闪光光解笼状化合物在嗅感觉神经元的纤毛
Summary
笼状化合物的光解允许快速和本地化的各种生理活性化合物的浓度增加生产。在这里，我们将展示如何获得光解笼中的cAMP结合或分离小鼠嗅感觉神经元的嗅觉传导的研究为笼钙膜片钳记录。
Abstract
笼状化合物的光解允许快速和本地化的各种生理活性化合物的浓度增加生产。笼中的化合物是由一种化学笼紫外线闪光灯可以打破生理不活跃分子。在这里，我们展示了如何获取结合分离小鼠嗅感觉神经元的嗅觉传导的研究笼化合物光解的膜片钳记录。嗅觉传导的过程（图1）在嗅感觉神经元，气味结合的受体导致cAMP的增加，打开环核苷酸门控（CNG）， 通道 2的纤毛。 CA通过CNG的渠道进入激活钙激活Cl通道。我们展示了如何脱离鼠标嗅上皮和如何CNG通道或钙激活的氯通道激活，光解笼中的 cAMP 4笼约5元</ SUP>。我们使用闪光灯6,7申请紫外线闪烁睫状肌地区uncage阵营或Ca，而膜片钳记录采取措施，目前在全细胞电压钳配置 8-11 。
Protocol
1. Instrumentation To measure the response of olfactory sensory neurons to photolysis of caged compounds we use a flash lamp in combination with a typical patch-clamp recording system including: a patch-clamp amplifier, a recording electrode and a reference electrode connected to the head-stage of a patch-clamp amplifier, a digitizer, a computer, software for data acquisition, micro-manipulators, an epifluorescence microscope, a perfusion system, an anti-vibration table and a Faraday cage (Figure 2). <li…
Discussion
闪存与膜片钳记录相结合的笼化合物的光解是一种有用的技术，获得快速和地方在内外细胞的生理活性分子的浓度跳跃。合成了几种类型的笼compounds1，这种技术可以适用于各种类型的细胞，包括培养细胞中表达，可激活或由一些可用的笼化合物11的光解调制的离子通道。
笼状化合物的光解需要高强度的近紫外线光脉冲uncage在很短的时间足够数量的分子。可用于各种光…
Disclosures
The authors have nothing to disclose.
Materials
| Equipment | Company | Catalogue number | Comments |
|---|---|---|---|
| Adapter module flash lamp to microscope | Rapp OptoElectronic | FlashCube 70 | |
| Air table | TMC | MICRO-g 63-534 | |
| Digitizer | Axon Instruments | Digidata 1322A | |
| Data Acquisition Software | Axon Instruments | pClamp 8 | |
| Data Analysis Software | WaveMetrics | Igor | |
| Mirror for adapter module | Rapp OptoElectronic | M70/100 | |
| Electrode holder | Axon Instruments | 1-HL-U | |
| Faraday’s cage | Custom made | ||
| Filter cube | Olympus | U-MWU | Excitation filter removed |
| Flash lamp | Rapp OptoElectronic | JML-C2 | |
| Forceps Dumont #55 | World Precision Instruments | 14099 | |
| Glass capillaries | World Precision Instruments | PG10165-4 | |
| Glass bottom dish | World Precision Instruments | FD35-100 | |
| Illuminator | Olympus | Highlight 3100 | |
| Inverted microscope | Olympus | IX70 | |
| Micromanipulators | Luigs & Neumann | SM I | |
| Micropipette Puller | Narishige | PP-830 | |
| Monitor | HesaVision | MTB-01 | |
| Neutral density filters | Omega Optical | varies | |
| Objective 100X | Zeiss | Fluar 440285 | Either Zeiss or Olympus |
| Objective 100X | Olympus | UPLFLN 100XOI2 | Either Zeiss or Olympus |
| Optical UV shortpass filter | Rapp OptoElectronic | SP400 | |
| Patch-clamp amplifier | Axon Instruments | Axopatch 200B | |
| Photo Diode Assembly | Rapp OptoElectronic | PDA | |
| Quartz light guide | Rapp OptoElectronic | varies | We use 600 μm diameter |
| Silver wire | World Precision Instruments | AGT1025 | |
| Silver ground pellet | Warner instruments | 64-1309 | |
| Xenon arc lamp | Rapp OptoElectronic | XBL-JML |
| Reagent | Company | Catalogue number |
|---|---|---|
| BCMCM-caged cAMP | BioLog | B016 |
| Bovine serum albumin (BSA) | Sigma | A8806 |
| CaCl2 standard solution 0.1 M | Fluka | 21059 |
| Caged Ca: DMNP-EDTA | Invitrogen | D6814 |
| Cysteine | Sigma | C9768 |
| Concanavalin A type V (ConA) | Sigma | C7275 |
| CsCl | Sigma | C4036 |
| DMSO | Sigma | D8418 |
| DNAse I | Sigma | D4527 |
| EDTA | Sigma | E9884 |
| EGTA | Sigma | E4378 |
| Glucose | Sigma | G5767 |
| HEPES | Sigma | H3375 |
| KCl | Sigma | P3911 |
| KOH | Sigma | P1767 |
| Leupeptin | Sigma | L0649 |
| MgCl2 | Fluka | 63020 |
| Papain | Sigma | P3125 |
| Poly-L-lysine | Sigma | P1274 |
| NaCl | Sigma | S9888 |
| NaOH | Sigma | S5881 |
| NaPyruvate | Sigma | P2256 |
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Tags
Flash PhotolysisCaged CompoundsOlfactory Sensory NeuronsPatch-clamp RecordingsPhotolysis Of Caged CompoundsOlfactory TransductionDissociated Mouse Olfactory Sensory NeuronsCiliaOdorant BindingReceptorsCAMPCyclic Nucleotide-gated (CNG) ChannelsCa EntryCa-activated Cl ChannelsMouse Olfactory EpitheliumFlash LampUltraviolet FlashesWhole-cell Voltage-clamp Configuration
Cite This Article
Boccaccio, A., Sagheddu, C., Menini, A. Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons. J. Vis. Exp. (55), e3195, doi:10.3791/3195 (2011).