Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

10–15 ms. (Note that the turnover time for cyclic guanosine monophosphate (cGMP), tcG, listed in Table 1 represents the value applicable during very intense illumination; under dark-adapted conditions, when the light-induced activity of the phosphodiesterase (PDE) is relatively low, this time constant is likely to be much longer.)

The shut-off time constants for the activated visual pigment (R*) and the activated G-protein/PDE (E*), tR and tE, are around 20-fold shorter in human cones than in mouse rods, with values of 80 and 200 ms having been reported in rods.

Cone Avoidance of Saturation

Work done in collaboration with Edward N. Pugh, Jr. has shown that the ability of the cones to avoid saturation is explicable in terms of the combination of these two 20-fold shorter time constants and the bleaching of cone visual pigment.

In human rod photoreceptors in vivo, the circulating current is halved at a steady intensity of 70 scotopic trolands (600 R* s 1), with complete saturation occurring at 1000 scotopic trolands ( 104 R* s 1). If the activation gain of transduction is the same in human cones as in human rods, then the two very short cone time constants would elevate the intensities required for half and full saturation by some 400 , to levels of 240 000 and4 106 R* s 1 in cones. An additional factor is that the cGMP-gated channels of mammalian cones (in contrast to those of rods) show increased cGMP-binding affinity when Ca2+ falls, thereby further increasing the R* rate required for saturation.

How do these estimated rates of isomerization compare with the maximum rate at which the cone visual pigment can be bleached during steady illumination? At steady state, the rate of photoisomerization equals the rate of pigment regeneration, which is set by the delivery of 11-cis retinal to opsin. For human L/M cones, the maximal rate of regeneration has been measured as45% min–1, or 0.75% s–1. If the outer segment contains40 million pigment molecules, then the maximal rate of photoisomerization during intense steady light will be300 000 R* s–1. This rate cannot be exceeded in the steady state because a higher rate of isomerization would lead to such a low level of cone pigment available to absorb light that the rate could simply not be maintained.

Hence, from the numbers in the preceding paragraphs, the rate of photoisomerization required to saturate the human cone current exceeds the highest rate of isomerization of cone pigment molecules ( 300 000 R* s 1) that can be elicited by a steady light of arbitrarily high intensity. Therefore, the human cone photoreceptor cannot be saturated by steady lights, no matter how bright they are. This is not to say that the cone can never be saturated;

if an intense light is presented from dark-adapted conditions (when the cone initially has a full complement of visual pigment), then it will transiently be driven into saturation until bleaching reduces the amount of visual pigment to a suitably low level.

Modeling of Human Cone Light Adaptation

A computational model of human cone light adaptation has been developed by Hans van Hateren and Herman Snippe, which puts factors of the type described in the preceding section into a comprehensive theoretical/ numerical model. They take a molecular description of the steps in cone phototransduction closely similar to that illustrated in Figure 5, including pigment bleaching, and

Figure 5 Reaction steps underlying the cone’s rapid recovery. Visual pigment (R) is formed by delivery of 11-cis retinal (11-cis RAL). Light (of intensity I) activates the visual pigment, and the activated pigment (R*) is inactivated with a time constant tR. R* activates the guanine-nucleotide-binding G protein to G*, which then binds to phosphodiesterase (E) to form active G*–E* (E*). The active G*–E* complex (E*) has a lifetime tE. E* hydrolyzes cGMP (cG) with a rate constant b. The increase in b is proportional to steady intensity I, and to the product of the R* and E* lifetimes; that is, b = bDark + (A/ncG) tR tE I (see Nikonov, S., Lamb, T. D., and Pugh, E. N., Jr. (2000). The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse. Journal of General Physiology 116: 795 824). cGMP is formed by guanylyl cyclase (GC), under regulation by the Ca2+-sensitive guanylyl-cyclase- activating proteins (GCAPs). When present, cG causes opening of ion channels (chans) in the plasma membrane, admitting Ca2+. Ca2+ has a powerful negative-feedback action through GCAPs onto the rate a of cGMP synthesis. Ca2+ is removed from the cytoplasm with a time constant tCa. Reproduced from Lamb,

T. D. and Pugh, E. N., Jr. (2006). Avoidance of saturation in human cones is explained by very rapid inactivation reactions and pigment bleaching. Investigative Ophthalmology and Visual Science 47, E-Abstract 3714, with permission from the Association for Research in Vision and Ophthalmology.

they express the system in terms of differential equations. Their simulations predict that human cones will indeed conform to Weber’s law over a very wide range of background intensities, and that they will not saturate with steady intensities of any level. Thus, there is now a comprehensive description of the process of light adaptation in human cones and, in particular, of the ability of human cones to avoid saturation.

See also: Phototransduction: Adaptation in Rods; Phototransduction: Phototransduction in Cones.

Further Reading

Burkhardt, D. A. (1994). Light adaptation and photopigment bleaching in cone photoreceptors in situ in the retina of the turtle. Journal of Neuroscience 14: 1091–1105.

Friedburg, C., Allen, C. P., Mason, P. J., and Lamb, T. D. (2004). Contribution of cone photoreceptors and post-receptoral mechanisms to the human photopic electroretinogram. Journal of Physiology 556: 819–834.

Kenkre, J. S., Moran, N. A., Lamb, T. D., and Mahroo, O. A. R. (2005). Extremely rapid recovery of human cone circulating current at the

extinction of bleaching exposures. Journal of Physiology 567: 95–112.

Lamb, T. D. and Pugh, E. N., Jr. (2006). Avoidance of saturation in human cones is explained by very rapid inactivation reactions and pigment bleaching. Investigative Ophthalmology and Visual Science

47, E-Abstract 3714.

Matthews, H. R., Fain, G. L., Murphy, R. L. W., and Lamb, T. D. (1990). Light adaptation in cones of the salamander: A role for cytoplasmic calcium concentration. Journal of Physiology 420: 447–469.

Nikonov, S., Lamb, T. D., and Pugh, E. N., Jr. (2000). The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse. Journal of General Physiology 116: 795–824.

Pugh, E. N., Jr., Nikonov, S., and Lamb, T. D. (1999). Molecular mechanisms of vertebrate photoreceptor light adaptation. Current Opinion in Neurobiology 9: 410–418.

Pugh, E. N., Jr. and Lamb, T. D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: Stavenga, D. G., de Grip, W. J., and Pugh, E. N., Jr. (eds.) Handbook of Biological Physics. Molecular Mechanisms of Visual Transduction vol. 3, ch. 5, pp. 183–255. Amsterdam: Elsevier.

van Hateren, J. H. and Lamb, T. D. (2006). The photocurrent response of human cones is fast and monophasic. BMC Neuroscience 7: 34.

van Hateren, J. H. and Snippe, H. P. (2007). Simulating human cones from mid-mesopic up to high-photopic luminances. Journal of Vision 7(4): 1.

Glossary

Dark adaptation – The very slow recovery of visual sensitivity that occurs upon the return to darkness following exposure of the eye to extremely intense (and possibly prolonged) illumination. Full recovery of the human visual system takes about 45 min, after a total bleach of all the rhodopsin.

Light adaptation – The rapid adjustment of sensitivity and kinetics (of the entire visual system or of the photoreceptors) that occurs in response to altered ambient light intensity. The adjustment is rapid irrespective of whether the change is an increase or decrease in intensity, provided that the change is not too great.

Saturation – The failure of the photoreceptors (or of the visual system) to respond to incremental illumination in the presence of appropriately bright illumination. The rod photoreceptors saturate at a relatively low background intensity; in contrast, the cone photoreceptors avoid saturation by steady backgrounds, no matter how bright the light is. Weber’s law – The reduction in visual sensitivity that occurs in inverse proportion to the intensity of the ambient background illumination. This corresponds to a rise in visual threshold in direct proportion to the ambient illumination.

Vision over a Billion-Fold Range of Light Intensities

Our visual system operates effectively over an enormously wide range of intensities, of at least a billionfold, from around 10 4 cd m 2 under shaded starlight conditions to around 105 cd m 2 under intense sunlight. Changes in pupil area account for only about 1 log unit of this >9-log-unit range, since the pupil diameter changes from a maximum of 8 mm to a minimum of 2.5 mm, corresponding to a 10-fold reduction in area. Instead, the great bulk of the operational range is achieved by the combination of, first, a switch between the rod (scotopic) and cone (photopic) pathways in our duplex visual system and, second, the ability of each of these photoreceptor systems to operate over a range of 5 log units (100 000-fold) or more.

This ability of the visual system (or of any of its component parts, such as the photoreceptors) to adjust its performance to the ambient level of illumination is known as light adaptation; the adjustment typically occurs very rapidly (within seconds), whether the light intensity is increasing or decreasing. The term dark adaptation is reserved for the special case of recovery in darkness, following exposure of the eye to extremely bright and/ or prolonged illumination that activates (bleaches) a substantial fraction of the visual pigment, rhodopsin. Dark adaptation occurs slowly, and the full recovery of the scotopic visual system after a very large bleach can take as much as an hour.

The changes that accompany light adaptation are beneficial to the possessor of the eye. At very low intensities, the sensitivity is increased to the utmost that is possible so that the rod photoreceptors reliably signal the arrival of individual photons and the scotopic visual system operates in a photon-counting mode. The ability of the scotopic system to operate at incredibly low intensities is enhanced by two deliberate trade-offs – of reduced spatial resolution (increased spatial summation) and reduced temporal resolution (increased temporal integration) – that permit more reliable detection of small signals in the presence of noise. Similar trade-offs are used in the photopic system so that as the ambient illumination decreases from daylight levels toward twilight levels, one’s spatial and temporal resolution deteriorate; this is why, in cricket, bad light stops play.

In contrast, the changes that characterize dark adaptation are disadvantageous. To be essentially blind to dim stimuli, for some considerable time following intense light exposure, cannot in any way be useful to an organism. Indeed, for a caveman, entering a cave from bright sunshine, it may have been a serious handicap to have been unable to see well for tens of minutes. Why should such an apparently unsatisfactory situation have persisted? A possible reason could be because it represents an unfortunate downside that has somehow resulted from the enhancements that were needed in order to enable the scotopic system to detect individual photons, and thereby be able to function at incredibly low light levels.

Performance of the Scotopic (Rod) System

For the rod pathway, the dominant mechanisms of scotopic light adaptation result from alterations of signal processing at postsynaptic stages within the retina, and the

rods themselves adapt over only a modest range of intensities before being driven into saturation. This is illustrated in Figure 1, which compares the changes in sensitivity of the rod photoreceptors and of the overall visual system during scotopic light adaptation.

The blue symbols and curve plot the relative sensitivity of the overall visual system, measured psychophysically, while the red symbols and curve plot the relative sensitivity of primate rod photoreceptors. Importantly, the overall scotopic visual system begins desensitizing at intensities around 1000 times lower than those required to begin desensitizing the rod photoreceptors. This occurs because the postreceptoral scotopic system is able to integrate photon signals from large numbers of rod photoreceptors, thereby gaining increased sensitivity, while

Log background (isoms/s)

Figure 1 Sensitivity of the human scotopic visual system (blue) and of monkey rod photoreceptors (red) as functions of background intensity in double logarithmic coordinates. The blue symbols are from human psychophysical measurements and the blue curve plots Weber law desensitization in conjunction with saturation, as described by eqn [1], with parameters

I0 ¼ 0.016 photoisomerizations per second (blue arrow)

and Isat ¼ 2500 photoisomerizations per second (black arrow). The red symbols are from suction pipette measurements from isolated rod photoreceptors of monkeys (Macaca fascicularis); these symbols have been shifted vertically to align with the blue symbols in the upper intensity range. The red curve also plots eqn [1] with the same value of Isat, but with I0 ¼ 50 photoisomerizations per second (red arrow). Data for the blue symbols are from Figure 3 of Aguilar, M. and Stiles, W. S. (1954). Saturation of the rod mechanism of the retina at high levels of stimulation. Optica Acta 1: 59–65. Their troland values were converted using a factor of K ¼ 8.6 photoisomerizations per second per troland. Data for the filled symbols are from Figure 9A and Table III of Tamura, T., Nakatani, K., and Yau, K.-W. (1991). Calcium feedback and sensitivity regulation in primate rods.

Journal of General Physiology 98: 95–130.

introducing the need to begin desensitizing at much lower background intensities in order to avoid saturation. Hence, the rod photoreceptors maintain their maximal sensitivity over several log units of the lowest intensity regime (up to 10 isomerizations per second) where the visual system needs to exhibit gradual desensitization.

When the background intensity is reduced from relatively high scotopic intensities (moving from right to the left along the x-axis in Figure 1), the sensitivity of rods, and of the scotopic visual system, steadily rises. However, below the intensities indicated by the blue and red arrows, the sensitivity of, first, the rods and, second, the visual system fails to continue increasing, as if the respective mechanism were experiencing a phenomenon equivalent to light. Accordingly, the arrowed intensities for the rods and for the scotopic visual system have been referred to as equivalent background intensities. Clearly, the equivalent background for the scotopic system (around 0.016 photoisomerizations per second) is several log units lower than the equivalent background intensity for the rods (around 50 isomerizations per second).

The curves in Figure 1 plot desensitization according to the combination of Weber’s law with saturation at high intensities, as described by

where S is flash sensitivity, SD is its dark-adapted value, and I is the background intensity. The first term on the right-hand side expresses Weber’s law, where I0 is the equivalent background intensity mentioned above. This first term indicates that, at low background intensities (when I << I0) the sensitivity S approaches a constant level (its dark-adapted value, SD), while for brighter backgrounds (when I >> I0) the sensitivity declines inversely with background intensity.

At higher scotopic intensities, both the rods and the overall scotopic system exhibit saturation, characterized by a steep decline in sensitivity with increasing background intensity. This behavior is described by the second term on the right-hand side in eqn [1], where Isat is termed the saturation intensity of around 2500 isomerizations per second. It is almost certain that saturation of the overall scotopic system results directly from saturation of the rods.

The span of intensities from I0 (the equivalent background) to Isat (the saturation intensity) is known as the Weber region and, in this range of background intensities, the sensitivity declines inversely with background intensity; that is, S / 1/I. Since the contrast in a visual stimulus is, likewise, inversely proportional to background intensity (i.e., contrast = I/I ), this Weber region is characterized by a fixed level of contrast sensitivity; that is, a given level of contrast elicits a fixed size of response. Thus, an important feature of Weber’s law light adaptation is that it provides automatic extraction of visual contrast.

For mammalian rods, the Weber region encompasses only 1–2 log units of intensity, though for the larger rods of lower vertebrates, it may encompass a slightly wider range of about 3 log units. On the other hand, for the overall scotopic system, the Weber region covers a much wider range of at least 5 log units (i.e., over 100 000-fold). In addition, for cone photoreceptors, it extends over an even wider range.

The Purpose of Light Adaptation:

Optimization of Performance

The purpose of light adaptation is to permit the visual system (or any neuron within it) to provide the best performance possible at that particular level of illumination. However, it is not always clear what constitutes best. For example, for the rod photoreceptors, it is clear that at very low ambient levels of illumination, their sensitivity should be as high as possible. However, we cannot readily anticipate the time course of their response that will be optimal.

Avoidance of Saturation: Range Extension

As the ambient light intensity increases, it is important that the rod (or any other cell) should avoid saturating, or else it will be unable to signal. By preventing saturation, light adaptation permits a photoreceptor to extend the range of intensities over which it operates. Although the rods achieve light adaptation over a limited range of intensities, the cones excel, and are able to avoid saturation no matter how bright the steady illumination becomes. Why has evolution permitted the rods to be driven into saturation by relatively low intensities? In part, it is because the photopic (cone) system is functional at these intensities, so that there is no disadvantage if the rods saturate. Not only is there no disadvantage – in fact, there is a distinct advantage when the rods saturate, in conserving energy during daylight conditions. Maintenance of the rod circulating current, in darkness and at low light levels, represents an extremely high metabolic load on the cells, and the elimination of this load when the cones are functional provides a major benefit to retinal metabolism. From this perspective, the limited range of rod light adaptation is beneficial, whereas an extended range (as occurs in cones) would be detrimental.

Extraction of Contrast Information and

Optimization of Response Kinetics

In addition to the very important function of extending the operating range of the photoreceptor, there are two other ways in which photoreceptor light adaptation

optimizes the cell’s response. First, as described above in relation to eqn [1], it permits the extraction of contrast in the visual scene, independent of the absolute level of illumination. Second, it provides real-time adjustment of the time course of the response to an incremental flash of light, in a manner that is presumed to be optimal for the visual system. Thus, at very low background intensities, the response is sluggish, and postreceptoral elements are able to integrate visual signals over relatively long times. At progressively higher background intensities, the response becomes progressively accelerated, thereby improving the time resolution of the system. However, we do not have sufficient information yet to be able to describe exactly how it is that kinetic changes of this kind are actually optimal for the visual system.

Light Adaptation of the Rod

Photoreceptors: Range Extension,

Desensitization, and Acceleration

In the presence of background illumination, it is not only the overall visual system that adapts, but also the rod photoreceptors themselves display light adaptation, characterized by an extension of their operating range and by desensitization and acceleration of the incremental flash response.

Prevention of Rod Photoreceptor Saturation:

Range Extension

The response of a salamander rod to the onset of steady illumination at different intensities is illustrated in Figure 2. At the beginning of the step of light, the rod’s response begins rising according to what is predicted from the time integral of the flash response, but very soon deviates, falling well below the linear prediction (upper panel). Characteristically, the response to such a step of light typically exhibits an early peak followed by a sag. This deviation from the simplest linear prediction is a crucial aspect of light adaptation – if this deviation did not occur, then the rod would be driven into saturation by lights of very low intensity. Such saturation can be induced by exposing the rod to a solution that clamps the cytoplasmic calcium concentration; in the presence of calcium-clamping solution (lower panel), the responses of the rod follow the predictions of the smooth theoretical curves, and a very low intensity (labeled 2) saturates the rod. This result shows that at least a part of the rod’s ability to continue operating in backgrounds of moderate intensity (i.e., the extension of its operating range) is a consequence of changes in cytoplasmic calcium concentration; the molecular mechanisms that contribute to this will be discussed below.

Light

4

3

2

1

2

1

D

Desensitization and Acceleration

The manner in which background illumination modifies the rod’s response to a dim test flash is illustrated in Figure 3. The uppermost trace is for a dim flash presented in darkness, while the remaining traces are for exactly the same flash presented on backgrounds of successively higher intensity. Characteristically, the flash response becomes progressively more desensitized and accelerated with backgrounds of higher intensity. Thus, the peak of the incremental flash response moves downward and leftward as the background intensity increases.

By plotting the peak amplitude of the flash response as a function of the background intensity upon which it was elicited, one obtains a plot of the kind indicated by the red symbols in Figure 1, where sensitivity declines according to Weber’s law, given above in eqn [1].

Unaltered Rising Phase, but Accelerated Recovery

For the incremental flash responses in Figure 3, the vertical scale has been adjusted to take account of changes

Figure 3 Dim flash responses of a salamander rod obtained in the dark (top trace) or in the presence of backgrounds of progressively higher intensity. Each trace was obtained by taking the raw response and dividing by the circulating current, and then dividing by the flash intensity. Reproduced from Pugh, E. N., Jr., Nikonov, S., and Lamb, T. D. (1999). Molecular mechanisms of vertebrate photoreceptor light adaptation. Current Opinion in Neurobiology 9: 410–418.

in the level of circulating current remaining in the presence of the different background intensities. Thus, rather than plotting raw sensitivity (response per photoisomerization), Figure 3 instead plots the fractional response

(i.e., the incremental response as a fraction of the circulating current at that background) per photoisomerization. This has been done in order to provide a direct measure of the level of activation of the guanine nucleo- tide-binding protein (G protein) cascade of phototransduction; thus, it can be shown that the level of cascade activation is best measured by the fractional channel opening, which in turn is measured by the incremental response expressed as a fraction of the existing circulating current. When plotted in this manner, the incremental responses in Figure 3 demonstrate the remarkable property that the onset phase of the response is invariant; that is, the traces for different background intensities exhibit a common rise at early times, indicated by the smooth gray trace. This behavior indicates that the amplification parameter describing the activation steps in phototransduction is unaltered during light adaptation; in other words, light adaptation causes no change in the efficacy of the activation steps in phototransduction. Instead, it is clear that light adaptation causes a marked speeding up of the shut-off steps in the transduction cascade. The molecular identity of the steps that are accelerated is analyzed below.

Saturation of the Rod Photocurrent at

Higher Background Intensities

At higher background intensities, the rod circulating current is completely suppressed. Thus, in the upper panel of Figure 2, intensities higher than those labeled 4 cause the response simply to rise to its maximum level (corresponding to the closure of all cyclic guanosine monophosphate (cGMP)-gated channels in the outer segment) and, as a result, incremental stimuli are unable to elicit any incremental response so that the cell’s response is saturated. Typically, such saturation sets in exponentially with increasing background intensity, as described by the second term on the right of eqn [1].

Calcium-Dependent Mechanisms of

Rapid Light Adaptation in Rod

Photoreceptors

The mechanisms that contribute to light adaptation in photoreceptors (i.e., to the alteration in response properties of the photoreceptors upon exposure to background illumination) are closely associated with the mechanisms of response recovery. These mechanisms of adaptation can be classified broadly as (1) those that are calcium dependent and (2) those that do not involve calcium. Both categories are important; yet, the noncalcium-dependent mechanisms have frequently been overlooked.

Role of Calcium: Resensitization through Prevention of Saturation

When cGMP-gated ion channels in the outer segment are closed in response to light, the cytoplasmic concentration of calcium drops. This drop in Ca2þ concentration is vitally important to light adaptation, though it is crucial to emphasize that it does not cause the desensitization that characterizes photoreceptor light adaptation. Quite the contrary: the drop in Ca2þ concentration actually rescues the rod from the saturation that would otherwise be induced by light, and thereby prevents the onset of massive desensitization at relatively low intensities of background illumination. Thus, the light-induced drop in Ca2þ acts to increase the rod’s sensitivity above the drastically reduced level that would occur either if the Ca2þ concentration did not alter or if the rod’s calciumdependent mechanisms were inoperative.

Powerful Negative-Feedback Loop Mediated by Calcium

Calcium is the cytoplasmic messenger for a very powerful negative-feedback loop that tends to stabilize the rod’s circulating current. If ever the Ca2þ concentration drops (e.g., in response to light, or as a result of some other perturbation), then, as described below, a number of changes occur very rapidly. These changes are stimulated by the unbinding of Ca2þ from at least three classes of calcium-sensitive protein: (1) guanylyl cyclase activator proteins (GCAPs) 1 and 2, which activate guanylyl cyclase; (2) recoverin, which regulates the lifetime of activated rhodopsin; and (3) calmodulin, which modulates the opening of cGMP-gated channels. Calcium’s action via each of these pathways leads to the opening of cGMPgated channels, thereby increasing the circulating current and admitting Ca2þ ions from the extracellular medium. This influx of Ca2þ ions tends to counteract the initial reduction in Ca2þ concentration, thereby completing a negative-feedback loop.

Each of these molecular mechanisms contributing to the calcium negative-feedback loop contributes toward extending the rod’s operational range of light intensities by helping prevent saturation of the circulating current. Thus, each of these three molecular mechanisms assists in rescuing the rod from saturation and hence increasing, rather than decreasing, the rod’s sensitivity compared with the case that would exist if the mechanism were absent. Each of the three mechanisms is most effective over some range of calcium levels, and a corresponding range of light intensities. Overall, the most powerful of the three (at least in rods) is the GCAPs’ activation of guanylyl cyclase.

Since the various components of the calcium negativefeedback loop act quite rapidly, they contribute to determining not only the photoreceptor’s sensitivity in the

presence of background illumination, but also the kinetics of its response to an incremental flash presented on the background. The importance of altered Ca2þ concentration in setting the incremental flash response kinetics can be demonstrated by incorporating a calcium buffer (such as 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA)) into the outer segment. Although the flash response begins rising exactly as in control conditions, it does not begin recovering as soon and, instead, rises to a substantially larger and later peak with slower final recovery (see also Figure 4).

Three Calcium-Sensitive Molecular Pathways

Guanylyl cyclase activation

In response to a drop in calcium concentration, Ca2þ will unbind from the GCAP proteins (GCAP1 and GCAP2), thereby activating guanylyl cyclase and stimulating the production of cGMP at a greatly increased rate, leading to the opening of cGMP-gated channels.

The cyclase activity increases roughly as the fourth power of the drop in Ca2þ concentration, and furthermore (as also applies for the other two routes considered below), the number of channels open increases approximately as the cube of the cGMP concentration. Because of the cascading of two such steep dependencies, any small fractional change in Ca2þ concentration stimulates a large and opposite fractional change in channel opening; that is, the fractional change in channel opening is opposite in

sign to, and up to 12 the magnitude of, the originating fractional change in Ca2þ concentration. As a result, this molecular mechanism is the most potent of the three that contribute to the calcium negative-feedback loop and, hence, to setting the adaptational state in rods. It is especially dominant at relatively bright background intensities, corresponding to low Ca2þ concentrations, and is

pA 1

WT

0

Figure 4 Single-photon responses from rods of wild-type (WT) and GCAPs knock-out (GCAPs / ) mice. Suction pipette recordings from single rods, analyzed to extract the mean response to a single photoisomerization. Circulating current in rods of both strains averaged 12 pA. Reproduced from Burns, M. E., Mendez, A., Chen, J., and Baylor, D. A. (2002). Dynamics of cyclic GMP synthesis in retinal rods. Neuron 36: 81–91.

therefore the most important in extending the rod’s operating range to high intensities.

The role of the GCAPs/guanylyl cyclase component of the Ca2þ feedback loop in setting the waveform of the incremental flash response is illustrated in Figure 4, where averaged responses are shown for two classes of rod: rods from wild-type (WT) mice and rods from GCAPs knock-out mice. In a manner very similar to that seen in rods containing the calcium buffer BAPTA, the response in the GCAPs knockout case begins rising exactly as for the control (WT) case, but it does not recover as soon; therefore, the response continues rising and reaches a larger and later peak.

Shortened R* lifetime

Activated rhodopsin (R*) is inactivated by multiple phosphorylation steps mediated by rhodopsin kinase (GRK1) followed by binding of arrestin. It is generally assumed that the decline in R* activity follows exponential kinetics, and can therefore be described by a characteristic lifetime, tR; however, it is worth bearing in mind that there is no direct evidence for this assumption. It was established by Satoru Kawamura that GRK1’s phosphorylation of R* is calcium dependent and that the effect is mediated by the calcium-binding protein recoverin. The molecular mechanism of this dependence is not entirely clear; however, some evidence suggests that the calcium-bound form of recoverin binds to GRK1, thereby preventing it from interacting with R*. In any case, it is proposed that a reduction in Ca2þ concentration leads to a shortened R* lifetime, tR.

The slowest time constant in the phototransduction cascade (the so-called dominant time constant, tdom) can be estimated from the steepness of the relationship between the duration that the rod is held in saturation by a bright flash and the flash intensity. Over the years, there has been considerable debate as to whether this dominant time constant is set by the R* lifetime, tR, or, instead, by the lifetime tE of the transducin–phosphodiesterase (PDE) complex (the effector). The situation may be species dependent; however, in mouse rods, it has now been clearly established by Marie Burns’ group that, under dark resting conditions, the dominant time constant is that of transducin–PDE, with tE 200 ms, while the R* lifetime is shorter, with tR 80 ms. In the scenario where the R* lifetime is shorter than the transducin–PDE lifetime, further light-induced shortening of tR is likely to have very little effect on the response kinetics, but will instead cause a reduction in sensitivity because fewer molecules of transducin will be activated during the R* lifetime.

Although it remains difficult to establish the effectiveness of any individual mechanism in an intact rod with a functional calcium feedback loop, it appears that the recoverinmediated reduction in R* lifetime plays a moderate role, especially at relatively low background intensities.

Channel reactivation

In response to a drop in calcium concentration, Ca2þ unbinds from calmodulin (in the case of the rods), leading to a lowered dissociation constant (K½) for the binding of cGMP to the channels. The effect of the lowered K½ is that any given concentration of cGMP will cause the opening of a larger fraction of the cGMP-gated channels, leading to an increase in circulating current and the influx of more calcium. However, the potency of this effect is low in rods, and the mechanism contributes only weakly to rod adaptation. In contrast, cones possess a much more powerful mechanism, mediated by a different calciumsensitive protein.

Rod Photoreceptor Light Adaptation

Independent of Calcium

There are at least three classes of noncalcium-dependent phenomena that represent mechanisms of light adaptation in rod photoreceptors, insofar as the properties of the response to light are altered in comparison with the dark-adapted state. First, there is response compression, whereby the reduced level of circulating current in the presence of steady background illumination reduces the size of the flash response. This phenomenon will not be discussed here, in part because it is both very well known and very simple and also because (in philosophical terms) it can be viewed as a failure of light adaptation; in comparison, cones cope much better and effectively avoid response compression by feedback mechanisms that maintain the circulating current. Second, there is pigment depletion. However, this is never relevant in rod light adaptation because the rods are driven into saturation even by very low levels of bleached pigment (see section titled ‘Dark adaptation of the rods: Very slow recovery from bleaching’). Third, there is a direct effect of PDE activation, which is now considered.

Accelerated Turnover of cGMP

In a rod outer segment in darkness, the activity of the PDE is low; therefore, the turnover rate constant for cGMP (denoted b) is low, with a correspondingly long turnover time constant for cGMP, tcGMP ¼ 1/b, of around 1 s in amphibian rods and around 200 ms in mammalian rods. The magnitude of this parameter has a major effect on both the sensitivity and the kinetics of the rod’s response to a flash. Thus, when the PDE activity increases in steady illumination, the shorter turnover time for cGMP contributes to both desensitization and acceleration of the photoresponse (compared with the case that would have applied, had the steady level of PDE activity not increased).

To provide an intuitive understanding of this mechanism, it is helpful to consider what we have referred to

previously as the bathtub analogy. Imagine a container of water, such as a tall cylinder, and let the height of water in the cylinder represent the level (concentration) of cGMP in the outer segment. The rate at which water runs out of the cylinder, through a drain hole at the base, is proportional both to the height of water and to the size of the opening, representing the cGMP level and the PDE activity, b, respectively. Likewise, the rate at which water flows in to the cylinder through a tap at the top represents the activity of guanylyl cyclase, a. When a steady state is reached, the height of water will equal the rate of influx divided by the size of the drain hole; that is, cGMP ¼ a/b. Importantly, whenever the water level is perturbed from this steady-state level (e.g., upon a brief opening of an additional drain hole), the level will re-equilibrate with a time constant tcGMP ¼ 1/b (provided that the rate of influx through the tap remains constant). Hence, if the drain hole is small (and the inflow via the tap correspondingly small), then any perturbation in water level elicited by a transient additional outflow will be corrected only slowly; if the drain hole is large (and the influx correspondingly large), then any perturbation will be rapidly corrected. Furthermore, though perhaps less intuitively, it can be shown that for a noninstantaneous perturbation, corresponding to the normal flash response, not only will the kinetics of recovery be faster, but the peak also will be smaller.

Hence, the effect of the increased PDE activity during steady illumination is both to accelerate the response kinetics and to reduce the peak amplitude (i.e., reduce the sensitivity) to an incremental flash. Calculations show that in rods the 20-fold increase in b during steady illumination provides the primary mechanism underlying the measured shortening of the time to peak and the decrease in flash sensitivity.

Slow Changes in Rods: Light Adaptation or Dark Adaptation?

In addition to the conventional features of rod photoreceptor light adaptation that occur extremely rapidly (on a subsecond time scale), other changes have been reported to occur over a time frame of minutes of exposure, in response to lights that saturate the cell’s response. As the effects of these changes are very slow, and can only be observed in darkness when the adapting exposure is extinguished, there is a semantic issue as to whether these phenomena should be thought of as light adaptation or as dark adaptation.

Light-Induced Change in the Dominant Time

Constant

It has recently been shown by Marie Burns’ group that exposure of mouse rods to a just-saturating intensity of

around 1000 photoisomerizations per second, for 1 min or more, leads to a persistent speeding of the bright-flash response upon extinction of the background. The change did not involve any reduction in the activation phase of transduction, but instead involved a reduction in the dominant time constant of response recovery; typically, the dominant time constant tdom dropped from around 200 ms under dark-adapted conditions to around 100 ms immediately after extinction of the saturating light. The adaptational effect developed relatively slowly, building up over 60 s or so, and it required a rhodopsin bleach level of around 2% for full effect. The effect was relatively long lasting, declining with a time constant of around 80 s.

The molecular mechanism giving rise to this adaptational effect is not known, though some evidence suggests that it corresponds to a reduction in lifetime of the activated transducin–PDE complex. If so, it represents a phenomenon distinct from the actions of dimmer adapting lights.

Light-Induced Translocation of Proteins

The light-induced translocation of transducin, recoverin, and arrestin in photoreceptors is dealt with in detail elsewhere in this encyclopedia and, therefore, mentioned only briefly here. Movements of protein are elicited only at quite bright intensities (generally in the saturating range) and occur over a time scale of many minutes. In mouse rods, intensities above 3000 photoisomerizations per second for 30 min (which bleach a substantial fraction of the rhodopsin) trigger the movement of transducin from the outer segment to the inner segment, while slightly lower intensities of 1000 photoisomerizations per second or more trigger the movement of arrestin in the opposite direction; recoverin also leaves the outer segment in bright light.

Protein movements of these kinds may well affect the adaptational state of the rod, though this is yet to be established clearly. Since the movements are triggered only by saturating light intensities, the electrical effects cannot readily be observed during the illumination because the circulating current is completely suppressed. One possibility is that the protein translocation contributes to some form of conservation – for example, lowering the guanosine 50-triphosphate (GTP) consumption involved in the continual (and maximal) activation of transducin during daylight conditions. Alternatively, it may be that the changes help prepare the rod for its return to lower intensities, as occurs around dusk. Interestingly, in one attempt that was made to detect any change in the amplification constant of human rods (using the electroretinogram (ERG)) following exposures to intensities that elicit transducin translocation in mouse rods, no change in amplification was detectable.

Dark Adaptation of the Rods: Very Slow Recovery from Bleaching

Following exposure of our eye to very intense illumination, our visual threshold is greatly elevated and may take tens of minutes to recover fully. Closely comparable effects can be measured in the overall visual system and at the level of the rod photoreceptors or the rod bipolar cells. The slow recovery of sensitivity is referred to as dark adaptation or bleaching adaptation; however, it should be noted that this use of the term adaptation is something of a misnomer. Adaptation normally refers to beneficial adjustments; yet, the changes that accompany intense illumination are distinctly disadvantageous – thus, there can be no advantage in being almost blind following exposure to intense light.

The recovery of visual threshold for a human subject is plotted in Figure 5, following the cessation of nine light exposures that bleached from 0.5% to 98% of the rhodopsin. For a bleach of 20%, the visual threshold was initially elevated by 3.5 log units. This indicates that the elevation of threshold is out of all proportion to the fraction of pigment remaining unbleached; even though 80% of the rhodopsin remained functional, the threshold was raised 3000-fold. Instead, there is overwhelming evidence that the phenomenon arises from the presence within the outer segment of unregenerated opsin (i.e., the presence of the protein part of the visual pigment, prior to its recombination with the regenerated 11-cis retinal).

Remarkably, the recovery of scotopic (rod-mediated) threshold exhibits a region of common slope across all the bleach levels, as indicated by the parallel red lines in Figure 5. This region is termed the S2 component of recovery, and has a slope CS2 = 0.24 log unit min 1 that is characteristic of dark adaptation recovery in normal (young adult) human eyes; also characteristic is the nature of the rightward shift of the recovery traces as a function of increasing bleach level – the form of this shift is as expected for a rate-limited (zero-order) recovery process, as distinct from an exponential (first-order) recovery process.

From a detailed analysis of results of this kind, in combination with knowledge of the retinoid cycle, Trevor Lamb and Edward Pugh developed a cellular model that can account for human dark adaptation behavior. They postulated that (1) the presence of opsin (without chromophore) gives rise to a phenomenon closely equivalent to light, through activation of the G protein cascade of transduction and (2) the elimination of opsin via its reconversion to rhodopsin follows rate-limited kinetics because of a limitation in the supply of 11-cis retinal that results from the movement of this substance from a pool in the retinal pigment epithelium.

Application of this cellular model has provided an accurate account of (1) the regeneration of visual pigment