A "melanopic" spectral efficiency function predicts the sensitivity of melanopsin photoreceptors to polychromatic lights

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photosensitive retinal ganglion cells expressing the photopigment melanopsin. These mRGCs are especially important contributors to circadian entrainment, the pupil light reflex, and other so-called nonimage-forming (NIF) responses. The spectral sensitivity of melanopsin phototransduction has been addressed in several species by comparing responses to a range of monochromatic stimuli. The resultant action spectra match the predicted profile of an opsin:vitamin A-based photopigment (nomogram) with a peak sensitivity (λ(max)) around 480 nm. It would be most useful to be able to use this spectral sensitivity function to predict melanopsin's sensitivity to broad-spectrum, including "white," lights. However, evidence that melanopsin is a bistable pigment with an intrinsic light-dependent bleach recovery mechanism raises the possibility of a more complex relationship between spectral quality and photoreceptor response. Here, we set out to empirically determine whether simply weighting optical power at each wavelength according to the 480-nm nomogram and integrating across the spectrum could predict melanopsin sensitivity to a variety of polychromatic stimuli. We show that pupillomotor and circadian responses of mice relying solely on melanopsin for their photosensitivity (rd/rd cl) can indeed be accurately predicted using this methodology. Our data therefore suggest that the 480-nm nomogram may be employed as the basis for a new photometric measure of light intensity (which we term "melanopic") relevant for melanopsin photoreception. They further show that measuring light in these terms predicts the melanopsin response to light of divergent spectral composition much more reliably than other methods for quantifying irradiance or illuminance currently in widespread use.     

Spectral modulation attenuates molecular, endocrine, and neurobehavioral disruption induced by nocturnal light exposure

The human eye serves distinctly dual roles in image forming (IF) and non-image-forming (NIF) responses when exposed to light. Whereas IF responses mediate vision, the NIF responses affect various molecular, neuroendocrine, and neurobehavioral variables. NIF responses can have acute and circadian phase-shifting effects on physiological variables. Both the acute and phase-shifting effects induced by photic stimuli demonstrate short-wavelength sensitivity peaking ≈450-480 nm. In the current study, we examined the molecular, neuroendocrine, and neurobehavioral effects of completely filtering (0% transmission) all short wavelengths <480 nm and all short wavelengths <460 nm or partially filtering (~30% transmission) <480 nm from polychromatic white light exposure between 2000 and 0800 in healthy individuals. Filtering short wavelengths <480 nm prevented nocturnal light-induced suppression of melatonin secretion, increased cortisol secretion, and disrupted peripheral clock gene expression. Furthermore, subjective alertness, mood, and errors on an objective vigilance task were significantly less impaired at 0800 by filtering wavelengths <480 nm compared with unfiltered nocturnal light exposure. These changes were not associated with significantly increased sleepiness or fatigue compared with unfiltered light exposure. The changes in molecular, endocrine, and neurobehavioral processes were not significantly improved by completely filtering <460 nm or partially filtering <480 nm compared with unfiltered nocturnal light exposure. Repeated light-dark cycle alterations as in rotating nightshifts can disrupt circadian rhythms and induce health disorders. The current data suggest that spectral modulation may provide an effective method of regulating the effects of light on physiological processes.     

Divergent photic thresholds in the non-image-forming visual system: entrainment, masking and pupillary light reflex

Light is the principal cue that entrains the circadian timing system, but the threshold of entrainment and the relative contributions of the retinal photoreceptors—rods, cones and intrinsically photosensitive retinal ganglion cells—are not known. We measured thresholds of entrainment of wheel-running rhythms at three wavelengths, and compared these to thresholds of two other non-image-forming visual system functions: masking and the pupillary light reflex (PLR). At the entrainment threshold, the relative spectral sensitivity and absolute photon flux suggest that this threshold is determined by rods. Dim light that entrained mice failed to elicit either masking or PLR; in general, circadian entrainment is more sensitive by 1–2 log units than other measures of the non-image-forming visual system. Importantly, the results indicate that dim light can entrain circadian rhythms even when it fails to produce more easily measurable acute responses to light such as phase shifting and melatonin suppression. Photosensitivity to one response, therefore, cannot be generalized to other non-image-forming functions. These results also impact practical problems in selecting appropriate lighting in laboratory animal husbandry.     

