July 2002
Volume 43, Issue 7
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Visual Neuroscience  |   July 2002
Correlating Retinal Function with Melatonin Secretion in Subjects with an Early or Late Circadian Phase
Author Affiliations
  • Marianne Rufiange
    From the Department of Ophthalmology, McGill University, Montreal Children’s Hospital Research Institute, Montreal, Quebec, Canada; and
    Department of Psychiatry, University of Montreal, Chronobiology Laboratory, Sacre-Coeur Hospital, Montreal, Quebec, Canada.
  • Marie Dumont
    Department of Psychiatry, University of Montreal, Chronobiology Laboratory, Sacre-Coeur Hospital, Montreal, Quebec, Canada.
  • Pierre Lachapelle
    From the Department of Ophthalmology, McGill University, Montreal Children’s Hospital Research Institute, Montreal, Quebec, Canada; and
Investigative Ophthalmology & Visual Science July 2002, Vol.43, 2491-2499. doi:
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      Marianne Rufiange, Marie Dumont, Pierre Lachapelle; Correlating Retinal Function with Melatonin Secretion in Subjects with an Early or Late Circadian Phase. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2491-2499.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Evaluate the diurnal variation of retinal function, as measured with the electroretinogram (ERG), in subjects with an early (morning type: M-type) or a late (evening type: E-type) circadian phase.

methods. Subjects (n = 24) were recruited according to their scores on a Morningness-Eveningness Questionnaire assessing preferences in, e.g., bedtime, waketime, and timing of performance. ERG testing was performed twice on each subject, at 22:30 and at 08:00. Luminance-response functions were obtained in scotopic (blue flashes) and in photopic conditions (white, blue, green, and red flashes). Salivary melatonin samples were taken every half-hour from 20:30 to 00:00 and from 06:30 to 09:30.

results. In scotopic conditions, both groups had lower ERG amplitudes and retinal sensitivity at 08:00. In photopic conditions, the two groups showed an opposite pattern of diurnal variations. The E-types demonstrated a significant reduction in ERG amplitudes at 08:00, whereas the M-types showed an increase in amplitude at the same time. In addition, negative correlations were found between both the cone ERG and mixed rod–cone ERG and the concentration of salivary melatonin, indicating that the ERG amplitude is lowest when melatonin concentration is highest.

conclusions. The reduction in scotopic ERG responses at 08:00 seen in both groups might be due to the peak of rod disc shedding that takes place, in some mammals, at around light onset. The strong correlation between the cone ERG and salivary melatonin could be attributable to a direct effect of retinal melatonin on the physiology of cones or of the circadian phase of the subjects.

