这项工作是支持“法新社NATIONALE DE LA RECHERCHE”授予的ANR - 09 - JCJC - 0117 - 01和“Neuropôle RECHERCHE Francilien NERF了”罗曼奇瑞授予。我们感谢弗朗索瓦勒菲弗尔，在C + + / Qt的软件开发和洛朗黑和巴蒂斯特让维耶在发展光学成像设置的帮助。

1。准备成像的动物（按照欧洲实验动物护理和使用建议，86/609/EEC指令） <ol><li> 6日至8周龄雄性小鼠C57BL6与intraperitonealy鸡尾酒注射氯胺酮（10mg/kg的）和甲苯噻嗪（100mg/kg）麻醉。手术开始，当鼠标不再响应hindpaw捏。在整个实验动物被放置在一个加热垫。不断监测和体温保持在37 ° C。麻醉深度维持整个手术和成像会议通过检查出肢体的情况下退出。否则管理的一个皮下注射20％的初始麻醉鸡尾酒。 </li><li>使用指甲钳，去除头发从头皮。从暴露的皮肤上残留的头发清洁，用无菌纱布，用生理盐水浸泡。 </li><li>鼠标放置在立体框架。口鼻部的后脑勺在同一平面上，为了设置嗅球表面的水平。安全牢固的耳朵和鼻子的酒吧，以防止在成像过程中的动作。 </li><li>应用动物的眼睛眼药膏，以防止干燥和疼痛。 </li><li>与70 °乙醇和连续扫描的优碘的头皮面积消毒手术器械。 </li><li>要删除的皮肤覆盖的头骨，开始通过在皮肤上，用剪刀在两耳之间的头回的一个切口。然后切在双方对基地的耳朵和眼睑对前额沿前后方向。完成切割吻靠近鼻子栏上的皮肤去除头皮。 </li><li>根据双眼观察，用生理盐水浸泡的棉签，轻轻取下的头骨上方的骨膜。使用一双镊子去除剩余的组织用手术刀刮去表面的头骨有一个干净的准备。 </li><li>OB的是位于眼睑之间的两个hemibulbs组成的对称结构。他们只限于在延髓尾方向由静脉窦，矢状缝分离。放置一个用蒸馏水浸泡在上述OB的骨片吸收明胶海绵。重要的是要保持这个骨面积在整个实验湿润。 </li></ol> 2。准备颅窗口<ol><li>取出明胶海绵和启动N ° 10手术刀刀片轻轻刮骨。保持45 °之间的刀片和骨不断的角度，从眼睑移动灯泡区矢状面刀片。不要适用于骨的垂直压力，或从头以上的静脉窦骨。 </li><li>在变薄的过程中，停止每5min和地方水合对骨明胶海绵降温的准备。棉签，用海绵去除骨灰尘的头骨。 </li><li>保持清扫和冷却或者直到可视化的小梁，松质骨层。 OB的细血管，必须在这个阶段中可见。停止刮伤骨，并开始使用手术刀的尖端垂直，以“画”一个矩形区域封闭的OB。在这个阶段，N ° 11手术刀刀片都可以使用。保持应该是安全的任何手术刀中风的静脉窦范围内的手术。 </li><li>逐步形成的矩形沟槽挖掘使用手术刀连续运动。擦拭手术刀定期清洁，并保持锋利的尖端。要格外小心的提示，以避免接触硬脑膜表面的深度。 </li><li>为了得到剩余的骨厚度感，一双镊子尖轻轻推。如果骨瓣褶皱的压力下，移动到下一个步骤。 </li><li>根据一滴生理盐水，用手术刀化的尖端水平抬起骨瓣。切除皮瓣做彗星arefully，以避免撕掉剩余的骨。 </li><li> OB的表面暴露后，检查没有任何出血或血管吻合。受伤的硬脑膜或组织表面会降低获取光信号的机会。用明胶海绵在盐水中浸泡，以保持灯泡湿润的地区。 </li><li>应用聚丙烯牙科水泥周围的骨形成一个很好的窗口。 </li><li>将低熔点琼脂糖（1.2％），硬脑膜下降，并把在窗口尺寸的无菌玻璃盖。成像会议期间，一个可以添加少量琼脂糖，以弥补的干燥。琼脂糖防止随呼吸移动的OB和光学成像将提供一个平坦的表面。 </li></ol> 3。嗅觉活动映射的光学成像装置<ol><li>在时间和强度，使用嗅觉的嗅觉刺激，必须准确界定。我们使用自定义修改的multivial灌注系统Valvebank 8II的自动化科学的一个基本的空气压缩机（压缩呼吸的空气将被视为适合）相关的版本。该系统可以准确，快速的外部阀门控制。在选定的浓度纯气味的解决方案是在矿物油稀释。要激活的醛，如背转播醛常用。一个精密量的稀释气味（20至50μL）装上滤纸，并放置在一个注射器水库。通过灌注系统，压力控制空气传送系统，确保恒定的速率odorized空气流量交付期间阀门的开度的动物鼻子。气味是透过一个聚乙烯的R - 3603真空管账面〜1000ml/min的流量空气（圣戈班公司）。避免油管之间的气味和残留量的气味污染。如果可用，气味刺激的重复性，可控器lled通过火焰离子化检测器的使用（microFID 2020 Photovac）。 </li><li>光学装置已开启。它由一个冷却隔行12位CCD相机（ORCA的AG滨松市）的荧光体视显微镜（莱卡MZ16），计算机控制的嗅觉和适当的带通干涉滤光片稳定的励磁灯。一个计划，说明我们的设置是在图2。对于内在的光信号成像（IOSI），一个200W的卤钨灯（奥丽尔QTH），加上一个光纤环光（肖特）插入显微镜围绕目标是用来提供稳定，甚至照明。对于一个150W的金属卤化物灯用5mm的核心液体光导（徕卡）黄素自体荧光信号成像（法祀），提供通过外延的立体显微镜照明端口甚至本地化激发的荧光。 定制软件实现图像采集和硬件同步。开放型的ource软件事无巨细，也可用于控制光的设置和收购。 </li></ol> 4。光学成像<ol><li>广场的体视显微镜下的立体框架，视野为中心的颅窗口（见图2A光学装置的原理）。 </li><li>调整显微镜专注于毛细血管。之前刺激试验（见说明5）灯泡的形象是560nm（绿色）的光（光纤环），它提供了一个良好的血管成像对比下。这张照片是用来作为解剖对照检查的准备状态，并在实验过程中获得了好几次。 </li><li>为iOS成像，反射光的强度是由CCD相机记录下630 + / -10纳米（红色）照明。图像采集全帧每秒5张，对应于约150毫秒的曝光时间（不分级）。光源功率调整，重新<html> ACH至少〜3000级灰度的OB区，接近CCD像素井的饱和度。这样做，使人们有可能采取“防治荒漠化公约”的12位动态的优势，在激活过程中捕捉到微弱的强度变化。最大的IOS幅度达到〜1％。 </li><li>对于FAS成像荧光激发480nm + / - 20nm的（蓝色）epiflurorescence光下获得的。在515nm的高通滤波器设置捕捉光线。在每秒5帧的图像采集与分级为4 4，最大限度地提高灵敏度。同样激发光功率调整到IOS上的OB区3000级灰度。最大FAS的幅度达到〜3％。 对于这两种成像方式，在受平面的领域深入是相同的，在0.5毫米测量放大4倍左右。 </li><li>光线应打开之间的成像试验，以避免加热和漂白的准备。 </li></ol>乐“&gt; 5。成像试验<ol><li>标准成像会议包括只有空气是交付10秒5的基准，其次是取决于所选择的气味的浓度，70至82S的10S，进一步记录为基线恢复恒定的空气流动（图3的气味刺激2A）。在试验结束时，光关闭，纯净的空气交付间审判间隔时间（1〜3分钟）洗出残留气味分子，并避免感官习惯。气味试验交错空白试验（空中传送）。 </li><li>气味诱发的激活区在地图上的球状地区的可视化和对应的个别肾小球的大小（80 至 200微米直径9）。图像处理解释图2B。嗅觉的地图是图3中。相反，任何其他感官系统，在OB信号信噪比足以解决在单一试验的气味诱发反应（图内部监督办公室和图3B。 FAS的3D）。 </li><li>小鼠安乐死立即按照欧洲实验动物护理和使用建议使用方法的成像会议结束。 </li></ol> 6。代表性的结果（参见图3中的嗅觉地图）： <img alt="图1" src="/files/ftp_upload/3336/3336fig1.jpg" /> 图1主要在啮齿类动物中的嗅球的组织结构。嗅感觉神经元，位于主要的嗅觉上皮的初级感觉细胞，表达了同样的气味受体和收敛到相同的肾小球在OB。嗅觉肾小球圆形neuropils（虚线圆圈），分别位于表面的OB。需要注意的是一个非常密集和复杂的血管网肾小球。缩写（顶/下）：ONL：嗅神经层; GL：肾小球层，英超：外部网状层; MCL：僧帽细胞层;保利协鑫：颗粒细胞层。 <img alt="图2" src="/files/ftp_upload/3336/3336fig2.jpg" /> 图2反射荧光信号，并记录在体内。 A.宽领域的光学成像设置。暴露于麻醉小鼠的大脑，任红（IOS）或蓝色（FAS）的光通过一个环形光纤环连接到光学镜头或显微镜EPI照明端口。气味装入密封的小瓶和odorized空气传递到动物的鼻子（绿灯：开阀）。 B.记录协议和数据处理。 IOS和FAS系列（工期90年代）个别试验记录。该图显示了审单的时间表：基准变化5至10秒，从3到10秒的刺激，并回到基线，​​从70到82S。图像处理要求像素由像素强度值的加减在基准强度值（FAS）的刺激期间Øř刺激加回到基线（IOS）的。这种差异是基准值再除以获得％的变化（图产生的图像 。） 。 <img alt="图3" src="/files/ftp_upload/3336/3336fig3.jpg" /> 图3中的气味诱发电位在OB活动地图使用IOS和FAS成像。 A.背OB血管可视化下大开绿灯。公元前。 IOS映像（审单，分别与三个平均试验）为20％的醛10S介绍。白色箭头表示这气味激活的球形地区的利益。这些激活地图使用帧平均气味刺激结束后，在第一第二（A和-0.52％，在B中最大的反射率变化-0.63％）。注意吸光度的气味激活发生的黑区。光盘。 FAS的收购顺序相同的气味（审单与三个平均试验分别在相同的鼠标LY）。这些激活地图使用帧平均气味刺激开始后，在第一秒（荧光变化0.72％，在D和0.66％，在E最大）。需要注意的是对应的自体荧光发射的黑色箭头所指的白色区域在IOS黑区。在FAS地图看到粒状方面是由于4分级要求的灵敏度提高到4。 FAS的图像不纠正，从自体荧光漂白。图像的实际尺寸：0.7 mm宽x长1.2毫米。

