这项工作是由国家科学基金会资助IOS 1121692，和大学的华盛顿研究基金会的支持。

1。挥发性Follection <ol><li>在这个例子中，我们使用挥发性样品M. lewisii花-高山野花原产于美国加州。收集挥发物，采用动态吸附方法，根据到Riffell 等。14。简单地说，该方法采用一个闭环特氟隆包括在花捕集系统。使用的惰性的真空泵，花的周围空气被吸入，通过一个“陷阱”巴斯德吸管包括充满PORAPAK Q矩阵。从泵的返回空气通过活性炭过滤。一规定时间周期之后，在我们的情况下24小时，PORAPAK Q矩阵，用一种非极性溶剂中，典型的己烷洗脱，收集的浓缩提取物。的提取物，然后储存于-80℃直至分析。如果有必要，样品可以被集中在分析之前，下的氮气流。除非样品良好的特点，运行等分，它通过一个气相色谱 - 质谱（GC-MS）使用样品之前确定挥发性成分。 </li></ol> 2。电准备<ol><li>剪切约1厘米，从1,000微升的移液管尖的末尾。放置一个熊蜂（ 熊蜂的凤仙花 ）到移液管尖端部的基础上，并轻轻推向相对端的前端，直到只有头部露出。 </li><li>牙科用蜡融化，并在外露的头，模具的蜡附着在安全的复眼，使蜜蜂的头部完全不动。请务必不要让任何到天线的蜜蜂蜡。 </li><li>一旦头是安全的，做成正方形，窗口等，切口进入头部胶囊用剃刀刀片断路器或适当大小的手术刀切角质层。使用刀片的断路器，从背侧的头壳，紧接后面的天线和相邻的一个的化合物眼。切一条直线上，从一个复眼对侧复眼。直线切割后到对面的复眼，开始做一个切口，背部，直到头壳曲线和其胸部两端附近。在这一点上，朝向头部胶囊的相对端开始削减。最后，一​​旦一条线被切断的相对端，切断回线的初始切口到起始位置。重要的是要除去角质层是相邻天线，因为这会妨碍插入电极。 </li><li>的角质层被切断后，使用钳子删除蜂角质层，然后公开蜜蜂的大脑，更重要的是，在触角叶。立即开始与昆虫盐水，superfusing大脑，使大脑不成为dessicated的。后大脑暴露出来，仔细一对非常细小的镊子以上的触角叶外周神经鞘立即删除。要非常小心，不蜜蜂的大脑穿刺的镊子。 </li></ol> 3。气相色谱与多声道录音<ol><li>蜜蜂“准备” - 管固定在它的大脑暴露 - 现在是准备好电生理记录。将蜜蜂，固定在夹具的磁基地，位于空气表。 </li><li>安排的输液袋，流量控制器，和油管（充满昆虫盐水），以便，盐水连续superfuses大脑。 </li><li>使用一台显微操作，insertareference电极，钨丝，到蜜蜂的眼睛。 </li><li>使用一个单独的微操纵器，如一个螺旋线的四极管，或硅的多通道电极（Neuronexus技术），插入的多通道电极， 到的蜜蜂触角叶。此电极被连接到一个预放大器，如的TDT系统的S-3 Z系列的TDT系统的Z-的总线bioamp处理器。气相色谱输出matogram经由屏蔽BNC电缆的探测器可以是接口与放大器和数据采集系统，使得两个神经和GC的信号是同步的。 </li><li>等待约30-60分钟，以稳定的神经录音。一旦在记录信道为单位的自发活动和波形形状已成为一致，使用的气味刺激的蜜蜂的注射器，并观察响应的记录信道的气味。 </li><li>记录神经元的细胞外尖峰通过自动阈值由3.5至5西格玛各个记录通道上的信号的记录信道。手动阈值，可能需要一些渠道，以避免电气噪声污染。从神经元的动作电位，会出现作为在记录通道的电压尖峰。当通道的电压超过阈值时，系统缓冲器并保存阈值交叉之前和之后的几毫秒，从而羚牛GA管理单元拍摄的波形，或尖峰。 </li><li>立即到空气表旁边是GC。花的提取物注射到GC之前，请务必升温的GC运行，该方法是正确的。在我们的例子中，我们使用一个开始温度的方法，在50℃，4分钟，随后在10℃/分钟的速率由一个增加的温度。至220°C，届时我们举办的GC 6分钟。我们使用DB-5气相色谱柱（J＆W科学，福尔瑟姆，CA，USA），用氦气作为载气。的入口是不分流，与设定为200℃的温度下火焰离子化检测器的温度被设定为230℃。 </li><li>的提取物样品的花香顶空进样到被加热的注射端口的GC释放到GC柱吸附的挥发物。列的污水分裂1:1之间火焰离子化检测器（FID）和蜜蜂天线使用玻璃“Y”型连接器（J＆W科学）。开始记录奥尔丁从电极注入到GC中的样本。 </li><li> GC运行完成后，让制备5〜15分钟的休息。然后或者另一个样品注入到GC或刺激使用单一挥发性化合物或化合物的混合物的制剂。在这后者的刺激的制备方法，从一个恒定的空气流的空气的脉冲改道通过含有该化合物已经沉积的一片滤纸的玻璃注射器。由一个螺线管激活的阀，由软件控制的装置，脉冲刺激气味。 </li><li>如果单位活动突然停止或更改，请检查吊盐水，让准备休息了15分钟。如果自发活动不恢复以前的水平，然后准备应被丢弃和另外一只蜜蜂（如果可用）。 </li><li>实验结束后，用5％福尔马林20分钟的探头还组织修复大脑。其次，消费的大脑并将其放置在2％的戊二醛，4小时，并随后做一个梯度乙醇脱水系列和清除与水杨酸甲酯的脑。基于的组织，其中电极刺破组织应该是清晰可辨的，通过共聚焦显微镜在AL的位置上的固定和结算。 </li></ol> 4。数据分析<ol><li>分析所收集的数据，实验后的分离和识别所记录的神经单位。使用典型的软件程序（离线的分拣，MClust，并SClust）独立波形，或“尖峰”，根据穗的形状，如峰值或谷值振幅，半峰宽等，或减少的措施（主要成分）17 ，18（ 图2）。只有那些使用集群的尖峰，在 ​​三维空间中分离（PC1-PC3），在统计学上彼此不同的（多元方差分析，P &lt;0.05）（ 图2），以供进一步分析。请参阅引用＃S 17-19的四极记录和穗排序方法的完整描述。 </li><li>在每个群集高峰时间戳记，并导出这些数据进行分析，使用MATLAB或Neuroexplorer的（NEX），北卡罗来纳州温斯顿-塞勒姆，创建栅格图和射击频率响应（ 图2，图3A）。 </li><li>识别使用同时记录GC数据的挥发物的保留时间。使用的挥发物的保留时间，确定从色谱图中的峰的顶点，在这些时间点检查单元响应。 </li><li>要检查个别单位的反应，通过GC运行，BIN数的峰值在100毫秒的间隔和检查过程中，发射率参考洗脱挥发物的保留时间。在100毫秒的时间间隔的像素合并的尖峰提供足够的细节，或信号，有关的时间过程中神经元的响应于从GC洗脱加臭剂。 </li><li>要EXA开采的人口不同洗脱挥发物的反应，集成的单个的单元的发射率响应超过3秒的采样窗口，1.5秒前，和1.5秒后，在保留时间为易失性的（ 图3）。此时间段典型的洗脱挥发性从GC的持续时间。我们发射率响应的单位按颜色编码（红色的是射速高，反应，蓝色是一种低响应），并安排他们的活动矩阵，每一行代表合奏GC污水（列）（ 图3）。 </li></ol>

