Dark-adapted Retina Research Articles

Immediate Reaction of the Red-Eared Turtle (Pseudemys scripta elegans) to X-Irradiation

The possibility of X-ray perception was discussed by Roentgen in 1895. In the following years a wealth of information was collected, proving the existence of the X-ray phosphene and the radium phosphene, leaving open, however, the question into the mechanisms involved. Lipetz (1) in 1955 published a critical and thorough review of the problem and tested in 1955 to what extent the X-ray phospene could be related to a direct action of the X-rays on the photochemistry of the retina and of the cones by using the electrical discharges from a single ganglion cell of a frog retina in situ as an objective means of the retinal response. His findings were consistent with, but did not prove, the hypothesis that the X-ray phosphene in the dark-adapted retina is produced by a direct action of the rays on the visual purple of the retina (2). Since then numerous reports on the ability of organisms to sense X-rays have been published: Baylor and Smith (3) studied perception of X-rays by daphnids; Olivard and Hungate (4) reported on experiments with Drosophila; Dawson and Smith (5) investigated the effects of low-level irradiation on the threshold shift in the visual receptor of the horseshoe crab (Limulus polyphenus) ; Hug (6) observed immediate reaction to very small doses of X-rays and a-rays in snails; Andrews and Cameron (7) found indications for avoidance reactions of mice to X-ray beams; Garcia et al. (8) influenced taste perception in rats by X-irradiation; and Logie et al. (9) observed biased performance in rats, suggesting perception of moderate-intensity X-radiation. Following the trend of present research (10) to collect as much information as possible for different species and different sensory systems, some observations on the reaction of the red-eared turtle to X-irradiation appear of interest.

Analysis of Retinal Function by a Two-Filter Method

Any two colored lights are effective in some characteristic energy ratio for stimulating cones, and some other characteristic ratio for stimulating rods, owing to the different spectral sensitivities of these receptors. Such ratios can be used to assess precisely the contributions of rods and cones to visual responses. In these experiments a violet and a yellow test light are used in this way to analyze cone and rod participation in dark adaption, and in the thresholds of the dark-adapted retina at various distances from the fixation point (“retinal profiles”). Also, since the human macular pigment absorbs the violet light and not the yellow, these lights can map the extent and depth of the yellow patch. A functional cross section of the retina results, measuring separately rod and cone activities and macular pigmentation, that agrees reasonably well with histological descriptions. A considerable section of dark adaptation is shown to involve mixed cone and rod responses. Thresholds of the dark adapted eye near the foveal border are similarly mixed, Color sensations at the threshold desaturate with the first participation of rods, and are wholly lost while cone activity is still prominent. The macular pigmentation becomes appreciable at some distance from the fixation point, and in this subject increases regularly in depth to a maximum in the central fovea.

Studies on dark adaptation. IV. Pre-exposure tolerance of the dark-adapted peripheral retina.

The effects of low-brightness short-duration pre-exposures on peripheral dark adaptation were investigated. Three peripheral locations: 2 deg, 6 deg, and 18 deg, were tested with a square 1-deg test patch of 0.033 sec duration. Pre-exposure was to a centrally fixated circular field which subtended 55-deg diam. Monocular curves were obtained after pre-exposures of 0.01, 0.1, 1, and 10 ft-L each presented for 1 and 10 sec. All pre-exposure conditions resulted in some loss of sensitivity at the 2-deg location; pre-exposure combinations of 0.01 and 0.1 ft-L-sec resulted in little if any loss of sensitivity at the 6-deg and 18-deg locations.

The thermal stability of rhodopsin and opsin.

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E(a)) and entropies of activation (DeltaSdouble dagger) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species -frog, squid, cattle-presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.

The visual pigment of the conger eel.

New methods of studying the spectral absorption of intact retinae are described. Using these methods the retina of Conger conger (L.) has been studied and the retinal spectral absorption curves are compared with those obtained on retinal extracts made with digitonin solution. The retina of the conger like that of deep-sea fish is golden in colour, its absorption curve being similar in shape to that of frog rhodopsin but with its maximum displaced about 16 m µ towards the blue end of the spectrum. The absorption curve of unbleached retinae is displaced about 4 m µ towards the red end of the spectrum from the absorption curve of unbleached retinal extract, but, when an estimated correction for possible yellow impurities in the extract is made, this displacement is only one of 2 m µ . The change in optical density of the dark-adapted retina on bleaching with strong white light is 0.6 at λ = 484 m µ : this probably represents a retinal density for unbleached pigment of about 0.8. The visual pigment in the intact retina is approximately twice as effective as simple calculations based on extracts would predict. The absorption of light by the retina is dominated by the principal photosensitive pigment, whilst the screening of the rods, due to the absorption of light by the layers of retina lying between the rods and the internal limiting membrane, is trivial.

