Ch. Baumann

Kinetics of rhodopsin bleaching in the isolated human retina

1. Pieces of human retina were dissected from eyes enucleated because of malignant tumours. The isolated retinas were perfused by an ionic medium (36° C) and investigated spectrophotometrically.

The dark adaptation of single units in the isolated frog retina following partial bleaching of rhodopsin.

Abstract The dark adaptation of single units following partial bleaching of rhodopsin has been studied in isolated perfused retinac of frogs ( Rana esculenta ). This dark adaptation occurs without regeneration of rhodopsin, as absence of regeneration is a feature of the perfused retina. The shape of the dark adaptation curves depends on the amount of pigment bleached. If less than 35–40% of rhodopsin have been bleached, the curves show two phases separated by a distinct kink. Bleaching more than 35–40% of the pigment is followed by a recovery process that exhibits one phase only. If a second phase initiated by a kink appears, the spectral sensitivity curve is finally found to be scotopic. In the case of single branched curves, spectral sensitivity distributions show up eventually that are similar to Granits narrow shaped modulator curves. The analysis of scotopic thresholds finally reached shows that, within a certain range, the scotopic sensitivity is proportional to the fraction of light absorbed. This result suggests the dominance of photochemical processes over nervous ones in setting the sensitivity level.

Lichtabhängige langsame Potentiale aus dem Stirnorgan des Frosches

Zusammenfassung1. Langsame Potentiale werden vom durchtrennten, zentralen Ende des Pinealnerven bei optischer Reizung des Stirnorgans abgeleitet.2. Depolarisierende langsame Potentiale des Stirnorgans gehen mit einer Aktivierung, hyperpolarisierende Potentiale mit einer Hemmung der Impulsentladungen des Pinealnerven einher. Unter der Wirkung von Lokalanaesthetica erlischt die Spike-Aktivität, ohne daß die langsamen Potentiale alteriert werden.3. Die Polarität der langsamen Potentiale ändert sich mit der Wellenlänge des Reizlichtes. Rein hyperpolarisierende Potentiale entstehen bei spektralen Reizen unterhalb 425 mμ, rein depolarisierende Potentiale oberhalb 520 mμ. In einem mittleren Wellenlängenbereich (435–517 mμ) sind sowohl hyper- als auch depolarisierende Anteile in den Potentialabläufen erkennbar; die Depolarisationen lassen sich durch Adaptation an ultraviolettes Dauerlicht verdeutlichen.4. Die rein de- und hyperpolarisierenden Potentiale sind durch minutenlange Reiznachwirkungen und starke Erschöpfbarkeit gekennzeichnet. Bei wiederholter Reizung sind langsame Antworten nur ableitbar, wenn de- und hyperpolarisierende Reize miteinander alternieren und eine bestimmte Relation an Reizenergie untereinander aufweisen.5. Die Herkunft der langsamen Potentiale und ihre Rolle bei der Erregungsübertragung im Stirnorgan werden diskutiert.

Regeneration of rhodopsin in the isolated retina of the frog (Rana esculenta).

Abstract Isolated retinae of the frog (Rana esculenta) were exposed to quasimonochromatic radiation of 450, 475, 500 or 540 nm wavelength, respectively. These coloured lights were formed by a prism monochromator whose band-width was set to 12–16 nm. More than 90 per cent of rhodopsin was found to be in the bleached state after exposure times of 30–50 min. A fraction of the bleached pigment was regenerated during a subsequent dark period of 90 min. The fraction regenerated (7–24 per cent) was found to depend on the spectral content of the bleaching light in such a way that wavelengths shorter than 500 nm favoured the regeneration process. Regeneration occured from a substance with maximum absorption at 380 nm. This substance is assumed to be retinaldehyde in which the correct isomer for regeneration (11-cis) is formed via photoisomerization.

Sehpurpurbleichung und Stäbchenfunktion in der isolierten Froschnetzhaut

Summary1. The visual purple content of the isolated frogs retina has been diminished by partial bleaching. After cessation of the bleaching light, the rhodopsin concentration in the rods underwent no further change since bleached rhodopsin was not regenerated by the preparation. Threshold changes in the retina following the partial bleach have been recorded by measuring the light intensities required to elicit a constant small response from the electro-retinogram.2. If the fraction of pigment bleached does not exceed 45% of the full rhodopsin content the dark adaptation curves obtained by the above method exhibit two branches and reach steady values of sensitivity after about 45 min. Bleaching of more than 45% is followed by a shorter recovery process occuring in one stage only. Scotopic and mesopic spectral sensitivity curves with λmax at 502 mμ are connected with two branches in the dark adaptation curve whereas single branchea curves are accompanied by photopic spectral sensitivity distributions (λmax=560–570 mμ).3. In experiments showing two branches in the recovery curve the logarithm of threshold measured after completion of the dark adaptation is linearly related to the fraction of pigment bleached.Zusammenfassung1. Der Sehpurpur der isolierten Froschnetzhaut wurde partiell gebleicht und die Erregbarkeit der Präparation vor und nach der Bleichung elektroretinographisch bestimmt (Methode der konstanten Antwort).2. Die nach der Einwirkung des bleichenden Lichts zweiphasisch ablaufende Dunkeladaptation dauert 45 min, wenn weniger als 45% des Sehpurpurs gebleicht werden. Die spektrale Empfindlichkeit am Ende dieser Dunkeladaptation folgt unterhalb 520 mμ der Sehpurpurabsorption. Werden mehr als 45% des Pigments gebleicht, dann verkürzt sich die Dauer der Dunkeladaptation auf 25–28 min und wird einphasisch. Die spektrale Sensitivität zeigt in diesem Fall ein Maximum zwischen 560 und 570 mμ.3. Nach partieller Bleichung bis 44% steigt der Logarithmus der Dunkelschwelle linear mit der gebleichten Sehpurpurfraktion an. Bei noch weitergehender Bleichung wird die Dunkelschwelle unabhängig vom Ausmaß der Sehpurpurbleichung. Die Erregbarkeit der Netzhaut wird dann allein vom photopischen System bestimmt.

The equilibrium between metarhodopsin I and metarhodopsin II in the isolated frog retina.

1. Rapid and slow changes in the absorbance of isolated frog retinae produced by exposure to brief flashes were studied at temperatures between 5 and 30 °C.

Flash photolysis of rhodopsin in the isolated frog retina

Abstract The flash photolysis of rhodopsin was studied by exposing the isolated frog retina to intense white flashes from a 500 J xenon-filled discharge tube. The flash duration (i.e. the time necessary for 90 per cent of the flash energy to dissipate) was between 200 and 380 μsec. With increasing quantities of the flash light, the fraction of pigment bleached by a single flash rose to a maximum of 55 per cent. By applying Poisson statistics, it has been shown that the bleached fraction arises from those rhodopsin molecules that had absorbed an odd number of quanta. The 55 per cent figure is an upper limit which can not be exceeded by increasing the light energy. The residual fraction of 45 per cent in flash-exposed retinae was found to consist of a 1:1 mixture of rhodopsin and isorhodopsin, both formed by photoregeneration.

Boll's phenomenon

in 1876, Franz Christian Boll (1849-79) submitted a short note to the Royal Academy of Sciences in Berlin. The paper was a contribution “to the Anatomy and Physiology of the Retina” (Boll, 1876) and described a discovery which formed the starting point for all work on visual pigments. Boll observed that the retina of a dark-adapted frog eye exhibits a purple colour which fades away upon illumination. This colour phenomenon is situated in the rod outer segments. Boll illustrated this in a painted colour plate published in his second and more elaborate paper on the subject (Boll, 1877). The Figs. 5-9 of Boll’s plate show mosaics of red and green dots, the distal ends of the retinal rods. Light causes bleaching which is more obvious in the red rods. It should be possible with the help of modem high speed colour film to produce photographs documenting Boll’s observation. Retinae of dark-adapted frogs (Rona es&en&x) were isolated and put, receptor-side down, on a coverglass. A little trough of 1 mm depth filled with the bathing solution was mounted on a microscope slide, and the coverglass with the retina was placed on top of this trough so that the receptors pointed upward, i.e. onto the microscope objective. The preparations were illuminated with deep red light (cut-off filter Schott RG 715) of very weak intensity. This light was hardly visible and had to be intensified by a conventional i.r. image conversion tube (type no. 6929). The retinae were inspected at an overall magnification of 300x and those parts where the receptors were oriented parallel to the optical axis were selected for photography. The photographs were taken with the aid of a xenon flash lasting 1Sms (time for 90% light dissipation). Plate l(a) shows the result of a flash exposure on an unbleached preparation. The red and green rods of the frog retina can be clearly distinguished from one another. Some outer segments are isolated from the retina. They float on top of the preparation and form long shadows of some 50-6Om length. The question arises as to what extent the visual pigments are affected by the flash exposure. From sensitometric data of the film material used and from the geometry of the photomicrographic system it can be estimated that the illumination at the object plane is between 10’ and 1031m~s~m-2 per flash. In previous experiments carried out with the same flash tube, flashes of this strength bleached between 1 and 5% rhodopsin in isolated frog retinae (Baumann, 1970). The flashes in this previous study were shorter (0.2 ms) than the present one (1.5 ms) so that the conditions are not exactly comparable. Nevertheless, one may safely assume that one flash will not bleach more than about 10% of the rhodopsin. About one third of the bleached pigment will be in the state of metarhodopsin I while the rest is metarhodopsin II (other intermediates can be ignored). These figures are derived from a kinetic model recently worked out to describe the meta I to meta II transition in frog rods (Baumann, 1976) The red colour seen in Plate l(a) is then mostly due to unbleached rhodopsin as only a small fraction of this pigment is bleached by the flash, and the small amount of metarhodopsin II present will not significantly interfere with the colour of the original pigment. Similar considerations apply to the photosensitive pigment of the green rods. Plate l(b) was obtained after exposure of the preparation to bright white xenon light lasting about one minute. Another five min elapsed from the end of this period until the second photographic flash exposure. The red rods have now lost their colour. The green ones, however, retain most of their green ColOUr. Black and white photographs of the receptor mosaic as shown in Plate 1 have been published earlier (Denton and Wyllie, 1955). When such photographs are taken from bleached retinae illuminated with red light, the “green” rods appear quite dark (Baumann, 1967). Apparently, it is primarily this photostable absorption at long wavelengths which together with the blue-absorbing visual pigment (P 433,; cf. Dartnall, 1957; 1967) determines the green appearance of these receptors. Boll did not believe that a photochemical process alone accounted for his observations. In his paper of 1877, he suggests that, in addition to the photochemical action of light, a physical-optical effect might contribute to the colour of the rods. With respect to the green rods, his view is apparently correct (Liebman and Entine, 1968) although the mechanism of the postulated physical effect is still obscure. The term “Boll’s phenomenon” used as the title of this note was introduced after Boll’s death by Kiihne (Kilhne and Sewall, 1880).

Empfindlichkeit und Sehpurpurgehalt der isolierten menschlichen Netzhaut

SummaryPieces of human retinas were dissected from dark adapted eyes enucleated because of malignant tumours. The preparations were perfused at 36°C with a glucose-containing ionic medium of pH 7.4. The rhodopsin content was determined by means of the absorbance changes due to bleaching. Electroretino-graphic responses were recorded transretinally.Scotopicb-waves (8 preparations) or slow P III responses (19 preparations) were obtained at stimulus strengths corresponding to approx. one quantum absorbed per rod per flash (20 ms). Photopic potentials appeared when the stimuli were 1.5 log units more intense.Partial bleaching of rhodopsin was followed by an amplitude decrease of scotopic responses. This decrease was a temporary one from which the retinas fully recovered if less than 6% of the rhodopsin had been bleached. Bleaching of larger amounts of the pigment resulted in a permanent loss of potential amplitude and of retinal sensitivity. During the recovery process after small bleaches, the reduction of potential amplitude was proportional to the concentration of metarhodopsin380 in the rods, and may indicate a link between metarhodopsin380 and the temporary desensitization of the retina.

Properties of the frog's retinal ganglion cells as revealed by substitution of chromatic stimuli.

Abstract By means of the method of stimulus substitution, single ganglion cells of the isolated perfused retina of Rana esculenta were investigated. The scotopic system was eliminated (a) by a permanent background illumination, (b) by bleaching more than 90 per cent of rhodopsin. The non-appearance of the impulse response during and after light substitution was taken as a criterion for an invariant spectral stimulus-response behaviour (univariance). Only some of the ganglion cells investigated showed this property. The spectral sensitivity of the other cells could be described only by several disconnected branches of functions. Therefore, it was concluded that there exist at least two photopic (or mesopic) mechanisms in the frog retina.