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1 Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK
2 Division of Neuroscience, John Curtin School of Medical Research, The Australian National University, Canberra, ACT 0200, Australia
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Abstract |
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90%) of the cone photopigment. During steady-state exposures of this intensity, the amplitude of the bright-flash response declined to 50% of its dark-adapted level. After the intense background was turned off, the amplitude of the bright-flash response recovered substantially, for flashes presented within 20 ms of background extinction, and fully, for flashes presented 100 ms after extinction. In addition, a prominent ‘background-off a-wave’ was observed, beginning within 5–10 ms of background extinction. We interpret these results to show, firstly, that human cones are able to preserve around half of their circulating current during steady-state illumination that bleaches 90% of their pigment and, secondly, that following extinction of such illumination, the cone circulating current is restored within a few tens of milliseconds. This behaviour is in stark contrast to that in human rods, where the circulating current is obliterated by a background that bleaches only a few percent of the pigment, and where full recovery following a large bleach takes at least 20 min, some 50 000 times more slowly than shown here for human cones.
(Received 13 April 2005;
accepted after revision 27 May 2005;
first published online 2 June 2005)
Corresponding authors O. A. R. Mahroo and T. D. Lamb: Division of Neuroscience, John Curtin School of Medical Research, The Australian National University, Canberra, ACT 0200, Australia. Email: omar.mahroo{at}cantab.net and trevor.lamb{at}anu.edu.au
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Introduction |
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Boynton & Whitten (1970) and Valeton & van Norren (1983) made extracellular recordings of the cone ‘late receptor potential’ in the eyes of macaques and rhesus monkeys, respectively, and reported that at least 50% of the maximal dark-adapted response remained, even on backgrounds of up to 106 trolands (Td). Schnapf et al. (1990) made suction pipette recordings from the outer segments of macaque cones, but as these were not in contact with the retinal pigment epithelium (RPE), it is difficult to relate the results to in vivo behaviour in the case of exposures bright enough to bleach a significant fraction of cone pigment. Furthermore, none of the cited papers investigated recovery at the cessation of illumination, and nor did they study human cones. More recently, Paupoo et al. (2000) used electroretinogram (ERG) bright-flash responses to monitor human cone circulating current during steady illumination. They reported that the current appeared to fall to around 20% of its dark-adapted level in steady light of 20 000 Td, but here we will argue that this was an underestimate, and that the true level is around 50%, consistent with previous studies on monkey cone late receptor potential.
The speed of recovery of human cone circulating current following intense exposures was also investigated by Paupoo et al. (2000). They reported that, at cessation of a steady light that bleached 50% of the pigment, the cone circulating current recovered within 3 s – that value, however, was the minimum time resolution of their method, which used a manually switched incandescent light as the bleaching source.
The present work was designed to extend the experiments of Paupoo et al. (2000), by studying the responses of human cones during and after very intense illumination. A main aim was to improve the time resolution, by using electronic extinction of illumination from intense light emitting diodes (LEDs). Our novel finding is that, upon extinction of a steady background bleaching 90% of the cone pigment, the cone circulating current recovers almost completely within 20–100 ms. For comparison, Thomas & Lamb (1999) found that full recovery of rod current after a similar bleach took at least 20 min. A preliminary account of some of this work has been given by Moran et al. (2003) and Mahroo et al. (2005).
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Methods |
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Subjects
The subjects were two of the authors: N.A. (recordings from her amblyopic right eye) and O.A.R.M (recordings from his slightly myopic left eye). Ethical approval was obtained from the Cambridge Human Biology Research Ethics Committee, and informed written consent was obtained from each subject following detailed explanation of the procedures and risks.
Pupil dilatation
The subject's pupil was dilated by application of two drops of 1% tropicamide at an interval of 5–10 min, followed if necessary by further drops about 90 min later. The diameter of the pupil, which was typically close to 8 mm, was monitored continuously under infrared illumination, and the image was recorded on video tape.
Ganzfeld
Light stimuli were delivered in a ganzfeld apparatus, and were viewed by the subject through a small monocular port (Smith & Lamb, 1997). Light calibrations were performed using an IL-1700 photometer (International Light, Newburyport, MA, USA) with photopic (Y) and scotopic (Z-CIE) filters. Unless otherwise specified, all intensities are given in photopic units of cd m–2 (for steady illumination) and cd m–2 s (for flashes).
Cone isolation
For isolation of the photopic system, a rod-saturating steady background was provided continuously by a blue LED (470 nm peak) that delivered 2.9 photopic cd m–2 and 34 scotopic cd m–2. This level (1700 scotopic Td with a dilated pupil) was sufficient to saturate the rods, but caused negligible effect on the cone system (Mahroo & Lamb, 2004). In this paper the expression ‘the dark-adapted state’, when applied to the cone system, refers to experiments conducted in the presence of the rod-saturating background.
