Why rods and cones?

Under twenty-first-century metropolitan conditions, almost all of our vision is mediated by cones and the photopic system, yet cones make up barely 5% of our retinal photoreceptors. This paper looks at reasons why we additionally possess rods and a scotopic system, and asks why rods comprise 95% of our retinal photoreceptors. It considers the ability of rods to reliably signal the arrival of individual photons of light, as well as the ability of the retina to process these single-photon signals, and it discusses the advantages that accrue. Drawbacks in the arrangement, including the very slow dark adaptation of scotopic vision, are also considered. Finally, the timing of the evolution of cone and rod photoreceptors, the retina, and the camera-style eye is summarised.

Does EBV alter the pathogenesis of malaria?

Plasmodium falciparum infections have been implicated in immune deficiencies resulting in ineffective control of Epstein-Barr virus, thereby increasing the risk of endemic Burkitt lymphoma in children. However, the impact of Epstein-Barr virus infections on the development of immunity to P. falciparum has not been studied in depth. In this review, we examine novel findings from animal co-infection models and human immuno-epidemiologic studies to speculate on the impact of acute gammaherpesvirus co-infection on malarial disease severity. Children are often concurrently or sequentially infected with multiple pathogens, and this has implications for understanding the development of protective immunity as well as in the evaluation of vaccine efficacy.

The impact of supplementing lambs with algae on growth, meat traits and oxidative status.

The current study examined the effect of supplementing lambs with algae. Forty, three month old lambs were allocated to receive a control ration based on oats and lupins (n=20) or the control ration with DHA-Gold™ algae (~2% of the ration, n=20). These lambs came from dams previously fed a ration based on either silage (high in omega-3) or oats and cottonseed meal (OCSM: high in omega-6) at joining (dam nutrition, DN). Lamb performance, carcase weight and GR fat content were not affected by treatment diet (control vs algae) or DN (silage vs OSCM). Health claimable omega-3 fatty acids (EPA+DHA) were significantly greater in the LL of lambs fed algae (125±6mg/100g meat) compared to those not fed algae (43±6mg/100g meat) and this effect was mediated by DN. Supplementing with algae high in DHA provides a means of improving an aspect of the health status of lamb meat.

Examination of the effect of ageing and temperature at rigor on colour stability of lamb meat.

A study of factors (ageing period, rigor temperature and vitamin E level) impacting on the colour stability of lamb m. longissimus thoracis et lumborum (LL) during 3 days of simulated retail display was undertaken. The LL were taken from 84 lambs from 3 slaughters. Slices of LL were measured fresh (24h post-mortem) or after ageing for 5 days in vacuum packaging. The oxy/met ratio (630/580 nm), declined with display time, and increased with increasing temperature at pH6.0. Redness (a*) values also declined with display time and a reduction in redness values was observed as LL pH at 24h post-mortem and/or pH at 18°C increased. There was no effect of ageing period or vitamin E level on the oxy/met ratio or a* values when the vitamin E level averaged 3.76 mg/kg LL. These results suggest that maximising vitamin E levels in lambs and achieving a moderate rate of pH decline will optimise colour stability irrespective of ageing period.

The interrelationship between sensory tenderness and shear force measured by the G2 Tenderometer and a Lloyd texture analyser fitted with a Warner-Bratzler head.

A comparison of peak shear force results for a Lloyd texture analyser fitted with a Warner Bratzler type of shearing head and a G2 Tenderometer was undertaken using sheep meat. The G2 is a new version of the Tenderometer that uses an electric linear motor to compress the sample, but still retains the blunt wedge-shaped "tooth". Analysis of sheep meat samples (n=121) revealed that the average G2 Tenderometer shear force results were approximately 1.2 times those for the Lloyd based on the following model; Lloyd=1.561 Tenderometer(0.84). Both instruments explained low amounts of the variation (less than 20%) in the sensory traits tenderness and overall liking. The high values for the sensory traits indicate that a wider range of samples, including samples with lower sensory scores, is required to develop robust threshold estimates so that either instrument could be use as an auditing instrument for the processing industry.