Diurnal variations in human retinal functions as measured with the electroretinogram (ERG) have been previously reported. Nozaki et al. 1 observed a diurnal rhythm in the scotopic mixed (rod–cone) b-wave amplitude in >60% of their subjects where the minimal amplitude was observed early in the morning. Birch et al. 2 3 reported a 24-hour rhythm for the rod b-wave amplitude in eyes entrained to a 14 hour-light/10 hour-dark cycle. Again, the lowest amplitude was observed at 09:30, a time that the authors associated with the diurnal peak of rod outer segment disc shedding. More recently, Hankins et al. 4 5 reported an increase in the implicit time of the photopic b-wave at night compared with daytime measurements for subjects kept in natural lighting conditions, a finding that is also consistent with the presence of diurnal variations in the human ERG. 
The abovementioned variations in ERG parameters could either be the consequences of the light–dark cycle or that of the sleep–wake rhythm. However, several studies 6 7 8 9 10 have shown diurnal variations of the ERG in animals kept under constant environmental conditions. It is therefore possible that the diurnal variations in retinal functions are under the control of an endogenous circadian oscillator. Among the several hypotheses proposed to explain this diurnal rhythm, there is the role of dopamine and melatonin within the retina. Dubocovich 11 suggested that interactions between retinal dopamine and melatonin could control the retinal sensitivity to light. A circadian rhythm in the secretion of melatonin has been demonstrated in isolated retinas of some mammals, including rats and mice, that was maintained in constant darkness, 12 13 14 showing that the rhythmic secretion of melatonin is endogenous to the retina itself. According to most studies, the rod and cone photoreceptors would be the most probable candidates for melatonin synthesis in the retina. 15  
Retinal melatonin was shown to be involved in cone elongation, 16 aggregation of melanin pigments in the pigmented epithelium, 17 18 suppression of light-adaptive horizontal cell spinule formation, 19 priming of the light-evoked rod outer segment disc shedding, 20 21 and inhibition of dopamine release. 22 23 24 Melatonin is considered by many as the major signal of dark adaptation or nighttime within the retina and dopamine as that of light adaptation or daytime. 15 19 25 26 It is therefore possible that the diurnal variations observed in retinal functions could in part be the consequences of the endogenous rhythm of retinal melatonin secretion. 
In humans, it is impossible to measure directly the secretion of retinal melatonin. However, with the development of highly sensitive radioimmunological assays, it is now possible to determine repeatedly and noninvasively the concentration of circulating melatonin in the saliva. This measure accurately reflects the plasmatic concentration of pineal melatonin. 27 Although the phase relationship between retinal and pineal melatonin rhythms is still uncertain, both episodes of melatonin secretion are essentially nocturnal and are acutely suppressed by exposing the retina to light. In addition, both circadian rhythms are similarly entrained by the light–dark cycle. 15 Therefore, it is reasonable to assume that the two rhythms of melatonin secretion will have the same circadian phase under a normal light–dark cycle. 
Consequently, the aim of this study was to assess the relationship between the timing of melatonin secretion and the diurnal variations in retinal function as measured with the ERG. We also examined if the variation in the cone response could be influenced by the wavelength of the stimulus because a previous report of ours on the photopic luminance-response function showed that the maximal amplitude of the cone b-wave was wavelength dependent, 28 a finding that suggests that the cone function can be further characterize with this technique. Retinal function was evaluated at the same clock time in the late evening and in the early morning, in two groups of healthy volunteers kept under the same sleep/dark schedule but differing as to their timing of melatonin secretion. 
Methods
Subjects
A French version of the Horne and Ostberg 29 questionnaire was used to characterize Morningness-Eveningness orientation in 569 university students. This questionnaire is composed of 19 questions from which a score (M/E score) between 16 and 86 is obtained. A score higher than 58 identifies morning type individuals (M-types), whereas an index lower than 42 identifies evening types (E-types). It is expected that M-types will have earlier habitual waketimes and bedtimes, and earlier onset and offset of melatonin secretion compared with E-types. 30 Among the respondents, 108 were M-types and 142 were E-types. From the above, 12 M-types (M/E scores: 60–74, mean, 66.6; age, 19–25 years; mean, 21.1 years; 9 women, 3 men), and 12 E-types (M/E scores: 22–38, mean, 27.8; age, 20–25 years; mean, 21.3 years; 8 women, 4 men) were selected for the study. All subjects had a regular sleep–wake schedule without sleep or vigilance complaints. Subjects were not using medications known to affect sleep, vigilance, or melatonin secretion (e.g., NSAIDs, β-blockers, anxiolytics, or hypnotics). No caffeine or tobacco products could be used during the experiment. A complete ophthalmologic examination was performed before the experiment to rule out any retinal disorders. All subjects had best corrected visual acuity of 20/20 or better. Finally, subjects were excluded if they had worked on night shifts during the past 6 months or had made a transmeridian flight in the past month. Each participant signed an informed consent form approved by the ethics committee of the Montreal Children’s Hospital and received a financial compensation. Furthermore, the research followed the tenets of the Declaration of Helsinki. 
Procedures
The experiment was performed from July 1999 to June 2000 and the subjects from the two groups were tested in random order. Ambulatory activity measures (Actiwatch; Mini-Mitter Co., Bend, OR) were obtained during 2 to 7 days before testing. Each participant also completed a 7-day sleep–wake diary. On the day of testing, the subjects were admitted to the Visual Electrophysiology Laboratory (Department of Ophthalmology, Montreal Children’s Hospital) at 19:30 and stayed until 09:30 the following morning. Each subject participated to two ERG sessions: the first one from 22:30 to 23:40 and the second from 08:00 to 09:10 on the following morning. The subjects were kept under dim illumination (<10 lux) from 19:30 to 09:30, except during the photopic part of the ERG, i.e., from 22:50 to 23:40 and from 08:20 to 09:10. Subjects slept in complete darkness from 00:00 to 06:00 and had to wear dark goggles (5 log units of attenuation) to go to the bathroom. 
To measure the concentration of melatonin, saliva samples were collected (using Salivettes; Sarstedt Inc., Newton, NC) every half-hour from 20:30 to 00:00 and from 06:30 to 09:30. Melatonin concentrations were determined by radioimmunoassay with a 125I-labeled tracer (Bühlmann Laboratories, Basel, Switzerland). With this method, the minimum detectable dose of melatonin (analytical sensitivity) is reported to be 0.2 pg/ml, whereas the functional least detectable dose is 0.65 pg/ml. 31 All samples from a given subject were assayed in the same run. Each sample was divided in two for the extraction, and sample duplicates with a coefficient of variation (CV) larger than 10% were rejected (3.2% of all samples). The intra-assay CVs for control samples of 1.9 and 14.7 pg/ml were 6.1% and 7.3%, respectively. 
Electrophysiological recordings were performed as previously reported 32 33 and in accordance with the ISCEV ERG standards. 34 The pupils were maximally (8–9 mm) dilated with tropicamide 1%, and the pupil size was measured at the beginning and at the end of the recording procedure. There were no pupil size differences noted either between the evening and morning sessions or between the beginning and end of the ERG procedure. DTL fiber electrodes (27/7 X-Static silver-coated nylon conductive yarn; Sauquoit Industries, Scranton, PA) were positioned deep into the inferior conjunctival bag and secured with double-sided adhesive tape at the external and internal canthi. Reference and ground electrodes (Grass gold cup electrode filled with Grass EC2 electrode cream) were pasted at the external canthi and forehead, respectively. ERGs (bandwidth, 0.3–500 Hz; amplification, 10,000× scotopic and 20,000× photopic; attenuation, 6 dB) and oscillatory potentials (OPs; bandwidth, 75–500 Hz; amplification, 10,000× scotopic and 20,000× photopic; attenuation, 6 dB) were recorded simultaneously from both eyes with an LKC UTAS-E-3000 system (LKC Systems Inc., Gaithersburg, MD) that included a Ganzfeld of 30 cm in diameter. 
