Authors would like to thank Peter Mombaerts for the generous gift of OR-GFP mice; Anne Lefranc and the CSGA animal facility for excellent animal care. Funding was provided by CNRS through an ATIP and ATIP Plus grants, by Conseil R&#233;gional de Bourgogne (FABER and PARI grants), by Universit&#233; de Bourgogne (BQR program).

该协议遵循UNIVERSITE勃艮第的动物护理准则和批准了UNIVERSITE勃艮第伦理委员会。 1.动物<ol><li>使用可在杰克逊实验室基因工程OR-IRES-tauGFP小鼠。这些小鼠以分析轴突靶向嗅觉系统19的和发展开发彼得Mombaerts博士的实验室。例如，MOR23-IRES-tauGFP行，股票号码006643，耐官方应变名B6; 129P2- Olfr16 tm2Mom / MomJ;同样，SR1-IRES-tauGFP行，股票编号6717负有正式名称B6; 129P2- Olfr124 tm1Mom / MomJ。 </li><li>关于动物的年龄:用于协议的一个更好的结果，使用2和4周龄之间的动物。在这个年龄组中，清扫容易（较软的骨头，更坚定的嗅上皮）和树突状旋钮都是双向gger比较年长的动物。 </li></ol> 2.制备电极和解决方案<ol><li>对于刺激移液器:购买prepulled刺激移液器。否则，手动准备他们。 <ol><li>使用火焰，弯曲61毫米玻璃吸管从尖端约1厘米。插入这六个移液器加直7 日在1.5厘米热缩管通过小孔加强。 </li><li>热缩管，以维护连接在一起的桶。附加一个额外的热缩管到的桶的另一端。用多管拉出器拉出此刺激吸管。 </li><li>添加一些白色液体胶水周围的小孔，以加强弯曲的提示。让干燥O / N。 </li></ol></li><li>制备1或2升的正常林格氏胞外溶液（以mM计:氯化钠124，氯化钾3，用MgSO 4 1.3， 氯化钙 2， 碳酸氢钠 26，将NaH 2 PO 4 1.25，葡萄糖15; pH值7.6和305毫渗透摩尔浓度）。保持在4℃直至使用。 </li><li>制备细胞内储备溶液（以mM计）:氯化钾70，KOH 53，甲磺酸30，EGTA 5，HEPES 10，蔗糖70; pH为7.2（KOH）和310毫渗透摩尔浓度。保持在4℃直至使用。好几个星期。 </li><li>准备用制霉菌素实验前细胞内的录音解决方案即兴（在最后一分钟）。 <ol><li>称取3毫克制霉菌素，加入50微升的DMSO;涡旋20秒然后超声处理2-3分钟，直到完全稀释。 </li><li>加入20微升的DMSO-制霉菌素溶液在5毫升细胞内贮备液。涡旋20秒，然后超声处理3分钟。保持此解决方案，在4°C和光线直射保护，制霉菌素是光敏感。此溶液可用于几个小时。更换每一天。 </li><li>一旦嗅觉上皮的准备是在显微镜下，需要一定的该溶液以1毫升注射器用火焰细长黄色尖端以填充电极;从光线直射保护。一旦在室温，制霉菌素液是不是稳定的;更换溶液在1ml注射器每小时或保持在冰上。 </li></ol></li><li>拉记录电极与一个车夫来获得长长的脖子和小头（〜2微米）以15-20MΩ与内部制霉菌素液阻力。 </li><li>制备在0.5M的在DMSO下通风柜加臭剂溶液;等分和保持在-20℃直到使用。稀臭气物质在林格氏溶液中，直至所希望的浓度。填写刺激吸管。 </li></ol> 3.准备嗅觉上皮注意:或-IRES-tauGFP小鼠表达的tauGFP的或启动子的控制之下。在这些小鼠中，表达感兴趣的或所有神经元都GFP标记的。该协议适用于表示在所有区域手术室。然而，解剖和录音是更容易表达在背区手术室。 <ol><li>通过注射氯胺酮和赛拉嗪（150毫克/公斤和10毫克/公斤生化需氧量的混合物麻醉动物Ÿ重，分别）。断头可以用锋利的剪刀对年轻小鼠或用适当的维护啮齿动物为铡老年小鼠进行。 <ol><li>采用环形解剖剪刀作一纵切口内侧通过背部皮肤。拉它拆开去除皮肤。使用剪刀，剪断下颚处下颚关节。由平行于牙齿冠状切口取出上前牙。 </li><li>使后面的眼睛头部的冠状切，并保持头部的仅前部。浸在了管制范围内的解剖冰冷的林格氏液。 </li></ol></li><li>范围下剖析:在腹侧，使沿上颚/牙齿的纵向切割。切背侧骨头纵向，鼻腔的dorso-侧面以下。除去大部分骨骼和味觉;在RT传送它相连的隔膜和上皮中含氧林格。 </li><li>对于最终的解剖:在开始录制前右会议上，剥离上皮从底层隔钳。分离钳和具有两个4-5毫米剪刀切口（使用microvannas剪刀）在隔膜，其中粘合力更强的前部的上皮。 <ol><li>小心地切割出来沿其背部连接到隔上皮去除犁鼻器。转移上皮至记录室朝上粘液层;保持平与竖琴室。 </li></ol></li><li>一个堂堂正正的显微镜配备荧光光学系统和感光摄像头安装在室内。可视化通过40X水浸物镜（数值孔径0.8）和一个额外的2X或4X放大率由放大透镜取得的制备在计算机屏幕上以高倍率。用含氧林格在室温（1-2毫升/分钟）连续灌注。 </li></ol> 4.记录会话<ol><li>搜索荧光细胞:激发准备在480纳米EGFP的，发射的光在530-550纳米范围内;针对一个树突状旋钮，这是在荧光下亮场可靠可见。 </li><li>填写与制霉菌素液内电极;轻轻拍打在电极去除气泡。 </li><li>在插入电极支架电极，适用于移​​液器正压;电阻应为15〜20兆欧。 </li><li>把电极接近细胞;一旦电阻达到〜40MΩ，释放正压和应用轻微负压达到千兆欧密封。 </li><li>一旦密封达到，设置膜电位约为-75毫伏; </li><li>一旦进入细胞被打开，继续刺激方案，药物治疗。为了测量响应单一的加臭剂的刺激，记录200-500毫秒自发性活动，刺激为500毫秒，并测量细胞的长达10秒13的响应。对于药物治疗，灌注药物制剂在所需的浓度20。 </li></ol> 5.数据分析<ol><li>在50％的最大幅值，上升时间（必要的时间，以达到90％的最大振幅的，在毫秒），总电流（在PAS的曲线下面积），时间:作为接着分析电流通过加臭剂刺激引起（发作和响应在最大振幅的50％的偏移量，在毫秒之间的时间）。 <ol><li>使用峰值传导电流（最大振幅达到）在不同浓度，绘制并使用希尔方程拟合剂量响应曲线为:I = I最大/（1+（K 1/2 / C）n），其中I表示峰值电流，IMAX在饱和浓度的最大响应，K 1/2的浓度在该半最大反应的达到，C加臭剂的浓度和n希尔系数。 </li></ol></li><li>在电流钳的最大去极化分析膜电位的录音中，托塔升势引起（在mVsec曲线下面积）;记录自发扣球活动在30秒到1分钟的录音;通过尖峰通过注入电流（5-10 PA）的记录引起兴奋。 </li></ol>