Explaining the variation in the shear force of lamb meat using sarcomere length, the rate of rigor onset and pH.

The temperature when the pH=6.0 (temp@pH6) impacts on the tenderness and eating quality of sheep meat. Due to the expense, sarcomere length is not routinely measured as a variable to explain variation in shear force, but whether measures such as temp@pH6 are as useful a parameter needs to be established. Measures of rigor onset in 261 carcases, including the temp@pH6, were evaluated in this study for their ability to explain some of the variation in shear force. The results show that for 1 day aged product combinations of the temp@pH6, the pH at 18 °C and the pH at 24 h provided a larger reduction (almost double) in total shear force variation than sarcomere length alone, with pH at 24 h being the single best measure. For 5 day aged product, pH at 18 °C was the single best measure. Inclusion of sarcomere length did represent some improvement, but the marginal increase would not be cost effective.

Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses.

To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7(giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods.

Relationship between consumer ranking of lamb colour and objective measures of colour.

Given the lack of data that relates consumer acceptance of lamb colour to instrument measures a study was undertaken to establish the acceptability thresholds for fresh and displayed meat. Consumers (n=541) were asked to score 20 samples of lamb loin (m.longissimus thoracis et lumborum; LL) on an ordinal scale of 1 (very acceptable) to 5 (very unacceptable). A sample was considered acceptable by a consumer if it scored three or less. Ten samples were used for testing consumer response to fresh colour and 10 to test consumer response to colour during display of up to 4days. The colour of fresh meat was measured using a Minolta chromameter with a closed cone and a Hunter Lab Miniscan was used for measuring meat on display. For fresh meat when the a( *) (redness) and L( *) (lightness) values are equal to or exceed 9.5 and 34, respectively, on average consumers will consider the meat colour acceptable. However a( *) and L( *) values must be much higher (14.5 and 44, respectively) to have 95% confidence that a randomly selected consumer will consider a sample acceptable. For aged meat, when the wavelength ratio (630/580nm) and the a( *) values are equal to or greater than 3.3 and 14.8, respectively, on average consumers will consider the meat acceptable. These thresholds need to be increased to 6.8 for ratio (630/580nm) and 21.7 for a( *) to be 95% confident that a randomly selected consumer will consider a sample acceptable.

Dark adaptation recovery of human rod bipolar cell response kinetics estimated from scotopic b-wave measurements.

We recorded ganzfeld scotopic ERGs to examine the responses of human rod bipolar cells in vivo, during dark adaptation recovery following bleaching exposures, as well as during adaptation to steady background lights. In order to be able to record responses at relatively early times in recovery, we utilized a 'criterion response amplitude' protocol in which the test flash strength was adjusted to elicit responses of nearly constant amplitude. In order to provide accurate and unbiased measures of response kinetics, we utilized a curve-fitting procedure to fit a smooth function to the measured responses in the vicinity of the peak, thereby extracting both the time-to-peak and the amplitude of the responses. Following bleaching exposures, the responses exhibited both desensitization and accelerated kinetics. During early post-bleach recovery, the flash sensitivity and time-to-peak varied according to a power-law expression (with an exponent of 6), as found in the presence of steady background light. This light-like phenomenon, however, appeared to be set against the backdrop of a second, more slowly recovering 'pure' desensitization, most clearly evident at late post-bleach times. The post-bleach 'equivalent background intensity' derived from measurements of flash sensitivity faded initially with an S2 slope of approximately 0.24 decades min(-1), and later as a gentle S3 tail. When calculated from kinetics, the results displayed only the S2 slope. While the recovery of rod bipolar cell response kinetics can be described accurately by a declining level of opsin in the rods, the sensitivity of these cells is reduced further than expected by this mechanism alone.

The pathology of Plasmodium chabaudi infection is not ameliorated by the secreted filarial nematode immunomodulatory molecule, ES-62.

ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes. This molecule has been shown to reduce the severity of inflammation in collagen-induced arthritis (CIA) in mice, a model of rheumatoid arthritis, via down-regulation of anti-collagen type 1 immune responses. Malaria parasites induce a pro-inflammatory host immune response and many of the symptoms of malaria are immune system-mediated. Therefore we have asked whether the immunomodulatory properties of ES-62 can down-regulate the severity of malaria infection in BALB/c mice infected with Plasmodium chabaudi. We have found that ES-62 has no significant effect on the course of P. chabaudi parasitaemia, and does not significantly affect any of the measures of malaria-induced pathology taken throughout infection.

Dark adaptation of human rod bipolar cells measured from the b-wave of the scotopic electroretinogram.

To examine the dark adaptation of human rod bipolar cells in vivo, we recorded ganzfeld ERGs to (a) a family of flashes of increasing intensity, (b) dim test flashes presented on a range of background intensities, and (c) dim test flashes presented before, and up to 40 min after, exposure to intense illumination eliciting bleaches from a few per cent to near total. The dim flash ERG was characterized by a prominent b-wave response generated principally by rod bipolar cells. In the presence of background illumination the response reached peak earlier and desensitized according to Weber's Law. Following bleaching exposures, the response was initially greatly desensitized, but thereafter recovered slowly with time. For small bleaches, the desensitization was accompanied by acceleration, in much the same way as for real light. Following a near-total bleach, the response was unrecordable for >10 min, but after approximately 23 min half-maximal sensitivity was reached, and full sensitivity was restored between approximately 35 and 40 min. With smaller bleaches, recovery commenced earlier. We converted the post-bleach measurements of desensitization into 'equivalent background intensities' using a Crawford transformation. Across the range of bleaching levels, the results were described by a prominent 'S2' component (0.24 decades min(-1)) together with a smaller and slower 'S3' component (0.06 decades min(-1)), as is found for dark adaptation of the scotopic visual system. We attribute the S2 component to the presence of unregenerated opsin, and we speculate that the S3 component results from ion channel closure by all-trans retinal.

The photocurrent response of human cones is fast and monophasic.

BACKGROUND: The precise form of the light response of human cone photoreceptors in vivo has not been established with certainty. To investigate the response shape we compare the predictions of a recent model of transduction in primate cone photoreceptors with measurements extracted from human cones using the paired-flash electroretinogram method. As a check, we also compare the predictions with previous single-cell measurements of ground squirrel cone responses. RESULTS: The predictions of the model provide a good description of the measurements, using values of parameters within the range previously determined for primate retina. The dim-flash response peaks in about 20 ms, and flash responses at all intensities are essentially monophasic. Three time constants in the model are extremely short: the two time constants for inactivation (of visual pigment and of transducin/phosphodiesterase) are around 3 and 10 ms, and the time constant for calcium equilibration lies in the same range. CONCLUSION: The close correspondence between experiment and theory, using parameters previously derived for recordings from macaque retina, supports the notion that the electroretinogram approach and the modelling approach both provide an accurate estimate of the cone photoresponse in the living human eye. For reasons that remain unclear, the responses of isolated photoreceptors from the macaque retina, recorded previously using the suction pipette method, are considerably slower than found here, and display biphasic kinetics.

Toward a unified model of vertebrate rod phototransduction.