Subjects were first dark-adapted for 30 minutes (from 22:00 to 22:30 and from 07:30 to 08:00, respectively). Scotopic luminance-response functions were then obtained (from 22:30 to 22:50 and from 08:00 to 08:20, respectively) with the use of 11 intensities of blue (GamColor filter 850, λmax = 410 nm; GAM Products, Hollywood, CA) flashes ranging from −5.01 to −0.96 log cd/m2 · sec. Each flash had a duration of 20 μsec, and the interstimulus interval was fixed at 10 seconds. Five responses were recorded and averaged at each flash intensity. To avoid the light adaptation effect previously reported, 35 the subjects were then light-adapted for 10 minutes (from 22:50 to 23:00 and from 08:20 to 08:30, respectively) to a white light background of 17 cd/m2, after which photopic ERGs were recorded against the same background light (from 23:00 to 23:40 and from 08:30 to 09:10, respectively). The interstimulus interval was reduced to 2.3 seconds for the photopic ERG. Ten responses were recorded and averaged at each flash intensity. Luminance–response functions were obtained with the use of 15 intensities of white (−0.8–2.84 log cd/m2 · sec), 8 intensities of blue (GamColor filter 850, λmax = 410 nm; −2.01–1.24 log cd/m2 · sec), 6 intensities of green (GamColor filter 650, λmax = 510 nm; −1.31–1.14 log cd/m2 · sec) and 6 intensities of red (GamColor filter 250, λmax = 640 nm; −1.43–1.02 log cd/m2 · sec) light. Flash intensities and background luminance were measured with a research radiometer (IL 1700; International Light, Newburyport, MA). 
Data Analysis
Of the 24 subjects, two had saliva samples with insufficient volume for accurate melatonin measurements. Therefore, melatonin results are available only for 11 M-type and 11 E-type subjects. Because there are large interindividual variations in melatonin concentration, 36 all analyses were performed on data transformed into percent of the maximum concentration observed in each individual subject. The onset of melatonin secretion was defined as the clock time of the first evening saliva sample with a melatonin concentration ≥33% of the maximum. Similarly, melatonin offset was defined as the clock time of the first morning sample with a melatonin concentration ≤33% of maximum concentration. For comparisons with ERG parameters, relative melatonin concentrations were averaged among the samples collected during the evening ERG session (at 22:30, 23:00, and 23:30) and during the morning ERG session (at 08:00, 08:30, and 09:00). For each subject, habitual bedtime and waketime were estimated by averaging the times reported in the sleep–wake diary for the 5 days before the experiment. These estimates were validated with the activity data recorded with the ambulatory monitor. 
Figure 1 illustrates typical scotopic and photopic ERG recordings evoked to progressively brighter flashes (from bottom to top). The corresponding luminance–response functions (mean of both eyes) are graphically reported at the bottom part of the figure. In scotopic conditions, the amplitudes of the a-wave (▪) and the b-wave (□) increase gradually with flash intensity. The maximal amplitude of the scotopic b-wave or rod V max was determined by fitting (GraphPad Software, San Diego, CA) the b-wave amplitude data—from the lowest intensity to the intensity at which an a-wave of twice the baseline noise was obtained (here, at −2.22 log cd/m2 · sec)—to a sigmoidal curve (solid line). 32 37 The point of maximal amplitude on this curve was identified as the rod V max. In addition, we also considered the ERG recorded at the maximal intensity (−0.96 log cd/m2 · sec) used in scotopic conditions, which we identified as the scotopic mixed rod–cone response. 34 In comparison, the luminance–response curve of the photopic ERG also shows that the a-wave augments in amplitude with intensity, but the behavior of the b-wave differs significantly. With progressively brighter flashes, the amplitude of the b-wave first increases to reach a maximum (V max) and then gradually decreases with higher flash intensities to finally form a plateau. This unique luminance-response function was previously described as the Photopic Hill. 28 38 39  
The analysis of the ERG included peak time and amplitude measurements of the a-, b-, and i-waves. The data from both eyes were averaged to yield a single data point. The amplitude of the a-wave was measured from baseline (which was recorded for 40 msec before flash onset) to trough and that of the b-wave from the trough of the a-wave to peak of the b-wave. The i-wave, which is the small positive peak after the b-wave, 40 was measured from the trough after the peak of the b-wave to the peak of the i-wave. The amplitude of each of the three major photopic OPs was also calculated from the preceding trough to the peak, except for OP2, which was measured from the baseline to the peak. Peak times were measured from flash onset to the peak of each wave. The rod sensitivity (K s) was determined as the flash intensity necessary to produce a b-wave 50% of the scotopic V max, whereas the cone sensitivity (K p) was defined as the flash intensity needed to produce the photopic V max (see Fig. 1 ). It should be noted that one outlier (> ±2 SD) was rejected for the analyses for the amplitude of the scotopic mixed a-wave and three outliers that were also rejected for the peak time of the scotopic mixed b-wave. 
Statistical comparisons between groups and times of testing were performed with 2 × 2 analyses of variance (ANOVAs), with the factor Group (M-types and E-types) and the factor Time-of-Day (22:30 and 08:00, repeated measures). Contrast analyses (planned comparisons) were performed when interaction effects were found. Melatonin onset and offset, as well as habitual bedtime and waketime were compared between groups with Student’s t-tests for independent samples. Correlations between diurnal variations in melatonin concentration and ERG parameters were computed with the Pearson test on the ratio of the results obtained during the evening session over the results obtained during the morning session. Melatonin ratios were log-transformed to normalized their distribution for the statistical analyses. 
Results
Melatonin Secretion
As expected, M-types had earlier habitual bedtimes and waketimes compared with E-types and also showed an earlier circadian phase, as estimated with the onset and offset of melatonin secretion (Table 1) . Averaged relative melatonin concentrations measured during the ERG sessions showed a strong Group-by-Time interaction (F 1,20 = 14.18, P < 0.001). The relative melatonin concentration was significantly higher in the M-type group compared with the E-type group during the evening session (F 1,20 = 8.69, P < 0.01), whereas the reverse was seen during the morning session (F 1,20 = 10.13, P < 0.01). Group differences in profiles of melatonin secretion in relation with the timing of the ERG recordings are illustrated at Figure 2 . The episode of melatonin secretion occurred earlier in the M-type group compared with the E-type group as revealed by the earlier increase in concentration in the evening as well as the earlier decrease in the morning in former group. A drop in melatonin concentration was observed at 23:30 in the E-type group, which was 40 minutes after the background light had been turned on. This decrease was seen in 8 of the 11 E-type subjects. No similar decreases were detected with the M-types during the evening session or during the morning session for either group. 
Retinal Function
Figure 3 shows representative ERG responses (i.e., scotopic V max, scotopic mixed response, photopic V max and photopic OPs) obtained from one E-type and one M-type subject during the evening (22:30, dash line) and the morning (08:00, solid line) sessions. It can be observed that the overall morphology of the ERG responses remained the same irrespective of the recording session. However, there are suggestions of amplitude changes that are most prominent for the E-type subject. This is best visualized at Table 2 , where the mean (±1 SD) of the scotopic and photopic results obtained from all 24 subjects are given along with their respective diurnal variations, where 08:00 ERG measurements are expressed as a percentage (%) of the 22:30 measurements. 
In photopic conditions (Table 2) , irrespective of the wavelength of the stimulus, the amplitude of the a- and b-waves at V max intensity as well as the sum OPs amplitude showed an opposite pattern in diurnal variation between the two groups of subjects. The E-type group demonstrated a 4% to 16% reduction in amplitude at 08:00 compared with 22:30, whereas the M-type group showed a 3% to 13% increase in amplitude. Furthermore, all Group-by-Time interactions resulted from a significant reduction in the amplitude at 08:00 for the E-types. For the M-types, none of the amplitude increases at 08:00 was significant. Finally, no diurnal changes were observed for either group in the cone sensitivity (K p), in the i-wave maximal amplitude (see Table 2 ) or in the photopic a- and b-wave peak times at V max intensity. 
In scotopic conditions (Table 2) , both groups showed a 08:00 decrease in rod sensitivity (K s) and in amplitude of the b-wave V max as well as in the amplitude of the mixed a- and b-waves. The magnitude of these diurnal changes, however, was greater in the E-type group. The amplitudes of the a- and b-waves in scotopic mixed ERGs both showed a Group-by-Time interaction. Compared with 22:30, the amplitudes of the a- and b-waves of the E-type group measured at 08:00 were significantly reduced by 24% and 17%, respectively, whereas the M-type group showed no significant diurnal changes (<5%). In contrast, the two groups could not be differentiated with their diurnal variations in K s and b-wave V max amplitude (Time effect, see Table 2 ) despite the fact that E-types showed a slightly greater morning decrease (14% for V max and 6% for K s) compared with M-types (7% for V max and 2% for K s). No diurnal variations in peak times were found for E-types and M-types except for the a- and b-waves of the scotopic mixed response (see Table 2 ). 
Correlations between Melatonin Secretion and Retinal Function
Figure 4 presents the evening session over morning session ratios of ERG amplitude results plotted against the corresponding log melatonin ratios obtained for each M-type and E-type subjects. In both scotopic and photopic conditions, the ERG amplitude ratios decreased as the ratios of melatonin concentration increased. The negative correlation was significant for the scotopic mixed a- and b-waves (r = −0.52, P < 0.01 and r = −0.45, P < 0.05, respectively) as well as for the photopic b-wave V max obtained with the white flash (r = −0.44, P < 0.05; see Fig. 4 ). However, significance was not reached for the scotopic b-wave V max or for the photopic a-wave at V max intensity. Although not shown at Figure 4 , the correlation was also significant for the photopic a- and b-waves at V max obtained with the green (r = −0.42 and r = −0.50, P < 0.05, respectively) and red (r = −0.49 and r = −0.43, P < 0.05, respectively) stimuli. Furthermore, a higher peak time ratio (scotopic mixed a- and b-waves) was associated with a higher ratio of salivary melatonin (r = +0.50 and r = +0.51, P < 0.05, respectively). No correlations were found with the K s or with photopic sum OPs. 
Discussion
Our results provide support to the hypothesis that parameters of the human ERG change according to the time of day and that melatonin (pineal and/or retinal) is associated with this diurnal variation. For E-types, the amplitudes of the a- and b-waves in photopic conditions were significantly lower in the morning ERG compared with the evening. This decrease was accompanied by a higher concentration of salivary melatonin compared with the level obtained during the evening session. In contrast, in the M-type group, ERG tended to increase as melatonin concentration decreased in the morning session relative to the evening session. Furthermore, the significant correlations obtained in photopic (cone response) and scotopic mixed (rod–cone response) conditions between the daily variation in amplitude and peak time of the a- and b-waves and that of the salivary melatonin concentration suggest a close relationship between the two phenomena. Two hypotheses arise from the above assertion: either melatonin has a direct effect on the visual physiology or the same circadian oscillator regulates both rhythms. 
It appears from the results reported in this study that the presence of melatonin in the organism is associated with a decrease in the amplitude and an increase in the peak time of the cone response. Our findings thus confirm those of Emser et al., 41 who reported a decrease in the amplitude of the human ERG after oral administration of melatonin in the afternoon. Unfortunately, the authors did not distinguish the cone from the rod effect. Similarly, Lu et al. 8 also showed that an intramuscular injection of melatonin during the day decreased the amplitude of the photopic b-wave of chickens. The above effects of exogenous melatonin on the ERG amplitude could be due to the fact that this hormone was shown to stimulate cone elongation 16 and suppress the light-adaptive horizontal cell spinule formation. 19 These two phenomena could contribute to the decrease in the cone response, the former by reducing the photon catch and the latter by diminishing the efficiency of synapses between the cones and the horizontal cells. 
On the other hand, it has been suggested that a circadian oscillator could in part regulate retinomotor movements of the cone inner segments. 42 This hypothesis is supported with the observation that the initiation of the cone contraction precedes the actual light onset at dawn and that this rhythm persists even in constant darkness conditions. 42 Therefore, we might be looking here at the impact that the circadian phase of our subjects exerted on the photoreceptor physiology rather than at the direct effect of melatonin on the cone ERG. On the other hand, the retinal secretion of melatonin could be the mediator by which the circadian oscillator modulates the retinomotor movements. 
In this study we also examined if the diurnal variation of the cone response could be wavelength dependent because a previous report of ours 28 showed that the color of the flash stimuli had an impact on the maximal amplitude of the cone b-wave as revealed with the photopic luminance-response curve or Photopic Hill. Our results do not reveal wavelength-dependent effects either in the pattern of diurnal variation or in the correlation with melatonin concentration. There is however a trend for the green and red stimuli to show a greater diurnal variation and a stronger correlation with melatonin compared with the white and blue stimuli. 
For the scotopic response, both groups of subjects showed similar diurnal variations, that is, a decrease in ERG amplitudes and retinal sensitivity at the morning session compared with the evening session. Furthermore, there was no correlation between melatonin and ERG parameters. A similar reduction in rod ERG at around the usual light onset was previously reported. Birch et al. 2 3 observed the lowest rod ERG amplitudes in humans 1.5 hours after light onset. Similarly, a study conducted in albino rabbits showed that the scotopic b-wave demonstrated a 16% reduction in amplitude some 30 minutes after the onset of light. 6 This reduction correlated with an increase in the phagosome count within the pigmented epithelium that is a marker of the disc shedding activity. In pigmented rats, ERG analysis also revealed a decrease in rod sensitivity occurring some 1.5 hours after light onset, a finding also highly correlated with the phagosome count. 43  
Photopigment renewal shows a circadian rhythm controlled intraocularly, the timing of the peak of disc shedding being determined by the light history of each eye. 6 The daily peak of disc shedding activity takes place between 30 minutes and 1.5 hours after the usual light onset. In our subjects, the mean light onset (waketime) during the 5 days before the ERG sessions was 09:04 for the E-type group and 07:40 for the M-type group (see Table 1 ). Consequently, we would have expected the disc shedding effect to be less important for the E-type group because only 2 of the 12 subjects were usually awake at the time of the morning ERG (08:00). Instead, the decrease in ERG amplitude and retinal sensitivity measured in scotopic conditions tended to be larger in E-types compared with M-types. Interestingly, Grace et al. 44 recently reported evidence suggesting that the disc shedding process might actually begin before the expected light onset and consequently would extend over a relatively longer period than originally postulated. This could suggest that the reduction in amplitude and in sensitivity of the scotopic ERG that we report might in fact result from the rod disc shedding. This decreased response could be the consequence of a momentary shortening of the rod outer segments and/or a decrease in the amount of functional rhodopsin. However, although melatonin was previously suggested to be responsible for the circadian regulation of the disc shedding and phagocytosis activities, 15 we believe that the difference in circadian phase between our two groups (∼1.2 hours, on average) may have been insufficient to precisely reveal the impact of melatonin secretion on those two phenomena. 
The results obtained in scotopic mixed conditions, where both rods and cones contribute to the response, support those obtained in both pure rod (scotopic ERG) and cone (photopic ERG) conditions. The largest reduction in amplitude, which we observed in the morning measurements for the E-type group (24% for a-wave and 17% for b-wave), could reflect the combined contribution of both the rod disc shedding process and the higher concentration of melatonin in the morning. In contrast, the opposite effects of rod disc shedding and low concentration of melatonin in the morning for the M-types resulted in a smaller decrease in amplitude (5% for b-wave). A similar explanation could also be offered to elucidate the larger diurnal variation observed in the E-type group compared with the M-type group in photopic conditions (see Table 2 , contrast analyses). The rod disc shedding could not only contribute to the morning decrease seen in the rod ERG but also to that of the cone ERG. The used disks and the phagosomes might diminish the rebound of light on the pigmented epithelium thus reducing both the rod and the cone responses. 
In conclusion, our study brings new evidence supportive of a diurnal variation in the human ERG. Our results further suggest that the circadian oscillator and/or the hormone melatonin would play a key role in this rhythm. This is consistent with several animal studies showing the circadian nature of the diurnal variation of the ERG. 6 7 8 9 10 In our study, melatonin was measured in saliva and thus reflects systemic pineal melatonin. It is impossible to state at this point if the level of retinal melatonin in humans follows the plasmatic one. However, based on immunocytochemical results, it was recently suggested that retinal and pineal melatonin would cooperatively regulate the retinal ganglion cell activity, 45 thus supporting our claim that melatonin (retinal and/or pineal) would be necessary to the normal processing of the visual information. 
 