The contribution of single and double cones to spectral sensitivity in budgerigars during changing light conditions

Bird colour vision is mediated by single cones, while double cones and rods mediate luminance vision in bright and dim light, respectively. In daylight conditions, birds use colour vision to discriminate large objects such as fruit and plumage patches, and luminance vision to detect fine spatial detail and motion. However, decreasing light intensity favours achromatic mechanisms and eventually, in dim light, luminance vision outperforms colour vision in all visual tasks. We have used behavioural tests in budgerigars (Melopsittacus undulatus) to investigate how single cones, double cones and rods contribute to spectral sensitivity for large (3.4°) static monochromatic stimuli at light intensities ranging from 0.08 to 63.5 cd/m². We found no influences of rods at any intensity level. Single cones dominate the spectral sensitivity function at intensities above 1.1 cd/m², as predicted by a receptor noise-limited colour discrimination model. Below 1.1 cd/m², spectral sensitivity is lower than expected at all wavelengths except 575 nm, which corresponds to double cone function. We suggest that luminance vision mediated by double cones restores visual sensitivity when single cone sensitivity quickly decreases at light intensities close to the absolute threshold of colour vision.     

Visual pigments, oil droplets, ocular media and cone photoreceptor distribution in two species of passerine bird: the blue tit (Parus caeruleus L.) and the blackbird (Turdus merula L.)

The spectral absorption characteristics of the retinal photoreceptors of the blue tit (Parus caeruleus) and blackbird (Turdus merula) were investigated using microspectrophotometry. The retinae of both species contained rods, double cones and four spectrally distinct types of single cone. Whilst the visual pigments and cone oil droplets in the other receptor types are very similar in both species, the wavelength of maximum sensitivity (λ_max) of long-wavelength-sensitive single and double cone visual pigment occurs at a shorter wavelength (557 nm) in the blackbird than in the blue tit (563 nm). Oil droplets located in the long-wavelength-sensitivesingle cones of both species cut off wavelengths below 570–573 nm, theoretically shifting cone peak spectral sensitivity some 40 nm towards the long-wavelength end of the spectrum. This raises the possibility that the precise λ_max of the long-wavelength-sensitive visual pigment is optimised for the visual function of the double cones. The distribution of cone photoreceptors across the retina, determined using conventional light and fluorescence microscopy, also varies between the two species and may reflect differences in their visual ecology. Accepted: 8 January 2000     

Visual adaptations in a diurnal rodent,<Emphasis Type="Italic"> Octodon degus</Emphasis>

The degu (Octodon degus) is a diurnal rodent, native to Chile. Basic features of vision and visual organization in this species were examined in a series of anatomical, electrophysiological and behavioral experiments. The lens of the degu eye selectively absorbs short-wavelength light and shows a progressive increase in optical density as a function of age. Electroretinograms recorded using a flicker-photometric procedure reveal three spectral mechanisms: a rod with peak sensitivity of about 500 nm and two types of cone having respective spectral peaks of about 362 nm and 507 nm. Opsin antibody labeling was used to determine the retinal distributions of the three receptor types. A total of about one-third of the approximately 9 million photoreceptors of the degu retina are cones with the two types (507 nm/362 nm) represented in a ratio of about 13:1. The contributions to vision of all three receptor types were examined in a series of behavioral experiments. A consistent feature of both the electrophysiological and behavioral results is that relatively high levels of light adaptation are required to effect the full transition from rod-based to cone-based vision. In behavioral tests degus were shown to be able to make color discriminations between ultraviolet and visible lights.     

CONES ARE REQUIRED FOR NORMAL TEMPORAL RESPONSES TO LIGHT OF PHASE SHIFTS AND CLOCK GENE EXPRESSION

In mammals, non-visual responses to light involve intrinsically photosensitive melanopsin-expressing retinal ganglion cells (ipRGCs) that receive synaptic inputs from rod and cone photoreceptors. Several studies have shown that cones also play a role in light entrainment, photic responses of the suprachiasmatic nucleus (SCN), pupil constriction, and sleep induction. These studies suggest that cones are mainly involved in the initial response to light, whereas melanopsin provides a sustained input for non-visual responses during continued light exposure. Based on this idea, we explored the effects of the absence of middle-wavelength (MW)-cones on the temporal responses of circadian behavior and clock gene expression in light. In mice lacking MW-cones, our results show a reduction in behavioral phase shifts in response to light stimulations of short duration at 480 and 530?nm, but no alteration for short-wavelength (360-nm) light exposures. Similarly, induction of the period gene mPer1 and mPer2 mRNAs in the SCN are attenuated in response to light exposures of mid to long wavelengths. Modeling of the photoresponses shows that mice lacking MW-cones have an overall reduction in sensitivity that increases with longer wavelengths. The differences in photic responsiveness are consistent with the idea that cones provide a strong initial phasic input to the circadian system at light-onset and may confer a priming effect on ipRGC responses to sub-threshold light exposures. In summary, the contribution of MW-cones is essential for the normal expression of phase shifts and clock gene induction by light in mammals. (Author correspondence: ouria.benyahya@inserm.fr)     

Spectral sensitivity of the cod,Gadus morhua L.

