Summary

Подготовка Жизнь Изолированные позвоночных фоторецепторных клеток для флуоресценции

Published: June 22, 2011
doi:

Summary

Описан способ подготовки одной живой клетки фоторецепторов из разных видов позвоночных для флуоресцентной визуализации. Метод может быть использован для изображений флуоресценции эндогенных флуорофоров, таких как NADH или витамин А, или, что из экзогенно добавил флуоресцентные красители чувствительны к Са<sup> 2 +</sup> Или других факторов.

Abstract

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.

Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all- trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).

Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single photoreceptor cells17-19.

Protocol

1. Подготовка Sylgard покрытых блюд, экспериментальной камеры, и бритвенных лезвий 35 мм Сокол чашки Петри покрытые эластомера Sylgard необходимы для надлежащего колки изолированной сетчатки получить отдельные клетки фоторецепторов. Эластомер получают в соответствии с инструкциями по?…

Discussion

Если здоровый изолированных клеток не получено, проблема заключается либо с изоляцией или здоровья сетчатки или с ее измельчать. Как правило, после удаления передней части глаза и стекловидное тело, сетчатку легко отрывается от земли пигментного эпителия. Если этого не произойдет, поп…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

Поддержка НОУ грант EY014850.

Materials

Name Company Catalogue number Comments
Dark room (100-150 ft2)      
Red lights19     Online stores
Infrared light sources and infrared image viewers FJW Optical Systems, Inc.    
Dissecting microscope19     Outfitted with infrared viewers
Epifluorescence microscope enclosed in a light-tight cage19      
Dissecting tools (scissors, forceps, blade holder) Roboz or Fine Science Tools    
Sylgard elastomer Essex (Charlotte, NC) Sylgard 184 elastomer kit  
Poly-L-ornithine (0.01%) Sigma-Aldrich P4957  
Poly-L-lysine (0.1%) Sigma-Aldrich P8920 Dilute to 0.01%
Experimental chambers Warner Instruments (Hamden, CT) D3512P  
Petri dishes, plastic pipettes Fisher Scientific    

Referenzen

  1. Burns, M. E., Arshavsky, V. Y. Beyond Counting Photons: Trials and Trends in Vertebrate Visual Transduction. Neuron. 48, 387-401 (2005).
  2. Ebrey, T., Koutalos, Y. Vertebrate Photoreceptors. Prog Retin Eye Res. 20, 49-94 (2001).
  3. Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. Adaptation in Vertebrate Photoreceptors. Physiol Rev. 81, 117-151 (2001).
  4. Palczewski, K. G Protein-Coupled Receptor Rhodopsin. Annu Rev Biochem. 75, 743-767 (2006).
  5. Saari, J. C. Biochemistry of Visual Pigment Regeneration: The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 41, 337-348 (2000).
  6. Lamb, T. D., Pugh, E. N. Dark Adaptation and the Retinoid Cycle of Vision. Prog Retin Eye Res. 23, 307-380 (2004).
  7. Imanishi, Y., Lodowski, K. H., Koutalos, Y. Two-Photon Microscopy: Shedding Light on the Chemistry of Vision. Biochemie. 46, 9674-9684 (2007).
  8. Sampath, A. P., Matthews, H. R., Cornwall, M. C., Fain, G. L. Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca2+ in Salamander Rods. J Gen Physiol. 111, 53-64 (1998).
  9. Sampath, A. P., Matthews, H. R., Cornwall, M. C., Bandarchi, J., Fain, G. L. Light-Dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors. J Gen Physiol. 113, 267-277 (1999).
  10. Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., Fain, G. L. Measurement of Cytoplasmic Calcium Concentration in the Rods of Wild-Type and Transducin Knock-out Mice. J Physiol. 542, 843-854 (2002).
  11. Leung, Y. T., Fain, G. L., Matthews, H. R. Simultaneous Measurement of Current and Calcium in the Ultraviolet-Sensitive Cones of Zebrafish. J Physiol. 579, 15-27 (2007).
  12. Matthews, H. R., Fain, G. L. Laser Spot Confocal Technique to Measure Cytoplasmic Calcium Concentration in Photoreceptors. Methods Enzymol. 316, 146-163 (2000).
  13. Szikra, T., Cusato, K., Thoreson, W. B., Barabas, P., Bartoletti, T. M., Krizaj, D. Depletion of Calcium Stores Regulates Calcium Influx and Signal Transmission in Rod Photoreceptors. J Physiol. 586, 4859-4875 (2008).
  14. Krizaj, D., Copenhagen, D. R. Compartmentalization of Calcium Extrusion Mechanisms in the Outer and Inner Segments of Photoreceptors. Neuron. 21, 249-256 (1998).
  15. Chen, C., Nakatani, K., Koutalos, Y. Free Magnesium Concentration in Salamander Photoreceptor Outer Segments. J Physiol. 553, 125-135 (2003).
  16. Chen, C., Jiang, Y., Koutalos, Y. Dynamic Behavior of Rod Photoreceptor Disks. Biophys J. 83, 1403-1412 (2002).
  17. Tsina, E., Chen, C., Koutalos, Y., Ala-Laurila, P., Tsacopoulos, M., Wiggert, B., Crouch, R. K., Cornwall, M. C. Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors. J Gen Physiol. 124, 429-443 (2004).
  18. Ala-Laurila, P., Kolesnikov, A. V., Crouch, R. K., Tsina, E., Shukolyukov, S. A., Govardovskii, V. I., Koutalos, Y., Wiggert, B., Estevez, M. E., Cornwall, M. C. Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology. J Gen Physiol. 128, 153-169 (2006).
  19. Koutalos, Y., Cornwall, M. C. Microfluorometric Measurement of the Formation of All-Trans-Retinol in the Outer Segments of Single Isolated Vertebrate Photoreceptors. Methods Mol Biol. 652, 129-147 (2010).
  20. Wu, Q., Blakeley, L. R., Cornwall, M. C., Crouch, R. K., Wiggert, B. N., Koutalos, Y. Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments. Biochemie. 46, 8669-8679 (2007).
  21. Kolesnikov, A. V., Ala-Laurila, P., Shukolyukov, S. A., Crouch, R. K., Wiggert, B., Estevez, M. E., Govardovskii, V. I., Cornwall, M. C. Visual Cycle and Its Metabolic Support in Gecko Photoreceptors. Vision Res. 47, 363-374 (2007).
  22. Chen, C., Blakeley, L. R., Koutalos, Y. Formation of All-Trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors. Invest Ophthalmol Vis Sci. 50, 3589-3595 (2009).

Play Video

Diesen Artikel zitieren
Boyer, N. P., Chen, C., Koutalos, Y. Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging. J. Vis. Exp. (52), e2789, doi:10.3791/2789 (2011).

View Video