Dark-adapted Frog Research Articles

Obtaining absorbance spectra from turbid retinal cell and tissue suspensions - Beating the light-scatter problem

Light scattering and inability to uniformly expose the cuvette contents to an incident light beam are significant limitations of traditional spectrophotometers. The first of these drawbacks limits their usefulness in studies of turbid cellular and tissue suspensions; the second limits their use in photodecomposition studies. Our strategy circumvents both problems. Although we describe its potential usefulness in vision sciences, application of spherical integrating cuvettes has broad application. Absorbance spectra of turbid bovine rod outer segments and dispersed living frog retina were studied using a standard single-pass 1 cm cuvettes, or a spherical integrating cuvette (DeSa Presentation Chamber, DSPC). The DSPC was mounted on an OLIS Rapid Scanning Spectrophotometer configured to generate 100 spectral scans/sec. To follow rhodopsin bleaching kinetics in living photoreceptors, portions of dark-adapted frog retina were suspended in the DSPC. The incoming spectral beam at 2 scans/sec entered the chamber through a single port. Separate ports contained a 519 nm light emitting diode (LED), or window to the photomultiplier tube. The surface of the DSPC was coated with a highly reflective coating allowing the chamber to act as a multi-pass cuvette. The LED is triggered to flash and the PMT shutter temporarily closed during a “Dark-Interval” between each spectral scan. By interleafing scans with LED pulses, spectra changes can be followed in real time. Kinetic analysis of the 3-dimensional data was performed by Singular Value Decomposition. For crude bovine rod outer segment suspensions, the 1 cm single-pass traditional cuvette gave non-informative spectra dominated by high absorbances and Rayleigh scattering. In contrast, spectra generated using the DSPC showed low overall absorbance with peaks at 405 and 503 nm. The later peak disappeared with exposure to white light in presence of 100 mM hydroxylamine. For the dispersed living retinal, the sample was pulsed at 519 nm between the spectra. The 495 nm rhodopsin peak gradually reduced in size concomitant with the emergence of a 400 nm peak, probably representing Meta II. A conversion mechanism of two species, A → B with rate constant of 0.132 sec−1 was fit to the data. To our knowledge this is the first application of integrating sphere technology to retinal spectroscopy. Remarkably, the spherical cuvette designed for total internal reflectance to produce diffused light was efffectively immune to light scattering. Furthermore, the higher effective path length enhanced sensitivity and could be accounted for mathematically allowing determination of absorbance/cm. The approach, which complements the use of the CLARiTy RSM 1000 for photodecomposition studies (Gonzalez-Fernandez et al. Mol Vis 2016, 22:953), may facilitate studies of metabolically active photoreceptor suspensions or whole retinas in physiological assays.

The color purple: milestones in photochemistry

Earlier this year, the Illinois Eye and Ear Infirmary, a venerable institution that was later incorporated into the University of Illinois at Chicago College of Medicine, celebrated its 150th anniversary. Asked to deliver a lecture to the distinguished group of physicians and scientists that had assembled to celebrate this historic event, I was struck by the fact that in 1858, when the Infirmary opened its doors to the public, absolutely nothing was known about any aspect of the visual process. There were, to be sure, significant advances in ocular surgery, a growing number of medical treatments for eye disease, and vastly improved recognition of retinal disorders due in no small measure to the arrival two years earlier of Helmholtz’s ophthalmoscope. But knowledge of how the visual signal was generated had not progressed much further than that shown in Descartes’ fanciful cartoon (Fig. 1). The situation changed dramatically some twenty years later, when Franz Boll, a German physiologist working in Rome, removed the retina from the eye of a dark-adapted frog, and reported the color changes he observed as the tissue was exposed to light. Boll had witnessed the light-

Effects of osmotic and light stimulation on 3H-taurine efflux from isolated rod outer segments and synthesis of tauret in the frog retina.