The spectral sensitivity of the cod was determined under both dark adapted and light adapted conditions in the laboratory. Cod were trained by cardiac conditioning to detect a difference in radiance between an image of spots and the background radiance of a screen. Thresholds for this response were measured for a range of different wavelengths, and expressed as quantum adjusted values. Electroretino‐graphic studies were also performed on the eyes of cod, and spectral sensitivity curves prepared. Under dark adapted conditions both the behavioural and e.r.g. derived curves showed greatest sensitivity in the blue/green at 490 nm, matching the absorption curve for rhodopsin. A secondary peak in the behaviourally derived curve in the green/yellow at 550 nm indicated that a population of yellow cones may be implicated with the rods in scotopic vision. Under light adapted conditions the behavioural curves showed a shift to the blue, perhaps indicating an adaption to the high red content of the illuminating source. The e.r.g. curve showed greatest sensitivity to blue/green, as in the scotopic experiments but with an enhanced response at 550 nm, indicating greater cone activity. It is suggested that there is complex interaction between rods and cones in the cod retina, both types of receptor being active over a wide range of light intensities.     

Wavelength-dependent effects of evening light exposure on sleep architecture and sleep EEG power density in men

Light strongly influences the circadian timing system in humans via non-image-forming photoreceptors in the retinal ganglion cells. Their spectral sensitivity is highest in the short-wavelength range of the visible light spectrum as demonstrated by melatonin suppression, circadian phase shifting, acute physiological responses, and subjective alertness. We tested the impact of short wavelength light (460 nm) on sleep EEG power spectra and sleep architecture. We hypothesized that its acute action on sleep is similar in magnitude to reported effects for polychromatic light at higher intensities and stronger than longer wavelength light (550 nm). The sleep EEGs of eight young men were analyzed after 2-h evening exposure to blue (460 nm) and green (550 nm) light of equal photon densities (2.8 x 10(13) photons x cm(-2) x s(-1)) and to dark (0 lux) under constant posture conditions. The time course of EEG slow-wave activity (SWA; 0.75-4.5 Hz) across sleep cycles after blue light at 460 nm was changed such that SWA was slightly reduced in the first and significantly increased during the third sleep cycle in parietal and occipital brain regions. Moreover, blue light significantly shortened rapid eye movement (REM) sleep duration during these two sleep cycles. Thus the light effects on the dynamics of SWA and REM sleep durations were blue shifted relative to the three-cone visual photopic system probably mediated by the circadian, non-image-forming visual system. Our results can be interpreted in terms of an induction of a circadian phase delay and/or repercussions of a stronger alerting effect after blue light, persisting into the sleep episode.     

Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms

Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.     

SPECTRAL SENSITIVITY IN TWO SPECIES OF PINNIPEDS (PHOCA VITULINA AND OTARIA FLAVESCENS)

Spectral sensitivity was measured in air in the light adapted state in two harbor seals and a South American sea lion using a behavioral training technique. Increment thresholds were determined in a spectral range from 390 nm to 670 nm in a simultaneous two‐choice discrimination task. The spectral sensitivity curves show two maxima in sensitivity, one main peak with a maximum around 500 nm in the harbor seal and around 550 nm in the South American sea lion, and a second, smaller peak with a maximum in the range of 410 nm in both species. The broad shape and the position of the maximum of the spectral sensitivity curve of the harbor seals suggests that even under photopic conditions both rods and cones are contributing to the measurements since harbor seals possess only one cone type. The maximum sensitivity in the green part of the spectrum may indicate an adaptation to a specific underwater environment.     