Figure 1.
 
Representative example of individual scotopic (left) and photopic (right) ERG recordings (top) and their corresponding luminance-response functions (bottom). Each tracing represents an average of 5 responses for the scotopic and 10 for the photopic. Arrows indicate flash onset and letters a, b and i identify the a-, b- and i-waves, respectively. Scotopic and photopic V max and K measurements are indicated on the luminance-response curves. (▪), a-wave; (□), b-wave. The curve fitting line (solid line) in the scotopic graph was obtained with the Naka-Rushton equation.
Figure 1.
 
Representative example of individual scotopic (left) and photopic (right) ERG recordings (top) and their corresponding luminance-response functions (bottom). Each tracing represents an average of 5 responses for the scotopic and 10 for the photopic. Arrows indicate flash onset and letters a, b and i identify the a-, b- and i-waves, respectively. Scotopic and photopic V max and K measurements are indicated on the luminance-response curves. (▪), a-wave; (□), b-wave. The curve fitting line (solid line) in the scotopic graph was obtained with the Naka-Rushton equation.
Table 1.
 
Clock Times for Habitual Sleep Episode and Onset/Offset of Melatonin Secretion in Evening-type (E-Types) and Morning-type (M-Types) Subjects
Table 1.
 
Clock Times for Habitual Sleep Episode and Onset/Offset of Melatonin Secretion in Evening-type (E-Types) and Morning-type (M-Types) Subjects
Variables E-Types M-Types t-Tests
Habitual bedtime 01:36 ± 01:05 23:23 ± 00:29 P < 0.001
Habitual waketime 09:04 ± 01:25 07:40 ± 00:37 P < 0.01
Melatonin onset 23:25 ± 01:12 22:14 ± 00:56 P < 0.05
Melatonin offset 08:35 ± 01:01 07:25 ± 00:38 P < 0.01
Figure 2.
 