After injection of 3H-taurine into eyeballs of frogs and maintenance for 3 h in darkness by a gentle shaking, an almost homogenous fraction of rod outer segments (ROS) was prepared. About a 22% decrease in tonicity caused by reducing NaCl in isotonic 225 mOsm normal solution caused a rapid increase in the rate coefficient of efflux of 3H-taurine from the ROS fraction. The peak level of increased efflux rate coefficient was 7-times higher than the basal isotonic level. This indicates that taurine could contribute essentially to the volume regulation, either via selective channels or a carrier transporter-mediated pathways. For further clarifying if taurine fluxes in the ROS are sensitive to the light, other experiments were performed. Neither light stimulation of dark-adapted ROSs fractions or dark stimulation of weakly illuminated ROSs revealed any detectable changes in the efflux rate coefficient of 3H-taurine. These results indicate that light-induced taurine efflux, if present in the ROS, must be small, compared with hypoosmotic induced efflux. Thus the question of light-induced release of taurine from ROS still remains to be clarified. In the second part of this study, using TLC (thin layer chromatography) in combination with 3H-taurine measurements we have tried to clarify whether frogs (Rana ridibunda) eye structures can synthesize tauret (retinylidenetaurine). In isolated retinal preparations almost no any noticeable radioactivity was detected compared with background level. The capability of the eye structures to synthesize tauret from 3H-taurine was revealed in the second whole eye injection experiment. About 0.3% of the total 3H-taurine pool taken up was converted into 3H-tauret in the dark-adapted frog retina. In the retina of frogs adapted to light compared with those which were dark adapted tauret quantities were remarkable lower--on average about half. These results are in agreement with our recent data obtained by HPLC, which indicate tauret levels several times higher in the dark-adapted frog retinae compared with those after long lasting light adaption. Taking into account these results one can conclude that the main structure able to synthesize 3H-tauret is probably pigment epithelium rather than retina.

Projection structure of frog rhodopsin in two crystal forms.

Rhodopsin is the G protein-coupled receptor that upon light activation triggers the visual transduction cascade. Rod cell outer segment disc membranes were isolated from dark-adapted frog retinas and were extracted with Tween detergents to obtain two-dimensional rhodopsin crystals for electron crystallography. When Tween 80 was used, tubular structures with a p2 lattice (a = 32 A, b = 83 A, gamma = 91 degrees) were formed. The use of a Tween 80/Tween 20 mixture favored the formation of larger p22(1)2(1) lattices (a = 40 A, b = 146 A, gamma = 90 degrees). Micrographs from frozen hydrated frog rhodopsin crystals were processed, and projection structures to 7-A resolution for the p22(1)2(1) form and to 6-A resolution for the p2 form were calculated. The maps of frog rhodopsin in both crystal forms are very similar to the 9-A map obtained previously for bovine rhodopsin and show that the arrangement of the helices is the same. In a tentative topographic model, helices 4, 6, and 7 are nearly perpendicular to the plane of the membrane. In the higher-resolution projection maps of frog rhodopsin, helix 5 looks more tilted than it appeared previously. The quality of the two frog rhodopsin crystals suggests that they would be suitable to obtain a three-dimensional structure in which all helices would be resolved.

Optical probes of intradiskal processes in rod photoreceptors II: Light-scattering study of ATP-dependent light reactions

Rod outer segment (ROS) disks, either stacked or freely floating, respond to flash illumination to yield a specific, ATP-dependent, light-scattering signal A L. In broken ROS A L signals occur only when A D signals (see Part I) have preceded them, The degree to which the preceding A D signal has been completed determines the amplitude of the following A L signal. However, in freshly detached ROS from dark-adapted frogs A L signals with maximal size can be obtained without pre-incubation with exogenous ATP. The energized state, which is restored in broken ROS with the help of ATP, appears to prevail in the living retina and must therefore be considered to be “physiological”. A L signals require structurally intact disks. Neither peripheral ROS proteins nor connecting filaments between adjacent disks are necessary. Their structural origin is the same as that of the preceding A D signal, i.e. osmotic disk swelling. A L signals consist of a single slow kinetic component (half-life 10 s at room temperature) and multiphase fast kinetic component (70 ms). The slow phase corresponds to a light-stimulated resumption of ATPase activity (this has been dealt with in a previous paper) whereas the fast component reflects an immediate response of the energized disk to the metarhodopsin I to metarhodopsin II transition. The latter effect is the subject of this paper. A variety of experiments, using different ATPase inhibitors, ionophores and membrane-permeable salts, have been carried out; they are all consistent with the notion that A L originates in the disk interior and probes the existence of a proton electrochemical potential difference Δμ(H +) across the disk membrane. A model is presented which can explain all given properties of A L satisfactorily. According to this model the photolysis of rhodopsin causes a proton release in the disk lumen. This, in turn, results in osmotic swelling of the disks, provided that the internal buffer sites have been (at least partially) titrated with protons prior to the flash. Such conditions, i.e. a low internal pH, are provided by the proton transport across the disk membrane, which presumably takes place during the course of the preceding A D signal.