Scanning electron microscopy and microspectrophotometry of the photoreceptors of ictalurid catfishes

The retinal photoreceptors from larval channel catfish (Ictalurus punctatus) were studied using single cell, in situ microspectrophotometry. Rods appear at 5 days after hatch; cones are present from day one. The rods contain a visual pigment which absorbs light maximally at 540 nm. The cones contain either a green sensitive visual pigment with peak absorbance at 535 nm or a red sensitive visual pigment with peak absorbance at 608 nm. All pigments are based on vitamin A₂. Visual pigment complement does not change with age, as photoreceptors from adultI. punctatus, I. catus andI. melas contain visual pigments virtually identical to those of the larvalI. punctatus. Regardless of age, no visual pigment with peak absorbance in the short wavelength region of the spectrum was ever observed. Scanning electron microscopy of adultI. punctatus retinas showed large rods with long, cylindrical outer segments and smaller cones with short, tapered outer segments. The myoids of both rods and cones are extensable. The rods, embedded in a granular tapetal material, comprise from 50 to 60% of the photoreceptors. Only single cones are present. The data are consistent with the idea that the ictalurid catfishes spend their entire lives in an environment deficient in blue light.     

Spectral sensitivity of vision and bioluminescence in the midwater shrimp Sergestes similis

In the oceanic midwater environment, many fish, squid, and shrimp use luminescent countershading to remain cryptic to silhouette-scanning predators. The mid-water penaeid shrimp, Sergestes similis Hansen, responds to downward-directed light with a dim bioluminescence that dynamically matches the spectral radiance of oceanic down-welling light at depth. Although the sensory basis of luminescent countershading behavior is visual, the relationship between visual and behavioral sensitivity is poorly understood. In this study, visual spectral sensitivity, based on microspectrophotometry and electrophysiological measurements of photoreceptor response, is directly compared to the behavioral spectral efficiency of luminescent countershading. Microspectrophotometric measurements on single photoreceptors revealed only a single visual pigment with peak absorbance at 495 nm in the blue-green region of the spectrum. The peak electrophysiological spectral sensitivity of dark-adapted eyes was centered at about 500 nm. The spectral efficiency of luminescent countershading showed a broad peak from 480 to 520 nm. Both electrophysiological and behavioral data closely matched the normalized spectral absorptance curve of a rhodopsin with lambda(max) = 495 nm, when rhabdom length and photopigment specific absorbance were considered. The close coupling between visual spectral sensitivity and the spectral efficiency of luminescent countershading attests to the importance of bioluminescence as a camouflage strategy in this species.     

Photoreceptors and eyes of pikeperch Sander lucioperca,pike Esox lucius,perch Perca fluviatilis and roach Rutilus rutilus from a clear and a brown lake

The photoreceptors and eyes of four fish species commonly cohabiting Fennoscandian lakes with different light transmission properties were compared: pikeperch Sander lucioperca, pike Esox lucius, perch Perca fluviatilis and roach Rutilus rutilus. Each species was represented by individuals from a clear (greenish) and a humic (dark brown) lake in southern Finland: Lake Vesijärvi (LV; peak transmission around 570 nm) and Lake Tuusulanjärvi (LT; peak transmission around 630 nm). In the autumn, all species had almost purely A2-based visual pigments. Rod absorption spectra peaked at c.526 nm (S. lucioperca), c. 533 nm (E. lucius) and c. 540 nm (P. fluviatilis and R. rutilus), with no differences between the lakes. Esox lucius rods had remarkably long outer segments, 1.5–2.8-fold longer than those of the other species. All species possessed middle-wavelength-sensitive (MWS) and long-wavelength-sensitive (LWS) cone pigments in single, twin or double cones. Rutilus rutilus also had two types of short-wavelength sensitive (SWS) cones: UV-sensitive [SWS1] and blue-sensitive (SWS2) cones, although in the samples from LT no UV cones were found. No other within-species differences in photoreceptor cell complements, absorption spectra or morphologies were found between the lakes. However, E. lucius eyes had a significantly lower focal ratio in LT compared with LV, enhancing sensitivity at the expense of acuity in the dark-brown lake. Comparing species, S. lucioperca was estimated to have the highest visual sensitivity, at least two times higher than similar-sized E. lucius, thanks to the large relative size of the eye (pupil) and the presence of a reflecting tapetum behind the retina. High absolute sensitivity will give a competitive edge also in terms of short reaction times and long visual range.     

Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.     