Flashes
Flashes of white light were delivered by a xenon flash gun (Mecablitz 60CT4, Metz, Zirndorf, Germany), and passed through a heat filter, short-wavelength filter, and prismatic diffuser, before entering the ganzfeld.
Bleaching exposures
Bleaching was achieved using up to 12 ultra-bright ‘white’ LEDs (LuxeonTM LXHL-BW01, Lumileds, San Jose, CA, USA). These emitters use a blue LED with a phosphor, and exhibit a spectral peak near 455 nm together with a very broad peak near 570 nm; they have a quoted colour temperature of 5500 K. The maximum intensity of this ganzfeld LED illumination was 1900 photopic cd m–2 and 3100 scotopic cd m–2.
Signal processing
Stimulus timing and data collection were controlled by a custom program script running under the Signal package (Cambridge Electronic Design, Cambridge). ERG signals were filtered from 0.16 Hz to 1 kHz, and sampled at 5 kHz. Data analysis was performed subsequently using custom programs running under Matlab (The MathWorks Inc.). Criteria for trace rejection were based on those described by Paupoo et al. (2000), with a noise level exceeding 20 µV peak-to-peak being grounds for rejection. However, in experiments in which each response was averaged from a large number of traces (>50), the presence of noise in the raw traces had little effect, and in practice very few traces were rejected.
All plotted error bars represent ± 1 S.E.M. around the mean.
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Theory |
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In order to estimate the fraction of pigment present during a background exposure, one approach that we take in the Results section is to monitor the regeneration of pigment following extinction of the background. To accomplish this, we monitor the amplitude of responses to dim test flashes, because the response sensitivity should be directly proportional to the level of pigment (Mahroo & Lamb, 2004). In applying this method, we require three theoretical expressions developed in the previous work. Firstly, we need to describe the non-linearity of the observed response versus intensity relation; secondly, we need to compensate for this compressive effect, to generate an estimate of pigment level at the relevant time; and thirdly, we need an expression for the expected pigment regeneration kinetics, so that we can extrapolate back to time zero (i.e. when the background was on). Here we summarize the required relations, as developed by Mahroo & Lamb (2004).
Non-linearity of response amplitude
The amplitude of the dim-flash responses, measured at a fixed time after flash delivery and under dark-adapted conditions, can be described well by an exponential saturation function
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| (1) |
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With the assumption that this same non-linearity applies during recovery from a bleach, and with knowledge of the values of the constants in eqn (1), it is straightforward to ‘linearize’ the measured postbleach response amplitude and thereby obtain an estimate of the fractional level of visual pigment, P; see eqns (9) and (10) of Mahroo & Lamb (2004). Their equations may alternatively be expressed as
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| (2) |
Regeneration of visual pigment
The time-course, P(t), of regeneration of visual pigment in darkness following a bleach is predicted by the ‘MLP rate-limited model’ of Mahroo & Lamb (2004) and Lamb & Pugh (2004) to be given by
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| (3) |
is the initial rate of recovery following a total bleach, and W(x) denotes the Lambert W function of argument x (Corless et al. 1996). For the derivation of this equation, see the Appendix of Mahroo & Lamb (2004). We use this expression to estimate the fraction B that had been bleached during the exposure, i.e. at t
= 0, when the background was extinguished. For an example of the application of eqn (3), see Fig. 4D. Rate of isomerization of photopigment during illumination
During illumination at any intensity I, the rate of creation of activated pigment molecules (i.e. the rate of R* formation), denoted as 
' photoisomerizations s–1, is given by
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| (4) |
5 x 107 pigment molecules per human cone outer segment. The parameter 
is termed the photosensitivity, and must be specified in units that conform with those used for intensity I. The reciprocal of photosensitivity –1 is equivalent to the ‘bleaching constant’, Qe, the quantity of light required in a brief flash to reduce the pigment level to 1/e (this parameter was termed LRh by Thomas & Lamb, 1999). Recently, Mahroo & Lamb (2004) estimated the reciprocal of photosensitivity as –1
= 710 cd m–2 min, corresponding to
= 2.4 x 10–5 cd–1 m2 s–1 (in experiments with a dilated pupil). Full complement of pigment
Under dark-adapted conditions, with P
= 1, substitution in eqn (4) gives '
I
x (1170 photo-isomerizations s–1 (cd m–2)–1). The conversion factor in brackets should be equivalent to that obtained by converting first from cd m–2 to Td, and then to photoisomerizations s–1, though for human cones the magnitude of the latter factor is not well established. Conversion from cd m–2 to Td requires a value for the effective pupil area, taking into account the Stiles & Crawford (1933) directional effect, and Paupoo et al. (2000) estimated a value of 20 mm2 for a dilated pupil. Dividing the above factor of 1170 by this effective pupil area gives the troland conversion factor as Kcone
= 59 isomerizations s–1 Td–1 for the experiments of Mahroo & Lamb (2004). Given that they recorded cone responses from the entire retina, their responses were likely to be largely influenced by peripheral cones; although the cone density is greatest in the fovea, there are many more cones, in total, in the periphery. For comparison, Hood & Birch (1995) estimated a value of 20 isomerizations s–1 Td–1 for foveal cones, and the three-fold ratio of estimates would appear entirely plausible in view of the much larger inner segment area of peripheral cones.