Mean (±SEM) salivary melatonin concentration expressed as percentage of the maximum for each subject. (•), E-types; (○), M-types. The black rectangles on the X-axis represent the timing of scotopic ERGs and the white rectangles, the timing of photopic ERGs.
Figure 2.
 
Mean (±SEM) salivary melatonin concentration expressed as percentage of the maximum for each subject. (•), E-types; (○), M-types. The black rectangles on the X-axis represent the timing of scotopic ERGs and the white rectangles, the timing of photopic ERGs.
Figure 3.
 
Typical ERG responses (scotopic V max, scotopic mixed response, photopic V max and photopic OPs) of a E-type (top) and M-type (bottom) subject obtained during the evening (dashed line) and morning (full line) sessions. The Y-axis is in μV and the X-axis in msec, where 0 represents flash onset. Scotopic V max and mixed response were evoked to −2.22 and −0.96 log cd/m2 · sec, respectively. Photopic V max for the E-types and M-types were evoked to 0.39 and 0.17 log cd/m2 · sec, respectively. Photopic maximal OPs for the E-types and M-types were evoked to 0.64 and 0.39 log cd/m2 · sec, respectively. The OP2, OP3, and OP4 are identified on the upper right recording.
Figure 3.
 
Typical ERG responses (scotopic V max, scotopic mixed response, photopic V max and photopic OPs) of a E-type (top) and M-type (bottom) subject obtained during the evening (dashed line) and morning (full line) sessions. The Y-axis is in μV and the X-axis in msec, where 0 represents flash onset. Scotopic V max and mixed response were evoked to −2.22 and −0.96 log cd/m2 · sec, respectively. Photopic V max for the E-types and M-types were evoked to 0.39 and 0.17 log cd/m2 · sec, respectively. Photopic maximal OPs for the E-types and M-types were evoked to 0.64 and 0.39 log cd/m2 · sec, respectively. The OP2, OP3, and OP4 are identified on the upper right recording.
Table 2.
 
Scotopic and Photopic ERG Results
Table 2.
 
Scotopic and Photopic ERG Results
Variables E-Types M-Types ANOVAs Contrast Analyses
22:30 08:00 M/E (%) 22:30 08:00 M/E (%)
Scotopic
V max 192.4 ± 44.0 160.2 ± 34.0 86 ± 20 177.2 ± 37.5 164.2 ± 36.9 93 ± 15 Time effect: F 1,22 = 10.77, P < 0.005
K s −3.72 ± 0.31 −3.49 ± 0.38 94 ± 11 −3.75 ± 0.20 −3.65 ± 0.28 98 ± 10 Time effect: F 1,22 = 4.34, P < 0.05
Scotopic mixed
 b-wave ampl 235.8 ± 69.2 194.8 ± 61.4 83 ± 13 224.3 ± 51.9 212.3 ± 48.4 95 ± 12 Interaction: F 1,22 = 5.14, P < 0.05 E-Types: F 1,22 = 15.76, P < 0.001
 a-wave ampl 112.6 ± 27.1 86.4 ± 35.1 76 ± 25 86.3 ± 23.9 84.1 ± 19.9 102 ± 25 Interaction: F 1,22 = 6.58, P < 0.05 E-Types: F 1,22 = 20.43, P < 0.000
 b-wave time 46.1 ± 1.3 48.0 ± 3.1 104 ± 6 48.5 ± 2.6 47.9 ± 3.1 99 ± 5 Interaction: F 1,22 = 5.12, P < 0.05 E-Types: F 1,22 = 4.89, P < 0.05
 a-wave time 23.9 ± 0.8 25.1 ± 1.6 104 ± 6 24.3 ± 1.2 23.3 ± 1.5 96 ± 6 Interaction: F 1,22 = 12.48, P < 0.005 E-Types: F 1,22 = 7.23, P < 0.01
M-Types: F 1,22=5.32, P < 0.05
Photopic V max
 White 92.8 ± 23.0 85.9 ± 21.6 93 ± 8 85.3 ± 20.7 89.9 ± 18.3 108 ± 22 Interaction: F 1,22 = 7.41, P < 0.01 E-Types: F 1,22 = 5.28, P < 0.05
 Blue 94.0 ± 22.4 86.1 ± 21.5 91 ± 10 90.8 ± 19.3 95.2 ± 20.2 105 ± 11 Interaction: F 1,22 = 9.94, P < 0.005 E-Types: F 1,22 = 8.19, P < 0.01
 Green 92.1 ± 24.9 84.1 ± 22.8 92 ± 14 84.3 ± 21.4 91.8 ± 18.1 113 ± 30 Interaction: F 1,22 = 6.95, P < 0.01 E-Types: F 1,22 = 3.66, P < 0.10
 Red 71.8 ± 20.6 63.5 ± 18.6 89 ± 8 70.3 ± 19.3 74.9 ± 19.1 110 ± 29 Interaction: F 1,22 = 7.70, P < 0.01 E-Types: F 1,22 = 6.41, P < 0.05
Photopic a-wave
 White 25.9 ± 4.3 24.7 ± 6.6 96 ± 23 25.1 ± 5.1 25.8 ± 5.4 104 ± 19 n.s.
 Blue 34.9 ± 8.0 32.3 ± 10.5 92 ± 19 33.3 ± 10.7 33.4 ± 9.0 103 ± 21 n.s.
 Green 27.6 ± 5.9 24.3 ± 6.0 89 ± 19 26.6 ± 5.1 28.7 ± 6.6 110 ± 25 Interaction: F 1,22=5.45, P < 0.05 E-Types: F 1,22 = 4.05, P < 0.05
 Red 24.5 ± 5.2 20.3 ± 6.1 86 ± 31 22.9 ± 6.5 23.4 ± 6.6 105 ± 29 Interaction: F 1,22 = 4.28, P < 0.05 E-Types: F 1,22 = 6.86, P < 0.01
Photopic sum OPs
 White 59.9 ± 18.4 58.0 ± 20.0 96 ± 9 52.3 ± 16.4 54.8 ± 16.6 111 ± 37 n.s.
 Blue 65.8 ± 22.3 55.5 ± 21.5 84 ± 12 57.5 ± 18.6 58.8 ± 18.2 104 ± 15 Interaction: F 1,22 = 12.57, P < 0.005 E-Types: F 1,22 = 19.72, P < 0.000
 Green 62.2 ± 21.4 55.8 ± 20.1 90 ± 17 54.0 ± 19.9 53.6 ± 19.3 103 ± 28 n.s.
 Red 52.6 ± 16.0 48.7 ± 16.2 93 ± 13 44.3 ± 14.0 46.7 ± 15.8 109 ± 31 n.s.
i-wave
 White 30.5 ± 8.4 27.1 ± 8.4 90 ± 23 25.9 ± 8.8 24.8 ± 6.9 103 ± 40 n.s.
Figure 4.
 