The primary visual pathway in humans is regulated according to long-term light exposure through the action of a nonclassical photopigment

BACKGROUND: The mammalian eye shows marked adaptations to time of day. Some of these modifications are not acute responses to short-term light exposure but rely upon assessments of the photic environment made over several hours. In the past, all attempts at a mechanistic understanding have assumed that these adaptations originate with light detection by one or other of the classical photoreceptor cells (rods or cones). However, previous work has demonstrated that the mammalian eye contains non-rod, non-cone photoreceptors. This study aimed to determine whether such photoreceptors contribute to retinal adaptation. RESULTS: In the human retina, second-order processing of signals originating in cones takes significantly longer at night than during the day. Long-term light exposure at night is capable of reversing this effect. Here, we employed the cone ERG as a tool to examine the properties of the irradiance measurement pathway driving this reversal. Our findings indicate that this pathway (1) integrates irradiance measures over time periods ranging from at least 15 to 120 min; (2) responds to relatively bright light, having a dynamic range almost entirely outside the sensitivity of rods; (3) acts on the cone pathway primarily through a local retinal mechanism; and (4) detects light via an opsin:vitamin A photopigment (lambda(max) approximately 483 nm). CONCLUSIONS: A photopigment with a spectral sensitivity profile quite different from those of the classical rod and cone opsins but matching the standard profile of an opsin:vitamin A-based pigment drives adaptations of the human primary cone visual pathway according to time of day.     

Effects of Exposure to Intermittent versus Continuous Red Light on Human Circadian Rhythms,Melatonin Suppression,and Pupillary Constriction

Exposure to light is a major determinant of sleep timing and hormonal rhythms. The role of retinal cones in regulating circadian physiology remains unclear, however, as most studies have used light exposures that also activate the photopigment melanopsin. Here, we tested the hypothesis that exposure to alternating red light and darkness can enhance circadian resetting responses in humans by repeatedly activating cone photoreceptors. In a between-subjects study, healthy volunteers (n = 24, 21–28 yr) lived individually in a laboratory for 6 consecutive days. Circadian rhythms of melatonin, cortisol, body temperature, and heart rate were assessed before and after exposure to 6 h of continuous red light (631 nm, 13 log photons cm⁻² s⁻¹), intermittent red light (1 min on/off), or bright white light (2,500 lux) near the onset of nocturnal melatonin secretion (n = 8 in each group). Melatonin suppression and pupillary constriction were also assessed during light exposure. We found that circadian resetting responses were similar for exposure to continuous versus intermittent red light (P = 0.69), with an average phase delay shift of almost an hour. Surprisingly, 2 subjects who were exposed to red light exhibited circadian responses similar in magnitude to those who were exposed to bright white light. Red light also elicited prolonged pupillary constriction, but did not suppress melatonin levels. These findings suggest that, for red light stimuli outside the range of sensitivity for melanopsin, cone photoreceptors can mediate circadian phase resetting of physiologic rhythms in some individuals. Our results also show that sensitivity thresholds differ across non-visual light responses, suggesting that cones may contribute differentially to circadian resetting, melatonin suppression, and the pupillary light reflex during exposure to continuous light.     

Melanopsin contributions to irradiance coding in the thalamo-cortical visual system

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input to the ～40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.     

Distinct responses of cones and melanopsin-expressing retinal ganglion cells in the human electroretinogram

ABSTRACT: BACKGROUND: The discovery of the novel photoreceptor, melanopsin-expressing retinal ganglion cells (mRGCs), has raised researchers' interest in photoreceptive tasks performed by the mRGC, especially in non-image-forming visual functions. In a prior study, we investigated the mRGC response to light stimuli independent of rods and cones with the four-primary illumination system, which modulates stimulus levels to the mRGC and cones independently, and mRGC baseline responses were recorded in the electroretinogram (ERG). METHODS: In the present study, we used the same illumination system to compare independent responses of the mRGC and cones in five subjects (mean +/- SD age, 23.0 +/- 1.7 years). The ERG waveforms were examined as direct measurements of responses of the mRGCs and cones to stimulation (250 msec). Implicit times (the time taken to peaks) and peak values from 30 stimuli given to each subject were analyzed. RESULTS: Two distinct positive peaks appeared in the mRGC response, approximately 80 msec after the onset of the stimuli and 30 msec after their offset, while no such peaks appeared in the cone response. The response to the mRGC stimulus was significantly higher than that to the cone stimulus at ~80 msec (p < 0.05) and tended to be higher than the cone stimulus at ~280 msec (p = 0.08). CONCLUSIONS: Implicit time of the first peak was much longer than that to the b-wave and this delay might reflect mRGC's sluggish responses. This is the first report of amplitudes and implicit time in the ERG from the response of the mRGC that is independent of rods and cones and obtained using the four-primary illumination system.