Intense illumination
With increasing intensity I of steady illumination, the fraction P of unbleached pigment declines. In the limit of very bright steady illumination (when P
0), the rate of photoisomerization is set simply by the rate at which photopigment is regenerated. (A comparable result for exponential regeneration of pigment was noted by Pugh & Mollon, 1979; in their Appendix III, where they reported that the fractional rate of isomerization asymptoted towards the reciprocal of the regeneration time constant.) In this limiting case of high intensities, the rate of pigment isomerization (expressed as a fraction of the total number of pigment molecules, NP) is simply , so that eqn (4) can be replaced by
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| (5) |
= 0.48 min–1 (= 8 x 10–3 s–1) for human cone pigment regeneration (averaged for the two subjects in Mahroo & Lamb, 2004), we obtain a limiting rate of '
4 x 105 photoisomerizations s–1 cone–1, in the presence of intense illumination.
Furthermore, from comparison of eqns (4) and (5), we find that for intense steady illumination
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| (6) |
/ (=
Qe). Predicted kinetics of recovery of cone circulating current
By making several simplifying assumptions, it is possible to predict the expected kinetics of recovery of the cone circulating current at the cessation of steady illumination. The first assumption is that the transduction cascade functions according to the scheme in Fig. 1 of Nikonov et al. (1998), at an intensity that is not so high as to cause saturation. The three ‘active’ substances R* (activated photopigment), E* (activated phosphodiesterase, PDE), and cG (cyclic GMP) are each assumed to be removed by a first-order reaction, so that during the steady state their levels are characterized by fixed turn-over times, denoted 
R, E and cG, respectively; these life-times during intense illumination are likely to be shorter than the corresponding life-times under dark-adapted conditions. Our next assumption is that at early times during recovery (before the calcium concentration has changed appreciably), the activity of guanylyl cyclase remains unaltered, so that cG continues to be produced at a constant rate while being hydrolysed with a time constant that declines in proportion to the declining level of E*. A further assumption is that several short transition steps in phototransduction can be approximated as pure delay stages. Such delays include: the metarhodopsin II formation time, Meta; the mean time GE taken for each G* to activate an E*; the cGMP-to-channel unbinding time, chan; the cell's capacitive time constant, RC; and any electrical filtering delay, filter (see Lamb & Pugh, 1992). We think that the total delay time tot, obtained by summing these individual delays, will be in the region of 4 ms (see Results).
In considering the extinction of steady illumination, at time defined as t
= 0, we will normalize all variables to their steady levels during that illumination, so that the initial levels of activated photopigment, activated phosphodiesterase, and cyclic GMP are set to unity, i.e.
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| (7) |
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| (8) |
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| (9) |
Rh
E; in the case of equality, a simpler equation applies). In normalized form, the differential equation for cyclic GMP concentration becomes
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| (10) |
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| (11) |
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| (12) |
and F(t) at extinction of intense steady illumination, are sketched schematically in Fig. 1. In the Results section, responses predicted for F(t) will be compared with the experimental results in Figs 9 and 10.
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Results |
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Figure 2 shows families of photopic responses to white flashes at a range of intensities, obtained in the presence of either the dim blue rod-saturating background (top row) or the bright white background (middle row). All responses were obtained after a minimum of several minutes of adaptation, and were recorded on a single day for each subject. Inspection shows the maximal a-wave amplitude in the presence of the bright background to be around half that with the dim blue background (i.e. in the ‘dark-adapted’ state), and this is consistent with an approximately 50% reduction in circulating current.
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The bottom row of Fig. 2 shows a reasonably close correspondence between the dark-adapted responses and the corresponding scaled light-adapted responses, particularly at early times (less than 7 ms) when the signal is expected to originate primarily from the photoreceptors, i.e. prior to significant intrusion of postreceptoral signals (Robson et al. 2003). This similarity is consistent with the idea that, at a steady state in the presence of the bright background, around one tenth of the pigment remains unbleached, and approximately half of the dark current continues to circulate. We now test this notion further by examining the response–intensity relation in the two adaptational states.