Propagation Velocity and Total Reflection of Spreading Induction in the Light-Adapted Human Retina

A process called “spreading induction” is started at the margin of a retinal image and propagated into the surrounding retinal areas. Thus a field of retinal induction is established around a retinal image. 1. The velocity of propagation was determined by measuring the latencies of the propagating process at various distances from the margin of a retinal image, and it was shown that the velocity depends greatly on the adaptation state of the retina, but only little on the intensity and hue of the light stimulus. 2. The value of velocity was 1.7mm. per sec. in the dark-adapted retina and 42mm. per sec. in the retina adapted to an illumination stronger than 1 lux. 3. When spreading induction comes across a white retinal image, it can totally be reflected at the margin of the retinal image. The critical angle of incidence for total reflection was determined, and on the basis of the law of refraction the velocity of spreading induction in the light-adapted part, the retinal image, was computed from the critical angle determined experimentally and the known velocity in the dark-adapted retina. The so computed values were found to agree satisfactorily with those determined by the direct method.

Refraction of Spreading Induction in the Retina

It was shown by the method of electrostimulation of the eye that spreading induction was refracted when a prism-shaped retinal image was formed on the way of its propagation through the retina. 1. The degree of refraction depended on the intensity and also the wave-length of light illuminating a prism-shaped patch whose image was formed on the retina. 2. The propagation velocity of spreading induction was measured to investigate its dependence on the intensity and the wave-length of adapting light. In the retina adapted to white light the velocity was higher than in the dark-adapted retina, while in the retina adapted to colored light the velocity was found lower than in the dark-adapted retina. 3. The refractive index computed from propagation velocities was in satisfactory agreement with that determined by measurements of refraction. 4. It was shown by many examples that the law of geometrical optics holds for spreading induction in the human retina.

From color induction to brightness induction.

Retinal induction was studied in the light-adapted human retina by the method of electrostimulation. 1. The properties of retinal induction are qualitatively the same as in the dark-adapted retina except that its dissipation is more rapid. 2. There is an optimal intensity of adapting light for production of retinal induction. 3. Color induction occurs when the intensity of inducing colored light is above the color-threshold. 4. Below the color-threshold a colored patch may appear dark against the background. Then a curve of induction is found quite different from the curve of color induction. A similar curve is obtained by a sequence of stimuli, black and white (brightness induction). 5. The critical intensity of inducing light changing from color induction to brightness induction is higher for blue and red lights and lower for yellow and green lights. 6. In the transitory range of intensity from color induction to bright-ness induction a curve of certain type belonging to neither of those stated above is obtained.

Retinal Induction in the Light-Adapted Human

Retinal induction of the light-adapted human retina was investigated. 1. When red, green and blue lights are used for preillumination in the light-adapted state of the retina, the peak of the curve of electrical excitability lies at 1, 2 and 3 seconds respectively just as in the dark-adapted retina. As the intensity of the adapting light increases from 0.004 lux to 30 lux, the magnitude of the curve decreases steadily. 2. The curves of retinal induction are not qualitatively different from those obtained from the dark-adapted eye. The retinal induction decays with time in about 5-6 seconds in the retina adapted to a weak general illumination. This is in contrast with the fact that the retinal induction of the dark-adapted retina remains unchanged for about 20 seconds. 3. The magnitude of retinal induction or contrast effect is smaller as the adaptation level of the retina is higher. 4. In the retina light-adapted to a general illumination of 0.004 lux, the white light of 32 lux interposed between the inducing light and the white test light does not affect the direct induction.

Cis-trans isomers of vitamin A and retinene in the rhodopsin system.

Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (lambda(max.) 500 mmicro). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (lambda(max.) 487 mmicro). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the isomerization can go further. The synthesis of rhodopsin in solution follows the course of a bimolecular reaction, as though one molecule of neoretinene b combines with one of opsin. The synthesis of isorhodopsin displays similar kinetics. The bleaching of rhodopsin, whether by chemical means or by exposure to yellow or orange (i.e., non-isomerizing) light, yields primarily or exclusively all-trans retinene. The same appears to be true of isorhodopsin. The process of bleaching is therefore intrinsically irreversible. The all-trans retinene which results must be isomerized to active configurations before rhodopsin or isorhodopsin can be regenerated. A cycle of isomerization is therefore an integral part of the rhodopsin system. The all-trans retinene which emerges from the bleaching of rhodopsin must be isomerized to neoretinene b before it can go back; or if first reduced to all-trans vitamin A, this must be isomerized to neovitamin Ab before it can regenerate rhodopsin. The retina obtains new supplies of the neo-b isomer: (a) by the isomerization of all-trans retinene in the eye by blue or violet light; (b) by exchanging all-trans vitamin A for new neovitamin Ab from the blood circulation; and (c) the eye tissues may contain enzymes which catalyze the isomerization of retinene and vitamin A in situ. When the all-trans retinene which results from bleaching rhodopsin in orange or yellow light is exposed to blue or violet light, its isomerization is accompanied by a fall in extinction and a shift of absorption spectrum about 5 mmicro toward shorter wave lengths. This is a second photochemical step in the bleaching of rhodopsin. It converts the inactive, all-trans isomer of retinene into a mixture of isomers, from which mixtures of rhodopsin and isorhodopsin can be regenerated. Isorhodopsin, however, is an artefact. There is no evidence that it occurs in the retina; nor has isovitamin Aa or b yet been identified in vivo. In rhodopsin and isorhodopsin, the prosthetic groups appear to retain the cis configurations characteristic of their retinene precursors. In accord with this view, the beta-bands in the absorption spectra of both pigments appear to be cis peaks. The conversion to the all-trans configuration occurs during the process of bleaching. The possibility is discussed that rhodopsin may represent a halochromic complex of a retinyl ion with opsin. The increased resonance associated with the ionic state of retinene might then be responsible both for the color of rhodopsin and for the tendency of retinene to assume the all-trans configuration on its release from the complex. A distinction must be made between the immediate precursor of rhodopsin, neovitamin Ab, and the vitamin A which must be fed in order that rhodopsin be synthesized in vivo. Since vitamin A isomerizes in the body, it is probable that any geometrical isomer can fulfill all the nutritional needs for this vitamin.

Acetylcholine in the Light and Dark Adapted Frog Retina

SummaryExtraction of frog retinas in acidified Ringer solution reveals an average Ach content of 3.4 γ/g in light-adapted and 3.6 γ/g in dark-adapted frogs. Direct comparison of Ach activity in extracts of lightand dark-adapted retinas, without reference to absolute magnitudes, also reveals no difference to absolute magnitudes, also reveals no difference greater than the error of the method. The lack of significant difference is in accord with the work of Therman, but not with that of Lange who finds a tenfold increase upon dark adaptation.

Preparation of Retinene in Vitro

RETINENE, obtained by Wald from dark-adapted retinas, is defined as the chromogen responsible for a maximum at 664 mµ in the absorption spectrum of the blue solution obtained by mixing retinal extracts with antimony trichloride in chloroform. Such extracts show (in chloroform) an ultra-violet absorption band with λmax.385 mµ. It has recently been suggested1 that the chromogen is vitamin A aldehyde, and that it can be obtained by direct oxidation of vitamin A alcohol.

Notes on the Distribution of Pigment Granules in the Retinal Pigment Epithelium of Light- and Dark-Adapted Rana Temporaria, and on the Nature of the Eye Pigment in the Frog and the Ox

ABSTRACT About two years ago, the writer made a study of the retinal pigment epithelium of twenty-two English frogs which had been subjected to various experimental conditions and found that the dispositions of the pigment granules could not be explained adequately in terms of the oft-described migration which is believed to take place during light- and dark-adaptation of the eye. The results were anomalous, in so far as the amount of pigment present under certain conditions seemed to be greater than at other times.

Colour sensations produced by ultra-violet light

The crystalline lens of the author's left eye has, as the result of an accident, been destroyed. The eye is now sensitive to ultra-violet light of quite low intensity. The sensation between 3600 and 3100 A. is blue, not violet. The sensations produced by extremely faint ultra-violet light on the dark-adapted retina are discussed; the behaviour is essentially similar to that in normal scotopic vision, the light appearing colourless and the peripheral region being more sensitive than the fovea.

The quantitative analysis of the photochemical bleaching of visual purple solutions in monochromatic light

In 1851 Muller noticed that under certain condition the rods of the frog's retina were coloured red. In 1876 Boll found that the retina bleached on exposure to light. This bleaching is now known to be due to the decolouration of a light sensitive pigment, visual purple or rhodopsin. It is generally accepted that this pigment place an important part in scotopic vision, i. e ., vision in dim light. Visual purple can be extracted from dark-adapted retinae by a number of reagents, including solutions of the salts and digitonin. The solutions of the pigments so obtained have been the subject of several spectrophotometric investigations. Trendelenburg in 1904 determined the adsorption curve of rabbits visual purple (maximum 507 mμ .) and also the relative efficiencies of different wave-lengths in bleaching the solutions. He established the similarity between the relative efficiencies and the human scotopic luminosity curve, as determined by him, for the same source of light. The adsorption curve has been determined by a number of other observers who find the maximum of adsorption of visual purple to be at about 500 for mammals, amphibia, etc., and at a variable wave-length for fish. Recently Hecht has studied the bleaching of visual purple solutions in white light. He showed that weak solutions bleach according to the Kinetics of a monomolecular reaction, the velocity constant being proportional to the intensity of the bleaching light and independent of the temperature within experimental error. In the present work the bleaching of visual purple in monochromatic light has been studied and, by means of absolute determinations of the light intensity, it has been found possible to apply fundamental photochemical principles to this reaction.

The Achromatic Sensitivity of the Dark-Adapted Retina in Relation to Stimulus-Distance

The Achromatic Sensitivity of the Dark-Adapted Retina in Relation to Stimulus-Distance