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M.D. Jacobs, C. Soeller, M.B. Cannell, P.J. Donaldson; Quantitative Real-Time Imaging of Local Fiber Cell Gap Junction Coupling in the Rat Lens . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4265.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate how the observed differentiation-dependent changes in gap junction morphology that occur with increasing depth into the lens affect the local patterns of cell-cell coupling. Methods: Lenses were extracted from adult rats, cut in half through the equator and placed in a perfusion chamber containing intracellular medium and 1mM CMNB-caged fluorescein. The chamber was mounted on the stage of a confocal microscope modified for two-photon excitation. The two-photon laser beam bypassed the scanning system of the microscope and was focused inside a selected fiber cell to uncage the fluorescein by flash photolysis. Movement of the fluorescein away from this point source, both within and between cells, was imaged in real-time using confocal optics in x-y and line-scan modes. Subsequent imaging of the fiber cell membranes and gap junctions by bright field and fluorescence labeling allowed cell coupling data to be correlated with local cell structure and gap junction morphology. Data were written to hard disk and quantitative analysis of dye movement was performed using custom-written software. Results: In the lens periphery the spread of the uncaged fluorescein was highly directional, corresponding to rows of fiber cells. Deeper in the lens (>300 µm) the cell-cell coupling was approximately isotropic around the cell targeted for photorelease. Steady-state fluorescence levels were reached in target cells ~10 s after continuous photorelease was initiated. In both peripheral and deep regions of the lens, fluorescence was easily detected 3 cells away from the target cell at steady-state. However, at the lens periphery the fluorescence among equivalent target cell neighbors was non-uniform and varied by up to a factor of 7. The directional cell-cell coupling observed at the periphery corresponded to the local expression pattern of gap junctions on opposite broad sides of the hexagonal fiber cells. The isotropic coupling deeper in the lens corresponded to the dispersal of gap junctions in older fiber cells. Quantitative image analysis allowed characterization of the time courses of differential dye transfer between neighboring fiber cells in different regions of the lens. Conclusion: Local fiber cell coupling in the lens was non-uniform at the equatorial periphery but became isotropic with age-dependent changes in fiber cell structure and gap junction morphology. These observations are consistent with a lens micro-circulation model which predicts that gap junctions at the lens equator direct fluxes to the lens surface where appropriate ion channels and transporters are concentrated.
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