>
	<li>剪切30µm 节并转移到幻灯片上 (请参阅材料表)。</li>
	<li>从伏隔核区域选择节, 大约2.76 毫米到2.28 毫米, 前 Bregma14。</li>
	<li>根据大鼠的立体定向脑结构, 将大鼠脑从嗅球中连续切片到伏隔核区域。</li>
	<li>将示例装载到幻灯片上。将各节保持在-20 摄氏度, 使其干燥10分钟。</li>
	<li>立即浸泡在预冷4% 多聚甲醛的幻灯片1小时在4摄氏度。</li>
	<li>将幻灯片置于室温 (RT) 的乙醇梯度增加: 50% 乙醇为5分钟;70% 乙醇为5分钟;100% 乙醇5分钟。</li>
	<li>重复用新鲜的100% 乙醇。</li>
	<li>将幻灯片放在吸水纸上, 风干。</li>
	<li>使用障碍笔在每个节周围绘制一个屏障 (请参阅材料表)。让屏障完全干燥1分钟。</li>
</ol>2. 脑部前处理
<ol><li>打开烤箱, 将温度设置为摄氏40摄氏度。</li>
	<li>添加3滴 (90 µL) 预处理4试剂 (参见材料表) 在每个脑部 (每张幻灯片一节)。</li>
	<li>在 RT 中孵化出30分钟的切片。</li>
	<li>将幻灯片在 1x pbs 中浸没1分钟, 用新鲜的 1x pbs 重复。 注: 幻灯片不应停留在 1x PBS 超过15分钟。</li>
</ol>3. RNA 原位杂交荧光复用试验
<ol><li>在水浴中, 将目标探头加热10分钟至40摄氏度, 然后冷却至 RT。</li>
	<li>将 RNA 试剂 (例如, 安培 1-4 FL) 放在 RT 上。</li>
	<li>从带有吸水纸的幻灯片中取出多余的液体, 然后放回幻灯片架上 (请参阅材料表)。</li>
	<li>在样品切片上添加3滴 (90 µL) 的 Drd1α探针 (C1)。在40摄氏度孵育2小时。</li>
	<li>浸泡在1x 洗涤缓冲器在 RT 2 分钟的幻灯片. 用新鲜的1x 洗涤缓冲器重复。</li>
	<li>从幻灯片上取出多余的液体, 用吸水纸将其放回幻灯片架上 (请参阅材料表)。</li>
	<li>在样品片上添加3滴 (90 µL) 的安培 1-FL。孵育30分钟, 在40摄氏度。</li>
	<li>浸泡在1洗涤缓冲的幻灯片2分钟的 RT. 重复使用新鲜的1wash 缓冲区。</li>
	<li>从幻灯片上取出多余的液体, 用吸水纸将其放回幻灯片架上 (请参阅材料表)。</li>
	<li>在样品片上添加3滴 (90 µL) 的安培 2-FL。孵育15分钟, 在40摄氏度。</li>
	<li>浸泡在1x 洗涤缓冲器在 RT 2 分钟的幻灯片. 用新鲜的1x 洗涤缓冲器重复。</li>
	<li>从幻灯片上取出多余的液体, 用吸水纸将其放回幻灯片架上 (请参阅材料表)。</li>
	<li>在样品片上添加3滴 (90 µL) 的安培 3-FL。孵育30分钟, 在40摄氏度。</li>
	<li>浸泡在1x 洗涤缓冲器的幻灯片在 RT 2 分钟. 用新鲜的1x 洗涤缓冲器重复。</li>
	<li>从幻灯片上取出多余的液体, 用吸水纸将其放回幻灯片架上 (请参阅材料表)。</li>
	<li>在样本切片上添加3滴 (90 µL) 安培 4-FL Alt A。孵育15分钟, 在40摄氏度。</li>
	<li>浸泡在1x 洗涤缓冲器在 RT 2 分钟的幻灯片. 用新鲜的1x 洗涤缓冲器重复。</li>
	<li>从幻灯片中取出多余的液体, 并立即将2滴安装试剂 (请参阅材料表) 放到每一节上。</li>
	<li>在每个脑部放置一个22毫米22毫米盖玻片 (参见材料表)。</li>
	<li>在黑暗中存放幻灯片2-8 °c 直到干燥。</li>
	<li>打开共焦显微镜, 切换到60X 目标。</li>
	<li>用共焦显微镜获取 Z 栈图像。有关共聚焦成像的详细过程, 请参阅补充视频。</li>
	<li>在伏隔核地区获取 Drd1α标记细胞的图像 (激发: 488 nm;发射: 515/30 nm)。 注意: 不要让大脑切片在孵化步骤之间进行干燥。</li>
</ol>4. 数据分析
<ol><li>半定量分析染色模式: "离散点"。<ol>
<li>要识别图像中的单个细胞, 请执行 DAPI 染色作为核标记。然而, 分析脑组织的某些部位仍然很难, 因为它有多种类型的细胞, 它们的细胞核和/或细胞形态不规则。在这里, 两位经验丰富的研究人员分别分配图像中的每个细胞来定义细胞区域。</li>
		<li>根据每个单元格使用特定条件 (图 1B) 的点数, 直观地评分代表共焦图像中的每个单元格。</li>
		<li>确定每个类别中的总单元格数并除以总单元格编号 x100 创建相对频率图, 以显示所有组织部分 (图 2A) 和男性和女性在每个类别中单元格的百分比频率。单独 (图 2E)。</li>
		<li>将所有以前类别中的单元格总和除以单元格编号 x100, 以确定所有组织节 (图 2B) 和男性和女性的累计频率 (图 2F)。</li>
	</ol>
</li>
	<li>统计分析 "离散点" 染色模式 (可选)。<ol>
<li>检查每个单元格的点数, 以提供一个在本质上为序数的分类变量 (即从最低单元表达式 (1) 到最高单元格表达式 (9) 的单元格数, 以进行进一步的统计分析。</li>
		<li>在对描述性统计进行检查后, 使用三主要方法: 壁炉 Haenszel 统计, 曼-惠特尼 U 测试和克鲁斯卡尔. 沃利斯测试。虽然精确的统计方法将依赖于实验设计和感兴趣的研究问题, 但统计决策树 (图 4) 可能有助于对分析方法做出决策。</li>
	</ol>
</li>
	<li>量化在感兴趣区域的信号强度 (ROI) 的染色模式: "簇"。<ol>
<li>使用以下公式计算每个图像的信号强度: 信号强度 = ROI 图像的总强度 (平均背景强度 x 总图像区域)</li>
		<li>计算 ROI 图像内的单元格总数, 以便对每个单元格的近似信号表达式求值。在信号强度/图像中的组差异以及单元格/图像的数目可能会引起关注, 考虑可能有助于在信号表达式/单元格中看到的组差异的机制 (图 3B-3D)。</li>
	</ol>
</li>
	<li>统计分析 "簇" 染色模式 (可选)。<ol>
<li>检查信号表达式/单元格, 以提供连续变量以进行进一步的统计分析。使用两种方法, 包括一个独立的样本 t 检验和方差分析, 根据在设计中包含的主体因素的数量。统计决策树 (图 4) 可以帮助做出有关最适当的统计方法的决策。</li>
	</ol>
</li>
</ol>