Recently, we introduced a phototransduction model that was able to account for the reproducibility of vertebrate rod single-photon responses (SPRs) (Hamer et al., 2003). The model was able to reproduce SPR statistics by means of stochastic activation and inactivation of rhodopsin (R*), transducin (G alpha ), and phosphodiesterase (PDE). The features needed to capture the SPR statistics were (1) multiple steps of R* inactivation by means of multiple phosphorylations (followed by arrestin capping) and (2) phosphorylation dependence of the affinity between R* and the three molecules competing to bind with R* (G alpha, arrestin, and rhodopsin kinase). The model was also able to account for several other rod response features in the dim-flash regime, including SPRs obtained from rods in which various elements of the cascade have been genetically disabled or disrupted. However, the model was not tested under high light-level conditions. We sought to evaluate the extent to which the multiple phosphorylation model could simultaneously account for single-photon response behavior, as well as responses to high light levels causing complete response saturation and/or significant light adaptation (LA). To date no single model, with one set of parameters, has been able to do this. Dim-flash responses and statistics were simulated using a hybrid stochastic/deterministic model and Monte-Carlo methods as in Hamer et al. (2003). A dark-adapted flash series, and stimulus paradigms from the literature eliciting various degrees of light adaptation (LA), were simulated using a full differential equation version of the model that included the addition of Ca2+-feedback onto rhodopsin kinase via recoverin. With this model, using a single set of parameters, we attempted to account for (1) SPR waveforms and statistics (as in Hamer et al., 2003); (2) a full dark-adapted flash-response series, from dim flash to saturating, bright flash levels, from a toad rod; (3) steady-state LA responses, including LA circulating current (as in Koutalos et al., 1995) and LA flash sensitivity measured in rods from four species; (4) step responses from newt rods ( Forti et al., 1989) over a large dynamic range; (5) dynamic LA responses, such as the step-flash paradigm of Fain et al. (1989), and the two-flash paradigm of Murnick and Lamb (1996); and (6) the salient response features from four knockout rod preparations. The model was able to meet this stringent test, accounting for almost all the salient qualitative, and many quantitative features, of the responses across this broad array of stimulus conditions, including SPR reproducibility. The model promises to be useful in testing hypotheses regarding both normal and abnormal photoreceptor function, and is a good starting point for development of a full-range model of cone phototransduction. Informative limitations of the model are also discussed.

Co-infected C57BL/6 mice mount appropriately polarized and compartmentalized cytokine responses to Litomosoides sigmodontis and Leishmania major but disease progression is altered.

This study examines the capacity of the mammalian host to fully compartmentalize the response to infection with type 1 vs. type 2 inducing organisms that infect different sites in the body. For this purpose, C57BL/6 mice were infected with the rodent filarial nematode Litomosoides sigmodontis followed by footpad infection with the protozoan parasite Leishmania major. In this host, nematode infection is established in the thoracic cavity but no microfilariae circulate in the bloodstream. We utilized quantitative ELISPOT analysis of IL-4 and IFN-gamma producing cells to assess cytokine bias and response magnitude in the lymph nodes draining the sites of infection as well as more systemic responses in the spleen and serum. Contrary to other systems where co-infection has a major impact on bias, cytokine ratios were unaltered in either local lymph node. The most notable effect of co-infection was an unexpected increase in the magnitude of the IFN-gamma response to L. major in mice previously infected with L. sigmodontis. Further, lesion development was significantly delayed in these mice. Thus, despite the ability of the immune system to appropriately compartmentalize the immune response, interactions between responses at distinct infection sites can alter disease progression.

Extremely rapid recovery of human cone circulating current at the extinction of bleaching exposures.

We used a conductive fibre electrode placed in the lower conjunctival sac to record the a-wave of the human photopic electroretinogram elicited by bright white flashes, delivered during, or at different times after, exposure of the eye to bright white illumination that bleached a large fraction (approximately 90%) of the cone photopigment. During steady-state exposures of this intensity, the amplitude of the bright-flash response declined to approximately 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.

Inverted photocurrent responses from amphibian rod photoreceptors: role of membrane voltage in response recovery.