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The authors declare that they have no competing financial interest.

这个协议的正确监测健康嗅觉神经元的性质的能力在很大程度上依赖于制剂的质量。因此，清扫步骤是关键的。首先关键是要注重质量（pH值，渗透压摩尔浓度），氧和温度（冰冷的，但不冻结）夹层介质。第二，与解剖工具上皮的操作必须尽可能限制，以避免损坏。最后，这是至关重要的，以获得以访问最大OSN人口尽可能制剂尽可能平坦。 解剖质量是根本，获得一个健康的...

嗅觉感觉神经元（OSN）代表嗅觉感知的第一步骤。位于嗅觉上皮衬里啮齿动物的鼻腔，他们变换气味物质的化学信息转换成通过他们的轴突传送到大脑的动作电位。为了更好地理解嗅觉编码机制，有必要表征嗅觉神经元的转导和膜特性。直到最近，大多数用于表征哺乳动物的嗅觉神经元的性能的技术进行了分离上1-4嗅觉神经元。解离过程中使用的各种机械和化学（ 即酶）进程从环境释放的嗅觉神经元。这些过程可引起细胞的记录数量较少。这种低数量可以甚至在GFP标记的细胞的情况下更加重要。离解也除去嗅觉神经元和嗅觉上皮的可以提高surviv其它细胞之间的局部细胞 - 细胞相互作用人与嗅觉神经元"性质的调制。为了绕过分离程序，一个完好的制剂被开发5。 每个OSN表达一种嗅觉受体（OR）从一个大的多基因家族6中选择。有〜表示在小鼠的主要嗅觉上皮1000 OR值。由于在野生型动物的大量或，机会来记录嗅觉神经元表达相同或非常低。为了克服这些限制，基因靶向的小鼠可在其中表达所识别或标记有荧光蛋白7-9所有嗅觉神经元。这些标记的嗅觉神经元被用来做功能分析分离的筹备与7,10,11前面提到的缺点。因此，一个完整的上皮细胞的准备从5标记基因小鼠规避这些问题。它允许嗅觉神经元表达精确定义的OR ​​值的环境中尽可能接近在vi的活动的监控沃越好。此外，嗅觉神经元的膜片钳记录也允许膜性能，转导途径药理学，配体/或相互作用的精确分析。所有这些话题几乎不能使用外记录进行分析。我们使用这种技术来监控嗅觉神经元表达气味受体SR1和MOR23 12,13的响应。该技术的可行性，通过MOR23其他群体表达嗅觉神经元14以及上表达神经元15,16等手术室证实。限定嗅觉神经元的人口的监视可以导致它们的性质在许多不同的情况下，例如显影14的分析，老化17，臭气物质诱发塑性18，和变化中的气味受体的序列中的气味编码15的作用。这个协议从而提供了一个有力的工具在膜级监测定义嗅觉神经元的功能特性。

<table><tbody><tr><td>vibration table with Faraday cage</td><td>TMC</td><td>63-500 SERIES</td><td>required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment</td></tr><tr><td>optics</td><td></td><td></td><td></td></tr><tr><td>microscope</td><td>Olympus</td><td>BX51WI</td><td>upright microcope equipped with epifluorescence; fixed or moving stage depending on the user&#039;s preference</td></tr><tr><td>objectives</td><td>Olympus</td><td>LUMPLFL40XW</td><td>at least 2 objectives required: a 4X or 10X for coarse approach to the cell; and a 40X immersion long distance example Olympus LUMPLFL40XW / IR /0,8 / WD:3.3 MM</td></tr><tr><td>magnifier</td><td>Olympus</td><td>U-TVCAC</td><td>ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode</td></tr><tr><td>camera</td><td>Olympus</td><td>DP72</td><td>a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary</td></tr><tr><td>filters</td><td>Olympus/Chroma</td><td></td><td>depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m</td></tr><tr><td>amplifier</td><td>HEKA</td><td>EPC10 USB</td><td>monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is controled by computer</td></tr><tr><td>software</td><td>HEKA</td><td>Patchmaster</td><td>controls the amplifier during the experiment</td></tr><tr><td>micromanipulator</td><td>Sutter</td><td>MP225</td><td>precision micromanipulator, allows precise movements down to 1/10th of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift</td></tr><tr><td>electrode puller</td><td>Sutter</td><td>P97</td><td>with a FT345-B wide trough filament;&amp;nbsp; to prepare recording pipets of about 2&amp;micro;m diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution</td></tr><tr><td>glass</td><td>Sutter</td><td>BF120-69-10</td><td>in our recording conditions, this glass is ideal for recording pipets</td></tr><tr><td>recording chamber</td><td>Warner Instruments</td><td>RC-26G</td><td>a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/anchor should be used.</td></tr><tr><td>stimulation</td><td></td><td></td><td></td></tr><tr><td>glass</td><td>WPI</td><td>TW100F-4</td><td>attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes</td></tr><tr><td>multibarrel puller</td><td>MDI</td><td>PMP-107-Z</td><td>by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels</td></tr><tr><td>precision pressure injector&amp;nbsp;</td><td>Toohey Company</td><td>P/N T25-1-900 Single Channel&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>this precision pressure injector&amp;nbsp; controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V&amp;nbsp; TTLs</td></tr><tr><td>micromanipulator</td><td>Narishige</td><td>YOU-1</td><td>a coarse manipulator is enough to bring the puffing electrode close to the recording site</td></tr><tr><td>tubings</td><td>N/A</td><td></td><td>tygon tubing to bring the pressure from the puffer to the puffing pipette</td></tr><tr><td>solutions/perfusion/chemicals</td><td></td><td></td><td></td></tr><tr><td>vacuum pump</td><td>gardner denver</td><td>300 series</td><td>a vibrating membrane pump is more quiet and efficient than other types of pumps</td></tr><tr><td>perfusion system</td><td>N/A</td><td>N/A</td><td>gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber</td></tr><tr><td>nystatin</td><td>Sigma-Aldrich</td><td>N3503</td><td>mandatory to perpare internal solution for perforated patch clamp</td></tr><tr><td>DIMETHYL SULFOXIDE</td><td>Sigma-Aldrich</td><td>D5879</td><td>used to disolve nystatin for internal solution for perforated patch&amp;nbsp;</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9625</td><td>extracellular solution</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P4504</td><td>intracellular/extracellular solution</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>extracellular solution</td></tr><tr><td>Sodium phosphate monobasic monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S9638</td><td>extracellular solution</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt; 7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>63140</td><td>extracellular solution</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>extracellular solution</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td>extracellular solution</td></tr><tr><td>EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid)</td><td>Sigma-Aldrich</td><td>E3889</td><td>internal solution</td></tr><tr><td>Potassium hydroxyde</td><td>Sigma-Aldrich</td><td>P1767</td><td>internal solution</td></tr><tr><td>Methyl Sulfoxide</td><td>Sigma-Aldrich</td><td>W387509</td><td>intracellular solution</td></tr><tr><td>Hepes-Na</td><td>Sigma-Aldrich</td><td>H7006</td><td>intracellular solution</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td>intracellular solution</td></tr></tbody></table>