Correlations between the evening session over morning session ratios ([22:30/08:00] ×100) of ERG amplitude results and log melatonin concentrations obtained from E-type (filled symbols) and M-type (open symbols) subjects. Numbers higher than 100 on the Y-axis and than 0 on the X-axis identify measurements which were higher during the evening session compared with the morning one. See text for statistical description.
Figure 4.
 
Correlations between the evening session over morning session ratios ([22:30/08:00] ×100) of ERG amplitude results and log melatonin concentrations obtained from E-type (filled symbols) and M-type (open symbols) subjects. Numbers higher than 100 on the Y-axis and than 0 on the X-axis identify measurements which were higher during the evening session compared with the morning one. See text for statistical description.
The authors thank Francois Champoux for his technical assistance with the testing of subjects, Jean Paquet for his help with statistical analyses, and Olga Dembinska for her help with testing procedure and data analysis software. 
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Figure 1.
 
Representative example of individual scotopic (left) and photopic (right) ERG recordings (top) and their corresponding luminance-response functions (bottom). Each tracing represents an average of 5 responses for the scotopic and 10 for the photopic. Arrows indicate flash onset and letters a, b and i identify the a-, b- and i-waves, respectively. Scotopic and photopic V max and K measurements are indicated on the luminance-response curves. (▪), a-wave; (□), b-wave. The curve fitting line (solid line) in the scotopic graph was obtained with the Naka-Rushton equation.
Figure 1.
 
Representative example of individual scotopic (left) and photopic (right) ERG recordings (top) and their corresponding luminance-response functions (bottom). Each tracing represents an average of 5 responses for the scotopic and 10 for the photopic. Arrows indicate flash onset and letters a, b and i identify the a-, b- and i-waves, respectively. Scotopic and photopic V max and K measurements are indicated on the luminance-response curves. (▪), a-wave; (□), b-wave. The curve fitting line (solid line) in the scotopic graph was obtained with the Naka-Rushton equation.
Figure 2.
 
Mean (±SEM) salivary melatonin concentration expressed as percentage of the maximum for each subject. (•), E-types; (○), M-types. The black rectangles on the X-axis represent the timing of scotopic ERGs and the white rectangles, the timing of photopic ERGs.
Figure 2.
 
Mean (±SEM) salivary melatonin concentration expressed as percentage of the maximum for each subject. (•), E-types; (○), M-types. The black rectangles on the X-axis represent the timing of scotopic ERGs and the white rectangles, the timing of photopic ERGs.
Figure 3.
 
Typical ERG responses (scotopic V max, scotopic mixed response, photopic V max and photopic OPs) of a E-type (top) and M-type (bottom) subject obtained during the evening (dashed line) and morning (full line) sessions. The Y-axis is in μV and the X-axis in msec, where 0 represents flash onset. Scotopic V max and mixed response were evoked to −2.22 and −0.96 log cd/m2 · sec, respectively. Photopic V max for the E-types and M-types were evoked to 0.39 and 0.17 log cd/m2 · sec, respectively. Photopic maximal OPs for the E-types and M-types were evoked to 0.64 and 0.39 log cd/m2 · sec, respectively. The OP2, OP3, and OP4 are identified on the upper right recording.
Figure 3.
 
Typical ERG responses (scotopic V max, scotopic mixed response, photopic V max and photopic OPs) of a E-type (top) and M-type (bottom) subject obtained during the evening (dashed line) and morning (full line) sessions. The Y-axis is in μV and the X-axis in msec, where 0 represents flash onset. Scotopic V max and mixed response were evoked to −2.22 and −0.96 log cd/m2 · sec, respectively. Photopic V max for the E-types and M-types were evoked to 0.39 and 0.17 log cd/m2 · sec, respectively. Photopic maximal OPs for the E-types and M-types were evoked to 0.64 and 0.39 log cd/m2 · sec, respectively. The OP2, OP3, and OP4 are identified on the upper right recording.
Figure 4.
 
Correlations between the evening session over morning session ratios ([22:30/08:00] ×100) of ERG amplitude results and log melatonin concentrations obtained from E-type (filled symbols) and M-type (open symbols) subjects. Numbers higher than 100 on the Y-axis and than 0 on the X-axis identify measurements which were higher during the evening session compared with the morning one. See text for statistical description.
Figure 4.
 
Correlations between the evening session over morning session ratios ([22:30/08:00] ×100) of ERG amplitude results and log melatonin concentrations obtained from E-type (filled symbols) and M-type (open symbols) subjects. Numbers higher than 100 on the Y-axis and than 0 on the X-axis identify measurements which were higher during the evening session compared with the morning one. See text for statistical description.
Table 1.
 