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没有利益冲突的声明。

昆虫的嗅觉介导的行为驱动许多不同的流程，包括复制，主机选址，并确定适当的食物资源。这些过程的研究需要从源头抓起，以及识别能力，调解行为的化合物，这些化合物的挥发物排放的识别能力。复杂的问题是，气味是由几十到几百，共同创造一个独特的气味，被认为比单个的成分6,7,13,19,20不同的单个化合物。 ，最初研究中的性信息素系统12进行的，最近更是在食品和产卵...

<table border="1"><tbody><tr><td> 项目名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> PORAPAK Q型80-100目</td><td>沃特斯</td><td> WAT027060 </td><td></td></tr><tr><td>雷诺烤箱袋</td><td>雷诺</td><td></td><td></td></tr><tr><td> GC </td><td>安捷伦</td><td> 7820A </td><td></td></tr><tr><td> GC柱</td><td> J＆W科学，福尔瑟姆，CA，USA </td><td> DB-5（30米，0.25毫米，0.25微米） </td><td></td></tr><tr><td>分析氦载气</td><td>普莱克斯</td><td> HE K表</td><td> 1毫升/分钟</td></tr><tr><td> 16通道硅电极</td><td> Neuronexus技术部历史ologies </td><td> a4x4-3mm50-177 </td><td></td></tr><tr><td>细金属丝镍铬，0.012毫米直径） </td><td>山特维克山特维克HP里德</td><td> PX000004 </td><td>为了使自定义的四极管和stereotrodes </td></tr><tr><td>前置放大器</td><td>塔克 - 戴维斯系统</td><td> PZ-2的</td><td></td></tr><tr><td>扩音器</td><td>塔克 - 戴维斯系统</td><td> RZ-2 </td><td></td></tr><tr><td>数据采集​​系统 - OpenEx套件</td><td>塔克 - 戴维斯系统</td><td></td><td></td></tr><tr><td>在线穗排序软件 - SpikePac </td><td>塔克 - 戴维斯系统</td><td></td><td></td></tr><tr><td>离线穗排序软件 - Mclust穗排序工具箱</td><td>大卫雷迪希，大学神经科学系美国明尼苏达州</td><td>免费在<a href="http://redishlab.neuroscience.umn.edu/MClust/MClust.html" target="_blank">http://redishlab.neuroscience.umn.edu/MClust/MClust.html</a>下载</td><td> MATLAB工具箱</td></tr></tbody></table>

identification of olfactory volatiles using gas chromatography-multi-unit recordings (gcmr) in the insect antennal lobe

