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。

<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

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

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 ...

科研

神经科学

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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 ...

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 ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

一种免费呼吸功能磁共振成像方法研究人类嗅觉功能

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

有效的人工剔骨方法编写完整的鼠标鼻组织与保存的解剖组织

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

生物学

气味对通过呼吸同步嗅觉计评估的大脑兴奋性的影响

Combining a Breath-Synchronized Olfactometer with Brain Simulation to Study the Impact of Odors on Corticospinal Excitability and Effective Connectivity

It is widely accepted that olfactory stimulation elicits motor behaviors, such as approaching pleasant odorants and avoiding unpleasant ones, in animals and humans. Recently, studies using electroencephalography and transcranial magnetic stimulation (TMS) have demonstrated a strong link between processing in the olfactory system and activity in the motor cortex in humans. To better understand the interactions between the olfactory and the motor systems and to overcome some of the previous methodological limitations, we developed a new method combining an olfactometer that synchronizes the random order presentation of odorants with different hedonic values and the TMS (single- and dual-coil) triggering with nasal breathing phases. This method allows probing the modulations of corticospinal excitability and effective ipsilateral connectivity between the dorsolateral prefrontal cortex and the primary motor cortex that could occur during pleasant and unpleasant odor perception. The application of this method will allow for objectively discriminating the pleasantness value of an odorant in a given participant, indicating the biological impact of the odorant on brain effective connectivity and excitability. In addition, this could pave the way for clinical investigations in patients with neurological or neuropsychiatric disorders who may exhibit&#160;odor hedonic alterations and maladaptive approach-avoidance behaviors.

作者聚焦：探索嗅觉对皮质脊髓兴奋性的影响 - 神经学研究的见解和创新 

This paper describes using a breath-synchronized olfactometer to trigger single- and dual-coil transcranial magnetic stimulation (TMS) during odorant presentation synchronized to human nasal breathing. This combination allows us to objectively investigate how pleasant and unpleasant odors impact corticospinal excitability and brain-effective connectivity in a given individual.

将呼吸同步嗅觉计与大脑模拟相结合，研究气味对皮质脊髓兴奋性和有效连接性的影响

It is widely accepted that olfactory stimulation elicits motor behaviors, such as approaching pleasant odorants and avoiding unpleasant ones, in animals ...

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将呼吸同步嗅觉计与大脑模拟相结合，研究气味对皮质脊髓兴奋性和有效连接性的影响