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 border="0" cellpadding="0" cellspacing="0" width="857">
	<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>
</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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker，创新的自动化到动眼神经分析方法

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

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 the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

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

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 ...

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

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

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

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

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

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, ...

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

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

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

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

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

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

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) ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...