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
Summary
嗅觉受体神经元（ORNs）气味信号转换成一个受体的电流，从而触发动作电位，被输送到第二个神经元在嗅球中。这里，我们描述的吸入移液管的技术，同时记录从小鼠ORNs电流和动作电位的加臭剂诱导的受体。
Abstract
动物采样的臭味通过化学感受系统位于鼻腔周围环境。化学感受信号的复杂行为的影响，如食物的选择，捕食，同种和配偶识别和其他社会有关的线索。位于嗅觉受体神经元（ORNs）中嵌入鼻腔的嗅觉上皮细胞背侧部分。这些双极神经元的轴突发送到嗅球（见图1，Reisert钊1，最初发表于普通生理学杂志“）和扩展的单枝晶的纤毛辐射到的粘液覆盖的嗅觉上皮细胞的边界上皮细胞。的纤毛包含的信号转导机制，最终导致兴奋性通过睫状转导通道，环核苷酸门控通道（CNG）的Ca 2 +活化的Cl -的信道（ 图1）流入的电流。随之而来的depola三丙触发在电池主体2-4的动作电位的产生。
在这个视频中，我们描述了使用的“吸吸液管技术”，记录的气味引起的反应ORNs。该方法最初发展到记录从杆感光体5和jove.com修改，以记录从鼠标锥感光体6，可以发现在该方法的一个变种。后来被改编吸入吸液管技术也记录ORNs 7,8。简要地说，在解离的嗅觉上皮细胞和细胞分离后，整个细胞的ORN体被吸入到的记录吸管的前端。枝晶和纤毛仍然暴露于沐浴液，从而解决方案的变化，例如，使气味或药物受体阻滞剂的应用。在此配置中，还没有进入到细胞内环境获得（的没有全细胞电压钳）和细胞内的电压保持自由变化。这一切都OWS受体电流缓慢，起源于发射的细胞体9纤毛和快动作电位的同时记录。动力学这两个信号之间的差异在允许它们被分开使用不同的过滤器设置。这种技术可以用在任何野生型或基因敲除小鼠，或选择性地从记录ORNs也表达GFP标记的特定子集ORNs，例如表示一个给定的加臭剂的受体或离子通道。
Protocol
1。录像设置的记录室被安装在尼康TE2000U Eclipse的倒置显微镜相衬嵌合的光学上的气动工作台和电屏蔽使用一个法拉第笼。有机玻璃录音室包括两个部分，由阻挡层部分分离，并粘附到硅烷化的载玻片。该室的部分之一是用来解决细胞，而另一种是用于在录制过程中，以尽量减少刺激曝光过早曝光的结算，但尚未使用的单元格，以加臭剂。的记录设置包括的吸入移液管（见下文…
Discussion
<p class="jove_content">抽吸移液器技术是一个电的方法，该方法被用于记录气味诱导慢受体电流和快速的双相动作电位同时从ORN。由于细胞的质膜不违反，此方法离开确保没有改变的加臭剂的反应，由于细胞质离子浓度或稀释的细胞内因素变化的细胞内环境不受干扰。细胞可以记录从较长的持续时间（在小鼠在青蛙和1小时至4小时）。这种技术的另一个优点是，因为在单元主体内的记录移液器，即纤毛和树突状旋钮?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院DC009613，人类前沿科学计划和莫雷护理奖学金（JR）的支持。
Materials
| Name of the material | Type | Company | Catalogue / Model number |
Comments |
| Air table | equipment | Newport | ||
| Air Pump | equipment | Newport | ACGP | |
| Pipette Puller | equipment | Sutter | P-97 | |
| Borosilicate glass | equipment | WPI | 1B150-4 | |
| Nikon Eclipse Inverted microscope | equipment | Nikon | TE2000U | Equipped with Hg lamp, GFP filter and objectives 20X and 5X at least |
| Amplifier PC-501A | equipment | Warner | 64-0008 | Headstage 1 GΩ |
| Diamond knife | Equipment | Custom-made | ||
| Digitizer Mikro1401 A/D | equipment | Cambridge Electronic Design | ||
| Filter unit 3382 | equipment | Krohn Hite corporation | ||
| Signal | software | Cambridge Electronic Design | ||
| Molded Ag/AgCl Pellet | equipment | WPI | 64-1297 | |
| Pipette holder | equipment | Warner | 64-0997 | Custom modified to fit headstage |
| Recording chamber | Equipment | Custom-made | ||
| Micromanipulator MP85-1028 |
equipment | Sutter Instrument | Micromanipulator MP85-1028 |
|
| Mineral oil | Solution | Sigma | 330779-1L | |
| Oscilloscope TDS 1001 | equipment | Tektronix | ||
| Three-barreled square glass tube | Equipment | Warner | 64-0119 | 0.6 mm ID , 5 cm long |
| Valve | equipment | The Lee Company | ||
| Valvelink 8.2 | equipment | Automate Scientific | ||
| SF-77B Perfusion fast step | equipment | Warner |
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Tags
Odorant-induced ResponsesOlfactory Receptor NeuronsSuction Pipette TechniqueChemosensory SystemsNasal CavityOlfactory EpitheliumSignal Transduction MachineryExcitatory Current InfluxCiliary Transduction ChannelsCyclic Nucleotide-gated ChannelCa2+-activated Cl- ChannelDepolarizationAction Potential GenerationSuction Pipette Technique For Recording ORN Responses
Cite This Article
Ponissery Saidu, S., Dibattista, M., Matthews, H. R., Reisert, J. Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique. J. Vis. Exp. (62), e3862, doi:10.3791/3862 (2012).