Author Produced

闪光光解笼状化合物在嗅感觉神经元的纤毛

Neuroscience

Your institution must subscribe to JoVE's Neuroscience section to access this content.

Fill out the form below to receive a free trial or learn more about access:

 

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。

Cite this Article

Copy Citation | Download Citations

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).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

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 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 。

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。可用于各种光源:一个不断汞或氙弧灯控制快门,再加啶端口显微镜,氙气闪光灯,紫外激光,和最近开发的高功率紫外线灯发光二极管(LED )。每个类型的光源优势和disadvan产品关键词根据具体应用和设备的成本。一个闪光灯相比,连续运行的灯有一个较低的光照强度,因此快门控制的光脉冲的持续时间,需要增加多达数百毫秒uncaged分子获得足够数量。紫外激光是非常昂贵的。高功率紫外发光二极管闪光光解14最近市售其他方法可以提供一个很好的选择。然而,闪光灯的优势是,他们有一个更广泛的发射光谱比紫外发光二极管,允许使用uncaging在我们的应用程序使用一个氙气闪光灯的主要优点是与不同的光谱特征笼化合物的几种类型:一个好的时间分辨率,确实光脉冲的持续时间约1毫秒;一个广泛的紫外光谱,是适合光化学性质不同的分子光解的可能性选择毛钱nsion的光点照亮睫状肌的地区,可以方便地选择不同的光照强度 6 。此外,氙气闪光灯的灯有一个合理的成本,这是很容易实现电设置,不需要特殊的维护。

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

没有利益冲突的声明。

Materials

Name Company Catalog 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 Corporation U-MWU Excitation filter removed
Flash lamp Rapp OptoElectronic JML-C2
Forceps Dumont #55 World Precision Instruments, Inc. 14099
Glass capillaries World Precision Instruments, Inc. PG10165-4
Glass bottom dish World Precision Instruments, Inc. FD35-100
Illuminator Olympus Corporation Highlight 3100
Inverted microscope Olympus Corporation IX70
Micromanipulators Luigs & Neumann SM I
Micropipette Puller Narishige International PP-830
Monitor HesaVision MTB-01
Neutral density filters Omega Optical varies
Objective 100X Carl Zeiss, Inc. Fluar 440285 Either Zeiss or Olympus
Objective 100X Olympus Corporation 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, Inc. 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-Aldrich A8806
CaCl2 standard solution 0.1 M Fluka 21059
Caged Ca: DMNP-EDTA Invitrogen D6814
Cysteine Sigma-Aldrich C9768
Concanavalin A type V (ConA) Sigma-Aldrich C7275
CsCl Sigma-Aldrich C4036
DMSO Sigma-Aldrich D8418
DNAse I Sigma-Aldrich D4527
EDTA Sigma-Aldrich E9884
EGTA Sigma-Aldrich E4378
Glucose Sigma-Aldrich G5767
HEPES Sigma-Aldrich H3375
KCl Sigma-Aldrich P3911
KOH Sigma-Aldrich P1767
Leupeptin Sigma-Aldrich L0649
MgCl2 Fluka 63020
Papain Sigma-Aldrich P3125
Poly-L-lysine Sigma-Aldrich P1274
NaCl Sigma-Aldrich S9888
NaOH Sigma-Aldrich S5881
NaPyruvate Sigma-Aldrich P2256

DOWNLOAD MATERIALS LIST

References

  1. Ellis-Davies, G. C. R. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods. 4, 619-628 (2007).
  2. Pifferi, S., Boccaccio, A., Menini, A. Cyclic nucleotide-gated ion channels in sensory transduction. FEBS Lett. 580, 2853-2859 (2006).
  3. Bozza, T. C., Kauer, J. S. Odorant response properties of convergent olfactory receptor neurons. J. Neurosci. 18, 4560-4569 (1998).
  4. Hagen, V., Bendig, J., Frings, S., Eckardt, T., Helm, S., Reuter, D. Highly Efficient and Ultrafast Phototriggers for cAMP and cGMP by Using Long-Wavelength UV/Vis-Activation. Angew. Chem. Int. Ed. Engl. 40, 1045-1048 (2001).
  5. Kaplan, J. H., Ellis-Davies, G. C. Photolabile chelators for the rapid photorelease of divalent cations. Proc. Natl. Acad. Sci. 85, 6571-6575 (1988).
  6. Rapp, G. Flash lamp-based irradiation of caged compounds. Methods. Enzymol. 291, 202-222 (1998).
  7. Gurney, A. M. Flash photolysis of caged compounds. Microelectrodes: Theory and Applications. Montenegro, I., Queiros, M. A., Daschbach, J. L. Proc. NATO Adv. Study Inst. Portugal. (1991).
  8. Lagostena, L., Menini, A. Whole-cell recordings and photolysis of caged compounds in olfactory sensory neurons isolated from the mouse. Chem. Senses. 28, 705-716 (2003).
  9. Boccaccio, A., Lagostena, L., Hagen, V., Menini, A. Fast adaptation in mouse olfactory sensory neurons does not require the activity of phosphodiesterase. J. Gen. Physiol. 128, 171-184 (2006).
  10. Boccaccio, A., Menini, A. Temporal development of cyclic nucleotide-gated and Ca2+ -activated Cl- currents in isolated mouse olfactory sensory neurons. J. Neurophysiol. 98, 153-160 (2007).
  11. Sagheddu, C., Boccaccio, A., Dibattista, M., Montani, G., Tirindelli, R., Menini, A. Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16B-transfected HEK 293T cells. J. Physiol. 588, 4189-4204 (2010).
  12. Balana, B., Taylor, N., Slesinger, P. A. Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells. J. Vis. Exp. (44), e2189-e2189 (2010).
  13. Cygnar, K. D., Stephan, A. B., Zhao, H. Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording. J. Vis. Exp. (37), e1850-e1850 (2010).
  14. Bernardinelli, Y., Haeberli, C., Chatton, J. Y. Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution. Cell. Calcium. 37, 565-572 (2005).

Comments

0 Comments


    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Usage Statistics