Equivalence of background and bleaching desensitization in isolated rod photoreceptors of the larval tiger salamander

Psychophysical experiments have shown an equivalence between sensitivity reduction by background light and by bleaches for the human scotopic system. We have compared the effects of backgrounds and bleaches on the light-sensitive membrane-current responses of isolated rod photoreceptors from the salamander Ambystoma tigrinum. The quantum catch loss was factored out from the desensitization due to bleaching to give the fraction of "extra" desensitization due to adaptation. For backgrounds, desensitization is well described by the Weber/Fechner equation. The extra desensitization after bleaches can also be described by the Weber/Fechner equation, if an "equivalent" background produced by bleaching is made linearly proportional to the fraction of pigment bleached. A background which produces an extra desensitization of a factor of two is equivalent to a fractional bleach of approximately 6%. Equivalent background and bleaching desensitizations were associated with similar reductions in circulating current. There is a linear relation between log flash sensitivity and decrease in circulating current. Equivalent background and bleaching desensitizations were associated with similar increases in cGMP phosphodiesterase and guanylate cyclase activity. These were inferred from membrane current changes after steps into lithium or IBMX solutions. There were also similar reductions in the integration times of dim flash responses for equivalent desensitizations produced by backgrounds and bleaches. These results suggest that the equivalence between background and bleaching found psychophysically may arise at the very earliest stages of visual processing and that these two processes of desensitization have similar underlying mechanisms.     

Visual pigment bleaching in isolated salamander retinal cones. Microspectrophotometry and light adaptation

Visual pigment bleaching desensitizes rod photoreceptors greatly in excess of that due to loss of quantum catch. Whether this phenomenon also occurs in cone photoreceptors was investigated for isolated salamander red-sensitive cones. In parallel experiments, (a) visual pigment depletion by steps of bleaching light was measured by microspectrophotometry, and (b) flash sensitivity was measured by recording light-sensitive membrane current. In isolated cones, visual pigment bleaching permanently reduced flash sensitivity significantly below that due to the reduction in quantum catch, and there was little spontaneous recovery of visual pigment. The "extra" desensitization due to bleaching was most prominent up to bleaches of approximately 80% visual pigment and reached a level approximately 1 log unit beyond that due to loss of quantum catch. At higher bleaches, the effect of loss of quantum catch became more important. Bleaching did not greatly reduce the maximum light-suppressible membrane current. A 99% reduction of the visual pigment permanently reduced the circulating current by only 30%. Visual pigment bleaching speeded up the kinetics of dim flash responses. All electrical effects of bleaching were reversed on exposure to 11-cis retinal, which probably caused visual pigment regeneration. Light adaptation in photopic vision is known to involve significant visual pigment depletion. The present results indicate that cones operate with a maintained circulating current even after a large pigment depletion. It is shown how Weber/Fechner behavior may still be observed in photopic vision when the contributions of bleaching to adaptation are included.     

Visual pigment and photoreceptor sensitivity in the isolated skate retina

Photoreceptor potentials were recorded extracellularly from the aspartate-treated, isolated retina of the skate (Raja oscellata and R. erinacea), and the effects of externally applied retinal were studied both electrophysiologically and spectrophotometrically. In the absence of applied retinal, strong light adaptation leads to an irreversible depletion of rhodopsin and a sustained elevation of receptor threshold. For example, after the bleaching of 60% of the rhodopsin initially present in dark-adapted receptors, the threshold of the receptor response stabilizes at a level about 3 log units above the dark-adapted value. The application of 11-cis retinal to strongly light-adapted photoreceptors induces both a rapid, substantial lowering of receptor threshold and a shift of the entire intensity-response curve toward greater sensitivity. Exogenous 11-cis retinal also promotes the formation of rhodopsin in bleached photoreceptors with a time-course similar to that of the sensitization measured electrophysiologically. All-trans and 13-cis retinal, when applied to strongly light-adapted receptors, fail to promote either an increase in receptor sensitivity or the formation of significant amounts of light-sensitive pigment within the receptors. However, 9-cis retinal isin. These findings provide strong evidence that the regeneration of visual pigment in the photoreceptors directly regulates the process of photochemical dark adaptation.     