perforated patch-clamp recording of mouse olfactory sensory neurons in intact neuroepithelium: functional analysis of neurons expressing an identified odorant receptor

当与特定的配体刺激的判断嗅感觉神经元的生理反应（OSN）关键是理解嗅觉驱动行为和其调制的基础。这些编码特性在很大程度上依赖于臭味分子和嗅觉受体（OR）之间的初始相互作用表达在嗅觉神经元。的表达，或者关键的身份，特异性和配体谱。找到或配体的可能性表示在内的上皮随机选择一个OSN很低。为了应对这一挑战，这个协议使用基因表达定义的OR的启动子控制下的荧光蛋白标记的小鼠。嗅觉神经元位于在紧张的和有组织的上皮衬里鼻腔，与邻近细胞影响他们的成熟和功能。这里，我们描述的方法来分离完整嗅上皮，并通过膜片钳记录记录嗅觉神经元e的特性xpressing定义的气味受体。该协议允许一个同时保持相邻组织的影响表征OSN膜特性。膜片钳的结果分析产生的配体/或相互作用，转导途径和药理学，嗅觉神经元"编码性质和它们在膜级调制的精确定量。

本协议的结果取决于解剖的质量。此清扫步骤必须短（少于10至15分钟）和精确的（ 即，避免了上皮的损害）。 图1说明了一个理想的准备看起来像在不同的放大级别。在明亮的下一个领域低倍率不同的细胞类型（如嗅觉神经元的旋钮，支持细胞）是有区别的（ 图1A）。在最高放大级别，通常为80X 160X，在明亮的领域，嗅觉神经元的树突状旋钮应该是从支持细胞（ <s...

Watch this Scientific Journal Video about Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant

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

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 ...

perforated-patch-clamp-recording-mouse-olfactory-sensory-neurons

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

26.6K 次观看.  Université de Bourgogne. 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.

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

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

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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 ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

纤毛对小鼠的主要嗅觉系统整装标签

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

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 ...

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

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 ...

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

Source: Jarriault, D., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52652">Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor.</a> J. Vis. Exp. (2015).
This video demonstrates electrophysiological recording of olfactory sensory neurons (OSNs) in mice using a perforated patch-clamp technique. The olfactory epithelium, containing OSNs with fluorophore-tagged odor receptors, is isolated from a mouse and secured in a recording chamber. A recording pipette is clamped to the dendritic knob of an OSN, and nystatin within the pipette perforates the membrane to form electrical connectivity with the cell. Finally, another pipette is used to deliver an odor stimulant, and the electrophysiological activity of the patched cell is recorded.

小鼠嗅觉感觉神经元的穿孔膜片钳记录

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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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

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此视频中的章节

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

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

多单位记录方法来描述蝗虫的神经活动（

纤毛对小鼠的主要嗅觉系统整装标签

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

实时 cAMP 检测气味受体激活的活细胞测量

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

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

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

小鼠嗅觉感觉神经元的穿孔膜片钳记录