We recorded photocurrent responses of retinal rods isolated from cane toads Bufo marinus and clawed frogs Xenopus laevis. With the outer segment drawn part way into the suction pipette, presentation of flashes to the base of the outer segment (outside the pipette) elicited a slow inverted response. Stimulation of the same region, with the outer segment drawn fully in, gave a response of conventional polarity. For moderate to bright flashes a fast transient preceded the slow inverted response. Upon bleaching the tip of the outer segment, the slow inverted response was abolished but the fast initial transient remained, and we attribute this fast component to a capacitive current. Experiments employing simultaneous whole-cell patch-clamp and suction pipette recording revealed that both the fast and slow components of the inverted responses were absent in voltage-clamped cells. In current-clamped cells the slow inverted current response was delayed substantially with respect to the voltage response. We present a computational model for the slow component, in which hyperpolarization leads to increased activity of the Na+ -Ca2+, K+ exchanger, hence lowering the cytoplasmic Ca2+ concentration, activating guanylyl cyclase, raising cyclic GMP concentration, opening cyclic nucleotide-gated channels, and increasing circulating current in the unstimulated region. For the measured voltage response to stimulation of the base, we solve these equations to predict the photocurrent in the tip, and obtain an adequate explanation of the inverted responses. Our work suggests a novel role for membrane voltage in accelerating the inactivation phase of the response to light.

Dark adaptation and the retinoid cycle of vision.

Following exposure of our eye to very intense illumination, we experience a greatly elevated visual threshold, that takes tens of minutes to return completely to normal. The slowness of this phenomenon of "dark adaptation" has been studied for many decades, yet is still not fully understood. Here we review the biochemical and physical processes involved in eliminating the products of light absorption from the photoreceptor outer segment, in recycling the released retinoid to its original isomeric form as 11-cis retinal, and in regenerating the visual pigment rhodopsin. Then we analyse the time-course of three aspects of human dark adaptation: the recovery of psychophysical threshold, the recovery of rod photoreceptor circulating current, and the regeneration of rhodopsin. We begin with normal human subjects, and then analyse the recovery in several retinal disorders, including Oguchi disease, vitamin A deficiency, fundus albipunctatus, Bothnia dystrophy and Stargardt disease. We review a large body of evidence showing that the time-course of human dark adaptation and pigment regeneration is determined by the local concentration of 11-cis retinal, and that after a large bleach the recovery is limited by the rate at which 11-cis retinal is delivered to opsin in the bleached rod outer segments. We present a mathematical model that successfully describes a wide range of results in human and other mammals. The theoretical analysis provides a simple means of estimating the relative concentration of free 11-cis retinal in the retina/RPE, in disorders exhibiting slowed dark adaptation, from analysis of psychophysical measurements of threshold recovery or from analysis of pigment regeneration kinetics.

Contribution of cone photoreceptors and post-receptoral mechanisms to the human photopic electroretinogram.

We recorded the electroretinogram (ERG) from human subjects with normal vision, using ganzfeld stimulation in the presence of rod-suppressing blue background light. In families of responses to flashes of increasing intensity, we investigated features of both receptoral and post-receptoral origin. Firstly, we found that the oscillatory potentials (OPs, that have long been known to be post-receptoral) exhibited a time course that was invariant over a range of bright flash intensities. Secondly, we found that the photopic b-wave (which probably originates in cone ON bipolar cells) was most pronounced after test flashes of around 20 Td s, and could be suppressed either by increasing the test flash intensity or by applying a second flash after the test flash. We obtained estimates of the time course of the cone photoreceptor response using the paired-flash technique, in which an intense 'probe' flash was delivered at different times after a test flash. The response to the probe flash was recorded and, its amplitude was measured at early times after the probe flash. Estimates obtained in this way were of normalized amplitude, but could be scaled to an absolute amplitude by making an assumption about the level of probe-flash response that corresponded to complete suppression of photoreceptor current. For moderately bright test flashes the estimated cone photoreceptor response at early times coincided closely with the a-wave of the test flash ERG. However, the maximal size of this estimated response accounted for only about 70% of the peak a-wave amplitude in the case of bright flashes, and for an even smaller proportion after flashes of lower intensity, and we take this to indicate the existence of a third substantial post-receptoral contribution to the a-wave. For dim flashes, the time-to-peak of the cone response was around 15-20 ms, and for saturating flashes the dominant time constant of recovery was about 18 ms. The intensity dependence of the estimated cone response amplitude at fixed times followed an exponential saturation relation. We provide a comparison between our estimates of photoreceptor responses from human cones, and recent estimates from monkey cones obtained using related ERG approaches, and earlier single-cell measurements from isolated primate cones.

Recovery of the human photopic electroretinogram after bleaching exposures: estimation of pigment regeneration kinetics.

We used a fibre electrode in the lower conjunctival sac of the human eye to record the a-wave of the photopic electroretinogram elicited in response to dim red flashes, delivered in the presence of a rod-saturating blue background, before and after exposure of the eye to bright white illumination that bleached a significant fraction of cone photopigment. Responses were recorded from two normal subjects whose pupils were maximally dilated. A range of intensities of bleaching light were used, from 500 to 3000 photopic cd m(-2), and exposures were made sufficiently long in duration to achieve a steady-state bleach. In addition, responses were also recorded following shorter durations of exposures to the highest intensity (3000 cd m(-2)); these durations ranged from 5 to 60 s. The amplitude of the a-wave response to dim flashes was reduced following the exposures, with brighter or longer exposures causing greater reduction. The amplitude then recovered within about 4 min to the prebleach level. The amplitudes measured at ca 15 ms after the flash were used to derive the effective intensity of the flashes, thereby quantifying the fraction of photopigment available at the time of delivery of each flash. Recovery from all exposures in both subjects followed a common time course, which could be described well by a model of pigment kinetics based on rate-limited regeneration, where the initial rate of recovery following a total bleach was ca 50% of the total pigment per minute, and the residual pigment level for half the maximal rate was ca 20% of the total pigment. The same parameters, together with a fixed photosensitivity, could account for the steady-state pigment levels seen at each bleaching intensity, and also for the fraction of pigment bleached following exposures of different duration at the highest intensity. The dim-flash ERG thus provides a novel method for assessing pigment regeneration in vivo. Our finding that pigment regeneration follows rate-limited kinetics may explain previous reports of pigment regeneration deviating from first order kinetics. We present a model of regeneration in which the rate limit arises from a limitation in the delivery of 11-cis-retinoid to the photoreceptor outer segments.

Multiple steps of phosphorylation of activated rhodopsin can account for the reproducibility of vertebrate rod single-photon responses.

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K., J.H. Parkes, and P.A. Liebman. 2000. Biochemistry. 39:5738-5749.). We evaluated the model by means of Monte-Carlo simulations of dim-flash responses, and compared the response statistics derived from them with those obtained from empirical dim-flash data (Whitlock, G.G., and T.D. Lamb. 1999. Neuron. 23:337-351.). The model accounts for four quantitative measures of SPR reproducibility. It also reproduces qualitative features of rod responses obtained with altered nucleotide levels, and thus contradicts the conclusion that such responses imply that phosphorylation cannot dominate R* inactivation (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Moreover, the model is able to reproduce the salient qualitative features of SPRs obtained from mouse rods that had been genetically modified with specific pathways of R* inactivation or Ca2+ feedback disabled. We present a theoretical analysis showing that the variability of the area under the SPR estimates the variability of integrated R* activity, and can provide a valid gauge of the number of R* inactivation steps. We show that there is a heretofore unappreciated tradeoff between variability of SPR amplitude and SPR duration that depends critically on the kinetics of inactivation of R* relative to the net kinetics of the downstream reactions in the cascade. Because of this dependence, neither the variability of SPR amplitude nor duration provides a reliable estimate of the underlying variability of integrated R* activity, and cannot be used to estimate the minimum number of R* inactivation steps. We conclude that multiple phosphorylation-dependent decrements in R* activity (with Arr-quench) can confer the observed reproducibility of rod SPRs; there is no compelling need to invoke a long series of non-phosphorylation dependent state changes in R* (as in Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Our analyses, plus data and modeling of others (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.), also argue strongly against either feedback (including Ca2+-feedback) or depletion of any molecular species downstream to R* as the dominant cause of SPR reproducibility.