<ol>
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声明没有利益冲突。

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"。

<table><tbody><tr><td>HybEZ Oven system</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>310010</td><td></td></tr><tr><td>RNAscope Probe - Rn-Drd1a</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>317031</td><td>Color channel 1, Green</td></tr><tr><td>RNAscope Probe - Rn-Th-C2</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>314651-C2</td><td>Color channel 2, Orange</td></tr><tr><td>RNAscope Fluorescent Multiplex Reagent Kit</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>320850</td><td></td></tr><tr><td>ImmEdge Hydrophobic Barrier Pen</td><td>Vector Laboratory</td><td>H-4000</td><td></td></tr><tr><td>SuperFrost Plus Slides</td><td>Fisher Scientific</td><td>12-550-154%&amp;nbsp;</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127-500G</td><td></td></tr><tr><td>Sevoflurane</td><td>Merritt Veterinary Supply</td><td>347075</td><td></td></tr><tr><td>Tissue-Tek vertical 24 slide rack</td><td>Fisher Scientific</td><td>NC9837976</td><td></td></tr><tr><td>Tissue-Tek staining dish</td><td>Fisher Scientific</td><td>NC0731403</td><td></td></tr><tr><td>Precision General Purpose Baths</td><td>ThermoFisher Scientific</td><td>TSGP28</td><td></td></tr><tr><td>Pretreatment 4</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>320850</td><td>parts of kits</td></tr><tr><td>ProLong Gold anti-fade reagent</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Amp 1-FL</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>320850</td><td>parts of kits</td></tr><tr><td>Amp 2-FL</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>320850</td><td>parts of kits</td></tr><tr><td>Amp 3-FL</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>320850</td><td>parts of kits</td></tr><tr><td>Amp 4-FL-Alt A</td><td>Advanced Cell Diagnostics&amp;nbsp;</td><td>320850</td><td>parts of kits</td></tr><tr><td>EZ-C1 software package</td><td>Nikon Instruments</td><td></td><td>version 3.81b</td></tr><tr><td>SAS/STAT Software</td><td>SAS Institute, Inc.,</td><td></td><td>version 9.4</td></tr></tbody></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 ...

identification-dopamine-d1-alpha-receptor-within-rodent-nucleus

Research

JoVE Journal

Neuroscience

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

8.5K 次观看.  University of South Carolina. 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。

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

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

科研

神经科学

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

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

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

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

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

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 ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven 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, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

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, ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

生物学

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

使用多重 原位 杂交检测小鼠迷走神经传入神经元中G蛋白偶联受体表达

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...

Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation

Source: Wickham, R. J., et al. <a href="https://app.jove.com/v/60886">Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine.</a> J. Vis. Exp. (2020).
This video demonstrates the implantation of electrodes and cannulas in an anesthetized rat to measure dopamine release in the nucleus accumbens using a potentiostat and the infusion of drugs to modulate dopaminergic activity in the ventral tegmental area.

使用 VTA 多巴胺能神经元受体作监测大鼠模型中的多巴胺释放

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

摘要

探索更多视频

此视频中的章节

在黑质和腹侧被盖区多巴胺调控的综合性能分析

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

在脑切片检测轴突本地化mRNAs的高空间分辨率

快速

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

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

增强Northern印迹检测小RNA种类的

使用多重原位杂交检测小鼠迷走神经传入神经元中G蛋白偶联受体表达

使用 VTA 多巴胺能神经元受体作监测大鼠模型中的多巴胺释放

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

摘要

探索更多视频

此视频中的章节

在黑质和腹侧被盖区多巴胺调控的综合性能分析

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

在脑切片检测轴突本地化mRNAs的高空间分辨率

快速

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

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

增强Northern印迹检测小RNA种类的

使用多重 原位 杂交检测小鼠迷走神经传入神经元中G蛋白偶联受体表达

使用 VTA 多巴胺能神经元受体作监测大鼠模型中的多巴胺释放

使用多重原位杂交检测小鼠迷走神经传入神经元中G蛋白偶联受体表达