Response–intensity relation
Figure 3 plots the response amplitudes measured from the experiment of Fig. 2 against flash intensity, for the two subjects, at a fixed early measurement time of 5 ms, chosen so as to exclude postreceptoral signals (Robson et al. 2003). The ‘dark-adapted’ values, obtained in the presence of the dim blue background, have been plotted using filled symbols, while the light-adapted values have been plotted using open symbols, after scaling the amplitudes up by a factor of 1.9x, and the intensities down by a factor of 10x. The curve plots the exponential saturation relation, eqn (1), expected for photoreceptor response amplitude measured at a fixed time after the flash (see Lamb et al. 1981; Lamb & Pugh, 1992).
Inspection of the open symbols in Fig. 3 shows that, during application of the intense background, the brightest flashes almost saturated the response at the measurement time of 5 ms; thus, the right-most open symbols in Fig. 3 are at about 80–90% of the maximal level obtained in ‘dark-adapted’ conditions. Although the use of a later measurement time gave slightly larger responses during the background (see red traces in Fig. 2), it was not appropriate to compare these values with ‘dark-adapted’ values, because of the possible intrusion of postreceptoral signals; note the sloping behaviour of the black traces at times later than 5 ms, in Fig. 2C and F (see also Robson et al. 2003). Delivery of brighter flashes did not help, because the flash artefact (visible, for example, in Fig. 2C) became much more prominent, and prevented clear estimation of the response amplitude.
Our interpretation of the results of Figs 2 and 3 is that the factor of 1.9x used to scale the measurements in the presence of the intense bleaching light was broadly appropriate for bringing the two sets of response amplitudes into coincidence, and is unlikely to be in error by more than about 10–20%. Accordingly, our results indicate that in the presence of the intense background the cone circulating current was reduced to about 52 ± 10% of its level in darkness (see also Fig. 10 subsequently).
Estimation of bleach level from recovery of dim-flash response postexposure
In the analysis of Fig. 3, we obtained an approximate value for the extent of pigment bleaching by examining the horizontal scaling required to bring the two sets of symbols into coincidence. Another, arguably better, method is to monitor recovery of the dim-flash response for several minutes after extinction of the bleaching background, as described recently by Mahroo & Lamb (2004), and summarized in the Theory section. Figure 4 illustrates the use of this approach for subject N.A.M.
In order to determine the degree of response non-linearity under ‘dark-adapted’ conditions, Fig. 4A presents a family of responses to relatively dim flashes. The response–intensity relation obtained from these traces is plotted in Fig. 4B, using the measurement interval of 13–14 ms indicated by the broken vertical lines in Fig. 4A. As expected, the non-linearity in the experimental results is well described by the exponential saturation relation of eqn (1), and hence it was possible to estimate the intensity constant in eqn (1) as Q0 = 0.46 cd m–2 s at the measurement time of 13–14 ms used in this experiment. (Note that, for these experiments it is entirely appropriate to use a later measurement time, even though activity in OFF bipolar cells will be contributing, because all that is needed is a defined relationship between light absorption and response amplitude; see Mahroo & Lamb, 2004. In contrast, the experiments of Figs 2 and 3 were designed to probe the currents of the cone photoreceptors alone.)
Recovery of the dim-flash response after bleaching is illustrated in the right-hand panels of Fig. 4. Flashes of a fixed intensity, Q = 0.4 cd m–2 s, that elicited the response indicated by the thick black trace in Fig. 4A, were delivered both before and after a minimum of 3 min exposure to the same intense white light (1900 cd m–2) as previously, and the resulting responses are shown in Fig. 4C. The smallest responses were recorded shortly after extinction of the bleach, while the larger responses were recorded at progressively later postbleach times. The amplitudes of these responses, r, again measured over the indicated interval of 13–14 ms after the flash, were used to estimate the fraction of photopigment by substitution in eqn (2) of the Theory, and the resulting estimates are plotted as a function of postbleach time in Fig. 4D; estimates from responses recorded prior to the bleach are plotted at negative postbleach times. Thus the values of r/rDA from Fig. 4C were simply substituted in eqn (2), together with the fixed value of Q/Q0, where Q was the intensity of the flash and where Q0 had been determined in Fig. 4B. rDA was estimated from the response-amplitude recorded at late postbleach times, i.e. upon full recovery.
