Rejection of the biophoton hypothesis on the origin of photoreceptor dark noise.

Rod photoreceptors of the vertebrate retina produce, in darkness, spontaneous discrete current waves virtually identical to responses to single photons. The waves comprise an irreducible source of noise (discrete dark noise) that may limit the threshold sensitivity of vision. The waves obviously originate from acts of random activation of single rhodopsin molecules. Until recently, it was generally accepted that the activation occurs due to the rhodopsin thermal motion. Yet, a few years ago it was proposed that rhodopsin molecules are activated not by heat but rather by real photons generated within the retina by chemiluminescence. Using a high-sensitive photomultiplier, we measured intensities of biophoton emission from isolated retinas and eyecups of frogs (Rana ridibunda) and fish (sterlet, Acipenser ruthenus). Retinal samples were placed in a perfusion chamber and emitted photons collected by a high-aperture quartz lens. The collected light was sent to the photomultiplier cathode through a rotating chopper so that a long-lasting synchronous accumulation of the light signal was possible. The absolute intensity of bio-emission was estimated by the response of the measuring system to a calibrated light source. The intensity of the source, in turn, was quantified by measuring rhodopsin bleaching with single-rod microspectrophotometry. We also measured the frequency of discrete dark waves in rods of the two species with suction pipette recordings. Expressed as the rate constant of rhodopsin activation, it was 1.2 × 10-11/s in frogs and 7.6 × 10-11/s in sterlets. Approximately two thirds of retinal samples of each species produced reliably measurable biophoton emissions. However, its intensity was ≥100 times lower than necessary to produce the discrete dark noise. We argue that this is just a lower estimate of the discrepancy between the hypothesis and experiment. We conclude that the biophoton hypothesis on the origin of discrete dark noise in photoreceptors must be rejected.

Origins of the phototransduction delay as inferred from stochastic and deterministic simulation of the amplification cascade.

PURPOSE: To identify steps of the phototransduction cascade responsible for the delay of the photoresponse. METHODS: Electrical responses of fish (Carassius) cones and Rana ridibunda frog rods and cones were recorded with a suction pipette technique and as an aspartate-isolated mass receptor potential from isolated perfused retinas. Special attention was paid to sufficiently high temporal resolution (1-ms flash, 700 Hz amplification bandpass). Stochastic simulation of the activation steps from photon absorption to the formation of catalytically active phosphodiesterase (PDE) was performed. In addition, a deterministic mathematical model was fit to the experimental responses. The model included a detailed description of the activation steps of the cascade that enabled identification of the role of individual transduction stages in shaping the initial part of the response. RESULTS: We found that the apparent delay of the photoresponse gets shorter with increasing stimulus intensity and reaches an asymptotic value of approximately 3 ms in cones and greater than or equal to 10 ms in rods. The result seems paradoxical since it is suggested that the delay occurs in the chain of steps from photon absorption to the formation of active transducin (T*) which in cones is, on average, slower than in rods. Stochastic simulation shows that actually the steps from photon absorption to T* may not contribute perceptibly to the delay. Instead, the delay occurs at the stage that couples the cycle of repetitive activation of T by rhodopsin (R*) with the activation of PDE. These steps include formation of T* (= T α GTP) out of T αßγ GTP released from the activation cycle and the subsequent interaction of T* with PDE. This poses a problem. The duration of an average cycle of activation of T in rods is approximately 5 ms and is determined by the frequency of collisions between R* and T in the photoreceptor membrane. The frequency is roughly proportional to the surface packing density of T in the membrane. As the packing density of PDE is approximately 12 times lower than that of T, it could be expected that the rate of the T*-PDE interaction were an order of magnitude slower than that of R* and T. As modeling shows, this is the case in rods. However, the delay in cones is approximately 3 ms which could be achieved only at a T*-PDE interaction time of less than or equal to 5 ms. This means that either the frequency of the collisions of T* and PDE, or the efficiency of collisions, or both in cones are approximately ten times higher than in rods. This may be a challenge to the present model of the molecular organization of the photoreceptor membrane. CONCLUSIONS: The delay of the photoresponse is mainly set by the rate of interaction of T* with PDE. In cones, the delay is shorter than in rods and, moreover, shorter than the duration of the cycle of repetitive activation of T by R*. This poses a problem for the present model of diffusion interaction of phototransduction proteins in the photoreceptor membrane.

Elevated cAMP improves signal-to-noise ratio in amphibian rod photoreceptors.

The absolute sensitivity of vertebrate retinas is set by a background noise, called dark noise, which originates from several different cell types and is generated by different molecular mechanisms. The major share of dark noise is produced by photoreceptors and consists of two components, discrete and continuous. Discrete noise is generated by spontaneous thermal activations of visual pigment. These events are undistinguishable from real single-photon responses (SPRs) and might be considered an equivalent of the signal. Continuous noise is produced by spontaneous fluctuations of the catalytic activity of the cGMP phosphodiesterase. This masks both SPR and spontaneous SPR-like responses. Circadian rhythms affect photoreceptors, among other systems by periodically increasing intracellular cAMP levels ([cAMP]in), which increases the size and changes the shape of SPRs. Here, we show that forskolin, a tool that increases [cAMP]in, affects the magnitude and frequency spectrum of the continuous and discrete components of dark noise in photoreceptors. By changing both components of rod signaling, the signal and the noise, cAMP is able to increase the photoreceptor signal-to-noise ratio by twofold. We propose that this results in a substantial improvement of signal detection, without compromising noise rejection, at the rod bipolar cell synapse.

Why do green rods of frog and toad retinas look green?

Amphibian "green" rods express a blue-sensitive cone visual pigment, and should look yellow. However,when observing them axially under microscope one sees them as green. We used single-cell microspectrophotometry (MSP) to reveal the basis of the perceived color of these photoreceptors. Conventional side-on MSP recording of the proximal cell segments reveals no selective longwave absorbing pigment explaining the green color. End-on MSP recording shows, in addition to the green rod visual pigment, an extra 2- to 4-fold attenuation being almost flat throughout the visible spectrum. This attenuation is absent in red (rhodopsin) rods, and vanishes in green rods when the retina is bathed in high-refractive media, and at wide illumination aperture. The same treatments change the color from green to yellow. It seems that the non-visual pigment attenuation is a result of slender green rod myoids operating as non-selective light guides. We hypothesize that narrow myoids, combined with photomechanical movements of melanin granules, allow a wide range of sensitivity regulation supporting the operation of green rods as blue receptors at mesopic-to low-photopic illumination levels.End-on transmittance spectrum of green rods looks similar to the reflectance spectrum of khaki military uniforms. So their greenness is the combined result of optics and human color vision.

cAMP controls rod photoreceptor sensitivity via multiple targets in the phototransduction cascade.

In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide-gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca(2+) exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca(2+)](in). Analysis by a complete model of rod phototransduction suggests that an increase of [Ca(2+)](in) might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca(2+)](in) and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions.

G-protein betagamma-complex is crucial for efficient signal amplification in vision.

A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. Although much is now known about the role of the α-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary ßγ-complex in this process remains a mystery. Here, we show that elimination of the transducin γ-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin ßγ-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions.

Lateral diffusion of rhodopsin in photoreceptor membrane: a reappraisal.

PURPOSE: In a series of works between 1972 and 1984, it was established that rhodopsin undergoes rotational and lateral Brownian motion in the plane of photoreceptor membrane. The concept of free movement of proteins of phototransduction cascade is an essential principle of the present scheme of vertebrate phototransduction. This has recently been challenged by findings that show that in certain conditions rhodopsin in the membrane may be dimeric and form extended areas of paracrystalline organization. Such organization seems incompatible with earlier data on free rhodopsin diffusion. Thus we decided to reinvestigate lateral diffusion of rhodopsin and products of its photolysis in photoreceptor membrane specifically looking for indications of possible oligomeric organization. METHODS: Diffusion exchange by rhodopsin and its photoproducts between bleached and unbleached halves of rod outer segment was traced using high-speed dichroic microspectrophotometer. Measurements were conducted on amphibian (frog, toad, and salamander) and gecko rods. RESULTS: We found that the curves that are supposed to reflect the process of diffusion equilibration of rhodopsin in nonuniformly bleached outer segment largely show production of long-lived bleaching intermediate, metarhodopsin III (Meta III). After experimental elimination of Meta III contribution, we observed rhodopsin equilibration time constant was threefold to tenfold longer than estimated previously. However, after proper correction for the geometry of rod discs, it translates into generally accepted value of diffusion constant of approximately 5 x 10(-9) cm(2) s(-1). Yet, we found that there exists an immobile rhodopsin fraction whose size can vary from virtually zero to 100%, depending on poorly defined factors. Controls suggest that the formation of the immobile fraction is not due to fragmentation of rod outer segment discs but supposedly reflects oligomerization of rhodopsin. CONCLUSIONS: Implications of the new findings for the present model of phototransduction are discussed. We hypothesize that formation of paracrystalline areas, if controlled physiologically, could be an extra mechanism of cascade regulation.

The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones.

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore-opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.

Kinetics of turn-offs of frog rod phototransduction cascade.

The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs. The two processes are among the key factors that set the speed and sensitivity of the photoresponse and whose regulation contributes to light adaptation. The aim of this study was to determine time courses of flash-induced PDE activity in frog rods that were dark adapted or subjected to nonsaturating steady background illumination. PDE activity was computed from the responses recorded from solitary rods with the suction pipette technique in Ca(2+)-clamping solution. A flash applied in the dark-adapted state elicits a wave of PDE activity whose rising and decaying phases have characteristic times near 0.5 and 2 seconds, respectively. Nonsaturating steady background shortens both phases roughly to the same extent. The acceleration may exceed fivefold at the backgrounds that suppress approximately 70% of the dark current. The time constant of the process that controls the recovery from super-saturating flashes (so-called dominant time constant) is adaptation independent and, hence, cannot be attributed to either of the processes that shape the main part of the PDE wave. We hypothesize that the dominant time constant in frog rods characterizes arrestin binding to rhodopsin partially inactivated by phosphorylation. A mathematical model of the cascade that considers two-stage rhodopsin quenching and transducin inactivation can mimic experimental PDE activity quite well. The effect of light adaptation on the PDE kinetics can be reproduced in the model by concomitant acceleration on both rhodopsin phosphorylation and transducin turn-off, but not by accelerated arrestin binding. This suggests that not only rhodopsin but also transducin shut-off is under adaptation control.

Visual cycle: Dependence of retinol production and removal on photoproduct decay and cell morphology.

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

The identity of metarhodopsin III.

A fast-scanning dichroic microspectrophotometer was used to trace products of rhodopsin photolysis (metarhodopsins I/II/III and later) in structurally intact amphibian rod outer segments (ROSs) and metabolically active rods. The instrument allows the recording of absorbance spectra with a time resolution better than 1 s, and to discriminate between products with similar absorbance spectra that differ with respect to the orientation of their chromophore in the photoreceptor membrane. We demonstrate that metarhodopsin III is in a pH-reversible equilibrium with metarhodopsin II and that the metarhodopsin III chromophore is orientated with respect to the membrane plane even more strictly than the 11-cis retinal in "dark" rhodopsin. This indicates that all-trans retinal in metarhodopsin III is still attached to its native binding site on opsin. The kinetic scheme of the decay of metarhodopsins is presented in which metarhodopsin III lies in a shunt pathway from metarhodopsin II to retinal. Formation of metarhodopsin III was detected at bleaches as low as approximately 3%, contrary to previous reports that it is not formed at below 10% bleaches. Another product that is spectrally similar to metarhodopsin III, termed P440, appears at later stages of photolysis as the result of the decay of metarhodopsin II and metarhodopsin III. The chromophoric group in P440 is orientated preferentially across the disk membrane. The final product(s) in isolated ROS, where the reduction of retinal to retinol is blocked, consists of a mixture of a free retinal and retinal possibly attached to different binding sites in the membrane. In metabolically active rods the later products are quickly converted to retinol. We conclude that metarhodopsin III represents a specific conformational state of metarhodopsin where the chromophoric binding site is still occupied by all-trans retinal. Hence, the formation and decay of metarhodopsin III may be limiting for the rate of rhodopsin regeneration and photoreceptor dark adaptation.

pH and rate of "dark" events in toad retinal rods: test of a hypothesis on the molecular origin of photoreceptor noise.

Thermal activation of the visual pigment constitutes a fundamental constraint on visual sensitivity. Its electrical correlate in the membrane current of dark-adapted rods are randomly occurring discrete "dark events" indistinguishable from responses to single photons. It has been proposed that thermal activation occurs in a small subpopulation of rhodopsin molecules where the Schiff base linking the chromophore to the protein part is unprotonated. On this hypothesis, rates of thermal activation should increase strongly with rising pH. The hypothesis has been tested by measuring the effect of pH changes on the frequency of discrete dark events in red rods of the common toad Bufo bufo. Dark noise was recorded from isolated rods using the suction pipette technique. Changes in cytoplasmic pH upon manipulations of extracellular pH were quantified by measuring, using fast single-cell microspectrophotometry, the pH-dependent metarhodopsin I-metarhodopsin II equilibrium and subsequent metarhodopsin III formation. These measurements show that, in the conditions of the electrophysiological experiments, changing perfusion pH from 6.5 to 9.3 resulted in a cytoplasmic pH shift from 7.6 to 8.5 that was readily sensed by the rhodopsin. This shift, which implies an 8-fold decrease in cytoplasmic [H(+)], did not increase the rate of dark events. The results contradict the hypothesis that thermal pigment activation depends on prior deprotonation of the Schiff base.

Two temporal phases of light adaptation in retinal rods.

Vertebrate rod photoreceptors adjust their sensitivity as they adapt during exposure to steady light. Light adaptation prevents the rod from saturating and significantly extends its dynamic range. We examined the time course of the onset of light adaptation in bullfrog rods and compared it with the projected onset of feedback reactions thought to underlie light adaptation on the molecular level. We found that adaptation developed in two distinct temporal phases: (1) a fast phase that operated within seconds after the onset of illumination, which is consistent with most previous reports of a 1-2-s time constant for the onset of adaptation; and (2) a slow phase that engaged over tens of seconds of continuous illumination. The fast phase desensitized the rods as much as 80-fold, and was observed at every light intensity tested. The slow phase was observed only at light intensities that suppressed more than half of the dark current. It provided an additional sensitivity loss of up to 40-fold before the rod saturated. Thus, rods achieved a total degree of adaptation of approximately 3,000-fold. Although the fast adaptation is likely to originate from the well characterized Ca(2+)-dependent feedback mechanisms regulating the activities of several phototransduction cascade components, the molecular mechanism underlying slow adaptation is unclear. We tested the hypothesis that the slow adaptation phase is mediated by cGMP dissociation from noncatalytic binding sites on the cGMP phosphodiesterase, which has been shown to reduce the lifetime of activated phosphodiesterase in vitro. Although cGMP dissociated from the noncatalytic binding sites in intact rods with kinetics approximating that for the slow adaptation phase, this hypothesis was ruled out because the intensity of light required for cGMP dissociation far exceeded that required to evoke the slow phase. Other possible mechanisms are discussed.