Clock Times for Habitual Sleep Episode and Onset/Offset of Melatonin Secretion in Evening-type (E-Types) and Morning-type (M-Types) Subjects
Table 1.
 
Clock Times for Habitual Sleep Episode and Onset/Offset of Melatonin Secretion in Evening-type (E-Types) and Morning-type (M-Types) Subjects
Variables E-Types M-Types t-Tests
Habitual bedtime 01:36 ± 01:05 23:23 ± 00:29 P < 0.001
Habitual waketime 09:04 ± 01:25 07:40 ± 00:37 P < 0.01
Melatonin onset 23:25 ± 01:12 22:14 ± 00:56 P < 0.05
Melatonin offset 08:35 ± 01:01 07:25 ± 00:38 P < 0.01
Table 2.
 
Scotopic and Photopic ERG Results
Table 2.
 
Scotopic and Photopic ERG Results
Variables E-Types M-Types ANOVAs Contrast Analyses
22:30 08:00 M/E (%) 22:30 08:00 M/E (%)
Scotopic
V max 192.4 ± 44.0 160.2 ± 34.0 86 ± 20 177.2 ± 37.5 164.2 ± 36.9 93 ± 15 Time effect: F 1,22 = 10.77, P < 0.005
K s −3.72 ± 0.31 −3.49 ± 0.38 94 ± 11 −3.75 ± 0.20 −3.65 ± 0.28 98 ± 10 Time effect: F 1,22 = 4.34, P < 0.05
Scotopic mixed
 b-wave ampl 235.8 ± 69.2 194.8 ± 61.4 83 ± 13 224.3 ± 51.9 212.3 ± 48.4 95 ± 12 Interaction: F 1,22 = 5.14, P < 0.05 E-Types: F 1,22 = 15.76, P < 0.001
 a-wave ampl 112.6 ± 27.1 86.4 ± 35.1 76 ± 25 86.3 ± 23.9 84.1 ± 19.9 102 ± 25 Interaction: F 1,22 = 6.58, P < 0.05 E-Types: F 1,22 = 20.43, P < 0.000
 b-wave time 46.1 ± 1.3 48.0 ± 3.1 104 ± 6 48.5 ± 2.6 47.9 ± 3.1 99 ± 5 Interaction: F 1,22 = 5.12, P < 0.05 E-Types: F 1,22 = 4.89, P < 0.05
 a-wave time 23.9 ± 0.8 25.1 ± 1.6 104 ± 6 24.3 ± 1.2 23.3 ± 1.5 96 ± 6 Interaction: F 1,22 = 12.48, P < 0.005 E-Types: F 1,22 = 7.23, P < 0.01
M-Types: F 1,22=5.32, P < 0.05
Photopic V max
 White 92.8 ± 23.0 85.9 ± 21.6 93 ± 8 85.3 ± 20.7 89.9 ± 18.3 108 ± 22 Interaction: F 1,22 = 7.41, P < 0.01 E-Types: F 1,22 = 5.28, P < 0.05
 Blue 94.0 ± 22.4 86.1 ± 21.5 91 ± 10 90.8 ± 19.3 95.2 ± 20.2 105 ± 11 Interaction: F 1,22 = 9.94, P < 0.005 E-Types: F 1,22 = 8.19, P < 0.01
 Green 92.1 ± 24.9 84.1 ± 22.8 92 ± 14 84.3 ± 21.4 91.8 ± 18.1 113 ± 30 Interaction: F 1,22 = 6.95, P < 0.01 E-Types: F 1,22 = 3.66, P < 0.10
 Red 71.8 ± 20.6 63.5 ± 18.6 89 ± 8 70.3 ± 19.3 74.9 ± 19.1 110 ± 29 Interaction: F 1,22 = 7.70, P < 0.01 E-Types: F 1,22 = 6.41, P < 0.05
Photopic a-wave
 White 25.9 ± 4.3 24.7 ± 6.6 96 ± 23 25.1 ± 5.1 25.8 ± 5.4 104 ± 19 n.s.
 Blue 34.9 ± 8.0 32.3 ± 10.5 92 ± 19 33.3 ± 10.7 33.4 ± 9.0 103 ± 21 n.s.
 Green 27.6 ± 5.9 24.3 ± 6.0 89 ± 19 26.6 ± 5.1 28.7 ± 6.6 110 ± 25 Interaction: F 1,22=5.45, P < 0.05 E-Types: F 1,22 = 4.05, P < 0.05
 Red 24.5 ± 5.2 20.3 ± 6.1 86 ± 31 22.9 ± 6.5 23.4 ± 6.6 105 ± 29 Interaction: F 1,22 = 4.28, P < 0.05 E-Types: F 1,22 = 6.86, P < 0.01
Photopic sum OPs
 White 59.9 ± 18.4 58.0 ± 20.0 96 ± 9 52.3 ± 16.4 54.8 ± 16.6 111 ± 37 n.s.
 Blue 65.8 ± 22.3 55.5 ± 21.5 84 ± 12 57.5 ± 18.6 58.8 ± 18.2 104 ± 15 Interaction: F 1,22 = 12.57, P < 0.005 E-Types: F 1,22 = 19.72, P < 0.000
 Green 62.2 ± 21.4 55.8 ± 20.1 90 ± 17 54.0 ± 19.9 53.6 ± 19.3 103 ± 28 n.s.
 Red 52.6 ± 16.0 48.7 ± 16.2 93 ± 13 44.3 ± 14.0 46.7 ± 15.8 109 ± 31 n.s.
i-wave
 White 30.5 ± 8.4 27.1 ± 8.4 90 ± 23 25.9 ± 8.8 24.8 ± 6.9 103 ± 40 n.s.
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