Adaptation in Skate Photoreceptors

Receptor potentials were recorded extracellularly from the all-rod retina of the skate after the application of sodium aspartate. This agent suppresses the responses of proximal elements, but leaves relatively unaffected the electrical activity of the photoreceptors (a-wave) and pigment epithelium (c-wave). Since the latter develops too slowly to interfere with the receptor response, it was possible to isolate receptor potentials and to compare their behavior in light and dark adaptation with earlier observations on the S-potential, b-wave, and ganglion cell discharge. The results show that the photoreceptors display the full complement of adaptational changes exhibited by cells proximal to the receptors. Thus, it appears that visual adaptation in the skate is governed primarily by the photoreceptors themselves. Of particular interest was the recovery of sensitivity in the presence of background fields that initially saturate the receptor potential. Analysis of this recovery phase indicates that a gain-control mechanism operates within the receptors, at a distal stage of the visual process.     

Peroxidase uptake by photoreceptor terminals of the skate retina

The photoreceptors of dark-adapted skate retinas bathed in a Ringer solution containing horseradish peroxidase (HRP) incorporate the tracer into membrane-bound compartments within the synaptic terminal of the cell; after 1 or 2 h of incubation, approx. 10-38% of the synaptic vesicles were labeled. The receptors appeared to be functioning normally throughout the incubation period, since electrical potentials of normal amplitude could be elicited in response to dimphotic stimuli. However, it was possible to block the uptake of peroxidase by a regimen of light adaptation that effectively suppressed light-induced activity in the electroretinogram. If, during incubation with peroxidase, retinas were exposed at 10-min intervals to an intense 1-ms flash from a xenon discharge tube, the receptor terminals were almost completely devoid of peroxidase; fewer than 2% of the vesicles were labeled. The suppression of HRP uptake could also be achieved in dark-adapted retinas by adding magnesium to the bathing solution, suggesting that calcium is necessary for transmitter release from vesicles in the receptor terminals. These findings are consistent with the view that vertebrate photoreceptors discharge a neurotransmitter in darkness, and that light decreases the release of this substance. It seems likely that the incorporation of peroxidase into vesicles of physiologically active receptor terminals reflects a mechanism for the retrieval of vesicle membrane after exocytosis.     

Light adaptation in retinal rods of the rabbit and two other nonprimate mammals

The responses of rabbit rods to light were studied by drawing a single rod outer segment projecting from a small piece of retina into a glass pipette to record membrane current. The bath solution around the cells was maintained at near 40 degrees C. Light flashes evoked transient outward currents that saturated at up to approximately 20 pA. One absorbed photon produced a response of approximately 0.8 pA at peak. At the rising phase of the flash response, the relation between response amplitude and flash intensity (IF) had the exponential form 1-e-kappa FIF (where kappa F is a constant denoting sensitivity) expected from the absence of light adaptation. At the response peak, however, the amplitude-intensity relation fell slightly below the exponential form. At times after the response peak, the deviation was progressively more substantial. Light steps evoked responses that rose to a transient peak and rapidly relaxed to a lower plateau level. The response-intensity relation again indicated that light adaptation was insignificant at the early rising phase of the response, but became progressively more prominent at the transient peak and the steady plateau of the response. Incremental flashes superposed on a steady light of increasing intensity evoked responses that had a progressively shorter time-to-peak and faster relaxation, another sign of light adaptation. The flash sensitivity changed according to the Weber-Fechner relation (i.e., inversely) with background light intensity. We conclude that rabbit rods adapt to light in a manner similar to rods in cold-blooded vertebrates. Similar observations were made on cattle and rat rods.     

Visual Adaptation in the Retina of the Skate

The electroretinogram (ERG) and single-unit ganglion cell activity were recorded from the eyecup of the skate (Raja erinacea and R. oscellata), and the adaptation properties of both types of response compared with in situ rhodopsin measurements obtained by fundus reflectometry. Under all conditions tested, the b-wave of the ERG and the ganglion cell discharge showed identical adaptation properties. For example, after flash adaptation that bleached 80% of the rhodopsin, neither ganglion cell nor b-wave activity could be elicited for 10–15 min. Following this unresponsive period, thresholds fell rapidly; by 20 min after the flash, sensitivity was within 3 log units of the dark-adapted level. Further recovery of threshold was slow, requiring an additional 70–90 min to reach absolute threshold. Measurements of rhodopsin levels showed a close correlation with the slow recovery of threshold that occurred between 20 and 120 min of dark adaptation; there is a linear relation between rhodopsin concentration and log threshold. Other experiments dealt with the initial unresponsive period induced by light adaptation. The duration of this unresponsive period depended on the brightness of the adapting field; with bright backgrounds, suppression of retinal activity lasted 20–25 min, but sensitivity subsequently returned and thresholds fell to a steady-state value. At all background levels tested, increment thresholds were linearly related to background luminance.     

Desensitization of skate photoreceptors by bleaching and background light

Through extracellular measurements of photoreceptor responses to flashed stimuli, we examined how the bleaching of rhodopsin affects increment receptor threshold in the isolated retina of the skate (Raja oscellata and R. erinacea). Both initially unbleached and previously bleached photoreceptors, when exposed to full-field luminous backgrounds of fixed intensity, attain approximately stable levels of increment threshold that vary with the intensity of the background light. Values of stabilized increment thresholds measured after various extents of bleaching (less than approximately 50%), when plotted against background intensity in log-log coordinates, tend to converge with increasing intensity of the background; this relationship of the increment threshold functions resembles that which Blakemore and Rushton (1965b) found to describe the transient effect of bleaching on psychophysical increment threshold for the human rod mechanism. Our data are consistent with the possibility that related photochemical processes govern the stabilized levels of receptor sensitivity exhibited by the isolated retina (a) during steady illumination and (b) long after substantial bleaching.     

The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20-3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity approximately 30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different intensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the inactivation of activated phosphodiesterase (E*, representing the complex G(alpha)-PDE of phosphodiesterase with the transducin alpha-subunit); or (3) the hydrolysis of cGMP, with rate constant beta. Our measurements show that, over the range of background intensities we used, beta increased on average to approximately 20 times its dark-adapted value; and our theoretical analysis indicates that this increase in beta is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.     

Physiological roles of Na+/Ca2+ exchange in Limulus ventral photoreceptors

In previous work we have presented evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors (1989. J. Gen. Physiol. 93:473-492). This article assesses the contributions to photoreceptor physiology from Na+/Ca2+ exchange. Four separate physiological processes were considered: maintenance of resting sensitivity, light-induced excitation, light adaptation, and dark adaptation. (a) Resting sensitivity: reduction of [Na+]o caused a [Ca2+]o-dependent reduction in light sensitivity and a speeding of the time courses of the responses to individual test flashes; this effect was dependent on the final value to which [Na+]o was reduced. The desensitization caused by Na+ reduction was dependent on the initial sensitivity of the photoreceptor; in fully dark-adapted conditions no desensitization was observed; in light-adapted conditions, extensive desensitization was observed. (b) Excitation: Na+ reduction in fully dark-adapted conditions caused a Ca2+o-dependent depolarizing phase in the receptor potential that persisted beyond the stimulus duration and was evoked by a bright adapting flash. (c) Light adaptation: the degree of desensitization induced by a bright adapting flash was Na+o dependent, being larger with lower [Na+]o. Na+ reduction enhanced light adaptation only at intensities brighter than 4 x 10(-6) W/cm2. In addition to being Na+o dependent, light adaptation was Ca2+o dependent, being greater at higher [Ca2+]o. (d) Dark adaptation: the recovery of light sensitivity after adapting illumination was Na+o dependent. Dark adaptation after bright illumination in voltage-clamped and in unclamped conditions was faster in normal-Na+ saline than in reduced Na+ saline. The final sensitivity to which photoreceptors recovered was lower in reduced-Na+ saline when bright adapting illumination was used. The results suggest the involvement of Na+/Ca2+ exchange in each of these physiological processes. Na+/Ca2+ exchange may contribute to these processes by counteracting normal elevations in [Ca2+]i.     

Light Adaptation in the Ventral Photoreceptor of Limulus

Light adaptation in both the ventral photoreceptor and the lateral eye photoreceptor is a complex process consisting of at least two phases. One phase, which we call the rapid phase of adaptation, occurs whenever there is temporal overlap of the discrete waves that compose a light response. The recovery from the rapid phase of adaptation follows an exponential time-course with a time constant of approximately 75 ms at 21°C. The rapid phase of adaptation occurs at light intensities barely above discrete wave threshold as well as at substantially higher light intensities with the same recovery time-course at all intensities. It occurs in voltage-clamped and unclamped photoreceptors. The kinetics of the rapid phase of adaptation is closely correlated to the photocurrent which appears to initiate it after a short delay. The rapid phase of adaptation is probably identical to what is called the "adapting bump" process. At light intensities greater than about 10 times discrete wave threshold another phase of light adaptation occurs. It develops slowly over a period of ½ s or so, and decays even more slowly over a period of several seconds. It is graded with light intensity and occurs in both voltage-clamped and unclamped photoreceptors. We call this the slow phase of light adaptation.     

Role of intracellular calcium and sodium in light adaptation in the retina of the honey bee drone (Apis mellifera, L)

In the honey bee drone, the decrease in sensitivity to light of a retinula cell exposed to background illumination was found to be accurately reflected by the difference in amplitude between the initial transient depolarization and the lowest steady depolarization evoked by the background light. It is shown that both the decrease in sensitivity to light and the accompanying drop in potential from the transient to the plateau can be prevented by injecting EGTA intracellularly. A decrease in duration and amplitude of responses to short test flashes such as observed immediately after illumination was found to occur too when Ca or Na, but not K, Li, or Mg injected into dark-adapted retinula cells. Injection of EGTA into a retinula cell maintained a steady state of light adaptation, was found to cause an increase in amplitude and duration of the response to a short test flash, thus producing the effects of dark adaptation. It is suggested that, in the retina of the honey bee drone, an increase in intracellular calcium concentration plays a central role in light adaptation and that an increase in intracellular sodium concentration, resulting from the influx of sodium ions during the responses to light, could lead to this increase in intracellular free calcium.     

Relationship between light sensitivity and intracellular free Ca concentration in Limulus ventral photoreceptors. A quantitative study using Ca-selective microelectrodes

The possible role of Ca ions in mediating the drop in sensitivity associated with light adaptation in Limulus ventral photoreceptors was assessed by simultaneously measuring the sensitivity to light and the intracellular free Ca concentration (Cai); the latter was measured by using Ca-selective microelectrodes. In dark-adapted photoreceptors, the mean resting Cai was 3.5 +/- 2.5 microM SD (n = 31). No correlation was found between resting Cai and absolute sensitivity from cell to cell. Typically, photoreceptors are not uniformly sensitive to light; the Cai rise evoked by uniform illumination was 20-40 times larger and faster in the most sensitive region of the cell (the rhabdomeral lobe) than it was away from it. In response to a brief flash, the Cai rise was barely detectable when 10(2) photons were absorbed, and it was saturated when approximately 10(5) photons were absorbed. During maintained illumination, starting near the threshold of light adaptation, steady Cai increases were associated with steady desensitizations over several log units of light intensity: a 100-fold desensitization was associated with a 2.5-fold increase in Cai. After a bright flash, sensitivity and Cai recovered with different time courses: the cell was still desensitized by approximately 0.5 log units when Cai had already recovered to the prestimulus level, which suggests that under those conditions Cai is not the rate-limiting step of dark adaptation. Ionophoretic injection of EGTA markedly decreased the light-induced Cai rise and increased the time to peak of the light response, but did not alter the resting Cai, which suggests that the time to peak is affected by a change in the capacity to bind Ca2+ and not by resting Cai. Lowering the extracellular Ca2+ concentration (Cao) first decreased Cai and increased sensitivity. Longer exposure to low Cao resulted in a further decrease of Cai but decreased rather than increased sensitivity, which suggests that under certain conditions it is possible to uncouple Cai and sensitivity.     

Dim light adaptation attenuates acute melatonin suppression in humans

Abstract Studies in rodents with retinal degeneration indicated that neither the rod nor the cone photoreceptors obligatorily participate in circadian responses to light, including melatonin suppression and photoperiodic response. Yet there is a residual phase-shifting response in melanopsin knockout mice, which suggests an alternate or redundant means for light input to the SCN of the hypothalamus. The findings of Aggelopoulos and Meissl suggest a complex, dynamic interrelationship between the classic visual photoreceptors and SCN cell sensitivity to light stimuli, relative to various adaptive lighting conditions. These studies raised the possibility that the phototransductive physiology of the retinohypothalamic tract in humans might be modulated by the visual rod and cone photoreceptors. The aim of the following two-part study was to test the hypothesis that dim light adaptation will dampen the subsequent suppression of melatonin by monochromatic light in healthy human subjects. Each experiment included 5 female and 3 male human subjects between the ages of 18 and 30 years, with normal color vision. Dim white light and darkness adaptation exposures occurred between midnight and 0200 h, and a full-field 460-nm light exposure subsequently occurred between 0200 and 0330-h for each adaptation condition, at 2 different intensities. Plasma samples were drawn following the 2-h adaptation, as well as after the 460-nm monochromatic light exposure, and melatonin was measured by radioimmunoassay. Comparison of melatonin suppression responses to monochromatic light in both studies revealed a loss of significant suppression after dim white light adaptation compared with dark adaptation (p < 0.04 and p < 0.01). These findings indicate that the activity of the novel circadian photoreceptive system in humans is subject to subthreshold modulation of its sensitivity to subsequent monochromatic light exposure, varying with the conditions of light adaptation prior to exposure.     

Cellular mechanisms that underlie bleaching and background adaptation.

Experiments were performed on rod photoreceptors isolated from the eye of the larval tiger salamander to determine if the same or different mechanisms underlie the desensitization produced by dim background light (background adaptation) and that which persists in the steady state in darkness after a significant fraction of the photopigment is bleached (bleaching adaptation). We have examined adaptational effects after light that bleached between approximately 50% and 95% of the photopigment under conditions which preclude pigment regeneration. The steady-state desensitization, far greater than that predicted by quantum-catch loss, is relieved upon regeneration of the visual pigment with 11-cis retinal. We measured the spread of desensitization along the long axis of the rod after a local bright bleach at one end by comparing responses to dim local test flashes elicited in different regions of the outer segment, before and after bleaching. The space constant for this spread was less than 2.5 microns. We have previously measured the space constant for the longitudinal spread of desensitization during a local dim background in Ambystoma rods to be 7 microns. This is similar to a space constant of 6 microns measured under similar conditions in Bufo rods by Lamb et al. (1981. J. Physiol. 319:463-496). If calcium carries the signal for background desensitization, this difference in space constant for background and bleaching adaptation precludes it as the messenger for the steady component of bleaching adaptation. Experiments with isobutylmethyl xanthine (IBMX) also indicate that Ca2+ as well as c-GMP are unlikely regulators of bleaching desensitization, since elevation of cytosolic levels of both of these internal messengers by IBMX has little effect on sensitivity in bleach-adapted cells. All of our findings are consistent with the notion that bleaching adaptation is not mediated by a freely diffusible cytoplasmic messenger.     

Retinal mechanisms of visual adaptation in the skate

Electrical potentials were recorded from different levels within the skate retina. Comparing the adaptive properties of the various responses revealed that the isolated receptor potential and the S-potential always exhibited similar changes in sensitivity, and that the b-wave and ganglion-cell thresholds acted in concert. However, the two sets of responses behaved differently under certain conditions. For example, a dimly iluminated background that had no measurable effect on the senitivities of either of the distal responses, raised significantly the thresholds of both the b-wave and the ganglion cell responses. In addition, the rate of recovery during the early, "neural" phase of dark adaptation was significantly faster for the receptor and S-potentials than for the b-wave or ganglion cell discharge. These results indicate that there is an adaptive ("network") mechanism in the retina which can influence significantly b-wave and gaglion cell activity and which behaves independently of the receptors and horizontal cells. We conclude that visual adaptation in the skate retina is regulated by a combination of receptoral and network mechanisms.     

A quantitative comparison of the effects of intracellular calcium injection and light adaptation on the photoresponse of Limulus ventral photoreceptors

Calcium ions were iontophoretically injected into ventral photoreceptors of Limulus by passing current between two intracellular pipettes. Changes in sensitivity and photoresponse time course were measured for both light adaptation and Ca++ injection. We found for some photoreceptors that there was no significant difference in the photoresponse time course for desensitization produced by light adaptation or by Ca++ injection. In other photoreceptors, the time delay of photoresponse for Ca++ injection was slightly longer than for light adaptation. The variability of threshold response amplitude and time delay decreases when the photoreceptor is desensitized by either light adaptation or Ca++ injection. The peak amplitude versus log stimulus intensity relationships for controls, light adaptation, and Ca++ injection all could be described very closely by a single template curve shifted along the log intensity axis. A 40- to 50-fold change in sensitivity is associated with a 2-fold change in photoresponse time delay for both light adaptation and Ca++ injection.     

Local adaptation in the ventral photoreceptors of Limulus

Local adaptation was demonstrated in the ventral photoreceptors of Lumulus using either flashes or continuous illumination. Spots of light locally desensitized the region of the photoreceptor on which they were focused. In dark-adapted photoreceptors where "quantum bumps" were clearly discernible, local adaptation of the quantum bumps was observed. Local adaptation could induce differences of threshold of 1 decade over distances of 50-80 mum. With continuous local illumination these gradients could be maintained from 2 s to 30 min. In addition, the decrease in time scale associated with light adaptation was also found to be localized to the region of illumination.     

S-Potentials in the Skate Retina : Intracellular recordings during light and dark adaptation

The S-potentials recorded intracellularly from the all-rod retina of the skate probably arise from the large horizontal cells situated directly below the layer of receptors. These cells hyperpolarize in response to light, irrespective of stimulus wavelength, and the responses in photopic as well as scotopic conditions were found to be subserved by a single photopigment with λ_max = 500 nm. The process of adaptation was studied by recording simultaneously the threshold responses and membrane potentials of S-units during both light and dark adaptation. The findings indicate that the sensitivity of S-units, whether measured upon steady background fields or in the course of dark adaptation, exhibits changes similar to those demonstrated previously for the ERG b-wave and ganglion cell discharge. However, the membrane potential level of the S-unit and its sensitivity to photic stimulation varied independently for all the adapting conditions tested. It appears, therefore, that visual adaptation in the skate retina occurs before the S-unit is reached, i.e., at the receptors themselves.     

鲤属鱼L-型外水平细胞所接收的锥细胞信号

在保持正常血液循环的眼杯标本上,用细胞内微电极技术记录了 L-型外水平细胞(LHC)的反应,并且用颜色光适应方法分析了它所接收的锥细胞输入。结果表明,在白光明适应条件下,LHC 依然接收红敏和绿敏锥细胞的输入,但两者的相对贡献与暗视时有所不同。用蓝适应光可以基本上分离红敏锥细胞的信号;而在红色背景光下,绿敏锥细胞主导了反应的上升相和撤光后回跳相(off-rebound),但在反应平台处却是红敏锥细胞的信号作出主要贡献,表明不同的锥细胞信号显然有不同的潜伏期和时程。