所有的生物体生活在一个世界充满感官刺激，确定其所处的环境行为和生理反应。嗅觉是特别重要的昆虫，它用自己的嗅觉系统响应和歧视之中，复杂的刺激气味。这些气味引起的行为，调解过程，如繁殖和栖息地选择1-3。此外，化学传感由昆虫介导的行为，农业和人类健康是非常重要的，其中包括4-6，草食动物的粮食作物7，授粉和传播疾病8,9。因此，在昆虫行为的嗅觉信号，其作用是识别重要的，了解生态过程和人类的食物资源和福祉。 至目前为止，已经难以识别驱动昆虫行为的挥发性物质，往往是乏味。目前的技术包括：气相色谱耦合电生理记录（GC-EAG）和气相色谱耦合的单感器录音（GC-SSR）10-12。这些技术被证明是重要的生物活性化合物的鉴定。我们已经开发出一种方法，使用气相色谱多通道电生理的录音（称为“GCMR”）的神经元在触角叶（AL;昆虫的嗅觉中枢）13,14。这个国家的最先进的技术使我们能够探测气味信息是如何表示的昆虫的大脑。此外，因为在这个级别的嗅觉处理神经元对气味的反应是非常敏感的，因为天线AL神经元的受体神经元到收敛的程度，AL录音可以让活性成分自然的​​气味，高效和高灵敏度的检测。在这里，我们描述GCMR举一个例子，它的使用。 一些一般的步骤是国际富豪的ved检测生物活性挥发物和昆虫响应。挥发物，首先需要收集的利益来源（在这个例子中，我们使用花沟酸浆属 （属Phyrmaceae）），其特征为需要使用标准的GC-MS技术14-16。昆虫准备用最小的解剖研究，之后记录电极插入触角叶，多渠道的神经开始录制的。处理后的神经数据，然后显示特定的气味分子的昆虫的神经系统造成显着的神经反应。 虽然我们在这里给出的例子中特定授粉研究，GCMR可以扩大到范围广泛的研究生物和挥发性来源。例如，此方法可用于在吸引或排斥矢量昆虫和作物害虫的加臭剂的识别。此外，GCMR也可以被用来识别有益昆虫的引诱剂，如婆llinators。该技术可以扩展到非昆虫科目以及。

在使用M. GCMR分析的lewisii花香的味道，我们3微升提取物注入到GC。挥发物的纯化，通过GC的总数通常为60-70挥发物。香味M. lewisii是主要组成的单萜类化合物，包括β-月桂烯（非周期性）和α-蒎烯，用剩余六碳挥发物，如2 -己醇，倍半萜类化合物，包括&lt;1％的顶空组成的气味。 GCMR利用触角叶神经元的敏感性，以及神经元的处理用的重要的生物...

Watch this Scientific Journal Video about Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe at JoVE.com

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

嗅觉介导许多不同的昆虫的行为，并经常由数十至数百种挥发性化合物组成的复杂混合物。气相色谱法与多通道记录在昆虫触角叶中，我们描述的生物活性化合物的鉴定方法。

用气相色谱 - 多单元的录音（GCMR）昆虫触角叶嗅觉挥发物的鉴定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

identification-olfactory-volatiles-using-gas-chromatography-multi

Research

JoVE Journal

Neuroscience

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

14.2K 次观看.  University of Washington. 嗅觉介导许多不同的昆虫的行为，并经常由数十至数百种挥发性化合物组成的复杂混合物。气相色谱法与多通道记录在昆虫触角叶中，我们描述的生物活性化合物的鉴定方法。

视频: Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

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

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

在自主机器人以昆虫触角电位传感器的嗅觉搜索

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

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 ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

光感受器细胞光谱灵敏度在从昆虫模型决定<em&gt;在体内</em&gt;细胞内记录

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

昆虫控制的机器人：移动机器人平台来评估昆虫的气味跟踪能力

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor ...

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 ...

Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined curved surfaces, and images of real-world objects (with and without disparity) in a similar manner as described in the ventral visual stream. In addition, parieto-frontal areas are believed to convert visual object information into appropriate motor outputs, such as the pre-shaping of the hand during grasping. To better characterize object selectivity in the cortical network involved in visuomotor transformations, we provide a battery of tests intended to analyze the visual object selectivity of neurons in parieto-frontal regions.

A detailed protocol to analyze object selectivity of parieto-frontal neurons involved in visuomotor transformations is presented.

用单单元记录法研究猕猴背视流中的对象表示

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined ...