Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

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.