George Wald

The molecular basis of visual excitation.

In 1967 Professor Wald, together with Professors H. K. Hartline and R. Granit, received the Nobel Prize for Medicine. The article that follows consists of most of the lecture delivered by Professor Wald last December when he received the prize in Stockholm.

Visual Pigments in Single Rods and Cones of the Human Retina

Difference spectra of the visual pigments have been measured in single rods and cones of a parafoveal region of the human retina. Rods display an absorption maximum (λmax) at about 505 m� associated with rhodopsin. Three kinds of cones were measured: a blue-sensitive cone with Amaxe about 450 mpf; two green-sensitive cones with Xmaa about 525 mu�; and a red-sensitive cone with λmax about 555 m� These are presumably samples of the three types of cone responsible for human color vision.

The Problem of Visual Excitation

This paper attempts to come to grips with the problem, how the action of light on a visual pigment results in a nervous excitation. The only action of light in vision is to isomerize retinene, the chromophore of the visual pigments, from the 11-cis to the all-trans configuration. This change triggers a progressive opening-up of the protein structure, exposing new reactive groups. Since the absorption of one photon by one molecule of visual pigment may stimulate a dark-adapted rod, some large amplification process is needed between the act of absorption and the response. This may be an enzymatic catalysis, or the consequence of puncturing a critical membrane. A microspectrophotometric study of retinas and single rods shows the outer segment to have a quasi-crystalline structure, in which the visual pigments are almost perfectly oriented, and even “free” molecules capable of diffusion maintain a degree of orientation. Examination of mud puppy retinas in the electron microscope has revealed several new aspects of structure: (1) A system of cytoplasmic filaments (“dendrites”) springing from the inner segments of the rods and cones and standing like palisades around the outer segments. These may facilitate exchanges of material between the inner and outer segments. (2) Systems of particles in the membranes of the dendrites and pigment epithelium processes, which may be involved in interchanges of material with the outer segments. (3) A system of particles in crystalline array in the rod lamellae, which may contain the visual pigment. If so, measurements of the visual pigment in situ show that each particle should contain about 50 molecules of pigment. Such typically solid-state processes as exciton migration and photoconduction probably have at most very limited scope in the outer segments of rods and cones. The seat of excitation is probably the plasma membrane which envelops rod outer segments and composes also the lamellae in cones.

The change in refractive power of the human eye in dim and bright light.

It has been reported that the human eye behaves as though relatively short-sighted in dim light. Observers tend to compensate for this change by setting optical instruments more negatively in dim than in bright light. New measurements of telescope settings by 21 observers reveal an average increase in power of the eye in dim light of 0.59 diopter (range +1.4 diopters to −3.4 diopters). The dilation of the pupil in dim light does not contribute significantly to this phenomenon. The chromatic aberration of the eye was measured in 14 observers with a specially designed spectral stigmatoscope. The refractive power of the eye increases about 3.2 diopters between 750 and 365 mμ. For this reason the Purkinje shift of maximum visual sensitivity from 560 mμ in bright light to 505 mμ in dim light produces a relative myopia in dim light of 0.35 to 0.40 diopter. Persons who display changes larger or smaller than this do so because of involuntary changes in the accommodation in bright and dim light. In dim light the eye enters a state of relatively fixed focus, little different from its condition when the accommodation is paralyzed with homatropine. In this fixed state the accommodation may be relaxed, or it may add as much as 3 diopters to the refractive power of the eye. Experienced observers focus optical instruments in dim light close to the optimal settings determined objectively. Departures of more than 0.5 diopter in either direction from the optimal focus depress the visual sensitivity and acuity. It is concluded that setting optical instruments about 0.4 diopter more negatively in dim than in bright light is justified on the basis of the chromatic aberration of the eye. Many observers gain a further advantage from individual adjustments of focus in dim light, appropriate to their accommodative behavior.

The origin of optical activity.