Lithium decreases retinal melatonin levels in the frog

The effect of the acute i.p. administration of lithium chloride (1 mg/kg) and of haloperidol (1 mg/kg) on retinal melatonin levels was studied in light-adapted and dark-adapted frogs of the species Rana esculenta. Two hours following drug administration, animals were killed by decapitation and a single retina from each frog was collected and homogenized in 1 ml of chilled 0.1 N HCl. After centrifugation, the pH of the supernatant was adjusted to 7.0. Melatonin was extracted by diethylether and assayed by double antibody radioimmunoassay (RIA). Lithium induced a significant decrease of retinal melatonin levels both in light-adapted ( P < 0.006) and in dark-adapted ( P < 0.01) animals, whercas no change was observed after haloperidol treatment. These results suggest that the scleral aggregation of pigment granules induced by lithium in the frog retina is not mediated by a stimulation of melatonin synthesis.

Retinoid changes in the in vitro regeneration of frog visual pigments.

To investigate the regeneration of visual pigment, the changes in composition and quantity of retinoids were measured by high-performance liquid chromatography (HPLC). Eye cups or eye cup sections of dark-adapted frogs were exposed to light (greater than 500 nm) and incubated in the dark for several hours (pH 7.4, 27 +/- 1 degree C). Retinoids extracted by the oxime method before and after illumination were analysed by HPLC. In the dark, every eye cup or eye cup section contained 11-cis and all-trans isomers of retinyl palmitate (the proportion of 11-cis was nearly 40%). The amount of retinyl palmitate was 1-1.5 mol equiv of visual pigment. After 80% of the visual pigment had been bleached by illumination, eye cups or eye cup sections were incubated in the dark for 10 h. During the incubation, 70% of the bleached pigment was regenerated, and the proportion of 11-cis retinyl ester decreased from 40% to 13%. These results indicate that stored 11-cis retinyl ester is used for the regeneration. The regeneration rate of A2-pigment (half time = 75 min) was faster than that of A1-pigment (half time = 90 min), consistent with the result of Tsin & Flores' (1986) in vivo experiment with goldfish.

Retinal noise, the performance of retinal ganglion cells, and visual sensitivity in the dark-adapted frog.

By extracellular recording of action potentials from single ganglion cells in the dark-adapted eyecup of the frog, we show that four different characteristics of a cell, the frequency-of-response function, the variability in responses to low suprathreshold stimulation, the maintained discharge, and the susceptibility to desensitization by weak background lights, all seem to reflect the same intrinsic noise. In a small fraction of reliable cells this noise is close to that expected from dark isomerizationlike events in rods, but it differs by orders of magnitude between cells. We have also determined a threshold intensity for the phototactic jumping behavior of frogs in darkness. This intensity, while below the human threshold, is still above the threshold for an appreciable fraction of frog retinal ganglion cells, and the sensitivity of this visually guided behavior at 16 degrees C is not strictly limited by dark events in rods.

Lithium and melatonin: morphological modifications induced in frog retina pigment screening.

Pigment screening (PS) is a phenomenon occurring in the retina of lower vertebrates, which consists in the dispersion, induced by the light, of melatonin granules into processes of the pigment epithelium which extend between photoreceptors, and in the aggregation, induced by the dark, of the same granules within the cell bodies of the pigment epithelium. It has been hypothesized that this phenomenon might be modulated by melatonin, which is present not only in the pineal but also in the retina, and whose retinal content is high in the dark and low in the light. In the present study we demonstrate that a single intraperitoneal injection of lithium chloride affects in a peculiar way PS of light-adapted frogs and of frogs subjected to natural day/night conditions, but not that of dark-adapted frogs. The implications of this findings are briefly discussed, also in view of the reported effect of lithium on the 24-hour rhythm of retinal melatonin, and of the possible relationship between this effect and the altering properties of lithium on other biological rhythms.

Rapid declines in cyclic GMP of rod outer segments of intact frog photoreceptors after illumination.

Considerable disagreement has resulted from experiments designed to test whether light-induced falls in cGMP in outer segment (OS) of photoreceptors precede their light-induced electrical responses. Different studies have reported initial declines at 50 ms, at s, or not at all for physiological stimuli. Such studies have employed whole retinas, isolated rod OS, or isolated rod OS with attached inner segments and involved a variety of techniques. We developed an apparatus that illuminates intact pieces of dark-adapted frog retinas at 22 degrees C for known brief durations and then rapidly (47 ms) presses their OS surface against a copper mirror cooled by liquid helium. Freezing occurs in less than 2 ms. Cyclic GMP was then assayed in cryostat sections of the OS layer. Six illumination intensities that bleached from 90 to 9 X 10(8) rhodopsin molecules per s were delivered for durations of 0.1-2 s. Compared to dark-adapted values, progressive losses of cGMP were seen with all illumination intensities. Because a significant loss in cGMP was seen after a 100 ms exposure to our dimmest stimulus, it appears that a loss of cGMP could play a role in rod visual transduction.

The specific inhibition of 11- cis-retinyl palmitate formation in the frog eye by diaminophenoxypentane, an inhibitor of rhodopsin regeneration

The antischistosomal drug 1,5-di-(p-aminophenoxy)pentane (DAPP), an inhibitor of rhodopsin regeneration in the vertebrate retina, is shown to completely block the production of 11- cis-retinyl palmitate in the frog eye. An untreated frog generates a large amount of 11- cis-retinyl palmitate during 1–2 days in the dark after a strong bleach. Also, it is demonstrated that DAPP can deplete the stores of 11- cis-retinyl palmitate in the dark-adapted frog eye. The specificity of DAPP's inhibition of dark-adaptation is explored, and the usefulness of employing retinotoxic drugs to investigate the physiology and biochemistry of rhodopsin regeneration is discussed.

Drugs and the frog retina: Effect of dopaminergic agents on the pigment screening of light- and dark-adapted frogs

This is a study, using the light and electron microscope, of the action of a dopamine agonist (apomorphine) and of a dopamine antagonist (haloperidol) on the retinal pigment screening (PS) of lightand dark-adapted frogs. Pigment screening is a phenomenon which consists of the migration of melanin granules into processes of the pigment epithelium that extend between photoreceptors, in response to changes in the conditions of illumination. In the light the pigment migrates vitreally, in the dark it aggregates sclerally. A single intravenous injection of apomorphine (0.15 mg/kg) and of haloperidol (1 mg/kg) did not induce substantial modifications in the pattern of pigment screening which was similar to that of controls both in light- and dark-adapted frogs. This suggests that dopamine is probably not directly involved in the phenomenon of pigment screening in the retina of the frog.

Studies on the off-response of the rod photoreceptors in the isolated frog retina

Transretinal potential changes induced by a 30 sec exposure to 503 nm light were studied in the dark-adapted frog isolated retina. The retina was treated with aspartate and 0·5 m m Ba 2+ to suppress the PII and slow PIII components of the electroretinogram, and therefore the response to the light stimulus consisted of the on-response (fast PIII response) and the off-response. The amplitude of the off-response was proportional to that of the on-response when the stimulus intensity was weak. The amplitude ratio of the off-response to the on-response was unaffected by partial bleachings of rhodopsin. In the presence of 700 nm background illumination, the amplitude of the on-response was decreased, whereas that of the off-response was increased. The amplitude of the off-response increased to about four-fold that of the original response at 3 hr after turning on the background illumination, but the effects of 480 nm background light were less remarkable. Both the on- and off-response, however, had a peak spectral sensitivity at about 500 nm, regardless of the presence of background light. From these findings, it was suggested that the red rods contribute to the development of the off-response, but the cones would also contribute through small focal gap junctions between the cones and the red rods.

Light-related changes in electron-dense material in photoreceptor synaptic clefts of the frog, Rana catesbeiana.

Lumps of electron-dense material were observed in synaptic clefts associated with all types of photoreceptors, in the vicinity of the synaptic ribbons, in the retinae of dark-adapted frogs. Frogs were reared under a cyclic illumination (light on at 8:00; light off at 20:00) and then exposed to one of two courses of dark adaptation: one started from 11:00 in the morning, and the other started from 20:00 in the evening. The synaptic clefts of red rods became wider at some places where spherical or polygonal lumps of dense material were accumulated. The frequency and sectional area of the lumps increased faster for the first hour in the regime starting from 20:00 than in the regime starting from 11:00, then they reached the similar saturation levels of about 0.6 (per ribbon) and 1.6 to 1.8 X 10(4) (nm2) in both the regimes. In green-rod synapses, plate-shaped lumps of dense material were present in synaptic clefts and interspaces between the processes of second-order neurons. In cone synapses at the end of about 1 h darkness, the frequency and area of the lumps reached maximum values of about 0.12 (per ribbon) and 9 X 10(3) (nm2) in the regime starting from 11:00 and, about 0.08 (per ribbon) and 4 X 10(3) (nm2) in the regime starting from 20:00. On exposure to light, the dense material abruptly disappeared from all types of photoreceptor synaptic clefts. Large dense-core vesicles, occasionally observed in light-adapted rod photoreceptor terminals, seem to participate in exocytosis of the dense material. The number of dense-core vesicles per synaptic ribbon in a terminal was about 0.55 at the end of 3 h light in the morning and about 1.28 at the end of 12 h light in the evening. The increased number of dense-core vesicles during the daytime may contribute to the faster accumulation of dense material in the synaptic clefts. Although the chemical identification or the functional significance of the electron-dense material remains unknown, it is interesting that this material showed a rise and fall in response to darkness and illumination. Also the fact that this material is clearly visible will be helpful for future analysis of frog photoreceptor synapses.

Effect of pH on the formation and decay of the metarhodopsins of the frog.

1. Suspensions of membrane vesicles were prepared by sonication of dark-adapted frog rod outer segments. The pH was adjusted to one of twelve values between 5.0 and 9.8; the temperature was 15 degrees C. Each preparation was exposed to a single yellow flash of 120 microseconds duration, and rapid slow changes of absorbance were measured at 475 nm wavelength. 2. Rapid changes consist of a transient rise followed by a diphasic decay to a new level of absorbance which is lower than that before the light exposure. The level of absorbance reached at the end of the rapid changes is lower at lower pH. 3. Kinetic analysis reveals that three reactants take part in the rapid reactions, viz. metarhodopsin I and two isochromic forms of metarhodopsin II named metarhodopsin II' and metarhodopsin II". The kinetics of the reversible transition from metarhodopsin I to metarhodopsin II' are not measurably influenced by the pH of the medium. However, the reversible reaction of metarhodopsin II' with metarhodopsin II" is dependent on pH because metarhodopsin II" is encountered either as a protonated or as a deprotonated compound. 4. Slow reactions are due to metarhodopsin I and to metarhodopsin III. A combined quantitative analysis of both rapid and slow reactions involves a reversible reaction between metarhodopsin II' and metarhodopsin III and a hydrolysis of metarhodopsin II' to retinal and opsin. The scheme employed accounts for the pH dependence of all equilibria although only one of the metarhodopsins reacts directly with protons.

Influence of calcium on guanosine 3',5'-cyclic monophosphate levels in frog rod outer segments.

In the presence of 10(-9) M calcium, rod outer segments freshly detached from dark-adapted frog retinas contain between 0.01 and 0.02 moles of guanosine 3',5'-cyclic monophosphate (cyclic GMP) per mole of rhodopsin. The dark level of cyclic GMP is reduced approximately 50% by illumination that bleaches 5 x 10(5) rhodopsin molecules/outer segments. The dark levels of cyclic GMP also can be suppressed to approximately 0.007 mol/mol of rhodopsin by increasing the concentration of calcium from 10(-9) M to 2 x 10(-9) M, and they remain at this level as calcium concentration is raised to 10(-3) M. The final level to which illumination reduces cyclic GMP in unaffected by the calcium concentration between 10(-9) and 10(-3) M. The maximal light-induced decrease in cyclic GMP occurs within 1 s from the onset of illumination at all calcium concentrations. The magnitude and time-course of the light-induced decrease in cyclic GMP measured in these experiments are comparable to values obtained previously (Woodruff et al. 1977. J. Gen. Physiol. 69:677-679; Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). The data are consistent with a role for cyclic GMP in visual transduction irrespective of the calcium concentration.

Alteration of metabolism of retinal taurine following prolonged light and dark adaptation: a quantitative comparison with gamma-aminobutyric acid (GABA).

Alteration of metabolism of taurine in prolonged light- and dark-adapted frog retinae were studied in comparison with that of gamma-aminobutyric acid (GABA) and the following results were obtained. (1) Statistically significant alterations in retinal taurine, an increase in dark-adapted, and a decrease in light-adapted states, respectively, occurred when frogs were adapted continuously to light or dark for more than 3 weeks. Under the same experimental conditions, no alteration in retinal GABA was noted. (2) At 3 weeks and thereafter, a significant increase of retinal cysteine sulfinic acid decarboxylase (CSD; EC 4.1.1.12) activity, an enzyme involved in the biosynthetic pathway of taurine, also occurred in the dark, whereas the activity in the light-adapted retina was reduced. On the other hand, the retinal activity of L-glutamate decarboxylase (GAD; EC 1.1.1.15), the rate-limiting enzyme of GABA biosynthesis, was not altered in dark- as well as light-adapted state. Similarly, retinal GABA-transaminase (GABA-T; EC 2.6.1.19)-succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.16) was unaltered. (3) These alterations in retinal taurine were, however, unaccompanied by any changes in factors related to transmitter actions such as evoked release, high affinity uptake, and specific binding to synaptic membranes. The above results suggest that, different from GABA as a potent candidate for inhibitory neurotransmitter, retinal taurine may act as neuromodulator and/or may play an important role as a basic factor for maintaining cellular integrity under certain pathophysiological conditions.

Stimulatory effect of taurine on Ca-uptake by disc membranes from photoreceptor cell outer segments

Effects of taurine and related compounds on Ca-uptake by the disc membranes prepared from dark-adapted frog retina were studied. Taurine stimulated ATP-dependent Ca-uptake and the turnover of 45Ca in the disc membranes without affecting basal activity, but it was not observed with the synaptic plasma membranes from rat brain. The stimulatory effect appears to be specific to taurine, since cysteine sulfinic acid, hypotaurine, isethionic acid, β-alanine and γ-aminobutyric acid (GABA) did not stimulate Ca-uptake. The maximal activation, observed at about 30 mM taurine, was about 3 fold, and the Km value for taurine was 10 mM. These results might suggest that taurine modifies translocation of Ca ion in the rod outer segment.

Guanine nucleotide concentrations In vivo in outer segments of dark and light adapted frog retina

Cyclic GMP, GTP, and GDP were measured in outer segments of dark-adapted frog retina, and following exposure to 2 min and 2 hr of light. GTP concentrations decreased after 2 min of light, and there was a concomitant increase in GDP. After 2 hr of light GTP and GDP levels were like those of dark-adapted retinas. Cyclic GMP concentrations decreased to 30% of the values for dark-adapted retinas after 2 min or 2 hr of light exposure. The changes in GTP and GDP were transitory, and apparently associated with light adaptation; the decrease in cyclic GMP occurred within 2 min and remained low at 2 hr, attaining a new steady-state level.

Relation of the e-wave to ganglion cell activity and rod responses in the frog

The e-wave of the electroretinogram was recorded intraretinally from dark-adapted frog eyecups. e-Wave latency was roughly proportional to log stimulus intensity, varying from 2 to 65 sec. The delayed response of ganglion cell units (types 3 and 4) began simultaneously with the e-wave. The e-wave response had similar latencies as the onset of the decay phase of the saturated rod response (determined by recording the mass receptor response and by monitoring b-wave threshold). These results suggest that the e-wave and the delayed ganglion cell response represent closely-linked retinal phenomena which are initiated by rod response decay.