Isolated Frog Retina Research Articles

The effect of flash illumination on the endogenous cyclic GMP content of isolated frog retinae

In isolated, intact frog retinae high concentrations of cyclic 3′,5′-guanosine monophosphate were found. These levels decline rapidly upon flash-illumination. A significant drop of the endogenous cyclic 3′,5′-guanosine monophosphate content was obtained only 3 sec after a weak light flash which bleached a percentage of the rhodopsin not detectable by customary spectrophotometric methods. No effect of light on retinal cyclic 3′,5′-adenosine monophosphate was found. Cyclic 3′,5′-guanosine monophosphate levels in dark or light were not influenced by 10 m m-aspartate indicating that the light-effect did occur in the photoreceptor cells.

The formation of metarhodospin380 in the retinal rods of the frog.

1. The formation of metarhodopsin380 (metarhodopsin II) was studied in isolated frog retinas exposed to intense flashes of 120 mus duration. 2. A rapid increase in absorbance at 375 nm during the flash was followed by a slower absorbance increase in the subsequent dark period. The slower increase showed virtual completion after 5 ms. 3. The fast absorbance increase during the flash was due to the formation of metarhodopsin478. The rate of this reaction was dependent on the time course of the flash and on the decay rate of lumirhodopsin. 4. Kinetic analysis indicates that three consecutive reactions occur: the light-controlled formation of lumirhodopsin, its first-order decay to metarhodopsin478 and the conversion of metarhodopsin478 into metarhodopsin380. At 21 degrees C, the decay constants were 2 X 10(4) S-1 (lumirhodopsin) and 1 X 10(3) S-1 (metarhodopsin478), respectively.

Synaptic activity of frog retinal photoreceptors. A peroxidase uptake study.

The uptake of horseradish peroxidase (HRP) into membranous structures, detectable by light and electron microscopy, is used here to monitor the synaptic activity of photoreceptors of isolated frog retinas maintained in the dark or under various illumination conditions. The major findings are: (a) Neurotransmission from photoreceptor terminals seems to involve the same types of endocytic membrane-retrieval processes that occur at other nerve terminals. Presumably, the endocytic processes compensate for exocytic events associated with neurotransmission. The retrieved membrane is "recycled" to form vesicles. Some of these accumulate near the synaptic ribbons, perhaps indicating reutilization for exocytosis. On the other hand, some retrieved membrane evidently is degraded via multivesicular bodies that appear to undergo "retrograde" transport from the receptor synapses to the myoid regions. (b) Photoreceptor terminals take up much HRP in the dark. Steady illumination markedly decreases uptake by rods. Uptake by cones is notably reduced only at illumination intensities higher than those that have maximal effects on rods. (c) The decrease in rod HRP uptake with light is reversible when retinas are allowed to adapt to the dark, if the light exposures used were at intensities that bleach very little visual pigment. Such "recovery" is not observed after light exposures that bleach a considerable amount of visual pigment. The cones recover their dark levels of HRP uptake even after light exposures that bleach considerable amounts of visual pigment. The changes in HRP uptake that we observe parallel expectations for photoreceptor synaptic neurotransmission derived from indirect physiological evidence.

Reduction of the retinal-opsin linkage in isolated frog retinas

A method has been developed which allows the specific reduction of the retinal-opsin Schiff base linkage in the photoreceptor cell disk membrane, using the reducing agent borane dimethylamine. The reaction is carried out in the dark at pH 1·5, conditions which minimize transiminization reactions. The product of this reaction, N-retinylopsin, can be isolated from reduced rod outer segments by column chromatography in detergent solution, or by electrophoresis in sodium dodecylsulfate polyacrylamide gels. The method is applicable to retinas which have been fixed with formaldehyde, thus allowing subsequent autoradiography of radioactive tissue. One such autoradiographic experiment showed that in the frog retina, injected [ 3H]retinol is incorporated exclusively in the outer segments of rods and cones.

Rhodopsin in the rod outer segment plasma membrane.

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.

