This work was supported by USPHS grants MH-091460 (E.N.), MH-084018 (A.S.), MH-094268 Silvo O. Conte center (A.S.), MH-069853 (A.S.), MH-085226 (A.S.), MH-088753 (A.S.), MH-092443 (A.S.), and MH-096208 (K.I.), grants from DANA (E.N.), Stanley (A.S.), RUSK (A.S.), S-R foundations (A.S.), NARSAD (A.S. and K.I.), and Maryland Stem Cell Research Fund (A.S. and K.I.).
We sincerely appreciate the efforts and contributions of Pearl Kim, Maria Papapavlou, Nao Gamo, Youjin Chung, Yukiko Lema and Mark Christie towards coordination of the biopsy process.

注：所有研究志愿者在这项研究中给予批准的霍华德大学和约翰斯·霍普金斯大学的机构审查委员会知情同意的文件。只有谁同意签署知情同意书的文件参与者被纳入研究。该研究基地本分析组成如下：20名受试者（12 BD与10控制; 30％男性）和平均38.2（14.1）岁（SD）。 注：喷雾用RNA酶扎普所有表面由玻璃和塑料表面去除RNase污染。 1.活检程序<ol><li>在鼻研究参与者的腔喷利多卡因（利多卡因）的液体4％和盐酸羟甲唑啉0.05％，让麻木的时间为15分钟。 </li><li>浸泡8.5×3英寸外科cottonoids在利多卡因和羟甲唑啉的混合物中，并插入在鼻腔沿鼻中隔优越部腔，并允许静置15分钟。 </li><li>使用30°的刚性鼻内窥镜定位鼻腔的上部，取出使用从鼻中隔的优越部向上咬鼻组织钳，考虑几个小试样鼻粘膜​​。 </li></ol> 2.准备组织块<ol><li>紧接活检，填补标准尺寸cryomolds与组织冷冻介质。 </li><li>使用高压灭菌镊子，安装新鲜组织的冷冻介质冻结干冰。填冷冻块的剩余部分与所述冷冻介质，冷冻冷冻块的干冰上，并在-80℃存储块。 </li></ol> 3. Cryosectioning <ol><li>以cryosectioning准备聚萘二甲酸（PEN）膜的载玻片立即之前。喷载玻片用5分钟的RNase扎普溶液。然后用清水冲洗在酸二乙酯（DEPC）水滑梯，让空气干燥30分钟。激活在UV光下干燥载玻片30分钟。 </li><li>冷冻切片每一块完全由30微米。继续干我的幻灯片CE，和部分冷冻尽可能。商店载玻片在-80ºC完成时。 </li></ol> 4.激光捕获显微切割<ol><li>通过紧接显微脱水幻灯片开始。让冷冻载玻片解冻持续10秒，然后将下面的乙醇系列应用到每个幻灯片：60％（2毫升，15秒），80％（2毫升，15秒），95％（3毫升，25秒），100 ％（7毫升，60秒）。 </li><li>转光活化定位显微镜（PALM）显微镜上，并设置为满足以下条件：对于10X放大率设置能量至82，聚焦到74，和速度，以25 - 30。20X放大倍率，设定能量到63，聚焦到67，和速度，以5 - 6根据需要调整，以获得最佳的切割效果。 </li><li>视觉识别使用20X或10X的放大倍率的神经元。区分从底层子黏膜在显微镜下对薄表面嗅神经元层，而无需使用染色过程。观察神经元的密集柱状外观18。</li><li>在步骤4.2中使用的上述显微镜铅笔功能，微量周围神经元层。追溯稍微远离神经元以防止激光神经元的燃烧。这是为了得到高品质的RNA是必不可少的。然后启动激光制导切割。 </li><li>拿起用细尖镊子显微解剖切片，并把组织中含冰100微升裂解液微管。重复从单个样品获得的所有神经节。每个样品的总清扫时间不应超过50分钟。 </li><li>一旦剥离完成后，立即涡样本裂解物，并保持在干冰。样品储存在-80℃总RNA分离。 </li></ol> 5.提取总RNA和质量控制分析<ol><li>解冻样品裂解物在冰上。进行总RNA分离与RNAqueous微套件用DNase-I灭活按照制造商的协议，没有修改（见&lt;STRONG&gt;图1）。总洗脱体积大约为20微升。 </li><li>测量RNA的浓度和评价RNA质量与使用生物分析仪RNA完整性数（RIN）。符合图2中的质量控制标准样品可以用于cDNA的合成。 </li></ol> 6. cDNA合成<ol><li>使用经过验证的商业试剂盒合成cDNA（ 见表试剂 ）。执行通过组合0.1退火工序 - 1.0纳克的RNA（根据情况而定）和无RNA酶的水，以每管8微升的总体积。然后加入1μl的寡聚d（T）20引物和1μl的10mM的的dNTP每管混合，为10微升的总体积。 </li><li>轻轻涡旋并离心样本和退火的样品在65℃下5分钟。在反应完成后，立即凉爽样品在冰上。 </li><li>根据表1的规格制备预混溶液。然后加入9.5μ升主混合物和0.5μl的商业制备的逆转录酶（RT）到每个管中。加入0.5微升无RNase水，以控制管代替室温。 </li><li>运行与第一温育，在50ºC一个RT反应50分钟，然后85ºC5分钟，并最后4ºC保持，没有骑自行车。 </li><li>稀释所有的样品通过用5倍的水和分装。样品储存在-80ºC。 </li></ol> 7.实时PCR - 确认神经元富集<ol><li>根据表2的规格制备预混溶液。使用内部对照如GAPDH。比较嗅觉标记蛋白（OMP）的表达与undissected组织OMP表达显微组织，以确定神经元的浓缩。 注：OMP是一个标记为嗅觉神经元。 OMP序列：前进，5&#39;-CTGTCGGACCTGGCCAAG-3&#39; 反向，5&#39;-CACCCTCGCCAAAGGTGA-3&#39; GAPDH序列：前进，5&#39;-CATGAGAAGTATGACAACAGCCT-3&#39; 反向，5&#39;-AGTCCTTCCACGATACCAAAGT-3&#39;。 </li><li>在每孔和3微升cDNA的增加7μl反应混合液建立PCR板。 </li><li>离心板5分钟，在300×g离心（1300转）。采用用于商业supermix诸如SYBR更环保的qPCR所指定的默认​​热循环条件运行实时PCR反应。 </li><li>分析数据（ 见图2规格）。 </li></ol> 8.实时PCR - 目的基因的表达<ol><li>执行上显示的2倍以上的神经元浓缩样品。 </li><li>准备在冰上每个探针（FAM标记的探针的兴趣和VIC标记的GAPDH）在根据表3的规格单独管主混合物。 </li><li>设置在各个孔和2μlcDNA的PCR板8微升主混合物。 </li><li>离心板5分钟，在300×g离心（1300转）。运行实时PCR反应使用def行凶热循环条件为每按生产厂家提供了基因表达的主混合物协议。分析表达式的结果（ 见图2规格）。 </li></ol>

