Name: olfactory neurons obtained through nasal biopsy combined with laser-capture microdissection: a potential approach to study treatment response in mental disorders
Uploaded: 2014-12-04T13:00:00.000+00:00
Duration: 8 min 33 s
Description: 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

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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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。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Tissue Preparation&lt;/strong&gt;</td></tr><tr><td>Tissue-Tek Cryomold Molds</td><td>Sakura Finetek</td><td>4557</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cryosectioning&lt;/strong&gt;</td></tr><tr><td>Membrane Slide 1.0 PEN (D)</td><td>Carl Zeiss Microscopy</td><td>415190-9041-000</td><td></td></tr><tr><td>Rnase Zap</td><td>Ambion</td><td>AM9780</td><td></td></tr><tr><td>DEPC Treated Water</td><td>Quality Biological</td><td>351-068-131</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Microdissection&lt;/strong&gt;</td></tr><tr><td>Microscope: PALM Series MicroLaser System</td><td>Carl Zeiss Microscopy</td><td></td><td>Model: Axiovert 200M&lt;br /&gt;
Software: Robo v3.2</td></tr><tr><td>No. 5 Dumont Microdissction Forceps</td><td>Roboz</td><td>RS-49085</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;RNA Extraction&lt;/strong&gt;</td></tr><tr><td>RNAqueous Micro Kit</td><td>Ambion</td><td>AM1931</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;cDNA Synthesis&lt;/strong&gt;</td></tr><tr><td>SuperScript III First Strand Synthesis Kit</td><td>Invitrogen</td><td>18080-051</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;OMP qPCR&lt;/strong&gt;</td></tr><tr><td>SYBR GreenER qPCR SuperMix</td><td>Invitrogen</td><td>11760-500</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Taqman qPCR&lt;/strong&gt;</td></tr><tr><td>TaqMan Expression Assay Probes</td><td>Applied Biosystems</td><td>Various</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Applied Biosystems</td><td>4369016</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

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 ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular phenotype diversity. Single-cell experiments investigating the network dynamics of biological systems demonstrate cellular and tissue responses to various conditions at biologically meaningful resolution. Herein, we explain our methods for gathering single cells from anatomically specific locations and accurately measuring a subset of their gene expression profiles. We combine laser capture microdissection (LCM) with microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). We also use this microfluidic RT-qPCR platform to measure the microbial abundance of gut contents.

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

将激光捕获微切片和微流体qPCR相结合，分析单细胞的转录曲线:阿片类药物依赖的系统生物学方法

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular ...

遗传学

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 ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

新生大鼠于A气味偏侧学习模型解剖神经回路托换记忆的形成

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

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 ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

小鼠嗅觉学习和鉴别的客观和可重复性试验

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

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

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鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

隔离啮齿类动物或人类的鼻嗅干细胞

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

将激光捕获微切片和微流体qPCR相结合，分析单细胞的转录曲线:阿片类药物依赖的系统生物学方法

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

新生大鼠于A气味偏侧学习模型解剖神经回路托换记忆的形成

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

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

小鼠嗅觉学习和鉴别的客观和可重复性试验

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization