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Electroolfactogram (EOG) Recording in the Mouse Main Olfactory Epithelium
小鼠主嗅上皮中的嗅电图(EOG)记录   

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Abstract

Olfactory sensory neurons in the main olfactory epithelium (MOE) are responsible for detecting odorants and EOG recording is a reliable approach to analyze the peripheral olfactory function. However, recently we revealed that rodent MOE can also detect the air pressure caused by airflow. The sensation of airflow pressure and odorants may function in synergy to facilitate odorant perception during sniffing. We have reported that the pressure-sensitive response in the MOE can also be assayed by EOG recording. Here we describe procedures for pressure-sensitive as well as odorant-stimulated EOG measurement in the mouse MOE. The major difference between the pressure-sensitive EOG response and the odorant-stimulated response was whether to use pure air puff or use an odorized air puff.

Keywords: Olfaction(嗅觉), Epithelium(上皮), EOG(EOG), Mouse(老鼠), Odorants(气味)

Materials and Reagents

  1. 3-heptanone (Sigma-Aldrich)
  2. Forskolin (Sigma-Aldrich)
  3. IBMX (3-isobutyl-1-methylxanthine) (Sigma-Aldrich)
  4. SCH202676 (Sigma-Aldrich)
  5. Compressed pure nitrogen air (Praxair Inc)
  6. Thin-wall glass capillary (OD 1.0 mm ID 0.78 mm) (Harvard Apparatus)
  7. C57Bl/6 mice (Charles River or Jackson Lab)
    Note: Mice used were 2.5-5 months age-matched males or females. Mice were maintained on a 12 h light/dark cycle at 22 °C, and had access to food and water ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington and performed in accordance with their guidelines.
  8. Ringer’s solution (see Recipes)

Equipment

  1. Dissecting microscope
  2. Faraday cage
  3. Air table
  4. Specimen stage
  5. Nitrogen air tank
  6. Air puff valve (ASCO scientific, catalog number: 330224S303 )
  7. Glass cylinder
  8. Air delivery tube
  9. Oscilloscope
  10. CyberAmp 320 (an electric amplifier) (Axon Instruments)
  11. Recording electrode and reference electrode
  12. Digidata 1332A (Axon Instrument)
  13. MiniDigi 1A processor (Axon Instruments)
  14. S48 Stimulator (Glass Technologies)
  15. Hum Bug (a line frequency noise eliminator) (Quest scientific)
  16. Flow meter (Praxair Inc, Seattle, WA, catalot number: PRS FM43504 )
  17. Horizontal electrode puller, Model p-97 (Sutter Instruments)
  18. Computer

Software

  1. Clampex 10, Clampfit 10, Axoscope 10 (All from Axon Instruments Foster City)

Procedure


A.   Preparation of electrodes

  1. Glass capillary electrodes were pulled using a micropipette puller, then filled with Ringer’s solution and connected to the head stage of amplifier.
  2. Silver wire of reference grounding electrode, which was an agar- and Ringer’s solution-filled, was connected to the head stage.


B. MOE dissection

  1. Mice were sacrificed by decapitation. Skin overlying skull and lower jaw were removed with a small scissor.
  2. The rostral part of head was separated from the caudal part with a scissor and was bisected sagittally among midline with a sharp razor blade.
  3. Under a stereomicroscope, the septal cartilage and septum was carefully removed to expose the MOE, one of which was then put on the recording specimen stage. The other side was kept under moist condition for subsequent use.


C. Configuration of EOG recording

  1. A filter paper immersed in Ringer’s solution was used to hold the sample on a plastic specimen stage during recording.
  2. The filter paper was connected to Ringer’s bath solution and also served to connect the recording circuit as the reference electrode was immersed in Ringer’s bath solution.
  3. Humidified nitrogen puff (nitrogen passing over dsH2O in a horizontal glass cylinder) was used because olfactory tissue remained viable for a longer period of time with humidified air. The air-puff was driven by a pressure tank containing compressed ultra-pure nitrogen gas.
  4. Air-puffs were applied to the exposed MOE using an automated four-way slider valve that was controlled by a computer via a S48 stimulator. The duration of air puff was usually 100-200 ms. The tip of the puff application tube was directly pointed to the recording site on the MOE. The distance from tip of the air-puff application tube to surface of the recording turbinate was 1.5-2.0 centimeter.
  5. A flow meter was installed in line to regulate and measure the flow rate of air-puffs.
  6. An oscilloscope was required to calibrate the scale of EOG amplitude. EOG recordings could be performed using various application flow rates (0.03-2.4 L/min), but low flow rate (0.03-0.5 L/min) was physiological relevant in mouse EOG recording.
  7. If studying odorant-stimulated EOG response in the MOE, odorized air was generated by blowing nitrogen air through a horizontal glass cylinder that was half-filled with an odorant, i.e. 3-heptanone at variable concentrations.


D. EOG measurement

  1. The EOG field potential was detected with a Ringer’s solution-filled glass microelectrode in contact with the apical surface of the olfactory epithelia in an open circuit configuration.
  2. Electrophysiological EOG signals were amplified (normally 100x) with a CyberAmp 320 and digitized at 10 kHz or 1 kHz by means of a Digidata 1332A processor or simultaneously through a MiniDigi 1A processor; the signals were acquired online with software pClamp 10.3 and simultaneously with Axoscope 10.


E. Exclusion of artifacts from EOG recording of pressure-sensitive response

Occasionally, artifacts were seen in the EOG recordings due to damaged tissue preparations or other unpredicted reasons. Artifacts could be excluded from pressure-sensitive EOG recording on the basis of following criteria.

  1. Artifacts usually had symmetric rising and decay phases while pressure-sensitive signals had a fast rising phase (about 100 msec) with a relative slow decay phase. The decay phase of pressure-stimulated EOG signals were readily fitted with a mono-exponential function, giving a deactivation time constant of 1,400 msec. Artifacts usually lacked the mono-exponential deactivation phase.
  2. The half-width of maximum response of symmetric artifacts was about 200 msec, which is much shorter than the airflow-sensitive signal (about 600 msec).
  3. Artifacts did not demonstrate amplitude adaptation upon repetitive stimulation, while the air pressure-sensitive response showed adaptation upon rapid repetitive stimulations.
  4. The amplitude of pressure-sensitive responses was much larger than that of artifacts. Pressure-sensitive responses were sensitive to odorants, forskolin/IBMX (that elevate cellular cAMP level), or SCH202676 (a general inhibitor of GPCRs) while artifacts were insensitive to these chemical treatments. Artifacts were more easily to be excluded from odorant-sensitive EOG recording because odorant-sensitive EOG recording was about several folds larger than pressure-sensitive EOG measurement.


F. Data analysis

  1. Data were analyzed with Clampfit 10, and GraphPad Prism 5. The latency and rise time of EOG response could be analyzed with Clampfit 10. The desensitization and deactivation phases of the EOG field potential were fitted with a mono-exponential function f(t)= A0 x exp(-t/τ) + a, where τ is the time constant; A0 is the maximal response, a is residual response. Depending on stimulation protocols (i.e. inter-stimulation interval), olfaction adaption or recovery could be assayed using EOG amplitudes of repetitive odorant/air-pressure stimulation.
  2. The kinetic and amplitude of EOG recording can provide some useful information about how olfactory signals are processed in olfactory sensory neurons.


G.Comparison of pressure-sensitive EOG response with odorant-stimulated EOG response

  1. EOG measurement can be used to study both odorant- and air pressure-stimulated responses in the MOE.
  2. Procedurally the major difference between pressure-sensitive EOG response and odorant-stimulated response was whether to use pure air puff or to use odorized air puff.
  3. Two measurements also have several functional distinctions:
    1. Most of odorant-stimulated EOG measurement more or less contained some portion of pressure-sensitive response because air-phase odorants need an air puff (which exert an air pressure) to be blown onto the surface of MOE.
    2. Odorant-stimulated EOG response is generally higher than pressure-sensitive response although it may depend on dosage of stimulation (i.e. odorant concentration vs. flow rate of air puff).
    3. Pressure-sensitive EOG response was positively correlated with odorant-stimulated EOG response. Most of EOG field potential amplitude varies from 0.5-50 mV depending the odorant concentration, application flow rate and tissue quality.
    4. At high odorant dosage, decay phase of odorant-stimulated EOG response is much slower than that of the pressure-sensitive response.
    5. Pressure-sensitive response and odor-evoked response in the MOE share a common signal pathway, both of which may function synergistically to promote olfaction. 

Recipes

  1. Ringer’s solution
    125 mM NaCl
    2.5 mM KCl
    1 mM MgCl2
    2.5 mM CaCl2
    1.25 mM NaH2PO4
    20 mM HEPES
    15 mM D-Glucose
    pH 7.3
    Osmolarity 305
    Filter sterilized

Acknowledgments

The EOG recording method described in this protocol was published in Chen et al. (2013). This research was supported by a National Institutes of Health Grant DC0415 (To Dr Daniel R. Storm).

References

  1. Chen, X., Xia, Z. and Storm, D. R. (2012). Stimulation of electro-olfactogram responses in the main olfactory epithelia by airflow depends on the type 3 adenylyl cyclase. J Neurosci 32(45): 15769-15778.

简介

所有陆地植物的主要空间表面都涂有脂质角质层,限制非气孔水分流失,保护植物免受病原体,食草动物和紫外线辐射角质层由两部分组成:角质,羟基和环氧长链脂肪酸衍生物的聚合物和甘油,以及作为非常长链脂肪酸衍生物的角质蜡。虽然角质层的化学分析可能是一个漫长而技术上具有挑战性的任务,但是角质层蜡的分析相对简单,可以常规用于表征不同植物物种,给定物种适应环境条件或突变体表型。在这里,我们提出了一种针对拟南芥模拟生物表面上的角质层蜡分析的方案。因为在植物的最外表面 上发现角质蜡,蜡提取过程非常简单,样品处理可在不到一天的时间内完成。化学分析包括气相色谱与火焰离子化检测(GC / FID)联用对单体进行定量,以及通过质谱法或单个蜡组分与已知标准物的保留时间的比较来鉴定蜡单体。

关键字:嗅觉, 上皮, EOG, 老鼠, 气味

材料和试剂

  1. (Sigma-Aldrich)
  2. 福司柯林(Sigma-Aldrich)
  3. IBMX(3-异丁基-1-甲基黄嘌呤)(Sigma-Aldrich)
  4. SCH202676(Sigma-Aldrich)
  5. 压缩纯氮气(Praxair公司)
  6. 薄壁玻璃毛细管(OD 1.0mm ID 0.78mm)(Harvard Apparatus)
  7. C57Bl/6小鼠(Charles River或Jackson Lab)
    注意: 使用的小鼠是2.5-5个月龄匹配的男性或女性。 将小鼠在22℃下在12小时光/暗循环下维持,并且随意获得食物和水。 所有动物程序由华盛顿大学的机构动物护理和使用委员会批准并根据他们的指导进行。
  8. 林格的解决方案(参见配方)

设备

  1. 解剖显微镜
  2. 法拉第笼
  3. 气垫
  4. 样品阶段
  5. 氮气罐
  6. 空气阀(ASCO scientific,目录号:330224S303)
  7. 玻璃圆筒
  8. 空气输送管
  9. 示波器
  10. CyberAmp 320(电放大器)(Axon Instruments)
  11. 记录电极和参考电极
  12. Digidata 1332A(Axon Instrument)
  13. MiniDigi 1A处理器(Axon Instruments)
  14. S48刺激器(玻璃技术)
  15. Hum Bug(线频率消声器)(Quest science)
  16. 流量计(Praxair Inc,Seattle,WA,catalot number:PRS FM43504)
  17. 水平电极拉出器,型号p-97(Sutter Instruments)
  18. 电脑

软件

  1. Clampex 10,Clampfit 10,Axoscope 10(全部来自Axon Instruments Foster City)

程序


A.   电极的制备

  1. 使用微量吸移管拉出玻璃毛细管电极,然后填充林格氏溶液并连接到放大器的头级。
  2. 参考接地电极的银线,其是填充琼脂和林格溶液的,连接到头部阶段。


B. MOE解剖

  1. 通过断头处死小鼠。 用小剪刀去除皮肤覆盖的颅骨和下颌。
  2. 用剪刀将头部的头部部分与尾部分开,并用锋利的剃刀刀片在中线之间横切。
  3. 在立体显微镜下,小心地去除中隔软骨和隔膜以暴露MOE,其中之一然后放在记录样品台上。 另一侧保持在潮湿条件下,以备后续使用


C. EOG记录的配置

  1. 浸渍在林格氏溶液中的滤纸在记录期间用于将样品保持在塑料样品台上。
  2. 将滤纸连接到林格氏浴溶液,并且当参考电极浸入林格氏浴溶液中时,还用于连接记录电路。
  3. 使用加湿的氮气抽吸(在水平玻璃圆筒中通过dsH 2 O的氮气),因为嗅觉组织在加湿空气下保持存活更长时间。该气泡由含有压缩的超纯氮气的压力罐驱动
  4. 使用由计算机通过S48刺激器控制的自动四通滑阀将空气喷射施加到暴露的MOE。气泡的持续时间通常是100-200毫秒。吹气管的尖端直接指向MOE上的记录位置。从吹气管的尖端到记录鼻甲的表面的距离为1.5-2.0厘米。
  5. 流量计安装在线,以调节和测量空气泡的流量。
  6. 需要一个示波器来校准EOG振幅的标度。可以使用各种应用流速(0.03-2.4L/min)进行EOG记录,但是低流速(0.03-0.5L/min)在小鼠EOG记录中是生理上相关的。
  7. 如果研究MOE中的气味刺激的EOG反应,通过吹入氮气通过水平玻璃圆筒产生加味空气,该水平玻璃圆筒半充满加味剂,即不同浓度的3-庚酮。


D.EOG测量

  1. 使用与开路配置中的嗅觉上皮的顶面接触的林格氏溶液填充的玻璃微电极检测EOG场电位。
  2. 电生理EOG信号用CyberAmp 320放大(通常为100x),并通过Digidata 1332A处理器或者同时通过MiniDigi 1A处理器在10kHz或1kHz下数字化;用软件pClamp 10.3在线获取信号,并与Axoscope 10同时进行


E.从压力敏感反应的EOG记录中排除伪像

偶尔,由于受损的组织准备或其他不可预测的原因,在EOG记录中看到伪影。根据以下标准,可以从压敏EOG记录中排除伪像。

  1. 伪像通常具有对称的上升和衰减相位,而压敏信号具有相对慢的衰减相位的快速上升阶段(大约100毫秒)。压力刺激的EOG信号的衰减阶段容易地用单指数函数拟合,给出1400msec的失活时间常数。人工产物通常缺乏单指数失活期
  2. 对称伪影的最大响应的半宽度为大约200毫秒,这比气流敏感信号(大约600毫秒)短得多。
  3. 人工制品在重复性刺激时没有表现出振幅适应,而空气压力敏感性反应在快速反复刺激时表现出适应性。
  4. 压力敏感反应的幅度远大于人工制品的幅度。压力敏感反应对气味剂,毛喉素/IBMX(提高细胞cAMP水平)或SCH202676(GPCRs的一般抑制剂)敏感,而人工制品对这些化学处理不敏感。人工制品更容易从气味敏感的EOG记录中排除,因为气味敏感的EOG记录比压敏式EOG测量大几倍。


F.数据分析

  1. 用Clampfit 10和GraphPad Prism 5分析数据。可以用Clampfit 10分析EOG反应的潜伏期和上升时间.EOG场电位的脱敏和失活阶段与单指数函数f(t)= (-t /τ)+ a,其中τ是时间常数; 0 是最大响应,a是残余响应。根据刺激方案(即刺激间隔),可以使用重复加臭剂/空气压力刺激的EOG振幅来测定嗅觉适应或恢复。
  2. EOG记录的动力学和振幅可以提供关于嗅觉感觉神经元如何处理嗅觉信号的一些有用信息。


压敏EOG响应与气味刺激的EOG反应的比较

  1. EOG测量可用于研究MOE中加臭剂和空气压力刺激的反应。
  2. 在程序上,压敏EOG反应和气味刺激反应之间的主要区别是是使用纯空气喷射还是使用加味空气喷烟。
  3. 两个测量也有几个功能区别:
    1. 大多数气味刺激的EOG测量或多或少包含一些部分的压敏响应,因为空气气味气味剂需要吹到MOE表面上的空气喷雾(其施加空气压力)。
    2. 气味刺激的EOG反应通常高于压敏反应,尽管它可能取决于刺激的剂量(即气味浓度与气泡的流速)。
    3. 压力敏感的EOG反应与气味刺激的EOG反应呈正相关。 大多数EOG场电位振幅根据加臭剂浓度,应用流速和组织质量而在0.5-50mV之间变化。
    4. 在高气味剂用量下,气味刺激的EOG反应的衰变阶段比压敏反应慢得多。
    5. MOE中的压力敏感反应和气味诱发反应共享一个共同的信号通路,两者都可以协同作用以促进嗅觉。

食谱

  1. 林格的解决方案
    125 mM NaCl 2.5mM KCl
    1mM MgCl 2
    2.5mM CaCl 2·h/v 1.25mM NaH 2 PO 4>/
    20 mM HEPES
    15mM D-葡萄糖 pH 7.3
    渗透压305
    过滤灭菌

致谢

该协议中描述的EOG记录方法在Chen等人(2013)中公开。 这项研究得到国家卫生研究院拨款DC0415(Dr Daniel R. Storm)的支持。

参考文献

  1. Chen,X.,Xia,Z.and Storm,D.R。(2012)。 通过气流刺激主嗅上皮中的电嗅觉反应取决于3型腺苷酸环化酶。 J Neurosci 32(45):15769-15778。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, X., Xia, Z. and Storm, D. R. (2013). Electroolfactogram (EOG) Recording in the Mouse Main Olfactory Epithelium. Bio-protocol 3(11): e789. DOI: 10.21769/BioProtoc.789.
  2. Chen, X., Xia, Z. and Storm, D. R. (2012). Stimulation of electro-olfactogram responses in the main olfactory epithelia by airflow depends on the type 3 adenylyl cyclase. J Neurosci 32(45): 15769-15778.
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