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作者报告没有与商业利益的财务关系。

通过合并鼻腔活检和LCM获得丰富的嗅觉神经细胞层一个新的平台，并提出了在这项研究中已得到验证。这种技术可以有广泛的影响。它可以对生物标志物的研究，包括对治疗的反应，以及其他神经精神状况的药物发现的努力得到应用，使得该领域的更广泛的影响。 为了获得高质量的神经元服从下游应用，一些关键和故障排除步骤必须注意。首先，获得4 - 6片每名患者嗅觉组?...

双相情感障碍（BD）是一种严重的神经精神疾病，其特点是情绪的病理变化，驱动器和认知1。用于治疗BD的锂已经显示出改变的在动物研究2的大量基因的稳态mRNA水平，但它仍然是未知的，如果任何这些分子与在人体2的临床响应相关联。理解锂反应的机制将需要调查在神经组织​​锂诱​​导分子变化。不幸的是，它是不实用的，得到了​​BD患者的脑活检前后锂疗法，以确定锂反应的分子签名。验尸脑组织已经被用于研究在BD中的生物标记物，但是，它们不能被用来评估与情绪，认知和驱动的动态变化有关的分子标记;回顾性确定的锂治疗反应的有效性可能会产生问题4。淋巴细胞和其他血细胞可能是有用的，但在血细胞的分子变化可能不反映神经元的变化3-5。脑脊液可能不足以获得对细胞内的分子，可能反映疾病相关和药物可逆固有变化的信息。 嗅觉上皮（OE）是中枢神经系统（CNS）的一个独特的部分，胚胎学相关于边缘结构6;它是通过鼻腔活检方便。它由胶质状的发展7-9的不同阶段支撑（ 即 sustentacular）细胞，基底细胞增生，和嗅觉受体神经元。因此，OE提供了通俗易懂的学习与神经精神疾病的7动态变化的病人中枢神经系统的独特机会。研究，展示了OE的效用作为替代组织调查反映这些OCC相关疾病事件urring在大脑神经元8,9。例如，有研究利用OE调查与精神疾病相关的10-14分子型材。嗅觉系统也可用来识别临床endophenoytpes如与精神分裂症15阴性症状相关的气味赤字。此外，神经发育过程中的OE持续终生，提供了一个有用的途径精神病条件8,9的基本病理生理学模型。 然而，一个缺点使用这种组织的是嗅觉活检与非神经元细胞16的实质性的污染。例如，在先前的OE研究中所用的基因表达研究中的总RNA含有从整个鼻腔活检组织，包括从非神经细胞17的RNA提取的RNA。因此，先前的方法已经由小区的质量的限制。为了克服这个问题，一种新颖的方法，得到<html>丰富的神经元细胞群结合鼻腔活检使用激光捕获显微切割（LCM）已开发18。 LCM是一种技术，其允许使用紫外激光切割结合红外线激光19-21细胞的选择性分离。结合LCM使用OE方法由非神经元细胞减少的OE大量污染，从而提高神经元细胞18的富集。此外，神经元层，可以从下层层在显微镜下区分，从而省去了染色。从其他细胞群使用其通过感兴趣7的细胞类型表达的初级抗体的神经元细胞类型可以进一步进行区分。因此，该过程建立了几乎纯的神经元细胞群可用于基因表达研究，免疫组织化学等形态学调查富集更简单的方法。 ve_content“&gt;这项研究的目的是建立一个实验平台，进行调查与疾病状态和治疗反应相关的嗅觉神经元的分子变化。为了解决这个问题，一小套非吸烟患者谁见了DSM-IV诊断标准BD的基础上诊断访谈为遗传研究（DIG）22被招募进行两次经鼻活检：1的活检预处理与锂和第二活检，6周每日口服锂治疗后此外，合格的BD患者必须是：症状为抑郁症的基础上，给予蒙哥马利-艾森贝格量表（MADRS）23，临床医生得分≥10出60;对于有症状或轻躁狂躁狂，根据得分≥10出来的56对管理扬躁狂量表临床医生（YMRS） 24;或≥10两个MADRS和两个尺度医生之间的协议YMRS评价者-间信系数&gt; 0.96活检后，神经元ENRI。通过LCM从OE地交付。以下附加的质量控制措施，以确保从组织和神经元的富集提取高质量的RNA，实时RT-PCR进行，调查所关注的基因的前后的治疗表达水平。接下来的部分包含了这种方法的有效性的描述，突出了协议的优化，并申请了故障排除的协议的策略。

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			<td height="20" style="height:20px;">Microscope: PALM Series MicroLaser System</td>
			<td>Carl Zeiss Microscopy</td>
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			<td height="20" style="height:20px;">Model: Axiovert 200M</td>
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			<td>Applied Biosystems</td>
			<td>Various</td>
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			<td height="21" style="height:21px;">TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
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olfactory neurons obtained through nasal biopsy combined with laser-capture microdissection: a potential approach to study treatment response in mental disorders

双相情感障碍（BD）是一种严重的神经精神障碍与了解甚少病理生理学和通常用的情绪稳定，碳酸锂。动物研究和人类遗传学研究表明，锂影响参与神经元生长，存活和成熟，以及特别是分子参与Wnt信号传导该分子靶点。定的伦理挑战获得脑活检调查与锂反应在中枢神经系统（CNS）有关的动态分子改变，可以考虑使用从嗅觉组织获得的神经元来实现这一goal.The嗅觉上皮包含嗅觉受体神经元在开发和胶质状的支持细胞的不同阶段。这提供了研究中的患者的神经精神性疾病的中枢神经系统的动态变化一个独特的机会，使用从鼻腔活检安全地获得嗅觉组织。为了克服由SUBST带来的缺点活检嗅觉组织与非神经元细胞antial污染，一种新的方法，以获得富集的神经元细胞群体通过结合鼻活检与激光捕获显微切割的开发。在这项研究中，用于研究治疗相关的动态中神经元组织的分子变化的系统的开发和验证，使用了BD患者招募的锂治疗反应的分子机制的研究的小试验样品。

在分子签名的研究的协议和故障诊断策略被成功地进行了优化。样品RNA的质量和神经元富集的标准中更下游的实时PCR分析中使用了标准化。 RIN和RNA的浓度进行检测的干扰因素，以检测表达水平。基于几个样品与RINS从1分析 - 10，它被确定的〜3.0和高于最小RIN足以用于本研究中的下游分析。其结果是，用于cDNA合成的RNA量的标准化为1.0纳克。因此，高品质的样品与RIN以上〜3.0和RNA的量为1.0毫微克被转...

Watch this Scientific Journal Video about Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disord

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

在这项研究中，一种新的平台来调查在躁郁症（BD）的治疗的反应的突触体分子签名被开发和验证。从BD患者嗅上皮是通过鼻腔活检获得。那么激光捕获显微切割是结合实时RT PCR，调查BD锂反应的分子签名。

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元：一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

olfactory-neurons-obtained-through-nasal-biopsy-combined-with-laser

Research

JoVE Journal

Neuroscience

9.7K 次观看.  Johns Hopkins University. 在这项研究中，一种新的平台来调查在躁郁症（BD）的治疗的反应的突触体分子签名被开发和验证。从BD患者嗅上皮是通过鼻腔活检获得。那么激光捕获显微切割是结合实时RT PCR，调查BD锂反应的分子签名。 

视频: Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

针对嗅球神经元采用组合在体内电穿孔和GAL4基于增强陷阱斑马鱼线

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

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 ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.