No other chemical characteristic is as distinctive of living organisms as is optical activity. Outside of organisms, all syntheses of disymmetric molecules produce equal numbers of optical antipodes (racemic mixtures) unless deliberate means are employed to bias the result by the use of asymmetric reagents or forces. Inside living organisms, however, all syntheses and degradations of such molecules involve one enantiomorph alone. Only the fact that chemistry is learned from the plane surfaces of paper and blackboard makes such selectivity seem strange. We tend to think of optical isomers as very much alike, but in fact they represent profound differences in shape; and, in the types of reaction upon which life depends, involving the ceaseless, intimate fitting together of molecular surfaces, shape is all-important. Organisms made the choice between optical antipodes long ago. To tamper with that choice now would be like trying to draw a left glove on a right hand. A start in either direction might be self perpetuating, but how was the original choice made? I n the past I think discussions of this problem have been misdirected, in the sense that an attempt was made to propose ways in which inorganic devices might have produced populations of organic molecules of predominantly one configuration or the other which, on their later incorporation into living organisms, conferred their optical activity on the latter. It is enormously more probable, of course, that all geochemical syntheses of organic molecules produced racemic mixtures. I think that organisms acquired optical activity, not as a gift from the inorganic world, but through processes of selection out of originally racemic mixtures. In what follows I shall try to outline the nature of such processes. How was this choice made initially?

Photo-labile pigments of the chicken retina.

THOUGH visual purples of the retinal rods have been known for sixty years1,2, nothing has been learned directly of analogous substances in the cones. In the chicken retina, which contains principally cones, attempts to identify photo-labile pigments heretofore have failed2. I have extracted from it two such pigments.

Visual Excitation and Blood Clotting

Both processes involve extremely large amplification between the stimulus and the response. In vision it had been suggested that thismight be achieved by a chain of successive proenzyme-enzyme activations. Such a chain has now been found to underlie the mechanism of blood clotting. Methods are suggested for pursuing this comparison further.

The role of vitamin A acid.

Publisher Summary The part that vitamin A plays in vision is not its principle activity. No animal dies of night blindness. It is clear that vitamin A must play some very general role in cellular metabolism or cell structure, a role perhaps particularly associated with epithelial cells, because these undergo such marked changes early in vitamin A deficiency. Yet, until now the visual role of vitamin A is the only one that has been clearly understood. In a recent attempt to pursue this further, in particular to clarify the mechanism of night blindness in vitamin A deficiency, this chapter follows simultaneously a complex of biochemical, physiological, and anatomical changes in single groups of rats, held on a vitamin A-deficient diet. It was observed that after the initial stores of vitamin A in the liver and blood had been exhausted, the level of rhodopsin in the retina began to fall, the visual threshold reciprocally rising, marking the beginning of night blindness.

The Sensitivity of the Human Eye to Infra-Red Radiation

The spectral sensitivity of human vision has been measured in the near infra-red, in two areas of the dark adapted eye: the central fovea (cones) to 1000 mμ, and a peripheral area, in which the responses are primarily caused by rods, to 1050 mμ. In both cases the estimates of spectral sensitivity are based upon determinations of the visual thresholds for radiation passing through a series of infra-red filters. By successive approximation, sensitivity functions were chosen which were consistent with the observed thresholds.The spectral sensitivity of the fovea determined in this way is consistent with previous measurements of Goodeve on the unfixated eye. At wave-lengths beyond 800 mμ the periphery becomes appreciably more sensitive than the fovea. This tendency increases at longer wave-lengths, so that at the longest wave-lengths studied, the radiation appeared colorless at the threshold and stimulated only rods.Lengthening the exposure time increases the sensitivity of the peripheral retina relative to the fovea. Our measurements involved exposures of 1 second and fields subtending a visual angle of 1 degree. With shorter exposures or smaller fields the fovea is favored, so that under such circumstances the fovea may become more sensitive than the periphery well into the infra-red.At 1050 mμ the sensitivity of the peripheral retina is only 3×10−13 times its maximum value at 505 mμ. A computation shows that by 1150 or 1200 mμ radiation should be more readily felt as heat by the skin than seen as light by the eye.

Vision in Annelid Worms

In a first electrophysiological study of worm vision, electroretinograms were measured in two alciopid worms: Torrea, taken at the surface, and deep-sea Vanadis. Both forms possess a primary retina in the focal plane of the lens, and accessory retinas lying beside the lens. Such accessory retinas occur also in deep sea fishes and cephalopods. In Torrea the primary retina peaks in sensitivity at 400 nanometers, the secondary retina at 560 nanometers. Both together could serve as a depth guage, since 560 nanometers attenuates much faster in seawater than 400 nanometers. The Vanadis eyes peaked in sensitivity at 460 to 480 nanometers, a property shared with deep-sea forms of other phyla; and appropriate, since these wavelengths penetrate seawater most deeply, and also are the wavelengths of maximum bioluminescence.