Properties of isolated frog retinae in reflecting non-polarized and polarized light

Using incident polarized and non-polarized parallel light, the light which is reflected from isolated frog retinae ( Rana esculenta, Rana temporaria) has been investigated. The angular distribution and state of polarization of the reflected light has been measured for several angles of incidence. With nonpolarized incident light, the reflected light was partially polarized. On the other hand, linear polarized incident light was partially depolarized. Both effects showed a strong dependence on the reflection angle. The polarization effects and angular characteristics are interpreted as resulting from different reflection and scattering properties of the retinal layers of receptors and ganglion cells. The results are discussed in relation to the waveguide theory of receptors.

Storage, distribution and utilization of vitamins A in the eyes of adult amphibians and their tadpoles

This study has investigated the quantities, isomeric configuration and ocular distribution of the two retinols (vitamins A 1 and A 2) in the eyes of goldfish. Xenopus. frogs and tadpoles, with particular emphasis on the shifting relationships of rhodopsin, porphyropsin. retinol and 3-dehydroretinol in tadpoles kept in light and darkness. The amounts of retinols (mainly esterified) stored in the dark-adapted eyes range from 41 moles per mole of visual pigment in the goldfish down to 0.4 mole in the Xenopus. The cleanly isolated frog retina has 0.07 mole of 11- cis and all- trans vitamin A (50 per cent esterified) per mole of ROS rhodopsin, the remainder (1 per cent esterified) being found in the pigment epithelium, 80 per cent in the oil-droplets separated by centrifugal flotation. A maximum of 3 per cent of the rhodopsin in the ROS is apparently in the pigment epithelium. During light-adaptation, retinol formed by rhodopsin bleaching in the ROS leaves the retina, enters the pigment epithelium where most of it passes into the oil-droplets. The dark-adapted stores always contain 11- cis isomer, which is present in least amount in eyes that have undergone one cycle of light- and dark-adaptation. During prolonged periods in the dark, there was an increase of 11- cis at the expense of all- trans retinyl ester. In illuminated tadpoles retinol and 3-dehydroretinol are utilized for visual pigment regeneration in the proportions present in the pigment epithelium. This is not observed in the dark, where the situation is dominated by the visual pigment renewal process. The prosthetic group used for visual pigment biosynthesis in the tadpole retina is mainly retinol, even though there is an overwhelming preponderance of 3-dehydroretinol in the pigment epithelium.

Effects of rhodopsin and its photoproducts on the late receptor potential of the isolated frog retina

The relation between the changes in amplitude oflate receptor potentialand the concentrations ofrhodopsinintermediates were studied in the isolatedbullfrog retinaby simultaneous measurement of the electrical response and spectral change. As a result, the amplitude ofrodreceptor potential in the time course of response decay is proportional to the log of 380-nm absorbing product concentration and inversely proportional to the log of the fraction of bleached rhodopsin. Possible mechanisms generating the rod response are discussed and it is suggested that the decay of rod receptor potential is related to the decay of rhodopsin intermediates.

Phosphorylation and dephosphorylation of frog rod outer segment membranes as part of the visual process.

The light-activated phosphorylation of rod outer segment membranes may be an intermediary process controlling photoreceptor responses. We have measured phosphorylation of the opsin moiety of rhodopsin in isolated frog retinas and in rod outer segment suspensions and have demonstrated a phosphorylation-dephosphorylation sequence in the suspensions. The results indicate that these reactions take place in vivo and may be physiologically relevant. Extraction of a protein kinase activity from rod outer segment membranes renders the membranes incapable of phosphorylation, but the light-activated reaction can be reconstituted by mixing the soluble extract and the "depleted" membranes. Illumination of the extract is without effect. Thus the light activation mechanism resides in the membranes. Regeneration of rhodopsin from opsin and 11-cis retinal does not influence the phosphorylation. Once activated, the reaction may use either rhodopsin or opsin as the substrate. Furthermore, 11-cis retinal regenerates rhodopsin from phosphorylated opsin without releasing bound phosphate. The isolated rod outer segment which contains regenerated rhodopsin thus differs from one that is dark adapted in that phosphate can remain bound and the phosphorylation reaction remains activated. Dark adaptation in vivo must include at least two membrane associated reactions beyond regeneration of rhodopsin's spectral properties: dephosphorylation, and the inactivation of the phosphorylation process.

Temperature dependence of cone pigment regeneration in the isolated frog retina following flash and continuous bleaches

The rate of regeneration of frog 580 cone pigment (λ max = 580 nm) is measured at 23.11 and 4°C following both flash (1 msec) and continuous (5-min) bleaches. We find that: (1) at 23°C the cone pigment regenerates more rapidly following the flash bleach ( t 1 = 1.5 min ) than following the continuous bleach ( t 1 = 8.5min ): (2) regeneration following the flash bleach is more temperature-dependent than the regeneration following the continuous bleach. This difference in temperature dependence results in almost equal rates of regeneration when the temperature is lowered to 4°C (flash bleach, t 1 = 15 min : continuous bleach. t 1 = 14 min ): (3) more pigment regenerates at lower temperatures. Possible mechanisms responsible for these results are discussed.

X-ray diffraction studies of retinal rods. I. Structure of the disc membrane, effect of illumination

The structure of the retinal rod disc membrane and its modifications upon bleaching have been studied by X-ray diffraction. Three types of preparations are used: functioning isolated frog retina, isolated rods from frog retina, oriented by a magnetic field, and stacked discs from cattle retina. X-rays are detected by a position-sensitive linear counter. Diffraction spectra are obtained in 10–100 s. The electron density profile favors models where the rhodopsin molecule spans the whole thickness of the membrane. Upon bleaching, a small increase of electron density appears instantly at the cytoplasmic edge of the membrane. In the intact retina this structural change is accompanied by disorder and slow swelling reactions which are not observed in the isolated rod outer segment. The diffraction signal arising from the protein distribution in the plane of the membrane has been reinvestigated carefully. Patterns identical to those of Blasie (Blasie (1969) J. Mol. Biol. 39, 407 and Blasie (1972) Biophys. J. 12, 191) can be obtained but these are shown to be dominated by artefacts. The actual signal is a single broad band around (55 Å) -1, upon which bleaching has a negligible effect. No measurable displacement of rhodopsin in the thickness of the membrane occurs upon bleaching. Temperature effects on the protein distribution are found to be large only for disc membranes from cattle retina. In this material from a warm-blooded animal those effects are correlated with the occurrence, upon lowering the temperature, of a partial phase transition of the paraffin chains of the lipids. The position and the slope of the transition are not sensitive to bleaching.

Experimental Siderosis of the Isolated Retina Induced by Fe(III) Solutions

The effect of Fe(III) ions in the range from 3 to 100 μmol/l on the electroretinogram (ERG) of the isolated frog retina was investigated. The perfusion with the test solution containing iron lasted 45 min. After moist combustion of the tissue the iron content of the retinae was determined by photometry using bathophenanthroline as an indicator. The ERG changes during experimental siderosis showed some analogy to clinical ERG classification of siderosis of the eye. Low Fe(III) concentrations of the test solution (3 μmol/l) had no significant effect on the ERG. At 10 μmol/l a decrease of the ERG could be observed which was less pronounced at 18 and 24 μmol/l. At 30 μmol/l Fe(III) and even higher concentrations there was a rapid and irreversible drop in potential. Increasing Fe(III) concentration of the bathing solution brought about an accumulation of iron in the retina which could not be eluted by desferrioxamine B (DF). This agrees with the observation that DF did not improve the ERG after intoxication with 100 μmol/l Fe(III).

Intoxication, detoxication and copper-storage of central nervous tissue at different external Cu(II)-concentrations

The isolated frog retina was perfused with bathing-solutions of different CuCl2-concentrations in order to investigate the toxic influence of Cu(II)-ions on the central nervous tissue. The amplitude of the b-wave (ϕb) of the exposure potential (ERG) was used to characterize retinal function. During 30 min of application the Cu(II)-solutions effected a decrease in potential dependent on dosage which was reversible at 6 μM/l but irreversible at higher concentrations. The prolongation of the Cu(II)-application led to a progressive and irreversible loss of potential already at 6 μM/l. Furthermore, the influence of the complexing agents d-penicillamine, dimercaprol, Na-thiosulfate and EDTA on retinal Cu(II)-intoxication was investigated. Corresponding to the mechanism of intoxication, which may by related to the blockade of enzymes containing SH-groups, the SH-donators brought about the best detoxication. The influence of d-penicillamine was especially favorable, which is in agreement with clinical experience. The Cu-analysis by means of the atomic absorption spectrometry showed that the Cu-content of the retina was much reduced if perfusion with Tyrode solution followed Cu(II)-intoxication, inspite of the persistance of the corresponding decrease of the ERG. A logarithmic increase in the Cu(II)-concentration of the bathing solution was proportional to an increase in the retinal Cu-content as well as to a decrease in the exposure potential. A considerable drop in the Cu-content of the tissue could only be effected by d-penicillamine.

The Effect of Ca-Ions on the Kinetics of Rhodopsin Photoproduct Decay in the Living, Isolated Frog Retina

The Effect of Ca-Ions on the Kinetics of Rhodopsin Photoproduct Decay in the Living, Isolated Frog Retina

Isomerization of all-trans-Retinal to 11-cis-Retinal in Isolated Frog Retinae

Isomerization of all-<i>trans</i>-Retinal to 11-<i>cis</i>-Retinal in Isolated Frog Retinae

The thermodynamics of the peripheral process of vision

In a range from 6 to 30°C, the influence of temperature on the relation between the light intensity and the amplitude of the b-wave of the exposure potential has been investigated for the isolated frog retina. Between 10 and 25°C, the gross activation energy of the response to light leads to a temperature coefficient which shows that the formation of the b-wave is mainly checked by diffusion processes. While at 6°C the b-wave has vanished, a complete delayed off-response can still be registered.

Dark adaptation of the frog's rods

Using sodium aspartate Ringer's and isolated frog retinas we have recorded gross potentials from the frog's 502 (rhodopsin) rods. Substantial dark adaptation is seen at the receptor in the isolated retina in which little or no rod pigment regeneration occurs. Lights that bleach only a fraction of a per cent of rod pigment substantially decrease rod sensitivity, but sensitivity quickly returns to the prebleach level. With lights that bleach measurable amounts of pigment, the rods recover their sensitivity more slowly and show a permanent loss in sensitivity.

Regeneration of the green-rod pigment in the isolated frog retina

The early receptor potential is used to show that, like the principal cone pigment (λ max = 580nm), the frog's green rod pigment (λ max = 433nm) regenerates in the isolated retina. Regeneration of rhodopsin (λ max = 502nm) was not observed.

Light-induced changes in the electrical impedance of the isolated frog retina.

1. An isolated frog retina was mounted in an impedance chamber and superfused on its vitread surface.2. Changes in the real part of the impedance (DeltaR) and also in the imaginary part were measured using alternating current in the frequency range 1-300 kHz passed from one surface of the retina to the other.3. Under most conditions, the response to a flash of light, measured at frequencies below about 100 kHz, was a decrease in the real part of the impedance (DeltaR < 0).4. The geometry of the electrodes was such that the system was particularly sensitive to changes in the impedance of the layer of photo-receptor outer segments. It was confirmed that most of DeltaR did arise here and that it was mediated by the absorption of light in rod photo-pigment.5. The magnitude of DeltaR increased when the channels between the outer segments were constricted, e.g. by osmotic swelling of the outer segments. In addition to this increase, a further increase was seen following the commencement of recording in most of the experiments from which usable measurements were obtained.6. In such retinas, the magnitude of DeltaR was greatest when measured at a frequency in the range 3-32 kHz, the largest changes being of the order of 0.1% of the resting value. A light flash bleaching about 1% of the pigment was sufficient to produce this. The onset of DeltaR was apparent within 1 msec of the flash (at 15 degrees C); it reached a maximum in 0.5-4 sec and then returned towards the base line.7. The main component of DeltaR was attributed to a decrease in the resistance of the spaces between the outer segments. In addition, there was a resistance increase which occurred at some other site, probably the surface membrane. The first of these components had an onset slightly more rapid than the second, and both occurred irrespective of whether the major cation in the superfusate was sodium or potassium.

Adaptational changes in the cone system of the isolated frog retina

An intermittent light presented to a dark adapted frog eye produces a small “flicker ERG” (response to the individual light pulses). After light adaptation the flicker ERG increases drastically in amplitude. This study showed that a qualitatively similar increase is seen in an isolated retina in which the rods were inactivated by bleaching 80 per cent of their pigment, rhodopsin. This bleach does not permanently affect the cones since, unlike the rods, their pigment regenerates in the isolated retina. Consequently, in the isolated retina the increase involves a change within the cone system, not an inhibition from the rods. The next paper shows an additional mechanism is involved in the intact retina.