The filled squares (
) in Fig. 4D, representing our estimates of the pigment level, begin recovering roughly as a ramp with time after cessation of the bleach. The curve near these points plots the kinetics of pigment regeneration predicted by eqn (3), and with parameters B
= 0.88,
= 0.41 min–1, and Km
= 0.2. Thus, the curve describes the predicted recovery for a bleach of 88%, and provides an adequate description of the measurements; this estimate of bleaching level is close to the approximate value of 90% that we employed previously in Figs 2 and 3. The value of Km is the same as used by Mahroo & Lamb (2004) to fit their own and previous data, and the rate is close to the rates they used for their subjects (0.47 min–1 for T.D.L and 0.5 min–1 for O.A.R.M). For comparison, also plotted in Fig. 4D as 
, are estimates of pigment levels following the same bleaching intensity for the other subject, O.A.R.M. The data points are slightly more scattered than those for N.A.M. as they were derived from only three repetitions of the bleaching exposure, but they follow a similar time-course and are consistent with a similar bleach level of around 88% at postbleach time zero.
Other intensities
The results of Figs 2–4 support the notion that around 50% of the cone circulating current remains present during an intense steady background sufficiently bright to bleach around 90% of the photopigment. Using the same approaches, we also estimated the remaining level of circulating current and the bleaching level at other background intensities. Although the maximum intensity available from the LEDs was 1900 cd m–2, we could deliver higher intensities using an additional incandescent lamp. For steady intensities ranging from 500 to 5000 cd m–2, we estimated the remaining current to range from 65% to 45% of the current in darkness, and the bleach level to range from 40% to >95% (results not illustrated).
Recovery of bright-flash response amplitude following extinction of intense illumination
Our main purpose in this investigation was to measure the speed of recovery of cone circulating current upon extinction of a very intense background. To accomplish this, we repetitively switched the background off for a short interval, and during each such interval we presented an intense flash at one of several selected times. Hence our approach was the ‘background-off’ equivalent of the well-established paired-flash protocol pioneered by Pepperberg et al. (1997). In order to avoid perturbing the fractional bleach, we adjusted the flash intensity so as to balance the decrease in illumination caused by the gap in the background. For example, if a background of 2000 cd m–2 were to be extinguished for 0.5 s, then we would have selected a flash intensity of 1000 cd m–2 s to balance. A large number of repetitions was required, in part so that we could investigate a number of timings relative to extinction, but also to average out the substantial noise (presumed to be of muscular origin) that was present in the recordings during exposure to intense illumination. Prior to the repeated cycles of extinction and flash, we allowed the subject to adapt to the intense steady background for 2–3 min.
Extinction period of 660 ms. In the experiment shown in Fig. 5, we chose to investigate postextinction intervals of up to 600 ms, using a shut-off duration of 660 ms. The background intensity was our standard 1900 cd m–2, and so to balance the period of darkness we used a flash intensity of 1200 cd m–2 s. In any individual cycle, this flash was presented at either 50, 100, 200, 300, 400 or 600 ms after shut-off, or alternatively 20 ms prior to shut-off, during the background (i.e. at –20 ms relative to background extinction). The cycles of background extinction and flash delivery were repeated every 3 s. The subject in this experiment was N.A.M. The protocol in this experiment was broadly comparable to that used by Robson et al. (2003) in their Fig. 2, except that rather than the relatively dim rod-saturating background of 2500 scotopic Td that they were extinguishing, we were extinguishing an extremely bright background of 150 000 scotopic Td (40 000 photopic Td).
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Traces of the kind illustrated on a slow time-base in Fig. 5 are presented on a fast time-base in the upper row of Fig. 6, after synchronization to the time of flash delivery and subtraction of the response to background extinction. The right-hand column plots the results for subject N.A.M. shown in Fig. 5, while the left-hand column shows corresponding results for subject O.A.R.M.
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The larger traces were obtained in darkness, either at a series of intervals after extinction of the background (coloured traces), or under ‘dark-adapted’ conditions (continuous black trace). In the latter case, as there was a full complement of pigment present, rather than a level of only about 12% for the coloured traces, the test flash intensity was reduced (from 1200 cd m–2 s, as used for all the other traces) by a factor of about 8, to 140 cd m–2 s. Importantly, the coloured traces are closely similar to each other and to the dark-adapted (continuous black) trace, in both panels (Fig. 6A and C); the one exception might be the cyan trace for subject O.A.R.M., which appears intermediate between the traces on the background and the other traces in darkness. The similarity is especially close over the first 4–5 ms, when the response is least likely to be affected by postreceptoral intrusion, but in fact there is little difference even out to 10–12 ms. Thus, with the possible exception of O.A.R.M.'s trace obtained for a separation of 50 ms, it appears that the circulating current monitored by the test flashes is of substantially the same amplitude in each case. Furthermore, even the oscillatory potentials appear broadly similar in shape and timing, for all of the traces elicited in darkness (apart from that for N.A.M. at a separation of 50 ms).
For bright flashes that elicit a fixed number of photoisomerizations, the amplitude of the a-wave response at a fixed measurement time (prior to postreceptoral intrusion) should be proportional to the cone photoreceptor circulating current (see, e.g. Thomas & Lamb, 1999). In the upper panels of Fig. 6, the level of cone circulating current has been estimated by measuring at the indicated time of 4 ms, and the time course of recovery of current is plotted in the lower pair of panels. (Note that the accuracy of the estimate in ‘dark-adapted’ conditions relies on the number of photoisomerizations having been the same in that case). By way of reference, horizontal lines have been drawn at the levels obtained under ‘dark-adapted’ conditions (, continuous black line) and during steady light (
, broken black line), corresponding to measurements made from the continuous and broken black traces in the upper panels, respectively. Inspection of the lower panels suggests that the cone circulating current had recovered fully within 100 ms of background extinction for O.A.R.M., and within 50 ms for N.A.M.
Extinction period of 250 ms. Figure 7 shows responses obtained in an experiment similar to that in Fig. 5, but with a shorter extinction period of 250 ms, and where we used shorter intervals between extinction and flash (down to 20 ms postextinction). Since the period of extinction was shorter, the intensity of the flash was correspondingly reduced (to 490 cd m–2 s) to maintain a constant mean level of bleaching. In this experiment, the cycles of background extinction and flash delivery were repeated every 4 s; the subject was O.A.R.M.
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). The results from Fig. 7 are analysed in Fig. 8 (left-hand side) in a format similar to that used above in Fig. 6. The reference trace (the lowermost trace in Fig. 7) has been subtracted from each of the other traces in Fig. 8A, and the resulting responses have been synchronized to the time of flash delivery, and plotted on a faster time-base. Once again, the broken trace and the continuous black trace plot the responses obtained during the continuous background and in the ‘dark-adapted state’, respectively (with the flash intensity in the latter case again reduced to allow for the greater content of pigment). As was seen in Fig. 6, the responses obtained at different times after background extinction are very similar to each other, both for subject O.A.R.M. (left) and for subject N.A.M. (right). Indeed the responses are almost indistinguishable from each other over the first 5 ms, i.e. during the period before postsynaptic activity would be expected to begin contributing.
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The ‘background-off a-wave’ response at extinction of intense illumination
The rapid component of response apparent in Figs 5 and 7 at extinction of the background is examined in Fig. 9, for two experiments on each subject. The left-hand side plots the responses obtained with bright test flashes presented during the steady background, and the conventional a-wave responses provide a means of assigning the zero-level of photoreceptor current. The right-hand pair of ordinate scales shows normalization in two ways: firstly (inner scale) with respect to the steady level of current during the steady background, and secondly (outer scale) with respect to the dark current.
The traces on the right-hand side plot the ERGs obtained upon extinction of the background, with each trace averaged from at least 300 presentations. A corneal-positive signal begins within 5–10 ms of background extinction. Because of its rapid onset we refer to this corneal-positive response as a ‘background-off a-wave’, and we now investigate whether it might reflect recovery of the cone circulating current.
From our analysis in the Theory section, we can predict the expected shape of the recovery of cone circulating current, based on the simplifying assumptions that the activated substances (activated photopigment, G-protein/PDE, and cyclic GMP) are each removed by first-order processes, and that calcium feedback plays a negligible role in the period immediately following background extinction. On this basis, the recovery of cone circulating current, normalized to its level during steady illumination, can be predicted by solving eqn (10) and substituting in eqn (12). The shape predicted in this way for the recovery of current is plotted by the grey trace in Fig. 9. The parameter values used were Rh
= 5 ms, E
= 13 ms, cG
= 4 ms, and total delay time for all the other short stages, tot
= 4 ms, though we stress that these values are by no means unique. The grey line provides an adequate description of the early phase of the ERG, up until around 13 ms after extinction of the background.
The good description provided by the simple theory of eqns (8)–(12) lends support to the idea that the ‘background-off a-wave’ at early times indeed reflects the initial return of cone circulating current. However, at times later than about 13 ms after extinction, the prominent corneal-positive ‘spike’ seems most likely to reflect postreceptoral activity.
Collected estimates for time-course of recovery of circulating current
In Fig. 10, we have collected all our results for the recovery of amplitude of the bright-flash response, and we have compared these with our estimate of recovery kinetics obtained from the ‘background-off’ response. All values in this figure have been normalized to the dark-adapted level, and have been gathered from both subjects over multiple experiments. Although the error bars are moderately large, the results from the bright-flash experiments strongly suggest that the cone circulating current has fully recovered within 100 ms of background extinction. Indeed it has probably recovered substantially even at the earlier measurement times, between 15 and 55 ms, though the need to subtract the rapidly changing ‘background-off’ response leads us to be cautious in interpreting these early measurements.
The shape of cone current recovery obtained directly from the ‘background-off a-wave’ response appears consistent with the measurements obtained with intense flashes, suggesting that recovery of the cone circulating current begins within 10 ms of extinction, and that substantial recovery has occurred within 15 ms.
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Discussion |
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Suppression of current by intense steady backgrounds
The estimate of the level of circulating current during intense steady illumination is closely consistent with measurements made from monkey eyes, using extracellular recording; Boynton & Whitten (1970) and Valeton & van Norren (1983) both estimated steady levels of around 50%, in macaque and rhesus monkey, respectively. Broadly comparable results have been obtained for cones in other vertebrate species; thus Burkhardt (1994), measuring intracellular voltage in turtle cones, and Donner et al. (1998), measuring extracellularly from frog retina, both reported that intense backgrounds produced steady response levels of no more than 50% of maximal.
Not surprisingly, though, this behaviour differs substantially from what is seen at early times after onset of a step; for example, Schnapf et al. (1990) found monkey cone currents were greatly reduced at high intensities, when measured 2 s after the onset of illumination. Taken together, these results indicate that, even though primate cones may transiently be saturated at the onset of intense illumination, their circulating current recovers to around half its original dark level upon continued exposure. As proposed by previous authors (e.g. Alpern et al. 1970; Boynton & Whitten, 1970; Burkhardt, 1994; Donner et al. 1998), we think that a substantial component of this recovery during prolonged exposures is attributable to pigment bleaching, i.e. to a reduction in the quantity of photopigment available. Thus, when an intense background is first turned on, the rate of photoisomerization is very high, but, as pigment bleaches, the rate declines to a lower level. According to eqn (5), the limiting steady rate of activation for intense illumination should be given by NP, or around 4 x 105 isomerizations s–1 in a human cone; for comparison, Burkhardt (1994) estimated a value of 5 x 106 isomerizations s–1 in turtle cones. We conclude that cone photoreceptors are able to retain about half their original circulating current in the face of the continual creation of R*s at these high rates. A corollary of the bleaching explanation is that the equilibration of cone current towards its final level would be expected to occur more rapidly, for lights of higher intensity.
In the case of cones isolated from the RPE, continued illumination would cause the pigment level (and consequently the rate of photoisomerization) to drop towards zero. As a result, the current would eventually recover all the way to its dark level, even during the illumination, as has in fact been reported by Schnapf et al. (1990). Similar recovery is not seen in rods isolated from the RPE, because bleached pigment continues to activate the rod transduction cascade, thereby preventing recovery of rod photocurrent, even after the light is turned off.
In a recent study using a similar approach to that described here, Paupoo et al. (2000) reported that the human cone circulating current was reduced to about 20% by very bright backgrounds (20 000 Td). However, we think that their estimate was too low for several reasons. Firstly, their experiments were performed without dilating the pupil, so that the pronounced pupillary constriction, combined with pigment bleaching, may have led to responses of artificially small amplitude on the bright backgrounds. Secondly, the relatively late measurement time of 6 ms after the flash, combined with the dimmer blue background intensity that they used, may have caused an artificial increase in the amplitude obtained under dark-adapted conditions, as a result of a contribution from a slow postreceptoral component (see their Fig. 2, and also Robson et al. 2003). Together, these effects will have led to overestimation of the fractional reduction in current during intense backgrounds. A third (and possibly more interesting) effect is related to recovery of the circulating current during prolonged exposures (see previous paragraphs). In the study of Paupoo et al. (2000) the bright flashes were delivered beginning quite soon after onset of the background illumination, when it is likely that the circulating current was transiently reduced below its subsequent steady-state level. In the present study we addressed these issues by dilating the pupil, by using brighter probe flashes, and by waiting until at least 2 min after onset of the background. For the future, we think it would be interesting to make measurements of circulating current at a range of intervals after the onset of illumination, in order to track the time-course of recovery of current.
Recovery of current following background extinction
The results of two experimental approaches indicate that the human cone circulating current recovers very rapidly upon extinction of intense steady illumination – certainly it is fully recovered within 100 ms, and quite probably it is very substantially recovered with 15–20 ms.
The first approach used the delivery of intense flashes to probe the level of circulating current. At times later than 100 ms after background extinction, this approach was very straightforward because the ERG response to switching the background-off exhibited only a slow rate of change by that stage. However, at times earlier than 100 ms, the occurrence of rapid components of the ERG off-response meant that subtraction of this signal became important, as did the assumption that delivery of the probe flash caused no change in the postreceptoral signals up to the time of measurement (typically 4–5 ms after the flash). The results of Figs 5–8 and 10 indicate that the form of the bright-flash response was virtually indistinguishable, both in shape and in amplitude, at all the separation times we tested, from 10 to 600 ms after background extinction. Furthermore, these responses were closely similar to the response elicited by a flash presented under dark-adapted conditions, when the flash intensity was adjusted so that it should have elicited the same number of photoisomerizations as the flashes in the bleached condition. Thus, the very rapid re-establishment of full-sized bright-flash responses strongly supports the notion that the cone circulating current had fully recovered within 100 ms of background extinction, and, subject to the provisos related to subtracting the off-response at early times, it appears to indicate that the current had substantially recovered within about 15 ms of background extinction.
The second approach was based on further examination of the off-response (that we subtracted in the bright-flash experiments). The basic idea was that if the cone current recovers rapidly then it should be visible as a component of the ERG and, conversely, that any rapid off-response in the ERG may potentially reflect recovery of the cone circulating current. Examination of the ERG traces in Figs 5 and 7 shows a gradual build-up of current, between about 7 and 13 ms after background extinction, followed by a pair of corneal-positive ‘spikes’, the first of which occurs about 16 ms after background extinction.
Possible origin of the corneal-positive spikes. The corneal-positive spikes are almost certainly of postreceptoral origin, and there is suggestive evidence that they arise in the cone OFF bipolar cell pathway. Thus, in patients with complete congenital stationary night blindness (CSNB1), which involves disruption of the ON bipolar cell pathway, spike activity at background extinction remains present, as illustrated in Fig. 1 of Khan et al. (2005) and experiments with drugs that selectively affect the ON and OFF bipolar cell pathways appear consistent with this interpretation (e.g. Sieving et al. 1994).
Significance of the current build-up prior to the spikes. In relation to photoreceptor activity, a more important feature is the occurrence of a gradual build-up of corneal-positive signal prior to the first of these spikes. This activity is shown magnified in Fig. 9, where it is compared with the behaviour predicted theoretically (grey trace) for recovery of the cone current at background extinction.
The simplified theoretical analysis predicts that the cone current should begin recovering approximately as the cube of postextinction time, with a vertical scaling determined by the three time constants for turnover of the active molecular substances (see eqn (11)). In addition, a number of short steps (not explicitly dealt with in the model) should each contribute a small delay, so that the predicted recovery begins as a delayed cube-law with time. In fact, eqn (11) represents an approximation that holds only at the earliest times, and for times comparable with the time constants of turnover it is necessary to resort to numerical integration of eqn (10). Unfortunately we do not have a priori values for the three time constants, though we would certainly expect that none of them should exceed the ‘dominant time constant’ of recovery (Pepperberg et al. 1992) seen in the dark-adapted flash family, which has been measured as 18 ms (Friedburg et al. 2004; Fig. 9). Our approach therefore has been to try values of R, E and cG shorter than 18 ms, and observe the shape of recovery. The grey trace in Fig. 9 was obtained using Rh
= 5 ms, E
= 13 ms, cG
= 4 ms, with a total delay time for all the other short stages of tot
=
Meta
+
GE
+
chan
+
RC
+
filter
= 4 ms, and it clearly provides an acceptable description of the response up until about 13 ms after background extinction. We found that other combinations of values for the time constants could provide equally acceptable fits, and we certainly do not take the above combination to be more than an approximate indication of the true values.
Before accepting the adequacy of the similarity between the predictions of the model and the measured off-response, it is necessary to be aware of the finding of Robson et al. (2003), that postreceptoral contributions intrude in the conventional photopic a-wave within 5 ms of flash delivery in the case of bright stimuli. Although we are not aware of any reports of postreceptoral contribution to the ‘off a-wave’ , it is possible that a comparable phenomenon occurs, and that the responses shown on the right of Fig. 9 might include some component of postreceptoral activity. Nevertheless, our starting point is to assume that at suitably early times the ‘off a-wave’ is an approximate reflection of the recovery of cone circulating current.
Our conclusion is that a relatively simple molecular model of the G-protein cascade, based on that of Lamb & Pugh (1992) as extended by Nikonov et al. (1998), can generate an adequate description of the experimentally observed ‘background-off a-wave’ , if suitably short time constants are specified for the turnover of R*, G*–E* and cyclic GMP during intense illumination. From the traces in Fig. 9, we think it reasonable to conclude that the cone circulating current has recovered substantially towards its original dark level within about 15 ms of background extinction, and that this interpretation is consistent with the results from the bright-flash experiments. Beyond about 13 ms, though, the ERG off-response cannot provide direct information about the cone circulating current, because the spike-like activity would seem almost certain to arise from postreceptoral activity.
Cone current recovery is much faster than cone pigment regeneration or rod current recovery
The speed of recovery of cone circulating current is orders of magnitude faster than the regeneration of cone pigment, or the recovery of rod circulating current from comparable bleach
and F(t) predicted by eqns (8)–(10) and (12) are plotted, using the following values of parameters: 