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我们什么都没有透露。

在这篇文章中，我们目前在体内的气味诱发的活动，在鼠标的OB录音的IOS和FAS成像技术。为了实现这一目标的一个相对简单的和负担得起的的宽视场光学成像设置是必要的。成像数据采集要求的训练，以执行精细的外科手术，并避免任何损伤硬脑膜或脑组织。特别严重出血会吸收光子成像记录，并结束实验。 IOS和FAS成像的一个好处是避免注射荧光示踪剂，可能会导致细胞毒性，副作用。他们使人们有可能解决有关空间的感官刺激，从而编码嗅觉地图问题。 2 - 脱氧葡萄糖成像相反，他们提供了可能性，在一个单一的动物形象几个气味。然而，由于光子穿透组织的限制，内部监督办公室和FAS限制背部分的OB不能记录从腹地区。 内源性光信号成像提供了极好的体内成像的空间分辨率。技术改进所关注的血管成分的定量计算的反射信号在感官的活化10 8,9以及动态血氧和体积。多波段成像的IOS成像方法目前在我们的实验室开发，充分量化总血红蛋白浓度和氧合在OB在感官激活。这些添加到FAS的成像光谱光学测量，将给予回答的机会在感官激活 11,12之间的血管和细胞内动态未解的关系。

<table border="1"><tbody><tr><td> 丽晶酒店的名称</td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Imalgene </td><td>梅里亚</td><td></td></tr><tr><td> Rompun </td><td>拜耳</td><td></td></tr><tr><td>琼脂糖，类型III - A </td><td> Sigma - Aldrich公司</td><td> A9793 - 50G </td></tr><tr><td>醛98％ </td><td>爱秩序</td><td> 115606 - 100ML </td></tr></tbody></table>

imaging odor-evoked activities in the mouse olfactory bulb using optical reflectance and autofluorescence signals

在感官刺激大脑，激活分布式参与刺激的编码的功能模块之间的神经元的数量。功能光学成像技术，是有利的可视化这些模块的高空间分辨率的感觉皮层的激活。在这种情况下，挂钩neuroenergetics的分子机制产生的内源性光信号对比的宝贵来源，记录在啮齿动物的大脑宽领域的感官刺激的空间地图。 在这里，我们目前两种技术的基础上在激活过程中的脑组织内源性的光学特性的变化。首先，由于在红光反射的地方改建生产的内在（IOS）的光信号：（一）吸收血氧水平和血容量（二）光子散射的变化。 体内 IOS来记录空间地图observati在20世纪80年代中期开始晶须万桶的光学地图在大鼠和猫视觉皮层的方向列。后来证明拉里-卡茨的第2组的IOS到气味的啮齿动物的主要嗅球（OB），表面成像。第二种方法依赖于黄素蛋白自体荧光信号（FAS）由于在这些线粒体代谢中间体的氧化还原状态的变化。更确切地说，该技术是基于绿色荧光，因为当组织与蓝色光激发氧化状态flavoproteins。虽然这种信号可能布里顿机会和他的同事3先驱研究大脑活动的研究记录的第一个荧光分子之间，直到最近，他们已被用于脑激活体内的映射。 FAS的成像是首次应用在啮齿类动物的体感皮层hindpaw Katsuei Shibuki的第4组的刺激。 嗅觉系统是因为它可以有效的检测和鉴定环境中的化学物质（食物，天敌）的绝大多数生物物种的生存至关重要的。 OB的是大脑中的嗅觉信息处理的第一个继电器。它接收从检测挥发性气味分子的嗅觉主要的感觉神经元传入的预测。每个感觉神经元对气味受体和携带同一类型的受体的神经元，送他们的神经过程〜100μm的3相同的定义良好的微区， 嗅觉小球（图1）离散的神经纤维构成的一类。在过去的十年中，IOS成像促进OB 的5，6，7这已成为一个研究最多的感官结构功能的探索。转播活动与FAS成像的映射尚未执行。 在这里，W发送一个有效的协议显示IOS和FAS成像的连续步骤，在小鼠的OB地图的气味诱发的活动。

Watch this Scientific Journal Video about Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals at JoVE.com

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

本文介绍了内在的光信号和flavoproteins自体荧光信号成像的协议，在小鼠嗅球表面的气味诱发的地图活动。

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. ...

imaging-odor-evoked-activities-mouse-olfactory-bulb-using-optical

Research

JoVE Journal

Neuroscience

15.9K 次观看.  UMR8165 Université Paris Sud 11, Paris Diderot 7 – CNRS. 本文介绍了内在的光信号和flavoproteins自体荧光信号成像的协议，在小鼠嗅球表面的气味诱发的地图活动。

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

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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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 ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

脑血流动力学和的光散射特性的同步评估<em&gt;体内</em&gt;大鼠脑使用多光谱漫反射成像

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

用光学分馏塔和标准显微镜设备对小鼠黑质多巴胺能神经元数的视学估计

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...