The eye is a light-sensitive organ through which visual information about the external world is transmitted to the brain. The pupil, a black, circular opening centered in the front of the eyeball, controls the amount of light entering the eye, widening when surroundings are dark and constricting when they are bright. The lens of the eye focuses light from the external world onto the retina, a thin, multilayered region composed of photoreceptors (rods and cones) and interneurons that lines the back of the eyeball. The fovea, located in the retina, is the focus point of the lens and contains the highest density of photoreceptors (only cones), making it responsible for high-acuity vision. Retinal axons leave the eye through the optic disc, an area also known as the “blind spot” for its lack of photoreceptors, and merge into the optic nerve, which transmits visual information to the brain. The photoreceptor cells in the eye are categorized into rods and cones. The rods expresses rhodopsin as their photoreceptor molecule while cones express red, green, or blue opsins. Traditionally, each cone cell is considered to express only a single type of opsin. This theory is largely supported by morphological evidence in many primates and adult humans. Having more than one opsin molecule per cone suggests that each cone will respond to multiple wavelength of light, which seems illogical from the wiring point of view. However, recent stud-ies have found many mammalian examples that reject this one-opsin-one-cone theory. Such co-expression of multiple opsin photoreceptors was first discovered in mice. The mouse retina expresses the M-opsin and S-opsin in two gradients with the M-opsin forming a dorsal-ventral increasing gradient and S-opsin in a ventral-dorsal increasing gradient. There is a substancial region of overlap in the gradient where M/S-opsins are both expressed in the mouse retina. Such co-expression of more than one opsin in a single cone is also displayed in human retinal development.
Isolated Retinal Rod Cell
Lecture 8 Model Systems
Isolated retinal rod cell
Individual rod cells consist of a synaptic terminal, an inner segment which contains the nucleus and organelles, and a large outer segment made up of numerous membranous discs. The discs contain various photopigments, and is the site where phototransduction occurs. These purified outer rod segments provided clues to how rhodopsin results in channel closure.
Lecture 8 Techniques
Exciting specific areas of the retina
Shearing rod cells
The outer segments of rod cells are easily separated from the rest of the cell, allowing the biochemical machinery of transduction (which occurs in the outer segments) to be studied.
Sucrose density gradient
A sucrose density gradient is used to separate cellular components by density. The gradient is prepared by layering different concentrations of sucrose (usually from 15-35%) on top of each other in a centrifugation tube. The cell parts are placed in the gradient and centrifuged until their density matches that of the surrounding sucrose. Sucrose density gradients were used to purify outer segments of rod cells to study the biochemical composition of phototransduction machinery.